U.S. patent application number 15/182958 was filed with the patent office on 2017-03-16 for influenza hemagglutinin proteins and methods of use thereof.
The applicant listed for this patent is AVATAR MEDICAL, LLC. Invention is credited to Peter Joseph Alff, Claudio Bertuccioli, Christopher Patrick Marshall, Mark Andrew Yondola.
Application Number | 20170072045 15/182958 |
Document ID | / |
Family ID | 51905385 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170072045 |
Kind Code |
A1 |
Marshall; Christopher Patrick ;
et al. |
March 16, 2017 |
INFLUENZA HEMAGGLUTININ PROTEINS AND METHODS OF USE THEREOF
Abstract
In some embodiments the present invention provides influenza
hemagglutinin ("HA") polypeptides, proteins, and protein complexes
that comprise a stalk domain that is engineered to facilitate
maintenance of its native trimeric conformation, even if the head
domain of the HA protein is removed or disrupted. In some
embodiments, the present invention provides compositions comprising
such polypeptides, proteins, and protein complexes, and methods of
use of such proteins and compositions, for example as vaccine
immunogens.
Inventors: |
Marshall; Christopher Patrick;
(New York, NY) ; Alff; Peter Joseph; (New York,
NY) ; Bertuccioli; Claudio; (New York, NY) ;
Yondola; Mark Andrew; (Medford, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AVATAR MEDICAL, LLC |
New York |
NY |
US |
|
|
Family ID: |
51905385 |
Appl. No.: |
15/182958 |
Filed: |
June 15, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14450236 |
Aug 2, 2014 |
9393297 |
|
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15182958 |
|
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61861989 |
Aug 3, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/16 20180101;
C12N 2760/16071 20130101; C12N 7/00 20130101; G01N 2333/11
20130101; A61K 39/145 20130101; C07K 2319/50 20130101; C12N
2760/16122 20130101; C07K 2319/735 20130101; C07K 14/005 20130101;
C07K 2319/90 20130101; A61K 45/06 20130101; C12N 2760/16134
20130101; G01N 33/56983 20130101; C12N 2760/16034 20130101; C12N
2760/16151 20130101; G01N 2469/20 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 7/00 20060101 C12N007/00; C07K 14/005 20060101
C07K014/005 |
Claims
1. An influenza hemagglutinin (HA) polypeptide, protein or protein
complex comprising an amino acid sequence having at least 65%
sequence identity to amino acid residues 229 to 519 of SEQ ID NO:
1, wherein the amino acid sequence comprises a point mutation to
tyrosine at one or more of amino acid positions 403, 406, 411, 422,
429, 432, 433, and 435, or an amino acid residue corresponding
thereto.
2. An influenza hemagglutinin (HA) polypeptide, protein or protein
complex according to claim 1, wherein the amino acid sequence
comprises point mutations to tyrosine at two or more of amino acid
positions 403, 406, 411, 422, 429, 432, 433, and 435, or amino acid
residues corresponding thereto.
3. An influenza hemagglutinin (HA) polypeptide that consists
essentially of an amino acid sequence having at least 65% sequence
identity to amino acid residues 229 to 519 of SEQ ID NO: 1, wherein
the amino acid sequence comprises a point mutation to tyrosine at
one or more of amino acid positions 403, 406, 411, 422, 429, 432,
433, and 435, or an amino acid residue corresponding thereto.
4. An influenza HA polypeptide, protein or protein complex
according to claim 1, wherein the polypeptide, protein or protein
complex is folded into a trimeric stalk conformation and comprises
at least one di-tyrosine cross-link, wherein one or both tyrosines
of the at least one di-tyrosine cross-link originate from a point
mutation to tyrosine.
5. An influenza HA polypeptide according to claim 3, wherein the
polypeptide is comprised with an influenza HA protein complex that
is folded into a trimeric stalk conformation, and wherein the
influenza HA protein complex comprises at least one di-tyrosine
cross-link, wherein one or both tyrosines of the at least one
di-tyrosine cross-link originates from a point mutation to
tyrosine.
6. An influenza HA polypeptide, protein or protein complex
according to claim 4, wherein the cross-links are located between
one or more paired tyrosine residues, wherein the paired tyrosine
residues are selected from the group consisting of residues 403 and
433; 411 and 422, 403 and 429, 403 and 432, 433 and 435, and 406
and 433.
7. An influenza HA protein complex according to claim 5, wherein
the cross-links are located between one or more paired tyrosine
residues, wherein the paired tyrosine residues are selected from
the group consisting of residues 403 and 433; 411 and 422, 403 and
429, 403 and 432, 433 and 435, and 406 and 433.
8. An influenza HA polypeptide, protein or protein complex
according to claim 1, wherein the influenza HA polypeptide, protein
or protein complex has the amino acid sequence of SEQ ID NO: 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 86, 87, 88, 89, 90.
91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, or 117, or an amino acid sequence having 65% or
greater sequence identity to any of such sequences.
9. An influenza HA polypeptide, protein or protein complex
according to claim 1, wherein the polypeptide, protein or protein
complex is capable of binding to an HA-stalk-specific antibody.
10. An influenza HA polypeptide, protein or protein complex
according to claim 9, wherein the polypeptide, protein or protein
complex is capable of binding to antibody C179.
11. An influenza HA polypeptide, protein or protein complex
according to claim 1, wherein the polypeptide, protein or protein
complex is capable of folding into a trimeric stalk
conformation.
12. An influenza HA polypeptide, protein or protein complex
according to claim 1, wherein the polypeptide, protein or protein
complex further comprises a trimerization domain.
13. An influenza HA polypeptide, protein or protein complex
according to claim 12, wherein the trimerization domain is a foldon
domain.
14. A nucleic acid molecule encoding an influenza HA polypeptide,
protein or protein complex according to claim 1.
15. A composition comprising an influenza HA polypeptide, protein
or protein complex according to claim 1.
16. The composition of claim 16, wherein the composition is a
vaccine composition.
17. The composition of claim 17, wherein the composition further
comprises an adjuvant, a carrier, an immunostimulatory agent, or
any combination thereof.
18. A composition comprising an influenza HA polypeptide according
to claim 2.
19. A composition comprising an influenza HA polypeptide according
to claim 2 and an influenza HA polypeptide consisting essentially
of an amino sequence having at least 65% sequence identity to SEQ
ID NO: 94 or SEQ ID NO: 95.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
14/450,236, filed on Aug. 2, 2014, which claims the benefit of U.S.
Provisional Patent Application No. 61/861,989, filed Aug. 3, 2013,
the contents of each of which are hereby incorporated by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 1, 2014, is named Avatar_006_US2_Sequence_Listing.txt and
is 411,982 bytes in size.
COPYRIGHT AND INCORPORATION BY REFERENCE
[0003] A portion of the disclosure of this patent document contains
material that is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
[0004] For the purposes of only those jurisdictions that permit
incorporation by reference, the text of all documents cited herein
is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0005] The US and world populations continue to be at risk of a
pandemic influenza outbreak, analogous to the 1918 Spanish (H1N1)
outbreak that killed more than 50 million people. Similarly,
weaponized influenza virus remains a major bio-warfare threat.
Furthermore, antigenic drift requires individuals seeking
protection against influenza to be vaccinated annually, and recent
studies have shown that seasonal vaccine products are only weakly
efficacious if a mismatch occurs between vaccination strains and
circulating strains.
[0006] The development of an effective universal influenza vaccine
that provides protection across strains of influenza virus would be
of enormous value. Evidence that antibodies specific for the
conserved stalk domain of the influenza HA protein can protect
against infection has prompted a concerted effort to identify
additional and better monoclonal antibodies, and to develop a
protective vaccine to address this significant unmet medical and
public health need.
SUMMARY OF THE INVENTION
[0007] Some aspects of the present invention are summarized below.
Additional aspects are described in the Detailed Description of the
Invention, the Examples, the Figures and the Claims sections of the
present patent application.
[0008] The influenza HA protein is known to induce potent
neutralizing antibodies that correlate with protection against
influenza virus infection. Most existing influenza virus vaccines
provide protection based on the generation of antibodies against
the highly variable, immunodominant, head domain of the influenza
HA protein. However, the head domain is often strain-specific so
such vaccines are generally only effective against homologous
influenza strains, and do not provide protection against other
forms of influenza virus, such as homologous drift variants and
heterologous strains. Recently it has been shown that the stalk
domain of influenza HA can elicit antibodies that react across
influenza virus subtypes, due to the more conserved structure of
the stalk domain and the presence of epitopes presented on the
conserved stalk. Also, potent neutralizing antibodies (nAbs) have
been isolated that specifically bind to the native trimeric
conformation of the stalk domain. However, the stalk domain becomes
highly unstable and readily transitions to a non-native
conformation or disassembles upon removal of the HA head
domain--limiting usefulness of the stalk domain on its own (e.g.
without the head domain) as a vaccine immunogen. An influenza HA
protein having a stalk domain stabilized in its native trimeric
conformation could be very valuable--providing a candidate
influenza vaccine immunogen capable of providing protection across
influenza virus strains. Similarly, such a stabilized influenza HA
protein could also be useful for the generation of antibodies, such
as diagnostic and therapeutic antibodies.
[0009] Based on an extensive analysis of the structure of the
influenza HA protein, the present invention provides a variety of
novel design strategies and novel constructs to stabilize or "lock"
the stalk domain of the influenza HA protein in its native trimeric
conformation. The present invention also provides a variety of
engineered influenza HA polypeptides, proteins, and/or protein
complexes, such as those that comprise one or more targeted
cross-links (such as di-tyrosine cross-links), one or more
to-tyrosine mutations, and/or one or more artificially-introduced
protease cleavage sites/motifs. The engineered HA influenza HA
polypeptides, proteins, and/or protein complexes of the invention
can be made using any suitable influenza HA polypeptide or protein
as a starting point. For example, an influenza HA sequence from any
influenza type, sub-type, or strain can be used as a starting point
for generation of the engineered products described herein. In many
of the embodiments described herein, the influenza strain Puerto
Rico/8/1934 or "PR8" (which is a strain of the H1N1 influenza
subtype of influenza A) was used as the starting point. The amino
acid sequence of a wild-type PR8 strain is provided in FIG. 9 (SEQ
ID NO: 1). However, any other influenza HA sequence from any other
influenza type, sub-type, or strain could equally be used.
Non-limiting examples of other influenza HA sequences that can be
used as the starting point for generating the engineered HA
products described herein include, but are not limited to, those
illustrated in FIGS. 55, 56, 57, 58, 59, and 60, and those having
the sequences of SEQ ID NO:s 80, 81, 82, 83, 84, 85, 111, 112, 113,
114, and 115. Similarly, codon optimized versions of the nucleotide
sequences that encode influenza HA proteins can be used as starting
points for the generation of the engineered HA products described
herein. Non-limiting examples of codon-optimized HA sequences from
the PR8 influenza strain include those having the sequences of SEQ
ID NO:s 63, 64, 65, 66, 67, and 68.
[0010] In some embodiments, the present invention provides
influenza HA polypeptides, proteins, and/or protein complexes that
comprise one or more targeted cross-links in their stalk domain
which serve to stabilize or "lock" the stalk domain in its native
trimeric conformation. In some embodiments such targeted
cross-links are di-tyrosine cross-links. In some embodiments, the
present invention provides an influenza HA protein complex
comprising a trimeric stalk domain formed by the association of
three protomers, wherein the stalk domain comprises one or more
targeted cross-links, such as di-tyrosine cross-links, that
stabilize the stalk domain in its native trimeric conformation. In
some such embodiments, the influenza HA protein complex further
comprises one or more cross-links in the influenza HA head domain.
In some such embodiments, the influenza HA protein complex does not
comprise an intact head domain. In embodiments where di-tyrosine
cross-links are used, such cross-links can be made between two
tyrosine residues that are naturally present in an HA polypeptide,
protein, and/or protein complex, or between two tyrosine residues
that have been introduced by mutation, or between a first tyrosine
residue that is naturally present in an HA polypeptide, protein,
and/or protein complex and a second tyrosine residue that has been
introduced by mutation. In some embodiments, the present invention
also provides influenza HA polypeptides, proteins, and/or protein
complexes that comprise one or more "to-tyrosine" mutations in the
HA stalk domain at locations that have been determined to be
desirable locations for the formation of di-tyrosine cross-links to
stabilize the stalk domain in its native trimeric conformation. In
some embodiments, the influenza HA polypeptides, proteins, and/or
protein complexes of the invention (whether containing targeted
cross-links (such as di-tyrosine cross-links), or to-tyrosine
mutations, or both) are full length HA proteins comprising both the
HA stalk domain (with or without the signal peptide) and the HA
head domain, and optionally also the HA transmembrane domain. In
some embodiments the influenza HA polypeptides, proteins, and/or
protein complexes of the invention lack one or more of the HA head
domain, the transmembrane domain, and/or the signal peptide. In
some embodiments the influenza HA polypeptides, proteins, and/or
protein complexes of the invention comprise the HA stalk domain, or
at least a portion of the HA stalk domain that is sufficient to
assemble into, or form a part of, the normal trimeric stalk
conformation. Thus, in some embodiments, it may be possible to
remove, add, or substitute certain HA stalk domain amino acids
without compromising the ability of the HA polypeptide or protein
to assemble into its trimeric conformation.
[0011] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that comprise
to-tyrosine mutations at one or more of amino acid positions 403,
406, 411, 422, 429, 432, 433, and 435, where such amino acid
numbering is based upon the sequence shown in FIG. 9 (SEQ ID NO:
1), or at amino acid positions that correspond to such amino acid
positions, for example as determined by alignment of an HA amino
acid sequence to SEQ ID NO: 1. Non-limiting examples of influenza
HA amino acid sequences that comprise one or more of such
to-tyrosine mutations include SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110.
In some embodiments the present invention provides influenza HA
polypeptides, proteins, and/or protein complexes that comprise
di-tyrosine cross-links between one or more pairs of amino acids
selected from the following amino acid positions: 308, 403, 406,
411, 422, 429, 432, 433, 435, and 437, where such amino acid
numbering is based upon the sequence shown in FIG. 9 (SEQ ID NO:
1), or at amino acid positions that correspond to such amino acid
positions, for example as determined by alignment of an HA amino
acid sequence to SEQ ID NO: 1.
[0012] In some embodiments, the present invention provides
influenza HA polypeptides, proteins, and/or protein complexes that
comprise one or more artificially-introduced protease cleavage
sites that can be used to proteolytically remove the head domain of
an HA polypeptide, protein, and/or protein complex. In some
embodiments the present invention provides influenza HA
polypeptides, proteins, and/or protein complexes that comprise one
or more artificially-introduced protease cleavage sites inserted
after (e.g. immediately after) amino acid positions 48, 63, 228,
278, 282, 283, 286, and 291, where such amino acid numbering is
based upon the sequence shown in SEQ ID NO: 1, or at amino acid
positions that correspond to such amino acid positions, for example
as determined by alignment of an HA amino acid sequence to sequence
ID NO: 1. Non-limiting examples of influenza HA amino acid
sequences that comprise one or more of such artificially-introduced
protease cleavage sites include SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, and 30.
[0013] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that comprise
at least one pair of artificially-introduced protease cleavage
sites, such that cleavage at both of the pair of cleavage sites
will result in removal of the HA head domain. Non-limiting examples
of influenza HA amino acid sequences that comprise a pair of such
artificially-introduced protease cleavage sites include SEQ ID NOs:
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 26, 27, 28,
29, and 30. In some such embodiments, where there are a pair of
artificially-introduced protease cleavage sites, the first such
protease cleavage site is inserted after (e.g. immediately after)
amino acid position 48 or 63, and the second such protease cleavage
site is inserted after (e.g. immediately after) amino acid position
228, 278, 282, 283, 286, or 291, where such amino acid numbering is
based upon the sequence shown in SEQ ID NO: 1, or at amino acid
positions that correspond to such amino acid positions, for example
as determined by alignment of an HA amino acid sequence to sequence
ID NO: 1.
[0014] In some embodiments, the present invention also provides
influenza HA stalk domain polypeptides, proteins, and/or protein
complexes that do not comprise an intact HA head domain, such as
those generated by proteolytic removal of the influenza HA head
domain, for example by cleavage at one or more of the
artificially-introduced protease cleavage sites described herein.
The stalk domain sequences of influenza HA are discontinuous
because the HA protein comprises an N-terminal region comprising
stalk domain sequences, followed by a middle region comprising head
domain sequences, followed by a C-terminal region comprising
additional stalk domain sequences. Accordingly, in some
embodiments, proteolytic cleavage/removal of the HA head domain
results in the generation of two stalk domain polypeptide
fragments--an N-terminal fragment and a C-terminal fragment. In
some embodiments the present invention provides such N- and
C-terminal stalk domain polypeptides, and/or polypeptides,
proteins, or protein complexes that comprise such N- and C-terminal
stalk domain polypeptides. In some embodiments such N- and
C-terminal stalk domain polypeptides are present in an HA stalk
domain protein complex having a native trimeric stalk domain
conformation. Non-limiting examples of influenza HA N-terminal
stalk domain polypeptides include SEQ ID NOs: 94 and 95.
Non-limiting examples of influenza HA C-terminal stalk domain
polypeptides include SEQ ID NOs: 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, and 117. A further
non-limiting example of an influenza HA N-terminal stalk domain
polypeptide is one that consists of, consists essentially of, or
comprises, amino acids 1-228 of SEQ ID NO: 117, or amino acids 229
to 519 of SEQ ID NO: 1. In some embodiments the influenza HA
N-terminal stalk domain polypeptide comprises one or more
to-tyrosine mutations, for example at one or more of positions 403,
406, 411, 422, 429, 432, 433, or 435 of SEQ ID NO: 1, or positions
corresponding thereto (for example as determined by alignment to
SEQ ID NO: 1) or at one or more of positions 112, 115, 120, 131,
137, 141, 142, or 144 of SEQ ID NO: 117, or positions corresponding
thereto (for example as determined by alignment to SEQ ID NO:
117).
[0015] In some embodiments, the present invention provides
influenza HA polypeptides, proteins, and/or protein complexes that
comprise both (a) one or more targeted cross-links, such as
di-tyrosine cross-links in their stalk domain which serve to
stabilize or "lock" the stalk domain in its native trimeric
conformation, and/or one or more "to-tyrosine" mutations in the HA
stalk domain at locations that have been determined to be desirable
locations for the formation of di-tyrosine cross-links to stabilize
the stalk domain in its native trimeric conformation, for example
as described above and elsewhere throughout the present patent
specification, and (b) one or more artificially-introduced protease
cleavage sites that can be used to proteolytically remove the head
domain of the HA polypeptide, protein, and/or protein complex, for
example as described above and elsewhere throughout the present
patent specification. In some embodiments, the present invention
provides an influenza HA polypeptide, protein or protein complex
that comprises: (a) a trimeric stalk domain that comprises one or
more to-tyrosine mutations, and (b) a head domain that comprises
one or more artificially-introduced protease recognition motifs.
Non-limiting examples of influenza HA amino acid sequences that
comprise both a to-tyrosine mutation and an artificially-introduced
protease cleavage site include SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, and 17. In addition, any of the to-tyrosine
mutations and protease cleave site insertions described or
illustrated herein can be combined in the same HA polypeptide,
protein, or protein complex.
[0016] In some embodiments, the present invention provides an
influenza HA protein complex that comprises: (a) a trimeric stalk
domain formed by the association of three protomers, wherein the
stalk domain comprises one or more artificially-introduced targeted
cross links, such as di-tyrosine cross-links (for example, to
stabilize the stalk domain in its native trimeric conformation),
and (b) a head domain that comprises one or more
artificially-introduced protease recognition motifs.
[0017] In some embodiments, the present invention provides a method
of making a headless influenza HA polypeptide, protein or protein
complex, the method comprising: (a) obtaining or expressing an
influenza HA protein comprising (i) a stalk domain and (ii) a head
domain containing one or more artificially-introduced protease
recognition motifs, (b) allowing the soluble influenza HA protein
obtained or expressed in step (a) to fold into its native
conformation having a head domain and a trimeric stalk domain
comprised of three protomers, (c) introducing one more targeted
cross-links, such as di-tyrosine cross-links, into the trimeric
stalk domain in order to stabilize the stalk domain in its native
trimeric conformation, and (d) subsequently proteolytically
cleaving the head domain at the one or more artificially-introduced
protease recognition motifs, thereby producing a headless influenza
HA protein complex. In some such methods the stalk domain comprises
one or more "to-tyrosine" mutations and step (c) comprises
introducing one or more di-tyrosine cross-links into the trimeric
stalk domain. In some such methods, the locations of the
di-tyrosine cross-links, to-tyrosine mutations, and/or
artificially-introduced protease cleavage sites/motifs can be those
specified above and/or elsewhere throughout the present patent
specification. In some such methods, the influenza HA protein may
be expressed in any suitable cell type, including, but not limited
to, mammalian cells or insect cells.
[0018] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of the influenza HA amino acid sequences presented herein, or
any variants or fragments thereof, that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise a to-tyrosine mutation at one or more of
residues 403, 406, 411, 422, 429, 432, 433, and 435, where such
amino acid numbering is based upon the sequence shown in FIG. 9
(SEQ ID NO: 1), or at amino acid positions that correspond to such
amino acid positions, for example as determined by alignment of an
HA amino acid sequence to sequence ID NO: 1.
[0019] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of the influenza HA amino acid sequences presented herein, or
any variants or fragments thereof, that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise an artificially-introduced protease cleavage
site inserted after, for example immediately after, one or more of
the following residues: 48, 63, 228, 278, 282, 283, 286 and 291,
where such amino acid numbering is based upon the sequence shown in
FIG. 9 (SEQ ID NO: 1), or at amino acid positions that correspond
to such amino acid positions, for example as determined by
alignment of an HA amino acid sequence to sequence ID NO: 1.
[0020] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of the influenza HA amino acid sequences presented herein, or
any variants or fragments thereof, that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise two artificially-introduced protease cleavage
sites, the first such site introduced immediately after residue 48
or 63, and the second such site introduced immediately after
residue 228, 278, 282, 283, 286 or 291, where such amino acid
numbering is based upon the sequence shown in FIG. 9 (SEQ ID NO:
1), or at amino acid positions that correspond to such amino acid
positions, for example as determined by alignment of an HA amino
acid sequence to sequence ID NO: 1.
[0021] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of the influenza HA amino acid sequences presented herein, or
any variants or fragments thereof, that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise both (a) a tyrosine residue (whether naturally
occurring or arising from a mutation to-tyrosine), at one or more
of residues 308, 403, 406, 411, 422, 429, 432, 433, 435, or 437,
and (b) an artificially-introduced protease cleavage site inserted
immediately after one or more of the following residues: 48, 63,
228, 278, 282, 283, 286 and 291, where such amino acid numbering is
based upon the sequence shown in FIG. 9 (SEQ ID NO: 1), or at amino
acid positions that correspond to such amino acid positions, for
example as determined by alignment of an HA amino acid sequence to
sequence ID NO: 1.
[0022] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of the influenza HA amino acid sequences presented herein, or
any variants or fragments thereof, that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise both (a) a tyrosine residue (whether naturally
occurring or arising from a mutation to-tyrosine), at one or more
of residues 308, 403, 406, 411, 422, 429, 432, 433, 435, or 437,
and (b) two artificially-introduced protease cleavage sites--the
first such site introduced immediately after residue 48 or 63, and
the second such site introduced immediately after residue 228, 278,
282, 283, 286 or 291, where such amino acid numbering is based upon
the sequence shown in FIG. 9 (SEQ ID NO: 1), or at amino acid
positions that correspond to such amino acid positions, for example
as determined by alignment of an HA amino acid sequence to sequence
ID NO: 1.
[0023] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of amino
acid residues 229 to 519 of SEQ ID NO: 1, or 279 to 519 of SEQ ID
NO: 1, or 283 to 519 of SEQ ID NO: 1, or 284 to 519 of SEQ ID NO:
1, or 287 to 519 of SEQ ID NO: 1, or 292 to 519 of SEQ ID NO: 1, or
SEQ ID NO: 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, or 117, or amino acid residues 1-228 of SEQ ID NO:
117, or sequences that have at least about 40% or 50% or 60% or 65%
or 70% or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity
with such amino acid sequences, wherein the influenza HA
polypeptides, proteins, and/or protein complexes comprise a
tyrosine residue or to-tyrosine mutation at one or more of residues
308, 403, 406, 411, 422, 429, 432, 433, 435, or 437, where such
amino acid numbering is based upon the sequence shown in FIG. 9
(SEQ ID NO: 1), or at amino acid positions that correspond to such
amino acid positions, for example as determined by alignment of an
HA amino acid sequence to sequence ID NO: 1, or at one or more of
residues 112, 115, 120, 131, 137, 141, 142, or 144, where such
amino acid numbering is based upon the sequence shown in FIG. 89
(SEQ ID NO: 117), or at amino acid positions that correspond to
such amino acid positions, for example as determined by alignment
of an HA amino acid sequence to sequence ID NO: 117.
[0024] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of amino
acid residues 1 to 47 of SEQ ID NO: 1, or 1 to 62 of SEQ ID NO: 1,
or sequences that have at least about 40% or 50% or 60% or 65% or
70% or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity with
such amino acid sequences one or more of residues 308, 403, 406,
411, 422, 429, 432, 433, 435, and 437, where such amino acid
numbering is based upon the sequence shown in FIG. 9 (SEQ ID NO:
1), or at amino acid positions that correspond to such amino acid
positions, for example as determined by alignment of an HA amino
acid sequence to sequence ID NO: 1.
[0025] In some embodiments the present invention provides
compositions and/or influenza HA protein complexes that comprise,
consist essentially of, or consist of a first and a second
polypeptide, wherein (a) the first (C-terminal) polypeptide
comprises, consists essentially of, or consists of amino acid
residues 229 to 519 of SEQ ID NO: 1, or 279 to 519 of SEQ ID NO: 1,
or 283 to 519 of SEQ ID NO: 1, or 284 to 519 of SEQ ID NO: 1, or
287 to 519 of SEQ ID NO: 1, or 292 to 519 of SEQ ID NO: 1, or SEQ
ID NO: 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, or 117, or amino acid residues 1-228 of SEQ ID NO: 117,
or sequences that have at least about 40% or 50% or 60% or 65% or
70% or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity with
such amino acid sequences, and wherein the influenza HA
polypeptides, proteins, and/or protein complexes comprise a
tyrosine residue or to-tyrosine mutation, at one or more of
residues 308, 403, 406, 411, 422, 429, 432, 433, 435, or 437, such
amino acid numbering is based upon the sequence shown in FIG. 9
(SEQ ID NO: 1), or at amino acid positions that correspond to such
amino acid positions, for example as determined by alignment of an
HA amino acid sequence to sequence ID NO: 1, or at one or more of
residues 112, 115, 120, 131, 137, 141, 142, or 144, where such
amino acid numbering is based upon the sequence shown in FIG. 89
(SEQ ID NO: 117), or at amino acid positions that correspond to
such amino acid positions, for example as determined by alignment
of an HA amino acid sequence to sequence ID NO: 117, and wherein
(b) the second (N-terminal) polypeptide comprises, consists
essentially of, or consists of amino acid residues 1 to 47 of SEQ
ID NO: 1, or 1 to 62 of SEQ ID NO: 1, where such amino acid
numbering is based upon the sequence shown in FIG. 9 (SEQ ID NO:
1), or at amino acid positions that correspond to such amino acid
positions, for example as determined by alignment of an HA amino
acid sequence to sequence ID NO: 1, or sequences that have at least
about 40% or 50% or 60% or 65% or 70% or 75% or 80% or 85% or 90%
or 95% or 98% or 99% identity with such amino acid sequences.
[0026] In some embodiments, the present invention provides an
influenza hemagglutinin (HA) polypeptide, protein or protein
complex comprising, consisting essentially of, or consisting of, an
amino acid sequence having at least 50, 55, 60, 65, or 70% sequence
identity to amino acid residues 229 to 519 of SEQ ID NO: 1, wherein
the amino acid sequence comprises a point mutation to tyrosine at
one or more of amino acid positions 403, 406, 411, 422, 429, 432,
433, and 435, where such amino acid numbering is based upon the
sequence shown in FIG. 9 (SEQ ID NO: 1), or at amino acid positions
that correspond to such amino acid positions, for example as
determined by alignment of an HA amino acid sequence to sequence ID
NO: 1. In some embodiments, the present invention provides an
influenza hemagglutinin (HA) polypeptide, protein or protein
complex comprising, consisting essentially of, or consisting of, an
amino acid sequence having at least 50, 55, 60, 65, or 70% sequence
identity to amino acid residues 1 to 228 of SEQ ID NO: 117, wherein
the amino acid sequence comprises a point mutation to-tyrosine at
one or more of amino acid positions 112, 115, 120, 131, 137, 141,
142, or 144, where such amino acid numbering is based upon the
sequence shown in FIG. 89 (SEQ ID NO: 117), or at amino acid
positions that correspond to such amino acid positions, for example
as determined by alignment of an HA amino acid sequence to sequence
ID NO: 117. In some such embodiments the influenza HA polypeptide,
protein or protein complex forms a part of, and/or is folded into a
protein complex having, or capable of forming, a trimeric stalk
conformation, and that comprises at least one di-tyrosine
cross-link, wherein one or both tyrosines of the at least one
di-tyrosine cross-link originate from one of the to-tyrosine
mutations. In some such embodiments, the influenza HA polypeptide,
protein or protein complex comprises cross-links located between
one or more paired tyrosine residues, wherein the paired tyrosine
residues are selected from the group consisting of residues 403 and
433; 411 and 422, 403 and 429, 403 and 432, 433 and 435, and 406
and 433, where such amino acid numbering is based upon the sequence
shown in FIG. 9 (SEQ ID NO: 1), or at amino acid positions that
correspond to such amino acid positions, for example as determined
by alignment of an HA amino acid sequence to sequence ID NO: 1.
[0027] In some embodiments the HA polypeptides, proteins or protein
complexes described herein are capable of folding into a trimeric
stalk conformation. In some such embodiments, the influenza HA
polypeptides, proteins or protein complexes described herein
further comprise one or more point mutations to cysteine. In some
embodiments, the influenza HA polypeptides, proteins or protein
complexes described herein further comprise a trimerization domain,
such as a foldon domain.
[0028] Non-limiting examples of influenza HA polypeptides, proteins
and/or protein complexes of the invention include, but are not
limited to, those of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 86, 87, 88, 89, 90. 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, and 117.
[0029] In some embodiments the influenza HA polypeptides, proteins
or protein complexes described herein are capable of eliciting
production of influenza HA-specific antibodies in a subject. In
some embodiments, the influenza HA polypeptides, proteins or
protein complexes described herein are capable of binding to an
antibody that recognizes the trimeric stalk domain of influenza
HA.
[0030] In some embodiments the present invention provides nucleic
acid molecules encoding the influenza HA polypeptides, proteins or
protein complexes described herein.
[0031] In some embodiments, the present invention provides
compositions comprising the influenza HA polypeptides, proteins or
protein complexes described herein, including, but not limited to,
vaccine compositions. In some such embodiments, such compositions
may further comprise an adjuvant, a carrier, an immunostimulatory
agent, or any combination thereof.
[0032] In some embodiments the present invention provides a method
of vaccinating a subject against influenza, the method comprising
administering to a subject a composition comprising an effective
amount of an influenza HA polypeptide, protein or protein complex
as described herein.
[0033] These and other embodiments of the present invention are
described throughout the present patent specification.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0034] FIG. 1. Schematic representation of a headless universal
vaccine immunogen (PR8) presenting a QNE-bnAb complex (left), and
the same bnAb neutralizing HA of (a) homologous PR8, (b) drift
(NL09), (c) group 1 heterologous (VN04), and (d) group 2
heterologous (x31) virus on the right.
[0035] FIGS. 2A-2B. Schematic representation of DT-cross-links in
headless HA stabilizing the stalk trimer. A. DT bonds (top in
black) conformationally lock the stalk trimer. B. The stalk trimer
has fallen apart without conformational locking. The QNE is
lost.
[0036] FIG. 3. Schematic diagram of a top-down view of the stalk,
showing HA variant design: two amino acid substitutions per
protomer (black and white circles).
[0037] FIGS. 4A-4B. (A) DT-specific fluorescence measurement at 405
nm or WT (negative control, left), four HA variants with two amino
acid substitutions each, and insulin, as it forms DT bonds with
high efficiency (positive control, right). (B) Relative
fluorescence of dityrosine mutants. Data represents the average of
four replicates with standard deviation indicated by the error
bars.
[0038] FIG. 5. Annotated crystal structure of HA bound to CRC261.
The lower circle indicates the targeted area for DT bond formation,
the middle circle indicates the targeted area for stalk-proximal
proteolytic cleavage, and the upper circle indicates the targeted
area for variable loop proteolysis designed to unravel the head to
enable stalk-proximal cleavage site access.
[0039] FIG. 6. Immunofluorescent staining of cells expressing WT
and a headless HA protein without cross-linking to stabilize the
stalk domain demonstrated that the non-stabilized headless HA
protein dis not bind one of the most broadly reactive mAbs, C179.
A549 cells were transfected with plasmids for the expression of
either WT HA or a recombinantly-spliced headless construct without
any cross-linking in the stalk domain. 24 hrs post transfection,
cells were fixed, permeabilized, and the HA protein was detected
with both rabbit polyclonal, pAB (general expression) (upper
panels), and mAb C179 anti-stalk (conformational) (lower panels)
primary Abs followed by anti-rabbit Alexa 555-conjugated and
anti-mouse Alexa 488-conjugatedsecondary Abs.
[0040] FIGS. 7A-7B. DT crosslinks form in the PR8 stalk
efficiently, and C179 antigenicity is preserved before & after
crosslinking. A. DT-specific fluorescence measurement at
ex320/em405 nm of WT (neg. control, A), four HA variants with two
amino acid substitutions each (to-Tyr substitutions), at residues
403 and 429 (B), 406 and 433 (C), 403 and 433 (D), and 403 and 432
(E) and insulin, which forms DT bonds with high efficiency
(positive control, F). B. C179 binding to variants (B-E) before and
after DT crosslinking, as measured by sandwich ELISA using goat
polyclonal anti-HA antibody for capture (BEI catalog # NR-3148) and
the C179 conformational Ab for detection.
[0041] FIGS. 8A-8C. 293T cells were untransfected (-) or
transfected with WT NA and the indicated HA plasmids. 72 hours post
transfection, VLPs in supernatants and WCEs were analyzed by
sandwich ELISA (A, BEI catalog # NR-3148 goat polyclonal anti-HA
capture, C179 detection), western blot (B, left panel; PNGase
treated WCE), and HA assay (C). Panel B, right. Cells were
transfected as above as indicated with HA and NA. 72 hours post
transfection, VLPs were purified over a 30% sucrose-NTE cushion,
assayed for total protein, and either mock incubated (WT, 48G) or
digested with TEV protease (WT+TEV, 48G+TEV) and PNGase treated.
Percent cleavage was determined by western blot.
[0042] FIG. 9. Amino acid sequence (SEQ ID NO. 1) of HA protein
from PR8 strain of H1N1 influenza virus. Amino acids 59 through 291
comprise the head domain, which may be proteolytically removed or
disrupted in some embodiments. Amino acids 1 through 58 (or 18 to
58 without the signal peptide--which is located at residues 1-17)
and 292 through 566 (or 292 through 529 without the transmembrane
domain and cytoplasmic tail) comprise the stalk domain. The stalk
domain is discontinuous and comprises both an N-terminal and a
C-terminal portion of the HA protein. Amino acids 529 through 565
comprise the transmembrane region and cytoplasmic tail. The HA
ectodomain (i.e. the outer exposed/non-membrane bound portion)
comprises residues 1-528 (or 18 to 528 without the signal
peptide).
[0043] FIG. 10. Nucleic acid sequence (SEQ ID NO. 2) of DNA
encoding HA protein from PR8 strain of H1N1 influenza virus.
[0044] FIG. 11. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 278 (underlined), and to-tyrosine mutations at
positions 403 (N403Y) and 433 (D433Y) (underlined) (SEQ ID NO:3).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:3 is encoded by the nucleic
acid sequence of SEQ ID NO:31 shown in FIG. 28.
[0045] FIG. 12. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 278 (underlined), and to-tyrosine mutations at
positions 411 (K411Y) and 422 (N422Y) (underlined) (SEQ ID NO:4).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:4 is encoded by the nucleic
acid sequence of SEQ ID NO:32 shown in FIG. 29.
[0046] FIG. 13. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 278 (underlined), and to-tyrosine mutations at
positions 403 (N403Y), 411 (K411Y), 422 (N422Y), and 433 (D433Y)
(underlined) (SEQ ID NO:5). The boxed C-terminal sequence comprises
the transmembrane region. The amino acid sequence of SEQ ID NO:5 is
encoded by the nucleic acid sequence of SEQ ID NO:33 shown in FIG.
30.
[0047] FIG. 14. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 282 (underlined), and to-tyrosine mutations at
positions 403 (N403Y) and 433 (D433Y) (underlined) (SEQ ID NO:6).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:6 is encoded by the nucleic
acid sequence of SEQ ID NO:34 shown in FIG. 31.
[0048] FIG. 15. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 282 (underlined), and to-tyrosine mutations at
positions 411 (K411Y) and 422 (N422Y) (underlined) (SEQ ID NO:7).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:7 is encoded by the nucleic
acid sequence of SEQ ID NO:35 shown in FIG. 32.
[0049] FIG. 16. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 228 (underlined), and to-tyrosine mutations at
positions 403 (N403Y), 411 (K411Y), 422 (N422Y), and 433 (D433Y)
(underlined) (SEQ ID NO:8). The boxed C-terminal sequence comprises
the transmembrane region. The amino acid sequence of SEQ ID NO:8 is
encoded by the nucleic acid sequence of SEQ ID NO:36 shown in FIG.
33.
[0050] FIG. 17. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 283 (underlined), and to-tyrosine mutations at
positions 403 (N403Y) and 433 (D433Y) (underlined) (SEQ ID NO:9).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:9 is encoded by the nucleic
acid sequence of SEQ ID NO:37 shown in FIG. 34.
[0051] FIG. 18. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 283 (underlined), and to-tyrosine mutations at
positions 411 (K411Y) and 422 (N422Y) (underlined) (SEQ ID NO:10).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:10 is encoded by the nucleic
acid sequence of SEQ ID NO:38 shown in FIG. 35.
[0052] FIG. 19. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 63 and 283 (underlined), and to-tyrosine mutations at
positions 403 (N403Y), 411 (K411Y), 422 (N422Y), and 433 (D433Y)
(underlined) (SEQ ID NO:11). The boxed C-terminal sequence
comprises the transmembrane region. The amino acid sequence of SEQ
ID NO:11 is encoded by the nucleic acid sequence of SEQ ID NO:39
shown in FIG. 36.
[0053] FIG. 20. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 403 (N403Y) and 433 (D433Y) (underlined) (SEQ ID NO:12).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:12 is encoded by the nucleic
acid sequence of SEQ ID NO:43 shown in FIG. 40.
[0054] FIG. 21. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 411 (K411Y) and 422 (N422Y) (underlined) (SEQ ID NO:13).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:13 is encoded by the nucleic
acid sequence of SEQ ID NO:44 shown in FIG. 41.
[0055] FIG. 22. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 403 (N403Y), 411 (K411Y), 422 (N422Y), and 433 (D433Y)
(underlined) (SEQ ID NO:14). The boxed C-terminal sequence
comprises the transmembrane region. The amino acid sequence of SEQ
ID NO:14 is encoded by the nucleic acid sequence of SEQ ID NO:45
shown in FIG. 42.
[0056] FIG. 23. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 403 (N403Y) and 433 (D433Y) (underlined) (SEQ ID NO:15).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:15 is encoded by the nucleic
acid sequence of SEQ ID NO:46 shown in FIG. 43.
[0057] FIG. 24. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 411 (K411Y) and 422 (N422Y) (underlined) (SEQ ID NO:16).
The boxed C-terminal sequence comprises the transmembrane region.
The amino acid sequence of SEQ ID NO:16 is encoded by the nucleic
acid sequence of SEQ ID NO:47 shown in FIG. 44.
[0058] FIG. 25. Amino acid sequence of a modified PR8 influenza HA
protein comprising inserted TEV protease cleavage sites at
positions 48 and 291 (underlined), and to-tyrosine mutations at
positions 403 (N403Y), 411 (K411Y), 422 (N422Y), and 433 (D433Y)
(underlined) (SEQ ID NO:17). The boxed C-terminal sequence
comprises the transmembrane region. The amino acid sequence of SEQ
ID NO:17 is encoded by the nucleic acid sequence of SEQ ID NO:48
shown in FIG. 45.
[0059] FIGS. 26A-26B. Amino acid sequence alignment of modified PR8
influenza HA proteins comprising one inserted protease cleavage
site, and the sequence of wild-type PR8 HA from the PR8 strain of
influenza virus H1N1 (SEQ ID NO:1--identified as "PR8HA-WT" in the
figure). Underlined amino acid residues indicate protease cleavage
sites inserted into the wild-type sequence by substitution and/or
replacement of amino acids in SEQ ID NO. 1. Protease cleavage sites
are inserted immediately after the following amino acid residues:
291 (SEQ ID NO. 18 and SEQ ID NO. 19), 48 (SEQ ID NO. 20), 286 (SEQ
ID NO. 21), 278 (SEQ ID NO. 22), 282, (SEQ ID NO. 23), 63 (SEQ ID
NO. 24), or 283 (SEQ ID NO. 25). The inserted protease cleavage
sites are TEV protease recognition sequences. The C-terminal
sequences shown within the boxed portion of the alignment comprise
the transmembrane regions of the influenza HA proteins. The amino
acid sequences of SEQ ID NO. 18, 19, 20, 21, 22, 23, 24 and 25 are
encoded by the nucleic acid sequences of SEQ ID NO. 49, 50, 52, 56,
53, 54, 51 and 55, respectively as shown in FIG. 46.
[0060] FIGS. 27A-27B. Amino acid sequence alignment of modified PR8
influenza HA proteins comprising two inserted protease cleavage
sites, and the sequence of wild-type PR8 HA from the PR8 strain of
influenza virus H1N1 (SEQ ID NO:1--identified as "PR8HA-WT" in the
figure). Protease cleavage sites are inserted immediately after the
following amino acid residues: 63 and 278 (SEQ ID NO. 26), 63 and
282 (SEQ ID NO. 27), 63 and 283 (SEQ ID NO. 28), 48 and 291 (SEQ ID
NO. 29 and 30). The inserted protease cleavage sites are TEV
protease recognition sequences. Underlined amino acid residues
indicate the sequence located between the protease cleavage sites
that would be removed from the HA sequence upon cleavage by a
protease (here, TEV protease), for example to facilitate the
production of a "headless" HA protein where the head domain is
disrupted or removed. The C-terminal sequences shown within the
boxed portion of the alignment comprise the transmembrane regions
of the influenza HA proteins. Amino acid residues shown in bold
(N403, F406, K411, N422, D429, L432, D433 and W435) illustrate
positions where to-tyrosine mutations may be made so as to
facilitate the formation of dityrosine bonds in the influenza HA
stalk domain, as described herein. The amino acid sequences of SEQ
ID NO. 26, 27, 28, 29 and 30 are encoded by the nucleic acid
sequences of SEQ ID NO. 57, 58, 62, 60 and 61, respectively as
shown in FIG. 47.
[0061] FIG. 28. Nucleic acid sequence (SEQ ID NO:31) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 278 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0062] FIG. 29. Nucleic acid sequence (SEQ ID NO:32) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 278 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0063] FIG. 30. Nucleic acid sequence (SEQ ID NO:33) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 278 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0064] FIG. 31. Nucleic acid sequence (SEQ ID NO:34) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 282 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0065] FIG. 32. Nucleic acid sequence (SEQ ID NO:34) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 282 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0066] FIG. 33. Nucleic acid sequence (SEQ ID NO:36) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 282 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0067] FIG. 34. Nucleic acid sequence (SEQ ID NO:37) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 283 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0068] FIG. 35. Nucleic acid sequence (SEQ ID NO:38) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 283 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0069] FIG. 36. Nucleic acid sequence (SEQ ID NO:39) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 283 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0070] FIG. 37. Nucleic acid sequence (SEQ ID NO:40) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 286 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0071] FIG. 38. Nucleic acid sequence (SEQ ID NO:41) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 286 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0072] FIG. 39. Nucleic acid sequence (SEQ ID NO:42) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 63 and 286 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0073] FIG. 40. Nucleic acid sequence (SEQ ID NO:43) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0074] FIG. 41. Nucleic acid sequence (SEQ ID NO:44) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0075] FIG. 42. Nucleic acid sequence (SEQ ID NO:45) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0076] FIG. 43. Nucleic acid sequence (SEQ ID NO:46) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y)
and 433 (D433Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0077] FIG. 44. Nucleic acid sequence (SEQ ID NO:47) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 411 (K411Y)
and 422 (N422Y) in the protein. The boxed C-terminal sequence
comprises the nucleic acid sequence that encodes the transmembrane
region of the protein.
[0078] FIG. 45. Nucleic acid sequence (SEQ ID NO:48) encoding a
modified PR8 influenza HA protein comprising inserted nucleic acid
residues (shown in lower case) that encode TEV protease cleavage
sites at positions 48 and 291 in the protein, and to-tyrosine
mutations (shown in lower case) encoded at positions 403 (N403Y),
411 (K411Y), 422 (N422Y), and 433 (D433Y) in the protein. The boxed
C-terminal sequence comprises the nucleic acid sequence that
encodes the transmembrane region of the protein.
[0079] FIGS. 46A-46F. Alignment of nucleic acid sequences encoding
modified PR8 influenza HA proteins comprising one inserted protease
cleavage site, and the sequence of wild-type HA protein from the
PR8 strain of influenza virus H1N1 (SEQ ID NO:2--identified as
"RR8HA-WT" in the figure). Underlined nucleic acid residues encode
TEV protease cleavage sites by substitution and/or replacement of
nucleic acid residues of SEQ ID NO. 2. The nucleic acid residues
are inserted into the nucleic acid sequence such that the encoded
protein will have a protease cleavage site immediately after the
following amino acid residues: 291 (SEQ ID NO. 49 and SEQ ID NO.
50), 48 (SEQ ID NO. 52), 286 (SEQ ID NO. 56), 278 (SEQ ID NO. 53),
282 (SEQ ID NO. 54), 63 (SEQ ID NO. 51), or 283 (SEQ ID NO. 55).
The boxed C-terminal sequences comprise the sequence that encodes
the transmembrane region of the protein.
[0080] FIGS. 47A-47E. Alignment of nucleic acid sequences encoding
PR8 influenza HA proteins comprising two inserted protease cleavage
sites, and the sequence of wild-type HA from the PR8 strain of
influenza virus H1N1 (SEQ ID NO:2--identified as "PR8HA-WT" in the
figure). Underlined nucleic acid residues encode TEV protease
cleavage sites by substitution and/or replacement of nucleic acid
residues of SEQ ID NO. 2. The nucleic acid residues are inserted
into the nucleic acid sequence such that the encoded HA protein
will have protease cleavage sites immediately after the following
amino acid residues: 63 and 278 (SEQ ID NO. 57), 63 and 282 (SEQ ID
NO. 58), 63 and 286 (SEQ ID NO. 59), 48 and 291 (SEQ ID NO. 60 and
61), and 63 and 283 (SEQ ID NO. 62). The boxed C-terminal sequences
comprise the sequence that encodes the transmembrane region of the
protein. Boxed nucleic acid residues (corresponding to amino acid
positions N403, F406, K411, N422, D429, L432, D433 and W435 in the
encoded HA protein) illustrate positions where to-tyrosine
mutations may be made so as to facilitate the formation of
dityrosine bonds in the stalk domain of the encoded influenza HA
protein, as described herein.
[0081] FIG. 48. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Homo sapiens (SEQ ID
NO:63).
[0082] FIG. 49. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Cricetulus griseus (SEQ ID
NO:64).
[0083] FIG. 50. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Nicotiana benthamiana (SEQ
ID NO:65).
[0084] FIG. 51. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Pichia pastoris (SEQ ID
NO:66).
[0085] FIG. 52. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Saccharomyces cerevisiae
(SEQ ID NO:67).
[0086] FIG. 53. Nucleic acid sequence encoding HA protein of PR8
strain of influenza virus H1N1 with codon optimization for
expression of the encoded HA protein in Spodoptera frugiperda (SEQ
ID NO:68).
[0087] FIGS. 54A-54C. Alignment of amino acid sequences of
full-length versions of HA proteins from various strains of
influenza virus (Udorn 72 (SEQ ID NO:73), Hong Kong 68 (SEQ ID
NO:74), Panama 99 (SEQ ID NO:75), Wisconsin 05 (SEQ ID NO:76),
Shanghai 13 (SEQ ID NO:77), Singapore 57 (SEQ ID NO:78), Vietnam 04
(SEQ ID NO:79) and PR8 34 (SEQ ID NO:1), USSR 77 (SEQ ID NO:111),
Texas 91 (SEQ ID NO:112), WSN 33 (SEQ ID NO:113), South Carolina
1918 (SEQ ID NO:114), and California 09 (SEQ ID NO:115)). Boxed
amino acid residues (corresponding to amino acid positions 403,
406, 411, 422, 429, 432, 433 and 435 in the sequence of wild-type
HA from the PR8 strain of influenza virus H1N1 (SEQ ID
NO:1--identified as "WT-PR8-34" in the figure) represent positions
where a mutation to a tyrosine residue is contemplated to
facilitate the formation of dityrosine bonds in the stalk region of
the HA protein. The italicized C-terminal sequences comprise the
sequence that encodes the endogenous transmembrane region of the
protein, and which can be removed or disrupted so as to generate a
soluble version of influenza HA protein (see, for example, FIGS.
55-60).
[0088] FIG. 55. Amino acid sequence of a soluble version of HA
protein from the PR8 strain of influenza virus (SEQ ID NO:80).
Amino acids 520-565 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:1 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0089] FIG. 56. Amino acid sequence of a soluble version of HA
protein from the Hong Kong 68 strain of influenza virus (SEQ ID
NO:81). Amino acids 521-566 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:74 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0090] FIG. 57. Amino acid sequence of a soluble version of HA
protein from the Wisconsin 05 strain of influenza virus (SEQ ID
NO:82). Amino acids 521-566 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:76 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0091] FIG. 58. Amino acid sequence of a soluble version of HA
protein from the Vietnam 04 strain of influenza virus (SEQ ID
NO:83). Amino acids 522-568 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:79 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0092] FIG. 59. Amino acid sequence of a soluble version of HA
protein from the Shanghai 13 strain of influenza virus (SEQ ID
NO:84). Amino acids 515-560 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:77 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0093] FIG. 60. Amino acid sequence of a soluble version of HA
protein from the Singapore 57 strain of influenza virus (SEQ ID
NO:85). Amino acids 516-562 of the endogenous transmembrane region
(italicized C-terminal sequence of SEQ ID NO:78 in FIG. 54) have
been replaced by an optional tag (underlined) comprising a thrombin
cleavage domain, a T4 foldon trimerization motif, and a 6.times.His
tag (SEQ ID NO: 118).
[0094] FIG. 61. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 403 (N403Y)
and 429 (D429Y) (underlined) (SEQ ID NO:86).
[0095] FIG. 62. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 403 (N403Y)
and 432 (L432Y) (underlined) (SEQ ID NO:87).
[0096] FIG. 63. Amino acid sequence of a modified PR8 influenza HA
protein comprising one to-tyrosine mutations at position 403
(N403Y) (underlined) (SEQ ID NO:88).
[0097] FIG. 64. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 403 (N403Y)
and 433 (D433Y) (underlined) (SEQ ID NO:89).
[0098] FIG. 65. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 433 (D433Y)
and 435 (W435Y) (underlined) (SEQ ID NO:90).
[0099] FIG. 66. Amino acid sequence of a modified PR8 influenza HA
protein comprising one to-tyrosine mutations at position 435
(W435Y) (underlined) (SEQ ID NO:91).
[0100] FIG. 67. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 406 (F406Y)
and 433 (D433Y) (underlined) (SEQ ID NO:92).
[0101] FIG. 68. Amino acid sequence of a modified PR8 influenza HA
protein comprising to-tyrosine mutations at positions 411 (K411Y)
and 422 (N422Y) (underlined) (SEQ ID NO:93).
[0102] FIGS. 69A-69B. Amino acid sequence alignment of modified PR8
influenza HA proteins comprising one or more to-tyrosine mutations,
and the sequence of wild-type PR8 HA from the PR8 strain of
influenza virus H1N1 (SEQ ID NO:1--identified as "PR8HA-WT" in the
figure). Dityrosine bonds may be introduced between various
combinations of endogenous tyrosine residues (e.g. Y308 and Y437 of
SEQ ID NO:1, shown in bold) and residues comprising to-tyrosine
mutations (e.g. N403, F406, K411, N422, D429, L432, D433 and W435
of SEQ ID NO:1, shown as underlined), as described herein.
[0103] FIG. 70. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/278S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:96) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 120 and 150
(underlined; corresponding to amino acid positions 403 and 433,
respectively, in SEQ ID NO:1).
[0104] FIG. 71. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/278S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:97) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 128 and 139
(underlined; corresponding to amino acid positions 411 and 422,
respectively, in SEQ ID NO:1).
[0105] FIG. 72. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/278S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:98) is the C-terminal portion of the stalk domain comprising
four to-tyrosine mutations at amino acid positions 120, 128, 139
and 150 (underlined; corresponding to amino acid positions 403,
411, 422 and 433, respectively, in SEQ ID NO:1).
[0106] FIG. 73. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/282S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:99) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 122 and 152
(underlined; corresponding to amino acid positions 403 and 433,
respectively, in SEQ ID NO:1).
[0107] FIG. 74. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/282S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:100) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 130 and 141
(underlined; corresponding to amino acid positions 411 and 422,
respectively, in SEQ ID NO:1).
[0108] FIG. 75. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/282S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:101) is the C-terminal portion of the stalk domain comprising
four to-tyrosine mutations at amino acid positions 122, 130, 141
and 152 (underlined; corresponding to amino acid positions 403,
411, 422 and 433, respectively, in SEQ ID NO:1).
[0109] FIG. 76. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/283G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:102) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 121 and 151
(underlined; corresponding to amino acid positions 403 and 433,
respectively, in SEQ ID NO:1).
[0110] FIG. 77. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/283G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:103) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 129 and 140
(underlined; corresponding to amino acid positions 411 and 422,
respectively, in SEQ ID NO:1).
[0111] FIG. 78. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (63G/283G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:94) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:104) is the C-terminal portion of the stalk domain comprising
four to-tyrosine mutations at amino acid positions 121, 129, 140
and 151 (underlined; corresponding to amino acid positions 403,
411, 422 and 433, respectively, in SEQ ID NO:1).
[0112] FIG. 79. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:105) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 113 and 143
(underlined; corresponding to amino acid positions 403 and 433,
respectively, in SEQ ID NO:1).
[0113] FIG. 80. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:106) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 121 and 132
(underlined; corresponding to amino acid positions 411 and 422,
respectively, in SEQ ID NO:1).
[0114] FIG. 81. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291G) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:107) is the C-terminal portion of the stalk domain comprising
four to-tyrosine mutations at amino acid positions 113, 121, 132
and 143 (underlined; corresponding to amino acid positions 403,
411, 422 and 433, respectively, in SEQ ID NO:1).
[0115] FIG. 82. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:108) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 113 and 143
(underlined; corresponding to amino acid positions 403 and 433,
respectively, in SEQ ID NO:1).
[0116] FIG. 83. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:109) is the C-terminal portion of the stalk domain comprising
two to-tyrosine mutations at amino acid positions 121 and 132
(underlined; corresponding to amino acid positions 411 and 422,
respectively, in SEQ ID NO:1).
[0117] FIG. 84. Protein fragments comprising a `headless` influenza
HA protein generated after proteolysis at two protease cleavage
sites (48G/291S) inserted into the full-length starting sequence
(PR8 HA, SEQ ID NO:1). The first fragment (SEQ ID NO:95) is the
N-terminal portion of the stalk domain and the second fragment (SEQ
ID NO:110) is the C-terminal portion of the stalk domain comprising
four to-tyrosine mutations at amino acid positions 113, 121, 132
and 143 (underlined; corresponding to amino acid positions 403,
411, 422 and 433, respectively, in SEQ ID NO:1).
[0118] FIGS. 85A-85C. (A) 293T Cells were transfected with
constructs for the expression of the indicated HA dityrosine
mutants (403Y, 411Y-422Y, 403Y-433Y and 433Y-435Y) and soluble HA
protein (with a C-terminal foldon domain) was purified from
supernatants 72 hours post transfection by Ni.sup.2+ affinity
chromatography. Pure HA protein was then subjected to dityrosine
crosslinking conditions in the presence (+) or absence (-) of the
required ARP peroxidase enzyme and analyzed by reducing SDS-PAGE
followed by Coomassie blue staining. The arrow marks the migration
of the monomer and crosslinked trimer, as indicated. (B) To confirm
the formation of Dityrosine crosslinks, the purified crosslinked
and uncrosslinked samples, obtained as described in A, were
analyzed for DT-specific fluorescence: excitation wavelength: 320
nm, emission wavelength: 405 nm. (C) Binding of the soluble
403Y-433Y HA mutant, before and after crosslinking, to the broadly
neutralizing V.sub.H1-69 stalk-specific mAb 8D4 by direct capture
ELISA at 20 .mu.g/ml of 8D4.
[0119] FIGS. 86A-86B. (A) 293T cells were transfected with plasmids
for the expression of HA (WT and the indicated insertion mutants)
and NA. Virus-like particles were analyzed by direct capture ELISA
from transfected cell supernatants with a globular head antibody,
PY-102. (B) VLPs obtained as described in A were purified over a
30% Sucrose-NTE cushion. 10 ug of total protein was then incubated
in cleavage buffer in the presence (+) or absence (-) of TEV
protease (Promega), according to the manufacturer's instructions.
Cleavage efficiency was monitored by Western Blot using an anti-HA2
antibody. Arrows indicate the cleavage product of each of the
indicated insertions (48G, 63G, and 278S).
[0120] FIGS. 87A-87B. (A) 293T cells were transfected with plasmids
for the expression of HA (WT and the indicated double-insertion
mutants) and NA. Virus-like particles were analyzed by direct
capture ELISA from transfected cell supernatants with a globular
head antibody, PY-102. (B) Binding of the double-insertion HA
mutants (insertions at positions 63+278, and 63+286) to two broadly
neutralizing V.sub.H1-69 stalk-specific mAb by direct capture ELISA
at 50 .mu.g/ml (normalized for HA presence in supernatants).
[0121] FIG. 88. Amino acid sequences of influenza HA C-terminal
fragments generated following proteolysis at a protease cleavage
site inserted at position 291 of the wild-type PR8 HA amino acid
sequence (SEQ ID NO:1). The sequence of SEQ ID NO:108 comprises
to-tyrosine mutations at positions 113 and 143 (underlined;
corresponding to positions 403 and 433, respectively, of SEQ ID
NO:1). The sequence of SEQ ID NO:109 comprises to-tyrosine
mutations at positions 121 and 132 (underlined; corresponding to
positions 411 and 422, respectively, of SEQ ID NO:1). The sequence
of SEQ ID NO:110 comprises to-tyrosine mutations at positions 113,
121, 132 and 143 (underlined; corresponding to positions 403, 411,
422 and 433, respectively, of SEQ ID NO:1). The C-terminal
transmembrane region is underlined in each sequence.
[0122] FIG. 89. Amino acid sequence of an influenza HA protein
C-terminal fragment (SEQ ID NO: 117). This fragment is generated
following proteolysis at a protease cleavage site inserted at
position 291 of the wild-type PR8 HA amino acid sequence (SEQ ID
NO:1). Underlined amino acid residues N112, F115, K120, N131, D137,
L141, D142 and W144 illustrate positions in SEQ ID NO: 117 where
to-tyrosine mutations can be made to facilitate the formation
di-tyrosine bonds. The underlined residues correspond to positions
N403, F406, K411, N422, D429, L432, D433 and W435 respectively, of
SEQ ID NO:1. The C-terminal transmembrane region is underlined. In
some embodiments the transmembrane region is absent (i.e. the
fragment does not contain the last 46 amino acid residues (229-274
of SEQ ID NO: 117, but contains residues 1-228 of SEQ ID NO: 117).
Tyrosine residues at positions 17 and 146 (shown in bold italic)
are endogenous tyrosine residues that may be used in the formation
of dityrosine bonds. These endogenous residues correspond to
tyrosine residues at positions 308 and 437, respectively, of SEQ ID
NO:1.
DETAILED DESCRIPTION OF THE INVENTION
[0123] The present invention provides, in part, influenza HA
polypeptides, proteins and/or protein complexes (such as those that
comprise a stalk domain having its native conformation and that may
or may not comprise an intact head domain), methods of making such
polypeptides, proteins and/or protein complexes, compositions (such
as pharmaceutical compositions and vaccine compositions) comprising
such polypeptides, proteins and/or protein complexes, and methods
of use of such polypeptides, proteins and/or protein complexes, for
example in vaccination methods, therapeutic methods and other
methods. In some embodiments, the influenza HA polypeptides,
proteins and/or protein complexes may be useful as immunogens, for
example in influenza vaccines.
DEFINITIONS AND ABBREVIATIONS
[0124] As used in the present specification the terms "about" and
"approximately," when used in relation to numerical values, mean
within + or -20% of the stated value.
[0125] The abbreviation "HA" as used herein refers to a
hemagglutinin protein. The abbreviation "Ab" as used herein refers
to antibody. The abbreviation "bnAbs" as used herein refers to
broadly neutralizing antibodies. The abbreviation "QNE" as used
herein refers to quaternary neutralizing epitopes. The abbreviation
"DT" as used herein refers to di-tyrosine. As used herein the
phrase "full-length" when used in relation to an influenza HA
protein or polypeptide does not require an HA protein or
polypeptide that is as long as a wild-type influenza HA protein.
Rather the term is used to refer to an influenza HA protein or
polypeptide that comprises, at least, both a stalk domain and a
head domain. Such stalk and head domains may or may not be as long
as those found in a wild-type influenza HA protein or polypeptide.
For example, an influenza HA protein or polypeptide that is missing
the transmembrane domain found in a wild-type influenza HA protein
or polypeptide may still be referred to as a "full-length" HA
protein or polypeptide herein if it has a stalk domain and a head
domain. In some embodiments, the phrase "full-length," when used in
relation to an influenza HA protein or polypeptide, may refer to an
influenza HA protein or polypeptide that, in addition to a stalk
and head domain, also comprises a transmembrane domain. As used
herein the phrase "soluble" when used in relation to an influenza
HA protein or polypeptide refers to an influenza HA protein or
polypeptide that does not comprise a transmembrane domain. Such
soluble HA proteins or polypeptides may comprise either a stalk
domain and a head domain, or stalk domain in the absence of a head
domain.
[0126] As used herein the terms "protein" and "polypeptide" are
used interchangeably, unless otherwise stated. As used herein the
term "protein complex" refers to an assembly of two or more
proteins or protein subunits, such as two or more monomers. Unless
otherwise stated, all description herein that relates to proteins
and/or polypeptides applies equally to protein complexes, and vice
versa.
[0127] As used herein the terms "stabilized" and "locked" are used
interchangeably, for example in relation to the effect of
cross-linking in stabilizing or locking the stalk domain of an
influenza HA protein, polypeptide, or protein complex in its native
trimeric conformation. These terms do not require 100% stability.
Rather these terms denote a degree of improved or increased
stability. For example, in some embodiments, when the term
"stabilized" is used in relation to a stalk domain cross-linked in
its native trimeric conformation, the term denotes that the native
trimeric conformation of the stalk domain has greater stability
than it would have had prior to or without such cross-linking.
Stability, and relative stability, may be measured in various ways
as described in other sections of this application, for example
based on the half-life of the native trimeric conformation of the
stalk domain. The improvement or increase in stability may be to
any degree that is useful or significant for the intended
application. For example, in some embodiments stability may be
increased by about 10%, 25%, 50%, 100%, 200% (i.e. 2-fold), 300%
(i.e. 3-fold), 400% (i.e. 4-fold), 500% (i.e. 5-fold), 1000% (i.e.
10-fold), or more.
[0128] As used herein the terms "stem" and "stalk" are used
interchangeably to refer to a stalk domain, or portion thereof, of
an influenza HA protein or polypeptide.
[0129] As used herein the term "engineered" when used in relation
to the influenza HA polypeptides, proteins and/or protein complexes
of the invention refers generally to influenza HA polypeptides,
proteins and/or protein complexes that have been altered in some
way as compared to the wild-type versions of those polypeptides,
proteins and/or protein complexes, for example, but not limited to,
by way of removal or disruption of a particular portion or domain
of the wild-type polypeptide, protein and/or protein complex (such
as a transmembrane domain or a head domain) or by introduction of
one or more point mutations (such as those introduced to facilitate
formation of di-tyrosine bonds) or by way of introduction of one or
more protease recognition motifs not ordinarily present in the
wild-type polypeptide, protein and/or protein complex, or by any
other modification of the polypeptide, protein and/or protein
complex as compared to its wild-type form.
[0130] Other definitions and abbreviations are found throughout the
specification.
Influenza and Influenza Viruses
[0131] Influenza, commonly known as "the flu", is an infectious
disease of birds and mammals caused by RNA viruses of the family
Orthomyxoviridae, the influenza viruses. Influenza spreads around
the world in seasonal epidemics, resulting in about three to five
million yearly cases of severe illness and about 250,000 to 500,000
yearly deaths, rising to millions in some pandemic years. In the
20th century three influenza pandemics occurred, each caused by the
appearance of a new strain of the virus in humans, and killed tens
of millions of people. Often, new influenza strains appear when an
existing flu virus spreads to humans from another animal species,
or when an existing human strain picks up new genes from a virus
that usually infects birds or pigs.
[0132] There are three different types of influenza virus, type A,
type B, and type C, with various subtypes and strains within those
types.
[0133] Influenza type A viruses are the most virulent human
pathogens among the three influenza types and cause the most severe
disease. The influenza A virus can be subdivided into different
subtypes or serotypes including, but not limited to H1N1 (which
caused Spanish Flu in 1918, and Swine Flu in 2009), H2N2 (which
caused Asian Flu in 1957), H3N2 (which caused Hong Kong Flu in
1968), H5N1 (which caused Bird Flu in 2004), H7N7, H1N2 (which is
endemic in humans, pigs and birds), H9N2, H7N2, H7N3, H10N7, and
H7N9. Wild aquatic birds are the natural hosts for a large variety
of influenza A. However, domestic poultry, such as turkeys and
chickens, can also become very sick and die from avian influenza,
and some avian influenza A viruses also can cause serious disease
and death in wild birds.
[0134] Influenza type B almost exclusively infects humans and is
less common than influenza A. The only other animals known to be
susceptible to influenza B infection are the seal and the ferret.
Influenza type B mutates at a rate 2-3 times slower than type A and
consequently is less genetically diverse, with only one influenza B
serotype known. As a result of this lack of antigenic diversity, a
degree of immunity to influenza B is usually acquired at an early
age. However, influenza B mutates frequently enough that lasting
immunity is not possible.
[0135] Influenza type C virus infects humans, dogs and pigs,
sometimes causing both severe illness and local epidemics. However,
influenza C is less common than the other types and usually only
causes mild disease.
[0136] Influenza viruses A, B and C are very similar in their
overall structure. They each comprise a viral envelope containing
two main types of glycoproteins, and a central core containing the
viral RNA genome and other viral proteins. Hemagglutinin ("HA") and
neuraminidase ("NA") are the two large envelope glycoproteins. HA
is a lectin that mediates binding of the virus to target cells and
entry of the viral genome into the target cell. The various
influenza A subtypes are classified based on their antibody
responses to the HA and NA proteins. For example, an "H7N2 virus"
designates an influenza A subtype that has an HA 7 protein and an
NA 2 protein. Similarly an "H5N1" virus has an HA 5 protein and an
NA 1 protein. There are currently around 17 known HA subtypes and
around 10 known NA subtypes. Many different combinations of HA and
NA proteins are possible. Influenza A subtypes H1N1, H1N2, and H3N2
are currently the main types in general circulation in the human
population. There are also several prominent subtypes of the avian
influenza A viruses that are known to infect both birds and
humans--such as H5N1, H7N2, H7N7, H7N3, and H7N7 subtypes.
[0137] Within influenza type A, one can group the various different
influenza subtypes in a variety of different ways, if desired. For
example, influenza type A subtypes are frequently classified or
grouped into different antigenic groups and antigenic subgroups
based on their HA protein. Such groupings relate to the
antigenicity and degree of HA sequence identity between the
different subgroups. Influenza subtypes in the same antigenic group
or antigenic subgroup are more similar to each other in terms of
antigenicity and HA sequence than those in other antigenic groups.
Antigenic group 1 consists of H1, H2, H5, H6, H8, H9, H11, H12,
H13, and H16 influenza A subtypes. Antigenic group 2 consists of
H3, H4, H14, H7, H10, and H15 influenza A subtypes. Within
antigenic group 1, there are three antigenic subgroups, which will
be referred to herein as antigenic subgroup 1A, 1B, and 1C.
Antigenic subgroup 1A consists of H1, H2, H5 and H6 influenza A
subtypes. Antigenic subgroup 1B consists of H11, H13 and H16
influenza A subtypes. Antigenic subgroup 1C consists of H8, H9, and
H12 influenza A subtypes.
[0138] In some embodiments herein the HA polypeptides, proteins and
protein complexes of the invention are generated from HA sequences
from any influenza type--including type A, B, or C. In some
embodiments herein the HA polypeptides, proteins and protein
complexes of the invention are generated from HA sequences from
influenza type A. In some embodiments herein the HA polypeptides,
proteins and protein complexes of the invention are generated from
HA sequences from influenza type A, antigenic group 1. In some
embodiments herein the HA polypeptides, proteins and protein
complexes of the invention are generated from HA sequences from
influenza type A, antigenic group 1A.
[0139] In some embodiments herein the HA polypeptides, proteins and
protein complexes of the invention can be used to vaccinate a
subject, and provide protection against, any influenza
type--including type A, B, or C. In some embodiments herein the HA
polypeptides, proteins and protein complexes of the invention can
be used to vaccinate a subject, and provide protection against,
influenza type A. In some embodiments herein the HA polypeptides,
proteins and protein complexes of the invention can be used to
vaccinate a subject, and provide protection against, influenza type
A, antigenic group 1. In some embodiments herein the HA
polypeptides, proteins and protein complexes of the invention can
be used to vaccinate a subject, and provide protection against,
influenza type A, antigenic group 1A. In some embodiments herein
the HA polypeptides, proteins and protein complexes of the
invention can be used to vaccinate a subject, and provide
protection against, influenza subtype H1. In some embodiments
herein the HA polypeptides, proteins and protein complexes of the
invention can be used to vaccinate a subject, and provide
protection against, influenza subtypes H1 and H2. In some
embodiments herein the HA polypeptides, proteins and protein
complexes of the invention can be used to vaccinate a subject, and
provide protection against, influenza subtypes H1, H2 and H5. In
some embodiments herein the HA polypeptides, proteins and protein
complexes of the invention can be used to vaccinate a subject, and
provide protection against, influenza subtypes H1, H2, H5 and
H6.
[0140] Tables A and B below provide some examples of the sequence
identity between the HA protein of H1N1 strain PR8, or certain
fragments of the HA protein, and corresponding proteins or
fragments from other influenza subtypes and strains, including some
from antigenic groups 1 and 2.
TABLE-US-00001 TABLE A Percent identity of full-length influenza HA
amino acid sequences to the PR8 amino acid sequence of SEQ ID NO: 1
(as illustrated in FIG. 54). % identity to Full-length influenza HA
protein SEQ ID NO: 1 Antigenic group 1: HA-USSR-77.pro (SEQ ID NO:
111) (H1 subtype) 90.1 HA-Texas-91.pro (SEQ ID NO: 112) (H1
subtype) 87.8 HA-WSN-33.pro (SEQ ID NO: 113) (H1 subtype) 90.1
HA-SouthCarolina-1918.pro (SEQ ID NO: 114) (H1 88.2 subtype)
HA-California-09.pro (SEQ ID NO: 115) (H1 subtype) 81.1
HA-Singapore-57.pro (SEQ ID NO: 78) (H2 subtype) 65.5
HA-Vietnam-04.pro (SEQ ID NO: 79) (H5 subtype) 63.8 Antigenic group
2: HA-Udorn-72.pro (SEQ ID NO: 73) (H3 subtype) 39.8
HA-HongKong-68.pro (SEQ ID NO: 74) (H3 subtype) 40.5
HA-Panama-99.pro (SEQ ID NO: 75) (H3 subtype) 39.2
HA-Wisconsin-05.pro (SEQ ID NO: 76) (H3 subtype) 38.7
HA-Shanghai-13.pro (SEQ ID NO: 77) (H7 subtype) 40.4
TABLE-US-00002 TABLE B Percent identity of fragment that remains
after cleavage of influenza HA protein at cleavage sites 48 and
291. % identity to C- terminal fragment of Influenza HA protein
C-terminal fragment SEQ ID NO: 1 Antigenic group 1: HA-USSR-77 (H1
subtype) 92.4 HA-Texas-91 (H1 subtype) 90.2 HA-WSN-33 (H1 subtype)
89.6 HA-SouthCarolina-1918 (H1 subtype) 89.6 HA-California-09 (H1
subtype) 85.4 HA-Singapore-57 (H2 subtype) 71.6 HA-Vietnam-04 (H5
subtype) 70.4 Antigenic group 2: HA-Udorn-72 (H3 subtype) 43.9
HA-HongKong-68 (H3 subtype) 44.5 HA-Panama-99 (H3 subtype) 43.3
HA-Wisconsin-05 (H3 subtype) 43.0 HA-Shanghai-13 (H7 subtype)
44.2
[0141] In addition to the sequence identities shown in the above
table, the percent identity between PR8 (SEQ ID NO: 1) and
sequences of H6, H9, H11, and H13 subtypes over a C-terminal
fragment remaining after proteolytic cleavage of the influenza HA
protein, was found to be 68.2%, 54.7%, 56.2%, and 50.5%,
respectively.
Influenza HA Polypeptides, Proteins and Protein Complexes
[0142] In some embodiments the present invention provides
engineered influenza HA polypeptides, proteins and/or protein
complexes, compositions comprising such polypeptides, proteins
and/or protein complexes, and methods of use of such polypeptides,
proteins and/or protein complexes. Such proteins can be made using
any suitable influenza virus HA protein as a starting point. For
example, the proteins of the invention can be made using an
influenza HA protein from any suitable influenza type (such as A,
B, or C), subtype (including, but not limited to, H1N1, H1N2, and
H3N2 subtypes) or strain (e.g. the H1N1 A/Puerto Rico/8/1934
("PR8") strain (SEQ ID NO. 1)) of influenza virus as the starting
point. One of the important features of the influenza HA
polypeptides, proteins and/or protein complexes described herein is
that they comprise the trimeric stalk domain of the HA protein
which, unlike the highly variable head domain, is more conserved
between influenza types, subtypes and strains. Accordingly, in
addition to being useful as vaccine immunogens against homologous
types, subtypes, and strains of influenza virus (i.e. against
influenza viruses of the same type, subtype and/or strain as used
as the starting point for making the influenza HA polypeptides,
proteins and/or protein complexes described herein), the HA
polypeptides, proteins and/or protein complexes of the invention
may also be useful as vaccine immunogens against heterologous
types, subtypes, and strains of influenza virus (i.e. against
influenza viruses of a different type, subtype and/or strain to
that used as the starting point for making the engineered HA
polypeptides, proteins and/or protein complexes).
[0143] In some embodiments the present invention provides
approaches for stabilizing the stalk domain of an influenza HA
protein in its native trimeric conformation, including providing
specific locations within the influenza HA protein that can be or
should be cross-linked, and providing mutant forms of the HA
protein that can facilitate the formation of such cross-links. Such
cross-links and mutations can be used alone (e.g. in the context of
a wild type HA protein or in the context of an HA protein that does
not comprise any man-made mutations or other man-made
modifications), or can be used in combination with one or more
other man-made mutations, modifications, cross-links, or
stabilization strategies. Thus, for example, the approaches
described herein can be used in conjunction with the use of added
foldon trimerization domains, stabilizing antibodies (such as 6F12,
C179, CR6261, F10, A66 and D8), and/or other partially or
potentially stabilizing modifications or mutations.
[0144] The present inventors have performed extensive analysis of
the structure of the influenza HA protein and have developed a
variety of novel design strategies and novel engineered influenza
HA polypeptides, proteins, and/or protein complexes. The present
invention also provides methods for making and using such influenza
HA polypeptides, proteins, and/or protein complexes. In some
embodiments, the present invention provides specific locations
within the amino acid sequence of the influenza HA protein at
which, or between which, targeted cross-links can be made in order
to "lock" the stalk domain of the HA protein in its native trimeric
conformation. In some embodiments, the targeted cross-links are
di-tyrosine cross-links. Where di-tyrosine cross-links are used,
the present invention provides specific amino acid residues (or
pairs of amino acid residues) that either comprise a pre-existing
tyrosine residue or can be or are mutated to a tyrosine residue
such that di-tyrosine cross-links can be made.
[0145] The engineered influenza HA polypeptides, proteins and/or
protein complexes described herein can be made based on the
sequence of any suitable influenza HA polypeptide, protein and/or
protein complex, such as a wild-type (WT) influenza HA protein or
polypeptide, or mutant, homolog, derivative, analog, ortholog, or
any other derivative of an influenza HA polypeptide, protein and/or
protein complex, provided that the HA polypeptide, protein and/or
protein complex has a stalk domain, or a portion of a stalk domain,
that is capable of folding into, or forming a part of, an stalk
domain having a native trimeric conformation and/or is capable of
binding to one or more anti-stalk antibodies. Amino acid sequences
of suitable influenza HA polypeptides, proteins and/or protein
complexes, and nucleic acid sequences that encode such influenza HA
polypeptides, proteins and/or protein complexes, are known in the
art and any such amino acid or nucleic acid sequence may be used.
Furthermore, amino acid sequences of several suitable influenza HA
polypeptides, proteins and/or protein complexes, and nucleic acid
sequences that encode such influenza HA polypeptides, proteins
and/or protein complexes, are provided herein. While any suitable
influenza virus HA protein can be used as a starting point for
making the soluble influenza HA polypeptides, proteins and/or
protein complexes described herein, such an HA protein should at
least comprise a stalk domain, or a portion of a stalk domain, that
is capable of folding into a native trimeric conformation and/or
that is capable of binding to one or more anti-stalk antibodies,
such as neutralizing anti-stalk antibodies. In some embodiments the
HA protein used as a starting point is a full-length wild-type HA
protein comprising a head domain and a stalk domain, and optionally
also a transmembrane domain. In some embodiments the HA protein
used lacks a transmembrane domain or lacks a functional or intact
transmembrane domain. In some embodiments the HA protein comprises
a T4 foldon trimerization motif. In some embodiments the HA
proteins that are used as a starting point for making the influenza
HA polypeptides, proteins and/or protein complexes described
herein: (a) comprise a stalk domain, or a portion of a stalk
domain, that is capable of folding into a native trimeric
conformation and/or that is capable of binding to one or more
neutralizing anti-stalk antibodies, (b) comprise a T4 foldon
trimerization motif, and (c) lack a functional or intact
transmembrane domain.
[0146] Throughout the present patent specification, when reference
is made to specific amino acid residues or specific amino acid
regions in the influenza HA protein by referring to their amino
residue number or numbers (such as amino acid residues 403 and 422,
for example), and unless otherwise stated, the numbering is based
on the HA amino acid sequence provided herein in, FIG. 9 and SEQ ID
NO: 1--which is an amino acid sequence of a wild-type HA protein
from influenza strain PR8 (influenza type A-H1N1 subtype). However,
it should be noted, and one of skill in the art will understand,
that different HA sequences may have different numbering systems,
for example, if there are additional amino acid residues added or
removed as compared to SEQ ID NO: 1 (for example, as illustrated in
FIGS. 26 and 27 and many of the other Figures and sequences
herein). As such, it is to be understood that when specific amino
acid residues are referred to by their number, the description is
not limited to only amino acids located at precisely that numbered
position when counting from the beginning of a given amino acid
sequence, but rather that the equivalent/corresponding amino acid
residue in any and all HA sequences is intended--even if that
residue is not at the same precise numbered position, for example
if the HA sequence is shorter or longer than SEQ ID NO. 1, or has
insertions or deletions as compared to SEQ ID NO. 1. One of skill
in the art can readily determine what is the
corresponding/equivalent amino acid position to any of the specific
numbered residues recited herein, for example by aligning a given
HA sequence to SEQ ID NO. Thus, in embodiments where specific amino
acid residues of the influenza HA protein are referred to, it is to
be understood that the invention is not to be limited to sequences
having the specified amino acid properties (e.g. presence of a
tyrosine residue, a mutation, or an insertion of a protease
recognition site, etc.) at only those precise numbered amino acid
positions. Rather the specified amino acid properties may be
located at any position in any influenza HA protein that is
equivalent/corresponding to the numbered positions recited for the
PR8 influenza HA protein of SEQ ID NO:1. This description applies
equally where references are made to specific nucleic acid residues
or specific nucleic acid regions in a nucleotide sequence encoding
an influenza HA protein by referring to their nucleic acid residue
number or numbers. Thus, unless otherwise stated, the numbering is
based on the nucleotide sequence provided herein in FIG. 10 and SEQ
ID NO. 2.
[0147] In some embodiments, the influenza HA polypeptides, proteins
or protein complexes of the present invention can be derived from
(or can comprise, consist essentially of, or consist of) the amino
acid sequences of any suitable influenza HA polypeptide, protein or
protein complex sequence known in the art, including, without
limitation: the amino acid sequence of the PR8 strain of H1N1
influenza virus (for example, in a full-length form (SEQ ID NO:1)
or a soluble form (SEQ ID NO:80; or amino acid residues 1-519
thereof), the amino acid sequence of the Udorn 72 strain of H3N2
influenza virus (for example, in a full-length form (SEQ ID NO:73)
or a soluble form comprising amino acid residues 1-520 thereof),
the amino acid sequence of the Hong Kong 68 strain of H3N2
influenza virus (for example, in a full-length form (SEQ ID NO:74)
or a soluble form (SEQ ID NO:81; or amino acid residues 1-520
thereof)), the amino acid sequence of the Panama 99 strain of H3N2
influenza virus (for example, in a full-length form (SEQ ID NO:75)
or a soluble form comprising amino acid residues 1-520 thereof),
the amino acid sequence of the Wisconsin 05 strain of H3N2
influenza virus (for example, in a full-length form (SEQ ID NO:76)
or a soluble form (SEQ ID NO:82; or amino acid residues 1-520
thereof)), the amino acid sequence of the Shanghai 13 strain of
H7N9 influenza virus (for example, in a full-length form (SEQ ID
NO:77) or a soluble form (SEQ ID NO:84; or amino acid residues
1-514 thereof)), the amino acid sequence of the Singapore 57 strain
of H2N2 influenza virus (for example, in a full-length form (SEQ ID
NO:78) or a soluble form (SEQ ID NO:85; or amino acid residues
1-515 thereof)), the amino acid sequence of the Vietnam 04 strain
of H5N1 influenza virus (for example, in a full-length form (SEQ ID
NO:79) or a soluble form (SEQ ID NO:83; or amino acid residues
1-521 thereof)), the amino acid sequence of the USSR 77 strain of
H1N1 influenza virus (for example, in a full-length form (SEQ ID
NO:111) or a soluble form comprising amino acid residues 1-519
thereof), the amino acid sequence of the Texas 91 strain of H1N1
influenza virus (for example, in a full-length form (SEQ ID NO:112)
or a soluble form comprising amino acid residues 1-519 thereof),
the amino acid sequence of the WSN 33 strain of H1N1 influenza
virus (for example, in a full-length form (SEQ ID NO:113) or a
soluble form comprising amino acid residues 1-518 thereof), the
amino acid sequence of the South Carolina 1918 strain of H1N1
influenza virus (for example, in a full-length form (SEQ ID NO:114)
or a soluble form comprising amino acids 1-519), the amino acid
sequence of the California 09 strain of H1N1 influenza virus (for
example, in a full-length form (SEQ ID NO:115) or a soluble form
comprising amino acids 1-519), or any fragment thereof. In some
embodiments, the influenza HA proteins and polypeptides of the
present invention can be derived from (or can comprise, consist
essentially of, or consist of) amino acid sequences that have at
least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
or 99% sequence identity to any known influenza HA sequences or to
HA sequences from any known influenza groups, subgroups, families,
subfamilies, types, subtypes, genera, species, strains, and/or
clades, or any fragment thereof. Furthermore, in addition to the
large number of specific amino acid and nucleotide molecules and
sequences provided herein (including SEQ ID NO:s 1-110); the
present invention also provides and encompasses amino acid and
nucleotide molecules and sequences that have at least about 40%,
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence
identity to any such molecules and sequences. Thus, for every
embodiment herein that refers to a specific sequence or specific
SEQ ID NO (such as SEQ ID NO:s 1-110), the present invention also
includes variations of such embodiments that include amino acid and
nucleotide molecules and sequences that have at least about 40%,
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence
identity to such specific sequences or SEQ ID NOs.
[0148] In some embodiments the present invention provides
engineered influenza HA polypeptides, proteins and/or protein
complexes that comprise a stalk domain (for example, that has, or
is capable of forming, its native trimeric conformation) and that
do not comprise a head domain. Such polypeptides, proteins and/or
protein complexes can be referred to as "headless" influenza HA
polypeptides, proteins and/or protein complexes.
[0149] In some embodiments the present invention provides influenza
HA polypeptides, proteins and/or protein complexes that comprise a
stalk domain having its native trimeric conformation and a head
domain. Such proteins may be referred to as "head-on" influenza HA
polypeptides, proteins and/or protein complexes. In some
embodiments such polypeptides, proteins and/or protein complexes
may also comprise one or more engineered protease recognition
motifs that can be used for proteolytic disruption and/or removal
of the head domain. In some embodiments such head-on influenza HA
polypeptides, proteins and/or protein complexes may be useful as,
for example, intermediates in the production of "headless"
influenza HA polypeptides, proteins and/or protein complexes--as
described herein.
[0150] "Headless" HA variants can be obtained or generated by a
variety of methods. For example in some embodiments, headless HA
variants can be obtained by removal of all or part of the HA head
domain, for example by proteolytic removal of the head domain, or
by another other suitable means. In other embodiments headless HA
variants can be obtained by expression of a nucleotide sequence
encoding only the stalk domain. In some embodiments, "headless" HA
variants can be generated by proteolytic cleavage of a full-length
influenza HA protein at protease recognition motifs inserted into
the protein such that following cleavage, the head domain sequence
is cut out and at least two protein fragments comprising the stalk
domain remain. FIG. 27 illustrates examples of protease cleavage
motifs and shows intervening sequences of the head domain that are
cut out following protease treatment. Thus, in some embodiments,
for example as shown in FIG. 27, a "headless" influenza HA variant
comprises at least two protein fragments--e.g. an N-terminal
fragment and a C-terminal fragment--comprising the stalk domain. In
some embodiments one or more fragments of a "headless" influenza
protein comprise one or more to-tyrosine mutations, and/or one- or
more dityrosine crosslinks. Such mutations and/or crosslinks will
typically be present in the C-terminal fragment of the "headless"
HA protein. (See for example SEQ ID NOs: 96-110 and 117). FIGS.
70-84 and 89 illustrate examples of some such HA peptides. In some
embodiments such peptides (for example, SEQ ID NOs: 96-110 and 117)
may be comprised within a larger HA molecule comprising a head
domain, or they may be present in a "headless" HA protein. In some
embodiments, several such peptides may associate to form an HA
protein complex that is in, or is capable of forming, a trimeric
stalk domain. In some embodiments an influenza HA polypeptide,
protein and/or protein complex comprises the amino acid sequence of
SEQ ID NO: 108, 109, 110. In some embodiments an influenza HA
polypeptide, protein and/or protein complex comprises the amino
acid sequence of SEQ ID NO:94 and SEQ ID NO:96, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:97, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:98, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:99, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:100, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:101, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:102, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:103, or the amino acid
sequence of SEQ ID NO:94 and SEQ ID NO:104, or the amino acid
sequence of SEQ ID NO:95 and SEQ ID NO:106, or the amino acid
sequence of SEQ ID NO:95 and SEQ ID NO:107, or the amino acid
sequence of SEQ ID NO:95 and SEQ ID NO:108, or the amino acid
sequence of SEQ ID NO:95 and SEQ ID NO:109, or the amino acid
sequence of SEQ ID NO:95 and SEQ ID NO:110. In some embodiments an
influenza HA polypeptide, protein and/or protein complex comprises
an N-terminal HA peptide comprising, consisting essentially of, or
consisting of, SEQ ID NO:94 or SEQ ID NO: 95, and a C-terminal HA
peptide comprising, consisting essentially of, or consisting of,
SEQ ID NO:96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,
108, 109 or 110, or a C-terminal HA peptide comprising, consisting
essentially of, or consisting of, amino acid residues 229 to 519 of
SEQ ID NO:1 wherein the amino acid sequence comprises a point
mutation to tyrosine at one or more of amino acid positions 403,
406, 411, 422, 429, 432, 433, or 435, or a C-terminal HA peptide
comprising, consisting essentially of, or consisting of, amino acid
residues 1 to 228 of of SEQ ID NO:117 wherein the amino acid
sequence comprises a point mutation to tyrosine at one or more of
amino acid positions 112, 115, 120, 131, 137, 141, 142, or 144.
[0151] It should be noted that amino acid residues 1 through 58 (or
18 to 58 without the signal peptide--which is located at residues
1-17) and 292 through 566 (or 292 through 529 without the
transmembrane domain and cytoplasmic tail) of the PR8 HA amino acid
sequence (SEQ ID NO. 1) represent the influenza HA stalk domain
sequence. The stalk domain is discontinuous and comprises both an
N-terminal and a C-terminal portion of the HA protein. The amino
acid sequences provided here in may comprise additional domains
that may be present or partially present or absent in some
embodiments but not in others, for example the head domain (e.g.
amino acid residues 59-291 of the PR8 HA amino acid sequence (SEQ
ID NO. 1)), and/or the transmembrane and cytoplasmic region (e.g.
amino acid residues 529 or 530 to 565 of the PR8 HA amino acid
sequence (SEQ ID NO. 1)), and/or the signal peptide (e.g. amino
acid residues 1-17 of the PR8 HA amino acid sequence (SEQ ID NO:1),
and/or one or more optional exogenous (non-HA) sequences such as
epitope tags, foldon domains, and the like. For example, in some
embodiments an optional foldon trimerization domain, thrombin
cleavage site, 6.times.His-tag (SEQ ID NO: 118), and/or a strep tag
may be present. In some embodiments these additional sequences may
be absent, modified, rearranged or replaced. For example, in some
embodiments different trimerization domains may be used, or
different epitope tags may be used. In some embodiments these
additional sequences may be absent, modified, rearranged or
replaced, for example with different transmembrane or cytoplasmic
domains.
[0152] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of influenza HA amino acid sequences presented herein, or any
variants or fragments thereof that have at least about 40% or 50%
or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or
99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise a tyrosine residue (whether naturally occurring
or arising from a mutation to-tyrosine), at one or more of residues
308, 403, 406, 437, 411, 422, 429, 432, 433, and 435.
[0153] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of influenza HA amino acid sequences presented herein, or any
variants or fragments thereof that have at least about 40% or 50%
or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or
99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise an artificially-introduced protease cleavage
site inserted immediately after one or more of the following
residues: 48, 63, 228, 278, 282, 283, 286 and 291.
[0154] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of influenza HA amino acid sequences presented herein, or any
variants or fragments thereof that have at least about 40% or 50%
or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or
99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise two artificially-introduced protease cleavage
sites, the first such site introduced immediately after residue 48
or 63, and the second such site introduced immediately after
residue 228, 278, 282, 283, 286 or 291.
[0155] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of influenza HA amino acid sequences presented herein, or any
variants or fragments thereof that have at least about 40% or 50%
or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or
99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise both (a) a tyrosine residue (whether naturally
occurring or arising from a mutation to-tyrosine), at one or more
of residues 308, 403, 406, 437, 411, 422, 429, 432, 433, and 435,
and (b) an artificially-introduced protease cleavage site inserted
immediately after one or more of the following residues: 48, 63,
228, 278, 282, 283, 286 and 291.
[0156] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of any
one of influenza HA amino acid sequences presented herein, or any
variants or fragments thereof that have at least about 40% or 50%
or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or
99% identity with such amino acid sequences presented herein,
wherein the influenza HA polypeptides, proteins, and/or protein
complexes comprise both (a) a tyrosine residue (whether naturally
occurring or arising from a mutation to-tyrosine), at one or more
of residues 308, 403, 406, 437, 411, 422, 429, 432, 433, and 435,
and (b) two artificially-introduced protease cleavage sites--the
first such site introduced immediately after residue 48 or 63, and
the second such site introduced immediately after residue 228, 278,
282, 283, 286 or 291.
[0157] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of amino
acid residues 229 to 519 of SEQ ID NO: 1, or 279 to 519 of SEQ ID
NO: 1, or 283 to 519 of SEQ ID NO: 1, or 284 to 519 of SEQ ID NO:
1, or 287 to 519 of SEQ ID NO: 1, or 292 to 519 of SEQ ID NO: 1, or
sequences that have at least about 40% or 50% or 60% or 65% or 70%
or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity with such
amino acid sequences, wherein the influenza HA polypeptides,
proteins, and/or protein complexes comprise a tyrosine residue
(whether naturally occurring or arising from a mutation
to-tyrosine), at one or more of residues 308, 403, 406, 437, 411,
422, 429, 432, 433, and 435.
[0158] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that are
derived from, comprise, consist essentially of, or consist of amino
acid residues 1 to 47 of SEQ ID NO: 1, or 1 to 62 of SEQ ID NO: 1,
or sequences that have at least about 40% or 50% or 60% or 65% or
70% or 75% or 80% or 85% or 90% or 95% or 98% or 99% identity with
such amino acid sequences.
[0159] In some embodiments the present invention provides
compositions and/or influenza HA protein complexes that comprise,
consist essentially of, or consist of a first and a second peptide,
wherein (a) the first peptide comprises, consists essentially of,
or consists of amino acid residues 229 to 519 of SEQ ID NO: 1, or
279 to 519 of SEQ ID NO: 1, or 283 to 519 of SEQ ID NO: 1, or 284
to 519 of SEQ ID NO: 1, or 287 to 519 of SEQ ID NO: 1, or 292 to
519 of SEQ ID NO: 1, or sequences that have at least about 40% or
50% or 60% or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98%
or 99% identity with such amino acid sequences, and wherein the
influenza HA polypeptides, proteins, and/or protein complexes
comprise a tyrosine residue (whether naturally occurring or arising
from a mutation to-tyrosine), at one or more of residues 308, 403,
406, 437, 411, 422, 429, 432, 433, and 435, and wherein (b) the
second peptide comprises, consists essentially of, or consists of
amino acid residues 1 to 47 of SEQ ID NO: 1, or 1 to 62 of SEQ ID
NO: 1, or sequences that have at least about 40% or 50% or 60% or
65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or 99%
identity with such amino acid sequences.
[0160] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that comprise
one or more artificially-introduced cross-links, wherein at least
one of the following amino acid residues within the influenza HA
polypeptides, proteins, and/or protein complexes is artificially
cross-linked to another amino acid residue in the influenza HA
protein: Y308, N403, F406, Y437, K411, N422, D429, L432, D433, and
W435 In some such embodiments, where the indicated position is not
a tyrosine, that residue is mutated to tyrosine. In some such
embodiments the cross-link is a di-tyrosine cross-link.
[0161] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes that comprise
one or more artificially-introduced cross-links, wherein such
artificially introduced cross-links connect two of the following
amino acid residues: Y308, N403, F406, K411, Y437, N422, D429,
L432, D433, and W435. In some such embodiments, where the indicated
position is not a tyrosine, that residue is mutated to tyrosine. In
some such embodiment the cross-link is a di-tyrosine
cross-link.
[0162] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes in which the
amino acid residues in one or more of the following pairs of amino
residues are cross-linked to each other by an artificially
introduced cross-link: 308/403, 308/435, 403/437, 403/429, 403/432,
403/433, 406/429, 406/433, 411/422, 422/433, 433/435 and 437/435.
In some such embodiments, where the indicated position is not a
tyrosine, that residue is mutated to tyrosine. In some such
embodiments the cross-link is a di-tyrosine cross-link.
[0163] In some embodiments, the present invention contemplates the
targeted introduction of one or more cross-links at any suitable
position(s) in an influenza HA polypeptide, protein or protein
complex, for example, in the stalk domain where the cross-link will
or may stabilize the stalk domain in a native trimeric conformation
or other conformation capable of binding anti-stalk antibodies,
such as neutralizing or broadly neutralizing anti-stalk antibodies.
Such stabilization may be achieved, for example, by introducing
cross-links that stabilize interactions or folds within a stalk
monomer or stalk protomer (intramolecular cross-link), and/or
interactions between one or more stalk monomers or stalk protomers
(intermolecular cross-link), or any combination of such crosslinks.
In some such embodiments the cross-link is a di-tyrosine
cross-link. For example, in some embodiments intermolecular
di-tyrosine cross-links may be formed between tyrosine residues at
positions 403 and 433, 411 and 422, or 433 and 435. Similarly, in
some embodiments intermolecular di-tyrosine cross-links may be
formed between a tyrosine at residue 403 and another tyrosine
residue, and/or between a tyrosine at residue 433 and another
residue, such as, in particular, any of the other residues
described herein as potential sites for di-tyrosine cross-links,
such as tyrosines (whether natural or mutated) located at residues
308, 403, 406, 411, 422, 429, 432, 433, 435, or 437.
[0164] In some embodiments the present invention provides influenza
HA polypeptides, proteins, and/or protein complexes comprising an
artificially introduced cross-link between two of the following
regions: amino acid residues from about position 298 to about 318,
amino acid residues from about position 393 to about position 413,
amino acid residues from about position 396 to about position 416,
amino acid residues from about position 401 to about position 421,
amino acid residues from about position 412 to about position 432,
amino acid residues from about position 419 to about position 439,
amino acid residues from about position 422 to about position 442,
amino acid residues from about position 423 to about position 443,
amino acid residues from about position 425 to about position 445
and amino acid residues from about 427 to about 447. In some such
embodiments the cross-link is a di-tyrosine cross-link.
[0165] In embodiments where the influenza HA polypeptides,
proteins, and/or protein complexes of the invention comprise one or
more di-tyrosine cross-links, di-tyrosine cross-links may be
introduced between two endogenous tyrosine residues, between two
tyrosine residues originating from "to-tyrosine" mutations, or
between a tyrosine residue originating from a "to-tyrosine"
mutation and an endogenous tyrosine residue. In some embodiments,
more than one di-tyrosine cross-link is introduced into an
influenza HA protein or polypeptide.
[0166] In embodiments where the influenza HA polypeptides,
proteins, and/or protein complexes of the invention comprise one or
more di-tyrosine cross-links, non-limiting examples of amino acid
positions where a "to-tyrosine" mutation can be introduced include
N403, F406, K411, N422, D429, L432, D433, W435, or any combination
thereof.
[0167] In embodiments where the influenza HA polypeptides,
proteins, and/or protein complexes of the invention comprise one or
more di-tyrosine cross-links, non-limiting examples of preexisting
or endogenous tyrosine residues that can be used to form a
di-tyrosine cross-link include Y308 and Y437, or any combination
thereof.
[0168] In embodiments where the influenza HA polypeptides,
proteins, and/or protein complexes of the invention comprise one or
more di-tyrosine cross-links, non-limiting examples of residue
pairs between which a di-tyrosine cross-link can be introduced
include 403/429, 403/432, 403/433, 406/429, 406/433, 411/422 and
433/435, or any combination thereof
[0169] In embodiments where the influenza HA polypeptides,
proteins, and/or protein complexes of the invention comprise one or
more di-tyrosine cross-links, non-limiting examples of regions or
secondary structures of the influenza HA protein from which amino
acids may be selected for tyrosine substitution and/or di-tyrosine
cross-linking include the stalk domain (e.g. amino acid residues 1
(with the signal peptide) or 18 (without the signal peptide) to 58,
and 292 to 529 (without the transmembrane and cytoplasmic
domain(s)) or 566 (with the transmembrane domain). In some
embodiments the lower region of the stalk domain (comprising amino
acid residues 18-46, 334-343, 344-390 and 449-503 of SEQ ID NO:1),
and/or the head domain (e.g. amino acid residues 59 to 291 of SEQ
ID NO:1), of the influenza HA polypeptides, proteins, and/or
protein complexes of the invention may also comprise one or more
di-tyrosine cross-links and/or one or more to-tyrosine
mutations.
[0170] Non-limiting examples of other regions of influenza HA
proteins from which one or more amino acids may be selected for
tyrosine substitution and/or cross-linking include amino acid
residues from about position 298 to about position 313, amino acid
residues from about position 393 to about position 413, amino acid
residues from about position 396 to about position 416, amino acid
residues from about position 401 to about position 421, amino acid
residues from about position 412 to about position 432, amino acid
residues from about position 419 to about position 439, amino acid
residues from about position 422 to about position 442, amino acid
residues from about position 423 to about position 443, amino acid
residues from about position 425 to about position 445, and amino
acid residues from about position 427 to about position 447.
[0171] In some embodiments, the present invention provides
influenza HA polypeptides, proteins, and/or protein complexes that
are derived from, comprise, consist essentially of, or consist of,
the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109 or 110 (each of which are
mutants of the influenza HA amino acid sequence that comprise one
or more protease recognition sequences to facilitate proteolytic
cleavage of the head domain of the HA protein, and/or one or more
"to tyrosine" mutations to facilitate di-tyrosine cross-linking and
to facilitate "locking" of the stalk domain of the influenza HA
protein in a particular conformation, for example, in its native
trimeric conformation), or any fragment thereof, such as fragments
comprising amino acid the stalk domain of the influenza HA protein,
or any other fragments of the influenza HA protein that may be
generated proteolytically and/or that may be assembled into or form
part of a functional influenza HA protein. In some embodiments, the
present invention provides influenza HA polypeptides, proteins,
and/or protein complexes that are derived from, comprise, consist
essentially of, or consist of, an amino acid sequence having at
least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,
or 99% sequence identity to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 107, 108, 109 or 110, or any fragment
thereof.
[0172] Non-limiting examples of amino acid positions in an
influenza HA protein or polypeptide to which di-tyrosine
cross-links may be targeted include positions Y308
(pre-existing/endogenous Tyr residue) and N403Y (to-Tyr
substitution), the positions Y308 (pre-existing/endogenous Tyr
residue) and W435Y (to-Tyr substitution), the positions N403Y
(to-Tyr substitution) and Y437 (pre-existing/endogenous Tyr
residue), the positions N403Y (to-Tyr substitution) and D429Y
(to-Tyr substitution), the positions N403Y (to-Tyr substitution)
and L432Y (to-Tyr substitution), the positions N403Y (to-Tyr
substitution) and D433Y (to-Tyr substitution), the positions N406Y
(to-Tyr substitution) and D429Y (to-Tyr substitution), the
positions N406Y (to-Tyr substitution) and D433Y (to-Tyr
substitution), the positions D433Y (to-Tyr substitution) and W435Y
(to-Tyr substitution), the positions K411Y (to-Tyr substitution)
and W422Y (to-Tyr substitution), and the positions Y437
(pre-existing/endogenous Tyr residue) and W435Y (to-Tyr
substitution). In some embodiments, the influenza HA polypeptides,
proteins and/or protein complexes of the invention comprise one of
the above listed di-tyrosine cross-links. In some embodiments, the
influenza HA polypeptides, proteins and/or protein complexes of the
invention comprise two of the above listed di-tyrosine cross-links
(for example, SEQ ID NO: 5, 8, 11, 14, and 17). In some
embodiments, the influenza HA polypeptides, proteins and/or protein
complexes of the invention comprise three of the above listed
di-tyrosine cross-links. In some embodiments, the influenza HA
polypeptides, proteins and/or protein complexes of the invention
comprise four of the above listed di-tyrosine cross-links. In some
embodiments, the influenza HA polypeptides, proteins and/or protein
complexes of the invention comprise five or more of the above
listed di-tyrosine cross-links. In some embodiments, the influenza
HA polypeptides, proteins and/or protein complexes of the invention
comprise any combination or one or more of the above listed
di-tyrosine cross-links.
[0173] Non-limiting examples of influenza HA polypeptides, proteins
and/or protein complexes designed to have more than one di-tyrosine
cross-link include influenza HA proteins with two "to-tyrosine"
mutations, where each such tyrosine residue forms a crosslink with
different endogenous/preexisting tyrosine residues, or influenza HA
proteins with four "to-tyrosine" mutations, e.g.
N403Y/K411Y/N422Y/D433Y, as illustrated by SEQ ID NOs: 5, 8, 11, 14
and 17 where the tyrosine at position 403 is designed to pair with
the tyrosine at position 411, and the tyrosine at position 422 is
designed to pair with the tyrosine at position 433, thus
stabilizing the stalk domain of the HA protein by the formation of
two di-tyrosine cross-links.
[0174] A bond between a first HA polypeptide and second HA
polypeptide within the same protein complex (e.g. monomers that
arrange to form a trimer) is an example of an inter-molecular bond.
The invention provides exemplary influenza HA proteins and
polypeptides comprising cross-links designed to stabilize
inter-molecular interactions, as well as influenza HA polypeptides,
proteins or protein complexes derived from such sequences and
including the specific "to-tyrosine" mutations present in such
sequences. For example, one introduced tyrosine in one monomer is
designed to pair with the other introduced tyrosine on the adjacent
monomer.
[0175] In some embodiments, an HA polypeptide is intra-molecularly
cross-linked (e.g., both tyrosines of the cross-link are located
within the same HA polypeptide). The invention provides exemplary
influenza HA proteins and polypeptides comprising cross-links
designed to stabilize intra-molecular interactions, including
without limitation, SEQ ID NO: as well as influenza HA
polypeptides, proteins or protein complexes derived from such
sequences and including the specific "to-tyrosine" mutations
present in such sequences.
[0176] In some embodiments (including all of those described above,
and those involving influenza HA polypeptides, proteins, and/or
protein complexes having any of the specific amino acid sequences
recited herein, and those involving variants or fragments of such
influenza HA polypeptides, proteins, and/or protein complexes
having less than 100% identity to the specific amino acid sequences
provided herein), the influenza HA polypeptides, proteins, and/or
protein complexes of the invention should have one or more desired
properties, such as being capable of (1) forming a native trimeric
conformation of the stalk domain, (2) having the stalk domain
"locked" in a native trimeric conformation by cross-linking, (3)
binding to an influenza HA stalk-specific antibody, (4) binding to
a neutralizing antibody, (5) binding to a broadly neutralizing
antibody, (6) binding to an antibody selected from the group
consisting of 6F12, C179, CR6261, F10, A66, and D8, (7) binding to
and/or activating a B cell receptor, (8) eliciting an antibody
response in an animal, (9) eliciting a protective antibody response
in an animal, (10) eliciting production of neutralizing antibodies
in an animal, (11) eliciting production of broadly neutralizing
antibodies in an animal, (12) eliciting production of antibodies
that recognize quaternary neutralizing epitopes (QNEs) in an
animal, and/or (13) eliciting a protective immune response in an
animal. In some embodiments the influenza HA polypeptides,
proteins, and/or protein complexes described herein are capable of
eliciting a protective immune response against one or more
influenza virus strains in an animal and/or capable of eliciting a
protective immune response against both homologous and heterologous
influenza virus strains in an animal.
[0177] Unless otherwise stated, all description herein that relates
to specific influenza HA polypeptides, proteins, and protein
complexes, relates equally to all homologs, orthologs, analogs,
derivatives, mutant forms, fragments, chimeras, fusion proteins
etc. thereof, such as those that have certain desired properties or
features (for example those that have a stalk domain, or a portion
of a stalk domain, that is capable of folding into a native
trimeric conformation, or that have desired functional properties,
including, but not limited to, being capable of binding to, or
eliciting the production of, one or more anti-HA antibodies, such
as antibodies that are specific to the influenza HA stalk
domain).
[0178] Similarly, all description herein that relates to specific
polypeptides, proteins, and/or protein complexes polypeptides,
proteins, and/or protein complexes (e.g. those having specific
amino acid sequences or those from a specific influenza type,
subtype, or strain) relates equally to other related forms of such
polypeptides, proteins, and/or protein complexes that may exist in
nature (for example in different influenza types, subtypes or
strains) or that are related to the specific sequences provides
herein but have been altered artificially in some way, such as by
recombinant means, chemical means, or any other means. The
influenza HA polypeptides, proteins, and/or protein complexes
described herein can have, or can be derived from, the nucleotide
and/or amino acid sequences of any suitable influenza HA
polypeptides, proteins, and/or protein complexes known in the art.
In some embodiments, the influenza HA polypeptides, proteins,
and/or protein complexes of the invention may be, or may be derived
from, derivatives and/or analogs of specific influenza HA
polypeptides, proteins, and/or protein complexes described herein
or known in the art, including proteins that are substantially
homologous to any such proteins, or fragments thereof (e.g., in
various embodiments, those having at least about 50% or 55% or 60%
or 65% or 70% or 75% or 80% or 85% or 90% or 95% or 98% or 99%
identity with an amino acid or nucleic acid sequence of any
specific influenza HA polypeptides, proteins, and/or protein
complexes described herein or known in the art, when aligned using
any suitable method known to one of ordinary skill in the art, such
as, for example, using a computer homology program known in the
art) or whose encoding nucleic acid is capable of hybridizing to a
coding nucleic acid sequence of a protein of the invention, under
high stringency, moderate stringency, or low stringency
conditions.
[0179] In some embodiments, the invention provides fragments of the
influenza HA polypeptides, proteins, and/or protein complexes
described herein, such as those comprising, consisting essentially
of, or consisting of, at least about 10 amino acids, 20 amino
acids, 50 amino acids, 100 amino acids, 200 amino acids, or 500
amino acids.
[0180] In some embodiments one or more amino acid residues within a
specific influenza HA polypeptide, protein, or protein complex as
described herein, or as known in the art, can be deleted, added, or
substituted with another amino acid. In embodiments where such
mutations are introduced, the influenza HA polypeptides, proteins,
or protein complexes can be micro-sequenced to determine a partial
amino acid sequence. In other embodiments the nucleic acid
molecules encoding the influenza HA polypeptides, proteins, and/or
protein complexes can be sequenced to identify and/or confirm the
introduction of mutations.
[0181] In some embodiments, one or more amino acid residues can be
substituted by another amino acid having a similar polarity and
that may acts as a functional equivalent, resulting in a silent
alteration. In some embodiments substitutions for an amino acid
within the sequence may be selected from other members of the class
to which the amino acid belongs e.g. to create a conservative
substitution. For example, the nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Such substitutions are generally understood to be
conservative substitutions.
[0182] In some embodiments artificial, synthetic, or non-classical
amino acids or chemical amino acid analogs can be used to make the
influenza HA polypeptides, proteins, and/or protein complexes of
the invention. Non-classical amino acids include, but are not
limited to, the D-isomers of the common amino acids, fluoro-amino
acids, and "designer" amino acids such as .beta.-methyl amino
acids, C.gamma.-methyl amino acids, N.gamma.-methyl amino acids,
and amino acid analogs in general. Additional non-limiting examples
of non-classical amino acids include, but are not limited to:
.alpha.-aminocaprylic acid, Acpa; (S)-2-aminoethyl-L-cysteine/HCl,
Aecys; aminophenylacetate, Afa; 6-amino hexanoic acid, Ahx;
.gamma.-amino isobutyric acid and .alpha.-aminoisobytyric acid,
Aiba; alloisoleucine, Aile; L-allylglycine, Alg; 2-amino butyric
acid, 4-aminobutyric acid, and .alpha.-aminobutyric acid, Aba;
p-aminophenylalanine, Aphe; b-alanine, Bal; p-bromophenylalaine,
Brphe; cyclohexylalanine, Cha; citrulline, Cit;
.beta.-chloroalanine, Clala; cycloleucine, Cle;
p-cholorphenylalanine, Clphe; cysteic acid, Cya; 2,4-diaminobutyric
acid, Dab; 3-amino propionic acid and 2,3-diaminopropionic acid,
Dap; 3,4-dehydroproline, Dhp; 3,4-dihydroxylphenylalanine, Dhphe;
p-flurophenylalanine, Fphe; D-glucoseaminic acid, Gaa;
homoarginine, Hag; .delta.-hydroxylysine/HCl, Hlys;
DL-.beta.-hydroxynorvaline, Hnvl; homoglutamine, Hog;
homophenylalanine, Hoph; homoserine, Hos; hydroxyproline, Hpr;
p-iodophenylalanine, Iphe; isoserine, Ise; .alpha.-methylleucine,
Mle; DL-methionine-S-methylsulfoniumchloide, Msmet; 3-(1-naphthyl)
alanine, 1Nala; 3-(2-naphthyl) alanine, 2Nala; norleucine, Nle;
N-methylalanine, Nmala; Norvaline, Nva; O-benzylserine, Obser;
O-benzyltyrosine, Obtyr; O-ethyltyrosine, Oetyr; O-methylserine,
Omser; O-methylthreonine, Omthr; O-methyltyrosine, Omtyr;
Ornithine, Orn; phenylglycine; penicillamine, Pen; pyroglutamic
acid, Pga; pipecolic acid, Pip; sarcosine, Sar; t-butylglycine;
t-butylalanine; 3,3,3-trifluroalanine, Tfa; 6-hydroxydopa, Thphe;
L-vinylglycine, Vig; (-)-(2R)-2-amino-3-(2-aminoethylsulfonyl)
propanoic acid dihydroxochloride, Aaspa;
(2S)-2-amino-9-hydroxy-4,7-dioxanonanoic acid, Ahdna;
(2S)-2-amino-6-hydroxy-4-oxahexanoic acid, Ahoha;
(-)-(2R)-2-amino-3-(2-hydroxyethylsulfonyl) propanoic acid, Ahsopa;
(-)-(2R)-2-amino-3-(2-hydroxyethylsulfanyl) propanoic acid, Ahspa;
(2S)-2-amino-12-hydroxy-4,7,10-trioxadodecanoic acid, Ahtda;
(2S)-2,9-diamino-4,7-dioxanonanoic acid, Dadna;
(2S)-2,12-diamino-4,7,10-trioxadodecanoic acid, Datda;
(S)-5,5-difluoronorleucine, Dfnl; (S)-4,4-difluoronorvaline, Dfnv;
(3R)-1-1-dioxo-[1,4]thiaziane-3-carboxylic acid, Dtca;
(S)-4,4,5,5,6,6,6-heptafluoronorleucine, Hfnl;
(S)-5,5,6,6,6-pentafluoronorleucine, Pfnl;
(5)-4,4,5,5,5-pentafluoronorvaline, Pfnv; and
(3R)-1,4-thiazinane-3-carboxylic acid, Tca. Furthermore, the amino
acid can be D (dextrorotary) or L (levorotary). For a review of
classical and non-classical amino acids, see Sandberg et al., 1998
(Sandberg et al., 1998. New chemical descriptors relevant for the
design of biologically active peptides. A multivariate
characterization of 87 amino acids. J Med Chem 41(14): pp.
2481-91).
Nucleic Acids
[0183] In addition to providing certain influenza HA polypeptides,
proteins, and/or protein complexes, as described herein, the
present invention also provides nucleic acids encoding such
influenza HA polypeptides, proteins, and/or protein complexes, and
compositions and vectors comprising such nucleic acids. Such
nucleic acids can be obtained or made using any suitable method
known in the art. For example, nucleic acid molecules encoding
influenza HA polypeptides, proteins, and/or protein complexes may
be obtained from cloned DNA or made by chemical synthesis. In some
embodiments the nucleic acids may be obtained by reverse
transcribing RNA prepared by any of the methods known to one of
ordinary skill in the art, such as random- or poly A-primed reverse
transcription. Whatever the source, a nucleic acid molecule
encoding an influenza HA polypeptide, protein, and/or protein
complex of the present invention can be cloned into any suitable
vector, such as those to be used for propagation of the nucleic
acid molecule or those to be used for expression of the nucleic
acid molecule. The nucleic acid may be cleaved at specific sites
using various restriction enzymes, if needed. In embodiments
requiring expression, the nucleic acid can be operatively linked to
a promoter suitable for directing expression in the desired cell
type, such as a mammalian cell or an insect cell, and may be
incorporated into any suitable expression vector, such as a
mammalian or insect expression vector. A nucleic acid molecule
encoding an influenza HA polypeptide, protein, and/or protein
complex of the present invention optimized by methods known in the
art to improve expression levels of the protein expressed
therefrom. For example, codon optimization may be used to minimize
or eliminate variations in codon usage between species. In some
embodiments an influenza HA polypeptide, protein, and/or protein
complex of the present invention is derived from a nucleic acid
molecule that has been codon optimized for expression in humans
(see, for example, SEQ ID NO. 63 and FIG. 48), Cricetulus griseus
(see, for example, SEQ ID NO.64 and FIG. 49), Nicotiana benthamiana
(see, for example, SEQ ID NO.65 and FIG. 50), Pichia pastoris (see,
for example, SEQ ID NO.66 and FIG. 51), Saccharomyces cerevisiae
(see, for example, SEQ ID NO.67 and FIG. 52) or Spodoptera
frugiperda (see, for example, SEQ ID NO.68 and FIG. 53).
[0184] In some embodiments, the present invention provides nucleic
acids that are derived from, comprise, consist essentially of, or
consist of, the nucleic acid sequence of SEQ ID NO: 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, or 62 (each of which encodes a mutant of
the influenza HA amino acid sequence that comprises one or more
protease recognition sequences to facilitate proteolytic cleavage
of the head domain of the HA protein, and/or one or more "to
tyrosine" mutations to facilitate di-tyrosine cross-linking and to
facilitate "locking" of the stalk domain of the influenza HA
protein in a particular conformation, for example, in its native
trimeric conformation), or any fragment thereof, such as fragments
encoding the stalk domain of the influenza HA protein.
[0185] Furthermore, one or skill in the art can readily visualize,
or make, nucleic acid molecules that comprise any one or more of
the specific "to-tyrosine" mutations described herein, for example,
by locating the nucleotide codon that encodes the specific amino
acid residue to be mutated, and mutating the nucleotides at that
codon as necessary to result in a tyrosine-encoding codon.
Cross-Linking
[0186] In some embodiments the influenza HA polypeptides and/or
proteins of the invention are assembled into protein complexes
having a desired conformational structure, such as the native
trimeric conformation of the stalk domain, and are cross-linked in
order to stabilize that conformation. Details of particular regions
of the influenza HA protein that can be cross-linked, as well as
particular influenza HA mutants designed to facilitate such
cross-linking, are described in other sections of this application.
In some embodiments the cross-links may be used to stabilize the
tertiary and/or quarternary structures of the influenza HA protein.
In some embodiments, the cross-linking may be intra- and/or
intermolecular cross-linking. In some embodiments, the cross-links
that are used are targeted cross-links. In some embodiments, the
cross-links that are used are stable under physiological
conditions. In some embodiments, the cross-links that are used do
not lead to aggregate formation of the influenza HA protein, for
example during expression and/or during storage (such as storage of
compositions comprising high concentrations of the influenza HA
protein). In some embodiments the introduction of such cross-links
may enhance the effectiveness of the influenza HA polypeptides,
proteins and proteins of the invention as immunogens, such as
vaccine immunogens. In some embodiments the introduction of such
cross-links may stabilize epitopes within the influenza HA protein,
for example, epitopes in the stalk domain, such that the epitopes
can be recognized by particular antibodies, elicit production of
antibodies, and/or activate B cell receptors upon antibody
binding.
[0187] In some embodiments targeted cross-linking can be used. A
targeted cross-link is one that can be made to form at a particular
position or positions within the influenza HA protein or protein
complex. Several strategies may be used to target cross-links to
specific locations in an influenza HA protein or polypeptide, such
as the specific locations described herein. The present invention
provides residue pairs within the influenza HA protein that, when
cross-linked, can or may stabilize an influenza HA polypeptide,
protein, or protein complex in a conformation that is capable of
binding to, or eliciting the production of, neutralizing
antibodies, and/or that is capable of generating a neutralizing
antibody response in an animal. A targeted cross-link may be
introduced at one or more of the locations or positions specified
herein by exploiting the physical and/or chemical properties of
certain amino acid side chains, for example by making use of
enzymatic reactions that recognize specific amino acid sequences or
three-dimensional structures, or by incorporating non-natural amino
acids that have the ability to form cross-links in a folded protein
or protein complex.
[0188] Cross-links or modifications may be targeted to specific
sites in the structure of the influenza HA protein or polypeptide,
for example the stalk domain, in order to achieve the desired
outcome, e.g. stabilization of the stalk domain in its native
trimeric conformation. The present invention contemplates the
targeted introduction of one or more cross-links and/or other
stabilizing modifications at any suitable position(s) in an
influenza HA protein or polypeptide, preferably where the
cross-link or modification stabilizes the stalk domain in its
native trimeric conformation, or provides enhanced stabilization of
the native trimeric conformation of the stalk domain. The invention
contemplates that any influenza HA protein amino acid residue,
residue pair, secondary structure or other region described herein
for di-tyrosine cross-linking may also be used in the formation of
other targeted cross-links or bonds or other modifications,
including but not limited to amino acid positions Y308, N403, N406,
K411, W422, D429, L432, D433, W435, and Y437 or any combination
thereof; residue pairs 308/403, 308/435, 403/437, 403/429, 403/432,
403/433, 406/429, 406/433, 411/422, 433/435 and 437/435, or any
combination thereof; regions or secondary structures including, for
example the HA protein stalk domain or head domain; and other
regions of influenza HA protein including the transmembrane domain
or the lower region of the stalk domain.
[0189] In some embodiments the influenza HA polypeptides, proteins
and protein complexes of the invention comprise cross-links in the
stalk domain, such cross-links need not be located only in the
stalk domain. In some embodiments cross-links may be located
anywhere throughout the influenza HA polypeptide, protein or
protein complex, including the head domain in "head-on"
polypeptides, proteins and/or protein complexes, as desired.
Preferably, an influenza HA polypeptide, protein and/or protein
complex comprising cross-links in other regions (e.g. outside of
the stalk domain) will retain one or more desired properties such
as being capable of (1) forming a native trimeric conformation of
the stalk domain, (2) having the stalk domain "locked" in a native
trimeric conformation by cross-linking, (3) binding to an influenza
HA stalk-specific antibody, (4) binding to a neutralizing antibody,
(5) binding to a broadly neutralizing antibody, (6) binding to an
antibody selected from the group consisting of 6F12, C179, CR6261,
F10, A66, and D8, (7) binding to and/or activating a B cell
receptor, (8) eliciting an antibody response in an animal, (9)
eliciting a protective antibody response in an animal, (10)
eliciting production of neutralizing antibodies in an animal, (11)
eliciting production of broadly neutralizing antibodies in an
animal, (12) eliciting production of antibodies that recognize
quaternary neutralizing epitopes (QNEs) in an animal, and/or (13)
eliciting a protective immune response in an animal.
[0190] A wide variety of methods of cross-linking proteins intra-
and inter-molecularly are known in the art, including those having
cross-links with varying lengths of spacer arms, and those with and
without fluorescent and functional groups for purification. Such
methods include, but are not limited to, the use of
heterobifunctional cross-linkers (e.g. succinimidyl
acetylthioacetate (SATA), trans-4-(maleimidylmethyl)
cyclohexane-1-carboxylate (SMCC), and succinimidyl
3-(2-pyridyldithio)propionate (SPDP)), homobifunctional
cross-linkers (e.g. succinimidyl 3-(2-pyridyldithio)propionate),
photoreactive cross-linkers (e.g.
4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt
(ATFB, STP ester), 4-azido-2,3,5,6-tetrafluorobenzoic acid,
succinimidyl ester (ATFB, SE), 4-azido-2,3,5,6-tetrafluorobenzyl
amine, hydrochloride, benzophenone-4-isothiocyanate,
benzophenone-4-maleimide, 4-benzoylbenzoic acid, succinimidyl
ester, N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (PEAS; AET),
thiol reactive cross-linkers (e.g. maleimides and iodoacetamides),
amine reactive cross-linkers (e.g. glutaraldyde, bis(imido esters),
bis(succinimidyl esters), diisocyanates and diacid chlorides).
Because thiol groups are highly reactive and relatively rare in
most proteins by comparison to amine groups, thiol-reactive
cross-linking may be used in some embodiments. In cases where thiol
groups are missing or not present at appropriate sites in the
structures of influenza HA protein, they can be introduced using
one of several thiolation methods. For example, Succinimidyl
trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate can be used to
introduce thiol-reactive groups at amine sites.
[0191] Several oxidative cross-links are known, such as disulfide
bonds (which form spontaneously and are pH and redox sensitive),
and di-tyrosine bonds (which are highly stable, and irreversible
under physiological conditions).
[0192] In some embodiments the cross-links stabilize the tertiary
structure of an influenza HA protein. In some embodiments the
cross-links stabilize the quaternary structure of an influenza HA
protein. In some embodiments the cross-links stabilize both the
tertiary and quaternary structure of an influenza HA protein.
[0193] In some embodiments an influenza HA polypeptide, protein
and/or protein complex of the invention has cross-links that are
thermostable. In some embodiments an influenza HA polypeptide,
protein and/or protein complex of the invention has cross-links
that are not toxic. In some embodiments an influenza HA
polypeptide, protein and/or protein complex of the invention has
cross-links that are targeted cross-links, or non-targeted
cross-links, or reversible cross-links, or irreversible
cross-links, or cross-links formed by use of homo-bifunctional
cross-linking agents, or cross-links formed by use of
hetero-bifunctional cross-linking agents, or cross-links formed by
use of reagents that react with amine groups, or cross-links formed
by use of reagents that react with thiol groups, or cross-links
formed by use of reagents that are photoreactive, or cross-links
formed between amino acid residues, or cross-links formed between
mutated amino acid residues incorporated into the structure of the
proteins or protein complexes, or oxidative cross-links, or
di-tyrosine bonds, or glutaraldehdye cross-links, or any
combination thereof. In some embodiments an influenza HA
polypeptide, protein and/or protein complex of the invention does
not have glutaraldehyde cross-links.
[0194] In some embodiments an influenza HA polypeptide, protein
and/or protein complex of the invention does not have any
artificially-introduced disulfide bonds, or if it does have such
disulfide bonds, also has additional artificially-introduced
cross-links. In some embodiments an influenza HA polypeptide,
protein and/or protein complex of the invention does not have any
artificially introduced disulfide bonds, but may have naturally
occurring disulfide bonds. Disulfide bonds can be introduced
artificially when cysteine side-chains are engineered by point
mutation. Disulfide bonds are, however, known to be pH sensitive
and to be dissolved under certain redox conditions, and the
preventative and/or therapeutic utility of proteins and/or protein
complexes engineered with disulfide cross-links, for example to be
used as immunogens in vivo, may therefore be compromised.
Furthermore, undesired disulfide bonds often form between proteins
with free sulfhydryl groups that mediate aggregate formation (see,
for example, Harris R J et al. 2004, Commercial manufacturing scale
formulation and analytical characterization of therapeutic
recombinant antibodies. Drug Dev Res 61 (3): 137-154; Costantino
& Pikal (Eds.), 2004. Lyophilization of Biopharmaceuticals,
editors Costantino & Pekal. Lyophilization of
Biopharmaceuticals. Series: Biotechnology: Pharmaceutical Aspects
II, see pages 453-454; Tracy et al., 2002, U.S. Pat. No.
6,465,425), which has also been reported as a problem with HIV
gp120 and gp41 (Jeffs et al. 2004. Expression and characterization
of recombinant oligomeric envelope glycoproteins derived from
primary isolates of HIV-1. Vaccine 22:1032-1046; Schulke et al.,
2002. Oligomeric and conformational properties of a proteolytically
mature, disulfide-stabilized human immunodeficiency virus type 1
gp140 envelope glycoprotein. J Virol 76:7760-7776). Thus, in many
embodiments it is preferred that disulfide bonding is not used, or
is not used as the sole method of cross-linking.
[0195] If the structure and/or immunogenicity of an influenza HA
polypeptide, protein and/or protein complex is compromised or
altered by a cross-link, maintaining its overall structure and
function can be achieved by controlling the availability of amino
acid side-chains for the cross-linking reaction or by introducing
additional cross-links or other stabilizing modifications. For
example, in the case of DT cross-linking, tyrosyl side-chains that
are available for reaction, but that lead to the distortion of the
structure of the complex, and that compromise the
immunogenicity/antigenicity of the influenza HA protein, can be
removed by mutating such residues to another amino acid such as,
for example, phenylalanine. Furthermore, point mutations may be
introduced at positions where the amino acid side-chains will react
with cross-linking agents or each other, such that the formation of
the bond(s) causes the most beneficial outcome. These positions may
also be identified as described herein.
[0196] When at a selected residue a reactive side-chain is not
already present, a point mutation may be introduced, for example
using molecular biological methods to introduce such a point
mutation into the cDNA of a nucleic acid directing its expression,
such that a reactive side-chain is present and available for the
reaction.
[0197] Cross-links that may be used include, but are not limited
to, reversible cross-links resulting from the use of homo- and
hetero-bifunctional cross-linking agents that react with amine
and/or thiol groups, photoreactive cross-link reagents, any
cross-links that may form between non-classical amino acids
incorporated into the structure of an influenza HA polypeptide,
protein and/or protein complex, any oxidative cross-links, such as,
but not limited to, di-tyrosine cross-links/bonds,
heterobifunctional cross-linkers (e.g. succinimidyl
acetylthioacetate (SATA), trans-4-(maleimidylmethyl)
cyclohexane-1-carboxylate (SMCC), and succinimidyl
3-(2-pyridyldithio)propionate (SPDP)), homobifunctional
cross-linkers (e.g. succinimidyl 3-(2-pyridyldithio)propionate),
photoreactive cross-linkers (e.g.
4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt
(ATFB, STP ester), 4-azido-2,3,5,6-tetrafluorobenzoic acid,
succinimidyl ester (ATFB, SE), 4-azido-2,3,5,6-tetrafluorobenzyl
amine, hydrochloride, benzophenone-4-isothiocyanate,
benzophenone-4-maleimide, 4-benzoylbenzoic acid, succinimidyl
ester, N-((2-pyridyldithio)ethyl)-4-azidosalicylamide (PEAS; AET),
thiol reactive cross-linkers (e.g. maleimides and iodoacetamides),
amine reactive cross-linkers (e.g. glutaraldyde, bis(imido esters),
bis(succinimidyl esters), diisocyanates and diacid chlorides).
[0198] The present invention also contemplates the introduction of
targeted non-covalent tyrosine-stacking interactions as
"cross-links" to stabilize protein-protein interactions and/or
desired protein or peptide conformations, such as the native
trimeric conformation of a stalk domain of an influenza HA
polypeptide, protein and/or protein complex. The cross-link
comprises a targeted pi stacking interaction including but not
limited to a T-shaped, sandwich, or parallel displaced pi stacking
interaction between the aromatic side chains of an
introduced/engineered tyrosine and an endogenous tyrosine,
phenylalanine, histidine, or tryptophan within the influenza HA
polypeptide, protein and/or protein complex, or between the
aromatic side chain of an introduced/engineered tyrosine and a
second introduced/engineered tyrosine within the influenza HA
polypeptide, protein and/or protein complex.
[0199] Irreversible cross-links, as used in the context of this
application, include those that are not significantly dissolved
under physiologically relevant conditions. It is preferred that the
type of cross-links used should not lead to aggregate formation
during expression or when the influenza HA polypeptides, proteins
and/or protein complexes of the invention are stored at high
concentrations. Disulfide bonds are not irreversible cross-links.
Rather they are reversible cross-links and may dissolve under
physiologically relevant conditions and/or lead to aggregate
formation during protein expression and/or production or when
stored in high concentrations.
[0200] In some embodiments cross-links may be targeted to the
specific regions of influenza HA polypeptides, proteins and/or
protein complexes described herein in order to achieve the desired
conformational stabilization and/or the desired immunogenic
properties (e.g. the ability to maintain the stalk domain in its
native trimeric conformation and/or to bind to broadly neutralizing
antibodies). Alternatively, proteins with the cross-links at the
locations specified herein may be isolated from a mixture of
cross-linked and un-cross-linked proteins with and without desired
modifications, for example based on chemical, physical, and/or
functional characteristics. Such characteristics may include, for
example, trimerization, the presence of a stalk domain having a
native trimeric conformation, and/or any desired antigenic,
immunogenic, or biochemical characteristics.
[0201] Alternatively, in some embodiments, cross-links may not be
targeted, and proteins with the desired cross-links or properties
may be isolated from a mixture of modified and unmodified proteins
made using a non-targeted cross-linking system.
[0202] In embodiments where influenza HA polypeptides, proteins or
protein complexes with the desired cross-links are to be isolated
from a mixture of cross-linked and un-cross-linked proteins, such
isolation or separation may be performed on the basis of one or
more characteristics including, but not limited to, molecular
weight, molecular volume, chromatographic properties, mobility in
electrophoresis, antigenic and biochemical characteristics,
fluorescence characteristics, solubility, binding to antibodies,
structural characteristics, immunological characteristics, or any
other suitable characteristics.
[0203] In addition to the specific cross-linking positions
described herein, additional positions within influenza HA
polypeptides, proteins or protein complexes can be identified at
which further cross-links can be made, for example where a reactive
side-chain would be able to form a bond with a reactive side-chain
elsewhere on the influenza HA polypeptide, protein or protein
complex. In some embodiments, such additional positions can be
selected, for example, to maintain or improve the
immunogenicity/antigenicity of the protein, polypeptide or protein
complex. In some embodiments, such additional positions to be
cross-linked can be selected in pairs.
Di-tyrosine (DT) Cross-Linking
[0204] In some embodiments the present invention provides influenza
HA polypeptides, proteins and/or protein complexes that comprise
di-tyrosine (DT) cross-links, and methods of making such
DT-cross-linked influenza HA polypeptides, proteins and/or protein
complexes.
[0205] Di-tyrosine cross-linking introduces one or more covalent
carbon-carbon bonds into proteins or protein complexes. This
provides a method for stabilizing proteins, protein complexes, and
conformations, by introduction of intra- and/or inter-polypeptide
di-tyrosine bonds while maintaining their structural and functional
integrity (See Marshall et al., US Patent Numbers 7,037,894 &
7,445,912, the contents of which are hereby incorporated by
reference). The minimally altering, and zero-length DT cross-link
is not hydrolyzed under physiological conditions, and has been
demonstrated to maintain proteins' structural integrity by liquid
chromatography/mass spectrometry (LC/MS). Di-tyrosine cross-links
are known to be safe, as they form naturally in vivo, both in the
context of proteins evolved to utililze their specific
characteristics (e.g. Elvin C M et al. 2005, Nature 437:999-1002;
Tenovuo J & Paunio K 1979, Arch Oral Biol.; 24(8):591-4), and
as a consequence of non-specific protein oxidation (Giulivi et al.
2003, Amino Acids 25(3-4):227-32), and as they are present in large
quantities in some of our most common foods: DT bonds form the
structure of wheat gluten--the quarternary protein structure
comprising the glutenin subunits--e.g. in bread dough during mixing
and baking (Tilley et al. 2001, Agric. Food Chem 49, 2627).
Di-tyrosine bonds do not form spontaneously in vitro. Rather, the
enzymatic cross-link reaction is carried out under optimized
conditions to preserve protein structure and function. Therefore,
non-specific bonding/aggregation does not occur (unlike with
disulfide bonding), and therefore large-scale manufacturing of a DT
stabilized immunogen may be economically more feasible.
[0206] Tyrosyl side-chains are present in many redox enzymes, and
catalysis of the enzyme-specific reactions often involves tyrosyl
radicals that are long-lived and have comparatively low reactivity.
Under optimized conditions radical formation is specific to tyrosyl
side-chains. In close proximity, tyrosyl side chains undergo
radical coupling and form a covalent, carbon-carbon bond. Tyrosyl
radicals that do not react revert to non-radicalized tyrosyl
side-chains (Malencik & Anderson, 2003. Di-tyrosine as a
product of oxidative stress and fluorescent probe. Amino Acids 25:
233-247). Therefore, tyrosyl side-chains must be situated in close
proximity to form DT bonds, either within a single folded
polypeptide chain, or on closely interacting protein domains within
a complex. Because a C.alpha.-C.alpha. separation of approximately
5-8 .ANG. is a prerequisite to bond formation (Brown et al., 1998.
Determining protein-protein interactions by oxidative cross-linking
of a glycine-glycine-histidine fusion protein. Biochemistry 37,
4397-4406; Marshall et al. 2006, U.S. Pat. No. 7,037,894), and
because no atom is added in the formation of these bonds, the
resulting "staple" is "zero length" and non-disruptive to the
protein structure.
[0207] Tyrosine residues to be cross-linked may be naturally
present in the primary structure of the protein to be cross-linked
or may be added by controlled point mutation. To form DT bonds,
proteins with tyrosyl side chains can be subjected to reaction
conditions that lead to the formation of DT bonds. Such conditions
are, or become, oxidative reaction conditions, as the DT bond
formation reaction is an oxidative cross-linking reaction. In some
embodiments the DT cross-linking reaction conditions yield proteins
that are otherwise not, or not detectably, modified. Such
conditions may be obtained by use of enzymes that catalyze the
formation of H.sub.2O.sub.2, such as peroxidases. DT bond formation
may be monitored by spectrophotometry with an excitation wavelength
of around 320 nm, and fluorescence measured at a wavelength of
around 400 nm (see, for example, FIG. 4A), while loss of tyrosyl
fluorescence is monitored also monitored by standard procedures.
When loss of tyrosyl florescence is no longer stoichiometric with
DT bond formation, the reaction may be stopped by any methods known
to one skilled in the art, such as, for example, by the addition of
a reducing agent and subsequent cooling (on ice) or freezing of the
sample. Further details of how to perform DT cross-linking are
known in the art and are described in, for example, Marshall et al.
2006, U.S. Pat. No. 7,037,894, the contents of which are hereby
incorporated by reference.
[0208] The major advantages of di-tyrosine cross-linking in protein
engineering include (i) the ability to target specific residues for
cross-linking (based on the primary, secondary, tertiary, and/or
quaternary structures of proteins and complexes), (ii) minimal
structural modification, (iii) specificity of the reaction
(tyrosine is the only amino acid known to form cross-links under
specific cross-linking conditions); (iv) stability of the linkage,
(v) zero length of the cross-link (no atom is added), and (vi) the
scalability of the cross-linking chemistry.
[0209] In some embodiments, targeted DT cross-links may be
introduced at one or more of the specific locations in the
influenza HA protein that are recited herein. In other embodiments,
additional positions within influenza HA polypeptides, proteins or
protein complexes can be identified at which DT cross-links can be
made. In some embodiments, di-tyrosine bonds or cross-links are
targeted to specific residue pairs within the structure of an
influenza HA polypeptide, protein and/or protein complex where DT
bonds will, or are expected to, form due to, for example, their
close proximity. In some embodiments tyrosyl side chains are
already present at amino acid residues to be cross-linked. In some
cases naturally occurring tyrosine residues may constitute either
one or both of the paired tyrosine residues necessary for
di-tyrosine bond formation. However, in other cases the influenza
HA polypeptides, proteins and/or protein complexes of the invention
are mutated or engineered to add one or more tyrosine residues, or
to substitute one or more non-tyrosine residues for tyrosine
residues. Such mutations are referred to herein as "to-tyrosine"
mutations, and can be introduced at locations where it is desirable
to form di-tyrosine cross-links/bonds. In some embodiments, the
present invention provides mutant influenza HA polypeptides,
proteins, and/or protein complexes in which tyrosyl side chains are
introduced at desired cross-linking positions by introducing point
mutations to tyrosine in a nucleic acid sequence encoding the
influenza HA polypeptide, protein, or protein complex.
Alternatively, in some embodiments influenza HA proteins,
polypeptides or protein complexes, or portions thereof, may be
synthesized to include tyrosine residues or amino acids having
tyrosyl side chains at desired cross-linking positions. Conversely,
in some embodiments the present invention provides mutant influenza
HA polypeptides, proteins, and/or protein complexes in which
tyrosyl side chains are removed at undesirable cross-linking
positions by introducing point mutations from tyrosine in a nucleic
acid sequence encoding the influenza HA polypeptide, protein, or
protein complex, or influenza HA polypeptides, proteins, or protein
complexes may be synthesized to exclude tyrosine residues or amino
acids having tyrosyl side chains at positions where cross-linking
is not desired. For example, at least one of the tyrosyl side
chains can be replaced with another side chain, such as a
phenylalanine side chain (see, for example, Marshall C P et al.,
U.S. Pat. No. 7,037,894, the contents of which are hereby
incorporated by reference). Accordingly, the influenza HA
polypeptides, proteins and protein complexes of the invention may
comprise point mutations "to tyrosine" or "from tyrosine." Such
mutations can be made by altering the nucleic acid sequences that
encode the influenza HA polypeptides, proteins and/or protein
complexes of the invention using any suitable mutagenesis methods
known in the art. Alternatively, mutant influenza HA polypeptides,
proteins and/or protein complexes may be synthesized, purified,
and/or produced by any other suitable methods known in the art.
[0210] In some embodiments, the present invention contemplates the
targeted introduction of one or more di-tyrosine cross-link at any
suitable position(s) in an influenza HA polypeptide, protein or
protein complex, for example, in the stalk domain where the
cross-link will or may stabilize the stalk domain in a native
trimeric conformation or other conformation capable of binding
anti-stalk antibodies, such as neutralizing or broadly neutralizing
anti-stalk antibodies. Such stabilization may be achieved, for
example, by introducing cross-links that stabilize interactions or
folds within a stalk monomer (intra-molecular cross-linking) and/or
interactions between one or more stalk monomers that comprise that
stalk trimer (inter-molecular cross-linking), or any combination of
intra- and/or inter-molecular crosslinks.
Proteolytic Cleavage
[0211] In some embodiments of the invention the influenza HA
polypeptides, proteins and/or protein complexes of the invention
(and/or intermediates in the synthesis thereof), comprise one or
more protease recognition motifs that can be used, for example, to
facilitate proteolytic removal of the head domain. Any suitable
protease recognition motifs known in the art can be used. Such
engineered protease recognition sites can be located at any
suitable location in the influenza HA polypeptide, protein, and/or
protein complex in which they will be useful for the disruption
and/or removal of the head domain but preferably will not disrupt
the native trimeric conformation of (and/or conformation of
neutralizing eptiopes in) the stalk domain. Such locations can be
determined using methods known in the art, including, but not
limited to, testing the effect of introducing engineered protease
recognition sites in functional assays, antibody binding assays,
antigenic assays, structural assays, and the like. In some
embodiments such engineered protease recognition motifs may be
located within a variable loop region--as such regions are known to
tolerate variations in amino acid sequence without significantly
altering the structure and/or function of the influenza HA protein.
The influenza HA proteins of the invention can be engineered to
introduce one or more protease recognition sequences by, for
example, inserting one or more amino acids that comprise, or
comprise part of, a protease recognition site (see for example SEQ
ID NO. 18, 19, 21, 23 and 25), or by substituting one or more amino
acids from the influenza HA protein with different amino acids that
comprise, or comprise part or, a protease recognition site (see for
example SEQ ID NO. 24), or by performing a combination of insertion
and substitution of amino acids (see for example SEQ ID NO. 20 and
22) in order to generate a protease recognition sequence within the
influenza HA protein sequence. The engineered protease recognition
site will typically consist of up to about 20 amino acid residues.
In some embodiments the influenza HA polypeptides, proteins and/or
protein complexes described herein comprise an engineered protease
recognition motif at one or more of the following primary
head-removal sites: amino acid residues 53-67, amino acid residues
60-76, amino acid residues 269-277, and amino acid residues
277-290, and may optionally also comprise an engineered protease
recognition motif at one or more of the following secondary
head-removal sites: amino acid residues 142-146, and amino acid
residues 155-164. In some embodiments the influenza HA
polypeptides, proteins and/or protein complexes of the present
invention comprise a protease recognition sequence that begins at
an amino acid residue position within one of the following regions
of the influenza HA protein: amino acid residues 40-68, amino acid
residues 60-76, amino acid residues 77-114, amino acid residues
120-141, amino acid residues 142-146, amino acid residues 148-178,
amino acid residues 182-188, amino acid residues 195-201, amino
acid residues 209-242, amino acid residues 250-255, amino acid
residues 260-285, amino acid residues 277-290, and amino acid
residues 286-320. In some embodiments such protease recognition
motifs may allow proteolytic cleavage at one or more of the Sa, Ca,
Sb and Cb antigenic sites in the influenza HA head domain. In some
embodiments the protease recognition motif is inserted into the
influenza HA protein immediately following the amino acid at
position 48, 63, 278, 282, 286, or 291. In some embodiments the
protease recognition motif is inserted into the influenza HA
protein within one or more of the following regions of the
influenza HA protein: amino acid residues 38-58, amino acid residue
53-73, amino acid residues 268-288, amino acid residues 272-292,
amino acid residues 276-296 and amino acid residues 281-301. In
some embodiments the protease recognition motifs may comprise a
PreScission Protease recognition sequence (for example, LEVLFQGP
(SEQ ID NO. 69)) or TEV recognition sequence, (for example, ENLYFQG
(SEQ ID NO. 70) or ENLYFQS (SEQ ID NO. 71)), or any combination
thereof. Nucleotide sequences encoding such protease recognition
sites can be engineered into the nucleic acids that encode the
influenza HA polypeptides, proteins, and/or protein complexes of
the invention using standard molecular biology techniques known in
the art.
Making and Analyzing Influenza HA Polypeptides, Proteins, and
Protein Complexes
[0212] In some embodiments the present invention provides methods
for making the influenza HA polypeptides, proteins, and protein
complexes of the invention. The influenza HA polypeptides,
proteins, and protein complexes of the invention can be made by any
suitable means known in the art. In some embodiments the influenza
HA polypeptides, proteins, and/or protein complexes of the
invention can be made by recombinant means. In some embodiments,
the influenza HA polypeptides, proteins, and/or protein complexes
of the invention, or any portion thereof, can be made by chemical
synthesis means. For example, a peptide corresponding to a portion
of a protein or protein complex as described herein can be
synthesized by use of a peptide synthesizer.
Recombinant Production Methods
[0213] In embodiments where the influenza HA polypeptides, proteins
and protein complexes of the invention are made by recombinant
means, nucleic acids encoding the influenza HA polypeptides,
proteins and protein complexes of the invention can be expressed in
any suitable cell type, including, but not limited to mammalian
cells, avian cells (such as EB66 duck cells) and insect cells (such
as SF9 or Hi5 cells, using a baculovirus expression system).
Methods for expressing polypeptides and proteins from nucleic acid
molecules are routine and well known in the art, and any suitable
methods, vectors, systems, and cell types known in the art can be
used. For example, typically nucleic acid sequences encoding the
influenza HA polypeptides, proteins and/or protein complexes of the
invention will be placed into a suitable expression construct
containing a suitable promoter, which will then be delivered to
cells for expression.
Chimeric/Fusion Proteins & Oligomerization Domains
[0214] In some embodiments it may be desirable to add chimeric
domains to the influenza HA polypeptides, proteins and/or protein
complexes described herein, to produce chimeric proteins/fusion
proteins, for example to facilitate the analysis and/or isolation
and/or purification of the influenza HA polypeptides, proteins
and/or protein complexes described herein. In some embodiments, the
influenza HA polypeptides, proteins and protein complexes of the
invention may comprise leader sequences, precursor polypeptide
sequences, secretion signals, localization signals, epitope tags,
protease cleavage sites, and the like. Epitope tags that can be
used include, but are not limited to, FLAG tags, glutathione
S-transferase (GST) tags, green fluorescent protein (GFP) tags,
hemagglutinin A (HA) tags, histidine (His) tags, luciferase tags,
maltose-binding protein (MBP) tags, c-Myc tags, protein A tags,
protein G tags, streptavidin (strep) tags, and the like.
[0215] In some embodiments it may be desirable to add
oligomerization domains to facilitate the assembly of influenza HA
polypeptides, proteins and/or protein complexes as described
herein, and/or to facilitate stabilization of stalk domain in a
native trimeric conformation, and/or to stabilize other structural
features of the influenza HA polypeptides, proteins and/or protein
complexes. In some embodiments the oligomerization domains are
trimerization motifs, including, but not limited to, the T4 foldon
motif. There are a wide variety of trimerization domains in natural
proteins that can be used for these purposes including, but not
limited to, those described in Habazettl et al., 2009 (Habazettl et
al., 2009. NMR Structure of a Monomeric Intermediate on the
Evolutionarily Optimized Assembly Pathway of a Small Trimerization
Domain. J. Mol. Biol.), Kammerer et al., 2005 (Kammerer et al.,
2005. A conserved trimerization motif controls the topology of
short coiled coils. Proc Natl Acad Sci USA 102 (39): 13891-13896),
Innamorati et al., 2006 (Innamorati et al., 2006. An intracellular
role for the Clq-globular domain. Cell signal 18(6): 761-770), and
Schelling et al., 2007 (Schelling et al., 2007. The reovirus
.sigma.-1 aspartic acid sandwich: A trimerization motif poised for
conformational change. Biol Chem 282(15): 11582-11589). Stabilizing
trimeric protein complexes can also be accomplished using the GCN4
and T4 fibrinitin motifs (Pancera et al., 2005. Soluble Mimetics of
Human Immunodeficiency Virus Type 1 Viral Spikes Produced by
Replacement of the Native Trimerization Domain with a Heterologous
Trimerization Motif: Characterization and Ligand Binding Analysis.
J Virol 79(15): 9954-9969; Guthe et al., 2004. Very fast folding
and association of a trimerization domain from bacteriophage T4
fibritin. J. Mol. Biol. v337 pp. 905-15; Papanikolopoulou et al.,
2008. Creation of hybrid nanorods from sequences of natural
trimeric fibrous proteins using the fibritin trimerization motif.
Methods Mol Biol 474:15-33). Heterologous oligomerization motifs
may be introduced by any recombinant methods known to one of
ordinary skill in the art in order to stabilize the protein-protein
interactions of the proteins of the present invention.
[0216] In some embodiments it may be desirable to add more than one
additional domain and/or tag to the influenza polypeptides,
proteins and/or protein complexes described herein, and any
combination of suitable chimeric and/or oligomerization domains may
be added to makedesired influenza HA polypeptides, proteins and/or
protein complexes. In some embodiments, the additional domains are
engineered at or in the transmembrane region of an influenza HA
protein, for example by insertion and/or substitution of one or
more amino acids in the transmembrane region such that all or a
portion of the transmembrane region is replaced by the additional
domains. In some embodiments the additional domains comprise a
thrombin cleavage site, a T4 foldon motif and a histidine tag (e.g.
a 6.times.His tag (SEQ ID NO: 118)). In some embodiments the
additional domains are encoded by a nucleic acid sequence
comprising
CGTTCTCTGGTTCCGCGTGGTTCTCCGGGTTCTGGTTACATCCCGGAAGCTCCGCGT
GACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTG
CACCACCACCACCACCACTGA (SEQ ID NO. 72). In some embodiments the
influenza HA polypeptides, proteins and/or protein complexes
comprise a tag comprising, consisting of, or consisting essentially
of the amino acid sequence
RSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLHHHHHH (SEQ ID NO:116).
[0217] Chimeric influenza HA polypeptides, proteins and/or protein
complexes can be made by any method known to one of ordinary skill
in the art, and may comprise, for example, one or several influenza
HA polypeptides, proteins and/or protein complexes of the
invention, and/or any fragment, derivative, or analog thereof (for
example, consisting of at least a domain of a polypeptide, protein,
or protein complex of the invention, or at least 6, and preferably
at least 10 amino acids of thereof) joined at its amino- or
carboxy-terminus via a peptide bond to an amino acid sequence of
another protein or other protein domain or motif. In some
embodiments such chimeric proteins can be produced by any method
known to one of ordinary skill in the art, including, but not
limited to, recombinant expression of a nucleic acid encoding a
chimeric protein (e.g. comprising a first coding sequence joined
in-frame to a second coding sequence); ligating the appropriate
nucleic acid sequences encoding the desired amino acid sequences to
each other in the proper coding frame, and expressing the chimeric
product.
Post-translational Modifications
[0218] In some embodiments, the influenza HA polypeptides, proteins
and protein complexes described herein may be altered by adding or
removing post-translational modifications, by adding or removing
chemical modifications or appendices, and/or by introducing any
other modifications known to those of ordinary skill in the art.
Included within the scope of the invention are influenza HA
polypeptides, proteins and protein complexes that are modified
during or after translation or synthesis, for example, by
glycosylation (or deglycosylation), acetylation (or deacetylation),
phosphorylation (or dephosphorylation), amidation (or
deamidization), pegylation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or buy any other
means known in the art. For example, in some embodiments the
influenza HA polypeptides, proteins and/or protein complexes may be
subjected to chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation,
oxidation, reduction, metabolic synthesis in the presence of
tunicamycin, etc. In some embodiments such post-translational
modifications can be used to render the influenza HA polypeptides,
proteins, and/or protein complexes of the present invention more
immunogenic, more stable, and/or more capable of binding to, or
eliciting the production of, neutralizing and broadly neutralizing
antibodies.
Obtaining Influenza HA Protein in Desired Conformations
[0219] In some embodiments the influenza HA polypeptides and/or
proteins of the invention are assembled into protein complexes
having a desired conformational structure, such as the native
trimeric structure of the stalk domain, and are cross-linked in
order to stabilize that conformation. As described elsewhere in the
present application, the influenza HA protein comprises a trimer
formed from three monomers. In some embodiments, prior to and/or
during the enzymatic cross-linking reaction, the influenza HA
protein may be obtained in (and/or maintained in) the desired
conformation, for example while cross-linking is performed. In some
embodiments the influenza HA protein may be produced and/or
isolated in such a way that most, or substantially all, of the
influenza HA molecules have a stalk domain present in a native
trimeric conformation. For example, when the HA protein is
expressed or obtained in a form that still comprises the head
domain, the stalk domain will typically assume its native trimeric
stalk confirmation. In some embodiments influenza HA molecules in a
desired conformation may be separated from a mixed population of
influenza HA protein molecules comprising some that are in the
desired conformation (e.g. native trimeric conformation of the
stalk domain) and some that are in other conformations (e.g. stalk
domain in a monomeric and/or dimeric conformation). In some
embodiments, the influenza HA protein is expressed in cells (for
example as its membrane bound or soluble form) and spontaneously
assembles into its normal conformation (e.g. having a stalk domain
in its native trimeric conformation). In some embodiments no
additional stabilization may be necessary to retain stalk domain
the influenza HA protein in its native trimeric form. In some
embodiments the expressed and assembled/folded influenza HA protein
may be kept under particular conditions, or in particular
compositions, that favor formation and/or maintenance of the native
trimeric conformation of the stalk domain. The influenza HA protein
may be obtained and/or isolated and/or maintained in the desired
conformation using any suitable method known in the art, including,
but not limited to, standard protein purification methods, such as
ion exchange chromatography, size exclusion chromatography, and/or
affinity chromatography methods. In some embodiments the influenza
HA protein may be expressed in the presence of, co-expressed with,
or contacted with, molecules that bind to the influenza HA protein
and stabilize it in its desired conformation, including, but not
limited to, antibodies, small molecules, peptides, and/or
peptidomimetics. Non-limiting examples of antibodies that bind to
the stalk domain in its native trimeric conformation include 6F12,
C179, CR6261, F10, A66, and D8. Other antibodies that can be used
to characterize or stabilize the HA polypeptides, proteins and
protein complexes of the invention include, but are not limited to,
18A3, 18C11, 18E7, 18E12, 18H9, 16B5, 10A14, 5K24, FI6v3, 6K14,
6J24, 8D4, anti-influenza human antibodies of the V.sub.H1-69 heavy
chain lineage, and anti-influenza human antibodies of the
V.sub.H3-30 heavy chain lineage. In some embodiments, the influenza
HA protein may be obtained, isolated, or maintained in its desired
conformation by controlling the ionic strength of the media/buffer
in which the protein is present (such as by using high or low ionic
strength media). In some embodiments the influenza HA protein may
be obtained, isolated, or maintained at one or more temperatures
that favor preservation of the desired conformation. In some
embodiments the influenza HA protein may be obtained, isolated, or
maintained over a period of time that diminishes the degree to
which the desired conformation lost.
[0220] In some embodiments analysis may be performed to confirm
that the desired conformation, such as the native trimeric
conformation of the stalk domain, has been formed and/or maintained
in the influenza HA protein. Such analysis may be performed prior
to cross-linking, during the cross-linking process, after the
cross-linking process, or at any combination of such stages. Such
analysis may comprise any suitable methods known in the art for
assessing the 3-dimensional structure of a protein or protein
complex, including functional analysis, crystallographic analysis,
and the like. In some embodiments such analysis may include
assessing binding of the influenza HA protein to certain
antibodies, such as those that are specific to the native trimeric
conformation of the stalk domain and/or those that are known to
bind to antigenic sites in the stalk domain or elsewhere in the
influenza HA protein, as described elsewhere herein, including, but
not limited to the 6F12, C179, CR6261, F10, A66, and D8
antibodies.
Protein Purification
[0221] In some embodiments the methods for making influenza HA
polypeptides, proteins, and protein complexes of the invention may
comprise purifying the influenza HA polypeptides, proteins, or
protein complexes before, during, or after, one or more steps in
the manufacturing process. For example, in some embodiments the
influenza HA polypeptides, proteins, and/or protein complexes of
the invention may be purified after completion of all of the
manufacturing steps. In some embodiments the influenza HA
polypeptides, proteins, and/or protein complexes of the invention
may be purified before commencing the cross-linking process or
after one or more of the intermediate method steps in the process,
for example, after expression of an influenza HA polypeptide or
protein, after assembly of a protein complex, after obtaining the
influenza HA protein in a desired conformation, during or after
performing a cross-linking reaction, or after removal of the head
domain. The influenza HA polypeptides, proteins, and/or protein
complexes of the invention may be isolated or purified using any
suitable method known in the art. Such methods include, but are not
limited to, chromatography (e.g. ion exchange, affinity, and/or
sizing column chromatography), ammonium sulfate precipitation,
centrifugation, differential solubility, or by any other technique
for the purification of proteins known to one of ordinary skill in
the art. In specific embodiments it may be necessary to separate
the desirable influenza HA polypeptides, proteins, and/or protein
complexes of the invention from those that were not sufficiently
cross-linked, or those in which the head domain was not
sufficiently removed. This can be done using any suitable system
known in the art. For example, influenza HA proteins having a stalk
domain in the native trimeric conformation can be separated from
those that have a stalk domain that is not in the native trimeric
conformation using antibody-based separation methods. The influenza
HA polypeptides, proteins, and/or protein complexes of the
invention may be purified from any source used to produce them. For
example, the influenza HA polypeptides, proteins, and/or protein
complexes of the invention may be purified from sources including
insect, prokaryotic, eukaryotic, mono-cellular, multi-cellular,
animal, plant, fungus, vertebrate, mammalian, human, porcine,
bovine, feline, equine, canine, avian, or tissue culture cells, or
any other source. The degree of purity may vary, but in various
embodiments, the purified influenza HA polypeptides, proteins,
and/or protein complexes of the invention are provided in a form in
which they comprise more than about 10%, 20%, 50%, 70%, 75%, 80%,
85%, 90%, 95%, 98%, 99%, or 99.9% of the total protein in the final
composition. In some embodiments the influenza HA polypeptides,
proteins, and/or protein complexes of the invention may be isolated
and purified from other proteins, or any other undesirable products
(such as non-cross-linked products or products where removal of the
head domain is insufficient or incomplete), by standard methods
including, but not limited to, chromatography, glycerol gradients,
affinity chromatography, centrifugation, ion exchange
chromatography, size exclusion chromatography, and affinity
chromatography, or by any other standard technique for the
purification of proteins known in the art. The influenza HA
polypeptides, proteins, and/or protein complexes to be isolated may
be expressed in high or low ionic media, or isolated in high or low
ionic buffers or solutions. The influenza HA polypeptides,
proteins, and/or protein complexes of the invention may also be
isolated at one or more temperatures that favor preservation of the
desired conformation. They may also be isolated over a period of
time that diminishes the degree to which a preparation would have
lost the desired conformation. The degree to which a preparation of
proteins retains one or more desired conformations (such as the
native trimeric conformation of the stalk domain and/or
conformations that favor binding to neutralizing antibodies, or
other desired properties) may be assayed by any suitable method
known in the art, including, for example, but not limited to,
biochemical, biophysical, immunologic, and virologic analyses. Such
assays include, for example, but are not limited to,
immunoprecipation, enzyme-linked immunosorbent assays (ELISAs), or
enzyme-linked immunosorbent spot (ELISPOT) assays, crystallographic
analysis (including co-crystallization with antibodies),
sedimentation, analytical ultracentrifugation, dynamic light
scattering (DLS), electron microscopy (EM), cryo-EM tomography,
calorimetry, surface plasmon resonance (SPR), fluorescence
resonance energy transfer (FRET), circular dichroism analysis, and
small angle x-ray scattering, neutralization assays,
antibody-dependent cellular cytotoxicity assays, and/or virologic
challenge studies in vivo.
[0222] The yield of the influenza HA polypeptides, proteins, and/or
protein complexes of the invention can be determined by any means
known in the art, for example, by comparing the amount of the final
engineered proteins (such as cross-linked influenza HA proteins) as
compared to the amount of the starting material, or as compared to
the amount of the materials present in any preceding step of the
production methods. Protein concentrations can determined by
standard procedures, such as, for example, Bradford or Lowry
protein assays. The Bradford assay is compatible with reducing
agents and denaturing agents (Bradford, M, 1976. Anal. Biochem. 72:
248). The Lowry assay has better compatibility with detergents and
the reaction is more linear with respect to protein concentrations
and read-out (Lowry, 0 J, 1951. Biol. Chem. 193: 265).
Exemplary Production Methods
[0223] In some embodiments the present invention provides methods
for making "headless" influenza HA polypeptides, proteins and/or
protein complexes as described herein. In some embodiments methods
for making "headless" influenza HA polypeptides, proteins and/or
protein complexes comprise: (a) expressing an influenza HA protein
having (i) both a stalk domain and a head domain, and (ii) one or
more engineered protease recognition motifs in or near its head
domain, (b) allowing the soluble influenza HA protein expressed in
step (a) to fold into its native conformation having a trimeric
stalk domain and a head domain, (c) introducing one more
cross-links into the trimeric stalk domain, wherein the cross-links
stabilize the stalk domain in its native trimeric conformation, and
(d) subsequently proteolytically disrupting or removing the head
domain, thereby producing a headless influenza HA protein. In some
such embodiments the cross-links are targeted cross-links, such as
di-tyrosine cross-links. In some embodiments the methods also
involve first (at least prior to step (c)) identifying one or more
regions in the HA protein in which the introduction of one or more
cross-links in step (c) could stabilize the conformation of the
stalk in its native trimeric conformation and/or stabilize the
stalk in a conformation that allows binding of one or more broadly
neutralizing anti-stalk antibodies. In some embodiments methods for
making "headless" influenza HA polypeptides, proteins and/or
protein complexes comprise: (a) expressing an influenza HA protein
having: (i) both a stalk domain and a head domain, (ii) one or more
"to-tyrosine mutations within its stalk domain, and (iii) one or
more engineered protease recognition motifs within or close to its
head domain, (b) allowing the influenza HA protein to fold into its
native conformation having a trimeric stalk domain and a head
domain, (c) introducing one or more di-tyrosine cross-links into
the trimeric stalk domain, wherein the di-tyrosine cross-links are
stable under physiological conditions and stabilize the stalk
domain in its native trimeric conformation, and (d) subsequently
proteolytically removing the head domain, thereby producing a
soluble headless influenza HA protein. In some embodiments the
method also involves identifying first (at least prior to step (c))
one or more regions in the HA protein in which the introduction of
one or more DT cross-links in step (c) could stabilize the
conformation of the stalk in its native trimeric conformation
and/or stabilize the stalk in a conformation that allows binding of
one or more broadly neutralizing anti-stalk antibodies. In such
methods the soluble influenza HA protein will typically comprises
one or more protease recognition motifs that can be used to
facilitate proteolytic removal of the head domain, as described
above and in other sections of this application.
[0224] In some embodiments the methods for making "headless"
influenza HA polypeptides, proteins and/or protein complexes
described herein may further comprise performing an analysis after
commencement or completion of the proteolytic cleavage step(s) to
confirm that the head domain of the influenza HA protein has been
sufficiently disrupted or removed. In some such embodiments this
analysis may comprise, for example, performing an SDS PAGE gel
mobility shift assay or using a head-specific antibody.
[0225] In some embodiments the present invention provides methods
for making "head-on" influenza HA polypeptides, proteins and/or
protein complexes as described herein. In some embodiments methods
for making "head-on" influenza HA polypeptides, proteins and/or
protein complexes comprise: (a) expressing an influenza HA protein
comprising a stalk domain and a head domain, (b) allowing the
expressed influenza HA protein to fold into its native conformation
having a trimeric stalk domain, and (c) introducing one more
physiologically stable cross-links into the HA protein in the
trimeric stalk domain and optionally also in the head domain,
thereby producing an engineered "head-on" influenza HA protein
having a cross-linked stalk domain. In some such embodiments the
cross-links are targeted cross-links, such as di-tyrosine
cross-links. In some embodiments methods for making "headless"
influenza HA polypeptides, proteins and/or protein complexes
comprise: (a) expressing an influenza HA protein having one or more
"to-tyrosine" mutations at targeted positions within its stalk
domain and optionally also in the head domain, (b) allowing the
influenza HA protein to fold into its native conformation having a
trimeric stalk domain and a head domain, and (c) performing a DT
cross-linking reaction to cross-link tyrosine residues in the stalk
domain and optionally also in the head domain, thereby producing an
engineered "head-on" influenza HA protein having a DT-cross-linked
stalk domain. In such methods the influenza HA protein may comprise
one or more protease recognition motifs that could be used, if
desired, to facilitate subsequent proteolytic removal of the head
domain of the "head-on" protein to generate a "headless" influenza
HA protein.
Properties of Influenza HA Polypeptides, Proteins and/or Protein
Complexes
[0226] In some embodiments, the influenza HA polypeptides, proteins
and/or protein complexes of the invention, including in particular
those that are cross-linked as described herein, have certain
structural, physical, functional, and/or biological properties.
Such properties may include one or more of the following, or any
combination of the following: presence or absence of a head domain,
existence of the stalk domain in its native trimeric conformation;
improved stability of the native trimeric conformation of the stalk
domain (as compared to non-cross-linked influenza HA proteins);
improved half-life of the influenza HA protein (as compared to
non-cross-linked influenza HA proteins); improved thermostability
(as compared to non-cross-linked influenza HA proteins); prolonged
shelf-life (as compared to non-cross-linked influenza HA proteins);
prolonged half-life inside the body of a subject (as compared to
non-cross-linked influenza HA proteins); ability to be stored in
solution without forming aggregates (including when present at a
high concentration in solution); reduced aggregation in solution
(as compared to non-cross-linked influenza HA proteins); binding to
an antibody; binding to a neutralizing antibody; binding to a
broadly neutralizing antibody; binding to a stalk-specific
antibody; binding to a conformationally-specific antibody; binding
to an antibody that recognizes a stalk domain epitope; binding to
an antibody selected from the group consisting of 6F12, C179,
CR6261, F10, A66, and D8; binding to a B cell receptor; activation
of a B cell receptor; eliciting an antibody response in vivo;
eliciting a protective antibody response in vivo; eliciting
production of neutralizing antibodies in vivo; eliciting production
of broadly neutralizing antibodies in vivo; eliciting production of
antibodies that recognize quaternary neutralizing epitopes (QNEs)
in vivo; eliciting a protective immune response in vivo; and/or
eliciting a humoral immune response in vivo. In the case of binding
to antibody molecules, in some embodiments the influenza HA
polypeptides, proteins, and/or protein complexes of the invention
bind to the antibodies (such as stalk-specific antibodies, and/or
6F12, C179, CR6261, F10, A66, and D8) with high specificity and/or
with high affinity.
Assays for Properties
[0227] In some embodiments the influenza HA polypeptides, proteins,
and/or protein complexes of the invention, or any intermediates in
their manufacture, may be analyzed to confirm that they have
desired properties, such as the desired structural, physical,
functional, and/or biological properties--such as those properties
listed above or identified elsewhere in this patent specification.
For example, in some embodiments in vitro or in vivo assays can be
performed to assess the influenza HA protein's conformational
structure, stability (e.g. thermostability), half-life (e.g. inside
the body of a subject), aggregation in solution, binding to an
antibody (such as a neutralizing antibody, broadly neutralizing
antibody; stalk-specific antibody; antibody that recognizes stalk
domain epitopes, conformationally-specific antibody, 6F12, C179,
CR6261, F10, A66, and Da), binding to a B cell receptor, activation
of a B cell receptor, antigenicity, immunogenicity, ability to
elicit an antibody response, ability to elicit a protective
antibody/immune response, ability to elicit production of
neutralizing antibodies, or ability to elicit production of broadly
neutralizing antibodies. In embodiments where the influenza HA
polypeptides, proteins, and/or protein complexes of the invention
are tested in an animal in vivo, the animal may be any suitable
animal species, including, but not limited to a mammal (such as a
rodent species (e.g. a mouse or rat), a rabbit, a ferret, a porcine
species, a bovine species, an equine species, an ovine species, or
a primate species (e.g. a human or a non-human primate), or an
avian species (such as a chicken)).
[0228] Assays for assessing a protein's conformational structure
are well known in the art and any suitable assay can be used,
including, but not limited to, crystallographic analysis (e.g.
X-ray crystallography or electron crystallography), sedimentation
analysis, analytical ultracentrifugation, electron microscopy (EM),
cryo-electron microscopy (cryo-EM), cryo-EM tomography, nuclear
magnetic resonance (NMR), small angle x-ray scattering,
fluorescence resonance energy transfer (FRET) assays, and the
like.
[0229] Assays for assessing a protein's stability are well known in
the art and any suitable assay can be used, including, but not
limited to, denaturing and non-denaturing electrophoresis,
isothermal titration calorimetry, and time-course experiments in
which proteins are incubated and analyzed over time at varying
protein concentrations, temperatures, pHs or redox conditions.
Proteins may also be analyzed for susceptibility to proteolytic
degradation.
[0230] Assays for assessing binding of proteins to antibodies are
well known in the art, and any suitable assay can be used,
including, but not limited to, immunoprecipation assays,
enzyme-linked immunosorbent assays (ELISAs), enzyme-linked
immunosorbent spot assays (ELISPOTs), crystallographic assays
(including co-crystallization with antibodies), surface plasmon
resonance (SPR) assays, fluorescence resonance energy transfer
(FRET) assays, and the like.
[0231] Assays for assessing neutralization activity are well known
in the art, and any suitable assay can be used. For example, assays
can be performed to determine the neutralizing activity of
antibodies or antisera generated by vaccination/immunization of
animals with the influenza HA polypeptides, proteins, and/or
protein complexes of the invention. Neutralization assays known in
the art include, but are not limited to, those described by Dey et
al. 2007 (Dey et al., 2007, Characterization of Human
Immunodeficiency Virus Type 1 Monomeric and Trimeric gp120
Glycoproteins Stabilized in the CD4-Bound State: Antigenicity,
Biophysics, and Immunogenicity. J Virol 81(11): 5579-5593) and
Beddows et al., 2006 (Beddows et al., 2007, A comparative
immunogenicity study in rabbits of disulfide-stabilized
proteolytically cleaved, soluble trimeric human immunodeficiency
virus type 1 gp140, trimeric cleavage-defective gp140 and momomeric
gp120. Virol 360: 329-340).
[0232] Assays for assessing whether a vaccine immunogen is capable
of eliciting an immune response and/or providing protective
immunity are well known in the art, and any suitable assay can be
used. For example, assays can be performed to determine whether
vaccination/immunization of animals with the influenza HA
polypeptides, proteins, and/or protein complexes of the invention
provide an immune response and/or protective immunity against
infection with influenza virus. In some embodiments comparisons may
be made between placebo and test vaccinated groups with regard to
their rates of infection or sero-conversion or viral loads.
[0233] Assays for assessing a protein's pharmacokinetics and
bio-distribution are also well known in the art, and any suitable
assay can be used to assess these properties of the influenza HA
polypeptides, proteins, and/or protein complexes of the
invention.
Compositions
[0234] In some embodiments the present invention provides
compositions comprising any of the influenza HA polypeptides,
proteins, and/or protein complexes described herein. In some
embodiments such compositions may be immunogenic compositions,
vaccine compositions and/or therapeutic compositions. In some
embodiments, such compositions may be administered to subjects. In
some embodiments the the influenza HA polypeptides, proteins,
and/or protein complexes described herein may be present in
virus-like particles or "VLPs."
[0235] In some embodiments the influenza HA polypeptides, proteins,
and/or protein complexes of the invention may be provided in a
composition that comprises one or more additional active
components, such as one or more additional vaccine immunogens or
therapeutic agents. In some embodiments the influenza HA
polypeptides, proteins, and/or protein complexes of the invention
may be provided in a composition that comprises one or more other
components, including, but not limited to, pharmaceutically
acceptable carriers, adjuvants, wetting or emulsifying agents, pH
buffering agents, preservatives, and/or any other components
suitable for the intended use of the compositions. Such
compositions can take the form of solutions, suspensions, emulsions
and the like. The term "pharmaceutically acceptable carrier"
includes various diluents, excipients and/or vehicles in which, or
with which, the influenza HA polypeptides, proteins, and/or protein
complexes of the invention can be provided. The term
"pharmaceutically acceptable carrier" includes, but is not limited
to, carriers known to be safe for delivery to human and/or other
animal subjects, and/or approved by a regulatory agency of the
Federal or a state government, and/or listed in the U.S.
Pharmacopeia, and/or other generally recognized pharmacopeia,
and/or receiving specific or individual approval from one or more
generally recognized regulatory agencies for use in humans and/or
other animals. Such pharmaceutically acceptable carriers, include,
but are not limited to, water, aqueous solutions (such as saline
solutions, buffers, and the like), organic solvents (such as
certain alcohols and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil), and the like. In some embodiments the
compositions of the invention also comprise one or more adjuvants.
Exemplary adjuvants include, but are not limited to, inorganic or
organic adjuvants, oil-based adjuvants, virosomes, liposomes,
lipopolysaccharide (LPS), molecular cages for antigens (such as
immune-stimulating complexes ("ISCOMS")), Ag-modified
saponin/cholesterol micelles that form stable cage-like structures
that are transported to the draining lymph nodes), components of
bacterial cell walls, endocytosed nucleic acids (such as
double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and
unmethylated CpG dinucleotide-containing DNA), AUM, aluminum
phosphate, aluminum hydroxide, and Squalene. In some embodiments
virosomes are used as the adjuvant. Additional commercially
available adjuvants that can be used in accordance with the present
invention include, but are not limited to, the Ribi Adjuvant System
(RAS, an oil-in-water emulsion containing detoxified endotoxin
(MPL) and mycobacterial cell wall components in 2% squalene (Sigma
M6536)), TiterMax (a stable, metabolizable water-in-oil adjuvant
(CytRx Corporation 150 Technology Parkway Technology Park/Atlanta
Norcross, Ga. 30092)), Syntex Adjuvant Formulation (SAF, an
oil-in-water emulsion stabilized by Tween 80 and pluronic
polyoxyethlene/polyoxypropylene block copolymer L121 (Chiron
Corporation, Emeryville, Calif.)), Freund's Complete Adjuvant,
Freund's Incomplete Adjuvant, ALUM--aluminum hydroxide, Al(OH)3
(available as Alhydrogel, Accurate Chemical & Scientific Co,
Westbury, N.Y.), SuperCarrier (Syntex Research 3401 Hillview Ave.
P.O. Box 10850 Palo Alto, Calif. 94303), Elvax 40W1,2(an
ethylene-vinyl acetate copolymer (DuPont Chemical Co. Wilmington,
Del.)), L-tyrosine co-precipitated with the antigen (available from
numerous chemical companies); Montanide (a manide-oleate, ISA
Seppic Fairfield, N.J.)), AdjuPrime (a carbohydrate polymer),
Nitrocellulose-absorbed protein, Gerbu adjuvant (C-C Biotech,
Poway, C A), and the like.
[0236] In some embodiments the compositions of the invention
comprise an "effective amount" of an influenza HA polypeptide,
protein, and/or protein complex of the invention. An "effective
amount" is an amount required to achieve a desired end result.
Examples of desired end results include, but are not limited to,
the generation of a humoral immune response, the generation of a
neutralizing antibody response, the generation of a broadly
neutralizing antibody response, and the generation of protective
immunity. The amount of an influenza HA polypeptide, protein,
and/or protein complex of the invention that is effective to
achieve the desired end result will depend on variety of factors
including, but not limited to, the type, subtype, and strain of the
influenza virus against which protection or some other therapeutic
effect is sought, the species of the intended subject (e.g. whether
a human or some other animal species), the age and/or sex of the
intended subject, the planned route of administration, the planned
dosing regimen, the seriousness of any ongoing influenza infection
(e.g. in the case of therapeutic uses), and the like. The effective
amount--which may be a range of effective amounts--can be
determined by standard techniques without any undue
experimentation, for example using in vitro assays and/or in vivo
assays in the intended subject species or any suitable animal model
species. Suitable assays include, but are not limited to, those
that involve extrapolation from dose-response curves and/or other
data derived from in vitro and/or in vivo model systems. In some
embodiments the effective amount may be determined according to the
judgment of a medical or veterinary practitioner based on the
specific circumstances.
Uses of the Influenza HA Polypeptides, Proteins & Protein
Complexes of the Invention
[0237] In some embodiments, the influenza HA polypeptides,
proteins, and protein complexes of the invention may be useful as
research tools, as diagnostic tools, as therapeutic agents, as
targets for the production of antibody reagents or therapeutic
antibodies, and/or as vaccines or components of vaccine
compositions. For example, in some embodiments the influenza HA
polypeptides, proteins, and protein complexes of the invention are
useful as vaccine immunogens in animal subjects, such as mammalian
subject, including humans. These and other uses of the influenza HA
polypeptides, proteins, and protein complexes of the invention are
described more fully below. Those of skill in the art will
appreciate that the influenza HA polypeptides, proteins, and
protein complexes of the invention may be useful for a variety of
other applications also, and all such applications and uses are
intended to fall within the scope of this invention.
Tools for Studying Influenza HA Antibodies
[0238] In one embodiment, the influenza polypeptides, proteins, and
protein complexes of the invention may be useful as analytes for
assaying and/or measuring binding of, and/or titers of, anti-HA
antibodies, for example in ELISA assays, Biacore/SPR binding
assays, and/or any other assays for antibody binding known in the
art. For example, the influenza HA polypeptides, proteins, and
protein complexes of the invention could be used to analyze, and/or
compare the efficacy of anti-HA antibodies.
Tools for Generation of Antibodies
[0239] The influenza HA polypeptides, proteins, and protein
complexes of the invention (including any intermediates and/or
variants produced during manufacture of the influenza HA
polypeptides, proteins, and protein complexes) may also be useful
for the generation of therapeutic antibodies and/or antibodies that
can be used as research tools or for any other desired use. For
example, the influenza HA polypeptides, proteins, and protein
complexes of the invention can be used for immunizations to obtain
antibodies to the influenza HA protein for use as research tools
and/or as therapeutics. In some embodiments the influenza HA
polypeptides, proteins, and protein complexes of the invention can
be used to immunize a non-human animal, such as a vertebrate,
including, but not limited to, a mouse, rat, guinea pig, rabbit,
goat, non-human primate, etc. in order to generate antibodies. Such
antibodies, which may be monoclonal or polyclonal, and/or cells
that produce such antibodies, can then be obtained from the animal.
For example, in some embodiments influenza HA polypeptides,
proteins, and protein complexes of the invention may be used to
immunize a mouse and to produce and obtain monoclonal antibodies,
and/or hybridomas that produce such monoclonal antibodies. Such
methods can be carried out using standard methods known in the art
for the production of mouse monoclonal antibodies, including
standard methods for hybridoma production. In some embodiments
influenza HA polypeptides, proteins, and protein complexes of the
invention may be used for the production of a chimeric (e.g.
part-human), humanized, or fully-human antibody, for example using
any of the methods currently known in the art for production of
chimeric, humanized and fully human antibodies, including, but not
limited to, CDR grafting methods, phage-display methods, transgenic
mouse methods (e.g. using a mouse that has been genetically altered
to allow for the production of fully human antibodies, such as the
Xenomouse) and/or any other suitable method known in the art.
Antibodies to the influenza HA polypeptides, proteins, and protein
complexes of the invention made using such systems can be
characterized antigenically using one or a set of several antigens,
preferably including the influenza HA polypeptides, proteins, and
protein complexes of the invention themselves. Additional
characterization of such antibodies may be carried out by any
standard methods known to one of ordinary skill in the art,
including, but not limited to, ELISA-based methods, SPR-based
methods, biochemical methods (such as, but not limited to,
iso-electric point determination), and methods known in the art for
studying biodistribution, safety, and efficacy of antibodies--for
example in preclinical and clinical studies.
Administration to Subjects
[0240] In some embodiments, the present invention provides methods
that comprise administering the influenza HA polypeptides, proteins
and/or protein complexes of the invention (or compositions
comprising such influenza HA polypeptides, proteins and/or protein
complexes) to subjects. Such methods may comprise methods for
treating individuals having influenza virus (i.e. therapeutic
methods) and/or methods for protecting individuals against future
influenza virus infection (i.e. prophylactic methods).
[0241] Subjects to which the influenza HA polypeptides, proteins
and/or protein complexes of the invention, or compositions
comprising such influenza HA polypeptides, proteins and/or protein
complexes, can be administered (for example in the course of a
method of treatment or a method of vaccination) include any and all
animal species, including, in particular, those that are
susceptible to influenza virus infection or that can provide model
animal systems for the study of influenza virus infection. In some
embodiments, the subjects are mammalian species. In some
embodiments, the subjects are avian species. Mammalian subjects
include, but are not limited to, humans, non-human primates,
rodents, rabbits, and ferrets. Avian subjects include, but are not
limited to chickens, such as those on poultry farms. In some
embodiments the subjects to which the influenza HA polypeptides,
proteins and/or protein complexes of the invention, or compositions
comprising such influenza HA polypeptides, proteins and/or protein
complexes are administered, either have influenza, or are at risk
of influenza infection, for example due to the subject's age and/or
underlying medical conditions. In some embodiments, the subject is
immuno-compromised. In some embodiments, the subject has heart
disease, lung disease, diabetes, renal disease, dementia, stroke
and/or rheumatologic disease. In some embodiments, the subject is a
human of greater than about 50 years in age, greater than about 55
years in age, greater than about 60 years in age, greater than
about 65 years in age, greater than about 70 years in age, greater
than about 75 years in age, greater than about 80 years in age,
greater than about 85 years in age, or greater than about 90 years
in age. In some embodiments, the subject is a human of less than
about 1 month in age, less than about 2 months in age, less than
about 3 months in age, less than about 4 months in age, less than
about 5 months in age, less than about 6 months in age, less than
about 7 months in age, less than about 8 months in age, less than
about 9 months in age, less than about 10 months in age, less than
about 11 months in age, less than about 12 months in age, less than
about 13 months in age, less than about 14 months in age, less than
about 15 months in age, less than about 16 months in age, less than
about 17 months in age, less than about 18 months in age, less than
about 19 months in age, less than about 20 months in age, less than
about 21 months in age, less than about 22 months in age, less than
about 23 months in age, or less than about 24 months in age.
[0242] Various delivery systems are known in the art and any
suitable delivery systems can be used to administer the
compositions of the present invention to subjects. Such delivery
systems include, but are not limited to, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral delivery systems. The compositions
of the present invention may be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local. Pulmonary administration can also be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent.
[0243] In some embodiments it may be desirable to administer the
pharmaceutical compositions of the invention locally to a tissue in
which the influenza HA polypeptide, protein or protein complex may
be most effective in generating a desirable outcome. This may be
achieved by, for example, local infusion, injection, delivery using
a catheter, or by means of an implant, such as a porous,
non-porous, or gelatinous implant or an implant comprising one or
more membranes (such as sialastic membranes) or fibers from or
through which the protein or protein complexes may be released
locally. In some embodiments a controlled release system may be
used. In some embodiments a pump may be used (see Langer, supra;
Sefton, 1987. CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al.,
1980. Surgery 88: 507; Saudek et al., 1989. N. Engl. J. Med. 321:
574). In some embodiments polymeric materials may be used to
facilitate and/or control release of the influenza HA polypeptide,
protein and/or protein complex (see Medical Applications of
Controlled Release, Langer and Wise (eds.), 1974. CRC Pres., Boca
Raton, Fla.; Controlled Drug Bioavailability, 1984. Drug Product
Design and Performance, Smolen and Ball (eds.), Wiley, New York;
Ranger & Peppas, 1983 Macromol. Sci. Rev. Macromol. Chem. 23:
61; see also Levy et al., 1985. Science 228:190; During et al,
1989. Ann. Neurol. 25: 351; Howard et al., 1989. J. Neurosurg
71:105). In some embodiments a controlled release system can be
placed in proximity to the tissue/organ to which the influenza HA
polypeptide, protein and/or protein complex is to be delivered
(see, e.g., Goodson, 1984. Medical Applications of Controlled
Release, supra, vol. 2: 115-138). Some suitable controlled release
systems that may be used in conjunction with the present invention
are described Langer, 1990, Science; vol. 249: pp. 527-1533
[0244] In some embodiments, administration of the influenza HA
polypeptide, protein and/or protein complex of the invention can be
performed in conjunction with administration of one or more
immunostimulatory agents. Non-limiting examples of such
immunostimulatory agents include various cytokines, lymphokines and
chemokines with immunostimulatory, immunopotentiating, and
pro-inflammatory activities, such as interleukins (e.g., IL-1,
IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g.,
granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and
other immunostimulatory agents, such as macrophage inflammatory
factor, Flt3 ligand, B7.1; B7.2. The immunostimulatory agents can
be administered in the same formulation as the influenza HA protein
or polypeptide, or can be administered separately.
[0245] In some embodiments, the influenza HA polypeptides,
proteins, and/or protein complexes of the invention, or
compositions comprising them, can be administered to subjects in a
variety of different influenza virus vaccination methods or
regimens. In some such embodiments, administration of a single dose
is preferred. However, in other embodiments, additional dosages can
be administered, by the same or different route, to achieve the
desired prophylactic effect. In neonates and infants, for example,
multiple administrations may be required to elicit sufficient
levels of immunity. Administration can continue at intervals
throughout childhood, as necessary to maintain sufficient levels of
protection against influenza virus infection. Similarly, adults who
are particularly susceptible to influenza virus infection, such as,
for example, the elderly and immunocompromised individuals, may
require multiple immunizations to establish and/or maintain
protective immune responses. Levels of induced immunity can be
monitored, for example, by measuring amounts of neutralizing
secretory and serum antibodies, and dosages adjusted or
vaccinations repeated as necessary to elicit and maintain desired
levels of protection.
[0246] In some embodiments, dosing regimens may comprise a single
administration/immunization. In other embodiments, dosing regimens
may comprise multiple administrations/immunizations. For example,
vaccines may be given as a primary immunization followed by one or
more boosters. In some embodiments of the present invention such a
"prime-boost" vaccination regimen may be used. For example, in some
such prime-boost regimens a composition comprising an influenza HA
polypeptide, protein or protein complex as described herein may be
administered to an individual on multiple occasions (such as two,
three, or even more occasions) separated in time, with the first
administration being the "priming" administration and subsequent
administrations being "booster" administrations. In other such
prime-boost regimens a composition comprising an influenza HA
polypeptide, protein or protein complex as described herein may be
administered to an individual after first administering to the
individual a composition comprising a viral or DNA vector encoding
an influenza HA polypeptide, protein or protein complex as a
"priming" administration, with one or more subsequent "booster"
administrations of a composition comprising an influenza HA
polypeptide, protein or protein complex as described herein.
Boosters may be delivered via the same and/or different route as
the primary immunization. Boosters are generally administered after
a time period after the primary immunization or the previously
administered booster. For example, a booster can be given about two
weeks or more after a primary immunization, and/or a second booster
can be given about two weeks or more after the first boosters.
Boosters may be given repeatedly at time periods, for example,
about two weeks or greater throughout up through the entirety of a
subject's life. Boosters may be spaced, for example, about two
weeks, about three weeks, about four weeks, about one month, about
two months, about three months, about four months, about five
months, about six months, about seven months, about eight months,
about nine months, about ten months, about eleven months, about one
year, about one and a half years, about two years, about two and a
half years, about three years, about three and a half years, about
four years, about four and a half years, about five years, or more
after a primary immunization or after a previous booster.
[0247] Preferred unit dosage formulations are those containing a
dose or unit (e.g. an effective amount), or an appropriate fraction
thereof, of the influenza HA polypeptides, proteins, and/or protein
complexes of the invention. In addition to such ingredients,
formulations of the present invention may include other agents
commonly used by one of ordinary skill in the art. Pharmaceutical
compositions provided by the invention may be conveniently
presented in preferred unit dosage formulations prepared using
conventional pharmaceutical techniques. Such techniques include the
step of bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s) or other ingredients. In
general, the formulations are prepared by uniformly and intimately
bringing into association the active ingredient with liquid
carriers. Formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets commonly used by one of ordinary skill in the
art.
Kits
[0248] The present invention further provides kits comprising
influenza HA polypeptides, proteins or protein complexes of the
invention, or compositions containing such polypeptides, proteins
or protein complexes. To facilitate use of the methods and
compositions of the invention, any of the components and/or
compositions described herein, and additional components useful for
experimental or therapeutic or vaccine purposes, can be packaged in
the form of a kit. Typically, the kit contains, in addition to the
above components, additional materials which can include, e.g.,
instructions for using the components, packaging material, a
container, and/or a delivery device.
[0249] Various embodiments of the present invention may also be
further described by the following non-limiting examples:
EXAMPLES
[0250] The numbers in square brackets/parentheses in the Examples
section of the present application are citations to the numbered
references provided as a reference list herein.
Example 1
[0251] The US and world populations continue to be at risk of a
pandemic Influenza outbreak, and weaponized influenza virus remains
a major bio-warfare/terrorism threat [23,24]. An influenza virus
HA-based vaccine immunogen capable of eliciting Ab responses to the
conserved stalk QNEs, instead of the immunodominant head of HA, is
expected to give rise to broadly neutralizing antibodies that could
protect from homologous (H1N1), as well as homologous drift
variant, group 1 heterologous (H5N1), and group 2 heterologous
challenge (H3N2). Thus, a single, universal immunogen could elicit
protective immune responses against seasonal, pandemic, and
weaponized influenza virus. Underscoring the commercial and public
health impact that influenza virus has on the population is the
fact that life insurance companies in the United States today are
required to hold capital against a potential reoccurrence of the
1918 Spanish Flu pandemic (Oliver, Wyman, & Co, 2012 &
[25]). The approach described herein has the potential to provide a
broadly protective influenza vaccine that could enable stockpiling
large amounts of vaccine product and eliminate real threats that
derive from the ramp-up times in manufacturing required to address
each new threat.
[0252] Current influenza virus vaccines protect mostly against
homologous virus strains, requiring new trivalent vaccine cocktails
to be matched seasonally to circulating strains. Protection is
primarily due to high affinity antibodies to hemagglutinin (HA),
and is often strain-specific due to a focusing of the immune
response predominantly against the highly variable, immunodominant
head domain of the HA protein. The HA stalk, however, is highly
conserved across influenza strains, and considerable evidence now
suggests that better responses to conserved regions of the stalk
would provide broader protection [1-4]. Immunization with DNA
coding for HA elicits predominantly stalk-specific Ab responses,
and data showing limited heterosubtypic protection by vaccination
with HA DNA by electroporation was recently described [5].
Furthermore, vaccination with a `headless` HA protein based
immunogen ("Headless HA", an HA construct from which the variable
head domain is removed) results in the induction of Ab responses
with significantly enhanced heterosubtypic binding activity [1,6].
A prime-boost combination of a viral or DNA vector encoding HA,
followed by a Headless protein boost holds the promise of
generating broadly heterospecific responses that yield long-lasting
protection. However, good protection against heterologous
challenges currently still remains elusive [7-10].
[0253] Significant attention has been focused on the identification
and characterization of broadly neutralizing antibodies ("bnAbs")
in order to reverse engineer an immunogen capable of eliciting
similar antibody responses [9,11]. A number of these bnAbs have
been described, and the most potent bind conserved,
complex/conformation-specific epitopes that are presented on the
conserved stalk of influenza HA trimers, but not on protomers of
the same complex [7,12,13]. Isolation of these human Abs proves
that a broadly protective vaccine is, in fact, an achievable goal
(a "protomer" is a subunit of the trimer, that itself is a HA1/HA2
heterodimer). These trimer/complex-specific epitopes are therefore
called quaternary neutralizing epitopes (QNEs), and they are
believed to represent key sites of vulnerability of influenza
viruses since they have the potential to elicit potent quaternary
bnAbs. [14,15]. Only the intact trimeric stalk exibits the broadly
protective QNE (see FIG. 2). A Headless construct that is locked in
its trimeric, native conformation, and that binds the potent and
broadly protective quaternary bnAbs could provide a universal
influenza immunogen and could elicit potent bnAbs in vaccinated
subjects.
[0254] Recently, a headless influenza hemagglutinin ("Headless HA")
immunogen has been shown to elicit antibody ("Ab") responses
focused on the highly conserved stalk region of influenza
hemagglutinin (HA) that are broadly cross-reactive. It has also
become clear that the most potent and broadly
neutralizing/protective Abs (bnAbs) against the stalk region are
trimer-specific (i.e. recognize the quaternary structure of the
stalk), and that their corresponding quaternary epitopes are not
displayed when the head of influenza HA is removed. In the absence
of the head domain, the stalk trimer apparently falls apart. The
present invention provides a Headless HA immunogen in which the
trimeric conformation of the stalk region is stabilized or
"conformationally locked"--for example by introduction of targeted
cross-links--before the head is proteolytically removed. This
Headless HA immunogen should retain binding to quaternary bnAbs and
present quaternary neutralizing epitopes ("QNEs") as an influenza
immunogen. Such a conformationally-locked Headless HA trimer may
enable the long-sought goal of broad protection against influenza
viruses from a single vaccination regimen.
[0255] Minimally modifying dityrosine ("DT") stabilization
technology enzymatically introduces safe, targeted, zero-length,
and irreversible DT bonds to lock proteins and complexes in native
conformations. Application of this technology fully preserves
protein structure and avoids aggregation because DT bonds do not
form spontaneously. Bonds only form between Tyr side-chains in very
close structural proximity, and are introduced after the protein
has fully folded and is in its native state. Targeted DT
crosslinking enables the design of an improved influenza vaccine
immunogen by conformationally locking QNEs to maximize broad
protection.
[0256] The methods described in the present example involve 3
steps. The first step involves expressing soluble, full-length
influenza HA with "to-Tyr" substitutions at targeted positions
within the stalk region. The second step involves introduction of
stabilizing DT crosslinks. And the third step involves
proteolytically removing the head domain of the influenza HA in
order to focus the immune responses on the DT-Headless HA QNEs.
[0257] Preliminary studies using a recombinant, soluble HIV Env
trimer have demonstrated that DT crosslinking can be used to
conformationally-lock the Env immunogen in its native, trimeric
conformation, so that it improves binding to the most potent HIV
quaternary bnAbs, analogous to the flu quaternary anti-stalk bnAbs,
demonstrating the feasibility of this approach. HIV Env and
influenza virus Headless are highly analogous in that both are
unstable trimers when expressed recombinantly; and in both, key
QNEs are only presented in the native trimeric complex. In other
preliminary studies targeted DT bonds have been successfully
introduced into the influenza HA stalk.
[0258] DT crosslinking of a recombinant PR8 HA construct in its
native, trimeric conformation can be performed to confirm binding
to key bnAbs, and subsequently the "head" domain can be removed by
engineering proteolytic cleavage sites, while maintaining the
DT-locked, native antigenic conformation of the stalk trimer. The
resulting Headless HA immunogen can be tested to confirm that it
elicits broad protection in a C57BL/6 mouse model. Pre-clinical
testing for efficacy can be performed in a highly predictive ferret
lethal challenge model. Pre-clinical testing for safety can be
performed in rabbits.
Targeted DT Cross-Linking
[0259] By generating native, soluble, and recombinant HA trimers
and applying targeted dityrosine (DT) "staples" to covalently
cross-link trimerizing interactions in the stalk of the trimer,
DT-stabilized HA trimmers will be engineered with fully preserved
antigenic profiles. Covalent stabilization of the trimer in the HA
stalk region will be engineered to render stable the quaternary
structure of the stalk, and this will allow subsequent proteolytic
removal of the head while preserving the QNEs of the stalk. DT
bonds are introduced to stabilize the complex after the
protein/complex is fully folded, and therefore locks the native
conformation, while maintaining structural functional integrity of
the protein [16-18]. These safe, irreversible, and zero-length
cross-links form only between Tyr residues in very close structural
proximity, and do not distort the structure of the protein. Nor do
they cause non-specific aggregate formation, as observed with
disulfide bonds [17,19-22]. Targeted DT cross-linking technology
can be applied to covalently stabilize a soluble HA trimer in its
correctly folded conformation, and then one can determine whether
it does, in fact, present key QNEs. Subsequently the immunodominant
head can be removed by introducing sequence-specific protease
cleavage sites--making use of variable loop tolerance for amino
acid variation and information gathered from transposon-based
mutagenesis analysis of HA. Presentation of QNEs on Headless HA is
expected to improve upon the breadth of protection in lethal
challenge studies with drift variant and heterologous viruses. The
inventors' prior work in HIV shows that highly glycosylated
multimers (e.g. HIV Env) can efficiently be locked together by DT
cross-linking at various locations within the cleaved Env
trimer--while maintaining the relevant quaternary structure and
antigenicity.
Conformationally Locking the Influenza Virus HA Trimeric
Complex
[0260] The HIV envelope spike is trimerized through well
characterized interactions at its base as well as interactions at
the spike's apex [33, 34]. In order to stabilize the trimerizing
interactions at the apex of the spike, tyrosine substitutions were
introduced, and the protein was expressed, purified, and DT
cross-linked. By fluorescence, 7 variants were identified that form
intermolecular, trimerizing cross-links with an average of
80%+efficiency prior to any optimization, as quantified using
DT-specific excitation (320 nm) and emission (405 nm) wavelengths.
The ability of these constructs to bind conformational and
trimer-specific bnAbs was assayed. DT crosslinking fully preserves
binding of the anti-CD4 binding site bnAb b12, which binds both
protomers and trimers, and the anti-V2 bnAb PG9, which
preferentially binds trimers, but also binds monomers. In addition,
conformational locking also significantly reduces binding to
non-neutralizing mAbs, such as b6 & b13, in ELISA assays. The
position of the DT bonds was confirmed by MS/MS of tryptic
fragments of the DT-Env trimer. More importantly, a
conformationally locked HIV Env trimer was found to bind
signficantly better to one of the most extremely broadly
neutralizing and potent anti-HIV Env bnAbs, PG16, by comparison to
the WT protomer; the PG16 epitope is only presented on the
native/functional HIV envelope trimer [28]. Improved PG16 binding
correlates with a significant reduction in binding to a poorly
neutralizing anti-V2 mAb, CH58, that binds an .alpha.-helical
conformer of an overlapping epitope that PG16 binds as a
.beta.-sheet. The next step with this DT-locked, soluble HIV Env
trimer will be to test it in animal immunogenicity experiments.
[0261] In influenza HA, the trimeric structure of the HA protein in
complex with the CR6261 bnAb was analyzed. Five examples of
possible HA variants (N403Y_D429Y; N403Y_L432Y; N403Y_D433Y;
N406Y_D429Y; and N406Y_D433Y) were initially identified, each with
two point mutations that were predicted to form intermolecular
bonds and stabilize the stalk trimer at the membrane distal/head
proximal end (see schematic for design in FIG. 3) without altering
the CR6261 quaternary epitope. Expression vectors encoding four of
these variants were generated, and the variants were expressed and
subjected to cross-linking conditions. Spectrofluorometry was used
to determine whether these variants were forming DT bonds using the
DT-specific excitation and emission wavelengths at which DT bonds
fluoresce powerfully in direct proportion to their molar
concentration. All four variants, but not wild-type HA, formed DT
bonds efficiently (FIG. 4). Based on comparison to the positive
control (insulin) and a DT standard, cross-linking efficiency of
>70% is estimated for all four of these constructs prior to any
optimization [35].
Removing the HA Head from the Conformationally Locked HA
[0262] Proteolytic removal of the HA head domain from the DT-locked
HA trimer requires engineering recognition motifs into the HA1 head
domain for a substrate-specific protease (e.g. TEV). Using a
transposon-based mutagenesis screen, four regions within the PR8
HA1 globular head have been identified that tolerate the insertion
of foreign sequences approximately the same size as an engineered
TEV protease site. Without further optimization, two of these
regions (located at amino acid residues 128 and 223) would allow
proteolytic cleavage of 3 of the 4 major antigenic sites in the PR8
globular head--the Sa, Ca, and Sb sites [36]. The remaining Cb site
will also be removed. Viruses with insertions at these sites in HA1
remain capable of fusion, and the HA complex thus remains
functionally intact. The proteolytic reaction will then be
performed.
[0263] These data demonstrate that the approach of locking together
the HA trimer in the stalk, and subsequently removing the
immunodominant head domain, will preserve vaccine-relevant QNEs of
headless HA, and will lock the immunogen in an antigenically
favorable conformation. This, in turn, suggests that the DT-locked
headless trimer(s) described herein are expected to induce broadly
protective antibody responses in vivo.
Conformationally Locking the Influenza Virus HA Trimeric
Complex
[0264] Experimental Design. Soluble forms (e.g. lacking the
transmembrane domain and possessing the T4 foldon trimerization
motif) of the WT HA and variants described above will be expressed
in SF9 or Hi5 cells as secreted proteins and purified by
well-established methods [37-38]. The antigenic effect of the
to-Tyr substitutions and the DT cross-linking will be determined in
ELISAs using a panel of anti-HA stalk broadly neutralizing mAbs
(e.g. 6F12, C179, CR6261, F10, A66 and D8), as structural changes
caused by to-Tyr substitutions may reduce or enhance binding to
some of these antibodies. Methods: Full-curve binding assays will
compare WT HA to the uncross-linked and cross-linked HA variants.
Changes in binding will be determined using non-linear regression
analysis (Graphpad software) of binding curves to calculate and
compare EC50 values for each construct with each mAb.
Intermolecular bond formation will be confirmed by gel-shift in
reducing SDS-PAGE (Western blot/Coomassie; DT bonds are not
reduced); DT cross-linking will be quantified by
spectrofluorometry, as described above. Such methods can be used to
produce HA variants that form intermolecular DT bonds, and that
retain binding to key anti-stalk quaternary bnAbs equal to
wild-type PR8 HA after cross-linking the engineered influenza
immunogen.
Proteolvtically Removing the HA Head from the Conformationally
Locked HA
[0265] PreScission Protease recognition sequences (LEVLFQGP (SEQ ID
NO:69) (cleavage between Q and G residues) and/or TEV recognition
sequences (ENLYFQG (SEQ ID NO:70) (cleavage between Q and G
residues) and ENLYFQS (SEQ ID NO:71) (cleavage between G and S
residues)) can be inserted at defined (e.g. amino acid residues 128
and 223) or additional positions to remove most of the globular
head of HA from the baculovirus expressed, purified, fully folded,
DT-stabilized, soluble HA precursor. Following antigenic
confirmation, amino acid analysis and mass spectrometry can be
performed to characterize the cross-linked molecule
biochemically.
[0266] Proteolysis of the head domain can be carried out by
standard biochemical procedures and assayed by SDS-PAGE
electro-mobility shift from a molecular weight corresponding to a
complete DT-HA trimer (225 kD) to that of a headless trimer (135
kDa) (Coomassie stain, Western blot). Removal of the head from the
DT cross-linked HA stalk can be confirmed with Head-specific Abs,
for example in Western blots and ELISA. The same bnAbs and assays
described above can be used to confirm preservation of the most
relevant QNEs in DT-Headless HA.
[0267] Amino acid analysis can be performed to assess any
non-specific changes to amino acid side chains, and to confirm the
presence of DT bonds (the DT moiety itself can be specifically
detected). In order to identify the position of the DT bonds in
DT-Headless, LC-MS/MS analysis of deglycosylated tryptic digests
can be performed, for example on a Thermo Scientific LTQ Mass
Spectrometer with a Michrom Paradigm HPLC and Vacuum Spray
ionization source.
[0268] Biochemical characterization can be performed to identify
variants of DT-stabilized, Headless HAs that retain binding to key
anti-stalk quaternary bnAbs equal to the wild-type soluble PR8 HA
trimer. If necessary, additional cleavage sites can be engineered
in order to first unravel the head, and thereby improve the
efficiency of proteolytic cleavage. Similarly, PreScission and/or
TEV proteases and their cleavage sites can be used as described
above.
Testing Protection Against Challenge with Drift and Heterologous
Viruses
[0269] A PR8 HA variant can be expressed in mg-quantities, DT
crosslinked, proteolyzed, purified, and antigenically
characterized. PR8, NL09, and VN04 HALO/PR8_6+2 mutant virus
preparations can be made. To establish the LD50 for each of the
challenge viruses, for each virus 4 groups of 4 C57BL/6 mice
(female, 6- to 8-week-old (Charles River Laboratories) can be
inoculated, using 10-fold dilutions of the indicated viruses for
each group around the published LD50 for each virus. To establish
the optimal dose of purified DT-locked Headless HA trimer immungen
that protects 80%+ of animals from 5.times. the LD50 dose of
homologous (PR8) challenge, 4 groups of 5 C57BL/6 mice (female, 6-
to 8-week-old (Charles River Laboratories) can be immunized with a
prime-boost strategy consisting of consecutive injections of
varying amounts of the purified DT-Headless HA immunogen with a
fixed amount of Poly I/C adjuvant (10 m). Briefly, each group can
be immunized with 0 .mu.g, 2.5 m, 5 .mu.g, and 10 .mu.g of
DT-locked Headless trimer formulated with Poly I/C as an adjuvant.
Three weeks later, the mice can be boosted, each with an equivalent
amount of the adjuvanted immunogen. Three weeks after the boost,
they can be challenged intranasally with a 5.times.LD50 dose of
homologous (PR8) influenza virus. Mice can be monitored and
evaluated for morbidity and mortality for a suitable time, such as
14 days. Mice losing more than 25% of their initial weight can be
sacrificed and scored as dead. Survival can be defined as <25%
weight loss. To test immunized mice for protection against a drift
variant and group 1 heterologous challenges, three groups of
C57BL/6 mice can be immunized with 10 .mu.g of Poly I/C adjuvant
only ("Adjuvant Only" control groups) and the remaining three
groups can be immunized according to the schedule described above
with the optimal dose of adjuvanted DT-Headless HA immunogen
identified above ("DT-locked Headless Trimer" groups).
[0270] Two weeks after the final immunization, one group of
Adjuvant Only and one group immunized with the optimized dose of
DT-locked Headless trimer each can be challenged intranasally with
a lethal dose of homologous virus (PR8 H1N1), the mouse-adapted
novel swine pandemic drift variant (NL/09, H1N1), and with
heterosubtypic, group 1 influenza virus (VN04 HALO/PR8_6+2 mutant
H5N1) (Table 1). Mice can be monitored and evaluated for morbidity
and mortality for 10 days and scored as described above.
TABLE-US-00003 TABLE 1 Immunization groups to assess breadth of
protection Adjuvant only Immunogen (negative controls) DT-locked
Headless trimer Challenge: A. Homologous B. Homologous homologus
(PR8) (PR8) (positive control) vs. C. Drift variant D. Drift
variant drift variant (NL09) (NL09) vs. E Group 1 Heterosubtypic F.
Group 1 Heterosubtypic heterosubtypic (VN04 HALO/PR8_6 + 2 (VN04
HALO/PR8_6 + 2 mutant) mutant)
[0271] Statistical Considerations: In view of the fact that both
the predictor (adjuvant only vs. adjuvant+DT Headless immunogen)
and the outcome (death vs. survival) are dichotomous, the null
hypothesis that the vaccine has no effect can be tested with
Fisher's Exact Test. To calculate the minimum number of animals per
group (equal numbers in all groups) necessary to detect an effect
at the 95% confidence level (r.05), the power can be set to 80% and
an assumed effect size of 50% can be used (80% lethality in the
control group, 30% lethality in the vaccinated groups).
Accordingly, each analyte and control group should use a minimum of
15 animals.
[0272] All methods can be carried out according to standard
procedures, for example as described in Steel et al. 2010 [1]. For
example, in ELISA assays the antigen (PR8 HA) can be immobilized
with an .alpha.-foldon mAb (e.g. 74550, Fibrogen Inc.) or an
.alpha.-stalk mAb to a non-quaternary epitope in order to optimize
presentation of its native structure. Antigen-specific Ig in serum
can be detected using labeled .alpha.-mouse Abs.
[0273] It is expected that DT-Headless will successfully induce
protection against drift virus (group D: NL09, H1N1), and/or a
heterologous strain (group F: H5N1). If need be the immunogen can
be reformulated with a different/additional adjuvant and/or the
doses tested can be increased, and immunogen dose-calibration
testing can be repeated. In addition, if need be the prime-boost
regimen can be altered to include a third boost with purified
DT-Headless HA antigen. The number of animals used in the final
challenge study can be altered/increased to achieve an acceptable
confidence level from homologous vs. drift and heterologous
challenges.
REFERENCES FOR EXAMPLE 1
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conserved hemagglutinin stalk domain. MBio 1, (2010). [0275] 2.
Pica, N. et al. Hemagglutinin stalk antibodies elicited by the 2009
pandemic influenza virus as a mechanism for the extinction of
seasonal H1N1 viruses. Proc. Natl. Acad. Sci. U.S.A. 109, 2573-2578
(2012). [0276] 3. Miller, M. S. et al. 1976 and 2009 H1N1 Influenza
Virus Vaccines Boost Anti-Hemagglutinin Stalk Antibodies in Humans.
J. Infect. Dis. (2012).doi:10.1093/infdis/jis652 [0277] 4. Krammer,
F., Pica, N., Hai, R., Tan, G. S. & Palese, P. Hemagglutinin
Stalk-Reactive Antibodies Are Boosted following Sequential
Infection with Seasonal and Pandemic H1N1 Influenza Virus in Mice.
J. Virol. 86, 10302-10307 (2012). [0278] 5. Wei, C.-J. et al.
Induction of broadly neutralizing H1N1 influenza antibodies by
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recognition of a highly conserved influenza virus epitope. Science
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I. A. Structural insights into key sites of vulnerability on HIV-1
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Dreyfus, C. et al. Highly conserved protective epitopes on
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Corti, D. et al. A neutralizing antibody selected from plasma cells
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A. S. Induction of unnatural immunity: prospects for a broadly
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& Wilson, I. A. Broadly neutralizing antibodies present new
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Anderson, S. R. Flexibility involving the intermolecular dityrosyl
cross-links of enzymatically polymerized calmodulin. Biochemistry
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Swanson, C. A. & Anderson, S. R. Dityrosine: preparation,
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18. Malencik, D. A. & Anderson, S. R. Dityrosine formation in
calmodulin: cross-linking and polymerization catalyzed by
Arthromyces peroxidase. Biochemistry 35, 4375-4386 (1996). [0292]
19. Rodriguez-Mateos, A., Millar, S. J., Bhandari, D. G. &
Frazier, R. A. Formation of dityrosine cross-links during
breadmaking. J. Agric. Food Chem. 54, 2761-2766 (2006). [0293] 20.
Horowitz, E. D., Finn, M. G. & Asokan, A. Tyrosine
cross-linking reveals interfacial dynamics in adeno-associated
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[0294] 21. Elvin, C. M. et al. Synthesis and properties of
crosslinked recombinant pro-resilin. Nature 437, 999-1002 (2005).
[0295] 22. Wang, W. Protein aggregation and its inhibition in
biopharmaceutics. International Journal of Pharmaceutics 289, 1-30
(2005). [0296] 23. Walker, L. M. et al. Broad and potent
neutralizing antibodies from an African donor reveal a new HIV-1
vaccine target. Science 326, 285-289 (2009). [0297] 24. Pejchal, R.
et al. Structure and function of broadly reactive antibody PG16
reveal an H3 subdomain that mediates potent neutralization of
HIV-1. Proc. Natl. Acad. Sci. U.S.A. 107, 11483-11488 (2010).
[0298] 25. Walker, L. M. et al. Broad neutralization coverage of
HIV by multiple highly potent antibodies. Nature 477, 466-470
(2011). [0299] 26. Harris, A. et al. Trimeric HIV-1 glycoprotein
gp140 immunogens and native HIV-1 envelope glycoproteins display
the same closed and open quaternary molecular architectures. Proc.
Natl. Acad. Sci. U.S.A. 108, 11440-11445 (2011). [0300] 27. Alff,
P. J. & Marshall, C. et al., C. Conformational-Locking of
Cleaved HIV-1 gp140 Trimers by Targeted Dityrosine Bonds.
Manuscript in Preparation [0301] 28. Palese, P. Influenza: old and
new threats. Nat. Med. 10, S82-7 (2004). [0302] 29. Madjid, M.
& Casscells, W. Influenza as a bioterror threat: the need for
global vaccination. Expert Opin Biol Ther 4,265-267 (2004). [0303]
30. Osterholm, M. T. Preparing for the next pandemic. N. Engl. J.
Med. 352, 1839-1842 (2005). [0304] 31. Avatar Medical, LLC
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Buckland, B. Vaccine process technology. Biotechnol. Bioeng. 109,
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Professor and Chair of Microbiology, Professor of Medicine,
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Aeschbach, R., Amado, R. & Neukom, H. Formation of dityrosine
cross-links in proteins by oxidation of tyrosine residues. Biochim.
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Recombinant protein vaccines produced in insect cells. Vaccine 30,
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Example 2
[0312] Recombinant, soluble protein immunogens represent a
significant opportunity in the fight against natural and weaponized
pathogens. Broadly neutralizing antibodies (bnAbs) against many
pathogens have been described in recent years, many of which bind
quaternary structures only displayed by protein complexes--which
themselves are often unstable. Therefore, an urgent need exists to
"lock" protein-based vaccine immunogens into the same native
quaternary conformation as they are presented by the pathogen
itself.
[0313] The present example relates to a headless
hemagglutinin-based universal flu vaccine made using a system that
includes (i) performing site-directed mutagenesis at positions
where resulting Tyr residues are predicted to be in close
structural proximity, (ii) expressing and purifying the mutant
protein, and (iii) subsequently enzymatically crosslinking/locking
of the fully-folded protein complex. DT crosslinking is targeted
and zero-length, DT bonds are irreversible and do not form
spontaneously, and, most importantly, introduction of DT-bonds
preserves protein structure and function, since it occurs once the
protein is fully folded.
[0314] At present, a universal influenza vaccine immunogen is not
available. Recently, however, headless HA constructs trimerized in
the transmembrane domain were described that focus Ab responses on
the highly conserved stalk, and that, indeed, elicit broadly
protective responses. Soluble headless HA trimerized by a foldon
motif, however misfolds, does not present key quaternary
neutralizing epitopes (QNEs), and elicits insufficiently protective
responses. The present invention provides an alternative system
that involves (i) DT-crosslinking a soluble HA construct in its
native, trimeric conformation, and (ii) removing the "head" domain
by engineering and cutting proteolytic cleavage sites in the
variable loops and at the base of the head. The interactions
between the subunits of the HA soluble stem (lacking the
transmembrane domain) can be locked by dityrosine crosslinking
while maintaining the structural integrity of HA trimers. Based on
crystal structures, constructs with Tyr side-chains in the stem and
in close structural proximity can be made, while avoiding bnAb
binding sites. These constructs can be expressed and the resulting
proteins purified by His-tag affinity chromatography. Tests can be
performed to determine whether the constructs form intermolecular
DT crosslinks by screening for DT-specific fluorescence and by gel
shift analyses (e.g. Western blots).
[0315] Using of a panel of anti-stem bnAbs, functional preservation
of DT crosslinked HA trimer can be measured by ELISA using
anti-stem bnAbs. Thermodynamic stabilization can be assayed to
confirm the positions of DT bonds and the constructs' structural
integrity after crosslinking biophysically. Constructs can be
selected based on favorable antigenic and/or biochemical profiles.
It is expected that binding to quaternary anti-stem bnAbs such as
6F12, C179, CR6261, F10, A66 and D8 will be fully maintained.
[0316] Proteolytic cleavage sites can be engineered to unravel and
remove the head of native, DT-stabilized HA trimers. Fully folded
HA constructs with 4 or more cleavage sites for 1 or 2 proteases
can be designed, generated, expressed, and DT crosslinked, and then
purified by His-tag affinity chromatography before digesting with
protease(s) to remove the head. Antigenic and biochemical and
biophysical analyses can be performed to confirm
preservation/integrity of QNEs in DT-headless HA after proteolytic
digestion and immunogenic analysis can be performed in mice.
[0317] Seasonal and pandemic influenza viruses remain a serious
threat to human health, due to their ability to evade immune
surveillance through rapid genetic drift and re-assortment. In the
US alone, influenza causes seasonal epidemics that contribute to
hundreds of thousands of hospitalizations and an average of 30,000
deaths annually, while creating a serious economic burden for
individuals and the economy as a whole [1-3]. Pandemic outbreaks
occur when a virulent strain of virus emerges that infects people
with little or no immunity, and rapidly spreads across the globe,
representing one of the most serious threats to human health. The
1918 Spanish Flu (H1N1) pandemic caused an estimated 50 million
deaths; the 1957 Asian influenza (H2N2) pandemic and the 1968 Hong
Kong (H3N2) pandemic each caused several million deaths [6].
Because influenza viruses are readily accessible and are easily
transmitted by aerosol, the possibility for genetic engineering
represents an enormous threat of weaponization, biowarfare, and
bioterrorism [7,8]. Vaccines hold the greatest promise of providing
protection in order to control infection.
[0318] Although highly effective when matched to circulating
strains, current influenza virus vaccines protect mostly against
homologous virus strains. Protection is primarily due to
high-avidity antibodies against the highly variable, immunodominant
head domain of the hemagglutinin (HA) protein, which is specific to
each strain of influenza. Therefore, new trivalent vaccine
cocktails must be tailored each year to the prevalent influenza
strains in circulation. Conventional, egg-based influenza vaccine
manufacturing requires that strains be selected 9 months before the
start of the season. Unfortunately, predictions of the circulating
strains are often inaccurate, resulting in vaccines that are poorly
matched, and therefore poorly protective [9-11]. A multitude of
development programs are underway to address this problem, many of
them in advanced stages, but the approach proposed herein has the
potential to move one or more programs beyond the safety and
efficacy hurdles, and enable a truly long-term broadly protective
vaccine product for both seasonal and pandemic influenza.
[0319] The stem of HA is highly conserved across a multitude of
influenza strains, and considerable evidence now suggests that
vaccination with a `headless` HA consisting primarily of the HA
stem results in the induction of antibody responses with
significantly enhanced heterosubtypic binding activity and broad
protection against lethal challenge [12-15, 16, 17]. As such,
headless HA holds significant promise as a universal vaccine
immunogen capable of protecting against all strains of influenza
[16] [17]. Interestingly, immunization with DNA coding for HA has
been observed to generate predominantly stalk-specific Ab
responses, and data describing heterosubtypic protection by
vaccination with HA DNA by electroporation was recently described
[50]. A prime-boost combination of an expression vector encoding
HA, followed by soluble headless protein boost holds the promise of
generating broadly heterospecific responses that yield long-lasting
protection.
[0320] The present invention provides a soluble `headless` HA
trimer covalently stabilized in its correctly folded conformation
that presents key quaternary neutralizing epitopes (QNEs). Targeted
dityrosine crosslinking technology is used to stabilize a
full-length HA trimer, and subsequently the head is removed using
sequence/substrate-specific proteases--making use of variable loop
tolerance for amino acid variation.
[0321] Dityrosine (DT) crosslinking provides a method for
stabilizing protein folds, complexes, and conformations by
enzymatically introducing zero-length crosslinks, while maintaining
structural and functional integrity of the protein [20,21].
Dityrosine bonds provide conformational stability and rigidity to
protein structures and have been described in many diverse natural
settings. DT crosslinks form naturally in vivo, both in the context
of proteins evolved to utilize their specific characteristics
[22-24], and as a consequence of protein oxidation [25]. DT bonds
form the structure of wheat gluten--the quaternary protein
structure comprising the glutenin subunits--and are present in
large quantities in some of our most common foods [26]. No other
amino acids form crosslinks or are modified when the reaction is
carried out under mild conditions, though the tyrosyl side-chains
themselves may oxidize if positioned too far apart, thus limiting
the efficiency of the reaction, particularly under sub-optimal
conditions. DT crosslinks are not hydrolyzed under normal
physiological conditions, and do not form spontaneously in vitro.
These features of the DT-crosslinking provide important advantages
over conventional S--S chemistry; namely spontaneous and/or
undesired protein products do not form and non-specific
bonding/aggregation does not occur on maturation and processing.
Because the reaction can be tightly controlled, development of a
large-scale high-yield process can be relatively straight-forward,
making the large-scale manufacturing a DT stabilized immunogen more
feasible economically.
[0322] One of the key features of DT crosslinking is that it is
highly dependent on the structural proximity of tyrosyl
side-chains, which must therefore be engineered within the
structure of a protein or protein complex. Because no carbons are
added in the formation of the bond, the resulting "staples" are
non-disruptive to the overall protein fold and, critically,
specific sites within the protein structure can be targeted with
high specificity. The necessary tyrosines may be present in the
primary structure of the protein or added by "to tyrosine" point
mutations, while Tyr residues that form undesirable DT bonds can be
mutated (to Phe, for example) to reduce background.
[0323] Protein immunogens are folded chains of amino acid
polypeptides, sometimes consisting of several polypeptide subunits.
The rate of spontaneous unfolding, conformational transition, and
dissociation determines a protein's functional half-life. Covalent
non-peptide bonds between non-adjacent amino acid side chains can
dramatically affect the rate of unfolding, and thus the half-life
of a protein or protein complex. At least two different chemistries
have evolved to accomplish covalent cross-links in proteins in vivo
to stabilize their conformations and/or retard unfolding: these are
disulfide bonds and dityrosine (DT) bonds.
[0324] One major advantage of a directed DT cross-linking approach
is that covalent bonds targeted to specific locations can reinforce
particular 3-D arrangements of epitopes' secondary, tertiary,
and/or quaternary structures, thereby preventing undesirable
conformational transitions, and have the potential to provide a
high degree of thermodynamic stabilization and conformational
locking without adversely affecting the antigenic properties of
protein immunogens.
[0325] Disulfide bonds have been found in many eukaryotic proteins
of diverse function. Intra-molecular S--S cross-links are often
essential in stabilizing protein domains, and inter-molecular S--S
bonds provide stability for the quaternary structure of protein
complexes. These bonds can form spontaneously, and therefore do not
require an additional manufacturing and purification process, but
also reduce manufacturing yields due to free sulfhydryl-mediated
aggregate formation. Furthermore, because they are formed as the
protein is folding in the ER/Golgi apparatus, they can lead to
structural distortions that would affect QNE presentation and the
breadth of immunogenic protection.
[0326] The C--C bond created by DT-crosslinking is stable under
virtually any physiological and/or operational conditions that are
likely to be used in accordance with the present invention,
including those used in the process of immunization and
vaccination. DT bonds are "zero length"--i.e. no atom is added. The
cross-linking catalyst simply initiates bond formation between two
tyrosines and is not incorporated into the product. Thus, no
undesirable chemical modification of the protein occurs. DT cross
linking is also very specific--no amino acids other than tyrosines
have been shown to form cross-links or to be modified when the
reaction is carried out under mild conditions. In addition, there
is a strict distance requirement between the tyrosine side-chains,
with the bond forming only when the two are in very close
proximity. Furthermore, DT crosslinks do not form spontaneously,
and, as described above, form only between Tyr residues in close
proximity. DT crosslinking a protein can therefore lock it in its
pre-exisiting native/functional conformation. In the context of
headless HA design, this allows one to (i) engineer headless in an
antigenically/immunogenicaly favorable conformation, e.g. by
introducing point mutations, and then (ii) lock it in this
preferred conformation by DT crosslinking.
[0327] Dityrosine bonds (DT bonds) that have important biological
functions have been identified in proteins of several species,
presumably in environments where disulfide bonds would be
unsuitable. Specific DT bonds have, for example, been described in
the cuticlin protein of Caenorhabditis elegans [27], the cell wall
proteins of bamboo shoots [28], and parchment collagen [29]. In all
of these cases, the proteins have evolved such that specifically
placed DT cross-links contribute to the structural rigidity
underlying the proteins' functionality. The importance of such
bonds is also evidenced by the fact that in yeast, for example, a
metabolic pathway has been described that leads to the formation of
DT bonds in specialized proteins [30].
[0328] Furthermore, due to the distinct fluorescent properties of
DT bonds, in the absence of atomic level structures, their
formation can easily be assayed using conventional 96- and 384-well
fluorescence plate readers. This also makes optimization of
cross-linking conditions simple and efficient.
[0329] The present methods involve (a) generating a DT stabilized
full-length HA molecule that retains a stalk-specific antigenic
profile equivalent to that of WT HA, (b) removing the head domain
from the fully folded DT-HA by proteolytic cleavage while retaining
the same `stalk-specific` antigenic profile as WT HA.
Immunogenicity may be confirmed in animal studies.
[0330] The present example utilizes HA from the H1N1 A/Puerto
Rico/8/1934 ("PR8") strain of influenza as the starting point. The
majority of influenza virus research in mice employs lab adapted
PR8 or the A/WSN/1933 (H1N1) [WSN] influenza viruses.
Immunogenicity and challenge studies can be carried out in BALB/c
mice with homologous and heterologous H1N1 PR8 and H3N2 X31
challenges. X31 is a reassortant virus carrying the HA and NA genes
of A/Hong Kong/1/1968 (H3N2) in the background of PR8 [35].
[0331] To identify HA constructs which allow dityrosine bonds to
form and stabilize the HA trimer, the trimeric HA crystal structure
is analyzed (pdb file 3GBN) and proximal residues are selected for
tyr-substitution away from the binding sites of quaternary
neutralizing antibodies (see FIG. 5). Once the in silico design of
"to-tyr" point mutants (2T-HAs) is complete, cDNA encoding the
ectodomain of wild-type HA (PR8) and to-tyr substitution mutants
can be generated and cloned into a baculovirus transfer vector
(pAcGP67A) using standard molecular biology techniques. WT and
2T-HA proteins can be expressed in SF9 or Hi5 cells and secreted HA
can be purified over lectin-based glyco-affinity columns and MonoQ
anion-exchange columns. Following purification, secreted HA
trimmers can be isolated from monomers and high molecular weight
aggregates by size exclusion chromatography (SEC) over a
Superdex200 column.
[0332] To evaluate whether or not the designed 2T-HA constructs
form intermolecular DT cross-links, the purified proteins can be
analyzed before and after exposure to DT crosslinking conditions by
gel-shift in reducing SDS-PAGE (Western blot and Coomassie stain)
and for DT-specific fluorescence. Constructs capable forming DT
cross-links with an efficiency of >50% can be taken forward for
further characterization. Based on preliminary studies with HIV env
trimers, it is believed that crosslinking efficiencies of greater
than 80% are attainable without significant process optimization.
Biochemical and biophysical analysis of DT-crosslinked HA trimers
(DT-HA) be can be performed to compare their thermostability with
that of uncrosslinked HA in normal human serum at 37.degree. C.
over a time-course of 1-30 days. Trimeric DT-HA and control
(uncrosslinked) trimeric HA can be analyzed each day for the
presence of retained trimer by Western blot. Likewise, a 60-day,
25.degree. C. time course in PBS (pH 7.4) of purified, trimeric
DT-HA and control (uncrosslinked) trimeric HA can be analyzed
weekly by SEC. The proportion of total material in the trimeric and
monomeric fractions can be quantified using standard
peak-integration software and the ratio of trimer to monomer in the
DT-HA and control samples can be determined. Given that DT-HA
constructs can be identified based on their stability in reducing
SDS-PAGE, it is expected that 100% of the DT crosslinked trimer
will remain trimeric under the experimental conditions described
above, while labile uncrosslinked HA trimers will dissociate into
monomeric subunits throughout the duration of the time course.
[0333] A central advantage of DT crosslinking technology over other
crosslinking methodologies is the ability to form covalent
intermolecular crosslinks without disrupting the antigenic profile
of vaccine immunogen candidates. The effect of the both the
"to-tyr" mutations and the DT crosslinking can be determined by
ELISA using a panel of anti-HA stem broadly neutralizing mAbs (e.g
6F12, C179, CR6261, F10, A66 and D8). Full-curve binding assays can
be used to compare WT HA trimers to the 2T-HA mutant trimers
(uncrosslinked) and to DT-HA trimers (crosslinked). Changes in
binding following the introduction of to-tyr mutations as well as
after DT crosslinking can be determined using non-linear regression
analysis of binding curves to calculate and compare EC50 values for
each construct with each mAb. The position of to-tyr mutations can
be distal to and non-overlapping with amino acids involved in
binding of the anti-stem bnAbs listed above. It is possible that
structural changes caused by tyrosine substitutions may reduce or
enhance binding to some of these antibodies. However, preliminary
studies using HIV suggest that DT crosslinking fully preserves a
protein candidate's antigenic profile and a similar degree of
antigenic preservation is expected following DT crosslinking of
influenza HA.
[0334] In order to assess non-specific changes to amino acid side
chains throughout the entire crosslinked protein, comparative amino
acid analysis (AAA) can be performed on uncrosslinked (control) and
crosslinked constructs. Amino acid analysis can also be used to
confirm the presence of DT bonds since dityrosine crosslinks
withstand even the acid-hydrolysis used to prepare samples for AAA
and dityrosine itself can be specifically detected in the analysis.
In order to directly identify the position of the dityrosine bonds
in DT-HA, mass spectrometry analysis of deglycosylated tryptic
digests can be used, for example by performing LC-MS/MS on a Thermo
Scientific LTQ Mass Spectrometer with a Michrom Paradigm HPLC and
Michrom Vacuum Spray ionization source. Collectively these studies
can be used to identify and characterize HA constructs capable of
forming trimerizing DT bonds. Such constructs may, even prior to
removal of the immune-dominant HA head domain, provide improved HA
immunogens stably presenting stalk specific QNEs.
[0335] Previously reported recombinant headless HA constructs do
not retain the fully native, quaternary structure of the HA stem
and thus, these constructs do not bind known quaternary specific
bnAbs. Following baculovirus expression and purification of DT-HA
construct(s) as described above the head domain can be removed
proteolytically--post-folding and after DT crosslinking--in order
to generate a stable headless HA which retains binding to broadly
protective, conformation-dependent quaternary antibodies. In order
to enable the proteolytic removal of the globular head domain of
HA, protease cleavage sites can be introduced into HA1.
Head-removal sites can be introduced at, for example, positions
60-76 (N-terminal site) and 277-290 (c-terminal site) through
standard molecular biology techniques [19]. Crystal structures of
HA indicate that these positions are solvent-exposed and could be
made further accessible to proteases by removing the structural
constraints that may hamper efficient proteolysis through the
introduction of additional cleavage sites into the HA1 variable
loop domains (AA positions 142-146 and 155-164) [37]. Unraveling
the head can be used to further improve protease substrate access,
if required. Introduction of cleavage sites into the HA variable
loops is not expected to alter the overall conformation of the HA
trimer as these sites are highly tolerant of amino acid
substitutions. Indeed, all of these amino acid positions (e.g.
142-146 and 155-164) have changed in infectious virus isolates
collected from 1968 through 1999 [38]. Cleaving HA1 in the variable
loops can be performed to destabilize the head's globular
structure, allowing complete exposure and efficient cleavage at the
primary head-removal sites (53-67 and 269-277). PreScission
Protease (GE Healthcare Life Sciences) recognition sequences
(LEVLFQGP (SEQ ID NO:69)) and TEV (Tobacco Etch Virus protease)
recognition sequences ENLYFQG (SEQ ID NO:70) and ENLYFQS (SEQ ID
NO:71)) can be used/introduced. TEV cleavage can be carried out at
a substrate to enzyme ratio of 1:50-200 w/w in a 25 mM Tris-HCl
buffer with 150-500 mM NaCl, and 14 mM .beta.-mercaptoethanol at pH
7.0. PreScission Protease cleavage can be performed in a 50 mM
Tris-HCl buffer, with 150 mM NaCl, 1 mM EDTA and 1 mM
dithiothreitol (DTT) at pH 7.0. Removal of the head can be assayed
by electro-mobility shift from a molecular weight corresponding to
a full-length DT-HA trimer (.about.225 kDa) to that of a headless
trimer (.about.135 kDa) by SDS-PAGE, followed by coomassie stain
and Western blot. Head-specific detection Abs can be used to
confirm removal of the head from the DT crosslinked HA stem by
Western blot and ELISA. If HA head-removal is incomplete, the
positions of Prescission Protease and TEV sites can be swapped, or,
only a single type of site can be introduced at all desired
cleavage positions.
[0336] In order to test the immunogenicity of the DT-headless
constructs, mouse immunogenicity studies can be performed. BALB/c
mice (6-8 weeks old) can be anesthetized with isoflurane 3-5% and
subsequently immunized in a prime-boost regime/schedule with two
intramuscular injections 3 weeks apart, first with DNA comprising
37.5 .mu.g of pGag-EGFP and 75 m of pDZ_PR8_HA followed by
electoporation pulsing (prime), and subsequently with 25 .mu.g of
WT HA, foldon/GCN4-stabilized HA trimers, or DT-headless protein
(boost). Protein (boost) immunogens can be formulated with Alum
(Aluminum phosphate, 300 .mu.g/dose). Two weeks following the
second injection (boost), serum can be collected and assayed for
anti-HA responses relative to pre-immunization serum and adjuvant
only controls. Overall anti-HA IgG and IgM titers for each group
can be determined by ELISA. Heterosubtypic reactivity of antisera
to 10 different purified group 1 and group 2 HAs can be determined
Western blot and ELISA. Immunogens from each group are expected to
elicit anti-HA antibody responses. In order to investigate the
heterosubtypic neutralization capacity of anti-serum from each
group, the ability of these sera to neutralize a panel of
heterologous influenza viruses (HK/68 H3, Bris/07 H3, Neth/03 H7,
Ca1/09 H1, Sing/57 H2, Viet/04 H5, HK/97 H6, HK/99 H9) can be
tested. Anti-serum can be serially diluted 2-fold, mixed with an
equal volume of virus, and incubated for 2 h at 37.degree. C.
Virus-serum mixtures can be added to target cells (MDCK) in serum
free media containing trypsin and incubated for 3 h prior to
replacement of the media. Cells can be monitored for cytopathic
effects 3-5 days following exposure to virus-serum mixtures.
[0337] A major objective of this immunogen design and development
process is to generate a DT-headless immunogen capable of eliciting
bnAbs and protecting against heterologous influenza challenge. To
directly investigate the ability of DT-headless to elicit
protective responses against influenza infection, 3 groups of 20
BALB/c mice can be immunized with WT HA, foldon/GCN4 headless, or
DT-headless, compared to non/pre-immunized and adjuvant-only
immunized controls (groups 4 and 5, 20 mice each), and challenged
intranasally with a lethal dose of homologous (PR8) or heterologous
(X31) virus--10 mice each--2 weeks following the second
immunization (boost). Mice can be anesthetized with an
intraperitoneal injection of ketamine (75 mg/kg) and xylazine (15
mg/kg) prior to challenge, and body weight can be monitored daily.
<20% weight loss can be used as a surrogate for survival. It is
expected that each immunogen (WT HA, foldon/GCN4 headless,
DT-headless) will provide some degree of protection against PR8
challenge. However, it is expected that immunization with a
DT-headless immunogen will provide significantly improved
protection against heterologous influenza challenge and that this
protection will correlate with the titers of bnAbs recognizing
conserved QNEs that presented on the native HA stem in its trimeric
form.
[0338] The baculovirus expression vector system (BEVS) can be used
for manufacturing of recombinant HA antigen as this system is well
established and suitable production/purification protocols have
been well described and validated [10]. Generally, such protocols
involve harvesting infected cells by centrifugation,
detergent-mediated protein solubilization, followed by purification
involving two chromatographic (IE and HIC columns) steps [10]. Due
to the large difference in MW of the trimeric stalk as compared to
the monomeric head, and the enzymes used in processing, gel
filtration can also be used. IE chromatography can also be
used.
[0339] Two enzymes are used in the processes described
herein--peroxidases to catalyze the formation of DT bonds and
proteases to cleave off the HA head after cross-linking. Both are
commercially available.
[0340] Purity of the finished immunogens can be ascertained by
conventional gel electrophoresis and HPLC. Cross-linking can be
assessed by a combination of gel electrophoresis under denaturing
conditions, fluorescence measurements, and amino acid analysis.
Immunogenicity can be assessed by profiling against a panel of
selected antibodies as described above. HPLC-based assays can be
used to identify and measure protein sugar compositions.
[0341] DT-Headless HA can be formulated with an adjuvant selected
based on technical specifications and other considerations.
Adjuvanted HA formulated with a variety of excipients and
stabilizing agents/preservatives can be lyophilized, and following
rehydration tested biophysically (dynamic light scattering) and
antigenically. The effect of storage at room temperature, 4.degree.
C. and -20.degree. C. can be tested to determine long-term storage
conditions, stability, and potency.
[0342] Animal efficacy studies (e.g. conducted in ferrets) can be
performed and acute and long-term animal safety studies can be
performed. Ferrets are susceptible to human influenza viruses and
develop some of the symptoms of influenza that are seen in humans;
furthermore, they are large enough to monitor clinical parameters
(e.g. temperature, pulse, and respiratory rate), and relatively
large amounts of sera can be obtained for use in serologic and
antigenic characterization.
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Example 3
[0393] Prior attempts at engineering headless HA have included
expressing HA proteins in which the globular head region was
recombinantly spliced out. Such prior headless HA constructs
generated considerable excitement in the field, because they
elicited improved, cross-reactive Ab responses. These Abs, however,
were not cross protective and only protected against homologous
challenge. These prior recombinant headless constructs do not bind
the current repertoire of cross-protective, conformational stalk
Abs, which suggests at least some degree of stalk mis-folding in
the absence of the intact globular head. These prior observations
were confirmed using one of the most broadly cross-reactive
anti-stalk Abs, C179, by immunofluorescence analysis (see FIG. 6).
Application of DT-based conformational locking will circumvent this
shortcoming, by holding together the stalk trimer in its native
conformation, prior to proteolytic removal of the head, and thus
result in a DT-locked Headless HA immunogen that will focus Ab
responses on the critical stalk QNEs.
Example 4
[0394] DT crosslinks were introduced into the PR8 HA stalk domain,
and the DT-cross-linked HA trimer maintained native antigenicity.
Based on the crystal structures of the 1918 H1N1 HA trimer in
complex with the cr6261 bnAb (pdb file: 3GBN) and of the PR8 HA
(pdb file: 1RU7), to-tyrosine substitutions were successfully
engineered into the HA stalk domain in order to enable DT crosslink
formation, which should maintain quaternary antigenicity upon
proteolytic removal of the globular head. 293T cells were
subsequently transfected with secreted variants of the to-tyrosine
mutants and measured at 405 nm fluorescence in transfected cell
supernatants, to determine the formation of DT bonds. A large
increase in 405 nm fluorescence (highly specific for DT bonds)
demonstrates robust crosslinking in several to-tyrosine mutants
(FIG. 7A). Based on comparison to the positive control (insulin)
and a DT standard, a cross-linking efficiency of >70% was
confirmed for four of these constructs, prior to any optimization.
As shown in FIG. 7B, C179 Ab binding is unchanged before and after
the crosslinking reaction. These data show that the PR8 HA stalk
can be cross-linked and that the key quaternary stalk epitope bound
by one of the most broadly cross-reactive, conformational mAbs,
C179 (2), is maintained.
[0395] Targeted protease cleavage sites were also successfully
introduced and used to cleave the PR8 HA head domain. Extensive
analysis of the PR8 HA structure and transposon-based mutagenesis
studies revealed multiple locations within the globular head region
that could tolerate insertion of proteolytic cleavage sites. Out of
20+ possible sites identified, two constructs that allow insertion
were generated. One site is located at the base of the globular
head domain ("48G"), while the other resides closer to the variable
loops of the protein ("128S"). Both insertions express well as
indicated by Western blot of whole cell extracts (FIG. 6B, left)
and form virus-like particles (VLPs) in sufficient quantity for
detection in transfected cell supernatants by C179 ELISA (FIG. 8A).
Of the two constructs generated, the 48G insertion is predicted to
be least accessible for the protease, yet would most completely
remove the Head domain, due to its location close to the base of
the head. In order to demonstrate that the 48G site is sufficiently
accessible, TEV protease cleavage was performed on the HA 48G
protein, using WT HA as a negative control. As shown in FIG. 8B,
right, TEV protease cleavage of the HA 48G protein results in the
removal of the first 48 AAs (6.5 kDa) of HA, but no cleavage occurs
in the WT HA protein. Furthermore, HA 48G also maintains
hemagglutination activity when assayed directly from transfected
cell supernatants, suggesting that it remains folded in its
functional conformation (FIG. 8C).
Example 5
[0396] Introduction: In designing a conformationally locked
headless HA, the atomic structures of the 1918 HA:cr6261 complex
(PDB:3GBN) and PR8 HA (PDB:1RU7) were analyzed to identify
positions that 1) enable dityrosine (DT) crosslinking in the stalk
at a sufficient distance from the cr6261 epitope to maintain stalk
bnAb binding; and 2) enable insertion of protease cleavage sites,
that can be used to remove the head.
PR8 HA Trimers were Successfully Locked in their Native Trimeric
Conformation Using DT Crosslinks at Several Locations in the HA
Stalk; and these DT-Locked HAs Maintain Native Stalk
Antigenicity
[0397] Several tyrosine mutations were engineered into the stalk of
PR8 HA that enable the trimers to be locked in their native
prefusion state, at high efficiency. FIG. 85A demonstrates a clear
shift to the trimeric state (reducing SDS-PAGE) following DT
crosslinking; and FIG. 85B confirms that di-tyrosine bonds have
formed by specific fluorescence at 405 nm. Densitometry of the
cross-linked species demonstrates greater than 80% conversion to
the trimeric state. Most importantly binding of 8D4, a
stalk-specific bnAb, is fully maintained (FIG. 85C).
Crystallographic analysis has shown that 8D4 binds the same epitope
as cr6261. These data confirm that the PR8 HA can be cross-linked
in its stalk while maintaining the native conformation of the key
V.sub.H1-69 quaternary stalk epitope.
Multiple C- and N-Terminal TEV Protease Recognition Sites have been
Successfully Engineered into the Head of PR8 HA, Individually and
in Combination
[0398] Regions were identified in the head of PR8 HA into which TEV
protease cleavage sites can be inserted without disrupting HA's
function. For structure-based design, PR8 HA and TEV protease
recognition site structural data were combined, and cleavage site
insertions were specifically targeted into regions of HA's head
based on the following criteria: i) proximity to the stalk apex, in
order to maximize removal of the immune-dominant head; ii)
similarity between the secondary structures of HA and the TEV
cleavage site, to minimize structural perturbation; iii) regions
identified as tolerant of insertion based on the transposon
mutagenesis screen with data from a transposon-based mutagenesis
screen (Heaton and Palese PNAS Vol. 110, No. 50; pp. 20248-53).
[0399] In total, approximately 50 insertion sites were screened
individually, and assayed for their ability to be incorporated into
VLPs by expressing only HA (WT or with insertion) and NA. This
assay encompasses several of HA's functional attributes, including
expression, cell surface accumulation, membrane microdomain
localization, and particle formation, and was therefore performed
with full length HA proteins. This approach identified several
C-terminal (e.g. at positions 278, 282, 283, 286, and 291) and 2
N-terminal (positions 48 and 63) where TEV insertions maintain the
function of WT HA in the VLP formation assay (FIG. 86A). Several of
these also maintain stalk bnAb binding, and cleave efficiently FIG.
86B). The single insertion HA mutants (insertion at positions 63,
278, and 286) bound to a broadly neutralizing V.sub.H1-69
stalk-specific mAb by direct capture ELISA at 50 .mu.g/ml
(normalized for HA presence in supernatants). Two C-terminal
insertions (positions 278 and 286) and 1 N-terminal insertion
(position 63) were prioritized for further analysis and testing in
combination. A second N-terminal insertion (position 48), binds
well to certain anti-stalk V.sub.H1-69 bnAbs (e.g. 18A3), but less
well to others, and therefore could nonetheless provide a
reasonable alternative to the insertion at position 63.
[0400] Some of the prioritized insertion sites have been tested in
various combinations (e.g. 63-278 and 63-286) and have shown that
both of the combinations of insertions also maintain efficient VLP
formation (FIG. 87A) and bind well to stalk bnAbs (FIG. 87B).
[0401] Going forward both components (to-tyrosine mutations and
proteolytic cleavage site insertions) can be introduced into a
single HA molecule. DT crosslinking can then be applied to lock the
stalk of HA in its trimeric, prefusion conformation, and following
this the head can be removed proteolytically to generate a fully
native, headless HA.
[0402] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. The invention may also be
further defined in terms of the following claims.
Sequence CWU 1
1
1181565PRTInfluenza A virus 1Met Lys Ala Asn Leu Leu Val Leu Leu
Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly
Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu
Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu
Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala
Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70
75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr
Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro
Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala
Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu
Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys
Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190
Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195
200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn
Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg
Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu
Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu
Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly
Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys Asn
Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser Leu
Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315
320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335 Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp
Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile Thr
Asn Lys Val Asn Thr Val Ile Glu 385 390 395 400 Lys Met Asn Ile Gln
Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Lys Arg
Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430 Asp
Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440
445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn
Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys
Met Glu Ser Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly Val
Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile Tyr
Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu Gly
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555 560
Cys Arg Ile Cys Ile 565 21698DNAInfluenza A virus 2atgaaggcaa
acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata 60tgtataggct
accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggaa tagccccact acaattgggg aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcatc aatgcatgag
tgtaacacga agtgtcaaac accgctcgga 900gctataaaca gcagtctccc
ttaccagaat atacacccag tcacaatagg agagtgccca 960aaatacgtca
ggagtgccaa attgaggatg gttacaggac taaggaacac tccgtccatt
1020caatccagag gtctatttgg agccattgcc ggttttattg aagggggatg
gactggaatg 1080atagatggat ggtatggtta tcatcatcag aatgaacagg
gatcaggcta tgcagcggat 1140caaaaaagca cacaaaatgc cattaacggg
attacaaaca aggtgaacac tgttatcgag 1200aaaatgaaca ttcaattcac
agctgtgggt aaagaattca acaaattaga aaaaaggatg 1260gaaaatttaa
ataaaaaagt tgatgatgga tttctggaca tttggacata taatgcagaa
1320ttgttagttc tactggaaaa tgaaaggact ctggatttcc atgactcaaa
tgtgaagaat 1380ctgtatgaga aagtaaaaag ccaattaaag aataatgcca
aagaaatcgg aaatggatgt 1440tttgagttct accacaagtg tgacaatgaa
tgcatggaaa gtgtaagaaa tgggacttat 1500gattatccca aatattcaga
agagtcaaag ttgaacaggg aaaaggtaga tggagtgaaa 1560ttggaatcaa
tggggatcta tcagattctg gcgatctact caactgtcgc cagttcactg
1620gtgcttttgg tctccctggg ggcaatcagt ttctggatgt gttctaatgg
atctttgcag 1680tgcagaatat gcatctga 16983566PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Glu Asn Leu Tyr Phe Gln Ser Asn Ala
Ser 275 280 285 Met His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn 290 295 300 Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys 305 310 315 320 Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg Met Val Thr Gly Leu Arg 325 330 335 Asn Thr Pro Ser Ile Gln
Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly
Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr 355 360 365 His His
Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380
Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile 385
390 395 400 Glu Lys Met Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe
Asn Lys 405 410 415 Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val
Asp Asp Gly Phe 420 425 430 Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Phe His Asp
Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Lys Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val 485 490 495 Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505
510 Asn Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr
515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val
Leu Leu 530 535 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser
Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile 565
4566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr
Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Glu Asn Leu
Tyr Phe Gln Ser Asn Ala Ser 275 280 285 Met His Glu Cys Asn Thr Lys
Cys Gln Thr Pro Leu Gly Ala Ile Asn 290 295 300 Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305 310 315 320 Pro Lys
Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg 325 330 335
Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340
345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly
Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys
Val Asn Thr Val Ile 385 390 395 400 Glu Lys Met Asn Ile Gln Phe Thr
Ala Val Gly Tyr Glu Phe Asn Lys 405 410 415 Leu Glu Lys Arg Met Glu
Tyr Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg
Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460
Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465
470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys Val Asp Gly Val Lys Leu
Glu Ser Met Gly Ile Tyr 515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr
Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val Ser Leu Gly Ala Ile
Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg
Ile Cys Ile 565 5566PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 5Met Lys Ala Asn Leu Leu Val Leu Leu
Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly
Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu
Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu
Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60 Asn
Leu Tyr Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70
75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr
Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro
Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser
Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu
Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala
Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu
Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys
Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190
Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195
200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn
Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg
Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu
Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu
Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly
Glu Asn Leu Tyr Phe Gln Ser Asn Ala Ser 275 280 285 Met His Glu Cys
Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn 290 295
300 Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys
305 310 315 320 Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr
Gly Leu Arg 325 330 335 Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe
Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile 385 390 395 400 Glu Lys
Met Tyr Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys 405 410 415
Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp Gly Phe 420
425 430 Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu
Asn 435 440 445 Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
Leu Tyr Glu 450 455 460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys
Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn Glu Cys Met Glu Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys
Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr 515 520 525 Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545
550 555 560 Gln Cys Arg Ile Cys Ile 565 6572PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Glu Asn Leu Tyr Phe
Gln 275 280 285 Ser Thr Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys
Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro Lys
Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu Arg
Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile
Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380
Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385
390 395 400 Lys Val Asn Thr Val Ile Glu Lys Met Tyr Ile Gln Phe Thr
Ala Val 405 410 415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu
Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Tyr Ile Trp
Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg
Thr Leu Asp Phe His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu
Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu
Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505
510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu
515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
Val Ala 530 535 540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile
Ser Phe Trp Met 545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg
Ile Cys Ile 565 570 7572PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50
55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu
Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys
Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr
Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175
Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile
Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn
Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys
Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Glu Asn Leu Tyr Phe Gln 275 280 285 Ser Thr
Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300
Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305
310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln
Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His
Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser
Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn
Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 405 410 415 Gly
Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys 420 425
430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu
Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met
Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser
Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550
555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570
8572PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr
Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Glu Asn Leu Tyr Phe Gln 275 280 285 Ser Thr Ser Asn Ala Ser Met
His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile
Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305 310 315 320 Val Thr
Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335
Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340
345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly
Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn Thr Val Ile Glu Lys
Met Tyr Ile Gln Phe Thr Ala Val 405 410 415 Gly Tyr Glu Phe Asn Lys
Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys 420 425 430 Lys Val Asp Asp
Gly Phe Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 450 455 460
Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465
470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser Ser Leu Val Leu Leu
Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550 555 560 Cys Ser Asn
Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 9572PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Glu Asn Leu
Tyr Phe 275 280 285 Gln Gly Ser Asn Ala Ser Met His Glu Cys Asn Thr
Lys Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro
Tyr Gln Asn Ile His Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro
Lys Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu
Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375
380 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn
385 390 395 400 Lys Val Asn Thr Val Ile Glu Lys Met Tyr Ile Gln Phe
Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met
Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Tyr Ile
Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu
Arg Thr Leu Asp Phe His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr
Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495
Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500
505 510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
Leu 515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser
Thr Val Ala 530 535 540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala
Ile Ser Phe Trp Met 545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys
Arg Ile Cys Ile 565 570 10572PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 10Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50
55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu
Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys
Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr
Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175
Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile
Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn
Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys
Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Thr Glu Asn Leu Tyr Phe 275 280 285 Gln Gly
Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300
Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305
310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln
Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His
Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser
Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn
Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 405 410 415 Gly
Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys 420 425
430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu
Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met
Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser
Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550
555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570
11572PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr
Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Thr Glu Asn Leu Tyr Phe 275 280 285 Gln Gly Ser Asn Ala Ser Met
His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile
Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305 310 315 320 Val Thr
Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335
Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340
345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly
Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn Thr Val Ile Glu Lys
Met Tyr Ile Gln Phe Thr Ala Val 405 410 415 Gly Tyr Glu Phe Asn Lys
Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys 420 425 430 Lys Val Asp Asp
Gly Phe Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 450 455 460
Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465
470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser Ser Leu Val Leu Leu
Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550 555 560 Cys Ser Asn
Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 12579PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
12Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln Gly Asn Leu Leu Glu Asp
Ser His Asn Gly Lys 50 55 60 Leu Cys Arg Leu Lys Gly Ile Ala Pro
Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Leu Leu Gly
Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90 95 Val Arg Ser Trp Ser
Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly 100 105 110 Ile Cys Tyr
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu
Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu 130 135
140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys Ser
145 150 155 160 His Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp
Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser
Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu Val Leu Val Leu Trp Gly
Ile His His Pro Pro Asn Ser 195 200 205 Lys Glu Gln Gln Asn Ile Tyr
Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215 220 Val Thr Ser Asn Tyr
Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg 225 230 235 240 Pro Lys
Val Arg Asp Gln Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu 245 250 255
Leu Lys Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile 260
265 270 Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly
Ile 275 280 285 Ile Thr Ser Asn Ala Ser Met His Glu Cys Glu Asn Leu
Tyr Phe Gln 290 295 300 Gly Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala
Ile Asn Ser Ser Leu 305 310 315 320 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 325 330 335 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 340 345 350 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 370 375 380
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 385
390 395 400 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu
Lys Met 405 410 415 Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
Lys Leu Glu Lys 420 425 430 Arg Met Glu Asn Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu Tyr Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 465 470 475 480 Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 500 505
510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu
515 520 525 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln
Ile Leu 530 535 540 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 545 550 555 560 Gly Ala Ile Ser Phe Trp Met Cys Ser
Asn Gly Ser Leu Gln Cys Arg 565 570 575 Ile Cys Ile
13579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln
Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys 50 55 60 Leu Cys Arg
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile
Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90
95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly
100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg
Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile
Phe Pro Lys Glu 130 135 140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly
Val Thr Ala Ala Cys Ser 145 150 155 160 His Glu Gly Lys Ser Ser Phe
Tyr Arg Asn Leu Leu Trp Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr
Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu
Val Leu Val Leu Trp Gly Ile His His Pro Pro Asn Ser 195 200 205 Lys
Glu Gln Gln Asn Ile Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215
220 Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg
225 230 235 240 Pro Lys Val Arg Asp Gln Ala Gly Arg Met
Asn Tyr Tyr Trp Thr Leu 245 250 255 Leu Lys Pro Gly Asp Thr Ile Ile
Phe Glu Ala Asn Gly Asn Leu Ile 260 265 270 Ala Pro Met Tyr Ala Phe
Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile 275 280 285 Ile Thr Ser Asn
Ala Ser Met His Glu Cys Glu Asn Leu Tyr Phe Gln 290 295 300 Gly Asn
Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu 305 310 315
320 Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr
325 330 335 Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn
Thr Pro 340 345 350 Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu 355 360 365 Gly Gly Trp Thr Gly Met Ile Asp Gly Trp
Tyr Gly Tyr His His Gln 370 375 380 Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Gln Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile Asn Gly Ile
Thr Asn Lys Val Asn Thr Val Ile Glu Lys Met 405 410 415 Asn Ile Gln
Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys Leu Glu Lys 420 425 430 Arg
Met Glu Tyr Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile 435 440
445 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr
450 455 460 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
Val Lys 465 470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly
Asn Gly Cys Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn Glu Cys
Met Glu Ser Val Arg Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro Lys Tyr
Ser Glu Glu Ser Lys Leu Asn Arg Glu 515 520 525 Lys Val Asp Gly Val
Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu 530 535 540 Ala Ile Tyr
Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu 545 550 555 560
Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg 565
570 575 Ile Cys Ile 14579PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Glu 35 40 45
Asn Leu Tyr Phe Gln Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys 50
55 60 Leu Cys Arg Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys Cys
Asn 65 70 75 80 Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro
Leu Leu Pro 85 90 95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr Pro
Asn Ser Glu Asn Gly 100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile Asp
Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe
Glu Arg Phe Glu Ile Phe Pro Lys Glu 130 135 140 Ser Ser Trp Pro Asn
His Asn Thr Asn Gly Val Thr Ala Ala Cys Ser 145 150 155 160 His Glu
Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu 165 170 175
Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 180
185 190 Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Pro Asn
Ser 195 200 205 Lys Glu Gln Gln Asn Ile Tyr Gln Asn Glu Asn Ala Tyr
Val Ser Val 210 215 220 Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro
Glu Ile Ala Glu Arg 225 230 235 240 Pro Lys Val Arg Asp Gln Ala Gly
Arg Met Asn Tyr Tyr Trp Thr Leu 245 250 255 Leu Lys Pro Gly Asp Thr
Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile 260 265 270 Ala Pro Met Tyr
Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile 275 280 285 Ile Thr
Ser Asn Ala Ser Met His Glu Cys Glu Asn Leu Tyr Phe Gln 290 295 300
Gly Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu 305
310 315 320 Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
Lys Tyr 325 330 335 Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu
Arg Asn Thr Pro 340 345 350 Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala
Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly Trp Thr Gly Met Ile Asp
Gly Trp Tyr Gly Tyr His His Gln 370 375 380 Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile Asn
Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys Met 405 410 415 Tyr
Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys Leu Glu Lys 420 425
430 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Tyr Ile
435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu
Arg Thr 450 455 460 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
Glu Lys Val Lys 465 470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn
Glu Cys Met Glu Ser Val Arg Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro
Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 515 520 525 Lys Val Asp
Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu 530 535 540 Ala
Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu 545 550
555 560 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys
Arg 565 570 575 Ile Cys Ile 15579PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 15Met Lys Ala Asn Leu
Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Glu 35 40
45 Asn Leu Tyr Phe Gln Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys
50 55 60 Leu Cys Arg Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys
Cys Asn 65 70 75 80 Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp
Pro Leu Leu Pro 85 90 95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr
Pro Asn Ser Glu Asn Gly 100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile
Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser
Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu 130 135 140 Ser Ser Trp Pro
Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys Ser 145 150 155 160 His
Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu 165 170
175 Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys
180 185 190 Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Pro
Asn Ser 195 200 205 Lys Glu Gln Gln Asn Ile Tyr Gln Asn Glu Asn Ala
Tyr Val Ser Val 210 215 220 Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr
Pro Glu Ile Ala Glu Arg 225 230 235 240 Pro Lys Val Arg Asp Gln Ala
Gly Arg Met Asn Tyr Tyr Trp Thr Leu 245 250 255 Leu Lys Pro Gly Asp
Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile 260 265 270 Ala Pro Met
Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile 275 280 285 Ile
Thr Ser Asn Ala Ser Met His Glu Cys Glu Asn Leu Tyr Phe Gln 290 295
300 Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu
305 310 315 320 Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys
Pro Lys Tyr 325 330 335 Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly
Leu Arg Asn Thr Pro 340 345 350 Ser Ile Gln Ser Arg Gly Leu Phe Gly
Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly Trp Thr Gly Met Ile
Asp Gly Trp Tyr Gly Tyr His His Gln 370 375 380 Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile
Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys Met 405 410 415
Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu Glu Lys 420
425 430 Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Tyr
Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
Glu Arg Thr 450 455 460 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu Lys Val Lys 465 470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys
Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu Ser Val Arg Asn Gly 500 505 510 Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 515 520 525 Lys Val
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu 530 535 540
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu 545
550 555 560 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln
Cys Arg 565 570 575 Ile Cys Ile 16579PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
16Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln Gly Asn Leu Leu Glu Asp
Ser His Asn Gly Lys 50 55 60 Leu Cys Arg Leu Lys Gly Ile Ala Pro
Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Leu Leu Gly
Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90 95 Val Arg Ser Trp Ser
Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly 100 105 110 Ile Cys Tyr
Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu
Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu 130 135
140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly Val Thr Ala Ala Cys Ser
145 150 155 160 His Glu Gly Lys Ser Ser Phe Tyr Arg Asn Leu Leu Trp
Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr Pro Lys Leu Lys Asn Ser
Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu Val Leu Val Leu Trp Gly
Ile His His Pro Pro Asn Ser 195 200 205 Lys Glu Gln Gln Asn Ile Tyr
Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215 220 Val Thr Ser Asn Tyr
Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg 225 230 235 240 Pro Lys
Val Arg Asp Gln Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu 245 250 255
Leu Lys Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile 260
265 270 Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly
Ile 275 280 285 Ile Thr Ser Asn Ala Ser Met His Glu Cys Glu Asn Leu
Tyr Phe Gln 290 295 300 Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala
Ile Asn Ser Ser Leu 305 310 315 320 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 325 330 335 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 340 345 350 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 370 375 380
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 385
390 395 400 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu
Lys Met 405 410 415 Asn Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn
Lys Leu Glu Lys 420 425 430 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu Asp Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 465 470 475 480 Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 500 505
510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu
515 520 525 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln
Ile Leu 530 535 540 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 545 550 555 560 Gly Ala Ile Ser Phe Trp Met Cys Ser
Asn Gly Ser Leu Gln Cys Arg 565 570 575 Ile Cys Ile
17579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln
Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys 50 55 60 Leu Cys Arg
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile
Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90
95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly
100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg
Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile
Phe Pro Lys Glu 130 135 140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly
Val Thr Ala Ala Cys Ser 145 150 155 160 His Glu Gly Lys Ser Ser Phe
Tyr Arg Asn Leu Leu Trp Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr
Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu
Val Leu Val Leu Trp
Gly Ile His His Pro Pro Asn Ser 195 200 205 Lys Glu Gln Gln Asn Ile
Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215 220 Val Thr Ser Asn
Tyr Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg 225 230 235 240 Pro
Lys Val Arg Asp Gln Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu 245 250
255 Leu Lys Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile
260 265 270 Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg Gly Phe Gly Ser
Gly Ile 275 280 285 Ile Thr Ser Asn Ala Ser Met His Glu Cys Glu Asn
Leu Tyr Phe Gln 290 295 300 Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 305 310 315 320 Pro Tyr Gln Asn Ile His Pro
Val Thr Ile Gly Glu Cys Pro Lys Tyr 325 330 335 Val Arg Ser Ala Lys
Leu Arg Met Val Thr Gly Leu Arg Asn Thr Pro 340 345 350 Ser Ile Gln
Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly
Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 370 375
380 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn
385 390 395 400 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile
Glu Lys Met 405 410 415 Tyr Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe
Asn Lys Leu Glu Lys 420 425 430 Arg Met Glu Tyr Leu Asn Lys Lys Val
Asp Asp Gly Phe Leu Tyr Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu Asp Phe His Asp
Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 465 470 475 480 Ser Gln
Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 500
505 510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg
Glu 515 520 525 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr
Gln Ile Leu 530 535 540 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val
Leu Leu Val Ser Leu 545 550 555 560 Gly Ala Ile Ser Phe Trp Met Cys
Ser Asn Gly Ser Leu Gln Cys Arg 565 570 575 Ile Cys Ile
18572PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu
Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys Glu Asn Leu Tyr
Phe Gln Gly Asn Thr Lys Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile
Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305 310 315 320 Val Thr
Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335
Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340
345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly
Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn Thr Val Ile Glu Lys
Met Asn Ile Gln Phe Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn Lys
Leu Glu Lys Arg Met Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp
Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 450 455 460
Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465
470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys
Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser Ser Leu Val Leu Leu
Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550 555 560 Cys Ser Asn
Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 19572PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser
Met 275 280 285 His Glu Cys Glu Asn Leu Tyr Phe Gln Ser Asn Thr Lys
Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro Lys
Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu Arg
Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile
Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380
Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385
390 395 400 Lys Val Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr
Ala Val 405 410 415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu
Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg
Thr Leu Asp Phe His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu
Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu
Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505
510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu
515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
Val Ala 530 535 540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile
Ser Phe Trp Met 545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg
Ile Cys Ile 565 570 20566PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 20Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Glu 35 40 45
Asn Leu Tyr Phe Gln Gly His Asn Gly Lys Leu Cys Arg Leu Lys Gly 50
55 60 Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu
Leu 65 70 75 80 Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser
Trp Ser Tyr 85 90 95 Ile Val Glu Thr Pro Asn Ser Glu Asn Gly Ile
Cys Tyr Pro Gly Asp 100 105 110 Phe Ile Asp Tyr Glu Glu Leu Arg Glu
Gln Leu Ser Ser Val Ser Ser 115 120 125 Phe Glu Arg Phe Glu Ile Phe
Pro Lys Glu Ser Ser Trp Pro Asn His 130 135 140 Asn Thr Asn Gly Val
Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser 145 150 155 160 Phe Tyr
Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro 165 170 175
Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val 180
185 190 Leu Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn
Ile 195 200 205 Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser
Asn Tyr Asn 210 215 220 Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro
Lys Val Arg Asp Gln 225 230 235 240 Ala Gly Arg Met Asn Tyr Tyr Trp
Thr Leu Leu Lys Pro Gly Asp Thr 245 250 255 Ile Ile Phe Glu Ala Asn
Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe 260 265 270 Ala Leu Ser Arg
Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser 275 280 285 Met His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn 290 295 300
Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305
310 315 320 Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly
Leu Arg 325 330 335 Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly
Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile
Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asn
Gly Ile Thr Asn Lys Val Asn Thr Val Ile 385 390 395 400 Glu Lys Met
Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys 405 410 415 Leu
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425
430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445 Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu 450 455 460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys Val
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr 515 520 525 Gln Ile Leu
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550
555 560 Gln Cys Arg Ile Cys Ile 565 21572PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
21Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165
170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val
Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln
Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr
Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg
Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr
Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala
Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser
Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Glu Asn 275 280 285
Leu Tyr Phe Gln Ser Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr 290
295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His
Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser
Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn Thr Pro Ser
Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr
His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Gln
Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390 395 400 Lys
Val Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 405 410
415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn Lys
420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala
Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe
His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys
Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys Met Glu Ser
Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu
Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515 520 525 Glu
Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 530 535
540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met
545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565
570 22566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu
Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Glu Asn Leu
Tyr Phe Gln Ser Asn Ala Ser 275 280 285 Met His Glu Cys Asn Thr Lys
Cys Gln Thr Pro Leu Gly Ala Ile Asn 290 295 300 Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305 310 315 320 Pro Lys
Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg 325 330 335
Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340
345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly
Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp
Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys
Val Asn Thr Val Ile 385 390 395 400 Glu Lys Met Asn Ile Gln Phe Thr
Ala Val Gly Lys Glu Phe Asn Lys 405 410 415 Leu Glu Lys Arg Met Glu
Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg
Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460
Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465
470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu
Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys Val Asp Gly Val Lys Leu
Glu Ser Met Gly Ile Tyr 515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr
Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val Ser Leu Gly Ala Ile
Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg
Ile Cys Ile 565 23572PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 23Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50
55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu
Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys
Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr
Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175
Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile
Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn
Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys
Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Glu Asn Leu Tyr Phe Gln 275 280 285 Ser Thr
Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300
Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305
310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln
Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His
Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser
Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn
Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 405 410 415 Gly
Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn Lys 420 425
430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu
Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met
Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser
Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550
555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570
24565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr
Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys Asn Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln
Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn 325 330 335
Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340
345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr
His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
Asn Thr Val Ile Glu 385 390 395 400 Lys Met Asn Ile Gln Phe Thr Ala
Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Lys Arg Met Glu Asn
Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465
470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly Val Lys Leu Glu
Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val
Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile
Cys Ile 565 25572PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Met Lys Ala Asn Leu Leu Val Leu
Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu
Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65
70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser
Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr
Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu
Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys
Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala
Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145
150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr
Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu
Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys
Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser
Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile
Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met
Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile
Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265
270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Glu Asn Leu Tyr Phe
275 280 285 Gln Gly Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys
Gln Thr 290 295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln
Asn Ile His Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr
Val Arg Ser Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn
Thr Pro Ser Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala
Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390
395 400 Lys Val Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala
Val 405 410 415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn
Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr
Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr
Leu Asp Phe His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys
Val Lys Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys
Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510
Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515
520 525 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
Ala 530 535 540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser
Phe Trp Met 545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
Cys Ile 565 570 26566PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50
55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu
Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys
Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr
Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175
Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile
Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn
Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys
Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Glu Asn Leu Tyr Phe Gln Ser Asn Ala Ser 275 280 285 Met His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn 290 295 300
Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305
310 315 320 Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly
Leu Arg 325 330 335 Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly
Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile
Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asn
Gly Ile Thr Asn Lys Val Asn Thr Val Ile 385 390 395 400 Glu Lys Met
Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys 405 410 415 Leu
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425
430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445 Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu 450 455 460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys Val
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr 515 520 525 Gln Ile Leu
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550
555 560 Gln Cys Arg Ile Cys Ile 565 27572PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Glu 50 55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Glu Asn Leu Tyr Phe
Gln 275 280 285 Ser Thr Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys
Cys Gln Thr 290 295 300 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro 305 310 315 320 Val Thr Ile Gly Glu Cys Pro Lys
Tyr Val Arg Ser Ala Lys Leu Arg 325 330 335 Met Val Thr Gly Leu Arg
Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile
Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly
Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380
Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385
390 395 400 Lys Val Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr
Ala Val 405 410 415 Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu
Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp
Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg
Thr Leu Asp Phe His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu
Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu
Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505
510 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu
515 520 525 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
Val Ala 530 535 540 Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile
Ser Phe Trp Met 545 550 555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg
Ile Cys Ile 565 570 28572PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 28Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50
55 60 Asn Leu Tyr Phe Gln Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu
Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys
Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr
Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175
Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile
Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn
Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys
Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Thr Glu Asn Leu Tyr Phe 275 280 285 Gln Gly
Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr 290 295 300
Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 305
310 315 320 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg 325 330 335 Met Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln
Ser Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Thr Gly Met Ile 355 360 365 Asp Gly Trp Tyr Gly Tyr His His
Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Gln Lys Ser
Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 385 390 395 400 Lys Val Asn
Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 405 410 415 Gly
Lys Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Asn Leu Asn Lys 420 425
430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu
Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn 485 490 495 Glu Cys Met Glu Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ser Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 515 520 525 Glu Ser Met
Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 530 535 540 Ser
Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met 545 550
555 560 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570
29579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln
Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys 50 55 60 Leu Cys Arg
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile
Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90
95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly
100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg
Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile
Phe
Pro Lys Glu 130 135 140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly Val
Thr Ala Ala Cys Ser 145 150 155 160 His Glu Gly Lys Ser Ser Phe Tyr
Arg Asn Leu Leu Trp Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr Pro
Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu Val
Leu Val Leu Trp Gly Ile His His Pro Pro Asn Ser 195 200 205 Lys Glu
Gln Gln Asn Ile Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215 220
Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg 225
230 235 240 Pro Lys Val Arg Asp Gln Ala Gly Arg Met Asn Tyr Tyr Trp
Thr Leu 245 250 255 Leu Lys Pro Gly Asp Thr Ile Ile Phe Glu Ala Asn
Gly Asn Leu Ile 260 265 270 Ala Pro Met Tyr Ala Phe Ala Leu Ser Arg
Gly Phe Gly Ser Gly Ile 275 280 285 Ile Thr Ser Asn Ala Ser Met His
Glu Cys Glu Asn Leu Tyr Phe Gln 290 295 300 Gly Asn Thr Lys Cys Gln
Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu 305 310 315 320 Pro Tyr Gln
Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr 325 330 335 Val
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Thr Pro 340 345
350 Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
355 360 365 Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His
His Gln 370 375 380 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys
Ser Thr Gln Asn 385 390 395 400 Ala Ile Asn Gly Ile Thr Asn Lys Val
Asn Thr Val Ile Glu Lys Met 405 410 415 Asn Ile Gln Phe Thr Ala Val
Gly Lys Glu Phe Asn Lys Leu Glu Lys 420 425 430 Arg Met Glu Asn Leu
Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile 435 440 445 Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 465 470
475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe
Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val
Arg Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser
Lys Leu Asn Arg Glu 515 520 525 Lys Val Asp Gly Val Lys Leu Glu Ser
Met Gly Ile Tyr Gln Ile Leu 530 535 540 Ala Ile Tyr Ser Thr Val Ala
Ser Ser Leu Val Leu Leu Val Ser Leu 545 550 555 560 Gly Ala Ile Ser
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg 565 570 575 Ile Cys
Ile 30579PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Glu 35 40 45 Asn Leu Tyr Phe Gln
Gly Asn Leu Leu Glu Asp Ser His Asn Gly Lys 50 55 60 Leu Cys Arg
Leu Lys Gly Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn 65 70 75 80 Ile
Ala Gly Trp Leu Leu Gly Asn Pro Glu Cys Asp Pro Leu Leu Pro 85 90
95 Val Arg Ser Trp Ser Tyr Ile Val Glu Thr Pro Asn Ser Glu Asn Gly
100 105 110 Ile Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg
Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile
Phe Pro Lys Glu 130 135 140 Ser Ser Trp Pro Asn His Asn Thr Asn Gly
Val Thr Ala Ala Cys Ser 145 150 155 160 His Glu Gly Lys Ser Ser Phe
Tyr Arg Asn Leu Leu Trp Leu Thr Glu 165 170 175 Lys Glu Gly Ser Tyr
Pro Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys 180 185 190 Gly Lys Glu
Val Leu Val Leu Trp Gly Ile His His Pro Pro Asn Ser 195 200 205 Lys
Glu Gln Gln Asn Ile Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val 210 215
220 Val Thr Ser Asn Tyr Asn Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg
225 230 235 240 Pro Lys Val Arg Asp Gln Ala Gly Arg Met Asn Tyr Tyr
Trp Thr Leu 245 250 255 Leu Lys Pro Gly Asp Thr Ile Ile Phe Glu Ala
Asn Gly Asn Leu Ile 260 265 270 Ala Pro Met Tyr Ala Phe Ala Leu Ser
Arg Gly Phe Gly Ser Gly Ile 275 280 285 Ile Thr Ser Asn Ala Ser Met
His Glu Cys Glu Asn Leu Tyr Phe Gln 290 295 300 Ser Asn Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu 305 310 315 320 Pro Tyr
Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr 325 330 335
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Thr Pro 340
345 350 Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu 355 360 365 Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr
His His Gln 370 375 380 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile Asn Gly Ile Thr Asn Lys
Val Asn Thr Val Ile Glu Lys Met 405 410 415 Asn Ile Gln Phe Thr Ala
Val Gly Lys Glu Phe Asn Lys Leu Glu Lys 420 425 430 Arg Met Glu Asn
Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile 435 440 445 Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 450 455 460
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 465
470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys
Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
Val Arg Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn Arg Glu 515 520 525 Lys Val Asp Gly Val Lys Leu Glu
Ser Met Gly Ile Tyr Gln Ile Leu 530 535 540 Ala Ile Tyr Ser Thr Val
Ala Ser Ser Leu Val Leu Leu Val Ser Leu 545 550 555 560 Gly Ala Ile
Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg 565 570 575 Ile
Cys Ile 311701DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 31atgaaggcaa acctactggt
cctgttaagt gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa
caattcaacc gacactgttg acacagtact cgagaagaat 120gtgacagtga
cacactctgt taacctgctc gaagacagcc acaacggaaa actatgtaga
180ttaaaaggag agaatctgta cttccaggga aaatgtaaca tcgccggatg
gctcttggga 240aacccagaat gcgacccact gcttccagtg agatcatggt
cctacattgt agaaacacca 300aactctgaga atggaatatg ttatccagga
gatttcatcg actatgagga gctgagggag 360caattgagct cagtgtcatc
attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca
acacaaacgg agtaacggca gcatgctccc atgaggggaa aagcagtttt
480tacagaaatt tgctatggct gacggagaag gagggctcat acccaaagct
gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg
gtattcatca cccgcctaac 600agtaaggaac aacagaatat ctatcagaat
gaaaatgctt atgtctctgt agtgacttca 660aattataaca ggagatttac
cccggaaata gcagaaagac ccaaagtaag agatcaagct 720gggaggatga
actattactg gaccttgcta aaacccggag acacaataat atttgaggca
780aatggaaatc taatagcacc aatgtatgct ttcgcactga gtagaggctt
tggggagaat 840ctgtacttcc agtcaaacgc atcaatgcat gagtgtaaca
cgaagtgtca aacaccgctc 900ggagctataa acagcagtct cccttaccag
aatatacacc cagtcacaat aggagagtgc 960ccaaaatacg tcaggagtgc
caaattgagg atggttacag gactaaggaa cactccgtcc 1020attcaatcca
gaggtctatt tggagccatt gccggtttta ttgaaggggg atggactgga
1080atgatagatg gatggtatgg ttatcatcat cagaatgaac agggatcagg
ctatgcagcg 1140gatcaaaaaa gcacacaaaa tgccattaac gggattacaa
acaaggtgaa cactgttatc 1200gagaaaatgt acattcaatt cacagctgtg
ggtaaagaat tcaacaaatt agaaaaaagg 1260atggaaaatt taaataaaaa
agttgatgat ggatttctgt acatttggac atataatgca 1320gaattgttag
ttctactgga aaatgaaagg actctggatt tccatgactc aaatgtgaag
1380aatctgtatg agaaagtaaa aagccaatta aagaataatg ccaaagaaat
cggaaatgga 1440tgttttgagt tctaccacaa gtgtgacaat gaatgcatgg
aaagtgtaag aaatgggact 1500tatgattatc ccaaatattc agaagagtca
aagttgaaca gggaaaaggt agatggagtg 1560aaattggaat caatggggat
ctatcagatt ctggcgatct actcaactgt cgccagttca 1620ctggtgcttt
tggtctccct gggggcaatc agtttctgga tgtgttctaa tggatctttg
1680cagtgcagaa tatgcatctg a 1701321701DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
32atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tggggagaat 840ctgtacttcc agtcaaacgc atcaatgcat
gagtgtaaca cgaagtgtca aacaccgctc 900ggagctataa acagcagtct
cccttaccag aatatacacc cagtcacaat aggagagtgc 960ccaaaatacg
tcaggagtgc caaattgagg atggttacag gactaaggaa cactccgtcc
1020attcaatcca gaggtctatt tggagccatt gccggtttta ttgaaggggg
atggactgga 1080atgatagatg gatggtatgg ttatcatcat cagaatgaac
agggatcagg ctatgcagcg 1140gatcaaaaaa gcacacaaaa tgccattaac
gggattacaa acaaggtgaa cactgttatc 1200gagaaaatga acattcaatt
cacagctgtg ggttacgaat tcaacaaatt agaaaaaagg 1260atggaatact
taaataaaaa agttgatgat ggatttctgg acatttggac atataatgca
1320gaattgttag ttctactgga aaatgaaagg actctggatt tccatgactc
aaatgtgaag 1380aatctgtatg agaaagtaaa aagccaatta aagaataatg
ccaaagaaat cggaaatgga 1440tgttttgagt tctaccacaa gtgtgacaat
gaatgcatgg aaagtgtaag aaatgggact 1500tatgattatc ccaaatattc
agaagagtca aagttgaaca gggaaaaggt agatggagtg 1560aaattggaat
caatggggat ctatcagatt ctggcgatct actcaactgt cgccagttca
1620ctggtgcttt tggtctccct gggggcaatc agtttctgga tgtgttctaa
tggatctttg 1680cagtgcagaa tatgcatctg a 1701331701DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
33atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tggggagaat 840ctgtacttcc agtcaaacgc atcaatgcat
gagtgtaaca cgaagtgtca aacaccgctc 900ggagctataa acagcagtct
cccttaccag aatatacacc cagtcacaat aggagagtgc 960ccaaaatacg
tcaggagtgc caaattgagg atggttacag gactaaggaa cactccgtcc
1020attcaatcca gaggtctatt tggagccatt gccggtttta ttgaaggggg
atggactgga 1080atgatagatg gatggtatgg ttatcatcat cagaatgaac
agggatcagg ctatgcagcg 1140gatcaaaaaa gcacacaaaa tgccattaac
gggattacaa acaaggtgaa cactgttatc 1200gagaaaatgt acattcaatt
cacagctgtg ggttacgaat tcaacaaatt agaaaaaagg 1260atggaatact
taaataaaaa agttgatgat ggatttctgt acatttggac atataatgca
1320gaattgttag ttctactgga aaatgaaagg actctggatt tccatgactc
aaatgtgaag 1380aatctgtatg agaaagtaaa aagccaatta aagaataatg
ccaaagaaat cggaaatgga 1440tgttttgagt tctaccacaa gtgtgacaat
gaatgcatgg aaagtgtaag aaatgggact 1500tatgattatc ccaaatattc
agaagagtca aagttgaaca gggaaaaggt agatggagtg 1560aaattggaat
caatggggat ctatcagatt ctggcgatct actcaactgt cgccagttca
1620ctggtgcttt tggtctccct gggggcaatc agtttctgga tgtgttctaa
tggatctttg 1680cagtgcagaa tatgcatctg a 1701341719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
34atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcgaga atctgtactt ccagagcacc
tcaaacgcat caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgtac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctgtac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719351719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 35atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcgaga atctgtactt ccagagcacc tcaaacgcat caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac
agcagtctcc cttaccagaa tatacaccca 960gtcacaatag gagagtgccc
aaaatacgtc aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca
ctccgtccat tcaatccaga ggtctatttg gagccattgc cggttttatt
1080gaagggggat ggactggaat gatagatgga tggtatggtt atcatcatca
gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg
ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgaac
attcaattca cagctgtggg ttacgaattc 1260aacaaattag aaaaaaggat
ggaatactta aataaaaaag ttgatgatgg atttctggac 1320atttggacat
ataatgcaga attgttagtt ctactggaaa atgaaaggac tctggatttc
1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa
gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt
gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta tgattatccc
aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa
attggaatca atggggatct atcagattct ggcgatctac 1620tcaactgtcg
ccagttcact ggtgcttttg gtctccctgg gggcaatcag tttctggatg
1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719361719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 36atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcgaga atctgtactt ccagagcacc tcaaacgcat caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgtac attcaattca
cagctgtggg ttacgaattc 1260aacaaattag aaaaaaggat ggaatactta
aataaaaaag ttgatgatgg atttctgtac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719371719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
37atgaaggcta acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt gaacctgctg gaagatagcc acaacggcaa
gctgtgccgg 180ctgaagggcg agaacctgta ttttcaaggc aagtgcaata
tcgccggctg gctgctgggc 240aaccccgagt gtgatcctct gctgcctgtg
cggagctggt cctacatcgt ggaaaccccc 300aacagcgaga acggcatctg
ctaccccggc gacttcatcg actacgagga actgcgcgag 360cagctgagca
gcgtgtccag cttcgagaga ttcgagatct tccccaaaga gagcagctgg
420cccaaccaca acaccaacgg cgtgacagcc gcctgtagcc acgagggcaa
gagcagcttc 480tacagaaacc tgctgtggct gaccgagaaa gagggcagct
accccaagct gaagaacagc 540tacgtgaaca agaaaggcaa agaggtgctg
gtgctgtggg gcatccacca cccccccaac 600tctaaagagc agcagaacat
ctaccagaac gagaacgcct acgtgtccgt cgtgaccagc 660aactacaacc
ggcggttcac ccccgagatc gccgagaggc ctaaagtgcg ggatcaggcc
720ggcagaatga actactactg gaccctgctg aagcccggcg acaccatcat
cttcgaggcc 780aacggcaacc tgatcgcccc tatgtacgcc ttcgccctga
gcagaggctt cggcagcggc 840atcatcaccg aaaacctgta cttccaagga
tccaacgcca gcatgcacga gtgcaacacc 900aagtgccaga ccccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgtac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctgtac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719381719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 38atgaaggcta acctgctggt gctgctgagc
gccctggctg ccgctgatgc cgataccatc 60tgcatcggct accacgccaa caacagcacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt
gaacctgctg gaagatagcc acaacggcaa gctgtgccgg 180ctgaagggcg
agaacctgta ttttcaaggc aagtgcaata tcgccggctg gctgctgggc
240aaccccgagt gtgatcctct gctgcctgtg cggagctggt cctacatcgt
ggaaaccccc 300aacagcgaga acggcatctg ctaccccggc gacttcatcg
actacgagga actgcgcgag 360cagctgagca gcgtgtccag cttcgagaga
ttcgagatct tccccaaaga gagcagctgg 420cccaaccaca acaccaacgg
cgtgacagcc gcctgtagcc acgagggcaa gagcagcttc 480tacagaaacc
tgctgtggct gaccgagaaa gagggcagct accccaagct gaagaacagc
540tacgtgaaca agaaaggcaa agaggtgctg gtgctgtggg gcatccacca
cccccccaac 600tctaaagagc agcagaacat ctaccagaac gagaacgcct
acgtgtccgt cgtgaccagc 660aactacaacc ggcggttcac ccccgagatc
gccgagaggc ctaaagtgcg ggatcaggcc 720ggcagaatga actactactg
gaccctgctg aagcccggcg acaccatcat cttcgaggcc 780aacggcaacc
tgatcgcccc tatgtacgcc ttcgccctga gcagaggctt cggcagcggc
840atcatcaccg aaaacctgta cttccaagga tccaacgcca gcatgcacga
gtgcaacacc 900aagtgccaga ccccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgaac attcaattca
cagctgtggg ttacgaattc 1260aacaaattag aaaaaaggat ggaatactta
aataaaaaag ttgatgatgg atttctggac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719391719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
39atgaaggcta acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt gaacctgctg gaagatagcc acaacggcaa
gctgtgccgg 180ctgaagggcg agaacctgta ttttcaaggc aagtgcaata
tcgccggctg gctgctgggc 240aaccccgagt gtgatcctct gctgcctgtg
cggagctggt cctacatcgt ggaaaccccc 300aacagcgaga acggcatctg
ctaccccggc gacttcatcg actacgagga actgcgcgag 360cagctgagca
gcgtgtccag cttcgagaga ttcgagatct tccccaaaga gagcagctgg
420cccaaccaca acaccaacgg cgtgacagcc gcctgtagcc acgagggcaa
gagcagcttc 480tacagaaacc tgctgtggct gaccgagaaa gagggcagct
accccaagct gaagaacagc 540tacgtgaaca agaaaggcaa agaggtgctg
gtgctgtggg gcatccacca cccccccaac 600tctaaagagc agcagaacat
ctaccagaac gagaacgcct acgtgtccgt cgtgaccagc 660aactacaacc
ggcggttcac ccccgagatc gccgagaggc ctaaagtgcg ggatcaggcc
720ggcagaatga actactactg gaccctgctg aagcccggcg acaccatcat
cttcgaggcc 780aacggcaacc tgatcgcccc tatgtacgcc ttcgccctga
gcagaggctt cggcagcggc 840atcatcaccg aaaacctgta cttccaagga
tccaacgcca gcatgcacga gtgcaacacc 900aagtgccaga ccccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgtac attcaattca cagctgtggg ttacgaattc 1260aacaaattag
aaaaaaggat ggaatactta aataaaaaag ttgatgatgg atttctgtac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719401719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 40atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcacct caaacgcaga gaatctgtac ttccagagct caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgtac attcaattca
cagctgtggg taaagaattc 1260aacaaattag aaaaaaggat ggaaaattta
aataaaaaag ttgatgatgg atttctgtac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719411719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
41atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcaga gaatctgtac
ttccagagct caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg ttacgaattc 1260aacaaattag
aaaaaaggat ggaatactta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719421719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 42atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcacct caaacgcaga gaatctgtac ttccagagct caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgtac attcaattca
cagctgtggg ttacgaattc 1260aacaaattag aaaaaaggat ggaatactta
aataaaaaag ttgatgatgg atttctgtac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719431740DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
43atgaaggcta acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aagggaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgta cattcaattc 1260acagctgtgg
gtaaagaatt caacaaatta gaaaaaagga tggaaaattt aaataaaaaa
1320gttgatgatg gatttctgta catttggaca
tataatgcag aattgttagt tctactggaa 1380aatgaaagga ctctggattt
ccatgactca aatgtgaaga atctgtatga gaaagtaaaa 1440agccaattaa
agaataatgc caaagaaatc ggaaatggat gttttgagtt ctaccacaag
1500tgtgacaatg aatgcatgga aagtgtaaga aatgggactt atgattatcc
caaatattca 1560gaagagtcaa agttgaacag ggaaaaggta gatggagtga
aattggaatc aatggggatc 1620tatcagattc tggcgatcta ctcaactgtc
gccagttcac tggtgctttt ggtctccctg 1680ggggcaatca gtttctggat
gtgttctaat ggatctttgc agtgcagaat atgcatctga 1740441740DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
44atgaaggcta acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aagggaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgaa cattcaattc 1260acagctgtgg
gttacgaatt caacaaatta gaaaaaagga tggaatactt aaataaaaaa
1320gttgatgatg gatttctgga catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtga aattggaatc aatggggatc
1620tatcagattc tggcgatcta ctcaactgtc gccagttcac tggtgctttt
ggtctccctg 1680ggggcaatca gtttctggat gtgttctaat ggatctttgc
agtgcagaat atgcatctga 1740451740DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 45atgaaggcta
acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc 60tgcatcggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aagggaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgta cattcaattc 1260acagctgtgg
gttacgaatt caacaaatta gaaaaaagga tggaatactt aaataaaaaa
1320gttgatgatg gatttctgta catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtga aattggaatc aatggggatc
1620tatcagattc tggcgatcta ctcaactgtc gccagttcac tggtgctttt
ggtctccctg 1680ggggcaatca gtttctggat gtgttctaat ggatctttgc
agtgcagaat atgcatctga 1740461740DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 46atgaaggcta
acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc 60tgcatcggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aaagcaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgta cattcaattc 1260acagctgtgg
gtaaagaatt caacaaatta gaaaaaagga tggaaaattt aaataaaaaa
1320gttgatgatg gatttctgta catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtga aattggaatc aatggggatc
1620tatcagattc tggcgatcta ctcaactgtc gccagttcac tggtgctttt
ggtctccctg 1680ggggcaatca gtttctggat gtgttctaat ggatctttgc
agtgcagaat atgcatctga 1740471740DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 47atgaaggcta
acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc 60tgcatcggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aaagcaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgaa cattcaattc 1260acagctgtgg
gttacgaatt caacaaatta gaaaaaagga tggaatactt aaataaaaaa
1320gttgatgatg gatttctgga catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtga aattggaatc aatggggatc
1620tatcagattc tggcgatcta ctcaactgtc gccagttcac tggtgctttt
ggtctccctg 1680ggggcaatca gtttctggat gtgttctaat ggatctttgc
agtgcagaat atgcatctga 1740481740DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 48atgaaggcta
acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc 60tgcatcggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aaagcaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgta cattcaattc 1260acagctgtgg
gttacgaatt caacaaatta gaaaaaagga tggaatactt aaataaaaaa
1320gttgatgatg gatttctgta catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtga aattggaatc aatggggatc
1620tatcagattc tggcgatcta ctcaactgtc gccagttcac tggtgctttt
ggtctccctg 1680ggggcaatca gtttctggat gtgttctaat ggatctttgc
agtgcagaat atgcatctga 1740491719DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 49atgaaggcaa
acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata 60tgtataggct
accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggaa tagccccact acaattgggg aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcatc aatgcatgag
tgtgagaatc tgtacttcca gggaaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719501719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 50atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggaa
tagccccact acaattgggg aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcacct caaacgcatc aatgcatgag tgtgagaatc tgtacttcca
gagcaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgaac attcaattca
cagctgtggg taaagaattc 1260aacaaattag aaaaaaggat ggaaaattta
aataaaaaag ttgatgatgg atttctggac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719511698DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
51atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcatc aatgcatgag
tgtaacacga agtgtcaaac accgctcgga 900gctataaaca gcagtctccc
ttaccagaat atacacccag tcacaatagg agagtgccca 960aaatacgtca
ggagtgccaa attgaggatg gttacaggac taaggaacac tccgtccatt
1020caatccagag gtctatttgg agccattgcc ggttttattg aagggggatg
gactggaatg 1080atagatggat ggtatggtta tcatcatcag aatgaacagg
gatcaggcta tgcagcggat 1140caaaaaagca cacaaaatgc cattaacggg
attacaaaca aggtgaacac tgttatcgag 1200aaaatgaaca ttcaattcac
agctgtgggt aaagaattca acaaattaga aaaaaggatg 1260gaaaatttaa
ataaaaaagt tgatgatgga tttctggaca tttggacata taatgcagaa
1320ttgttagttc tactggaaaa tgaaaggact ctggatttcc atgactcaaa
tgtgaagaat 1380ctgtatgaga aagtaaaaag ccaattaaag aataatgcca
aagaaatcgg aaatggatgt 1440tttgagttct accacaagtg tgacaatgaa
tgcatggaaa gtgtaagaaa tgggacttat 1500gattatccca aatattcaga
agagtcaaag ttgaacaggg aaaaggtaga tggagtgaaa 1560ttggaatcaa
tggggatcta tcagattctg gcgatctact caactgtcgc cagttcactg
1620gtgcttttgg tctccctggg ggcaatcagt ttctggatgt gttctaatgg
atctttgcag 1680tgcagaatat gcatctga 1698521719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
52atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt tgagaacctg tacttccagg gaaacctgct
cgaagacagc 180cacaacggaa aactatgtag attaaaagga atagccccac
tacaattggg gaaatgtaac 240atcgccggat ggctcttggg aaacccagaa
tgcgacccac tgcttccagt gagatcatgg 300tcctacattg tagaaacacc
aaactctgag aatggaatat gttatccagg agatttcatc 360gactatgagg
agctgaggga gcaattgagc tcagtgtcat cattcgaaag attcgaaata
420tttcccaaag aaagctcatg gcccaaccac aacacaaacg gagtaacggc
agcatgctcc 480catgagggga aaagcagttt ttacagaaat ttgctatggc
tgacggagaa ggagggctca 540tacccaaagc tgaaaaattc ttatgtgaac
aaaaaaggga aagaagtcct tgtactgtgg 600ggtattcatc acccgcctaa
cagtaaggaa caacagaata tctatcagaa tgaaaatgct 660tatgtctctg
tagtgacttc aaattataac aggagattta ccccggaaat agcagaaaga
720cccaaagtaa gagatcaagc tgggaggatg aactattact ggaccttgct
aaaacccgga 780gacacaataa tatttgaggc aaatggaaat ctaatagcac
caatgtatgc tttcgcactg 840agtagaggct ttgggtccgg catcatcacc
tcaaacgcat caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719531701DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 53atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggaa
tagccccact acaattgggg aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tggggagaat
840ctgtacttcc agtcaaacgc atcaatgcat gagtgtaaca cgaagtgtca
aacaccgctc 900ggagctataa acagcagtct cccttaccag aatatacacc
cagtcacaat aggagagtgc 960ccaaaatacg tcaggagtgc caaattgagg
atggttacag gactaaggaa cactccgtcc 1020attcaatcca gaggtctatt
tggagccatt gccggtttta ttgaaggggg atggactgga 1080atgatagatg
gatggtatgg ttatcatcat cagaatgaac agggatcagg ctatgcagcg
1140gatcaaaaaa gcacacaaaa tgccattaac gggattacaa acaaggtgaa
cactgttatc 1200gagaaaatga acattcaatt cacagctgtg ggtaaagaat
tcaacaaatt agaaaaaagg 1260atggaaaatt taaataaaaa agttgatgat
ggatttctgg acatttggac atataatgca 1320gaattgttag ttctactgga
aaatgaaagg actctggatt tccatgactc aaatgtgaag 1380aatctgtatg
agaaagtaaa aagccaatta aagaataatg ccaaagaaat cggaaatgga
1440tgttttgagt tctaccacaa gtgtgacaat gaatgcatgg aaagtgtaag
aaatgggact 1500tatgattatc ccaaatattc agaagagtca aagttgaaca
gggaaaaggt agatggagtg 1560aaattggaat caatggggat ctatcagatt
ctggcgatct actcaactgt cgccagttca 1620ctggtgcttt tggtctccct
gggggcaatc agtttctgga tgtgttctaa tggatctttg 1680cagtgcagaa
tatgcatctg a 1701541719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 54atgaaggcaa
acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata 60tgtataggct
accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggaa tagccccact acaattgggg aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcgaga atctgtactt ccagagcacc
tcaaacgcat caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719551719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 55atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggaa
tagccccact acaattgggg aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcaccg agaatctgta cttccaggga tcaaacgcat caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgaac attcaattca
cagctgtggg taaagaattc 1260aacaaattag aaaaaaggat ggaaaattta
aataaaaaag ttgatgatgg atttctggac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgaa attggaatca
atggggatct atcagattct ggcgatctac 1620tcaactgtcg ccagttcact
ggtgcttttg gtctccctgg gggcaatcag tttctggatg 1680tgttctaatg
gatctttgca gtgcagaata tgcatctga 1719561719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
56atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggaa tagccccact acaattgggg aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcaga gaatctgtac
ttccagagct caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgaa attggaatca atggggatct atcagattct ggcgatctac
1620tcaactgtcg ccagttcact ggtgcttttg gtctccctgg gggcaatcag
tttctggatg 1680tgttctaatg gatctttgca gtgcagaata tgcatctga
1719571695DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 57atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tggggagaat
840ctgtacttcc agtcaaacgc atcaatgcat gagtgtaaca cgaagtgtca
aacaccgctc 900ggagctataa acagcagtct cccttaccag aatatacacc
cagtcacaat aggagagtgc 960ccaaaatacg tcaggagtgc caaattgagg
atggttacag gactaaggaa cactccgtcc 1020attcaatcca gaggtctatt
tggagccatt gccggtttta ttgaaggggg atggactgga 1080atgatagatg
gatggtatgg ttatcatcat cagaatgaac agggatcagg ctatgcagcg
1140gatcaaaaaa gcacacaaaa tgccattaac gggattacaa acaaggtgaa
cactgttatc 1200gagaaaatga acattcaatt cacagctgtg ggtaaagaat
tcaacaaatt agaaaaaagg 1260atggaaaatt taaataaaaa agttgatgat
ggatttctgg acatttggac atataatgca 1320gaattgttag ttctactgga
aaatgaaagg actctggatt tccatgactc aaatgtgaag 1380aatctgtatg
agaaagtaaa aagccaatta aagaataatg ccaaagaaat cggaaatgga
1440tgttttgagt tctaccacaa gtgtgacaat gaatgcatgg aaagtgtaag
aaatgggact 1500tatgattatc ccaaatattc agaagagtca aagttgaaca
gggaaaaggt agatggagtg 1560cgttctctgg ttccgcgtgg ttctccgggt
tctggttaca tcccggaagc tccgcgtgac 1620ggtcaggctt acgttcgtaa
agacggtgaa tgggttctgc tgtctacctt cctgcaccac 1680caccaccacc actga
1695581713DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 58atgaaggcaa acctactggt cctgttaagt
gcacttgcag ctgcagatgc agacacaata 60tgtataggct accatgcgaa caattcaacc
gacactgttg acacagtact cgagaagaat 120gtgacagtga cacactctgt
taacctgctc gaagacagcc acaacggaaa actatgtaga 180ttaaaaggag
agaatctgta cttccaggga aaatgtaaca tcgccggatg gctcttggga
240aacccagaat gcgacccact gcttccagtg agatcatggt cctacattgt
agaaacacca 300aactctgaga atggaatatg ttatccagga gatttcatcg
actatgagga gctgagggag 360caattgagct cagtgtcatc attcgaaaga
ttcgaaatat ttcccaaaga aagctcatgg 420cccaaccaca acacaaacgg
agtaacggca gcatgctccc atgaggggaa aagcagtttt 480tacagaaatt
tgctatggct gacggagaag gagggctcat acccaaagct gaaaaattct
540tatgtgaaca aaaaagggaa agaagtcctt gtactgtggg gtattcatca
cccgcctaac 600agtaaggaac aacagaatat ctatcagaat gaaaatgctt
atgtctctgt agtgacttca 660aattataaca ggagatttac cccggaaata
gcagaaagac ccaaagtaag agatcaagct 720gggaggatga actattactg
gaccttgcta aaacccggag acacaataat atttgaggca 780aatggaaatc
taatagcacc aatgtatgct ttcgcactga gtagaggctt tgggtccggc
840atcatcgaga atctgtactt ccagagcacc tcaaacgcat caatgcatga
gtgtaacacg 900aagtgtcaaa caccgctcgg agctataaac agcagtctcc
cttaccagaa tatacaccca 960gtcacaatag gagagtgccc aaaatacgtc
aggagtgcca aattgaggat ggttacagga 1020ctaaggaaca ctccgtccat
tcaatccaga ggtctatttg gagccattgc cggttttatt 1080gaagggggat
ggactggaat gatagatgga tggtatggtt atcatcatca gaatgaacag
1140ggatcaggct atgcagcgga tcaaaaaagc acacaaaatg ccattaacgg
gattacaaac 1200aaggtgaaca ctgttatcga gaaaatgaac attcaattca
cagctgtggg taaagaattc 1260aacaaattag aaaaaaggat ggaaaattta
aataaaaaag ttgatgatgg atttctggac 1320atttggacat ataatgcaga
attgttagtt ctactggaaa atgaaaggac tctggatttc 1380catgactcaa
atgtgaagaa tctgtatgag aaagtaaaaa gccaattaaa gaataatgcc
1440aaagaaatcg gaaatggatg ttttgagttc taccacaagt gtgacaatga
atgcatggaa 1500agtgtaagaa atgggactta tgattatccc aaatattcag
aagagtcaaa gttgaacagg 1560gaaaaggtag atggagtgcg ttctctggtt
ccgcgtggtt ctccgggttc tggttacatc 1620ccggaagctc cgcgtgacgg
tcaggcttac gttcgtaaag acggtgaatg ggttctgctg 1680tctaccttcc
tgcaccacca ccaccaccac tga 1713591713DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
59atgaaggcaa acctactggt cctgttaagt gcacttgcag ctgcagatgc agacacaata
60tgtataggct accatgcgaa caattcaacc gacactgttg acacagtact cgagaagaat
120gtgacagtga cacactctgt taacctgctc gaagacagcc acaacggaaa
actatgtaga 180ttaaaaggag agaatctgta cttccaggga aaatgtaaca
tcgccggatg gctcttggga 240aacccagaat gcgacccact gcttccagtg
agatcatggt cctacattgt agaaacacca 300aactctgaga atggaatatg
ttatccagga gatttcatcg actatgagga gctgagggag 360caattgagct
cagtgtcatc attcgaaaga ttcgaaatat ttcccaaaga aagctcatgg
420cccaaccaca acacaaacgg agtaacggca gcatgctccc atgaggggaa
aagcagtttt 480tacagaaatt tgctatggct gacggagaag gagggctcat
acccaaagct gaaaaattct 540tatgtgaaca aaaaagggaa agaagtcctt
gtactgtggg gtattcatca cccgcctaac 600agtaaggaac aacagaatat
ctatcagaat gaaaatgctt atgtctctgt agtgacttca 660aattataaca
ggagatttac cccggaaata gcagaaagac ccaaagtaag agatcaagct
720gggaggatga actattactg gaccttgcta aaacccggag acacaataat
atttgaggca 780aatggaaatc taatagcacc aatgtatgct ttcgcactga
gtagaggctt tgggtccggc 840atcatcacct caaacgcaga gaatctgtac
ttccagagct caatgcatga gtgtaacacg 900aagtgtcaaa caccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgcg ttctctggtt ccgcgtggtt ctccgggttc tggttacatc
1620ccggaagctc cgcgtgacgg tcaggcttac gttcgtaaag acggtgaatg
ggttctgctg 1680tctaccttcc tgcaccacca ccaccaccac tga
1713601734DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 60atgaaggcta acctgctggt gctgctgagc
gccctggctg ccgctgatgc cgataccatc 60tgcatcggct accacgccaa caacagcacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt
ggaaaacctg tattttcaag gcaatctgct ggaagatagc 180cacaacggca
agctgtgccg gctgaaggga atcgcccctc tgcagctggg caagtgcaat
240atcgccggct ggctgctggg caaccccgag tgtgatcctc tgctgcctgt
gcggagctgg 300tcctacatcg tggaaacccc caacagcgag aacggcatct
gctaccccgg cgacttcatc
360gactacgagg aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag
attcgagatc 420ttccccaaag agagcagctg gcccaaccac aacaccaacg
gcgtgacagc cgcctgtagc 480cacgagggca agagcagctt ctaccggaac
ctgctgtggc tgaccgagaa agagggcagc 540taccccaagc tgaagaacag
ctacgtgaac aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc
acccccccaa ctctaaagag cagcagaaca tctaccagaa cgagaacgcc
660tacgtgtccg tcgtgaccag caactacaac cggcggttca cccccgagat
cgccgagagg 720cctaaagtgc gggatcaggc cggcagaatg aactactact
ggaccctgct gaagcccggc 780gacaccatca tcttcgaggc caacggcaac
ctgatcgccc ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg
catcatcacc agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc
aagggaacac caagtgccag accccgctcg gagctataaa cagcagtctc
960ccttaccaga atatacaccc agtcacaata ggagagtgcc caaaatacgt
caggagtgcc 1020aaattgagga tggttacagg actaaggaac actccgtcca
ttcaatccag aggtctattt 1080ggagccattg ccggttttat tgaaggggga
tggactggaa tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca
gggatcaggc tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg
ggattacaaa caaggtgaac actgttatcg agaaaatgaa cattcaattc
1260acagctgtgg gtaaagaatt caacaaatta gaaaaaagga tggaaaattt
aaataaaaaa 1320gttgatgatg gatttctgga catttggaca tataatgcag
aattgttagt tctactggaa 1380aatgaaagga ctctggattt ccatgactca
aatgtgaaga atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc
caaagaaatc ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg
aatgcatgga aagtgtaaga aatgggactt atgattatcc caaatattca
1560gaagagtcaa agttgaacag ggaaaaggta gatggagtgc gttctctggt
tccgcgtggt 1620tctccgggtt ctggttacat cccggaagct ccgcgtgacg
gtcaggctta cgttcgtaaa 1680gacggtgaat gggttctgct gtctaccttc
ctgcaccacc accaccacca ctga 1734611734DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
61atgaaggcta acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt ggaaaacctg tattttcaag gcaatctgct
ggaagatagc 180cacaacggca agctgtgccg gctgaaggga atcgcccctc
tgcagctggg caagtgcaat 240atcgccggct ggctgctggg caaccccgag
tgtgatcctc tgctgcctgt gcggagctgg 300tcctacatcg tggaaacccc
caacagcgag aacggcatct gctaccccgg cgacttcatc 360gactacgagg
aactgcgcga gcagctgagc agcgtgtcca gcttcgaaag attcgagatc
420ttccccaaag agagcagctg gcccaaccac aacaccaacg gcgtgacagc
cgcctgtagc 480cacgagggca agagcagctt ctaccggaac ctgctgtggc
tgaccgagaa agagggcagc 540taccccaagc tgaagaacag ctacgtgaac
aagaaaggca aagaggtgct ggtgctgtgg 600ggcatccacc acccccccaa
ctctaaagag cagcagaaca tctaccagaa cgagaacgcc 660tacgtgtccg
tcgtgaccag caactacaac cggcggttca cccccgagat cgccgagagg
720cctaaagtgc gggatcaggc cggcagaatg aactactact ggaccctgct
gaagcccggc 780gacaccatca tcttcgaggc caacggcaac ctgatcgccc
ctatgtacgc cttcgccctg 840agcagaggct tcggcagcgg catcatcacc
agcaacgcca gcatgcacga gtgcgagaac 900ctgtacttcc aaagcaacac
caagtgccag accccgctcg gagctataaa cagcagtctc 960ccttaccaga
atatacaccc agtcacaata ggagagtgcc caaaatacgt caggagtgcc
1020aaattgagga tggttacagg actaaggaac actccgtcca ttcaatccag
aggtctattt 1080ggagccattg ccggttttat tgaaggggga tggactggaa
tgatagatgg atggtatggt 1140tatcatcatc agaatgaaca gggatcaggc
tatgcagcgg atcaaaaaag cacacaaaat 1200gccattaacg ggattacaaa
caaggtgaac actgttatcg agaaaatgaa cattcaattc 1260acagctgtgg
gtaaagaatt caacaaatta gaaaaaagga tggaaaattt aaataaaaaa
1320gttgatgatg gatttctgga catttggaca tataatgcag aattgttagt
tctactggaa 1380aatgaaagga ctctggattt ccatgactca aatgtgaaga
atctgtatga gaaagtaaaa 1440agccaattaa agaataatgc caaagaaatc
ggaaatggat gttttgagtt ctaccacaag 1500tgtgacaatg aatgcatgga
aagtgtaaga aatgggactt atgattatcc caaatattca 1560gaagagtcaa
agttgaacag ggaaaaggta gatggagtgc gttctctggt tccgcgtggt
1620tctccgggtt ctggttacat cccggaagct ccgcgtgacg gtcaggctta
cgttcgtaaa 1680gacggtgaat gggttctgct gtctaccttc ctgcaccacc
accaccacca ctga 1734621713DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 62atgaaggcta
acctgctggt gctgctgagc gccctggctg ccgctgatgc cgataccatc 60tgcatcggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt gaacctgctg gaagatagcc acaacggcaa
gctgtgccgg 180ctgaagggcg agaacctgta ttttcaaggc aagtgcaata
tcgccggctg gctgctgggc 240aaccccgagt gtgatcctct gctgcctgtg
cggagctggt cctacatcgt ggaaaccccc 300aacagcgaga acggcatctg
ctaccccggc gacttcatcg actacgagga actgcgcgag 360cagctgagca
gcgtgtccag cttcgagaga ttcgagatct tccccaaaga gagcagctgg
420cccaaccaca acaccaacgg cgtgacagcc gcctgtagcc acgagggcaa
gagcagcttc 480tacagaaacc tgctgtggct gaccgagaaa gagggcagct
accccaagct gaagaacagc 540tacgtgaaca agaaaggcaa agaggtgctg
gtgctgtggg gcatccacca cccccccaac 600tctaaagagc agcagaacat
ctaccagaac gagaacgcct acgtgtccgt cgtgaccagc 660aactacaacc
ggcggttcac ccccgagatc gccgagaggc ctaaagtgcg ggatcaggcc
720ggcagaatga actactactg gaccctgctg aagcccggcg acaccatcat
cttcgaggcc 780aacggcaacc tgatcgcccc tatgtacgcc ttcgccctga
gcagaggctt cggcagcggc 840atcatcaccg aaaacctgta cttccaagga
tccaacgcca gcatgcacga gtgcaacacc 900aagtgccaga ccccgctcgg
agctataaac agcagtctcc cttaccagaa tatacaccca 960gtcacaatag
gagagtgccc aaaatacgtc aggagtgcca aattgaggat ggttacagga
1020ctaaggaaca ctccgtccat tcaatccaga ggtctatttg gagccattgc
cggttttatt 1080gaagggggat ggactggaat gatagatgga tggtatggtt
atcatcatca gaatgaacag 1140ggatcaggct atgcagcgga tcaaaaaagc
acacaaaatg ccattaacgg gattacaaac 1200aaggtgaaca ctgttatcga
gaaaatgaac attcaattca cagctgtggg taaagaattc 1260aacaaattag
aaaaaaggat ggaaaattta aataaaaaag ttgatgatgg atttctggac
1320atttggacat ataatgcaga attgttagtt ctactggaaa atgaaaggac
tctggatttc 1380catgactcaa atgtgaagaa tctgtatgag aaagtaaaaa
gccaattaaa gaataatgcc 1440aaagaaatcg gaaatggatg ttttgagttc
taccacaagt gtgacaatga atgcatggaa 1500agtgtaagaa atgggactta
tgattatccc aaatattcag aagagtcaaa gttgaacagg 1560gaaaaggtag
atggagtgcg ttctctggtt ccgcgtggtt ctccgggttc tggttacatc
1620ccggaagctc cgcgtgacgg tcaggcttac gttcgtaaag acggtgaatg
ggttctgctg 1680tctaccttcc tgcaccacca ccaccaccac tga
1713631698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 63atgaaggcca acctgctggt gctgctgtct
gccctggctg ccgccgatgc cgataccatc 60tgtatcggct accacgccaa caacagcacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt
gaacctgctg gaagatagcc acaacggcaa gctgtgccgg 180ctgaagggaa
tcgcccctct gcagctgggc aagtgcaata tcgccggctg gctgctgggc
240aaccccgagt gtgatcctct gctgcctgtg cggagctggt cctacatcgt
ggaaaccccc 300aacagcgaga acggcatctg ctaccccggc gacttcatcg
actacgagga actgcgcgag 360cagctgagca gcgtgtccag cttcgagaga
ttcgagatct tccccaaaga gagcagctgg 420cccaaccaca acaccaacgg
cgtgacagcc gcctgtagcc acgagggcaa gagcagcttc 480tacagaaacc
tgctgtggct gaccgagaaa gagggcagct accccaagct gaagaacagc
540tacgtgaaca agaaaggcaa agaggtgctg gtgctgtggg gcatccacca
cccccccaac 600tctaaagagc agcagaacat ctaccagaac gagaacgcct
acgtgtccgt cgtgaccagc 660aactacaacc ggcggttcac ccccgagatc
gccgagaggc ctaaagtgcg ggatcaggcc 720ggcagaatga actactactg
gaccctgctg aagcccggcg acaccatcat cttcgaggcc 780aacggcaacc
tgatcgcccc tatgtacgcc ttcgccctga gcagaggctt cggcagcggc
840atcatcacca gcaacgccag catgcacgag tgcaacacca agtgccagac
ccccctgggc 900gccatcaata gcagcctgcc ctaccagaat atccaccccg
tgaccatcgg cgagtgcccc 960aaatacgtgc ggagcgccaa gctgcggatg
gtcaccggcc tgagaaacac ccccagcatc 1020cagagcaggg gcctgtttgg
agccattgcc ggctttatcg agggcggctg gaccggcatg 1080atcgacgggt
ggtacggcta tcaccaccag aatgagcagg gcagcggcta cgccgccgat
1140cagaagtcta cccagaacgc catcaacggc atcaccaaca aagtgaacac
cgtgatcgag 1200aagatgaaca tccagttcac cgccgtgggc aaagagttca
acaagctgga aaaacggatg 1260gaaaacctga acaaaaaggt ggacgacggc
ttcctggaca tctggaccta caacgccgag 1320ctgctggtgc tgctggaaaa
cgagcggacc ctggacttcc acgacagcaa cgtgaagaac 1380ctgtacgaga
aagtgaagtc ccagctgaaa aacaacgcca aagagatcgg caacggctgc
1440ttcgagttct accacaagtg cgacaacgag tgcatggaaa gcgtgcggaa
cggcacctac 1500gactacccta agtacagcga ggaaagcaag ctgaaccgcg
aaaaagtgga cggcgtgaag 1560ctggaatcca tgggcatcta tcagatcctg
gccatctaca gcaccgtggc cagcagcctg 1620gtgctgctgg tgtctctggg
cgccatctca ttttggatgt gcagcaacgg ctccctgcag 1680tgccggatct gcatctga
1698641698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 64atgaaggcca acctgctggt gctgctgtct
gccctggctg ccgctgatgc cgacaccatc 60tgtatcggct accacgccaa caactccacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccactccgt
gaatctgctg gaagattccc acaacggcaa gctgtgccgg 180ctgaagggaa
tcgcccctct gcagctgggc aagtgcaata tcgctggctg gctgctgggc
240aaccccgagt gtgatcctct gctgcctgtg cggtcctggt cctacatcgt
ggaaaccccc 300aactccgaga acggcatctg ctaccccggc gacttcatcg
actacgagga actgcgcgag 360cagctgtcct ccgtgtccag cttcgagaga
ttcgagatct tccccaaaga gtcctcctgg 420cccaaccaca acaccaacgg
cgtgaccgcc gcctgttctc acgagggcaa gagcagcttc 480taccggaacc
tgctgtggct gaccgagaaa gagggctcct accccaagct gaagaactcc
540tacgtgaaca agaaaggcaa agaggtgctg gtgctgtggg gcatccacca
cccccctaac 600tccaaagagc agcagaacat ctaccagaac gagaatgcct
acgtgtccgt cgtgacctcc 660aactacaacc ggcggttcac ccccgagatc
gccgagaggc ctaaagtgcg ggatcaggcc 720ggcagaatga actactactg
gaccctgctg aagcccggcg ataccatcat cttcgaggcc 780aacggcaacc
tgatcgcccc tatgtacgcc ttcgccctgt ccagaggctt cggctccggc
840atcatcacct ccaacgcctc catgcacgag tgcaacacca agtgccagac
ccccctgggc 900gccatcaaca gctccctgcc ttaccagaac atccaccccg
tgaccatcgg cgagtgcccc 960aaatatgtgc ggagcgccaa gctgcggatg
gtcaccggcc tgagaaacac cccctccatc 1020cagtctagag gcctgttcgg
cgctatcgcc ggctttatcg agggcggctg gaccggcatg 1080atcgacgggt
ggtacggcta ccatcaccag aatgagcagg gctccggcta cgccgccgac
1140cagaagtcta cccagaacgc catcaatggc atcaccaaca aagtgaacac
cgtgatcgag 1200aagatgaaca tccagttcac cgccgtgggc aaagagttca
acaagctgga aaaacggatg 1260gaaaacctga acaaaaaggt ggacgacggc
ttcctggaca tctggaccta caacgccgag 1320ctgctggtgc tgctggaaaa
cgagcggacc ctggacttcc acgactccaa cgtgaagaac 1380ctgtacgaga
aagtgaagtc ccagctgaaa aacaacgcca aagagatcgg caacggctgc
1440ttcgagttct accacaagtg cgacaacgag tgcatggaat ccgtgcggaa
cggcacctac 1500gactacccta agtactccga ggaaagcaag ctgaaccgcg
aaaaagtgga cggcgtgaag 1560ctggaatcca tgggcatcta tcagatcctg
gccatctact ccaccgtggc ctccagcctg 1620gtgctgctgg tgtctctggg
cgccatctct ttctggatgt gctccaacgg ctctctgcag 1680tgccggatct gcatctga
1698651698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 65atgaaggcta accttctggt gcttctgtct
gctcttgctg ctgctgatgc tgataccatc 60tgcattggtt accacgctaa caacagcacc
gatactgtgg ataccgtgct tgagaagaat 120gtgaccgtga cccactctgt
gaacctgctt gaggattctc acaacggtaa gctgtgcagg 180cttaagggta
ttgctccact gcagcttggt aagtgcaata tcgctggttg gcttctgggt
240aaccctgagt gtgatccttt gcttcctgtg aggtcctggt cctacattgt
ggaaactcct 300aacagcgaga acggtatctg ctaccctggt gatttcatcg
attacgagga actgagagag 360cagctgtcca gcgtttcatc ttttgagagg
ttcgagatct tccctaaaga gtcctcttgg 420cctaaccaca acactaacgg
tgttaccgct gcttgctctc acgagggtaa gagcagcttc 480tataggaacc
ttctttggct gaccgagaaa gagggaagct accctaagct gaagaacagc
540tacgtgaaca agaagggtaa agaggttctg gttctttggg gtatccacca
ccctccaaac 600tctaaagaac agcagaacat ctaccagaac gagaacgctt
acgtgagcgt ggtgaccagc 660aattacaaca gaaggttcac ccctgagatc
gctgagaggc ctaaggttag agatcaggct 720ggtaggatga actactactg
gacccttctg aagcctggtg ataccattat cttcgaggct 780aacggtaacc
tgatcgctcc tatgtacgct ttcgctctgt ctaggggttt cggttctggt
840atcatcacca gcaacgcttc tatgcacgag tgcaatacca agtgccagac
tcctcttggt 900gctatcaaca gctctctgcc ttaccagaac attcaccctg
tgaccattgg tgagtgccct 960aagtatgtga ggtctgctaa gttgaggatg
gtgaccggtc ttaggaacac cccttctatc 1020cagtctaggg gacttttcgg
agctatcgct ggattcattg agggaggttg gaccggtatg 1080atcgatggtt
ggtacggtta ccaccatcag aacgaacagg gttctggata cgctgctgat
1140caaaagtcta cccagaacgc tatcaacgga atcaccaaca aggtgaacac
cgtgatcgag 1200aagatgaaca tccagttcac cgctgtggga aaagagttca
acaagttgga gaagaggatg 1260gaaaacctta acaagaaagt ggatgatggt
ttcctggata tctggaccta caacgctgag 1320ctgcttgtgc ttcttgagaa
cgagaggacc ctggatttcc acgatagcaa cgtgaagaac 1380ctgtacgaga
aggtgaagtc ccagcttaag aacaacgcta aagagatcgg taacggttgc
1440ttcgagttct accacaagtg cgataacgag tgcatggaaa gcgtgaggaa
cggaacctac 1500gattacccta agtacagcga agagtccaag ctgaacaggg
aaaaagttga tggtgtgaag 1560cttgagagca tgggaatcta tcagatcctg
gctatctaca gcaccgtggc ttctagcctt 1620gtgcttttgg ttagcctggg
tgctatctcc ttctggatgt gctctaacgg ttctctgcag 1680tgcaggatct gcatctag
1698661698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 66atgaaggcta acttgttggt tttgttgtcc
gctttggctg ctgctgatgc tgacactatc 60tgtattggtt accacgctaa caactccact
gacactgttg acacagtttt ggagaagaac 120gttactgtta ctcactccgt
taatttgttg gaggactccc acaacggaaa gttgtgtaga 180ttgaagggta
tcgctccatt gcagttggga aagtgtaaca ttgctggttg gttgttgggt
240aacccagagt gtgatccttt gttgccagtt agatcctggt cctacatcgt
tgagactcca 300aactccgaga acggtatctg ttacccaggt gacttcatcg
actacgaaga gttgagagag 360cagttgtcct ccgtttcctc attcgagaga
ttcgagatct tcccaaaaga gtcctcttgg 420ccaaaccaca acactaacgg
tgttactgct gcttgttccc acgagggaaa gtcctcattc 480tacagaaact
tgttgtggtt gactgagaaa gagggttcct acccaaagtt gaagaactcc
540tacgttaaca agaagggtaa agaggttttg gttttgtggg gtatccacca
cccaccaaac 600tccaaagagc aacagaacat ctaccagaac gagaacgctt
acgtttctgt tgttacttcc 660aactacaaca gaagattcac tccagagatc
gctgaaagac caaaggttag agatcaggct 720ggtagaatga actactactg
gactttgttg aagccaggtg acacaatcat cttcgaggct 780aacggtaact
tgatcgctcc aatgtacgct ttcgctttgt ccagaggttt cggttccggt
840atcatcactt ctaacgcttc catgcacgag tgtaacacta agtgtcagac
tccattgggt 900gctatcaact cctccttgcc ataccaaaac atccacccag
ttactatcgg tgagtgtcct 960aagtacgtta gatccgctaa gttgagaatg
gttactggtt tgagaaacac tccatccatc 1020cagtctagag gtttgttcgg
tgctatcgct ggtttcattg aaggtggttg gactggtatg 1080atcgacggtt
ggtacggtta ccaccaccaa aacgaacaag gttctggtta cgctgctgac
1140caaaagtcca ctcagaacgc tatcaacggt atcactaaca aggttaacac
tgttatcgag 1200aagatgaaca tccagttcac tgctgttggt aaagagttca
acaagttgga aaagagaatg 1260gaaaacttga acaagaaagt tgacgacggt
ttcttggaca tctggactta caacgctgag 1320ttgttggttt tgttggagaa
cgagagaact ttggacttcc acgactccaa cgttaagaac 1380ttgtacgaga
aggttaagtc ccaattgaag aacaacgcta aagagatcgg taacggttgt
1440ttcgagttct accacaagtg tgacaacgag tgtatggaat ccgttagaaa
cggtacttac 1500gactacccta agtactccga agagtccaag ttgaacagag
aaaaggttga tggtgttaag 1560ttggaatcca tgggtatcta ccaaatcttg
gctatctact ctactgttgc ttcctccttg 1620gttttgttgg tttccttggg
tgctatctcc ttctggatgt gttccaacgg ttccttgcag 1680tgtagaatct gtatctaa
1698671698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 67atgaaggcca acttgttggt tttgttgtct
gctttggctg ctgctgatgc tgatacaata 60tgtattggtt accatgccaa caactctacc
gatactgttg ataccgtttt ggaaaagaac 120gttaccgtta cccattccgt
caatttgttg gaagattccc ataacggtaa attgtgcaga 180ttgaaaggta
ttgccccatt gcaattgggt aaatgtaata ttgctggttg gttgttgggt
240aacccagaat gtgatccatt attgccagtt agatcttggt cctacatcgt
tgaaactcca 300aattccgaaa atggtatttg ctacccaggt gatttcatcg
actatgaaga attgagagaa 360caattgtcct ccgtttcctc attcgaaaga
ttcgaaatct tcccaaaaga atcctcttgg 420ccaaaccata acactaatgg
tgttactgct gcttgttccc atgaaggtaa aagttctttc 480tacagaaact
tgttgtggtt gaccgaaaaa gaaggttctt acccaaagtt gaagaactcc
540tacgttaaca agaagggtaa agaagttttg gtcttgtggg gtattcatca
tccaccaaac 600tctaaagaac aacaaaacat ctaccaaaac gaaaacgcct
acgtttctgt tgttacctct 660aactacaaca gaagattcac cccagaaatt
gctgaaagac caaaggttag agatcaagct 720ggtagaatga attactactg
gactttgttg aaaccaggtg acaccattat tttcgaagcc 780aacggtaatt
tgattgctcc aatgtatgct ttcgctttgt ctagaggttt tggttccggt
840attattactt ccaacgcttc tatgcatgaa tgcaacacta agtgtcaaac
tccattgggt 900gctatcaatt catctttgcc ataccaaaac atccacccag
ttactattgg tgaatgtcct 960aagtatgtta gatccgccaa attgagaatg
gttaccggtt tgagaaacac cccatctatt 1020caatcaagag gtttgtttgg
tgctattgcc ggttttattg aaggtggttg gactggtatg 1080attgatggtt
ggtatggtta ccaccatcaa aacgaacaag gttctggtta cgctgctgat
1140caaaagtcta ctcaaaatgc cattaacggt attaccaaca aggttaacac
cgtcatcgaa 1200aagatgaaca ttcaattcac cgccgtcggt aaagaattca
acaagttaga aaagagaatg 1260gaaaacttga acaaaaaggt cgacgatggt
ttcttggata tttggactta caacgccgaa 1320ttattggtct tattggaaaa
cgaaagaacc ttggatttcc acgattccaa cgttaagaac 1380ttgtacgaaa
aggttaagtc ccaattgaaa aacaacgcca aagaaattgg taacggttgc
1440tttgaattct accacaagtg tgataacgaa tgcatggaat ctgttagaaa
cggtacttac 1500gattacccta agtactccga agaatccaag ttgaacagag
aaaaagttga cggtgtcaag 1560ttggaatcta tgggtatcta tcaaatcttg
gccatctact ctactgttgc ctcttcatta 1620gttttgttgg ttagtttggg
tgccatctct ttttggatgt gttctaatgg ttccttgcaa 1680tgtagaatct gtatttga
1698681698DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 68atgaaggcta acctgctggt gctgctgtcc
gctctggctg ctgctgacgc tgacaccatc 60tgcatcggtt accacgctaa caactccacc
gacaccgtgg acaccgtcct cgagaagaac 120gtgaccgtga cccactccgt
gaacctgctc gaggactccc acaacggcaa gctgtgccgc 180ctgaagggta
tcgctcctct gcagctgggc aagtgcaaca tcgctggatg gctgctgggc
240aaccccgagt gcgaccctct gctgcctgtc cgttcctggt cctacatcgt
ggaaaccccc 300aactccgaga acggtatctg ctaccccggc gacttcatcg
actacgagga actgcgcgag 360cagctgtcct ccgtgtccag cttcgagcgt
ttcgagatct tccccaagga atcctcctgg 420cccaaccaca acaccaacgg
tgtcaccgct gcttgctccc acgagggcaa gtcatccttc 480taccgcaacc
tgctgtggct gaccgagaag gaaggctcct accccaagct gaagaactcc
540tacgtgaaca agaagggcaa ggaagtcctc gtcctctggg gtatccacca
cccccctaac 600tccaaggaac agcagaacat ctaccagaac gagaacgctt
acgtgtccgt ggtcacctcc 660aactacaacc gtcgtttcac ccccgagatc
gctgagcgtc ctaaagtgcg cgaccaggct 720ggccgtatga actactactg
gaccctgctg aagcccggcg acaccatcat cttcgaggct
780aacggcaacc tgatcgctcc tatgtacgct ttcgctctgt cccgtggttt
cggttccggt 840atcatcacct ctaacgcttc catgcacgag tgcaacacca
agtgccagac ccccctgggt 900gctatcaact ccagcctgcc ttaccagaac
atccaccccg tgaccatcgg cgagtgccct 960aaatacgtgc gttccgctaa
gctgcgtatg gtcaccggcc tgcgtaacac cccctccatc 1020cagtcccgtg
gcctgttcgg tgctatcgct ggtttcatcg agggcggctg gaccggcatg
1080atcgacggat ggtacggcta ccaccaccaa aacgagcagg gttccggtta
cgctgctgac 1140cagaagtcca cccagaacgc tatcaacggt atcaccaaca
aagtgaacac cgtgatcgaa 1200aagatgaaca tccagttcac cgccgtcggc
aaggaattca acaagctcga gaagcgcatg 1260gaaaacctca acaagaaggt
ggacgacggc ttcctggaca tctggaccta caacgctgag 1320ctgctggtcc
tgttggagaa cgagcgtacc ctggacttcc acgactccaa cgtgaagaac
1380ctgtacgaga aagtgaagtc ccagctcaag aacaacgcca aggaaatcgg
caacggttgc 1440ttcgagttct accacaagtg cgacaacgaa tgcatggaat
ccgtgcgtaa cggcacctac 1500gactacccta agtactccga ggaatctaag
ctgaaccgcg agaaggtcga cggcgtgaag 1560ctcgagtcca tgggcatcta
ccaaatcctg gctatctact ctaccgtggc ttcctccctg 1620gtgctcctgg
tgtctctggg cgctatctcc ttctggatgt gctccaacgg ttccctgcag
1680tgccgtatct gcatctaa 1698698PRTUnknownDescription of Unknown
PreScission Protease recognition site peptide 69Leu Glu Val Leu Phe
Gln Gly Pro 1 5 707PRTUnknownsource/note="Description of Unknown
TEV recognition site peptide" 70Glu Asn Leu Tyr Phe Gln Gly 1 5
717PRTUnknownsource/note="Description of Unknown TEV recognition
site peptide" 71Glu Asn Leu Tyr Phe Gln Ser 1 5 72135DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
72cgttctctgg ttccgcgtgg ttctccgggt tctggttaca tcccggaagc tccgcgtgac
60ggtcaggctt acgttcgtaa agacggtgaa tgggttctgc tgtctacctt cctgcaccac
120caccaccacc actga 13573566PRTInfluenza A virus 73Met Lys Thr Ile
Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Leu Gly 1 5 10 15 Gln Asp
Phe Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30
His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asn Asp 35
40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser
Thr 50 55 60 Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly
Ile Asp Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His
Cys Asp Gly Phe Gln 85 90 95 Asn Glu Thr Trp Asp Leu Phe Val Glu
Arg Ser Lys Ala Phe Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr
Leu Glu Phe Ile Ser Glu Gly Phe Thr Trp Thr 130 135 140 Gly Val Thr
Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp 145 150 155 160
Ile Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Ser Thr 165
170 175 Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp
Lys 180 185 190 Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Gln
Glu Gln Thr 195 200 205 Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr
Val Ser Thr Lys Arg 210 215 220 Ser Gln Gln Thr Ile Ile Pro Asn Ile
Gly Ser Arg Pro Trp Val Arg 225 230 235 240 Gly Leu Ser Ser Arg Ile
Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Val
Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe
Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285
Pro Ile Gly Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290
295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly
Ala 305 310 315 320 Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu
Ala Thr Gly Met 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly
Leu Phe Ser Ala Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu
Gly Met Ile Asp Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser
Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala
Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val 385 390 395 400 Ile
Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410
415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala
Leu Glu 435 440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met
Asn Lys Leu Phe 450 455 460 Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn
Ala Glu Asp Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His
Lys Cys Asp Asn Ala Cys Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr
Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg
Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys 515 520 525 Asp
Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys 530 535
540 Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560 Arg Cys Asn Ile Cys Ile 565 74566PRTInfluenza A
virus 74Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu
Gly 1 5 10 15 Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu
Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly Thr Leu Val Lys
Thr Ile Thr Asp Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu
Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Lys Ile Cys Asn Asn Pro
His Arg Ile Leu Asp Gly Ile Asp Cys 65 70 75 80 Thr Leu Ile Asp Ala
Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln 85 90 95 Asn Glu Thr
Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn 100 105 110 Cys
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120
125 Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly Phe Thr Trp Thr
130 135 140 Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly
Pro Gly 145 150 155 160 Asn Gly Phe Phe Ser Arg Leu Asn Trp Leu Thr
Lys Ser Gly Ser Thr 165 170 175 Tyr Pro Val Leu Asn Val Thr Met Pro
Asn Asn Asp Asn Phe Asp Lys 180 185 190 Leu Tyr Ile Trp Gly Val His
His Pro Ser Thr Asn Gln Glu Gln Thr 195 200 205 Ser Leu Tyr Val Gln
Glu Ser Gly Arg Val Thr Val Ser Thr Arg Arg 210 215 220 Ser Gln Gln
Ser Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg 225 230 235 240
Gly Gln Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245
250 255 Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg
Gly 260 265 270 Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser Ile Met Ser
Ser Asp Ala 275 280 285 Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr
Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val
Asn Lys Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Lys Tyr Val Lys
Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg Asn Val Pro
Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala 340 345 350 Gly Phe
Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly 355 360 365
Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370
375 380 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg
Val 385 390 395 400 Ile Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu
Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu
Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn
Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His Thr Ile Asp
Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu Lys Thr Arg
Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470 475 480 Gly
Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu Ser 485 490
495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly
Tyr Lys 515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys
Phe Leu Leu Cys 530 535 540 Val Val Leu Leu Gly Phe Ile Met Trp Ala
Cys Gln Arg Gly Asn Ile 545 550 555 560 Arg Cys Asn Ile Cys Ile 565
75566PRTInfluenza A virus 75Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile
Leu Cys Leu Val Phe Ala 1 5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn
Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Ser Asn
Gly Thr Leu Val Lys Thr Ile Thr Asn Asp 35 40 45 Gln Ile Glu Val
Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Arg
Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys 65 70 75 80
Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gln 85
90 95 Asn Lys Glu Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser
Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg
Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu
Ser Phe Asn Trp Thr 130 135 140 Gly Val Ala Gln Asn Gly Thr Ser Ser
Ala Cys Lys Arg Arg Ser Asn 145 150 155 160 Asn Ser Phe Phe Ser Arg
Leu Asn Trp Leu His Gln Leu Lys Tyr Lys 165 170 175 Tyr Pro Ala Leu
Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys 180 185 190 Leu Tyr
Ile Trp Gly Val His His Pro Ser Thr Asp Ser Asp Gln Ile 195 200 205
Ser Ile Tyr Ala Gln Ala Ser Gly Arg Val Thr Val Ser Thr Lys Arg 210
215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Ile Pro Trp Val
Arg 225 230 235 240 Gly Val Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile
Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn
Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile Arg Ser Gly Lys
Ser Ser Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Gly Lys Cys Asn
Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys
Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys
Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330
335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala
340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp
Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala
Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn
Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile Glu Lys Thr Asn Glu Lys
Phe His Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg
Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp
Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn
Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455
460 Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
465 470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys
Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr
Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val
Glu Leu Lys Ser Gly Tyr Lys 515 520 525 Asp Trp Ile Leu Trp Ile Ser
Phe Ala Ile Ser Cys Phe Leu Leu Cys 530 535 540 Val Val Leu Leu Gly
Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile 545 550 555 560 Arg Cys
Asn Ile Cys Ile 565 76566PRTInfluenza A virus 76Met Lys Thr Ile Ile
Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala 1 5 10 15 Gln Lys Leu
Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His
His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40
45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr
50 55 60 Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu
Asn Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys
Asp Gly Phe Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu Arg
Ser Lys Ala Tyr Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp
Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu
Glu Phe Asn Asp Glu Ser Phe Asn Trp Thr 130 135 140 Gly Val Thr Gln
Asn Gly Thr Ser Ser Ser Cys Lys Arg Arg Ser Asn 145 150 155 160 Asn
Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys 165 170
175 Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys
180 185 190 Leu Tyr Ile Trp Gly Val His His Pro Val Thr Asp Asn Asp
Gln Ile 195 200 205 Phe Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val
Ser Thr Lys Arg 210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly
Ser Arg Pro Arg Ile Arg 225 230 235 240 Asn Ile Pro Ser Arg Ile Ser
Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu Ile
Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys
Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285 Pro
Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295
300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala
305 310 315 320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala
Thr Gly Met 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile
Phe Gly Ala Ile Ala 340 345
350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly
355 360 365 Phe Arg His Gln Asn Ser Glu Gly Ile Gly Gln Ala Ala Asp
Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys
Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr Asn Glu Lys Phe His
Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln
Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His
Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu
Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470
475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly
Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp
Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu
Lys Ser Gly Tyr Lys 515 520 525 Asp Trp Ile Leu Trp Ile Ser Phe Ala
Ile Ser Cys Phe Leu Leu Cys 530 535 540 Val Ala Leu Leu Gly Phe Ile
Met Trp Ala Cys Gln Lys Gly Asn Ile 545 550 555 560 Arg Cys Asn Ile
Cys Ile 565 77560PRTInfluenza A virus 77Met Asn Thr Gln Ile Leu Val
Phe Ala Leu Ile Ala Ile Ile Pro Thr 1 5 10 15 Asn Ala Asp Lys Ile
Cys Leu Gly His His Ala Val Ser Asn Gly Thr 20 25 30 Lys Val Asn
Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr 35 40 45 Glu
Thr Val Glu Arg Thr Asn Ile Pro Arg Ile Cys Ser Lys Gly Lys 50 55
60 Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly
65 70 75 80 Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu
Ile Ile 85 90 95 Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly
Lys Phe Val Asn 100 105 110 Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu
Ser Gly Gly Ile Asp Lys 115 120 125 Glu Ala Met Gly Phe Thr Tyr Ser
Gly Ile Arg Thr Asn Gly Ala Thr 130 135 140 Ser Ala Cys Arg Arg Ser
Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp 145 150 155 160 Leu Leu Ser
Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser 165 170 175 Tyr
Lys Asn Thr Arg Lys Ser Pro Ala Leu Ile Val Trp Gly Ile His 180 185
190 His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn
195 200 205 Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe
Val Pro 210 215 220 Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Leu Ser
Gly Arg Ile Asp 225 230 235 240 Phe His Trp Leu Met Leu Asn Pro Asn
Asp Thr Val Thr Phe Ser Phe 245 250 255 Asn Gly Ala Phe Ile Ala Pro
Asp Arg Ala Ser Phe Leu Arg Gly Lys 260 265 270 Ser Met Gly Ile Gln
Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly 275 280 285 Asp Cys Tyr
His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln 290 295 300 Asn
Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln 305 310
315 320 Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile
Pro 325 330 335 Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu Asn Gly 340 345 350 Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe
Arg His Gln Asn Ala 355 360 365 Gln Gly Glu Gly Thr Ala Ala Asp Tyr
Lys Ser Thr Gln Ser Ala Ile 370 375 380 Asp Gln Ile Thr Gly Lys Leu
Asn Arg Leu Ile Glu Lys Thr Asn Gln 385 390 395 400 Gln Phe Glu Leu
Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile 405 410 415 Gly Asn
Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser 420 425 430
Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp 435
440 445 Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg
Gln 450 455 460 Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe
Glu Ile Phe 465 470 475 480 His Lys Cys Asp Asp Asp Cys Met Ala Ser
Ile Arg Asn Asn Thr Tyr 485 490 495 Asp His Ser Lys Tyr Arg Glu Glu
Ala Met Gln Asn Arg Ile Gln Ile 500 505 510 Asp Pro Val Lys Leu Ser
Ser Gly Tyr Lys Asp Val Ile Leu Trp Phe 515 520 525 Ser Phe Gly Ala
Ser Cys Phe Ile Leu Leu Ala Ile Val Met Gly Leu 530 535 540 Val Phe
Ile Cys Val Lys Asn Gly Asn Met Arg Cys Thr Ile Cys Ile 545 550 555
560 78562PRTInfluenza A virus 78Met Ala Ile Ile Tyr Leu Ile Leu Leu
Phe Thr Ala Val Arg Gly Asp 1 5 10 15 Gln Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Glu Lys Val Asp 20 25 30 Thr Ile Leu Glu Arg
Asn Val Thr Val Thr His Ala Lys Asp Ile Leu 35 40 45 Glu Lys Thr
His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro 50 55 60 Leu
Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro 65 70
75 80 Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met
Glu 85 90 95 Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser
Phe Asn Asp 100 105 110 Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val
Lys His Phe Glu Lys 115 120 125 Val Lys Ile Leu Pro Lys Asp Arg Trp
Thr Gln His Thr Thr Thr Gly 130 135 140 Gly Ser Arg Ala Cys Ala Val
Ser Gly Asn Pro Ser Phe Phe Arg Asn 145 150 155 160 Met Val Trp Leu
Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly 165 170 175 Ser Tyr
Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val 180 185 190
His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val 195
200 205 Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser
Thr 210 215 220 Pro Asp Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly
Ser Arg Met 225 230 235 240 Glu Phe Ser Trp Thr Leu Leu Asp Met Trp
Asp Thr Ile Asn Phe Glu 245 250 255 Ser Thr Gly Asn Leu Ile Ala Pro
Glu Tyr Gly Phe Lys Ile Ser Lys 260 265 270 Arg Gly Ser Ser Gly Ile
Met Lys Thr Glu Gly Thr Leu Glu Asn Cys 275 280 285 Glu Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro 290 295 300 Phe His
Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val 305 310 315
320 Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln
325 330 335 Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly 340 345 350 Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr
His His Ser Asn 355 360 365 Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys
Glu Ser Thr Gln Lys Ala 370 375 380 Phe Asp Gly Ile Thr Asn Lys Val
Asn Ser Val Ile Glu Lys Met Asn 385 390 395 400 Thr Gln Phe Glu Ala
Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg 405 410 415 Leu Glu Asn
Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp 420 425 430 Thr
Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 435 440
445 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met
450 455 460 Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe
Glu Phe 465 470 475 480 Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser
Val Lys Asn Gly Thr 485 490 495 Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu
Ser Lys Leu Asn Arg Asn Glu 500 505 510 Ile Lys Gly Val Lys Leu Ser
Ser Met Gly Val Tyr Gln Ile Leu Ala 515 520 525 Ile Tyr Ala Thr Val
Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala 530 535 540 Gly Ile Ser
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 545 550 555 560
Cys Ile 79568PRTInfluenza A virus 79Met Glu Lys Ile Val Leu Leu Phe
Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly
Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp Thr Ile Met
Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45 Leu Glu
Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn 65
70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr
Ile Val 85 90 95 Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro
Gly Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser
Arg Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro Lys Ser
Ser Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala
Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg Asn Val
Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170 175 Lys
Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185
190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220 Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn
Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr Ile Leu
Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly Asn Phe
Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys Gly Asp
Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn Cys Asn
Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300 Met
Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310
315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn
Ser 325 330 335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly Leu Phe
Gly Ala Ile 340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met
Val Asp Gly Trp Tyr 355 360 365 Gly Tyr His His Ser Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Lys 370 375 380 Glu Ser Thr Gln Lys Ala Ile
Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile Asp Lys
Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405 410 415 Asn Asn
Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420 425 430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met 435
440 445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
Leu 450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys
Glu Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His Lys Cys
Asp Asn Glu Cys Met Glu 485 490 495 Ser Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg Glu Glu
Ile Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520 525 Ile Tyr Gln Ile
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu Ala
Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555
560 Ser Leu Gln Cys Arg Ile Cys Ile 565 80563PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
80Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser
Met 275 280 285 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala
Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr
Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Thr Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370
375 380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile
Glu 385 390 395 400 Lys Met Asn Ile Gln Phe Thr Ala Val Gly Lys Glu
Phe Asn Lys Leu 405 410 415 Glu Lys Arg Met Glu Asn Leu Asn Lys Lys
Val Asp Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His
Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln
Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485 490
495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn
500 505 510 Arg Glu Lys Val Asp Gly Val Arg Ser Leu Val Pro Arg Gly
Ser Pro 515 520 525 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly
Gln Ala Tyr Val 530 535 540 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser
Thr Phe Leu His His His 545 550 555 560 His His His
81565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 81Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe
Cys Leu Ala Leu Gly 1 5 10 15 Gln Asp Leu Pro Gly Asn Asp Asn Ser
Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly
Thr Leu Val Lys Thr Ile Thr Asp Asp 35 40 45 Gln Ile Glu Val Thr
Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Lys Ile
Cys Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys 65 70 75 80 Thr
Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln 85 90
95 Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn
100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser
Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly
Phe Thr Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly Gly Ser Asn Ala
Cys Lys Arg Gly Pro Gly 145 150 155 160 Asn Gly Phe Phe Ser Arg Leu
Asn Trp Leu Thr Lys Ser Gly Ser Thr 165 170 175 Tyr Pro Val Leu Asn
Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys 180 185 190 Leu Tyr Ile
Trp Gly Val His His Pro Ser Thr Asn Gln Glu Gln Thr 195 200 205 Ser
Leu Tyr Val Gln Glu Ser Gly Arg Val Thr Val Ser Thr Arg Arg 210 215
220 Ser Gln Gln Ser Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg
225 230 235 240 Gly Gln Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val
Lys Pro Gly 245 250 255 Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu
Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Met Arg Thr Gly Lys Ser
Ser Ile Met Ser Ser Asp Ala 275 280 285 Pro Ile Asp Thr Cys Ile Ser
Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro
Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro
Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335
Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala 340
345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr
Gly 355 360 365 Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala
Asp Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly
Lys Leu Asn Arg Val 385 390 395 400 Ile Glu Lys Thr Asn Glu Lys Phe
His Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile
Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu
Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln
His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460
Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465
470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile
Glu Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg
Asp Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Gly Val Arg
Ser Leu Val Pro Arg Gly 515 520 525 Ser Pro Gly Ser Gly Tyr Ile Pro
Glu Ala Pro Arg Asp Gly Gln Ala 530 535 540 Tyr Val Arg Lys Asp Gly
Glu Trp Val Leu Leu Ser Thr Phe Leu His 545 550 555 560 His His His
His His 565 82565PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 82Met Lys Thr Ile Ile Ala Leu Ser
Tyr Ile Leu Cys Leu Val Phe Ala 1 5 10 15 Gln Lys Leu Pro Gly Asn
Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val
Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40 45 Gln Ile
Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60
Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys 65
70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly
Phe Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys
Ala Tyr Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe
Asn Asp Glu Ser Phe Asn Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly
Thr Ser Ser Ser Cys Lys Arg Arg Ser Asn 145 150 155 160 Asn Ser Phe
Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys 165 170 175 Tyr
Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys 180 185
190 Leu Tyr Ile Trp Gly Val His His Pro Val Thr Asp Asn Asp Gln Ile
195 200 205 Phe Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr
Lys Arg 210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg
Pro Arg Ile Arg 225 230 235 240 Asn Ile Pro Ser Arg Ile Ser Ile Tyr
Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn Ser
Thr Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile Arg
Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Gly
Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro
Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310
315 320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly
Met 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly
Ala Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val
Asp Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser Glu Gly Ile
Gly Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asn
Gln Ile Asn Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr
Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val
Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430
Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435
440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu
Phe 450 455 460 Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp
Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp
Asn Ala Cys Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His
Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile
Lys Gly Val Arg Ser Leu Val Pro Arg Gly 515 520 525 Ser Pro Gly Ser
Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala 530 535 540 Tyr Val
Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu His 545 550 555
560 His His His His His 565 83566PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 83Met Glu Lys Ile Val
Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp
Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40
45 Leu Glu Lys Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu
Gly Asn 65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp
Ser Tyr Ile Val 85 90 95 Glu Lys Ala Asn Pro Val Asn Asp Leu Cys
Tyr Pro Gly Asp Phe Asn 100 105 110 Asp Tyr Glu Glu Leu Lys His Leu
Leu Ser Arg Ile Asn His Phe Glu 115 120 125 Lys Ile Gln Ile Ile Pro
Lys Ser Ser Trp Ser Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser
Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe 145 150 155 160 Arg
Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile 165 170
175 Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp
180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu
Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr
Leu Asn Gln Arg 210 215 220 Leu Val Pro Arg Ile Ala Thr Arg Ser Lys
Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu Phe Phe Trp Thr
Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe Glu Ser Asn Gly
Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270 Val Lys Lys
Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285 Asn
Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295
300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu
Arg Asn Ser 325 330 335 Pro Gln Arg Glu Arg Arg Arg Lys Lys Arg Gly
Leu Phe Gly Ala Ile 340 345 350 Ala Gly Phe Ile Glu Gly Gly Trp Gln
Gly Met Val Asp Gly Trp Tyr 355 360 365 Gly Tyr His His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380 Glu Ser Thr Gln Lys
Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 385 390 395 400 Ile Ile
Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe 405 410 415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp 420
425 430 Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met 435 440 445 Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu 450 455 460 Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly 465 470 475 480 Asn Gly Cys Phe Glu Phe Tyr His
Lys Cys Asp Asn Glu Cys Met Glu 485 490 495 Ser Val Arg Asn Gly Thr
Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg
Glu Glu Ile Ser Gly Val Arg Ser Leu Val Pro Arg 515 520 525 Gly Ser
Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 530 535 540
Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 545
550 555 560 His His His His His His 565 84559PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
84Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr 1
5 10 15 Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly
Thr 20 25 30 Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val
Asn Ala Thr 35 40 45 Glu Thr Val Glu Arg Thr Asn Ile Pro Arg Ile
Cys Ser Lys Gly Lys 50 55 60 Arg Thr Val Asp Leu Gly Gln Cys Gly
Leu Leu Gly Thr Ile Thr Gly 65 70 75 80 Pro Pro Gln Cys Asp Gln Phe
Leu Glu Phe Ser Ala Asp Leu Ile Ile 85 90 95 Glu Arg Arg Glu Gly
Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn 100 105 110 Glu Glu Ala
Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys 115 120 125 Glu
Ala Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr 130 135
140 Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp
145 150 155 160 Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met
Thr Lys Ser 165 170 175 Tyr Lys Asn Thr Arg Lys Ser Pro Ala Leu Ile
Val Trp Gly Ile His 180 185 190 His Ser Val Ser Thr Ala Glu Gln Thr
Lys Leu Tyr Gly Ser Gly Asn 195 200 205 Lys Leu Val Thr Val Gly Ser
Ser Asn Tyr Gln Gln Ser Phe Val Pro 210 215 220 Ser Pro Gly Ala Arg
Pro Gln Val Asn Gly Leu Ser Gly Arg Ile Asp 225 230 235 240 Phe His
Trp Leu Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe 245 250 255
Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys 260
265 270 Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu
Gly 275 280 285 Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu
Pro Phe Gln 290 295 300 Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro
Arg Tyr Val Lys Gln 305 310 315 320 Arg Ser Leu Leu Leu Ala Thr Gly
Met Lys Asn Val Pro Glu Ile Pro 325 330 335 Lys Gly Arg Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly 340 345 350 Trp Glu Gly Leu
Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala 355 360 365 Gln Gly
Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile 370 375
380
Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln 385
390 395 400 Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys
Gln Ile 405 410 415 Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr
Glu Val Trp Ser 420 425 430 Tyr Asn Ala Glu Leu Leu Val Ala Met Glu
Asn Gln His Thr Ile Asp 435 440 445 Leu Ala Asp Ser Glu Met Asp Lys
Leu Tyr Glu Arg Val Lys Arg Gln 450 455 460 Leu Arg Glu Asn Ala Glu
Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe 465 470 475 480 His Lys Cys
Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr 485 490 495 Asp
His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile 500 505
510 Asp Pro Val Arg Ser Leu Val Pro Arg Gly Ser Pro Gly Ser Gly Tyr
515 520 525 Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys
Asp Gly 530 535 540 Glu Trp Val Leu Leu Ser Thr Phe Leu His His His
His His His 545 550 555 85560PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 85Met Ala Ile Ile Tyr Leu
Ile Leu Leu Phe Thr Ala Val Arg Gly Asp 1 5 10 15 Gln Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp 20 25 30 Thr Ile
Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu 35 40 45
Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro 50
55 60 Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn
Pro 65 70 75 80 Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr
Ile Met Glu 85 90 95 Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro
Gly Ser Phe Asn Asp 100 105 110 Tyr Glu Glu Leu Lys His Leu Leu Ser
Ser Val Lys His Phe Glu Lys 115 120 125 Val Lys Ile Leu Pro Lys Asp
Arg Trp Thr Gln His Thr Thr Thr Gly 130 135 140 Gly Ser Arg Ala Cys
Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn 145 150 155 160 Met Val
Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly 165 170 175
Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val 180
185 190 His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn
Val 195 200 205 Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys
Arg Ser Thr 210 215 220 Pro Asp Ile Ala Thr Arg Pro Lys Val Asn Gly
Leu Gly Ser Arg Met 225 230 235 240 Glu Phe Ser Trp Thr Leu Leu Asp
Met Trp Asp Thr Ile Asn Phe Glu 245 250 255 Ser Thr Gly Asn Leu Ile
Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys 260 265 270 Arg Gly Ser Ser
Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys 275 280 285 Glu Thr
Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro 290 295 300
Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val 305
310 315 320 Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val
Pro Gln 325 330 335 Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile Glu Gly 340 345 350 Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
Gly Tyr His His Ser Asn 355 360 365 Asp Gln Gly Ser Gly Tyr Ala Ala
Asp Lys Glu Ser Thr Gln Lys Ala 370 375 380 Phe Asp Gly Ile Thr Asn
Lys Val Asn Ser Val Ile Glu Lys Met Asn 385 390 395 400 Thr Gln Phe
Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg 405 410 415 Leu
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp 420 425
430 Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu
435 440 445 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
Arg Met 450 455 460 Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly
Cys Phe Glu Phe 465 470 475 480 Tyr His Lys Cys Asp Asp Glu Cys Met
Asn Ser Val Lys Asn Gly Thr 485 490 495 Tyr Asp Tyr Pro Lys Tyr Glu
Glu Glu Ser Lys Leu Asn Arg Asn Glu 500 505 510 Ile Lys Gly Val Arg
Ser Leu Val Pro Arg Gly Ser Pro Gly Ser Gly 515 520 525 Tyr Ile Pro
Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp 530 535 540 Gly
Glu Trp Val Leu Leu Ser Thr Phe Leu His His His His His His 545 550
555 560 86565PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 86Met Lys Ala Asn Leu Leu Val Leu
Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu
Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65
70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser
Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr
Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu
Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys
Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala
Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn
Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu
Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185
190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr
195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr
Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val
Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu
Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn
Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe
Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys
Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser
Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310
315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg
Asn 325 330 335 Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile
Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly
Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr
Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile
Thr Asn Lys Val Asn Thr Val Ile Glu 385 390 395 400 Lys Met Tyr Ile
Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Lys
Arg Met Glu Asn Leu Asn Lys Lys Val Asp Tyr Gly Phe Leu 420 425 430
Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435
440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu
Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly
Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu
Cys Met Glu Ser Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys
Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly
Val Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile
Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu
Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555
560 Cys Arg Ile Cys Ile 565 87565PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 87Met Lys Ala Asn Leu
Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile
50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu
Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser
Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile
Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu
Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe
Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val
Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr
Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170
175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu
180 185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn
Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser
Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro
Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp
Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn
Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg
Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285 His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295
300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly
Leu Arg Asn 325 330 335 Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly
Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile
Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn
Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu 385 390 395 400 Lys Met
Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Tyr 420
425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu Ser Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545
550 555 560 Cys Arg Ile Cys Ile 565 88565PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
88Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser
Met 275 280 285 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala
Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr
Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Thr Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu
385
390 395 400 Lys Met Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
Lys Leu 405 410 415 Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485 490 495 Asn
Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505
510 Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln
515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile Cys Ile 565
89565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 89Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu
Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys Asn Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln
Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn 325 330 335
Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340
345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr
His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
Asn Thr Val Ile Glu 385 390 395 400 Lys Met Tyr Ile Gln Phe Thr Ala
Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Lys Arg Met Glu Asn
Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430 Tyr Ile Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465
470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly Val Lys Leu Glu
Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val
Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile
Cys Ile 565 90565PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 90Met Lys Ala Asn Leu Leu Val Leu
Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu
Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65
70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser
Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr
Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu
Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys
Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala
Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn
Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu
Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185
190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr
195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr
Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val
Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu
Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn
Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe
Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys
Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser
Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310
315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg
Asn 325 330 335 Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile
Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly
Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr
Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile
Thr Asn Lys Val Asn Thr Val Ile Glu 385 390 395 400 Lys Met Asn Ile
Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Lys
Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430
Tyr Ile Tyr Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435
440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu
Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly
Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu
Cys Met Glu Ser Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys
Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly
Val Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile
Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu
Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555
560 Cys Arg Ile Cys Ile 565 91565PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 91Met Lys Ala Asn Leu
Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile
50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu
Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser
Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile
Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu
Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe
Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135 140 Thr Asn Gly Val
Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr
Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170
175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu
180 185 190 Trp Gly Ile His His Pro Pro Asn Ser Lys Glu Gln Gln Asn
Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser
Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro
Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp
Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn
Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg
Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser Met 275 280 285 His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295
300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly
Leu Arg Asn 325 330 335 Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly
Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile
Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn
Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu 385 390 395 400 Lys Met
Asn Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420
425 430 Asp Ile Tyr Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu Cys Met Glu Ser Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val
Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545
550 555 560 Cys Arg Ile Cys Ile 565 92565PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
92Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1
5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys
Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Pro Leu
Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn
Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Asn 130 135
140 Thr Asn Gly Val Thr Ala Ala Cys Ser His Glu Gly Lys Ser Ser Phe
145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Glu Gly Ser
Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys
Glu Val Leu Val Leu 180 185 190 Trp Gly Ile His His Pro Pro Asn Ser
Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln Asn Glu Asn Ala Tyr Val
Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro Glu
Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala 225 230 235 240 Gly Arg
Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly Asp Thr Ile 245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Met Tyr Ala Phe Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser
Met 275 280 285 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu Gly Ala
Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr
Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Thr Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu
385
390 395 400 Lys Met Asn Ile Gln Tyr Thr Ala Val Gly Lys Glu Phe Asn
Lys Leu 405 410 415 Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu 420 425 430 Tyr Ile Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe
Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 485 490 495 Asn
Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505
510 Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln
515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile Cys Ile 565
93565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 93Met Lys Ala Asn Leu Leu Val Leu Leu Ser Ala
Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser
His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala Pro Leu
Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65 70 75 80 Asn
Pro Glu Cys Asp Pro Leu Leu Pro Val Arg Ser Trp Ser Tyr Ile 85 90
95 Val Glu Thr Pro Asn Ser Glu Asn Gly Ile Cys Tyr Pro Gly Asp Phe
100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp
Pro Asn His Asn 130 135 140 Thr Asn Gly Val Thr Ala Ala Cys Ser His
Glu Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu
Thr Glu Lys Glu Gly Ser Tyr Pro Lys 165 170 175 Leu Lys Asn Ser Tyr
Val Asn Lys Lys Gly Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Ile
His His Pro Pro Asn Ser Lys Glu Gln Gln Asn Ile Tyr 195 200 205 Gln
Asn Glu Asn Ala Tyr Val Ser Val Val Thr Ser Asn Tyr Asn Arg 210 215
220 Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Asp Gln Ala
225 230 235 240 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Lys Pro Gly
Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro
Met Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile
Ile Thr Ser Asn Ala Ser Met 275 280 285 His Glu Cys Asn Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Ser 290 295 300 Ser Leu Pro Tyr Gln
Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn 325 330 335
Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340
345 350 Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr
His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
Asn Thr Val Ile Glu 385 390 395 400 Lys Met Asn Ile Gln Phe Thr Ala
Val Gly Tyr Glu Phe Asn Lys Leu 405 410 415 Glu Lys Arg Met Glu Tyr
Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465
470 475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
Val Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn 500 505 510 Arg Glu Lys Val Asp Gly Val Lys Leu Glu
Ser Met Gly Ile Tyr Gln 515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val
Ala Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile
Cys Ile 565 9469PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 94Met Lys Ala Asn Leu Leu Val Leu
Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu
Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Glu 50 55 60
Asn Leu Tyr Phe Gln 65 9553PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 95Met Lys Ala Asn Leu Leu
Val Leu Leu Ser Ala Leu Ala Ala Ala Asp 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Glu 35 40 45
Asn Leu Tyr Phe Gln 50 96282PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 96Ser Asn Ala Ser Met His
Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu 1 5 10 15 Gly Ala Ile Asn
Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val Thr 20 25 30 Ile Gly
Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val 35 40 45
Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly 50
55 60 Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp
Gly 65 70 75 80 Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly
Tyr Ala Ala 85 90 95 Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly
Ile Thr Asn Lys Val 100 105 110 Asn Thr Val Ile Glu Lys Met Tyr Ile
Gln Phe Thr Ala Val Gly Lys 115 120 125 Glu Phe Asn Lys Leu Glu Lys
Arg Met Glu Asn Leu Asn Lys Lys Val 130 135 140 Asp Asp Gly Phe Leu
Tyr Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val 145 150 155 160 Leu Leu
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys 165 170 175
Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu 180
185 190 Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu
Cys 195 200 205 Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys
Tyr Ser Glu 210 215 220 Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly
Val Lys Leu Glu Ser 225 230 235 240 Met Gly Ile Tyr Gln Ile Leu Ala
Ile Tyr Ser Thr Val Ala Ser Ser 245 250 255 Leu Val Leu Leu Val Ser
Leu Gly Ala Ile Ser Phe Trp Met Cys Ser 260 265 270 Asn Gly Ser Leu
Gln Cys Arg Ile Cys Ile 275 280 97282PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
97Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr Pro Leu 1
5 10 15 Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val
Thr 20 25 30 Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val 35 40 45 Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly 50 55 60 Ala Ile Ala Gly Phe Ile Glu Gly Gly
Trp Thr Gly Met Ile Asp Gly 65 70 75 80 Trp Tyr Gly Tyr His His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala 85 90 95 Asp Gln Lys Ser Thr
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val 100 105 110 Asn Thr Val
Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val Gly Tyr 115 120 125 Glu
Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys Lys Val 130 135
140 Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
145 150 155 160 Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser
Asn Val Lys 165 170 175 Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys
Asn Asn Ala Lys Glu 180 185 190 Ile Gly Asn Gly Cys Phe Glu Phe Tyr
His Lys Cys Asp Asn Glu Cys 195 200 205 Met Glu Ser Val Arg Asn Gly
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu 210 215 220 Glu Ser Lys Leu Asn
Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser 225 230 235 240 Met Gly
Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser 245 250 255
Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser 260
265 270 Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 275 280
98282PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 98Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys
Cys Gln Thr Pro Leu 1 5 10 15 Gly Ala Ile Asn Ser Ser Leu Pro Tyr
Gln Asn Ile His Pro Val Thr 20 25 30 Ile Gly Glu Cys Pro Lys Tyr
Val Arg Ser Ala Lys Leu Arg Met Val 35 40 45 Thr Gly Leu Arg Asn
Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly 50 55 60 Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly 65 70 75 80 Trp
Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala 85 90
95 Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
100 105 110 Asn Thr Val Ile Glu Lys Met Tyr Ile Gln Phe Thr Ala Val
Gly Tyr 115 120 125 Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu
Asn Lys Lys Val 130 135 140 Asp Asp Gly Phe Leu Tyr Ile Trp Thr Tyr
Asn Ala Glu Leu Leu Val 145 150 155 160 Leu Leu Glu Asn Glu Arg Thr
Leu Asp Phe His Asp Ser Asn Val Lys 165 170 175 Asn Leu Tyr Glu Lys
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu 180 185 190 Ile Gly Asn
Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys 195 200 205 Met
Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu 210 215
220 Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu Glu Ser
225 230 235 240 Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val
Ala Ser Ser 245 250 255 Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser
Phe Trp Met Cys Ser 260 265 270 Asn Gly Ser Leu Gln Cys Arg Ile Cys
Ile 275 280 99283PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 99Ser Thr Ser Asn Ala Ser Met His
Glu Cys Asn Thr Lys Cys Gln Thr 1 5 10 15 Pro Leu Gly Ala Ile Asn
Ser Ser Leu Pro Tyr Gln Asn Ile His Pro 20 25 30 Val Thr Ile Gly
Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 35 40 45 Met Val
Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 50 55 60
Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 65
70 75 80 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser
Gly Tyr 85 90 95 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn
Gly Ile Thr Asn 100 105 110 Lys Val Asn Thr Val Ile Glu Lys Met Tyr
Ile Gln Phe Thr Ala Val 115 120 125 Gly Lys Glu Phe Asn Lys Leu Glu
Lys Arg Met Glu Asn Leu Asn Lys 130 135 140 Lys Val Asp Asp Gly Phe
Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu 145 150 155 160 Leu Val Leu
Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn 165 170 175 Val
Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 180 185
190 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn
195 200 205 Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro
Lys Tyr 210 215 220 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp
Val Lys Leu Glu 225 230 235 240 Ser Met Gly Ile Tyr Gln Ile Leu Ala
Ile Tyr Ser Thr Val Ala Ser 245 250 255 Ser Leu Val Leu Leu Val Ser
Leu Gly Ala Ile Ser Phe Trp Met Cys 260 265 270 Ser Asn Gly Ser Leu
Gln Cys Arg Ile Cys Ile 275 280 100284PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
100Ser Thr Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr
1 5 10 15 Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile
His Pro 20 25 30 Val Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser
Ala Lys Leu Arg 35 40 45 Met Val Thr Gly Leu Arg Asn Thr Pro Ser
Ile Gln Ser Arg Gly Leu 50 55 60 Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly Gly Trp Thr Gly Met Ile 65 70 75 80 Asp Gly Trp Tyr Gly Tyr
His His Gln Asn Glu Gln Gly Ser Gly Tyr 85 90 95 Ala Ala Asp Gln
Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn 100 105 110 Lys Val
Asn Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val 115 120 125
Gly Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys 130
135 140 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu
Leu 145 150 155 160 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe
His Asp Ser Asn 165 170 175 Val Lys Asn Leu Tyr Glu Lys Val Lys Ser
Gln Leu Lys Asn Asn Ala 180 185 190 Lys Glu Ile Gly Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn 195 200 205 Glu Cys Met Glu Ser Val
Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 210 215 220 Ser Glu Glu Ser
Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu 225 230 235 240 Glu Ser
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala 245 250 255
Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met 260
265 270 Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 275 280
101284PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 101Ser Thr Ser Asn Ala Ser Met His Glu Cys
Asn Thr Lys Cys Gln Thr 1 5 10 15 Pro Leu Gly Ala Ile Asn Ser Ser
Leu Pro Tyr Gln Asn Ile His Pro 20 25 30 Val Thr Ile Gly Glu Cys
Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg 35 40 45 Met Val Thr Gly
Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu 50 55 60 Phe Gly
Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile 65 70 75 80
Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 85
90 95 Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr
Asn 100 105 110 Lys Val Asn Thr Val Ile Glu Lys Met Tyr Ile Gln Phe
Thr Ala Val 115 120 125 Gly Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met
Glu Tyr Leu Asn Lys 130 135 140 Lys Val Asp Asp Gly Phe Leu Tyr Ile
Trp Thr Tyr Asn Ala Glu Leu 145 150 155 160 Leu Val Leu Leu Glu Asn
Glu Arg Thr Leu Asp Phe His Asp Ser Asn 165 170 175 Val Lys Asn Leu
Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala 180 185 190 Lys Glu
Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 195 200 205
Glu Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 210
215 220 Ser Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys
Leu 225 230 235 240 Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr
Ser Thr Val Ala 245 250 255 Ser Ser Leu Val Leu Leu Val Ser Leu Gly
Ala Ile Ser Phe Trp Met 260 265 270 Cys Ser Asn Gly Ser Leu Gln Cys
Arg Ile Cys Ile 275 280 102283PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 102Gly Ser Asn Ala Ser
Met His Glu Cys Asn Thr Lys Cys Gln Thr Pro 1 5 10 15 Leu Gly Ala
Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His Pro Val 20 25 30 Thr
Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala Lys Leu Arg Met 35 40
45 Val Thr Gly Leu Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe
50 55 60 Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile Asp 65 70 75 80 Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly
Ser Gly Tyr Ala 85 90 95 Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn Lys 100 105 110 Val Asn Thr Val Ile Glu Lys Met
Tyr Ile Gln Phe Thr Ala Val Gly 115 120 125 Lys Glu Phe Asn Lys Leu
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys 130 135 140 Val Asp Asp Gly
Phe Leu Tyr Ile Trp Thr Tyr Asn Ala Glu Leu Leu 145 150 155 160 Val
Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val 165 170
175 Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys
180 185 190 Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp
Asn Glu 195 200 205 Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser 210 215 220 Glu Glu Ser Lys Leu Asn Arg Glu Lys Val
Asp Gly Val Lys Leu Glu 225 230 235 240 Ser Met Gly Ile Tyr Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala Ser 245 250 255 Ser Leu Val Leu Leu
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys 260 265 270 Ser Asn Gly
Ser Leu Gln Cys Arg Ile Cys Ile 275 280 103283PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
103Gly Ser Asn Ala Ser Met His Glu Cys Asn Thr Lys Cys Gln Thr Pro
1 5 10 15 Leu Gly Ala Ile Asn Ser Ser Leu Pro Tyr Gln Asn Ile His
Pro Val 20 25 30 Thr Ile Gly Glu Cys Pro Lys Tyr Val Arg Ser Ala
Lys Leu Arg Met 35 40 45 Val Thr Gly Leu Arg Asn Thr Pro Ser Ile
Gln Ser Arg Gly Leu Phe 50 55 60 Gly Ala Ile Ala Gly Phe Ile Glu
Gly Gly Trp Thr Gly Met Ile Asp 65 70 75 80 Gly Trp Tyr Gly Tyr His
His Gln Asn Glu Gln Gly Ser Gly Tyr Ala 85 90 95 Ala Asp Gln Lys
Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys 100 105 110 Val Asn
Thr Val Ile Glu Lys Met Asn Ile Gln Phe Thr Ala Val Gly 115 120 125
Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met Glu Tyr Leu Asn Lys Lys 130
135 140 Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
Leu 145 150 155 160 Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Phe His
Asp Ser Asn Val 165 170 175 Lys Asn Leu Tyr Glu Lys Val Lys Ser Gln
Leu Lys Asn Asn Ala Lys 180 185 190 Glu Ile Gly Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn Glu 195 200 205 Cys Met Glu Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser 210 215 220 Glu Glu Ser Lys
Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu Glu 225 230 235 240 Ser
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser 245 250
255 Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys
260 265 270 Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 275 280
104283PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 104Gly Ser Asn Ala Ser Met His Glu Cys Asn
Thr Lys Cys Gln Thr Pro 1 5 10 15 Leu Gly Ala Ile Asn Ser Ser Leu
Pro Tyr Gln Asn Ile His Pro Val 20 25 30 Thr Ile Gly Glu Cys Pro
Lys Tyr Val Arg Ser Ala Lys Leu Arg Met 35 40 45 Val Thr Gly Leu
Arg Asn Thr Pro Ser Ile Gln Ser Arg Gly Leu Phe 50 55 60 Gly Ala
Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp 65 70 75 80
Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala 85
90 95 Ala Asp Gln Lys Ser Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn
Lys 100 105 110 Val Asn Thr Val Ile Glu Lys Met Tyr Ile Gln Phe Thr
Ala Val Gly 115 120 125 Tyr Glu Phe Asn Lys Leu Glu Lys Arg Met Glu
Tyr Leu Asn Lys Lys 130 135 140 Val Asp Asp Gly Phe Leu Tyr Ile Trp
Thr Tyr Asn Ala Glu Leu Leu 145 150 155 160 Val Leu Leu Glu Asn Glu
Arg Thr Leu Asp Phe His Asp Ser Asn Val 165 170 175 Lys Asn Leu Tyr
Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys 180 185 190 Glu Ile
Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu 195 200 205
Cys Met Glu Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser 210
215 220 Glu Glu Ser Lys Leu Asn Arg Glu Lys Val Asp Gly Val Lys Leu
Glu 225 230 235 240 Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser
Thr Val Ala Ser 245 250 255 Ser Leu Val Leu Leu Val Ser Leu Gly Ala
Ile Ser Phe Trp Met Cys 260 265 270 Ser Asn Gly Ser Leu Gln Cys Arg
Ile Cys Ile 275 280 105275PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 105Gly Asn Thr Lys Cys
Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln
Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40
45 Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
50 55 60 Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His
His Gln 65 70 75 80 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys
Ser Thr Gln Asn 85 90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
Thr Val Ile Glu Lys Met 100 105 110 Tyr Ile Gln Phe Thr Ala Val Gly
Lys Glu Phe Asn Lys Leu Glu Lys 115 120 125 Arg Met Glu Asn Leu Asn
Lys Lys Val Asp Asp Gly Phe Leu Tyr Ile 130 135 140 Trp Thr Tyr Asn
Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170
175 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu
180 185 190 Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg
Asn Gly 195 200 205 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys
Leu Asn Arg Glu 210 215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met
Gly Ile Tyr Gln Ile Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala
Ser Ser Leu Val Leu Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile
275 106275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 106Gly Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40 45 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 50 55 60 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 65 70 75 80
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 85
90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys
Met 100 105 110 Asn Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys
Leu Glu Lys 115 120 125 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Asp Ile 130 135 140 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170 175 Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 180 185 190 Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 195 200 205
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 210
215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile
Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile 275
107275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 107Gly Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40 45 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 50 55 60 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 65 70 75 80
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 85
90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys
Met 100 105 110 Tyr Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys
Leu Glu Lys 115 120 125 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Tyr Ile 130 135 140 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170 175 Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 180 185 190 Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 195 200 205
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 210
215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile
Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile 275
108275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 108Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40 45 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 50 55 60 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 65 70 75 80
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 85
90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys
Met 100 105 110 Tyr Ile Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys
Leu Glu Lys 115 120 125 Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Tyr Ile 130 135 140 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170 175 Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 180 185
190 Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly
195 200 205 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn
Arg Glu 210 215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile
Tyr Gln Ile Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala Ser Ser
Leu Val Leu Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe Trp Met
Cys Ser Asn Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile 275
109275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 109Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40 45 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 50 55 60 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 65 70 75 80
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 85
90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys
Met 100 105 110 Asn Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys
Leu Glu Lys 115 120 125 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Asp Ile 130 135 140 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170 175 Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 180 185 190 Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 195 200 205
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 210
215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile
Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile 275
110275PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 110Ser Asn Thr Lys Cys Gln Thr Pro Leu Gly
Ala Ile Asn Ser Ser Leu 1 5 10 15 Pro Tyr Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro Lys Tyr 20 25 30 Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn Thr Pro 35 40 45 Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 50 55 60 Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Gln 65 70 75 80
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn 85
90 95 Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Thr Val Ile Glu Lys
Met 100 105 110 Tyr Ile Gln Phe Thr Ala Val Gly Tyr Glu Phe Asn Lys
Leu Glu Lys 115 120 125 Arg Met Glu Tyr Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Tyr Ile 130 135 140 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 145 150 155 160 Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 165 170 175 Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 180 185 190 Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg Asn Gly 195 200 205
Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg Glu 210
215 220 Lys Val Asp Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile
Leu 225 230 235 240 Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
Leu Val Ser Leu 245 250 255 Gly Ala Ile Ser Phe Trp Met Cys Ser Asn
Gly Ser Leu Gln Cys Arg 260 265 270 Ile Cys Ile 275
111566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 111Met Lys Ala Lys Leu Leu Val Leu Leu Cys
Ala Leu Ser Ala Thr Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr
His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu
Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp
Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile 50 55 60 Ala Pro
Leu Gln Leu Gly Lys Cys Ser Ile Ala Gly Trp Ile Leu Gly 65 70 75 80
Asn Pro Glu Cys Glu Ser Leu Val Ser Lys Lys Ser Trp Ser Tyr Ile 85
90 95 Ala Glu Thr Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Tyr
Phe 100 105 110 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val
Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Arg Ser
Trp Pro Lys His Asn 130 135 140 Val Thr Arg Gly Val Thr Ala Ser Cys
Ser His Lys Gly Lys Ser Ser 145 150 155 160 Phe Tyr Arg Asn Leu Leu
Trp Leu Thr Glu Lys Asn Gly Ser Tyr Pro 165 170 175 Asn Leu Ser Lys
Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val 180 185 190 Leu Trp
Gly Val His His Pro Ser Asn Ile Glu Asp Gln Lys Thr Ile 195 200 205
Tyr Arg Lys Glu Asn Ala Tyr Val Ser Val Val Ser Ser Asn Tyr Asn 210
215 220 Arg Arg Phe Thr Pro Glu Ile Ala Glu Arg Pro Lys Val Arg Gly
Gln 225 230 235 240 Ala Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu
Pro Gly Asp Thr 245 250 255 Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Trp His Ala Phe 260 265 270 Ala Leu Asn Arg Gly Phe Gly Ser
Gly Ile Ile Thr Ser Asn Ala Ser 275 280 285 Met Asp Glu Cys Asp Thr
Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn 290 295 300 Ser Ser Leu Pro
Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305 310 315 320 Pro
Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg 325 330
335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr
Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn
Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe
Thr Ala Val Gly Lys Glu Phe Asn Lys 405 410 415 Leu Glu Lys Arg Met
Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu
Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455
460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met
Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser
Glu Glu Ser Lys Leu 500 505 510 Asn Arg Glu Lys Ile Asp Gly Val Lys
Leu Glu Ser Met Gly Val Tyr 515 520 525 Gln Ile Leu Ala Ile Tyr Ser
Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val Ser Leu Gly Ala
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys
Arg Ile Cys Ile 565 112566PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 112Met Lys Ala Lys Leu
Leu Val Leu Leu Cys Ala Phe Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Arg Leu Lys Gly Ile
50 55 60 Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile
Leu Gly 65 70 75 80 Asn Pro Lys Cys Glu Ser Leu Phe Ser Lys Glu Ser
Trp Ser Tyr Ile 85 90 95 Ala Glu Thr Pro Asn Pro Glu Asn Gly Thr
Cys Tyr Pro Gly Tyr Phe 100 105 110 Ala Asp Tyr Glu Glu Leu Arg Glu
Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe
Pro Lys Glu Ser Ser Trp Pro Asn His Thr 130 135 140 Val Thr Lys Gly
Val Thr Thr Ser Cys Ser His Asn Gly Lys Ser Ser 145 150 155 160 Phe
Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lys Asn Gly Leu Tyr Pro 165 170
175 Asn Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val
180 185 190 Leu Trp Gly Val His His Pro Ser Asn Ile Arg Asp Gln Arg
Ala Ile 195 200 205 Tyr His Thr Glu Asn Ala Tyr Val Ser Val Val Ser
Ser His Tyr Ser 210 215 220 Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg
Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Ile Asn Tyr Tyr
Trp Thr Leu Leu Glu Pro Gly Asp Thr 245 250 255 Ile Ile Phe Glu Ala
Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe 260 265 270 Ala Leu Ser
Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Ser 275 280 285 Met
Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn 290 295
300 Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly Glu Cys
305 310 315 320 Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr
Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe
Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met
Ile Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile
Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys
Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys 405 410 415
Leu Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420
425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu
Asn 435 440 445 Gly Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn
Leu Tyr Glu 450 455 460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys
Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys
Asn Asn Glu Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp
Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510 Asn Arg Gly Lys
Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525 Gln Ile
Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545
550 555 560 Gln Cys Arg Ile Cys Ile 565 113565PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
113Met Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Thr Asp
1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Asp Thr 20 25 30 Val Asp Thr Ile Phe Glu Lys Asn Val Ala Val Thr
His Ser Val Asn 35 40 45 Leu Leu Glu Asp Arg His Asn Gly Lys Leu
Cys Lys Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Lys Cys
Asn Ile Ile Gly Trp Leu Leu Gly 65 70 75 80 Asn Pro Glu Cys Asp Ser
Leu Leu Pro Ala Arg Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro
Asn Ser Glu Asn Gly Ala Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp
Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Leu 115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 130
135 140 Phe Asn Gly Val Thr Ala Ser Cys Ser His Arg Gly Lys Ser Ser
Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Lys Lys Gly Asp
Ser Tyr Pro Lys 165 170 175 Leu Thr Asn Ser Tyr Val Asn Asn Lys Gly
Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Val His His Pro Ser Ser
Ser Asp Glu Gln Gln Ser Leu Tyr 195 200 205 Ser Asn Gly Asn Ala Tyr
Val Ser Val Ala Ser Ser Asn Tyr Asn Arg 210 215 220 Arg Phe Thr Pro
Glu Ile Ala Ala Arg Pro Lys Val Lys Asp Gln His 225 230 235 240 Gly
Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 245 250
255 Ile Phe Glu Ala Thr Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
260 265 270 Leu Ser Arg Gly Phe Glu Ser Gly Ile Ile Thr Ser Asn Ala
Ser Met 275 280 285 His Glu Cys Asn Thr Lys Cys Gln Thr Pro Gln Gly
Ser Ile Asn Ser 290 295 300 Asn Leu Pro Phe Gln Asn Ile His Pro Val
Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Thr Lys
Leu Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Ile Pro Ser Ile Gln
Tyr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly
Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 355 360 365 His
Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375
380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Ile Ile Glu
385 390 395 400 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe
Asn Asn Leu 405 410 415 Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val
Asp Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Gly 435 440 445 Arg Thr Leu Asp Phe His Asp
Leu Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470
475 480 Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser Val
Arg 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser
Lys Leu Asn 500 505 510 Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser
Met Gly Val Tyr Gln 515 520 525 Ile Leu Ala Ile Tyr Ser Thr Val Ala
Ser Ser Leu Val Leu Leu Val 530 535 540 Ser Leu Gly Ala Ile Ser Phe
Trp Met Cys Ser Asn Gly Ser Leu Gln 545 550 555 560 Cys Arg Ile Cys
Ile 565 114566PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 114Met Glu Ala Arg Leu Leu Val Leu
Leu Cys Ala Phe Ala Ala Thr Asn 1 5 10 15 Ala Asp Thr Ile Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu
Glu Asp Ser His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile 50 55 60
Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Leu Leu Gly 65
70 75 80 Asn Pro Glu Cys Asp Leu Leu Leu Thr Ala Ser Ser Trp Ser
Tyr Ile 85 90 95 Val Glu Thr Ser Asn Ser Glu Asn Gly Thr Cys Tyr
Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu
Ser Ser Val Ser Ser Phe 115 120 125 Glu Lys Phe Glu Ile Phe Pro Lys
Thr Ser Ser Trp Pro Asn His Glu 130 135 140 Thr Thr Lys Gly Val Thr
Ala Ala Cys Ser Tyr Ala Gly Ala Ser Ser 145 150 155 160 Phe Tyr Arg
Asn Leu Leu Trp Leu Thr Lys Lys Gly Ser Ser Tyr Pro 165 170 175 Lys
Leu Ser Lys Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val 180 185
190 Leu Trp Gly Val His His Pro Pro Thr Gly Thr Asp Gln Gln Ser Leu
195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Ser Val Gly Ser Ser Lys
Tyr Asn 210 215 220 Arg Arg Phe Thr Pro Glu Ile Ala Ala Arg Pro Lys
Val Arg Asp Gln 225 230 235 240 Ala Gly Arg Met Asn Tyr Tyr Trp Thr
Leu Leu Glu Pro Gly Asp Thr 245 250 255 Ile Thr Phe Glu Ala Thr Gly
Asn Leu Ile Ala Pro Trp Tyr Ala Phe 260 265 270 Ala Leu Asn Arg Gly
Ser Gly Ser Gly Ile Ile Thr Ser Asp Ala Pro 275 280 285 Val His Asp
Cys Asn Thr Lys Cys Gln Thr Pro His Gly Ala Ile Asn 290 295 300 Ser
Ser Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys 305 310
315 320 Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Ala Thr Gly Leu
Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala
Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp
Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Gln Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asp Gly
Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn
Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Asn 405 410 415 Leu Glu
Arg Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430
Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435
440 445 Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Arg Asn Leu Tyr
Glu 450 455 460 Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asp
Ala Cys Met Glu Ser Val 485 490 495 Arg Asn Gly Thr Tyr Asp Tyr Pro
Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp
Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525 Gln Ile Leu Ala
Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540 Val Ser
Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555
560 Gln Cys Arg Ile Cys Ile 565 115566PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
115Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn
1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr
His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu
Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys
Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser
Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro
Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp
Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130
135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys
Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly
Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys
Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser
Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala
Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys
Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu
Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250
255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe
260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp
Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys
Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro
Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr
Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile
Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu
Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His
His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375
380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu
Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys
Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His
Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln
Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500
505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile
Tyr 515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu
Val Leu Val 530 535 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys
Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile 565
11644PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 116Arg Ser Leu Val Pro Arg Gly Ser Pro Gly
Ser Gly Tyr Ile Pro Glu 1 5 10 15 Ala Pro Arg Asp Gly Gln Ala Tyr
Val Arg Lys Asp Gly Glu Trp Val 20 25 30 Leu Leu Ser Thr Phe Leu
His His His His His His 35 40 117274PRTInfluenza A virus 117Asn Thr
Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Ser Ser Leu Pro 1 5 10 15
Tyr Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro Lys Tyr Val 20
25 30 Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Thr Pro
Ser 35 40 45 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
Ile Glu Gly 50 55 60 Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly
Tyr His His Gln Asn 65 70 75 80 Glu Gln Gly Ser Gly Tyr Ala Ala Asp
Gln Lys Ser Thr Gln Asn Ala 85 90 95 Ile Asn Gly Ile Thr Asn Lys
Val Asn Thr Val Ile Glu Lys Met Asn 100 105 110 Ile Gln Phe Thr Ala
Val Gly Lys Glu Phe Asn Lys Leu Glu Lys Arg 115 120 125 Met Glu Asn
Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp 130 135 140 Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu 145 150
155 160 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Lys
Ser 165 170 175 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys
Phe Glu Phe 180 185 190 Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser
Val Arg Asn Gly Thr 195 200 205 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn Arg Glu Lys 210 215 220 Val Asp Gly Val Lys Leu Glu
Ser Met Gly Ile Tyr Gln Ile Leu Ala 225 230 235 240 Ile Tyr Ser Thr
Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly 245 250 255 Ala Ile
Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile 260 265 270
Cys Ile 1186PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 118His His His His His His 1 5
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