U.S. patent application number 15/051370 was filed with the patent office on 2016-09-01 for immunogenic polypeptides comprising a scaffold polypeptide and a l2 polypeptide or a fragment thereof.
This patent application is currently assigned to DKFZ Deutsches Krebsforschungszentrum. The applicant listed for this patent is DKFZ Deutsches Krebsforschungszentrum. Invention is credited to Angelo BOLCHI, Martin MUELLER, Simone OTTONELLO, Ivonne RUBIO, Massimo TOMMASINO.
Application Number | 20160250316 15/051370 |
Document ID | / |
Family ID | 40394027 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160250316 |
Kind Code |
A1 |
MUELLER; Martin ; et
al. |
September 1, 2016 |
IMMUNOGENIC POLYPEPTIDES COMPRISING A SCAFFOLD POLYPEPTIDE AND A L2
POLYPEPTIDE OR A FRAGMENT THEREOF
Abstract
The present invention relates to an immunogenic polypeptide
comprising a) a scaffold polypeptide, and b) a L2 polypeptide or a
fragment of said L2 polypeptide, wherein said scaffold polypeptide
constrains the structure of said L2 polypeptide, or of a fragment
of said L2 polypeptide. Moreover, the present invention relates to
a vaccine comprising said immunogenic polypeptide. The present
invention is also concerned with a method for producing an antibody
against human papillomavirus. Also encompassed by the present
invention is an antibody obtained by carrying out the said
method.
Inventors: |
MUELLER; Martin;
(Neckargemuend, DE) ; RUBIO; Ivonne; (Heidelberg,
DE) ; TOMMASINO; Massimo; (Charly-Lyon, IT) ;
OTTONELLO; Simone; (Parma, IT) ; BOLCHI; Angelo;
(Parma, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DKFZ Deutsches Krebsforschungszentrum |
Heidelberg |
|
DE |
|
|
Assignee: |
DKFZ Deutsches
Krebsforschungszentrum
Heidelberg
DE
|
Family ID: |
40394027 |
Appl. No.: |
15/051370 |
Filed: |
February 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13140793 |
Aug 11, 2011 |
9303082 |
|
|
PCT/EP2009/067422 |
Dec 17, 2009 |
|
|
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15051370 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 37/04 20180101;
C07K 2317/34 20130101; A61K 2039/55566 20130101; C07K 16/084
20130101; A61K 2039/6031 20130101; A61P 31/20 20180101; A61K
2039/505 20130101; C12N 2710/20022 20130101; C12N 2710/20034
20130101; C07K 14/005 20130101; C12N 7/00 20130101; A61K 39/12
20130101; A61K 39/385 20130101; C07K 2319/35 20130101; A61K
2039/585 20130101; C07K 2317/76 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C07K 16/08 20060101 C07K016/08; C12N 7/00 20060101
C12N007/00; C07K 14/005 20060101 C07K014/005; A61K 39/385 20060101
A61K039/385 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2008 |
EP |
08172349.6 |
Claims
1. An immunogenic polypeptide comprising a) a scaffold polypeptide,
and b) a L2 polypeptide comprising an amino acid sequence as shown
in SEQ ID NO:1, or a fragment thereof, wherein said scaffold
polypeptide constrains the structure of said L2 polypeptide, or of
said fragment of said L2 polypeptide.
2. The immunogenic polypeptide of claim 1, wherein the scaffold
polypeptide is selected from the group consisting of thioredoxin
polypeptides, thioredoxin polypeptides derived from thermophile
bacteria, capsid polypeptides of adeno-associated viruses, the
tenth type III module of fibronectin (FN3), lipocalins, a
catalytically inactive version of Staphylococcus nuclease,
alpha-amylase inhibitors, and stefin A.
3. The immunogenic polypeptide of claim 1, wherein the scaffold
polypeptide is a thioredoxin polypeptide selected from the group
consisting of: a) a polypeptide having a sequence as shown in SEQ
ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ ID
No: 28, and b) a variant polypeptide having a sequence at least 70%
identical to the sequence shown in SEQ ID No: 24, SEQ ID No: 25,
SEQ ID No: 26, SEQ ID No: 27, or SEQ ID No: 28, wherein said
polypeptide constrains the structure of the L2 polypeptide, or
which constrains the structure of a fragment of said L2
polypeptide.
4. The immunogenic polypeptide according to claim 1, wherein the
fragment or variant of the fragment of the L2 polypeptide is
selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 31 and 32.
5. The immunogenic polypeptide according to claim 1, wherein the
fragment of the L2 polypeptide has a sequence as shown in SEQ ID
NO:2.
6. The immunogenic polypeptide according to claim 1, wherein the
polypeptide comprises a multimer of said fragment.
7. The immunogenic polypeptide according to claim 1, wherein the
immunogenic polypeptide has a sequence as shown in SEQ ID NO: 29 or
30.
8. A polynucleotide encoding the immunogenic polypeptide according
to claim 1.
9-16. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/140,793, filed Aug. 11, 2011, which is the National
Phase of International Patent Application No. PCT/EP2009/067422,
filed Dec. 17, 2009, which claims priority from European Patent
Application No. 08172349.6, filed Dec. 19, 2008. The contents of
these applications are incorporated herein by reference in their
entirety.
[0002] The present invention relates to an immunogenic polypeptide
comprising a) a scaffold polypeptide, and b) a L2 polypeptide or a
fragment of said L2 polypeptide, wherein said scaffold polypeptide
constrains the structure of said L2 polypeptide, or of the fragment
of said L2 polypeptide. Moreover, the present invention relates to
a vaccine comprising said immunogenic polypeptide. The present
invention is also concerned with a method for producing an antibody
against human papillomavirus. Also encompassed by the present
invention is an antibody obtained by carrying out the said
method.
[0003] Cervical cancer is women's second most frequent cancer
worldwide. Clinical and molecular studies have shown that certain
types of human papillomavirus (HPV), referred to as high-risk
types, are the etiological agents of this disease. Two anti-HPV
vaccines for the prophylaxis of cervical cancer have been licensed
recently by Merck (Gardasil.TM.) and GlaxoSmithKline (Cervarix.TM.)
(Schmiedeskamp et al, (2006) Human papillomavirus vaccines. Ann
Pharmacother, 40, 1344-1352). Both vaccines rely on the major
capsid protein L1 in the form of virus-like particles (VLPs) as
antigen (Roden et al., (2006) How will HPV vaccines affect cervical
cancer? Nat Rev Cancer, 6, 753-763); they protect against the HPV
types from which the L1-VLPs were derived, yet are largely
ineffective against all but the most closely related HPV types. The
two most prominent high-risk HPV types, 16 and 18, are the major
targets of both vaccines, although there is evidence for partial
cross-protection against HPV types 31 and 45 (reviewed by Muller
and Gissmann, (2007) A long way: history of the prophylactic
papillomavirus vaccine. Dis Markers, 23, 331-336; Huh and Roden,
(2008) The future of vaccines for cervical cancer. Gynecol Oncol,
109, S48-56). The limited cross-protective capacity of L1-based
vaccines, which is the main reason for the continuing effort toward
the development of improved vaccination strategies, likely reflects
the HPV type specificity of L1 neutralizing epitopes (Giroglou et
al., (2001) Immunological analyses of human papillomavirus capsids.
Vaccine, 19, 1783-1793).
[0004] Antibodies against the minor capsid protein L2 also
neutralize HPV infection and are often capable to cross-neutralize
various non-cognate virions, although with varying efficiencies
(Kondo et al. 2007, Neutralization of HPV 16, 18, 31, and 58
pseudovirions with antisera induced by immunizing rabbits with
synthetic peptides representing segments of the HPV16 minor capsid
protein L2 surface region. Virology, 358, 266-272; Gambhira, R.,
(2007) A protective and broadly cross-neutralizing epitope of human
papillomavirus L2. J Virol, 81, 13927-13931). The N-terminal region
of L2 interacts with an as yet unidentified secondary receptor on
the surface of target cells (Yang et al. (2003) Cell
surface-binding motifs of L2 that facilitate papillomavirus
infection. J Virol, 77, 3531-3541) and this interaction can be
blocked by anti-L2 antibodies. The precise identity of the L2
region involved in HPV-cell surface interaction is still a matter
of debate. This was initially proposed as the region comprised of
amino acids (aa) 108-120, and antibodies targeting this particular
L2 region were indeed shown to block viral infection in vitro
albeit at low titers (Kawana et al. (2001) Nasal immunization of
mice with peptide having a cross-neutralization epitope on minor
capsid protein L2 of human papillomavirus type 16 elicit systemic
and mucosal antibodies. Vaccine, 19, 1496-1502; Kawana et al.
(2001b) Human papillomavirus type 16 minor capsid protein L2
N-terminal region containing a common neutralization epitope binds
to the cell surface and enters the cytoplasm. J Virol, 75,
2331-2336). Subsequent experiments identified additional
neutralizing epitopes in the aa 1-88 region (Pastrana et al. (2005)
Cross-neutralization of cutaneous and mucosal Papillomavirus types
with anti-sera to the amino terminus of L2. Virology, 337, 365-372)
as well as in more extended N-terminal regions comprised of aa
11-200 and aa 18-144 (Kondo loc. cit). Perhaps the most prominent
of these N-terminal epitopes is the one located between aa 17-36.
This was identified as the target of an HPV 16 neutralizing and
protective monoclonal antibody (RG-1) as well as the major
determinant of the neutralizing activity found in sera from rabbits
and humans immunized with extended versions of L2 (aa 1-88, 11-200
or the full-length protein) (Gambhira, 2007, loc cit.). Since it
had been found that mutation of L2 amino acids 18 and 19 or of
amino acids 20 and 21 disrupted both L2 binding to the cell surface
and viral infection (Yang, R., et al. (2003). Cell surface-binding
motifs of L2 that facilitate papillomavirus infection. J. Virol.
77:3531-3541), it was concluded that the epitope recognized by the
RG-1 antibody overlaps the surface-binding motif of HPV 16 L2.
[0005] Besides the lack of precise knowledge on the most relevant
(cross) neutralizing epitope(s), a major problem with the use of L2
as a tool for HPV prophylaxis is the poor immunogenicity of the L2
protein and peptides thereof, as compared to L1-VLPs. A substantial
increase in immunogenicity has been reported lately via chemical
coupling of the HPV16 L2 peptide (17-36) to a broadly recognized T
helper epitope and to the Toll-like receptor ligand dipalmitoyl
S-glyceryl cysteine (Alphs et al. (2008) Protection against
heterologous human papillomavirus challenge by a synthetic
lipopeptide vaccine containing a broadly cross-neutralizing epitope
of L2. Proc Natl Acad Sci USA, 105, 5850-5855). Alternatively, L2
peptides have been fused to Adenovirus surface proteins (WO
2008/140474) or to other HPV proteins to increase immunogenicity
(WO 2002/070004, de Jong et al. (2002), Enhancement of human
papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in
healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6
fusion protein vaccine, Vaccine, 20(29-30):3456-3464).
[0006] A recently developed alternative strategy for increasing
peptide immunogenicity relies on the use of thioredoxin (Trx) as a
scaffold protein with the ability to constrain the structure of
single-copy as well as multimeric (tandemly repeated) peptide
epitopes inserted within its surface-exposed active site loop
(Moreno et al. (2007) Conformation-sensitive antibodies against
Alzheimer amyloid-beta by immunization with a
thioredoxin-constrained B-cell epitope peptide. J Biol Chem, 282,
11436-11445).
[0007] Thus, the L1 polypeptide is highly immunogenic and
antibodies against it show only a limited cross-protective
capacity, whereas antibodies against the L2 polypeptide are capable
of cross-neutralizing various HPV genotypes. The L2 polypeptide,
however has only limited immunogenicity.
[0008] Therefore, immunogenic polypeptides that are highly
immunogenic and allow for a cross-neutralization of various HPV
genotypes without the drawbacks as referred to above are highly
required.
[0009] The technical problem underlying the present invention can
be seen as the provision of means and methods for complying with
the aforementioned needs.
[0010] The technical problem is solved by the embodiments
characterized in the claims and herein below.
[0011] Accordingly, the present invention relates to an immunogenic
polypeptide comprising [0012] a) a scaffold polypeptide, and [0013]
b) a L2 polypeptide having an amino acid sequence as shown in SEQ
ID NO:1, or a fragment of said L2 polypeptide, [0014] wherein said
scaffold polypeptide constrains the structure of said L2
polypeptide, or of the fragment of said L2 polypeptide.
[0015] The term "polypeptide" as used herein relates to a polymer
comprising amino acids linked together by peptide bonds. The term
"immunogenic polypeptide" is understood by the skilled person.
Immunogenic polypeptides, preferably, elicit protective immune
response in a host, preferably, in a human. The immunogenic
polypeptide in the context of the present invention, preferably,
shall allow for establishing or improving immunity to infection
with various HPV genotypes. Preferably, the immunogenic polypeptide
according to the present invention allows for establishing or
improving immunity to infection with human papillomavirus genotypes
16, 18, 31, 45 and 58. Preferably, the said polypeptide also allows
for establishing or improving immunity to infection with human
papillomavirus genotypes 6, 52, 2, 27, 57 and/or 11. Immunogenic
polypeptides are preferred reagents for vaccine compositions.
[0016] The term "L2 polypeptide", preferably, refers to the
N-terminal region of the full-length L2 polypeptide of HPV 16
(human papillomavirus 16). The full-length L2 is one of the two
capsid proteins of HPV 16 and is frequently also referred to as
minor capsid protein. Together with the major capsid protein, L1,
the full-length L2 polypeptide forms viral capsids. The L2
polypeptide in the context of the present invention, preferably,
comprises the N-terminal amino acids 1 to 120 of the HPV16 L2
polypeptide as shown in SEQ ID NO:1.
[0017] The term "fragment" as used herein, preferably, refers to a
sub-polypeptide of the L2 polypeptide (as shown in SEQ ID NO:1).
Preferably, said fragment comprises at least 7, at least 10, at
least 12, at least 15, or at least 20 consecutive amino acid
residues of said L2 polypeptide. Preferred fragments of the L2
polypeptide have an amino acid sequence as shown in SEQ ID NO:2
(KTCKQAGTCPPDIIPKVEG), as shown in SEQ ID NO:3 (KTCKQAGTCPPD), as
shown in SEQ ID NO:4 (TCKQAGTCPPD), as shown in SEQ ID NO:5
(CKQAGTCPPD), as shown in SEQ ID NO:6 (TCKQAGTCPP), as shown in SEQ
ID NO:7 (CKQAGTCPP), as shown in SEQ ID NO:8 (DIIPKVEGKT), as shown
in SEQ ID NO:9 (TGYIPLGTR).
[0018] The most preferred fragments in the context of the present
invention are fragments having a sequence as shown in SEQ ID NO:2
(KTCKQAGTCPPDIIPKVEG, amino acids 20 to 38 of the L2 polypeptide as
shown in SEQ ID NO:1)), or as shown in SEQ ID NO:5 (CKQAGTCPPD,
amino acids 22 to 31 of the L2 polypeptide as shown in SEQ ID
NO:1).
[0019] Preferably, the terms "polypeptide" "L2 polypeptide" and
"fragment of the L2 polypeptide", respectively, shall also
encompass variants of said polypeptide, L2 polypeptide or variants
of said fragment of said L2 polypeptide, respectively. Such
variants have essentially the same immunological properties as the
specific polypeptides, respectively. In particular, they share the
same immunological properties if they are detectable by the same
specific assays referred to in this specification, e.g., by ELISA
assays using polyclonal or monoclonal antibodies specifically
recognizing the said polypeptides, respectively. Moreover, it is to
be understood that a variant as referred to in accordance with the
present invention shall have an amino acid sequence which differs
due to at least one amino acid substitution, deletion and/or
addition wherein the amino acid sequence of the variant is still,
preferably, at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%,
98%, or 99% identical with the amino sequence of the specific
polypeptide. The degree of identity between two amino acid
sequences can be determined by algorithms well known in the art.
Preferably, the degree of identity is to be determined by comparing
two optimally aligned sequences over a comparison window, where the
fragment of amino acid sequence in the comparison window may
comprise additions or deletions (e.g., gaps or overhangs) as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment. The percentage is
calculated by determining the number of positions at which the
identical amino acid residue occurs in both sequences to yield the
number of matched positions, dividing the number of matched
positions by the total number of positions in the window of
comparison and multiplying the result by 100 to yield the
percentage of sequence identity. Optimal alignment of sequences for
comparison may be conducted by the local homology algorithm of
Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson and Lipman
Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual
inspection. Given that two sequences have been identified for
comparison, GAP and BESTFIT are preferably employed to determine
their optimal alignment and, thus, the degree of identity.
Preferably, the default values of 5.00 for gap weight and 0.30 for
gap weight length are used. Variants referred to above may be
allelic variants or any other species specific homologs, paralogs,
or orthologs. Further included are variants which differ due to
posttranslational modifications such as phosphorylation or
myristoylation.
[0020] As mentioned above, in a preferred embodiment of the present
invention the fragment of the L2 polypeptide comprised by the
scaffold polypeptide of the immunogenic polypeptide has a sequence
as shown in SEQ ID NO:2 (KTCKQAGTCPPDIIPKVEG), or as shown in SEQ
ID NO:3 (KTCKQAGTCPPD), or as shown in SEQ ID NO:4 (TCKQAGTCPPD),
or as shown in SEQ ID NO:5 (CKQAGTCPPD), or as shown in SEQ ID NO:6
(TCKQAGTCPP), or a sequence as shown in SEQ ID NO:7 (CKQAGTCPP), or
a sequence as shown in SEQ ID NO:31 (IIPKVEGKT), or a sequence as
shown in SEQ ID NO:32 (IPKVEGKT). Since it has been shown in the
context of the present invention that the Alanine (A) residue
comprised by the aforementioned fragments can be replaced with
other amino acid residues (particularly, with a Glycine (G)
residue) without significantly affecting the immunogenicity of the
polypeptide according to the invention as well as the neutralizing
capacity of the antibodies against the said immunogenic polypeptide
(see Examples), variants of the aforementioned fragments preferably
have the amino acid sequence as shown in SEQ ID NO:10
(KTCKQXGTCPPDIIPKVEG), or as shown in SEQ ID NO:11 (KTCKQXGTCPPD),
or as shown in SEQ ID NO:12 (TCKQXGTCPPD), or as shown in SEQ ID
NO:13 (CKQXGTCPPD), or as shown in SEQ ID NO:14 (TCKQXGTCPP), or a
sequence as shown in SEQ ID NO:15 (CKQXGTCPP). Preferably, X
represents a Glycine (G) or an Alanine (A) residue. Moreover,
experiments with the aforementioned fragments of the L2 polypeptide
have shown that the most crucial amino acid residues for
immunogenicity and for the generation of cross-neutralizing
antibodies were amino acid residues 22 to 24 (CKQ) and 26 to 31
(GTCPPD) of the L2 polypeptide as shown in SEQ ID NO:1 (see
Examples). Accordingly, the most preferred variant of a fragment of
the L2 polypeptide has a sequence as shown in SEQ ID NO: 13),
CKQXGTCPPD).
[0021] In one preferred embodiment of the present invention, the
immunogenic polypeptide comprises a multimer of the L2 polypeptide
or a fragment thereof (or a variant of said L2 polypeptide or a
variant of any fragment thereof). Thus, the immunogenic polypeptide
shall comprise more than one L2 polypeptide or more than one
fragment of the L2 polypeptide. It is particularly envisaged that
the immunogenic polypeptide comprises more than one fragment of the
L2 polypeptide (or variants thereof). Preferably, the immunogenic
polypeptide comprises multimers of 2 to 15 fragments of the L2
polypeptide, and more preferably multimers of 3 to 9 (and, thus, of
3, 4, 5, 6, 7, 8 or 9) fragments of the L2 polypeptide. Most
preferably, said immunogenic polypeptide comprises multimers of
three or four fragments of the L2 polypeptide. Preferably, said
fragments are directly linked together. More preferably, said
fragments are linked via a linker peptide (for an explanation of
the term "linker peptide", see herein below). Preferably, if the
immunogenic polypeptide comprises more than one fragment of the L2
polypeptide, the fragments shall have the same or essentially the
same sequence. However, it is also contemplated that the multimer
comprises various fragments (or variants thereof) of the L2
polypeptide.
[0022] Other preferred L2 fragments (or variants thereof) are
selected from the group consisting of SEQ ID NO: 33 to SEQ ID
NO:479. SEQ ID NO:33 to SEQ ID NO:479 are also shown in Table 1.
The sequences as shown in SEQ ID NO:33 to SEQ ID NO:79 and in SEQ
ID NO:486 to SEQ ID NO:489 are variants of the L2 fragment
KTCKQAGTCPPDIIPKVEG as shown in SEQ ID NO:2; the sequences as shown
in SEQ ID NO:80 to SEQ ID NO:112 and in SEQ ID NO: 490 are variants
of the L2 fragment KTCKQAGTCPPD as shown in SEQ ID NO:3; the
sequences as shown in SEQ ID NO:113 to SEQ ID NO:139 are variants
of the L2 fragment TCKQAGTCPPD as shown in SEQ ID NO:4; the
sequences as shown in SEQ ID NO:140 to SEQ ID NO:161 are variants
of the L2 fragment CKQAGTCPPD as shown in SEQ ID NO:5; the
sequences as shown in SEQ ID NO:162 to SEQ ID NO:188 are variants
of the L2 fragment TCKQAGTCPP as shown in SEQ ID NO:6; the
sequences as shown in SEQ ID NO:189 to SEQ ID NO:210 are variants
of the L2 fragment CKQAGTCPP as shown in SEQ ID NO:7; the sequences
as shown in SEQ ID NO:211 to SEQ ID NO:238 are variants of the L2
fragment DIIPKVEGKT as shown in SEQ ID NO:8; the sequences as shown
in SEQ ID NO:239 to SEQ ID NO:266 are variants of the L2 fragment
IIPKVEGKT as shown in SEQ ID NO:31; the sequences as shown in SEQ
ID NO:267 to SEQ ID NO:293 are variants of the L2 fragment IPKVEGKT
as shown in SEQ ID NO:32; the sequences as shown in SEQ ID NO:294
to SEQ ID NO:301 are variants of the L2 fragment TGYIPLGTR as shown
in SEQ ID NO:9; the sequences as shown in SEQ ID NO:302 to SEQ ID
NO:348 are variants of the L2 fragment KTCKQXGTCPPDIIPKVEG as shown
in SEQ ID NO:10; the sequences as shown in SEQ ID NO:349 to SEQ ID
NO:381 are variants of the L2 fragment KTCKQXGTCPPD as shown in SEQ
ID NO:11; the sequences as shown in SEQ ID NO: 382 to SEQ ID NO:
408 are variants of the L2 fragment TCKQXGTCPPD as shown in SEQ ID
NO:12; the sequences as shown in SEQ ID NO: 409 to SEQ ID NO: 430
are variants of the L2 fragment CKQXGTCPPD as shown in SEQ ID
NO:13; the sequences as shown in SEQ ID NO: 431 to SEQ ID NO: 457
are variants of the L2 fragment TCKQXGTCPP as shown in SEQ ID
NO:14; the sequences as shown in SEQ ID NO:458 to SEQ ID NO:479 are
variants of the L2 fragment CKQXGTCPP as shown in SEQ ID NO:15.
[0023] As mentioned above, the immunogenic polypeptide shall also
comprise a linker peptide or more than one linker peptide. Said
linker peptide, preferably, shall prevent the formation of
junctional epitopes. Preferably, the linker peptide is positioned
at the C- and/or N-Terminus of the L2 polypeptide, or of the
fragment (or of the variant thereof). If the immunogenic
polypeptide comprises more than one fragment of the L2 polypeptide
(or more than one variant of said fragment), it is particularly
contemplated that the immunogenic polypeptide comprises a linker
peptide between the various fragments (or variants thereof). For
example, SEQ ID NO:21 shows a multimer of L2 fragments with a
GGP-linker (SEQ ID NO:16) inserted between any one of the L2
fragments.
[0024] Preferably, said linker has a length of 1 to 5 amino acids.
The person skilled in the art knows how to select suitable linker
peptides. Preferably, said 1 to 5 amino acids comprised by said
linker peptide are selected from the group consisting of Glycine
(G), Proline (P) or Serine (S). A particularly preferred linker
peptide comprises the amino acid sequence GGP (SEQ ID NO: 16).
However, also other linkers can be used such as GPGP (SEQ ID NO:
17), GPGPG (SEQ ID NO: 18), or SGSG (SEQ ID NO: 19). Preferably,
said linker peptide is positioned at the junction of the scaffold
polypeptide and the fragment of the L2 polypeptide and/or at the
junction of two L2 fragments (or variants thereof). Thus, said
linker peptide can be positioned either N-terminally or
C-terminally from the L2 fragment (or variant thereof) or both.
[0025] A preferred multimer of a fragment of the L2 polypeptide
comprised by the immunogenic polypeptide according to the invention
has an amino acid sequence such as the one shown in SEQ ID NO: 20,
or in SEQ ID NO: 21, or a sequence as shown in SEQ ID NO: 22. Other
preferred multimers are multimers comprising combinations of
different homooligomers of fragments of the L2 polypeptide (e.g. a
trimer of SEQ ID NO:2 linked to a trimer of SEQ ID NO:487 linked to
a trimer of SEQ ID NO:487). More preferably, the L2 polypeptides
comprised in said multimers are separated by linker sequences, see
e.g. SEQ ID NO: 491. Also preferred are repeats of heterooligomers
of fragments of the L2 polypeptide. A heterooligomer comprises e.g.
SEQ ID NO:2 linked to SEQ ID NO:487 linked to SEQ ID NO:77, the
corresponding multimer comprising e.g. said heterooligomer repeated
three times. More preferably, the L2 polypeptides comprised in said
multimers are separated by linker sequences, see e.g. SEQ ID
NO:492.
[0026] The L2 polypeptide, or fragment thereof (or the variant of
said L2 polypeptide or of the fragment thereof, or the
corresponding multimers, see elsewhere herein) shall be comprised
by a scaffold polypeptide which constrains the structure of the L2
polypeptide, or the fragment thereof (or the respective
variants).
[0027] The term "constraining" as used herein, preferably, means
that the L2 polypeptide, or the fragment thereof (or the respective
variants) that is comprised by the scaffold protein is present in a
conformation that mimics its natural conformation. Preferably, said
L2 polypeptide, or the fragment thereof (or the respective variant)
is kept by the scaffold polypeptide in a fixed conformation, when
constrained.
[0028] Any scaffold polypeptide being capable of constraining the
structure of said L2 polypeptide, or of the fragment of said L2
polypeptide, preferably, can be used for the production of the
immunogenic polypeptide according to the invention.
[0029] Preferably, the scaffold polypeptide is selected from the
group consisting of thioredoxin, capsid polypeptides of
adeno-associated viruses (e.g. AAV2, GenBank Accession No.,
NC_001401.2, GI:110645916; AAV8 GenBank Accession No., NC_006261.1,
GI:51949963; AAV7 GenBank Accession No., NC_006260.1, GI:51949960),
the tenth type III module of fibronectin (FN3, GenBank Accession
No. 1TTF_A; GI:157834026, with insertion of the L2 polypeptide,
fragment or variant thereof within the exposed PAVTVR (SEQ ID NO:
480) or GRGDSPASS (SEQ ID NO: 481) loop sites), lipocalins
(particularly, the bilin-binding protein from Pieris brassicae,
GenBank Accession No. CAA54063.1, GI:434995, with insertion of the
L2 polypeptide, fragment or variant thereof within the PNSVEKY (SEQ
ID NO: 482), IHGKE (SEQ ID NO: 483), TYGGVTK (SEQ ID NO: 484)
and/or YDEDKKGH loop sites), a catalytically inactive version of
Staphylococcus nuclease (e.g., GenBank Accession No. 2SNS_A,
2SNS_A, GI:157836360, with peptide insertion within the YKGQP (SEQ
ID NO: 485) loop site); an alpha-amylase inhibitor, preferably
tendamistat (GenBank Accession No. CAA00655.1, GI:413044, with
peptide insertion within the EDD and/or IGSHG loop sites); or
stefin A (GenBank Accession No. P01040.1, GI:118177, with insertion
of the L2 polypeptide, fragment or variant thereof within the KSL
loop site).
[0030] In one preferred embodiment of the present invention,
however, the scaffold protein is a thioredoxin polypeptide or a
variant thereof.
[0031] Thioredoxin polypeptides are the major cellular disulfide
redox components and serve as electron donors for enzymes such as
ribonucleotide reductases, thioredoxin peroxidases and methionine
sulfoxide reductases. Thioredoxins have an alpha/beta structure
with two disulfide bondable cysteine residues. Thioredoxins are
ubiquitous polypeptides and were shown to be present in most
organisms (for a review see Amer and Holmgren, Physiological
functions of thioredoxin and thioredoxin reductase, European
Journal of Biochemistry, Volume 267 Issue 20, Pages 6102-6109). The
thioredoxin polypeptide in the context of the present invention may
be derived from any organism. Preferably, the thioredoxin
polypeptide comprises the so called thioredoxin display site CGPC
(SEQ ID NO: 23). The thioredoxin display site, also known as
thioredoxin motif or as dithiol/disulfide active site, is a highly
conserved motif amongst thioredoxin polypeptides.
[0032] Preferably, said thioredoxin polypeptide is selected from
the group consisting of prokaryotic and eukaryotic thioredoxin
polypeptides, or any other thioredoxin or thioredoxin-like protein,
or proteins harbouring a thioredoxin (TRX) Pfam domain, bearing the
conserved CGPC (SEQ ID NO: 23), or a CGXC, or a CXXC sequence motif
(e.g., gi|40253454; gi|77456671; gi|31543902). More preferably,
said thioredoxin polypeptide is selected from the group consisting
of bacterial, animal and plant thioredoxin polypeptides Even more
preferably, the thioredoxin polypeptide is a Escherichia coli
thioredoxin as shown in SEQ ID NO: 24 (which shows 100% identity
with the thioredoxin polypeptide of Salmonella typhi), or the
homologous thioredoxin polypeptides from Salmonella enterica (SEQ
ID NO: 25), mouse (SEQ ID NO: 26), rabbit (SEQ ID NO: 27), human
(SEQ ID NO: 28), or any other thioredoxin or thioredoxin-like
protein as shown in SEQ ID NO: 17. Also included are oligomers of
said thioredoxin polypeptides, i.e. fusion polypeptides comprising
at least two copies of thioredoxin polypeptides, e.g. dimers or
trimers, wherein the C-terminus of one copy of a thioredoxin
polypeptide is linked to the N-terminus of the following copy of a
thioredoxin polypeptide. Preferably, at least one of the
thioredoxin polypeptides comprises at least one L2 peptide inserted
within the display site. More preferably, in said oligomers the
thioredoxin polypeptides are separated by linker peptides, see e.g.
SEQ ID NO:497 and SEQ ID NO: 498.
[0033] Preferably, the L2 polypeptide, or the fragment of said L2
polypeptide (or multimer or fragment thereof) is positioned within
the so called "display site" of thioredoxin. Thus, the said L2
polypeptide or fragment thereof, preferably is positioned between
the C and the G, or between the G and the P, or between the P and
the C residues of the display site sequence CGPC (SEQ ID NO: 23) of
the thioredoxin polypeptide. Also contemplated by the present
invention is positioning the L2 polypeptide or fragment thereof
adjacent to the display site, preferably, between any pair of amino
acid residues located up to 20, up to 10, or up to 5 amino acid
residues upstream or downstream from the display site.
[0034] The term "thioredoxin polypeptide" also includes variants of
the thioredoxin polypeptide. The explanations of the term "variant"
made elsewhere applies mutatis mutandis.
[0035] In a preferred embodiment the thioredoxin polypeptide is
selected from the group consisting of [0036] a) a polypeptide
having a sequence as shown in SEQ ID No: 24, SEQ ID No: 25, SEQ ID
No: 26, SEQ ID No: 27, or SEQ ID No: 28 (or any other thioredoxin
polypeptide as recited herein); and [0037] b) a variant polypeptide
having a sequence at least 70% identical to the sequence shown in
SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, or SEQ
ID No: 28 (or any other thioredoxin polypeptide as recited herein),
[0038] wherein said polypeptide constrains the structure of the L2
polypeptide, or which constrains the structure of a fragment of
said L2 polypeptide (or of a variant thereof).
[0039] As set forth above the thioredoxin polypeptide in the
context of the present invention, preferably, shall comprise the
thioredoxin display site.
[0040] In another preferred embodiment the thioredoxin polypeptide
is derived from a thermophile bacterium. The use of a thioredoxin
polypeptide from a thermophile bacterium allows for storage of the
immunogenic polypeptides, e.g., at room temperature (instead of
storing said polypeptide, e.g., at 4.degree. C. or at even lower
temperatures). Storing the immunogenic polypeptide, e.g., at
20.degree. C. is, particularly, advantageous if said polypeptide is
used as a vaccine since it allows the distribution of the
polypeptide even in regions where cooling systems are not
available.
[0041] Thermophile bacteria are known to grow at elevated
temperatures (>50.degree. C.), particularly in and/or around
geothermal vents in marine or aquatic environments. A variety of
termophile bacteria is known in the art. Preferred thermophile
bacteria in the context of the present invention are
Archaebacteria, particularly Methanosaeta thermophila,
Archaeoglobus fulgidus, Metallosphaera sedula, Sulfolobus
solfataricus, Sulfolobus tokodaii, Sulfolobus acidocaldarius,
Metallosphaera sedula, Thermofilum pendens, Picrophilus torridus,
Caldivirga maquilingensis. The amino acid sequence of thioredoxin
polypeptides of a variety of thermophile bacteria is well known in
the art. Preferred thiorexodin polypeptides derived from
thermophile bacteria have an amino acid sequence as shown in
GenBank-Accession Numbers: Methanosaeta thermophila (gi|116754023,
YP_843141; gi|116754438, YP_843556); Archaeoglobus fulgidus
(gi|11498883, NP_070112; gi|11499727, NP_070969); Metallosphaera
sedula (gi|146304377, YP_001191693; gi|146303559, YP_001190875);
Sulfolobus solfataricus (gi|15897303, NP_341908; gi|15899007,
NP_343612); Sulfolobus tokodaii (gi|15922449, NP_378118;
gi|15921676, NP_377345); Sulfolobus acidocaldarius (gi|70605894,
YP_254764.1; gi|70607552, YP_256422.1; gi|70607229, YP_256099);
Thermofilum pendens (gi|119720035, YP_920530); Picrophilus torridus
(gi|48477193, YP_022899); Caldivirga maquilingensis (gi|159040636,
YP_001539888). Also included are thioredoxin polypeptides from
Pyrococcus furiosus (SEQ ID NO: 493), Thermococcus kodakarensis
(SEQ ID NO: 494), Thermococcus onnurineus (SEQ ID NO: 495), and
Thermococcus sibiricus (SEQ ID NO: 496).
[0042] In a preferred embodiment the immunogenic polypeptide
further comprises a polypeptide that further stimulates (enhances)
immunogenicity of said immunogenic polypeptides. Such polypeptides
stimulating immunogenicity are well known in the art. Preferred
stimulating polypeptides are C4 bp (Complement component 4 binding
protein) and MDC/CCL22 (Macrophage-Derived Chemokine_CC motif
ligand 22. It is to be understood that the immunogenic polypeptide
and the stimulating polypeptide are fused in frame. Preferably, the
stimulating polypeptide is fused to the N- or C-terminus of to the
immunogenic polypeptide
[0043] Preferably, the immunogenic polypeptide according to the
invention is a polypeptide having an amino acid sequence as shown
in SEQ ID NO: 29, or SEQ ID NO: 30.
[0044] The immunogenic polypeptide as shown in SEQ ID NO:29
comprises a multimer of 3 of the L2 fragment having a sequence as
shown in SEQ ID NO:2, said fragments being connected by a linker
peptide having a sequence as shown in SEQ ID NO:16.
[0045] The immunogenic polypeptide as shown in SEQ ID NO:30
comprises a multimer of 9 of the L2 fragment having a sequence as
shown in SEQ ID NO:2, said fragments being connected by a linker
peptide having a sequence as shown in SEQ ID NO:16.
[0046] The sequences as shown in SEQ ID NO: 29, and SEQ ID NO: 30
comprise two hexahistidine-tags for purification of said
polypeptides. It is to be understood that these tag do not
contribute to the immunogenicity of said polypeptide and, thus, can
be omitted.
[0047] Advantageously, it was shown in the studies underlying the
present invention that an immunogenic polypeptide comprising a
scaffold polypeptide and a L2 polypeptide or a fragment thereof,
wherein said scaffold protein constrains the structure of said
polypeptide or of said fragment, confers strong immunogenicity and
induces strong neutralizing responses against HPV16 as well as
strong cross-neutralizing responses against other HPV genotypes
such as HPV18, HPV31, HPV45 and HPV58. Particularly, it was shown
that a thioredoxin polypeptide that comprises within its display
site the L2 polypeptide or a fragment has a strong immunogenicity
and allows for strong neutralizing as well as cross-neutralizing
responses (see Examples). The immunogenicity and
(cross-)neutralizing response was further enhanced when multimers
of the L2 polypeptides or fragments thereof were inserted within
the display site of the thioredoxin polypeptide (see Examples).
[0048] The immunogenic polypeptide according to the present
invention is of advantage over prior art polypeptides, since the
polypeptides as disclosed in prior art have a low immunogenicity or
only induce strong but not cross-neutralizing responses. For
example, L2 based peptides that are disclosed in the art are poorly
immunogenic whereas L1 based peptides have a limited
cross-protective capacity. Thus, the immunogenic polypeptide
according to the present invention allows for the production of
vaccines against a broad range of HPV genotypes, particularly
high-risk HPV genotypes.
[0049] Moreover, the present invention relates to a polynucleotide
encoding the immunogenic polypeptide according to the present
invention.
[0050] The polynucleotides of the present invention may contain
further nucleic acid sequences as well. Specifically, the
polynucleotides of the present invention may encode fusion proteins
wherein one partner of the fusion protein is a polypeptide being
encoded by a nucleic acid sequence recited above. Such fusion
proteins may comprise as additional part peptide sequences for
monitoring expression (e.g., green, yellow, blue or red fluorescent
proteins, alkaline phosphatase and the like) or so called "tags"
which may serve as a detectable marker or as an auxiliary measure
for purification purposes. Tags for the different purposes are well
known in the art and comprise FLAG-tags, 6-histidine-tags, MYC-tags
and the like.
[0051] The term "polynucleotide" as used herein refers to a linear
or circular nucleic acid molecule. It encompasses DNA as well as
RNA molecules. The polynucleotide of the present invention shall be
provided, preferably, either as an isolated polynucleotide (i.e.
isolated from its natural context) or in genetically modified form.
The term encompasses single as well as double stranded
polynucleotides. Moreover, comprised are also chemically modified
polynucleotides including naturally occurring modified
polynucleotides such as glycosylated or methylated polynucleotides
or artificially modified derivatives such as biotinylated
polynucleotides. The polynucleotide of the present invention is
characterized in that it shall encode a polypeptide as referred to
above. The polynucleotide, preferably, has a specific nucleotide
sequence as mentioned above. Moreover, due to the degeneracy of the
genetic code, polynucleotides are encompassed which encode a
specific amino acid sequence as recited above.
[0052] Moreover, the term "polynucleotide" as used in accordance
with the present invention further encompasses variants of the
aforementioned specific polynucleotides. Said variants may
represent orthologs, paralogs or other homologs of the
polynucleotide of the present invention. The polynucleotide
variants, preferably, comprise a nucleic acid sequence
characterized in that the sequence can be derived from the
aforementioned specific nucleic acid sequences by at least one
nucleotide substitution, addition and/or deletion whereby the
variant nucleic acid sequence shall still encode a polypeptide
having the activity as specified above (constraining the L2
polypeptide or a fragment thereof). Variants also encompass
polynucleotides comprising a nucleic acid sequence which is capable
of hybridizing to the aforementioned specific nucleic acid
sequences, preferably, under stringent hybridization conditions.
These stringent conditions are known to the skilled worker and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example for
stringent hybridization conditions are hybridization conditions in
6.times. sodium chloride/sodium citrate (=SSC) at approximately
45.degree. C., followed by one or more wash steps in 0.2.times.SSC,
0.1% SDS at 50 to 65.degree. C. The skilled worker knows that these
hybridization conditions differ depending on the type of nucleic
acid and, for example when organic solvents are present, with
regard to the temperature and concentration of the buffer. For
example, under "standard hybridization conditions" the temperature
differs depending on the type of nucleic acid between 42.degree. C.
and 58.degree. C. in aqueous buffer with a concentration of 0.1 to
5.times.SSC (pH 7.2). If an organic solvent is present in the above
mentioned buffer, for example 50% formamide, the temperature under
standard conditions is approximately 42.degree. C. The
hybridization conditions for DNA:DNA hybrids are preferably for
example 0.1.times.SSC and 20.degree. C. to 45.degree. C.,
preferably between 30.degree. C. and 45.degree. C. The
hybridization conditions for DNA:RNA hybrids are preferably, for
example, 0.1.times.SSC and 30.degree. C. to 55.degree. C.,
preferably between 45.degree. C. and 55.degree. C. The above
mentioned hybridization temperatures are determined for example for
a nucleic acid with approximately 100 bp (=base pairs) in length
and a G+C content of 50% in the absence of formamide. The skilled
worker knows how to determine the hybridization conditions required
by referring to textbooks such as the textbook mentioned above, or
the following textbooks: Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985,
"Nucleic Acids Hybridization: A Practical Approach", IRL Press at
Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential
Molecular Biology: A Practical Approach", IRL Press at Oxford
University Press, Oxford. Alternatively, polynucleotide variants
are obtainable by PCR-based techniques such as mixed
oligonucleotide primer-based amplification of DNA, i.e. using
degenerated primers against conserved domains of the polypeptides
of the present invention. Conserved domains of the polypeptide of
the present invention may be identified by a sequence comparison of
the nucleic acid sequence of the polynucleotide or the amino acid
sequence of the polypeptide of the present invention with sequences
of other members of the enzyme families referred to in accordance
with this invention. Oligonucleotides suitable as PCR primers as
well as suitable PCR conditions are described in the accompanying
Examples. As a template, DNA or cDNA from bacteria, fungi, plants
or animals may be used. Further, variants include polynucleotides
comprising nucleic acid sequences which are at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98% or at least 99% identical to the specific nucleic acid
sequences. Moreover, also encompassed are polynucleotides which
comprise nucleic acid sequences encoding amino acid sequences which
are at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98% or at least 99% identical to
the specific amino acid sequences referred to herein. The percent
identity values are, preferably, calculated over the entire amino
acid or nucleotide sequence region. A series of programs based on a
variety of algorithms is available to the skilled worker for
comparing different sequences. In this context, the algorithms of
Needleman and Wunsch or Smith and Waterman give particularly
reliable results. To carry out the sequence alignments, the program
PileUp (Higgins 1989, CABIOS, 5 1989: 151-153) or the programs Gap
and BestFit (Needleman 1970, J. Mol. Biol. 48; 443-453 and Smith
198, Adv. Appl. Math. 2: 482-489), which are part of the GCG
software packet from Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711, version 1991, are to be used. The
sequence identity values recited above in percent (%) are to be
determined, preferably, using the program GAP over the entire
sequence region with the following settings: Gap Weight: 50, Length
Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000,
which, unless otherwise specified, shall always be used as standard
settings for sequence alignments.
[0053] Moreover, the present invention relates to a vaccine
comprising the immunogenic polypeptide according to the
invention.
[0054] The term "vaccine" as used herein, preferably, relates to a
composition which--when administered to an animal, preferably a
human--elicits an immune response against various HPV genotypes.
Thus, administering said vaccine would stimulate the immune system
and establish or improve immunity to infection with various HPV
genotypes. Preferably, the vaccine according to the present
invention allows for establishing or improving immunity to
infection with human papillomavirus genotypes 16, 18, 31, 45 and
58. Preferably, the vaccine according to the present invention also
allows for establishing or improving immunity to infection with
human papillomavirus genotypes 6, 52, 2, 27, 57 and/or 11. It is to
be understood that the vaccine according to the present invention
may comprise further components.
[0055] A preferred further component is an adjuvant. Adjuvants are
compounds which may not elicit an immune response when administered
to the host alone but which may further enhance the immune response
of the host when administered together with the immunogenic
polypeptides. It is known in the art that adjuvants may act as
surfactants which promote concentration of immunogenic polypeptides
over a large surface area, or may have immunostimulatory
properties.
[0056] Preferred adjuvants in the context of the present invention
are muramyl dipeptide, saponins such as QS21 and Quil A,
monophosphoryl lipid A, mineral oil/surfactant mixtures (e.g.,
Montanide), aluminum hydroxide, aluminum phosphate, hydroxyapatite,
complete and/or incomplete Freund's adjuvant, or cytokines such as
interleukins, macrophage derived chemokines, complement binding
proteins and tumor necrosis factor (either free or fused to the
scaffold protein), and human use-approved live microbial carriers
such as the live attenuated Salmonella enterica serovar Typhimurium
strain.
[0057] Moreover the present invention relates to the use of the
immunogenic polypeptide according the invention for the preparation
of a vaccine for immunization of a subject against infection with
HPV.
[0058] Preferably, said subject is an animal, more preferably, said
subject is a vertebrate, even more preferably, said subject is a
mammal and, most preferably, said subject is a human. Preferably,
the immunization of said subject, establishes or improves immunity
of said subject to various HPV genotypes as referred to elsewhere
herein. It is to be understood that the immunogenic polypeptide
according to the invention or vaccine according to the invention
has to be administered to said subject for immunization. Said
administration can be done by any method deemed appropriate such as
oral or parenteral administration.
[0059] Moreover, the present invention relates to a method for
producing an antibody against the immunogenic polypeptide according
to the invention, comprising the following steps:
[0060] a) providing the immunogenic polypeptide according to the
invention;
[0061] b) immunizing a host with said immunogenic polypeptide,
and
[0062] c) harvesting the antibody against said immunogenic
polypeptide.
[0063] Preferably, the host will be sacrificed after the method has
been carried out. It is to be understood that such a method is not
deemed to be a method of treatment of the human or animal body.
[0064] The "host" in the context may be any host deemed
appropriate. Preferably, the host is a non-human host. Preferred
host for the production of monoclonal antibodies is a mouse or a
rabbit. A host for the production of polyclonal antibodies is
preferably selected from the group consisting of rabbits, mice,
chickens, goats, guinea pigs, hamsters, horses, rats, and
sheep.
[0065] Antibodies against the immunogenic polypeptide according to
the present invention can be prepared by well-known methods using
said immunogenic polypeptide as an antigen. Preferably, the
produced antibody is a polyclonal antibody. More preferably, said
antibody is a monoclonal antibody.
[0066] Most preferably, said monoclonal antibody is produced by the
hybridoma cell line which has been deposited with the German
Collection of Microorganisms and Cell Cultures (DSMZ),
Braunschweig, Germany on Nov. 27, 2008 under deposit number DSM
ACC2983 according to the Budapest Treaty, or a fragment thereof
(preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv, scFv, or
Fc fragments), or said monoclonal antibody produced by the
hybridoma cell line which has been deposited with the German
Collection of Microorganisms and Cell Cultures (DSMZ),
Braunschweig, Germany on Nov. 27, 2008 under deposit number DSM
ACC2984 according to the Budapest Treaty, or a fragment thereof
(preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv, scFv, or
Fc fragments). The monoclonal antibody produced by the hybridoma
cell line which has been deposited with the German Collection of
Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on
Nov. 27, 2008 under deposit number DSM ACC2983 is herein also
referred to as K4L2(20-38)4.1B. The monoclonal antibody produced by
the hybridoma cell line which has been deposited with the German
Collection of Microorganisms and Cell Cultures (DSMZ),
Braunschweig, Germany on Nov. 27, 2008 under deposit number DSM
ACC2984 is herein also referred to as K18L2(20-38)XIII.5G.
[0067] It is also contemplated by the present invention that the
antibody is a single chain antibody, a recombinant, human or
humanized antibody or primatized, chimerized or a fragment of the
antibody according to the present invention.
[0068] Also comprised by the aforementioned method of the present
invention is the production of a synthetic antibody, an antibody
fragment, such as F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv,
scFv, or Fc fragments etc., or a chemically modified derivative of
any of these. The antibody may belong to any immunoglobulin class,
including IgM, IgG, IgD, IgE, IgA, or subclasses of IgG (such as
IgG1, IgG2, IgG2, IgG2a, IgG2b, IgG3 or IgGM).
[0069] How to produce and harvest the aforementioned antibodies and
fragments is well known in the art. Antibodies or fragments thereof
can be obtained by using methods which are described, e.g., in
Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold
Spring Harbor, 1988. Monoclonal antibodies can be prepared by the
techniques originally described in Kohler and Milstein, Nature 256
(1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which
comprise the fusion of mouse myeloma cells to spleen cells derived
from immunized mammals. It is also contemplated that monoclonal
antibodies are produced by fusing myeloma cells with the B-cells
from rabbits that have been immunized with the desired antigen.
[0070] It is to be understood that the antibody produced by the
aforementioned method shall specifically bind the immunogenic
polypeptide according to the invention. Specific binding can be
tested by various well known techniques. Preferably, the antibody
produced by the aforementioned method shall specifically bind the
L2 polypeptide or fragment thereof. More preferably, said antibody
shall specifically bind the L2 polypeptide or fragment thereof,
when comprised by the immunogenic polypeptide according to the
present invention (linked to the scaffold polypeptide), and thus
when being present in a constrained structure. Thus, the antibody
according to the present invention shall not specifically bind the
parts of the immunogenic polypeptide that are derived from the
scaffold polypeptide.
[0071] The aforementioned method of the present invention,
preferably, allows for the production of an antibody against human
papillomavirus. Preferably, said antibody binds the L2 polypeptide
or fragments thereof of various HPV genotypes. Preferably, said
antibody binds the L2 polypeptide or fragment thereof of HPV
genotypes 16, 18, 31, 45 and 58. Preferably, the said antibody also
binds the L2 polypeptide or fragments thereof of HPV genotypes 52,
2, 27, 57 and/or 11.
[0072] The present invention relates also to an antibody
obtainable/produced by the aforementioned method of the present
invention.
[0073] Said antibody of the present invention, preferably is a
polyclonal antibody and, more preferably, a monoclonal
antibody.
[0074] Most preferably, the antibody according to the present
invention is the monoclonal antibody K4L2(20-38)4.1B (see Examples)
produced by the hybridoma cell line which has been deposited with
the German Collection of Microorganisms and Cell Cultures (DSMZ),
Braunschweig, Germany on Nov. 27, 2008 under deposit number DSM
ACC2983 according to the Budapest Treaty, or a fragment thereof
(preferably, F(ab)2, F(ab')2, Fab, F (ab'), Dab, Fv, sFv, scFv, or
Fc fragments), or the antibody according to the present invention
is the monoclonal antibody K18L2(20-38)XIII.5G (see Examples),
which has been deposited with the German Collection of
Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on
Nov. 27, 2008 under deposit number DSM ACC2984 according to the
Budapest Treaty, or a fragment thereof (preferably, F(ab)2,
F(ab')2, Fab, F (ab'), Dab, Fv, sFv, scFv, or Fc fragments).
[0075] The antibodies according to the present invention can be
used, for example, for the immunoprecipitation and
immunolocalization of the immunogenic polypeptides of the present
invention as well as for monitoring the presence of said variant
polypeptides; for example, for the diagnosis of HPV infection,
particularly for diagnosing infection with HPV genotypes 16, 18,
31, 45 and/or 58. Preferably, said diagnosis is done by determining
the amount (or presence) of the L2 polypeptide in a biological
sample from a subject suspected to be infected with HPV genotype
16, 18, 31, 45 and/or 58 (e.g. in a Pap smear). The presence of the
L2 polypeptide (or increased amounts of the L2 polypeptide compared
with a reference amount, e.g. the amount of said polypeptide in a
sample from a subject not infected with HPV) indicates infection
with HPV, whereas the absence of the L2 polypeptide (or decreased
amounts of the L2 polypeptide compared with a reference amount,
e.g. the amount of said polypeptide in a sample of a subject not
infected with HPV) indicates that said subject is not infected with
HPV.
[0076] Moreover, the antibodies according to the present invention
can be used for the preparation of a pharmaceutical composition for
passive immunization against various HPV genotypes, particularly
against HPV genotypes 16, 18, 31, 45 and/or 58. For passive
immunization, the antibody according to the present invention is
administered to a subject in order to protect said subject against
infection with various HPV genotypes and/or to treat an existing
HPV infection, particularly infection with HPV genotypes 16, 18,
31, 45 or 58.
[0077] Also, the antibody of the present invention can be used for
the production of anti-idiotypic antibodies. An "anti-idiotypic
antibody" in the context of the present invention is an antibody
that specifically binds to the idiotypic region of the antibody
according to the present invention, or a fragment thereof. The
idiotypic region of the antibody according to the present invention
(or a fragment thereof) is, preferably, the unique part of its
variable region that specifically binds to the immunogenic
polypeptide according to the present invention. Preferably, the
anti-idiotypic antibody is a monoclonal antibody.
[0078] Anti-idiotypic antibodies as well as methods for their
production are well known in the art, see, e.g., US20080127359, or
U.S. Pat. No. 5,792,455; Dalgleish: An anti-idiotype vaccine for
AIDS based on the HIV receptor. Ann Ist Super Sanita. 1991;
27(1):27-31, or Attanasio, Int Rev Immunol. 1990; 7(1):109-19.
[0079] Preferably, said anti-idiotypic antibodies are produced by
a) providing an antibody according to the present invention
(preferably, a monoclonal antibody according to the invention, more
preferably, K4L2(20-38)4.1B, or a fragment thereof, or
K18L2(20-38)XIII.5G, or a fragment thereof), b) immunizing a host
with said antibody, and c) harvesting the resulting anti-idiotypic
antibody.
[0080] Accordingly, the present invention also relates to a method
for producing anti-idiotypic antibodies by carrying out the
aforementioned steps a) and b).
[0081] Moreover, the present invention relates to the use of the
hybridoma cell line which has been deposited with the German
Collection of Microorganisms and Cell Cultures (DSMZ),
Braunschweig, Germany on Nov. 27, 2008 under deposit number DSM
ACC2983, and to the use of the hybridoma cell line which has been
deposited with the German Collection of Microorganisms and Cell
Cultures (DSMZ), Braunschweig, Germany on Nov. 27, 2008 under
deposit number DSM ACC2984 for the production of a monoclonal
antibody that specifically binds the L2 peptide (as described
herein).
[0082] Finally, the present invention also relates to the hybridoma
cell line which has been deposited with the German Collection of
Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany on
Nov. 27, 2008 under deposit number DSM ACC2983 or under deposit
number DSM ACC2984 according to the Budapest Treaty.
[0083] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
[0084] The Figures show:
[0085] FIGS. 1A-1D. Trx-L2 peptides. (A) Schematic representation
of the HPV16 L2 peptides examined in this study. L2 aa 1-120
1.times.(1.times.SEQ ID NO:1+1.times.SEQ ID NO:24, thus the L2
polypeptide having a sequence as shown in SEQ ID NO1 inserted
within the display site of the thioredoxin polypeptide having a
sequence as shown in SEQ ID NO:24), 2.times.(SEQ ID NO:1
(2.times.)+SEQ ID NO:16(1.times.)+SEQ ID NO:24, thus two fragments
of the L2 polypeptide, said fragments having a sequence as shown in
SEQ ID NO:2, said fragments linked via one linker peptide having a
sequence as shown in SEQ ID NO:16, inserted within the display site
of the thioredoxin polypeptide having a sequence as shown in SEQ ID
NO:24), 3.times.(SEQ ID NO:1 (3.times.)+SEQ ID NO:16(2.times.)+SEQ
ID NO:24); L2 aa 20-38 1.times.(SEQ IS NO:2+SEQ ID NO:24),
3.times.(SEQ ID NO:29), 9.times.(SEQ ID NO:30), 15.times.(SEQ IS
NO:2 (15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID NO:24); L2 aa
28-42 1.times., 4.times., 8.times.; L2 aa 56-75 1.times., 4.times.;
L2 aa 64-81 1.times., 4.times., 8.times.; L2 aa 95-115 1.times.,
4.times., 8.times.. (B) Representative example of the expression
analysis of pTrx-L2(20-38)n constructs with varying L2 peptide
insert multiplicity (n). SDS-PAGE of total bacterial lysates from
different clones ordered according to the multiplicity of their
L2(20-38) peptide inserts; the peptide insert multiplicity (n) of
the various fusion proteins and the migration positions of
molecular mass markers are indicated on the right hand side and on
the left hand side, respectively. (C) Representative examples of
purified Trx-L2(20-38)n fusion proteins used for mice immunization
(1) L2 aa 20-38 1.times.(SEQ IS NO:2+SEQ ID NO:24), 3.times.(SEQ ID
NO:29), 9.times.(SEQ ID NO:30), 15.times.(SEQ IS NO:2
(15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID NO:24). (D) Comparison
of the immune responses elicited by the HPV16 L2(20-38) peptide
chemically conjugated to KLH and by the same peptide grafted to
Trx, both administered (100 .mu.g/dose) with the same CFA/IFA
immunization protocol described in `Materials and methods`. ELISA
data, obtained using GST-L2 as target antigen and expressed as A405
values, are presented as dot plots. The L2 binding activity of
individual sera as well as the mean binding activity of each group
(horizontal bars) are shown; KLH and Trx indicate the unconjugated
carrier proteins utilized as negative controls. Please note that
the numbers correspond to the amino acid positions of the L2
polypeptide as shown in SEQ ID NO:1. E.g., "L2(20-38)" stands for a
fragment of the L2 polypeptide as shown in SEQ ID NO:1 comprising
amino acids 20 to 28 of said polypeptide.
[0086] FIG. 2. Antibody titers of mice vaccinated with the Trx-L2
peptide fusions. GST-L2 ELISA was used to determine the antibody
titers of sera from mice immunized three times with the indicated
Trx-L2(peptide)n fusions, for group L2 aa 20-38 1.times.(SEQ IS
NO:2+SEQ ID NO:24), 3.times.(SEQ ID NO:29), 9.times.(SEQ ID NO:30),
15.times.(SEQ IS NO:2 (15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID
NO:24) and group Trx-L2 L2 aa 1-120 1.times.(1.times.SEQ ID
NO:1+1.times.SEQ ID NO:24), 2.times.(SEQ ID NO:1 (2.times.)+SEQ ID
NO:16(1.times.)+SEQ ID NO:24), 3.times.(SEQ ID NO:1 (3.times.)+SEQ
ID NO:16(2.times.)+SEQ ID NO:24); L2 aa 28-42 1.times., 4.times.,
8.times.; L2 aa 56-75 1.times., 4.times.; L2 aa 64-81 1.times.,
4.times., 8.times.; L2 aa 95-115 1.times., 4.times., 8.times. (100
.mu.g each, corresponding to 1.7-4.3 nmol of protein, depending on
the size of the peptide insert; n values are shown on the x-axis)
administered with the CFA/IFA immunization protocol described in
`Materials and methods`. Sera from mice immunized with the Trx
protein scaffold only (not shown) were used as negative controls
and were assayed in parallel in each set of ELISAs. Binding titers
are given as the reciprocal of the maximum antisera dilutions that
yielded A405 values higher than the mean absorbance plus four
standard deviations of sera from mice immunized with the Trx
scaffold only. Data are presented as log 10 dot-plots of the
titers; horizontal bars represent the geometric mean of the titers
for each of the indicated subgroups of Trx-L2 antisera. The P
values in each panel indicate the statistical significance of the
differences between the immune responses induced by monopeptide and
multipeptide Trx-L2 fusions in each group.
[0087] FIG. 3. Neutralization of HPV 16 infection by Trx-L2 peptide
antisera. Serial dilutions of antisera raised against the
monopeptide and multipeptide Trx-L2 fusions shown in FIG. 2 were
analyzed for their capacity to block infection of 293TT cells by
HPV16 pseudovirions using secreted alkaline phosphatase (SEAP)
activity as readout. Neutralization efficiency was determined
relative to that of mock-treated (0% neutralization) and HPV 16
L1-specific mAb-treated (100% neutralization) controls, which were
run in parallel in each assay (see `Materials and methods` for
details). Data for the monopeptide and the aggregated multipeptide
forms of each Trx-L2 immunogen L2 aa 20-38 1.times.(SEQ IS NO:2+SEQ
ID NO:24), 3.times.(SEQ ID NO:29), 9.times.(SEQ ID NO:30),
15.times.(SEQ IS NO:2 (15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID
NO:24); L2 aa 28-42 1.times., 4.times., 8.times.; L2 aa 56-75
1.times., 4.times.; L2 aa 64-81 1.times., 4.times., 8.times. and L2
aa 95-115 1.times., 4.times., 8.times. are expressed as the
reciprocal of the maximum dilution causing .gtoreq.70%
neutralization. The geometric means of the titers and the 95%
confidence intervals for each group of anti-Trx-L2 peptide antisera
plus the anti-Trx-L2(1-120) 1.times.(1.times.SEQ ID
NO:1+1.times.SEQ ID NO:24), 2.times.(SEQ ID NO:1 (2.times.)+SEQ ID
NO:16(1.times.)+SEQ ID NO:24), 3.times.(SEQ ID NO:1 (3.times.)+SEQ
ID NO:16(2.times.)+SEQ ID NO:24) reference are represented on a log
scale.
[0088] FIG. 4. Cross-neutralization of HPV 31, 45, and 58
pseudovirions. The crossneutralization activities of the indicated
subset of Trx-L2 peptide antisera were assayed at a fixed 1:200
dilution against three heterologous pseudovirions (HPV 31, 45 and
58) plus the cognate HPV16 type. Mock-treated 293TT cells and cells
treated with type-specific neutralizing antibodies served as
negative and positive controls, respectively (see FIG. 3) legend
and `Materials and methods` for details). Cumulative monopeptide
and multipeptide data are presented for each immunogen except
Trx-L2 aa 20-38 1.times.(SEQ IS NO:2+SEQ ID NO:24), 3.times.(SEQ ID
NO:29), 9.times.(SEQ ID NO:30), 15.times.(SEQ IS NO:2
(15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID NO:24), the only one
for which a trend toward a peptide multiplicity-dependent increase
in cross-neutralization activity was observed; they are represented
as the mean plus SD of the neutralization values for the various
Trx-L2 peptide antisera relative to those obtained with HPV
type-specific antibodies.
[0089] FIG. 5. Neutralization of homologous and heterologous
pseudovirions by Trx-L2(20-38)n antisera. The strongest HPV 16
neutralizing antisera from each group of Trx-L2(20-38)n antigens
(n=1, 3, 9, 15) 1.times.(SEQ IS NO:2+SEQ ID NO:24), 3.times.(SEQ ID
NO:29), 9.times.(SEQ ID NO:30), 15.times.(SEQ IS NO:2
(15.times.)+SEQ ID NO:16 (14.times.)+SEQ ID NO:24) were titrated
against homologous (HPV16) and heterologous (HPV18, 31, 45, 58)
pseudovirions (see `Materials and methods` and FIG. 3 legend for
details).
[0090] FIG. 6. Sequence comparison of the L2(20-38) region of the
examined HPV types. Multiple sequence alignment was performed with
CLUSTAL W [30]; amino acids identical to those of the cognate HPV16
type are indicated with dots. Conservative and non-conservative
substitutions are shown in standard and in bold characters,
respectively; non-conservative substitutions occurring in only one
of the five examined HPV types are boxed.
[0091] FIG. 7. Neutralizing titers of supernatants of monoclonal
antibodies against the aa 20-31 from HPV16 L2 protein
[0092] IgG concentration in supernatants was adjusted to 0.6
.mu.g/ml, titer was defined as the last dilution that can protect
70% of pseudovirions infection. There are not big differences in
the neutralization capacity of antibodies #4 (K4L2(20-38)4.1B) and
#18 (K18L2(20-38)XIII.5G) except for the neutralization of HPV31.
Antibody #18 can neutralize the infection although with low titer,
but, antibody #4 is unable to neutralize the infection even at low
dilution factor. Antibody #8 and #1 can neutralize only HPV16.
Antibody #1 can neutralize HPV 16 with high titer.
[0093] FIG. 8. Identification of epitopes recognized by
neutralizing (boxed) and non-neutralizing antibodies
[0094] The different monoclonal antibodies raised against different
regions of the HPV 16 N-terminus were tested for their reactivity
with a set of overlapping peptides (amino acids 1-15, 5-19,
106-120) in ELISA. All four neutralizing antibodies show a distinct
pattern in binding the peptides, different to the pattern of the
non-neutralizing antibodies. The two cross-neutralizing antibodies
(K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) are directed
against region 20-38. Antibody #15, which shows a similar binding
pattern compared to #18, has an about 30 fold lower affinity to its
target which explains its failure to neutralize HPV
pseudovirions.
[0095] FIG. 9. Epitopes for non-neutralizing, neutralizing and
cross-neutralizing antibodies (K4L2(20-38)4.1B and
K18L2(20-38)XIII.5G) within region 20-42 of HPV 16 L2. Scheme with
the recognition patron of all mAbs isolated against the region
20-42. Cross-neutralizing antibodies Mab K4L2(20-38)4.1B recognize
the sequence aa 21-30 SEQ ID NO:4 and K18L2(20-38)XIII.5G recognize
the sequence aa 22-30 SEQ ID NO:5. Neutralizing antibody anti HPV16
K8L2(28-42)12.4B recognize the sequence aa 32-39 SEQ ID NO:31.
[0096] FIGS. 10A-10B. Epitope mapping for the two neutralizing
antibodies (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G). To
determine the amino acids required for binding of the two
antibodies K4 (A9 and K18 (B) an peptide-alanine scan was
performed. Antibody #4 the five amino acids xTCKxxxxCPxx are
essential for binding while for K8 only the two cysteine residues
are crucial for binding, although the remainder residues might
contribute to the binding.
[0097] FIG. 11. Neutralization assay of HPV 16 and HPV 31
pseudovirions with modified L2 proteins. To determine why
antibodies #4 (K4L2(20-38)4.1B) and #18 (K18L2(20-38)XIII.5G) have
different abilities to neutralize HPV 31 we tested hybrid particles
composed of HPV 16 L1 HPV 31 L2 and vice versa. In addition, the
corresponding epitope in HPV 31 recognized by K4 and K18 was
modified. Results indicate that the ability of (K4L2(20-38)4.1B)
and #18 (K18L2(20-38)XIII.5G) antibodies to neutralize depends on
the epitope sequence as HPV 31 L1/16L2 pseudovirions can be
neutralized by both antibodies. Altering serine at position 30 into
proline restores the ability to neutralize HPV 31 pseudovirions
indicating that this residue is important in binding the
antibodies.
EXAMPLES
Example 1
[0098] Monopeptide (1.times.SEQ ID NO:2) and multipeptide ((SEQ ID
NO:2 (3.times.)+SEQ ID NO:16(2.times.)) or 3.times.(SEQ ID NO:2
(3.times.)+SEQ ID NO:16(2.times.)) immunogenic peptides are
inserted within the display site of the thioredoxin polypeptide
having a sequence as shown in SEQ ID NO: 493 to SEQ ID NO: 496.
Fusion proteins are produced in E. coli cells, purified from cell
extracts and used for immunization.
TABLE-US-00001 TABLE 1 List of L2 peptide immunogens (and variants
thereof) SEQ ID NO: 33 RGCKQAGTCPPDVINKVEQ SEQ ID NO: 34
RGCKASNTCPPDVINKVEQ SEQ ID NO: 35 RGCKAAGTCPPDVINKVEQ SEQ ID NO: 36
QSCKAAGTCPPDVLNKVEQ SEQ ID NO: 37 QSCKAAGTCPPDVVNKVEQ SEQ ID NO: 38
QTCKQAGTCPPDVINKVEQ SEQ ID NO: 39 QTCKQAGTCPPDVVNKVEQ SEQ ID NO: 40
RTCKQAGTCPPDVINKVES SEQ ID NO: 41 RTCKQAGTCPPDVINKVEQ SEQ ID NO: 42
KGCKASGTCPPDVINKVEQ SEQ ID NO: 43 RTCKQSGTCPPDVVPKVEG SEQ ID NO: 44
RTCKQAGTCPPDVIPKVEG SEQ ID NO: 45 RTCKVTGTCPADVVPKVEG SEQ ID NO: 46
RTCKATGTRPADVIPKVEG SEQ ID NO: 47 RTCKQSGTCPPDIIPRVEQ SEQ ID NO: 48
RTCKQAGTCPPDIIPRLEQ SEQ ID NO. 49 RTCKQAGTCPPDIIPRVEQ SEQ ID NO: 50
KTCKVAGTCPPDVIPKVEG SEQ ID NO: 51 KTCKAAGTCPPDVIPKVEG SEQID NO: 52
RTCKAAGTCPPDVIPKVEG SEQ ID NO: 53 RTCKASGTCPPDVIPKVEG SEQ ID NO: 54
STCKAAGTCPADVIPKVEG SEQ ID NO: 55 KTCKLSGTCPEDVINKVEQ SEQ ID NO: 56
KTCKQSGTCPPDIIPRVEG SEQ ID NO: 57 KTCKQAGTCPPDIVPKVEG SEQ ID NO: 58
QTCKASGTCPPDVIPKVEG SEQ ID NO: 59 KTCKQAGTCPPDVIPKVEG SEQ ID NO: 60
QTCKAAGTCPSDIIPKVEH SEQ ID NO: 61 QTCKASGTCPPDVIPKVEQ SEQ ID NO: 62
QTCKLTGTCPPDVIPKVEH SEQ ID NO: 63 QTCKAAGTCPSDVINKVEH SEQ ID NO: 64
KQCQLGADCPPDVRNKVEG SEQ ID NO: 65 AKCQLSGNCLPDVKNKVEA SEQ ID NO: 66
AKCQLSGDCLPDVKNKVEA SEQ ID NO: 67 RHCALSGTCPDDVKNKVEN SEQ ID NO: 68
KHCAGSGTCPEDVKNKVEQ SEQ ID NO: 69 KTCLQGGDCIPDVKNKFEN SEQ ID NO: 70
RSCLQGGDCIPDVQNKFEG SEQ ID NO: 71 QTCKATGTCPPDVIPKVEG SEQ ID NO. 72
KTCKQSGTCPPDVVPKVEG SEQ ID NO: 73 RTCKQSGTCPPDVINKVEG SEQ ID NO: 74
KTCKQAGTCPSDVINKVEG SEQ ID NO: 75 KTCKLSGTCPEDVVNKIEQ SEQ ID NO: 76
RTCKQSGTCPPDVVDKVEG SEQ ID NO: 77 STCKAAGTCPPDVVNKVEG SEQ ID NO: 78
PTCK1AGNCPADIQNKFEN SEQ ID NO: 79 PACKISNTCPPDIINKYEN SEQ ID NO: 80
RGCKQAGTCPPD SEQ ID NO: 81 RGCKASNTCPPD SEQ ID NO: 82 RGCKAAGTCPPD
SEQ ID NO: 83 QSCKAAGTCPPD SEQ ID NO: 84 QTCKQAGTCPPD SEQ ID NO: 85
RTCKQAGTCPPD SEQ ID NO: 86 KGCKASGTCPPD SEQ ID NO: 87 RTCKQSGTCPPD
SEQ ID NO: 88 RTCKVTGTCPAD SEQ ID NO: 89 RTCKATGTRPAD SEQ ID NO: 90
KTCKVAGTCPPD SEQ ID NO: 91 KTCKAAGTCPPD SEQ ID NO: 92 RTCKAAGTCPPD
SEQ ID NO: 93 RTCKASGTCPPD SEQ ID NO. 94 STCKAAOTCPAD SEQ ID NO: 95
KTCKLSGTCPED SEQ ID NO: 96 KTCKQAGTCPED SEQ ID NO: 97 QTCKASGTCPPD
SEQ ID NO: 98 QTCKAAGTCPSD SEQ ID NO: 99 QTCKLTGTCPPD SEQ ID NO:
100 KQCQLGADCPPD SEQ ID NO: 101 AKCQLSGNCLPD SEQ ID NO: 102
AKCQLSGDCLPD SEQ ID NO: 103 RHCALSGTCPDD SEQ ID NO: 104
KHCAGSGTCPED SEQ ID NO: 105 KTCLQGGDCIPD SEQ ID NO: 106
RSCLQGGDCIPD SEQ ID NO: 107 QTCKATGTCPPD SEQ ID NO: 108
KTCKQSGTCPPD SEQ ID NO: 109 KTCKQAGTCPSD SEQ ID NO: 110
STCKAAGTCPPD SEQ ID NO: 111 PTCKIAGNCPAD SEQ ID NO: 112
PACKISNTCPPD SEQ ID NO: 113 GCKQAGTCPPD SEQ ID NO: 114 GCKASNTCPPD
SEQ ID NO: 115 GCKAAGTCPPD SEQ ID NO: 116 SCKAAGTCPPD SEQ ID NO:
117 TCKQSGTCPSD SEQ ID NO: 118 GCKASGTCPPD SEQ ID NO: 119
TCKQSGTCPPD SEQ ID NO: 120 TCKVTGTCPAD SEQ ID NO: 121 TCKATGTRPAD
SEQ ID NO: 122 TCKVAGTCPPD SEQ ID NO: 123 TCKAAGTCPPD SEQ ID NO:
124 TCKASGTCPPD SEQ ID NO: 125 TCKAAGTCPAD SEQ ID NO: 126
TCKLSGTCPED SEQ ID NO: 127 TCKAAGTCPSD SEQ ID NO: 128 TCKLTGTCPPD
SEQ ID NO: 129 QCQLGADCPPD SEQ ID NO: 130 KCQLSGNCLPD SEQ ID NO:
131 KCQLSGDCLPD SEQ ID NO: 132 HCALSGTCPDD SEQ ID NO: 133
HCAGSGTCPED SEQ ID NO: 134 TCLQGGDCIPD SEQ ID NO: 135 SCLQGGDCIPD
SEQ ID NO: 136 TCKATGTCPPD SEQ ID NO: 137 TCKQAGTCPSD SEQ ID NO:
138 TCKIAGNCPAD SEQ ID NO: 139 ACKISNTCPPD SEQ ID NO: 140
CKQSGTCPDD SEQ ID NO: 141 CKASNTCPPD SEQ ID NO: 142 CKAAGTCPPD SEQ
ID NO: 143 CKASGTCPPD SEQ ID NO: 144 CKQSGTCPPD SEQ ID NO: 145
CKVTGTCPAD SEQ ID NO: 146 CKATGTRPAD SEQ ID NO: 147 CKVAGTCPPD SEQ
ID NO: 148 CKAAGTCPAD SEQ ID NO: 149 CKLSGTCPED SEQ ID NO: 150
CKAAGTCPSD SEQ ID NO: 151 CKLTGTCPPD SEQ ID NO: 152 CQLGADCPPD SEQ
ID NO: 153 CQLSGNCLPD SEQ ID NO: 154 CQLSGDCLPD
SEQ ID NO: 155 CALSGTCPDD SEQ ID NO: 156 CAGSGTCPED SEQ ID NO: 157
CLQGGDCIPD SEQ ID NO: 158 CKATGTCPPD SEQ ID NO: 159 CKQAGTCPSD SEQ
ID NO: 160 CKIAGNCPAD SEQ ID NO: 161 CKISNTCPPD SEQ ID NO: 162
GCKQAGTCPP SEQ ID NO: 163 GCKASNTCPP SEQ ID NO: 164 GCKAAGTCPP SEQ
ID NO: 165 SCKAAGTCPP SEQ ID NO: 166 TCKLAGTCPP SEQ ID NO: 167
GCKASGTCPP SEQ ID NO: 168 TCKQSGTCPP SEQ ID NO: 169 TCKVTGTCPA SEQ
ID NO: 170 TCKATGTRPA SEQ ID NO: 171 TCKVAGTCPP SEQ ID NO: 172
TCKAAGTCPP SEQ ID NO: 173 TCKASGTCPP SEQ ID NO: 174 TCKAAGTCPA SEQ
ID NO: 175 TCKLSGTCPE SEQ ID NO: 176 TCKAAGTCPS SEQ ID NO: 177
TCKLTGTCPP SEQ ID NO: 178 QCQLGADCPP SEQ ID NO: 179 KCQLSGNCLP SEQ
ID NO: 180 KCQLSGDCLP SEQ ID NO: 181 HCALSGTCPD SEQ ID NO: 182
HCAGSGTCPE SEQ ID NO: 183 TCLQGGDCIP SEQ ID NO: 184 SCLQGGDCIP SEQ
ID NO: 185 TCKATGTCPP SEQ ID NO: 186 TCKQAGTCPS SEQ ID NO: 187
TCKIAGNCPA SEQ ID NO: 188 ACKISNTCPP SEQ ID NO: 189 CKLAGTCPP SEQ
ID NO: 190 CKASNTCPP SEQ ID NO: 191 CKAAGTCPP SEQ ID NO: 192
CKASGTCPP SEQ ID NO: 193 CKQSGTCPP SEQ ID NO: 194 CKVTGTCPA SEQ ID
NO: 195 CKATGTRPA SEQ ID NO: 196 CKVAGTCPP SEQ ID NO: 197 CKAAGTCPA
SEQ ID NO: 198 CKLSGTCPE SEQ ID NO: 199 CKAAGTCPS SEQ ID NO: 200
CKLTGTCPP SEQ ID NO: 201 CQLGADCPP SEQ ID NO: 202 CQLSGNCLP SEQ ID
NO: 203 CQLSGDCLP SEQ ID NO: 204 CALSGTCPD SEQ ID NO: 205 CAGSGTCPE
SEQ ID NO: 206 CLQGGDCIP SEQ ID NO: 207 CKATGTCPP SEQ ID NO: 208
CKQAGTCPS SEQ ID NO: 209 CKIAGNCPA SEQ ID NO: 210 CKISNTCPP SEQ ID
NO: 211 DVINKVEQTT SEQ ID NO: 212 DVINKVEQST SEQ ID NO: 213
DVINKVEQKT SEQ ID NO: 214 DVLNKVEQTT SEQ ID NO: 215 DVVNKVEQTT SEQ
ID NO: 216 DVINKVESTT SEQ ID NO: 217 DVINKVEQNT SEQ ID NO: 218
DVVPKVEGDT SEQ ID NO: 219 DVIPKVEGDT SEQ ID NO: 220 DIIPRVEQNT SEQ
ID NO: 221 DIIPRLEQNT SEQ ID NO: 222 DIIPRVEQDT SEQ ID NO: 223
DVIPKVEGTT SEQ ID NO: 224 DIIPKVEQKT SEQ ID NO: 225 DVIPKVEGST SEQ
ID NO: 226 DIIPKVEHNT SEQ ID NO: 227 DVIPKVEQNT SEQ ID NO: 228
DVIPKVEHNT SEQ ID NO: 229 DVINKVEHTT SEQ ID NO: 230 DVRNKVEGTT SEQ
ID NO: 231 DVKNKVEADT SEQ ID NO: 232 DVKNKVEANT SEQ ID NO: 233
DVKNKVENNT SEQ ID NO: 234 DVKNKVEQTT SEQ ID NO: 235 DVKNKFENST SEQ
ID NO: 236 DVQNKFEGNT SEQ ID NO: 237 DIQNKIEQTT SEQ ID NO: 238
DVIKRYEQTT SEQ ID NO: 239 VINKVEQTT SEQ ID NO: 240 VINKVEQST SEQ ID
NO: 241 VINKVEQKT SEQ ID NO: 242 VLNKVEQTT SEQ ID NO: 243 VVNKVEQTT
SEQ ID NO: 244 VINKVESTT SEQ ID NO: 245 VINKVEQNT SEQ ID NO: 246
VVPKVEGDT SEQ ID NO: 247 VIPKVEGDT SEQ ID NO: 248 IIPRVEQNT SEQ ID
NO: 249 IIPRLEQNT SEQ ID NO: 250 IIPRVEQDT SEQ ID NO: 251 VIPKVEGTT
SEQ ID NO: 252 IIPKVEQKT SEQ ID NO: 253 VIPKVEGST SEQ ID NO: 254
IIPKVEHNT SEQ ID NO: 255 VIPKVEQNT SEQ ID NO: 256 VIPKVEHNT SEQ ID
NO: 257 VINKVEHTT SEQ ID NO: 258 VRNKVEGTT SEQ ID NO: 259 VKNKVEADT
SEQ ID NO: 260 VKNKVEANT SEQ ID NO: 261 VKNKVENNT SEQ ID NO: 262
VKNKVEQTT SEQ ID NO: 263 VKNKFENST SEQ ID NO: 264 VQNKFEGNT SEQ ID
NO: 265 IQNKIEQTT SEQ ID NO: 266 VIKRYEQTT SEQ ID NO: 267 INKVEQTT
SEQ ID NO: 268 INKVEQST SEQ ID NO: 269 INKVEQKT SEQ ID NO: 270
LNKVEQTT SEQ ID NO. 271 VNKVEQTT SEQ ID NO: 272 INKVESTT SEQ ID NO:
273 INKVEQNT SEQ ID NO: 274 VPKVEGDT SEQ ID NO: 275 IPKVEGDT SEQ ID
NO: 276 IPRVEQNT SEQ ID NO: 277 IPRLEQNT SEQ ID NO: 278 IPRVEQDT
SEQ ID NO: 279 IPKVEGTT SEQ ID NO: 280 IPKVEHKT
SEQ ID NO: 281 IPKVEGST SEQ ID NO: 282 IPKVEHNT SEQ ID NO: 283
IPKVEQNT SEQ ID NO: 284 INKVEHTT SEQ ID NO: 285 RNKVEGTT SEQ ID NO:
286 KNKVEADT SEQ ID NO: 287 KNKVEANT SEQ ID NO: 288 KNKVENNT SEQ ID
NO: 289 KNKVEQTT SEQ ID NO: 290 KNKFENST SEQ ID NO: 291 QNKFEGNT
SEQ ID NO: 292 QNKIEQTT SEQ ID NO: 293 IKRYEQTT SEQ ID NO: 294
TGYIPLQTR SEQ ID NO: 295 TGYVPLGST SEQ ID NO: 296 TGYVPLGNT SEQ ID
NO: 297 TGYVPLSTG SEQ ID NO: 298 TGYIPLQST SEQ ID NO: 299 TGYVPVGST
SEQ ID NO: 300 TGYVPLQTS SEQ ID NO: 301 TGYVPLTTG SEQ ID NO: 302
RGCKQXGTCPPDVINKVEQ SEQ ID NO: 303 RGCKAXNTCPPDVINKVEQ SEQ ID NO:
304 RGCKAXGTCPPDVINKVEQ SEQ ID NO: 305 QSCKAXGTCPPDVLNKVEQ SEQ ID
NO: 306 QSCKAXGTCPPDVVNKVEQ SEQ ID NO: 307 QTCKQXGTCPPDVINKVEQ SEQ
ID NO: 308 QTCKQXGTCPPDVVNKVEQ SEQ ID NO: 309 RTCKQXGTCPPDVINKVES
SEQ ID NO: 310 RTCKQXGTCPPDVINKVEQ SEQ ID NO: 311
RGCKAXGTCPPDVINKVEQ SEQ ID NO: 312 RTCKQXGTCPPDVVPKVEG SEQ ID NO:
313 RTCKQXGTCPPDVIPKVEG SEQ ID NO: 314 RTCKVXGTCPADVVPKVEG SEQ ID
NO. 313 RTCKAXGTRPADVIPKVEO SEQ ID NO: 316 STCKAXGTCPPDVIPKLEG SEQ
ID NO: 317 RTCKQXGTCPPDIIPRLEQ SEQ ID NO: 318 RTCKQXGTCPPDIIPRVEQ
SEQ ID NO: 319 KTCKVXGTCPPDVIPKVEG SEQ ID NO: 320
KTCKAXGTCPPDVIPKVEG SEQ ID NO: 321 STCKAXGTCPPDVIPKVEG SEQ ID NO:
322 RTCKAXGTCPPDVIPKVEG SEQ ID NO: 323 STCKAXGTCPADVIPKVEG SEQ ID
NO: 324 KTCKLXGTCPEDVINKVEQ SEQ ID NO: 325 KTCKQXGTCPPDIIPKIEG SEQ
ID NO: 326 KTCKQXGTCPPDIVPKVEG SEQ ID NO: 327 STCKQXGTCPPDIIPRVEQ
SEQ ID NO: 328 KTCKQXGTCPPDVIPKVEG SEQ ID NO: 329
QTCKAXGTCPSDIIPKVEH SEQ ID NO: 330 QTCKAXGTCPPDVIPKVEQ SEQ ID NO:
331 QTCKLXGTCPPDVIPKVEH SEQ ID NO: 332 QTCKAXGTCPSDVINKVEH SEQ ID
NO: 333 KQCQLXADCPPDVRNKVEG SEQ ID NO: 334 AKCQLXGNCLPDVKNKVEA SEQ
ID NO: 335 AKCQLXGDCLPDVKNKVEA SEQ ID NO: 336 RHCALXGTCPDDVKNKVEN
SEQ ID NO: 337 KHCAGXGTCPEDVKNKVEQ SEQ ID NO. 338
KTCLQXGDCIPDVKNKFEN SEQ ID NO: 339 RSCLQXGDCIPDVQNKFEG SEQ ID NO:
340 QTCKAXGTCPPDVIPKVEG SEQ ID NO: 341 KTCKQXGTCPPDVVPKVEG SEQ ID
NO: 342 RTCKQXGTCPPDVINKVEG SEQ ID NO: 343 KTCKQXGTCPSDVINKVEG SEQ
ID NO: 344 KTCKLXGTCPEDVVNKIEQ SEQ ID NO: 345 RTCKQXGTCPPDVVDKVEG
SEQ ID NO: 346 STCKAXGTCPPDVVNKVEG SEQ ID NO: 347
PTCKIXGNCPADIQNKFEN SEQ ID NO: 348 PACKIXNTCPPDIINKYEN ***X = Gly
(G) or Ala (A) SEQ ID NO: 349 RGCKQXGTCPPD SEQ ID NO: 350
RGCKAXNTCPPD SEQ ID NO: 351 RGCKAXGTCPPD SEQ ID NO: 352
QSCKAXGTCPPD SEQ ID NO: 353 QTCKQXGTCPPD SEQ ID NO: 354
RTCKQXGTCPPD SEQ ID NO: 355 KGCKAXGTCPPD SEQ ID NO: 356
PTCKAXGTCPPD SEQ ID NO: 357 RTCKVXGTCPAD SEQ ID NO: 358
RTCKAXGTRPAD SEQ ID NO: 359 KTCKVXGTCPPD SEQ ID NO: 360
KTCKAXGTCPPD SEQ ID NO: 361 RTCKAXGTCPPD SEQ ID NO: 362
STCKAXGTRPPD SEQ ID NO: 363 STCKAXGTCPAD SEQ ID NO: 364
KTCKLXGTCPED SEQ ID NO: 365 ATCKQXGTCPPD SEQ ID NO: 366
STCKQXGTCPPD SEQ ID NO: 367 QTCKAXGTCPSD SEQ ID NO: 368
QTCKLXGTCPPD SEQ ID NO: 369 KQCQLXADCPPD SEQ ID NO: 370
AKCQLXGNCLPD SEQ ID NO: 371 AKCQLXGDCLPD SEQ ID NO: 372
RHCALXGTCPDD SEQ ID NO: 373 KHCAGXGTCPED SEQ ID NO: 374
KTCLQXGDCIPD SEQ ID NO: 375 RSCLQXGDCIPD SEQ ID NO: 376
QTCKAXGTCPPD SEQ ID NO: 377 KTCKQXGTCPED SEQ ID NO: 378
KTCKQXGTCPSD SEQ ID NO: 379 STCKAXGTCPPD SEQ ID NO: 380
PTCKIXGNCPAD SEQ ID NO: 381 PACKIXNTCPPD ***X = Gly (G) or Ala (A)
SEQ ID NO: 382 GCKQXGTCPPD SEQ ID NO: 383 GCKAXNTCPPD SEQ ID NO:
384 ACKAXGTCPPD SEQ ID NO: 385 SCKAXGTCPPD SEQ ID NO: 386
KCKAXGTCIPD SEQ ID NO: 387 GCKAXGTCPPD SEQ ID NO: 388 KCKAXGTCPPD
SEQ ID NO: 389 TCKVXGTCPAD SEQ ID NO: 390 TCKAXGTRPAD SEQ ID NO:
391 TCKVXGTCPPD SEQ ID NO: 392 SCKLXGTCPPD SEQ ID NO: 393
SCKQXGTCPSD SEQ ID NO: 394 TCKAXGTCPAD SEQ ID NO: 395 TCKLXGTCPED
SEQ ID NO: 396 TCKAXGTCPSD SEQ ID NO: 397 TCKLXGTCPPD SEQ ID NO:
398 QCQLXADCPPD SEQ ID NO: 399 KCQLXGNCLPD SEQ ID NO: 400
KCQLXGDCLPD SEQ ID NO: 401 HCALXGTCPDD SEQ ID NO: 402 HCAGXGTCPED
SEQ ID NO: 403 TCLQXGDCIPD
SEQ ID NO: 404 SCLQXGDCIPD SEQ ID NO: 405 TCKAXGTCPPD SEQ ID NO:
406 TCKQXGTCPSD SEQ ID NO: 407 TCKIXGNCPAD SEQ ID NO: 408
ACKIXNTCPPD ***X = Gly (G) or Ala (A) SEQ ID NO: 409 CKQXGTCPDD SEQ
ID NO: 410 CKAXNTCPPD SEQ ID NO: 411 CLAXGTCPAD SEQ ID NO: 412
CLAXGTCPPD SEQ ID NO: 413 CKLXGTCPAD SEQ ID NO: 414 CKVXGTCPAD SEQ
ID NO: 415 CKAXGTRPAD SEQ ID NO: 416 CKVXGTCPPD SEQ ID NO: 417
CKAXGTCPAD SEQ ID NO: 418 CKLXGTCPED SEQ ID NO: 419 CKAXGTCPSD SEQ
ID NO: 420 CKLXGTCPPD SEQ ID NO: 421 CQLXADCPPD SEQ ID NO: 422
CQLXGNCLPD SEQ ID NO: 423 CQLXGDCLPD SEQ ID NO: 424 CALXGTCPDD SEQ
ID NO: 425 CAGXGTCPED SEQ ID NO: 426 CLQXGDCIPD SEQ ID NO: 427
CKAXGTCPPD SEQ ID NO: 428 CKQXGTCPSD SEQ ID NO: 429 CKIXGNCPAD SEQ
ID NO: 430 CKIXNTCPPD ***X = Gly (G) or Ala (A) SEQ ID NO: 486
KTCKQSGTCPSDVVNKVEG SEQ ID NO: 487 QTCKAAGTCPSDVIPKIEH SEQ ID NO:
488 KTCKQSGTCPPDVIDKVEG SEQ ID NO: 489 STCKAAGTCPPDVIPKVKG SEQ ID
NO: 490 KTCKQSGTCPSD SEQ ID NO: 491 ((SEQ ID NO: 2)x3 + (SEQ ID NO:
487)x3 + (SEQ ID NO: 77)x3 with a tripeptide (GGP) linker):
KTCKQAGTCPPDIIPKVEGGGPKTCKQAGTCPPDIIPKVEGGGPKTCKQA
GTCPPDIIPKVEGGGPQTCKAAGTCPSDVIPKIEHGGPQTCKAAGTCPSD
VIPKIEHGGPQTCKAAGTCPSDVIPKIEHGGPSTCKAAGTCPPDVVNKVE
GGGPSTCKAAGTCPPDVVNKVEGGGPSTCKAAGTCPPDVVNKVEG SEQ ID NO: 492 ((SEQ
ID NO: 2) + (SEQ ID NO: 487) + (SEQ ID NO: 77))x3 with a tripeptide
(GGP) linker KTCKQAGTCPPDIIPKVEGGGPQTCKAAGTCPSDVIPKIEHGGPSTCKAA
GTCPPDVVNKVEGGGPKTCKQAGTCPPDIIPKVEGGGPQTCKAAGTCPSD
VIPKIEHGGPSTCKAAGTCPPDVVNKVEGGGPKTCKQAGTCPPDIIPKVE
GGGPQTCKAAGTCPSDVIPKIEHGGPSTCKAAGTCPPDVVNKVEG
TABLE-US-00002 TABLE 2 List of thioredoxin variants SEQ ID NO: 493
(variant thiorexodin polypeptide from hyperthermophile
archaebacterium Pyrococcus furiosus)
MIIEYDGEIDFTKGRVVLWFSIPGCGPCRLVERFMTELSEYFEDIQIVHI
NAGKWKNIVDKFNILNVPTLVYLKDGREVGRQNLIRSKEEILKKLKELQE SEQ ID NO: 494
(variant thiorexodin polypeptide from hyperthermophile
archaebacterium Thermococcus kodakarensis)
MIVEYDENVDFTKGKAVLWFSIPGCGPCRLVEAFMKELSEEFGEIAIVHV
NAEKWSGLVEGFRILNVPTLVYLKDGKEVARQNLIRGKGEVLIKFEEPRE L SEQ ID NO: 495
(variant thiorexodin polypeptide from hyperthermophile
archaebacterium Thermococcus onnurineus)
MIREFDGDFGKVERAKYALLWFSSPGCGPCRMIEPFMHELSEEYKEVEFW
EVDVEKHLPLAEKFDVMNVPTLIYLKEGNEIARQNLVRKKEEVEEKLMML LGSDS SEQ ID NO:
496 (variant of thiorexodin polypeptide from hyperthermophile
archaebacterium Thermococcus sibiricus)
MIHEYDGKIDFNRGKVVLWFSIQGCGPCRLVESFMEEVSEEFSEIRFIHV
GAEKWSNIVKRFEVLNVPTLVYLKDGKEVARQNLIRSKEEVLAKIEELHE SEQ ID NO: 497
(Dimer of Escherichia coli thioredoxin variants)
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEY
QGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGGGSEGGGSEGGGSEGGGSEGGGSEGGGSEGGGMSDKIIH
LTDDSFDTDVLKADGAILVDFWAEWCGPGCKMIAPILDEIADEYQGKLTV
AKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDA NLA SEQ ID NO:
498 (Trimer of Escherichia coli thioredoxin variants)
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEY
QGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQL
KEFLDANLAGGGGSEGGGSEGGGSEGGGSEGGGSEGGGSEGGGMSDKIIH
LTDDSFDTDVLKADGAILVDFWAEWCGPGCKMIAPILDEIADEYQGKLTV
AKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDA
NLAGGGGSEGGGSEGGGSEGGGSEGGGSEGGGSEGGGMSDKIIHLTDDSF
DTDVLKADGAILVDFWAEWCLSCKMIAPILDEIADEYQGKLTVAKLNIDQ
NPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA
Sequence CWU 1
1
4981120PRTHuman papillomavirus type 16 1Met Arg His Lys Arg Ser Ala
Lys Arg Thr Lys Arg Ala Ser Ala Thr 1 5 10 15 Gln Leu Tyr Lys Thr
Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile 20 25 30 Ile Pro Lys
Val Glu Gly Lys Thr Ile Ala Glu Gln Ile Leu Gln Tyr 35 40 45 Gly
Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser 50 55
60 Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro Leu Gly Thr Arg Pro Pro
65 70 75 80 Thr Ala Thr Asp Thr Leu Ala Pro Val Arg Pro Pro Leu Thr
Val Asp 85 90 95 Pro Val Gly Pro Ser Asp Pro Ser Ile Val Ser Leu
Val Glu Glu Thr 100 105 110 Ser Phe Ile Asp Ala Gly Ala Pro 115 120
219PRTHuman papillomavirus type 16 2Lys Thr Cys Lys Gln Ala Gly Thr
Cys Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Val Glu Gly 312PRTHuman
papillomavirus type 16 3Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro
Asp 1 5 10 411PRTHuman papillomavirus type 16 4Thr Cys Lys Gln Ala
Gly Thr Cys Pro Pro Asp 1 5 10 510PRTHuman papillomavirus type 16
5Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp 1 5 10 610PRTHuman
papillomavirus type 16 6Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro 1 5
10 79PRTHuman papillomavirus type 16 7Cys Lys Gln Ala Gly Thr Cys
Pro Pro 1 5 810PRTHuman papillomavirus type 16 8Asp Ile Ile Pro Lys
Val Glu Gly Lys Thr 1 5 10 99PRTHuman papillomavirus type 16 9Thr
Gly Tyr Ile Pro Leu Gly Thr Arg 1 5 1019PRTartificialL2 fragment
10Lys Thr Cys Lys Gln Xaa Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys 1
5 10 15 Val Glu Gly 1112PRTartificialL2 fragment 11Lys Thr Cys Lys
Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10 1211PRTartificialL2 fragment
12Thr Cys Lys Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10
1310PRTartificialL2 fragment 13Cys Lys Gln Xaa Gly Thr Cys Pro Pro
Asp 1 5 10 1410PRTartificialL2 fragment 14Thr Cys Lys Gln Xaa Gly
Thr Cys Pro Pro 1 5 10 159PRTartificialL2 fragment 15Cys Lys Gln
Xaa Gly Thr Cys Pro Pro 1 5 163PRTartificiallinker peptide 16Gly
Gly Pro 1 174PRTartificiallinker peptide 17Gly Pro Gly Pro 1
185PRTartificiallinker peptide 18Gly Pro Gly Pro Gly 1 5
194PRTartificiallinker peptide 19Ser Gly Ser Gly 1
2063PRTartificialMultimers with (GGP) linker 20Lys Thr Cys Lys Gln
Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Val Glu Gly
Gly Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro 20 25 30 Pro
Asp Ile Ile Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys Lys 35 40
45 Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly 50
55 60 21195PRTartificialMultimers with (GGP) linker 21Lys Thr Cys
Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Val
Glu Gly Gly Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro 20 25
30 Pro Asp Ile Ile Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys Lys
35 40 45 Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu
Gly Gly 50 55 60 Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro
Pro Asp Ile Ile 65 70 75 80 Pro Lys Val Glu Gly Gly Gly Pro Lys Thr
Cys Lys Gln Ala Gly Thr 85 90 95 Cys Pro Pro Asp Ile Ile Pro Lys
Val Glu Gly Gly Gly Pro Lys Thr 100 105 110 Cys Lys Gln Ala Gly Thr
Cys Pro Pro Asp Ile Ile Pro Lys Val Glu 115 120 125 Gly Gly Gly Pro
Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp 130 135 140 Ile Ile
Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys Lys Gln Ala 145 150 155
160 Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Gly Gly Pro
165 170 175 Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile
Pro Lys 180 185 190 Val Glu Gly 195 2272PRTartificialMultimers with
(GGP) linker 22Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Gly
Gly Pro Lys 1 5 10 15 Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp
Gly Gly Pro Lys Thr 20 25 30 Cys Lys Gln Ala Gly Thr Cys Pro Pro
Asp Gly Gly Pro Lys Thr Cys 35 40 45 Lys Gln Ala Gly Thr Cys Pro
Pro Asp Gly Gly Pro Lys Thr Cys Lys 50 55 60 Gln Ala Gly Thr Cys
Pro Pro Asp 65 70 234PRTartificialThioredoxin display site 23Cys
Gly Pro Cys 1 24109PRTEscherichia coli 24Met Ser Asp Lys Ile Ile
His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala
Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly
Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50
55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu
Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly
Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn
Leu Ala 100 105 25109PRTSalmonella enterica 25Met Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser Phe Gly Thr Asp 1 5 10 15 Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys
Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40
45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr
Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val
Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala
Asn Leu Ala 100 105 26105PRTMus musculus 26Met Val Lys Leu Ile Glu
Ser Lys Glu Ala Phe Gln Glu Ala Leu Ala 1 5 10 15 Ala Ala Gly Asp
Lys Leu Val Val Val Asp Phe Ser Ala Thr Trp Cys 20 25 30 Gly Pro
Cys Lys Met Ile Lys Pro Phe Phe His Ser Leu Cys Asp Lys 35 40 45
Tyr Ser Asn Val Val Phe Leu Glu Val Asp Val Asp Asp Cys Gln Asp 50
55 60 Val Ala Ala Asp Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe
Tyr 65 70 75 80 Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn
Lys Glu Lys 85 90 95 Leu Glu Ala Ser Ile Thr Glu Tyr Ala 100 105
27105PRTRattus norvegicus 27Met Val Lys Gln Ile Glu Ser Lys Ser Ala
Phe Gln Glu Val Leu Asp 1 5 10 15 Ser Ala Gly Asp Lys Leu Val Val
Val Asp Phe Ser Ala Thr Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile
Lys Pro Phe Phe His Ala Leu Ser Glu Lys 35 40 45 Phe Asn Asn Val
Val Phe Ile Glu Val Asp Val Asp Asp Cys Lys Asp 50 55 60 Ile Ala
Ala Glu Cys Glu Val Lys Cys Met Pro Thr Phe Gln Phe Phe 65 70 75 80
Lys Lys Gly Gln Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 85
90 95 Leu Glu Ala Thr Ile Asn Glu Leu Leu 100 105 28105PRTHomo
sapiens 28Met Val Lys Gln Ile Glu Ser Lys Thr Ala Phe Gln Glu Ala
Leu Asp 1 5 10 15 Ala Ala Gly Asp Lys Leu Val Val Val Asp Phe Ser
Ala Thr Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile Lys Pro Phe Phe
His Ser Leu Ser Glu Lys 35 40 45 Tyr Ser Asn Val Ile Phe Leu Glu
Val Asp Val Asp Asp Cys Gln Asp 50 55 60 Val Ala Ser Glu Cys Glu
Val Lys Cys Met Pro Thr Phe Gln Phe Phe 65 70 75 80 Lys Lys Gly Gln
Lys Val Gly Glu Phe Ser Gly Ala Asn Lys Glu Lys 85 90 95 Leu Glu
Ala Thr Ile Asn Glu Leu Val 100 105 29216PRTartificialimmunogenic
polypeptide 29Met Gly Ser Ser His His His His His His Ser Ser Gly
Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Gly Asp Lys Ile Ile His
Leu Thr Asp Asp Ser 20 25 30 Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala Ile Leu Val Asp Phe 35 40 45 Trp Ala Glu Trp Cys Gly Pro
Lys Thr Cys Lys Gln Ala Gly Thr Cys 50 55 60 Pro Pro Asp Ile Ile
Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys 65 70 75 80 Lys Gln Ala
Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly 85 90 95 Gly
Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile 100 105
110 Ile Pro Lys Val Glu Gly Gly Gly Pro Cys Lys Met Ile Ala Pro Ile
115 120 125 Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val
Ala Lys 130 135 140 Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr Gly Ile Arg 145 150 155 160 Gly Ile Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr 165 170 175 Lys Val Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Phe Leu Asp Ala 180 185 190 Asn Leu Arg Asp Pro
Asn Ser Ser Ser Val Asp Lys Leu Ala Ala Ala 195 200 205 Leu Glu His
His His His His His 210 215 30348PRTartificialimmunogenic
polypeptide 30Met Gly Ser Ser His His His His His His Ser Ser Gly
Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Gly Asp Lys Ile Ile His
Leu Thr Asp Asp Ser 20 25 30 Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala Ile Leu Val Asp Phe 35 40 45 Trp Ala Glu Trp Cys Gly Pro
Lys Thr Cys Lys Gln Ala Gly Thr Cys 50 55 60 Pro Pro Asp Ile Ile
Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys 65 70 75 80 Lys Gln Ala
Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly 85 90 95 Gly
Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile 100 105
110 Ile Pro Lys Val Glu Gly Gly Gly Pro Lys Thr Cys Lys Gln Ala Gly
115 120 125 Thr Cys Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Gly Gly
Pro Lys 130 135 140 Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile
Ile Pro Lys Val 145 150 155 160 Glu Gly Gly Gly Pro Lys Thr Cys Lys
Gln Ala Gly Thr Cys Pro Pro 165 170 175 Asp Ile Ile Pro Lys Val Glu
Gly Gly Gly Pro Lys Thr Cys Lys Gln 180 185 190 Ala Gly Thr Cys Pro
Pro Asp Ile Ile Pro Lys Val Glu Gly Gly Gly 195 200 205 Pro Lys Thr
Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile Pro 210 215 220 Lys
Val Glu Gly Gly Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys 225 230
235 240 Pro Pro Asp Ile Ile Pro Lys Val Glu Gly Gly Gly Pro Cys Lys
Met 245 250 255 Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln
Gly Lys Leu 260 265 270 Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro
Gly Thr Ala Pro Lys 275 280 285 Tyr Gly Ile Arg Gly Ile Pro Thr Leu
Leu Leu Phe Lys Asn Gly Glu 290 295 300 Val Ala Ala Thr Lys Val Gly
Ala Leu Ser Lys Gly Gln Leu Lys Glu 305 310 315 320 Phe Leu Asp Ala
Asn Leu Arg Asp Pro Asn Ser Ser Ser Val Asp Lys 325 330 335 Leu Ala
Ala Ala Leu Glu His His His His His His 340 345 319PRTHuman
papillomavirus type 16 31Ile Ile Pro Lys Val Glu Gly Lys Thr 1 5
328PRTHuman papillomavirus type 16 32Ile Pro Lys Val Glu Gly Lys
Thr 1 5 3319PRTartificialL2 fragment 33Arg Gly Cys Lys Gln Ala Gly
Thr Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
3419PRTartificialL2 fragment 34Arg Gly Cys Lys Ala Ser Asn Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
3519PRTartificialL2 fragment 35Arg Gly Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
3619PRTartificialL2 fragment 36Gln Ser Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp Val Leu Asn Lys 1 5 10 15 Val Glu Gln
3719PRTartificialL2 fragment 37Gln Ser Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp Val Val Asn Lys 1 5 10 15 Val Glu Gln
3819PRTartificialL2 fragment 38Gln Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
3919PRTartificialL2 fragment 39Gln Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
4019PRTartificialL2 fragment 40Arg Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Ser
4119PRTartificialL2 fragment 41Arg Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
4219PRTartificialL2 fragment 42Lys Gly Cys Lys Ala Ser Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
4319PRTartificialL2 fragment 43Arg Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
4419PRTartificialL2 fragment 44Arg Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
4519PRTartificialL2 fragment 45Arg Thr Cys Lys Val Thr Gly Thr Cys
Pro Ala Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
4619PRTartificialL2 fragment 46Arg Thr Cys Lys Ala Thr Gly Thr Arg
Pro Ala Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
4719PRTartificialL2 fragment 47Arg Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Val Glu Gln
4819PRTartificialL2 fragment 48Arg Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Leu Glu Gln
4919PRTartificialL2 fragment 49Arg Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Val Glu Gln
5019PRTartificialL2 fragment 50Lys Thr Cys Lys Val Ala Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
5119PRTartificialL2 fragment 51Lys Thr
Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15
Val Glu Gly 5219PRTartificialL2 fragment 52Arg Thr Cys Lys Ala Ala
Gly Thr Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
5319PRTartificialL2 fragment 53Arg Thr Cys Lys Ala Ser Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
5419PRTartificialL2 fragment 54Ser Thr Cys Lys Ala Ala Gly Thr Cys
Pro Ala Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
5519PRTartificialL2 fragment 55Lys Thr Cys Lys Leu Ser Gly Thr Cys
Pro Glu Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
5619PRTartificialL2 fragment 56Lys Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Val Glu Gly
5719PRTartificialL2 fragment 57Lys Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Ile Val Pro Lys 1 5 10 15 Val Glu Gly
5819PRTartificialL2 fragment 58Gln Thr Cys Lys Ala Ser Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
5919PRTartificialL2 fragment 59Lys Thr Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
6019PRTartificialL2 fragment 60Gln Thr Cys Lys Ala Ala Gly Thr Cys
Pro Ser Asp Ile Ile Pro Lys 1 5 10 15 Val Glu His
6119PRTartificialL2 fragment 61Gln Thr Cys Lys Ala Ser Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gln
6219PRTartificialL2 fragment 62Gln Thr Cys Lys Leu Thr Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu His
6319PRTartificialL2 fragment 63Gln Thr Cys Lys Ala Ala Gly Thr Cys
Pro Ser Asp Val Ile Asn Lys 1 5 10 15 Val Glu His
6419PRTartificialL2 fragment 64Lys Gln Cys Gln Leu Gly Ala Asp Cys
Pro Pro Asp Val Arg Asn Lys 1 5 10 15 Val Glu Gly
6519PRTartificialL2 fragment 65Ala Lys Cys Gln Leu Ser Gly Asn Cys
Leu Pro Asp Val Lys Asn Lys 1 5 10 15 Val Glu Ala
6619PRTartificialL2 fragment 66Ala Lys Cys Gln Leu Ser Gly Asp Cys
Leu Pro Asp Val Lys Asn Lys 1 5 10 15 Val Glu Ala
6719PRTartificialL2 fragment 67Arg His Cys Ala Leu Ser Gly Thr Cys
Pro Asp Asp Val Lys Asn Lys 1 5 10 15 Val Glu Asn
6819PRTartificialL2 fragment 68Lys His Cys Ala Gly Ser Gly Thr Cys
Pro Glu Asp Val Lys Asn Lys 1 5 10 15 Val Glu Gln
6919PRTartificialL2 fragment 69Lys Thr Cys Leu Gln Gly Gly Asp Cys
Ile Pro Asp Val Lys Asn Lys 1 5 10 15 Phe Glu Asn
7019PRTartificialL2 fragment 70Arg Ser Cys Leu Gln Gly Gly Asp Cys
Ile Pro Asp Val Gln Asn Lys 1 5 10 15 Phe Glu Gly
7119PRTartificialL2 fragment 71Gln Thr Cys Lys Ala Thr Gly Thr Cys
Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
7219PRTartificialL2 fragment 72Lys Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
7319PRTartificialL2 fragment 73Arg Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gly
7419PRTartificialL2 fragment 74Lys Thr Cys Lys Gln Ala Gly Thr Cys
Pro Ser Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gly
7519PRTartificialL2 fragment 75Lys Thr Cys Lys Leu Ser Gly Thr Cys
Pro Glu Asp Val Val Asn Lys 1 5 10 15 Ile Glu Gln
7619PRTartificialL2 fragment 76Arg Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp Val Val Asp Lys 1 5 10 15 Val Glu Gly
7719PRTartificialL2 fragment 77Ser Thr Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp Val Val Asn Lys 1 5 10 15 Val Glu Gly
7819PRTartificialL2 fragment 78Pro Thr Cys Lys Ile Ala Gly Asn Cys
Pro Ala Asp Ile Gln Asn Lys 1 5 10 15 Phe Glu Asn
7919PRTartificialL2 fragment 79Pro Ala Cys Lys Ile Ser Asn Thr Cys
Pro Pro Asp Ile Ile Asn Lys 1 5 10 15 Tyr Glu Asn
8012PRTartificialL2 fragment 80Arg Gly Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp 1 5 10 8112PRTartificialL2 fragment 81Arg Gly Cys Lys
Ala Ser Asn Thr Cys Pro Pro Asp 1 5 10 8212PRTartificialL2 fragment
82Arg Gly Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp 1 5 10
8312PRTartificialL2 fragment 83Gln Ser Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp 1 5 10 8412PRTartificialL2 fragment 84Gln Thr Cys Lys
Gln Ala Gly Thr Cys Pro Pro Asp 1 5 10 8512PRTartificialL2 fragment
85Arg Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp 1 5 10
8612PRTartificialL2 fragment 86Lys Gly Cys Lys Ala Ser Gly Thr Cys
Pro Pro Asp 1 5 10 8712PRTartificialL2 fragment 87Arg Thr Cys Lys
Gln Ser Gly Thr Cys Pro Pro Asp 1 5 10 8812PRTartificialL2 fragment
88Arg Thr Cys Lys Val Thr Gly Thr Cys Pro Ala Asp 1 5 10
8912PRTartificialL2 fragment 89Arg Thr Cys Lys Ala Thr Gly Thr Arg
Pro Ala Asp 1 5 10 9012PRTartificialL2 fragment 90Lys Thr Cys Lys
Val Ala Gly Thr Cys Pro Pro Asp 1 5 10 9112PRTartificialL2 fragment
91Lys Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp 1 5 10
9212PRTartificialL2 fragment 92Arg Thr Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp 1 5 10 9312PRTartificialL2 fragment 93Arg Thr Cys Lys
Ala Ser Gly Thr Cys Pro Pro Asp 1 5 10 9412PRTartificialL2 fragment
94Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Ala Asp 1 5 10
9512PRTartificialL2 fragment 95Lys Thr Cys Lys Leu Ser Gly Thr Cys
Pro Glu Asp 1 5 10 9612PRTartificialL2 fragment 96Lys Thr Cys Lys
Gln Ala Gly Thr Cys Pro Glu Asp 1 5 10 9712PRTartificialL2 fragment
97Gln Thr Cys Lys Ala Ser Gly Thr Cys Pro Pro Asp 1 5 10
9812PRTartificialL2 fragment 98Gln Thr Cys Lys Ala Ala Gly Thr Cys
Pro Ser Asp 1 5 10 9912PRTartificialL2 fragment 99Gln Thr Cys Lys
Leu Thr Gly Thr Cys Pro Pro Asp 1 5 10 10012PRTartificialL2
fragment 100Lys Gln Cys Gln Leu Gly Ala Asp Cys Pro Pro Asp 1 5 10
10112PRTartificialL2 fragment 101Ala Lys Cys Gln Leu Ser Gly Asn
Cys Leu Pro Asp 1 5 10 10212PRTartificialL2 fragment 102Ala Lys Cys
Gln Leu Ser Gly Asp Cys Leu Pro Asp 1 5 10 10312PRTartificialL2
fragment 103Arg His Cys Ala Leu Ser Gly Thr Cys Pro Asp Asp 1 5 10
10412PRTartificialL2 fragment 104Lys His Cys Ala Gly Ser Gly Thr
Cys Pro Glu Asp 1 5 10 10512PRTartificialL2 fragment 105Lys Thr Cys
Leu Gln Gly Gly Asp Cys Ile Pro Asp 1 5 10 10612PRTartificialL2
fragment 106Arg Ser Cys Leu Gln Gly Gly Asp Cys Ile Pro Asp 1 5 10
10712PRTartificialL2 fragment 107Gln Thr Cys Lys Ala Thr Gly Thr
Cys Pro Pro Asp 1 5 10 10812PRTartificialL2 fragment 108Lys Thr Cys
Lys Gln Ser Gly Thr Cys Pro Pro Asp 1 5 10 10912PRTartificialL2
fragment 109Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp 1 5 10
11012PRTartificialL2 fragment 110Ser Thr Cys Lys Ala Ala Gly Thr
Cys Pro Pro Asp 1 5 10 11112PRTartificialL2 fragment 111Pro Thr Cys
Lys Ile Ala Gly Asn Cys Pro Ala Asp 1 5 10 11212PRTartificialL2
fragment 112Pro Ala Cys Lys Ile Ser Asn Thr Cys Pro Pro Asp 1 5 10
11311PRTartificialL2 fragment 113Gly Cys Lys Gln Ala Gly Thr Cys
Pro Pro Asp 1 5 10 11411PRTartificialL2 fragment 114Gly Cys Lys Ala
Ser Asn Thr Cys Pro Pro Asp 1 5 10 11511PRTartificialL2 fragment
115Gly Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp 1 5 10
11611PRTartificialL2 fragment 116Ser Cys Lys Ala Ala Gly Thr Cys
Pro Pro Asp 1 5 10 11711PRTartificialL2 fragment 117Thr Cys Lys Gln
Ser Gly Thr Cys Pro Ser Asp 1 5 10 11811PRTartificialL2 fragment
118Gly Cys Lys Ala Ser Gly Thr Cys Pro Pro Asp 1 5 10
11911PRTartificialL2 fragment 119Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro Asp 1 5 10 12011PRTartificialL2 fragment 120Thr Cys Lys Val
Thr Gly Thr Cys Pro Ala Asp 1 5 10 12111PRTartificialL2 fragment
121Thr Cys Lys Ala Thr Gly Thr Arg Pro Ala Asp 1 5 10
12211PRTartificialL2 fragment 122Thr Cys Lys Val Ala Gly Thr Cys
Pro Pro Asp 1 5 10 12311PRTartificialL2 fragment 123Thr Cys Lys Ala
Ala Gly Thr Cys Pro Pro Asp 1 5 10 12411PRTartificialL2 fragment
124Thr Cys Lys Ala Ser Gly Thr Cys Pro Pro Asp 1 5 10
12511PRTartificialL2 fragment 125Thr Cys Lys Ala Ala Gly Thr Cys
Pro Ala Asp 1 5 10 12611PRTartificialL2 fragment 126Thr Cys Lys Leu
Ser Gly Thr Cys Pro Glu Asp 1 5 10 12711PRTartificialL2 fragment
127Thr Cys Lys Ala Ala Gly Thr Cys Pro Ser Asp 1 5 10
12811PRTartificialL2 fragment 128Thr Cys Lys Leu Thr Gly Thr Cys
Pro Pro Asp 1 5 10 12911PRTartificialL2 fragment 129Gln Cys Gln Leu
Gly Ala Asp Cys Pro Pro Asp 1 5 10 13011PRTartificialL2 fragment
130Lys Cys Gln Leu Ser Gly Asn Cys Leu Pro Asp 1 5 10
13111PRTartificialL2 fragment 131Lys Cys Gln Leu Ser Gly Asp Cys
Leu Pro Asp 1 5 10 13211PRTartificialL2 fragment 132His Cys Ala Leu
Ser Gly Thr Cys Pro Asp Asp 1 5 10 13311PRTartificialL2 fragment
133His Cys Ala Gly Ser Gly Thr Cys Pro Glu Asp 1 5 10
13411PRTartificialL2 fragment 134Thr Cys Leu Gln Gly Gly Asp Cys
Ile Pro Asp 1 5 10 13511PRTartificialL2 fragment 135Ser Cys Leu Gln
Gly Gly Asp Cys Ile Pro Asp 1 5 10 13611PRTartificialL2 fragment
136Thr Cys Lys Ala Thr Gly Thr Cys Pro Pro Asp 1 5 10
13711PRTartificialL2 fragment 137Thr Cys Lys Gln Ala Gly Thr Cys
Pro Ser Asp 1 5 10 13811PRTartificialL2 fragment 138Thr Cys Lys Ile
Ala Gly Asn Cys Pro Ala Asp 1 5 10 13911PRTartificialL2 fragment
139Ala Cys Lys Ile Ser Asn Thr Cys Pro Pro Asp 1 5 10
14011PRTartificialL2 fragment 140Thr Cys Lys Gln Ser Gly Thr Cys
Pro Ser Asp 1 5 10 14110PRTartificialL2 fragment 141Cys Lys Ala Ser
Asn Thr Cys Pro Pro Asp 1 5 10 14210PRTartificialL2 fragment 142Cys
Lys Ala Ser Asn Thr Cys Pro Pro Asp 1 5 10 14310PRTartificialL2
fragment 143Cys Lys Ala Ser Gly Thr Cys Pro Pro Asp 1 5 10
14410PRTartificialL2 fragment 144Cys Lys Gln Ser Gly Thr Cys Pro
Pro Asp 1 5 10 14510PRTartificialL2 fragment 145Cys Lys Val Thr Gly
Thr Cys Pro Ala Asp 1 5 10 14610PRTartificialL2 fragment 146Cys Lys
Ala Thr Gly Thr Arg Pro Ala Asp 1 5 10 14710PRTartificialL2
fragment 147Cys Lys Val Ala Gly Thr Cys Pro Pro Asp 1 5 10
14810PRTartificialL2 fragment 148Cys Lys Val Ala Gly Thr Cys Pro
Pro Asp 1 5 10 14910PRTartificialL2 fragment 149Cys Lys Leu Ser Gly
Thr Cys Pro Glu Asp 1 5 10 15010PRTartificialL2 fragment 150Cys Lys
Ala Ala Gly Thr Cys Pro Ser Asp 1 5 10 15110PRTartificialL2
fragment 151Cys Lys Leu Thr Gly Thr Cys Pro Pro Asp 1 5 10
15210PRTartificialL2 fragment 152Cys Gln Leu Gly Ala Asp Cys Pro
Pro Asp 1 5 10 15310PRTartificialL2 fragment 153Cys Gln Leu Ser Gly
Asn Cys Leu Pro Asp 1 5 10 15410PRTartificialL2 fragment 154Cys Gln
Leu Ser Gly Asn Cys Leu Pro Asp 1 5 10 15510PRTartificialL2
fragment 155Cys Ala Leu Ser Gly Thr Cys Pro Asp Asp 1 5 10
15610PRTartificialL2 fragment 156Cys Ala Gly Ser Gly Thr Cys Pro
Glu Asp 1 5 10 15710PRTartificialL2 fragment 157Cys Leu Gln Gly Gly
Asp Cys Ile Pro Asp 1 5 10 15810PRTartificialL2 fragment 158Cys Lys
Ala Thr Gly Thr Cys Pro Pro Asp 1 5 10 15910PRTartificialL2
fragment 159Cys Lys Gln Ala Gly Thr Cys Pro Ser Asp 1 5 10
16010PRTartificialL2 fragment 160Cys Lys Ile Ala Gly Asn Cys Pro
Ala Asp 1 5 10 16110PRTartificialL2 fragment 161Cys Lys Ile Ser Asn
Thr Cys Pro Pro Asp 1 5 10 16210PRTartificialL2 fragment 162Gly Cys
Lys Gln Ala Gly Thr Cys Pro Pro 1 5 10 16310PRTartificialL2
fragment 163Gly Cys Lys Ala Ser Asn Thr Cys Pro Pro 1 5 10
16410PRTartificialL2 fragment 164Gly Cys Lys Ala Ala Gly Thr Cys
Pro Pro 1 5 10 16510PRTartificialL2 fragment 165Ser Cys Lys Ala Ala
Gly Thr Cys Pro Pro 1 5 10 16610PRTartificialL2 fragment 166Thr Cys
Lys Leu Ala Gly Thr Cys Pro Pro 1 5 10 16710PRTartificialL2
fragment 167Gly Cys Lys Ala Ser Gly Thr Cys Pro Pro 1 5 10
16810PRTartificialL2 fragment 168Thr Cys Lys Gln Ser Gly Thr Cys
Pro Pro 1 5 10 16910PRTartificialL2 fragment 169Thr Cys Lys Val Thr
Gly Thr Cys Pro Ala 1 5 10 17010PRTartificialL2 fragment 170Thr Cys
Lys Ala Thr Gly Thr Arg Pro Ala 1 5 10 17110PRTartificialL2
fragment 171Thr Cys Lys Val Ala Gly Thr Cys Pro Pro 1 5 10
17210PRTartifiicial 172Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro 1 5
10 17310PRTartificialL2 fragment 173Thr Cys Lys Ala Ser Gly Thr Cys
Pro Pro 1 5 10 17410PRTartificialL2 fragment 174Thr Cys Lys Ala Ala
Gly Thr Cys Pro Ala 1 5 10 17510PRTartificialL2 fragment 175Thr Cys
Lys Leu Ser Gly Thr Cys Pro Glu 1 5 10 17610PRTartificialL2
fragment 176Thr Cys Lys Ala Ala Gly Thr Cys Pro Ser 1 5 10
17710PRTartificialL2 fragment 177Thr Cys Lys Leu Thr Gly Thr Cys
Pro Pro 1 5 10 17810PRTartificialL2 fragment 178Gln Cys Gln Leu Gly
Ala Asp Cys Pro Pro 1 5 10 17910PRTartificialL2 fragment 179Lys Cys
Gln Leu Ser Gly Asn Cys Leu Pro 1 5 10 18010PRTartificialL2
fragment 180Lys Cys Gln Leu Ser Gly Asp Cys Leu Pro 1 5 10
18110PRTartificialL2 fragment 181His Cys Ala Leu Ser Gly Thr Cys
Pro Asp 1 5 10 18210PRTartificialL2 fragment 182His Cys Ala Gly Ser
Gly Thr Cys Pro Glu 1 5 10 18310PRTartificialL2 fragment 183Thr Cys
Leu Gln Gly Gly Asp Cys Ile Pro 1 5 10 18410PRTartificialL2
fragment 184Ser Cys Leu Gln Gly
Gly Asp Cys Ile Pro 1 5 10 18510PRTartificialL2 fragment 185Thr Cys
Lys Ala Thr Gly Thr Cys Pro Pro 1 5 10 18610PRTartificialL2
fragment 186Thr Cys Lys Gln Ala Gly Thr Cys Pro Ser 1 5 10
18710PRTartificialL2 fragment 187Thr Cys Lys Ile Ala Gly Asn Cys
Pro Ala 1 5 10 18810PRTartificialL2 fragment 188Ala Cys Lys Ile Ser
Asn Thr Cys Pro Pro 1 5 10 1899PRTartificialL2 fragment 189Cys Lys
Leu Ala Gly Thr Cys Pro Pro 1 5 1909PRTartificialL2 fragment 190Cys
Lys Ala Ser Asn Thr Cys Pro Pro 1 5 1919PRTartificialL2 fragment
191Cys Lys Ala Ala Gly Thr Cys Pro Pro 1 5 1929PRTartificialL2
fragment 192Cys Lys Ala Ser Gly Thr Cys Pro Pro 1 5
1939PRTartificialL2 fragment 193Cys Lys Gln Ser Gly Thr Cys Pro Pro
1 5 1949PRTartificialL2 fragment 194Cys Lys Val Thr Gly Thr Cys Pro
Ala 1 5 1959PRTartificialL2 fragment 195Cys Lys Ala Thr Gly Thr Arg
Pro Ala 1 5 1969PRTartificialL2 fragment 196Cys Lys Val Ala Gly Thr
Cys Pro Pro 1 5 1979PRTartificialL2 fragment 197Cys Lys Val Ala Gly
Thr Cys Pro Pro 1 5 1989PRTartificialL2 fragment 198Cys Lys Leu Ser
Gly Thr Cys Pro Glu 1 5 1999PRTartificialL2 fragment 199Cys Lys Ala
Ala Gly Thr Cys Pro Ser 1 5 2009PRTartificialL2 fragment 200Cys Lys
Leu Thr Gly Thr Cys Pro Pro 1 5 2019PRTartificialL2 fragment 201Cys
Gln Leu Gly Ala Asp Cys Pro Pro 1 5 2029PRTartificialL2 fragment
202Cys Gln Leu Ser Gly Asn Cys Leu Pro 1 5 2039PRTartifical 203Cys
Gln Leu Ser Gly Asp Cys Leu Pro 1 5 2049PRTartificialL2 fragment
204Cys Ala Leu Ser Gly Thr Cys Pro Asp 1 5 2059PRTartificialL2
fragment 205Cys Ala Gly Ser Gly Thr Cys Pro Glu 1 5
2069PRTartificialL2 fragment 206Cys Leu Gln Gly Gly Asp Cys Ile Pro
1 5 2079PRTartificialL2 fragment 207Cys Lys Ala Thr Gly Thr Cys Pro
Pro 1 5 2089PRTartificialL2 fragment 208Cys Lys Gln Ala Gly Thr Cys
Pro Ser 1 5 2099PRTartificialL2 fragment 209Cys Lys Gln Ala Gly Thr
Cys Pro Ser 1 5 2109PRTartificialL2 fragment 210Cys Lys Ile Ser Asn
Thr Cys Pro Pro 1 5 21110PRTartificialL2 fragment 211Asp Val Ile
Asn Lys Val Glu Gln Thr Thr 1 5 10 21210PRTartificialL2 fragment
212Asp Val Ile Asn Lys Val Glu Gln Ser Thr 1 5 10
21310PRTartificialL2 fragment 213Asp Val Ile Asn Lys Val Glu Gln
Lys Thr 1 5 10 21410PRTartificialL2 fragment 214Asp Val Leu Asn Lys
Val Glu Gln Thr Thr 1 5 10 21510PRTartificialL2 fragment 215Asp Val
Val Asn Lys Val Glu Gln Thr Thr 1 5 10 21610PRTartificialL2
fragment 216Asp Val Ile Asn Lys Val Glu Ser Thr Thr 1 5 10
21710PRTartificialL2 fragment 217Asp Val Ile Asn Lys Val Glu Gln
Asn Thr 1 5 10 21810PRTartificialL2 fragment 218Asp Val Val Pro Lys
Val Glu Gly Asp Thr 1 5 10 21910PRTartificialL2 fragment 219Asp Val
Ile Pro Lys Val Glu Gly Asp Thr 1 5 10 22010PRTartificialL2
fragment 220Asp Ile Ile Pro Arg Val Glu Gln Asn Thr 1 5 10
22110PRTartificialL2 fragment 221Asp Ile Ile Pro Arg Leu Glu Gln
Asn Thr 1 5 10 22210PRTartificialL2 fragment 222Asp Ile Ile Pro Arg
Val Glu Gln Asp Thr 1 5 10 22310PRTartificialL2 fragment 223Asp Ile
Ile Pro Arg Val Glu Gln Asp Thr 1 5 10 22410PRTartificialL2
fragment 224Asp Ile Ile Pro Lys Val Glu Gln Lys Thr 1 5 10
22510PRTartificialL2 fragment 225Asp Val Ile Pro Lys Val Glu Gly
Ser Thr 1 5 10 22610PRTartificialL2 fragment 226Asp Ile Ile Pro Lys
Val Glu His Asn Thr 1 5 10 22710PRTartificialL2 fragment 227Asp Val
Ile Pro Lys Val Glu Gln Asn Thr 1 5 10 22810PRTartificialL2
fragment 228Asp Val Ile Pro Lys Val Glu His Asn Thr 1 5 10
22910PRTartificialL2 fragment 229Asp Val Ile Asn Lys Val Glu His
Thr Thr 1 5 10 23010PRTartificialL2 fragment 230Asp Val Arg Asn Lys
Val Glu Gly Thr Thr 1 5 10 23110PRTartificialL2 fragment 231Asp Val
Lys Asn Lys Val Glu Ala Asp Thr 1 5 10 23210PRTartificialL2
fragment 232Asp Val Lys Asn Lys Val Glu Ala Asn Thr 1 5 10
23310PRTartificialL2 fragment 233Asp Val Lys Asn Lys Val Glu Asn
Asn Thr 1 5 10 23410PRTartificialL2 fragment 234Asp Val Lys Asn Lys
Val Glu Gln Thr Thr 1 5 10 23510PRTartificialL2 fragment 235Asp Val
Lys Asn Lys Phe Glu Asn Ser Thr 1 5 10 23610PRTartificialL2
fragment 236Asp Val Gln Asn Lys Phe Glu Gly Asn Thr 1 5 10
23710PRTartificialL2 fragment 237Asp Ile Gln Asn Lys Ile Glu Gln
Thr Thr 1 5 10 23810PRTartificialL2 fragment 238Asp Val Ile Lys Arg
Tyr Glu Gln Thr Thr 1 5 10 2399PRTartificialL2 fragment 239Val Ile
Asn Lys Val Glu Gln Thr Thr 1 5 2409PRTartificialL2 fragment 240Val
Ile Asn Lys Val Glu Gln Ser Thr 1 5 2419PRTartificialL2 fragment
241Val Ile Asn Lys Val Glu Gln Lys Thr 1 5 2429PRTartificialL2
fragment 242Val Leu Asn Lys Val Glu Gln Thr Thr 1 5
2439PRTartificialL2 fragment 243Val Val Asn Lys Val Glu Gln Thr Thr
1 5 2449PRTartificialL2 fragment 244Val Ile Asn Lys Val Glu Ser Thr
Thr 1 5 2459PRTartificialL2 fragment 245Val Ile Asn Lys Val Glu Gln
Asn Thr 1 5 2469PRTartificialL2 fragment 246Val Val Pro Lys Val Glu
Gly Asp Thr 1 5 2479PRTartificialL2 fragment 247Val Ile Pro Lys Val
Glu Gly Asp Thr 1 5 2489PRTartificialL2 fragment 248Ile Ile Pro Arg
Val Glu Gln Asn Thr 1 5 2499PRTartificialL2 fragment 249Ile Ile Pro
Arg Leu Glu Gln Asn Thr 1 5 2509PRTartificialL2 fragment 250Ile Ile
Pro Arg Val Glu Gln Asp Thr 1 5 2519PRTartificialL2 fragment 251Val
Ile Pro Lys Val Glu Gly Thr Thr 1 5 2529PRTartificialL2 fragment
252Ile Ile Pro Lys Val Glu Gln Lys Thr 1 5 2539PRTartificialL2
fragment 253Val Ile Pro Lys Val Glu Gly Ser Thr 1 5
2549PRTartificialL2 fragment 254Ile Ile Pro Lys Val Glu His Asn Thr
1 5 2559PRTartificialL2 fragment 255Val Ile Pro Lys Val Glu Gln Asn
Thr 1 5 2569PRTartificialL2 fragment 256Val Ile Pro Lys Val Glu His
Asn Thr 1 5 2579PRTartificialL2 fragment 257Val Ile Asn Lys Val Glu
His Thr Thr 1 5 2589PRTartificialL2 fragment 258Val Arg Asn Lys Val
Glu Gly Thr Thr 1 5 2599PRTartificialL2 fragment 259Val Lys Asn Lys
Val Glu Ala Asp Thr 1 5 2609PRTartificialL2 fragment 260Val Lys Asn
Lys Val Glu Ala Asn Thr 1 5 2619PRTartificialL2 fragment 261Val Lys
Asn Lys Val Glu Asn Asn Thr 1 5 2629PRTartificialL2 fragment 262Val
Lys Asn Lys Val Glu Gln Thr Thr 1 5 2639PRTartificialL2 fragment
263Val Lys Asn Lys Phe Glu Asn Ser Thr 1 5 2649PRTartificialL2
fragment 264Val Gln Asn Lys Phe Glu Gly Asn Thr 1 5
2659PRTartificialL2 fragment 265Ile Gln Asn Lys Ile Glu Gln Thr Thr
1 5 2669PRTartificialL2 fragment 266Val Ile Lys Arg Tyr Glu Gln Thr
Thr 1 5 2678PRTartificialL2 fragment 267Ile Asn Lys Val Glu Gln Thr
Thr 1 5 2688PRTartificialL2 fragment 268Ile Asn Lys Val Glu Gln Thr
Thr 1 5 2698PRTartificialL2 fragment 269Ile Asn Lys Val Glu Gln Lys
Thr 1 5 2708PRTartificialL2 fragment 270Leu Asn Lys Val Glu Gln Thr
Thr 1 5 2718PRTartificialL2 fragment 271Val Asn Lys Val Glu Gln Thr
Thr 1 5 2728PRTartificialL2 fragment 272Ile Asn Lys Val Glu Ser Thr
Thr 1 5 2738PRTartificialL2 fragment 273Ile Asn Lys Val Glu Gln Asn
Thr 1 5 2748PRTartificialL2 fragment 274Val Pro Lys Val Glu Gly Asp
Thr 1 5 2758PRTartificialL2 fragment 275Ile Pro Lys Val Glu Gly Asp
Thr 1 5 2768PRTartificialL2 fragment 276Ile Pro Arg Val Glu Gln Asn
Thr 1 5 2778PRTartificialL2 fragment 277Ile Pro Arg Leu Glu Gln Asn
Thr 1 5 2788PRTartificialL2 fragment 278Ile Pro Arg Val Glu Gln Asp
Thr 1 5 2798PRTartificialL2 fragment 279Ile Pro Lys Val Glu Gly Thr
Thr 1 5 2808PRTartificialL2 fragment 280Ile Pro Lys Val Glu His Lys
Thr 1 5 2818PRTartificialL2 fragment 281Ile Pro Lys Val Glu Gly Ser
Thr 1 5 2828PRTartificialL2 fragment 282Ile Pro Lys Val Glu His Asn
Thr 1 5 2838PRTartificialL2 fragment 283Ile Pro Lys Val Glu Gln Asn
Thr 1 5 2848PRTartificialL2 fragment 284Ile Asn Lys Val Glu His Thr
Thr 1 5 2858PRTartificialL2 fragment 285Arg Asn Lys Val Glu Gly Thr
Thr 1 5 2868PRTartificialL2 fragment 286Lys Asn Lys Val Glu Ala Asp
Thr 1 5 2878PRTartificialL2 fragment 287Lys Asn Lys Val Glu Ala Asn
Thr 1 5 2888PRTartificialL2 fragment 288Lys Asn Lys Val Glu Asn Asn
Thr 1 5 2898PRTartificialL2 fragment 289Lys Asn Lys Val Glu Gln Thr
Thr 1 5 2908PRTartificialL2 fragment 290Lys Asn Lys Phe Glu Asn Ser
Thr 1 5 2918PRTartificialL2 fragment 291Lys Asn Lys Phe Glu Asn Ser
Thr 1 5 2928PRTartificialL2 fragment 292Gln Asn Lys Ile Glu Gln Thr
Thr 1 5 2938PRTartificialL2 fragment 293Ile Lys Arg Tyr Glu Gln Thr
Thr 1 5 2949PRTartificialL2 fragment 294Thr Gly Tyr Ile Pro Leu Gln
Thr Arg 1 5 2959PRTartificialL2 fragment 295Thr Gly Tyr Val Pro Leu
Gly Ser Thr 1 5 2969PRTartificialL2 fragment 296Thr Gly Tyr Val Pro
Leu Gly Asn Thr 1 5 2979PRTartificialL2 fragment 297Thr Gly Tyr Val
Pro Leu Ser Thr Gly 1 5 2989PRTartificialL2 fragment 298Thr Gly Tyr
Ile Pro Leu Gln Ser Thr 1 5 2999PRTartificialL2 fragment 299Thr Gly
Tyr Val Pro Val Gly Ser Thr 1 5 3009PRTartificialL2 fragment 300Thr
Gly Tyr Val Pro Leu Gln Thr Ser 1 5 3019PRTartificialL2 fragment
301Thr Gly Tyr Val Pro Leu Thr Thr Gly 1 5 30219PRTartificialL2
fragment 302Arg Gly Cys Lys Gln Xaa Gly Thr Cys Pro Pro Asp Val Ile
Asn Lys 1 5 10 15 Val Glu Gln 30319PRTartificialL2 fragment 303Arg
Gly Cys Lys Ala Xaa Asn Thr Cys Pro Pro Asp Val Ile Asn Lys 1 5 10
15 Val Glu Gln 30419PRTartificialL2 fragment 304Arg Gly Cys Lys Ala
Xaa Gly Thr Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
30519PRTartificialL2 fragment 305Gln Ser Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Leu Asn Lys 1 5 10 15 Val Glu Gln
30619PRTartificialL2 fragment 306Gln Ser Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Val Asn Lys 1 5 10 15 Val Glu Gln
30719PRTartificialL2 fragment 307Gln Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
30819PRTartificialL2 fragment 308Gln Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
30919PRTartificialL2 fragment 309Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Ser
31019PRTartificialL2 fragment 310Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
31119PRTartificialL2 fragment 311Lys Gly Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
31219PRTartificialL2 fragment 312Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
31319PRTartificialL2 fragment 313Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
31419PRTartificialL2 fragment 314Arg Thr Cys Lys Val Xaa Gly Thr
Cys Pro Ala Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
31519PRTartificialL2 fragment 315Arg Thr Cys Lys Ala Xaa Gly Thr
Arg Pro Ala Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
31619PRTartificialL2 fragment 316Ser Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Leu Glu Gly
31719PRTartificialL2 fragment 317Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Leu Glu Gln
31819PRTartificialL2 fragment 318Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Val Glu Gln
31919PRTartificialL2 fragment 319Lys Thr Cys Lys Val Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32019PRTartificialL2 fragment 320Lys Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32119PRTartificialL2 fragment 321Ser Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32219PRTartificialL2 fragment 322Arg Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32319PRTartificialL2 fragment 323Ser Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Ala Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32419PRTartificialL2 fragment 324Lys Thr Cys Lys Leu Xaa Gly Thr
Cys Pro Glu Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gln
32519PRTartificialL2 fragment 325Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Ile Glu Gly
32619PRTartificialL2 fragment 326Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Ile Val Pro Lys 1 5 10 15 Val Glu Gly
32719PRTartificialL2 fragment 327Ser Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Ile Ile Pro Arg 1 5 10 15 Val Glu Gln
32819PRTartificialL2 fragment 328Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
32919PRTartificialL2 fragment 329Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Ser Asp Ile Ile Pro Lys 1 5 10 15 Val Glu His
33019PRTartificialL2 fragment 330Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gln
33119PRTartificialL2 fragment 331Gln Thr Cys Lys Leu Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu His
33219PRTartificialL2 fragment 332Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Ser Asp Val Ile Asn Lys 1 5 10
15 Val Glu His 33319PRTartificialL2 fragment 333Lys Gln Cys Gln Leu
Xaa Ala Asp Cys Pro Pro Asp Val Arg Asn Lys 1 5 10 15 Val Glu Gly
33419PRTartificialL2 fragment 334Ala Lys Cys Gln Leu Xaa Gly Asn
Cys Leu Pro Asp Val Lys Asn Lys 1 5 10 15 Val Glu Ala
33519PRTartificialL2 fragment 335Ala Lys Cys Gln Leu Xaa Gly Asp
Cys Leu Pro Asp Val Lys Asn Lys 1 5 10 15 Val Glu Ala
33619PRTartificialL2 fragment 336Arg His Cys Ala Leu Xaa Gly Thr
Cys Pro Asp Asp Val Lys Asn Lys 1 5 10 15 Val Glu Asn
33719PRTartificialL2 fragment 337Lys His Cys Ala Gly Xaa Gly Thr
Cys Pro Glu Asp Val Lys Asn Lys 1 5 10 15 Val Glu Gln
33819PRTartificialL2 fragment 338Lys Thr Cys Leu Gln Xaa Gly Asp
Cys Ile Pro Asp Val Lys Asn Lys 1 5 10 15 Phe Glu Asn
33919PRTartificialL2 fragment 339Arg Ser Cys Leu Gln Xaa Gly Asp
Cys Ile Pro Asp Val Gln Asn Lys 1 5 10 15 Phe Glu Gly
34019PRTartificialL2 fragment 340Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Glu Gly
34119PRTartificialL2 fragment 341Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Val Pro Lys 1 5 10 15 Val Glu Gly
34219PRTartificialL2 fragment 342Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gly
34319PRTartificialL2 fragment 343Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Ser Asp Val Ile Asn Lys 1 5 10 15 Val Glu Gly
34419PRTartificialL2 fragment 344Lys Thr Cys Lys Leu Xaa Gly Thr
Cys Pro Glu Asp Val Val Asn Lys 1 5 10 15 Ile Glu Gln
34519PRTartificialL2 fragment 345Arg Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp Val Val Asp Lys 1 5 10 15 Val Glu Gly
34619PRTartificialL2 fragment 346Ser Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp Val Val Asn Lys 1 5 10 15 Val Glu Gly
34719PRTartificialL2 fragment 347Pro Thr Cys Lys Ile Xaa Gly Asn
Cys Pro Ala Asp Ile Gln Asn Lys 1 5 10 15 Phe Glu Asn
34819PRTartificialL2 fragment 348Pro Ala Cys Lys Ile Xaa Asn Thr
Cys Pro Pro Asp Ile Ile Asn Lys 1 5 10 15 Tyr Glu Asn
34912PRTartificialL2 fragment 349Arg Gly Cys Lys Gln Xaa Gly Thr
Cys Pro Pro Asp 1 5 10 35012PRTartificialL2 fragment 350Arg Gly Cys
Lys Ala Xaa Asn Thr Cys Pro Pro Asp 1 5 10 35112PRTartificialL2
fragment 351Arg Gly Cys Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
35212PRTartificialL2 fragment 352Gln Ser Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp 1 5 10 35312PRTartificialL2 fragment 353Gln Thr Cys
Lys Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10 35412PRTartificialL2
fragment 354Arg Thr Cys Lys Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10
35512PRTartificialL2 fragment 355Lys Gly Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp 1 5 10 35612PRTartificialL2 fragment 356Pro Thr Cys
Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10 35712PRTartificialL2
fragment 357Arg Thr Cys Lys Val Xaa Gly Thr Cys Pro Ala Asp 1 5 10
35812PRTartificialL2 fragment 358Arg Thr Cys Lys Ala Xaa Gly Thr
Arg Pro Ala Asp 1 5 10 35912PRTartificialL2 fragment 359Lys Thr Cys
Lys Val Xaa Gly Thr Cys Pro Pro Asp 1 5 10 36012PRTartificialL2
fragment 360Lys Thr Cys Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
36112PRTartificialL2 fragment 361Arg Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp 1 5 10 36212PRTartificialL2 fragment 362Ser Thr Cys
Lys Ala Xaa Gly Thr Arg Pro Pro Asp 1 5 10 36312PRTartificialL2
fragment 363Ser Thr Cys Lys Ala Xaa Gly Thr Cys Pro Ala Asp 1 5 10
36412PRTartificialL2 fragment 364Lys Thr Cys Lys Leu Xaa Gly Thr
Cys Pro Glu Asp 1 5 10 36512PRTartificialL2 fragment 365Ala Thr Cys
Lys Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10 36612PRTartificialL2
fragment 366Ser Thr Cys Lys Gln Xaa Gly Thr Cys Pro Pro Asp 1 5 10
36712PRTartificialL2 fragment 367Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Ser Asp 1 5 10 36812PRTartificialL2 fragment 368Gln Thr Cys
Lys Leu Xaa Gly Thr Cys Pro Pro Asp 1 5 10 36912PRTartificialL2
fragment 369Lys Gln Cys Gln Leu Xaa Ala Asp Cys Pro Pro Asp 1 5 10
37012PRTartificialL2 fragment 370Ala Lys Cys Gln Leu Xaa Gly Asn
Cys Leu Pro Asp 1 5 10 37112PRTartificialL2 fragment 371Ala Lys Cys
Gln Leu Xaa Gly Asp Cys Leu Pro Asp 1 5 10 37212PRTartificialL2
fragment 372Arg His Cys Ala Leu Xaa Gly Thr Cys Pro Asp Asp 1 5 10
37312PRTartificialL2 fragment 373Lys His Cys Ala Gly Xaa Gly Thr
Cys Pro Glu Asp 1 5 10 37412PRTartificialL2 fragment 374Lys Thr Cys
Leu Gln Xaa Gly Asp Cys Ile Pro Asp 1 5 10 37512PRTartificialL2
fragment 375Arg Ser Cys Leu Gln Xaa Gly Asp Cys Ile Pro Asp 1 5 10
37612PRTartificialL2 fragment 376Gln Thr Cys Lys Ala Xaa Gly Thr
Cys Pro Pro Asp 1 5 10 37712PRTartificialL2 fragment 377Lys Thr Cys
Lys Gln Xaa Gly Thr Cys Pro Glu Asp 1 5 10 37812PRTartificialL2
fragment 378Lys Thr Cys Lys Gln Xaa Gly Thr Cys Pro Ser Asp 1 5 10
37912PRTartificialL2 fragment 379Lys Thr Cys Lys Gln Xaa Gly Thr
Cys Pro Ser Asp 1 5 10 38012PRTartificialL2 fragment 380Pro Thr Cys
Lys Ile Xaa Gly Asn Cys Pro Ala Asp 1 5 10 38112PRTartificialL2
fragment 381Pro Ala Cys Lys Ile Xaa Asn Thr Cys Pro Pro Asp 1 5 10
38211PRTartificialL2 fragment 382Gly Cys Lys Gln Xaa Gly Thr Cys
Pro Pro Asp 1 5 10 38311PRTartificialL2 fragment 383Gly Cys Lys Ala
Xaa Asn Thr Cys Pro Pro Asp 1 5 10 38411PRTartificialL2 fragment
384Ala Cys Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
38511PRTartificialL2 fragment 385Ser Cys Lys Ala Xaa Gly Thr Cys
Pro Pro Asp 1 5 10 38611PRTartificialL2 fragment 386Lys Cys Lys Ala
Xaa Gly Thr Cys Ile Pro Asp 1 5 10 38711PRTartificialL2 fragment
387Gly Cys Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
38811PRTartificialL2 fragment 388Lys Cys Lys Ala Xaa Gly Thr Cys
Pro Pro Asp 1 5 10 38911PRTartificialL2 fragment 389Thr Cys Lys Val
Xaa Gly Thr Cys Pro Ala Asp 1 5 10 39011PRTartificialL2 fragment
390Thr Cys Lys Ala Xaa Gly Thr Arg Pro Ala Asp 1 5 10
39111PRTartificialL2 fragment 391Thr Cys Lys Val Xaa Gly Thr Cys
Pro Pro Asp 1 5 10 39211PRTartificialL2 fragment 392Ser Cys Lys Leu
Xaa Gly Thr Cys Pro Pro Asp 1 5 10 39311PRTartificialL2 fragment
393Ser Cys Lys Gln Xaa Gly Thr Cys Pro Ser Asp 1 5 10
39411PRTartificialL2 fragment 394Thr Cys Lys Ala Xaa Gly Thr Cys
Pro Ala Asp 1 5 10 39511PRTartificialL2 fragment 395Thr Cys Lys Leu
Xaa Gly Thr Cys Pro Glu Asp 1 5 10 39611PRTartificialL2 fragment
396Thr Cys Lys Ala Xaa Gly Thr Cys Pro Ser Asp 1 5 10
39711PRTartificialL2 fragment 397Thr Cys Lys Leu Xaa Gly Thr Cys
Pro Pro Asp 1 5 10 39811PRTartificialL2 fragment 398Gln Cys Gln Leu
Xaa Ala Asp Cys Pro Pro Asp 1 5 10 39911PRTartificialL2 fragment
399Lys Cys Gln Leu Xaa Gly Asn Cys Leu Pro Asp 1 5 10
40011PRTartificialL2 fragment 400Lys Cys Gln Leu Xaa Gly Asp Cys
Leu Pro Asp 1 5 10 40111PRTartificialL2 fragment 401His Cys Ala Leu
Xaa Gly Thr Cys Pro Asp Asp 1 5 10 40211PRTartificialL2 fragment
402His Cys Ala Gly Xaa Gly Thr Cys Pro Glu Asp 1 5 10
40311PRTartificialL2 fragment 403Thr Cys Leu Gln Xaa Gly Asp Cys
Ile Pro Asp 1 5 10 40411PRTartificialL2 fragment 404Thr Cys Leu Gln
Xaa Gly Asp Cys Ile Pro Asp 1 5 10 40511PRTartificialL2 fragment
405Thr Cys Lys Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
40611PRTartificialL2 fragment 406Thr Cys Lys Gln Xaa Gly Thr Cys
Pro Ser Asp 1 5 10 40711PRTartificialL2 fragment 407Thr Cys Lys Ile
Xaa Gly Asn Cys Pro Ala Asp 1 5 10 40811PRTartificialL2 fragment
408Thr Cys Lys Ile Xaa Gly Asn Cys Pro Ala Asp 1 5 10
40910PRTartificialL2 fragment 409Cys Lys Gln Xaa Gly Thr Cys Pro
Asp Asp 1 5 10 41010PRTartificialL2 fragment 410Cys Lys Ala Xaa Asn
Thr Cys Pro Pro Asp 1 5 10 41110PRTartificialL2 fragment 411Cys Leu
Ala Xaa Gly Thr Cys Pro Ala Asp 1 5 10 41210PRTartificialL2
fragment 412Cys Leu Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10
41310PRTartificialL2 fragment 413Cys Lys Leu Xaa Gly Thr Cys Pro
Ala Asp 1 5 10 41410PRTartificialL2 fragment 414Cys Lys Val Xaa Gly
Thr Cys Pro Ala Asp 1 5 10 41510PRTartificialL2 fragment 415Cys Lys
Ala Xaa Gly Thr Arg Pro Ala Asp 1 5 10 41610PRTartificialL2
fragment 416Cys Lys Ala Xaa Gly Thr Arg Pro Ala Asp 1 5 10
41710PRTartificialL2 fragment 417Cys Lys Ala Xaa Gly Thr Cys Pro
Ala Asp 1 5 10 41810PRTartificialL2 fragment 418Cys Lys Leu Xaa Gly
Thr Cys Pro Glu Asp 1 5 10 41910PRTartificialL2 fragment 419Cys Lys
Ala Xaa Gly Thr Cys Pro Ser Asp 1 5 10 42010PRTartificialL2
fragment 420Cys Lys Leu Xaa Gly Thr Cys Pro Pro Asp 1 5 10
42110PRTartificialL2 fragment 421Cys Gln Leu Xaa Ala Asp Cys Pro
Pro Asp 1 5 10 42210PRTartificialL2 fragment 422Cys Gln Leu Xaa Gly
Asn Cys Leu Pro Asp 1 5 10 42310PRTartificialL2 fragment 423Cys Gln
Leu Xaa Gly Asp Cys Leu Pro Asp 1 5 10 42410PRTartificialL2
fragment 424Cys Ala Leu Xaa Gly Thr Cys Pro Asp Asp 1 5 10
42510PRTartificialL2 fragment 425Cys Ala Gly Xaa Gly Thr Cys Pro
Glu Asp 1 5 10 42610PRTartificialL2 fragment 426Cys Leu Gln Xaa Gly
Asp Cys Ile Pro Asp 1 5 10 42710PRTartificialL2 fragment 427Cys Lys
Ala Xaa Gly Thr Cys Pro Pro Asp 1 5 10 42810PRTartificialL2
fragment 428Cys Lys Gln Xaa Gly Thr Cys Pro Ser Asp 1 5 10
42910PRTartificialL2 fragment 429Cys Lys Ile Xaa Gly Asn Cys Pro
Ala Asp 1 5 10 43010PRTartificialL2 fragment 430Cys Lys Ile Xaa Asn
Thr Cys Pro Pro Asp 1 5 10 43110PRTartificialL2 fragment 431Gly Cys
Lys Gln Xaa Gly Thr Cys Pro Pro 1 5 10 43210PRTartificialL2
fragment 432Gly Cys Lys Ala Xaa Asn Thr Cys Pro Pro 1 5 10
43310PRTartificialL2 fragment 433Gly Cys Lys Ala Xaa Gly Thr Cys
Pro Pro 1 5 10 43410PRTartificialL2 fragment 434Ser Cys Lys Ala Xaa
Gly Thr Cys Pro Pro 1 5 10 43510PRTartificialL2 fragment 435Thr Cys
Lys Gln Xaa Gly Thr Cys Pro Pro 1 5 10 43610PRTartificialL2
fragment 436Gly Cys Lys Ala Xaa Gly Thr Cys Pro Pro 1 5 10
43710PRTartificialL2 fragment 437Thr Cys Lys Gln Xaa Gly Thr Cys
Pro Pro 1 5 10 43810PRTartificialL2 fragment 438Thr Cys Lys Val Xaa
Gly Thr Cys Pro Ala 1 5 10 43910PRTartificialXaa represents Gly or
Ala 439Thr Cys Lys Ala Xaa Gly Thr Arg Pro Ala 1 5 10
44010PRTartificialL2 fragment 440Thr Cys Lys Val Xaa Gly Thr Cys
Pro Pro 1 5 10 44110PRTartificialL2 fragment 441Thr Cys Lys Ala Xaa
Gly Thr Cys Pro Pro 1 5 10 44210PRTartificialL2 fragment 442Thr Cys
Lys Ala Xaa Gly Thr Cys Pro Pro 1 5 10 44310PRTartificialL2
fragment 443Thr Cys Lys Ala Xaa Gly Thr Cys Pro Ala 1 5 10
44410PRTartificialL2 fragment 444Thr Cys Lys Leu Xaa Gly Thr Cys
Pro Glu 1 5 10 44510PRTartificialL2 fragment 445Thr Cys Lys Ala Xaa
Gly Thr Cys Pro Ser 1 5 10 44610PRTartificialL2 fragment 446Thr Cys
Lys Leu Xaa Gly Thr Cys Pro Pro 1 5 10 44710PRTartificialL2
fragment 447Gln Cys Gln Leu Xaa Ala Asp Cys Pro Pro 1 5 10
44810PRTartificialL2 fragment 448Lys Cys Gln Leu Xaa Gly Asn Cys
Leu Pro 1 5 10 44910PRTartificialL2 fragment 449Lys Cys Gln Leu Xaa
Gly Asp Cys Leu Pro 1 5 10 45010PRTartificialL2 fragment 450His Cys
Ala Leu Xaa Gly Thr Cys Pro Asp 1 5 10 45110PRTartificialL2
fragment 451His Cys Ala Gly Xaa Gly Thr Cys Pro Glu 1 5 10
45210PRTartificialL2 fragment 452Thr Cys Leu Gln Xaa Gly Asp Cys
Ile Pro 1 5 10 45310PRTartificialL2 fragment 453Ser Cys Leu Gln Xaa
Gly Asp Cys Ile Pro 1 5 10 45410PRTartificialL2 fragment 454Thr Cys
Lys Ala Xaa Gly Thr Cys Pro Pro 1 5 10 45510PRTartificialL2
fragment 455Thr Cys Lys Gln Xaa Gly Thr Cys Pro Ser 1 5 10
45610PRTartificialL2 fragment 456Thr Cys Lys Ile Xaa Gly Asn Cys
Pro Ala 1 5 10 45710PRTartificialL2 fragment 457Ala Cys Lys Ile Xaa
Asn Thr Cys Pro Pro 1 5 10 4589PRTartificialL2 fragment 458Cys Lys
Gln Xaa Gly Thr Cys Pro Pro 1 5 4599PRTartificialL2 fragment 459Cys
Lys Ala Xaa Asn Thr Cys Pro Pro 1 5 4609PRTartificialL2 fragment
460Cys Lys Ala Xaa Gly Thr Cys Pro Pro 1 5 4619PRTartificialL2
fragment 461Cys Lys Ala Xaa Gly Thr Cys Pro Pro 1 5
4629PRTartificialL2 fragment 462Cys Lys Gln Xaa Gly Thr Cys Pro Pro
1 5 4639PRTartificialL2 fragment 463Cys Lys Val Xaa Gly Thr Cys Pro
Ala 1 5 4649PRTartificialL2 fragment 464Cys Lys Ala Xaa Gly Thr Arg
Pro Ala 1 5 4659PRTartificialL2 fragment 465Cys Lys Val Xaa Gly Thr
Cys Pro Pro 1 5 4669PRTartificialL2 fragment 466Cys Lys Ala Xaa Gly
Thr Cys Pro Ala 1 5 4679PRTartificialL2 fragment 467Cys Lys Leu Xaa
Gly Thr Cys Pro Glu 1 5 4689PRTartificialL2 fragment 468Cys Lys Ala
Xaa Gly Thr Cys Pro Ser 1 5 4699PRTartificialL2 fragment 469Cys Lys
Leu Xaa Gly Thr Cys Pro Pro 1 5 4709PRTartificialL2 fragment 470Cys
Gln Leu Xaa Ala Asp Cys Pro Pro 1 5 4719PRTartificialL2 fragment
471Cys Gln Leu Xaa Gly Asn Cys Leu Pro 1 5 4729PRTartificialL2
fragment 472Cys Gln Leu
Xaa Gly Asp Cys Leu Pro 1 5 4739PRTartificialL2 fragment 473Cys Ala
Leu Xaa Gly Thr Cys Pro Asp 1 5 4749PRTartificialL2 fragment 474Cys
Ala Gly Xaa Gly Thr Cys Pro Glu 1 5 4759PRTartificialL2 fragment
475Cys Leu Gln Xaa Gly Asp Cys Ile Pro 1 5 4769PRTartificialL2
fragment 476Cys Lys Ala Xaa Gly Thr Cys Pro Pro 1 5
4779PRTartificialL2 fragment 477Cys Lys Gln Xaa Gly Thr Cys Pro Ser
1 5 4789PRTartificialL2 fragment 478Cys Lys Ile Xaa Gly Asn Cys Pro
Ala 1 5 4799PRTartificialL2 fragment 479Cys Lys Ile Xaa Asn Thr Cys
Pro Pro 1 5 4806PRTartificialloop site of FN3 480Pro Ala Val Thr
Val Arg 1 5 4819PRTartificialloop site of FN3 481Gly Arg Gly Asp
Ser Pro Ala Ser Ser 1 5 4827PRTartificialloop site of bilin-binding
protein 482Pro Asn Ser Val Glu Lys Tyr 1 5 4835PRTartificialloop
site of bilin-binding protein 483Ile His Gly Lys Glu 1 5
4847PRTartificialloop-site of bilin-binding protein 484Thr Tyr Gly
Gly Val Thr Lys 1 5 4855PRTartificialloop-site 485Tyr Lys Gly Gln
Pro 1 5 48619PRTHuman papillomavirus type 18 486Lys Thr Cys Lys Gln
Ser Gly Thr Cys Pro Ser Asp Val Val Asn Lys 1 5 10 15 Val Glu Gly
48719PRTHuman papillomavirus type 31 487Gln Thr Cys Lys Ala Ala Gly
Thr Cys Pro Ser Asp Val Ile Pro Lys 1 5 10 15 Ile Glu His
48819PRTHuman papillomavirus type 68 488Lys Thr Cys Lys Gln Ser Gly
Thr Cys Pro Pro Asp Val Ile Asp Lys 1 5 10 15 Val Glu Gly
48919PRTHuman Paillomavirus Type 82 489Ser Thr Cys Lys Ala Ala Gly
Thr Cys Pro Pro Asp Val Ile Pro Lys 1 5 10 15 Val Lys Gly
49012PRTHuman papillomavirus type 18 490Lys Thr Cys Lys Gln Ser Gly
Thr Cys Pro Ser Asp 1 5 10 491195PRTArtificial Sequence(SEQ ID
NO2)x3+ (SEQ ID NO 487)x3 +(SEQ ID NO77)x3 with tripeptide (GGP)
linkers 491Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile Ile
Pro Lys 1 5 10 15 Val Glu Gly Gly Gly Pro Lys Thr Cys Lys Gln Ala
Gly Thr Cys Pro 20 25 30 Pro Asp Ile Ile Pro Lys Val Glu Gly Gly
Gly Pro Lys Thr Cys Lys 35 40 45 Gln Ala Gly Thr Cys Pro Pro Asp
Ile Ile Pro Lys Val Glu Gly Gly 50 55 60 Gly Pro Gln Thr Cys Lys
Ala Ala Gly Thr Cys Pro Ser Asp Val Ile 65 70 75 80 Pro Lys Ile Glu
His Gly Gly Pro Gln Thr Cys Lys Ala Ala Gly Thr 85 90 95 Cys Pro
Ser Asp Val Ile Pro Lys Ile Glu His Gly Gly Pro Gln Thr 100 105 110
Cys Lys Ala Ala Gly Thr Cys Pro Ser Asp Val Ile Pro Lys Ile Glu 115
120 125 His Gly Gly Pro Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro
Asp 130 135 140 Val Val Asn Lys Val Glu Gly Gly Gly Pro Ser Thr Cys
Lys Ala Ala 145 150 155 160 Gly Thr Cys Pro Pro Asp Val Val Asn Lys
Val Glu Gly Gly Gly Pro 165 170 175 Ser Thr Cys Lys Ala Ala Gly Thr
Cys Pro Pro Asp Val Val Asn Lys 180 185 190 Val Glu Gly 195
492195PRTArtificial Sequence((SEQ ID NO2)+ (SEQ ID NO487) +(SEQ ID
NO77))x3 with tripeptide (GGP) linkers 492Lys Thr Cys Lys Gln Ala
Gly Thr Cys Pro Pro Asp Ile Ile Pro Lys 1 5 10 15 Val Glu Gly Gly
Gly Pro Gln Thr Cys Lys Ala Ala Gly Thr Cys Pro 20 25 30 Ser Asp
Val Ile Pro Lys Ile Glu His Gly Gly Pro Ser Thr Cys Lys 35 40 45
Ala Ala Gly Thr Cys Pro Pro Asp Val Val Asn Lys Val Glu Gly Gly 50
55 60 Gly Pro Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile
Ile 65 70 75 80 Pro Lys Val Glu Gly Gly Gly Pro Gln Thr Cys Lys Ala
Ala Gly Thr 85 90 95 Cys Pro Ser Asp Val Ile Pro Lys Ile Glu His
Gly Gly Pro Ser Thr 100 105 110 Cys Lys Ala Ala Gly Thr Cys Pro Pro
Asp Val Val Asn Lys Val Glu 115 120 125 Gly Gly Gly Pro Lys Thr Cys
Lys Gln Ala Gly Thr Cys Pro Pro Asp 130 135 140 Ile Ile Pro Lys Val
Glu Gly Gly Gly Pro Gln Thr Cys Lys Ala Ala 145 150 155 160 Gly Thr
Cys Pro Ser Asp Val Ile Pro Lys Ile Glu His Gly Gly Pro 165 170 175
Ser Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp Val Val Asn Lys 180
185 190 Val Glu Gly 195 493100PRTPyrococcus furiosus 493Met Ile Ile
Glu Tyr Asp Gly Glu Ile Asp Phe Thr Lys Gly Arg Val 1 5 10 15 Val
Leu Trp Phe Ser Ile Pro Gly Cys Gly Pro Cys Arg Leu Val Glu 20 25
30 Arg Phe Met Thr Glu Leu Ser Glu Tyr Phe Glu Asp Ile Gln Ile Val
35 40 45 His Ile Asn Ala Gly Lys Trp Lys Asn Ile Val Asp Lys Phe
Asn Ile 50 55 60 Leu Asn Val Pro Thr Leu Val Tyr Leu Lys Asp Gly
Arg Glu Val Gly 65 70 75 80 Arg Gln Asn Leu Ile Arg Ser Lys Glu Glu
Ile Leu Lys Lys Leu Lys 85 90 95 Glu Leu Gln Glu 100
494101PRTThermococcus kodakarensis 494Met Ile Val Glu Tyr Asp Glu
Asn Val Asp Phe Thr Lys Gly Lys Ala 1 5 10 15 Val Leu Trp Phe Ser
Ile Pro Gly Cys Gly Pro Cys Arg Leu Val Glu 20 25 30 Ala Phe Met
Lys Glu Leu Ser Glu Glu Phe Gly Glu Ile Ala Ile Val 35 40 45 His
Val Asn Ala Glu Lys Trp Ser Gly Leu Val Glu Gly Phe Arg Ile 50 55
60 Leu Asn Val Pro Thr Leu Val Tyr Leu Lys Asp Gly Lys Glu Val Ala
65 70 75 80 Arg Gln Asn Leu Ile Arg Gly Lys Gly Glu Val Leu Ile Lys
Phe Glu 85 90 95 Glu Pro Arg Glu Leu 100 495105PRTThermococcus
onnurineus 495Met Ile Arg Glu Phe Asp Gly Asp Phe Gly Lys Val Glu
Arg Ala Lys 1 5 10 15 Tyr Ala Leu Leu Trp Phe Ser Ser Pro Gly Cys
Gly Pro Cys Arg Met 20 25 30 Ile Glu Pro Phe Met His Glu Leu Ser
Glu Glu Tyr Lys Glu Val Glu 35 40 45 Phe Trp Glu Val Asp Val Glu
Lys His Leu Pro Leu Ala Glu Lys Phe 50 55 60 Asp Val Met Asn Val
Pro Thr Leu Ile Tyr Leu Lys Glu Gly Asn Glu 65 70 75 80 Ile Ala Arg
Gln Asn Leu Val Arg Lys Lys Glu Glu Val Glu Glu Lys 85 90 95 Leu
Met Met Leu Leu Gly Ser Asp Ser 100 105 496100PRTThermococcus
sibiricus 496Met Ile His Glu Tyr Asp Gly Lys Ile Asp Phe Asn Arg
Gly Lys Val 1 5 10 15 Val Leu Trp Phe Ser Ile Gln Gly Cys Gly Pro
Cys Arg Leu Val Glu 20 25 30 Ser Phe Met Glu Glu Val Ser Glu Glu
Phe Ser Glu Ile Arg Phe Ile 35 40 45 His Val Gly Ala Glu Lys Trp
Ser Asn Ile Val Lys Arg Phe Glu Val 50 55 60 Leu Asn Val Pro Thr
Leu Val Tyr Leu Lys Asp Gly Lys Glu Val Ala 65 70 75 80 Arg Gln Asn
Leu Ile Arg Ser Lys Glu Glu Val Leu Ala Lys Ile Glu 85 90 95 Glu
Leu His Glu 100 497253PRTArtificial SequenceDimer of Escherichia
coli thioredoxin variants 497Met Ser Asp Lys Ile Ile His Leu Thr
Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala
Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys
Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln
Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro
Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70
75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu
Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala
Gly Gly Gly 100 105 110 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly
Ser Glu Gly Gly Gly 115 120 125 Ser Glu Gly Gly Gly Ser Glu Gly Gly
Gly Ser Glu Gly Gly Gly Met 130 135 140 Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp Val 145 150 155 160 Leu Lys Ala Asp
Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys 165 170 175 Gly Pro
Gly Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 180 185 190
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 195
200 205 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu
Leu 210 215 220 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly
Ala Leu Ser 225 230 235 240 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala
Asn Leu Ala 245 250 498396PRTArtificialTrimer of Escherichia coli
thioredoxin variants 498Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile
Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys
Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr
Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu
Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90
95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Gly Gly
100 105 110 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly
Gly Gly 115 120 125 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu
Gly Gly Gly Met 130 135 140 Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser Phe Asp Thr Asp Val 145 150 155 160 Leu Lys Ala Asp Gly Ala Ile
Leu Val Asp Phe Trp Ala Glu Trp Cys 165 170 175 Gly Pro Gly Cys Lys
Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 180 185 190 Glu Tyr Gln
Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 195 200 205 Pro
Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 210 215
220 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
225 230 235 240 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala
Gly Gly Gly 245 250 255 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly
Ser Glu Gly Gly Gly 260 265 270 Ser Glu Gly Gly Gly Ser Glu Gly Gly
Gly Ser Glu Gly Gly Gly Met 275 280 285 Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp Val 290 295 300 Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys 305 310 315 320 Leu Ser
Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu 325 330 335
Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro 340
345 350 Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
Leu 355 360 365 Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala
Leu Ser Lys 370 375 380 Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu
Ala 385 390 395
* * * * *