U.S. patent application number 10/984376 was filed with the patent office on 2005-11-03 for combination neisserial compositions.
This patent application is currently assigned to Chiron S.r.l.. Invention is credited to Giuliani, Marzia Monica, Pizza, Mariagrazia, Rappuoli, Rino.
Application Number | 20050244436 10/984376 |
Document ID | / |
Family ID | 27255587 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050244436 |
Kind Code |
A1 |
Giuliani, Marzia Monica ; et
al. |
November 3, 2005 |
Combination Neisserial compositions
Abstract
Compositions comprising a first biological molecule from a
Neisseria bacterium and a second biological molecule from a
Neisseria bacterium. The term "biological molecule" includes
proteins and nucleic acids. Preferred Neisseria species are
N.meningitidis and N.gonorrhoeae.
Inventors: |
Giuliani, Marzia Monica;
(Siena, IT) ; Pizza, Mariagrazia; (Signa, IT)
; Rappuoli, Rino; (Berardenda, IT) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron S.r.l.
|
Family ID: |
27255587 |
Appl. No.: |
10/984376 |
Filed: |
November 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10984376 |
Nov 9, 2004 |
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09979263 |
Aug 29, 2002 |
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09979263 |
Aug 29, 2002 |
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PCT/IB00/00828 |
May 19, 2000 |
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Current U.S.
Class: |
424/249.1 ;
435/252.3; 530/350 |
Current CPC
Class: |
A61K 2039/70 20130101;
A61K 39/00 20130101; A61P 37/04 20180101; A61K 38/164 20130101;
A61K 39/39 20130101; C07K 14/22 20130101; A61K 39/02 20130101; A61K
39/095 20130101; A61K 2039/55505 20130101; A61P 31/04 20180101;
A61K 2039/53 20130101 |
Class at
Publication: |
424/249.1 ;
530/350; 435/252.3 |
International
Class: |
A61K 039/095; C12N
001/20; C07K 014/22 |
Foreign Application Data
Date |
Code |
Application Number |
May 19, 1999 |
GB |
9911692.3 |
Aug 19, 1999 |
GB |
9919705.5 |
Mar 9, 2000 |
GB |
0005730.7 |
Claims
1. A composition comprising a first isolated biological molecule
from a Neisseria bacterium and a second isolated biological
molecule from a Neisseria bacterium, wherein the first biological
molecule is from ORF 287 and the second biological molecule is from
ORF 961.
2. The composition of claim 1, wherein said first biological
molecule and said second biological molecule are from the same
Neisseria species.
3. The composition of claim 1, wherein said first biological
molecule and said second biological molecule are from different
Neisseria species.
4. The composition of claim 1, wherein said first biological
molecule and said second biological molecule are from
N.meningitidis and/or N.gonorrhoeae
5. The composition of claim 1, wherein said first biological
molecule and said second biological molecule are from
N.meningitidis.
6. The composition of claim 1, wherein said first biological
molecule comprises an amino acid sequence with at least 65%
sequence identity to a contiguous sequence of amino acids selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, and an immunogenic fragment comprising at least 10
contiguous amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or
SEQ ID NO:4; and said second biological molecule comprises an amino
acid sequence with at least 65% sequence identity to a contiguous
sequence of amino acids selected from the group consisting of SEQ
ID NO:5, SEQ ID NO:6, and an immunogenic fragment comprising at
least 10 contiguous amino acids of SEQ ID NO:5 or SEQ ID NO:6:
7. The composition of claim 6, wherein said first biological
molecule is selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
8. The composition of claim 7, wherein said first biological
molecule is SEQ ID NO:1.
9. The composition of claim 6, wherein said second biological
molecule is selected from the group consisting of SEQ ID NO:5, and
SEQ ID NO:6.
10. The composition of claim 9, wherein said second biological
molecule is SEQ ID NO:6.
11. A composition comprising a first isolated biological molecule
from a Neisseria bacterium and a second isolated biological
molecule from a Neisseria bacterium, wherein (a) said first
biological molecule comprises an amino acid sequence with at least
65% identity to the contiguous sequence of amino acids of SEQ ID
NO:1 or an immunogenic fragment comprising at least 10 contiguous
amino acids of SEQ ID NO:1; and (b) said second biological molecule
comprises an amino acid sequence with at least 65% identity to the
contiguous sequence of amino acids of SEQ ID NO:6 or an immunogenic
fragment comprising at least 10 contiguous amino acids of SEQ ID
NO:6.
12. The composition of claim 11, wherein said first biological
molecule comprises the amino acid sequence of SEQ ID NO:1, and said
second biological molecule comprises the amino acid sequence of SEQ
ID NO:6.
13. The composition of claim 1, further comprising an adjuvant.
14. The composition of claim 12, further comprising an adjuvant.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
CROSS-REFERENCE TO RELATED APPLICATION
[0002] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/979,263, filed Nov. 19, 2001, which is a
.sctn.371 filing from PCT IB00/00828, filed May 19, 2000, which
claims priority from GB 9911692.3, filed May 19, 1999, GB
9919705.5, filed Aug. 19, 1999 and GB 0005730.7, filed Mar. 9,
2000, from which applications priority is claimed pursuant to the
provisions of 35 U.S.C. .sctn..sctn. 119/120, and which
applications are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0003] This invention relates to compositions comprising
combinations of biological molecules from Neisseria bacteria,
particularly N.meningitidis and N.gonorrhoeae.
BACKGROUND ART
[0004] Neisseria meningitidis and Neisseria gonorrhoeae are
non-motile, Gram negative diplococci that are pathogenic in
humans.
[0005] Based on the organism's capsular polysaccharide, 12
serogroups of N.meningitidis have been identified. Group A is the
pathogen most often implicated in epidemic disease in sub-Saharan
Africa. Serogroups B and C are responsible for the vast majority of
cases in the United States and in most developed countries.
Serogroups W135 and Y are responsible for the rest of the cases in
the United States and developed countries.
[0006] The meningococcal vaccine currently in use is a tetravalent
polysaccharide vaccine composed of serogroups A, C, Y and W135.
Meningococcus B remains a problem, however. The polysaccharide
approach cannot be used because the menB capsular polysaccharide is
a polymer of .alpha.(2-8)-linked N-acetyl neuraminic acid that is
also present in mammalian tissue. One approach to a menB vaccine
uses mixtures of outer membrane proteins (OMPs) To overcome the
antigenic variability, multivalent vaccines containing up to nine
different porins have been constructed [eg. Poolman J T (1992)
Development of a meningococcal vaccine. Infect. Agents Dis.
4:13-28]. Additional proteins to be used in outer membrane vaccines
have been the opa and opc proteins, but none of these approaches
have been able to overcome the antigenic variability [eg.
Ala'Aldeen & Borriello (1996) The meningococcal
transferrin-binding proteins 1 and 2 are both surface exposed and
generate bactericidal antibodies capable of killing homologous and
heterologous strains. Vaccine 14(1):49-53].
[0007] Given the propensity for meningococcal disease during
non-epidemic periods to be caused by multiple strains or strain
variants [Russel et al. (1998) Abstracts of 11th International
pathogenic Neisseria conference, page 281] together with frequent
temporal shifts in the predominant strains in a community, it seems
that a universal meningococcal B vaccine will require more than one
antigenic species.
DESCRIPTION OF THE INVENTION
[0008] Neisserial protein and nucleotide sequences are disclosed in
the following documents:
[0009] WO 99/24578
[0010] WO 99/36544
[0011] WO 99/57280
[0012] WO 97/28273
[0013] WO 96/29412
[0014] WO 95/03413
[0015] Tettelin et al. (2000) Science 287:1809-1815
[0016] The present invention provides compositions comprising a
first biological molecule from a Neisseria bacterium and a second
biological molecule from a Neisseria bacterium. The term
"biological molecule" includes proteins and nucleic acids.
[0017] The compositions may also comprise further biological
molecules, preferably also from Neisseria, that is to say the
compositions may comprise two or more biological molecules (eg. 3,
4, 5, 6, 7, 8 etc.), at least two of which are from a Neisseria
bacterium (eg. 3, 4, 5, 6, 7, 8 etc.). Such compositions include
those comprising (i) two or more different Neisserial proteins,
(ii) two or more different Neisserial nucleic acids, or (iii)
mixtures of one or more Neisserial protein and one or more
Neisserial nucleic acid.
[0018] In one preferred embodiment, the first and second biological
molecules are from different Neisseria species (eg. one is from
N.meningitidis and one is from N.gonorrhoeae), but they may be from
the same species. The biological molecules in the compositions may
be from different serogroups or strains of the same species, such
as from N.meningitidis serogroups A, B or C.
[0019] The first biological molecule is preferably selected from
ORF 287 of the Neisserial genome. Particular amino acid sequences
are shown in FIGS. 1A-1D (SEQ ID NOS:1-4, respectively). These
sequences, as well as nucleic acid molecules encoding these
sequences, are preferred. In particular, SEQ ID NOS: 1 and 4 are
sequences for the ORF 287 protein from N.meningitidis serogroup B.
SEQ ID NO:1 herein corresponds to SEQ ID NO:1202 of WO 99/57280;
SEQ ID NO:4 herein corresponds to NMB2132 of the serogroup B strain
MC58 (Tettelin et al., (2000) Science 287:1809-1815); SEQ ID NO:2
herein is the ORF 287 sequence for N.gonorrhoeae and corresponds to
SEQ ID NO:1200 of WO 99/57280; and SEQ ID NO:3 herein is the ORF
287 sequence for N.meningitidis serogroup A and corresponds to SEQ
ID NO:1204 of WO 99/57280. The biological molecule is preferably a
purified or isolated biological molecule.
[0020] The second biological molecule is preferably selected from
ORF 961 of the Neisserial genome. Particular amino acid sequences
are shown in FIGS. 2A and 2B (SEQ ID NOS:5 and 6, respectively).
These sequences, as well as nucleic acid molecules encoding these
sequences, are preferred. In particular, SEQ ID NOS:5 and 6 are
sequences for the ORF 961 protein from N.meningitidis serogroup B.
SEQ ID NO:5 herein corresponds to NMB1994 of the serogroup B strain
MC58 (Tettelin et al., (2000) Science 287:1809-1815) and SEQ ID
NO:6 herein corresponds to SEQ ID NO:2944 of WO 99/57280. The
biological molecule is preferably a purified or isolated biological
molecule.
[0021] One or both of the first and second biological molecules may
be a Neisserial biological molecule which is not specifically
disclosed herein, and which may not have been identified,
discovered, made available to the public or purified before this
patent application was filed.
[0022] Details as to how the ORF 287 and 961 molecules can be
produced and used can be found from the relevant international
applications detailed above and these details need not be repeated
here.
[0023] SEQ ID NOS:1-6 in the compositions of the invention or
nucleic acid encoding the same may be supplemented or substituted
with molecules comprising sequences homologous (ie. having sequence
identity) to SEQ ID NOS: 1-6 or nucleic acid molecules encoding the
same. Depending on the particular sequence, the degree of identity
is preferably greater than 50% (eg. 65%, 80%, 90%, or more), and
includes mutants and allelic variants. Sequence identity between
the proteins is preferably determined by the Smith-Waterman
homology search algorithm as implemented in the MPSRCH program
(Oxford Molecular), using an affine gap search with parameters gap
open penalty=12 and gap extension penalty=1.
[0024] SEQ ID NOS:1-6 in the compositions of the invention or
nucleic acid molecules encoding the same may be supplemented or
substituted with molecules comprising fragments of SEQ ID NOS:1-6
or nucleic acid molecules encoding the same. Such fragments should
comprise at least n consecutive monomers from the molecules and,
depending on the particular sequence, n is either (i) 7 or more for
protein molecules (eg. 8, 10, 12, 14, 16, 18, 20 or more),
preferably such that the fragment comprises an epitope from the
sequence, or (ii) 10 or more for nucleic acid molecules (eg 12, 14,
15, 18, 20, 25, 30, 35, 40 or more).
[0025] Where the composition includes a protein that exists in
different nascent and mature forms, the mature form of the protein
is preferably used. For example, the mature form of a protein
lacking the signal peptide may be used.
[0026] In the case of protein molecules, SEQ ID NOS:1-6 in the
compositions of the invention may be supplemented or substituted
with an antibody that binds to the protein. This antibody may be
monoclonal or polyclonal.
[0027] In the case of nucleic acid molecules encoding SEQ ID
NOS:1-6, the compositions of the invention may be supplemented or
substituted with nucleic acid which can hybridise to the Neisserial
nucleic acid, preferably under "high stringency" conditions (eg.
65.degree. C. in a 0.1.times.SSC, 0.5% SDS solution).
[0028] It will be appreciated that any nucleic acid in the
compositions can take various forms (eg. single stranded, double
stranded, vectors, probes etc.). In addition, the term "nucleic
acid" includes DNA and RNA, and also their analogues, such as those
containing modified backbones, and also peptide nucleic acids (PNA)
etc.
[0029] In certain embodiments, the composition comprises molecules
from different Neisseria species, such as one or more
N.meningitidis molecule and one or more N.gonorrhoeae molecule. In
some embodiments, the composition may comprise molecules from
different serogroups and/or strains of the same species, such as
strains A and B of N.meningitidis. Further embodiments comprise
mixtures of one or more N.meningitidis molecules from different
strains and also one or more N.gonorrhoeae molecules.
[0030] Many proteins are relatively conserved between different
species, serogroups and strains of N.meningitidis and
N.gonorrhoeae. PCT/IB00/00642 includes a more detailed experimental
analysis of conserved regions in these proteins. To ensure maximum
cross-strain recognition and reactivity, regions of proteins that
are conserved between different Neisserial species, serogroups and
strains can be used in the compositions of the present invention.
The invention therefore provides proteins which comprise stretches
of amino acid sequence that are shared across the majority of
Neisseria, particularly N.meningitidis and N.gonorrhoeae.
Preferably, therefore, the composition comprises a protein
comprising a fragment of a Neisserial protein (preferably a protein
from SEQ ID NOS:1-6), wherein said fragment consists of n
consecutive conserved amino acids. Depending on the particular
protein, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more).
The fragment preferably comprises an antigenic or immunogenic
region of the Neisserial protein. A "conserved" amino acid is one
that is present in a particular Neisserial protein in at least x %
of Neisseria (or, preferably, in at least x % of combined
N.meningitidis and N.gonorrhoeae strains). The value of x may be
50% or more eg. 66%, 75%, 80%, 90%, 95% or even 100% (ie. the amino
acid is found in the protein in question in all Neisseria). In
order to determine whether an amino acid is "conserved" in a
particular Neisserial protein, it is necessary to compare that
amino acid residue in the sequences of the protein in question from
a plurality of different Neisseria (a "reference population").
Suitable definitions of "reference populations" can be found in
PCT/IB00/00642. Amino acid sequences of different Neissieriae can
easily be compared using computers. This will typically involve the
alignment of a number of sequences using an algorithm such as
CLUSTAL [Thompson et al. (1994) Nucleic Acids Res 22:4673-4680;
Trends Biochem Sci (1998) 23:403-405] or, preferably, PILEUP [part
of the GCG Wisconsin package, preferably version 9.0]. Conserved
amino acids are readily apparent in a multiple sequence
alignment--at the amino acid position in question a majority of the
aligned sequences will contain a particular amino acid. Conserved
amino acids can be made more visually apparent by using a program
such as BOXSHADE [available, for instance, at the NIH on-line],
PRETTYBOX [GCG Wisconsin, version 10] or JALVIEW [available on-line
at EBI].
[0031] Specific compositions according to the invention therefore
include those comprising:
[0032] two or more biological molecules selected from SEQ ID
NOS:1-6;
[0033] one or more biological molecules selected from SEQ ID
NOS:1-4 combined with one or more biological molecules selected
from SEQ ID NOS:5 and 6;
[0034] one or more biological molecules selected from SEQ ID
NOS:1-6 combined with the NspA protein (as disclosed in W096/29412;
see also FIG. 3 herein), preferably in mature form;
[0035] one or more biological molecules selected from SEQ ID
NOS:1-6 combined with transferrin binding protein A (ThpA) and/or B
(TbpB), such as the ThpA and TbpB disclosed in WO00/25811 (or
immunogenic fragments thereof).
[0036] one or more fragments of proteins selected from SEQ ID
NOS:1-6, with the fragment preferably comprising a stretch of
conserved amino acids;
[0037] a combination of different proteins, wherein the combination
as a whole includes one or more proteins that is recognised by each
strain in a reference population, although each individual protein
in the combination may not itself be recognised by each strain in
the reference population ie. each member of a reference population
recognises at least one protein in the combination.
[0038] The invention also provides the compositions of the
invention for use as medicaments (eg. as immunogenic compositions
or vaccines) or as diagnostic reagents. It also provides the use of
the compositions in the manufacture of: (i) a medicament for
treating or preventing infection due to Neisserial bacteria; (ii) a
diagnostic reagent for detecting the presence of Neisserial
bacteria or of antibodies raised against Neisserial bacteria;
and/or (iii) a reagent which can raise antibodies against
Neisserial bacteria.
[0039] The invention also provides a method of treating a patient,
comprising administering to the patient a therapeutically effective
amount of a composition according to the invention.
[0040] The invention further provides a process for producing a
composition according to the invention, comprising the step of
bringing one or more of SEQ ID NOS:1-6 into combination with one or
more other of SEQ ID NOS:1-6.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A-1D (SEQ ID NOS:1-4) show representative ORF 268
proteins for use in the subject compositions.
[0042] FIGS. 2A-2B (SEQ ID NOS:5 and 6) show representative ORF 961
proteins for use in the subject compositions.
[0043] FIG. 3A-3C (SEQ ID NOS:7-14) show sequence variability of
NspA from various strains of meningococcus B. These sequences may
be used as alternatives to the NspA of WO 96/29412.
[0044] FIG. 4A-4B show the binding of polyclonal anti-rNspA by
indirect fluorescence flow cytometry to encapsulated and
non-encapsulated menB strains.
[0045] FIGS. 5A-5E show similar data for encapsulated strains 8047,
CU385 & M986 (31A) and non-encapsulated strains BZ232, MC58,
NG3/88 & NGB165 (31B).
[0046] FIG. 6 shows a model of NspA secondary structure.
[0047] FIG. 7A-7B show FACS analysis for ORF 919, 287 and 953
antigens and combinations thereof.
EXAMPLES
Example 1
[0048] Trivalent Mixture
[0049] Proteins ORF1 (e.g. example 77 of WO99/24578; see also
WO99/55873), `287` (e.g. FIG. 21 of WO99/57280) and `919` (e.g.
WO99/57280 FIG. 23 therein) were combined and adjuvanted with
Al(OH).sub.3. The proteins were from the 2996 strain of MenB.
[0050] This mixture was also combined with a MenC polysaccharide
conjugate antigen [e.g. Costantino et al. (1992) Vaccine
10:691-698]. OMVs were used as controls.
[0051] The mixture was used in a bactericidal assay against the
homologous strain and also heterologous MenB strains. Titres were
as follows:
1 2996 BZ133 BZ232 1000 MC58 NGH38 Trivalent 2048 2048 4 <4 64 4
+MenC 2048 >32000 4 128 1024 128 Control 32765 4096 8192 16384
16384 8192
Example 2
[0052] Proteins 287, 919 and 953
[0053] Proteins 287, 919 and 953 are disclosed in WO99/57280. These
proteins from N.meningitidis serogroup B strain 2996 were expressed
and tested in a bactericidal assay against strain 2996, alone and
in combinations. OMVs from 2996 were used as a positive
control.
2 Antigen 287 919 953 Control Titre 8192 2048 128 65536 Combination
287 + 919 287 + 953 919 + 953 287 + 919 + 953 Titre 32000 8192 8192
8192
[0054] FIG. 7 shows FACS data for the individual antigens and for
the four combinations.
[0055] It is evident that the antigen mixtures are more effective
than the antigens in isolation and, in particular, that
combinations of 919+953 give surprisingly good results.
[0056] The individual antigens from 2996 and combinations were also
tested against different serogroup A, B & C strains (i.e.
heterologous challenge). Bactericidal titres were as follows:
3 Anti- Serogroup B (MenB) strains MenA MenC gen 2996 BZ133 BZ232
MC58 NGH38 F6124 C11 287 8192 >4096 256 1024 2048 1024 2048 919
2048 -- 1024 -- -- -- -- 953 128 -- -- -- -- -- -- 287 + 32000
>4096 512 512 1024 512 >2048 919 287 + 8192 >4096 1024 512
2048 2048 >2048 953 919 + 8192 -- 8192 -- -- -- -- 953 Tri- 8192
>2048 256 -- 1024 >2048 >2048 va- lent Con- 65536 -- 8192
2048 -- 2048 32768 trol
[0057] It is apparent that the antigen mixtures are useful in
conferring cross-strain activity.
[0058] In a second set of experiments, titres for the individual
antigens were as follows:
4 Anti- Serogroup B (MenB) strains MenA MenC gen 2996 BZ133 BZ232
MC58 NGH38 F6124 C11 287 16000 2048 16 512 >2048 64 1024 919
16000 -- 2048 -- -- -- -- 953 2048 -- 16 -- -- -- --
[0059] The three proteins used in this example were expressed and
used in the following forms:
[0060] (1) Protein 287 was expressed in E.coli as a GST fusion;
[0061] (2) Protein 919 was expressed in E.coli without its leader
peptide, without its mature N-terminal cysteine, and without any
fusion partners ("919-untag"); and
[0062] (3) Protein 953 was expressed using a histidine tag.
[0063] Three immunisations were administered with Freund's
adjuvants--the first included CFA, and the final two included
IFA.
Example 3
[0064] Further Polyvalent Combinations
[0065] Further combinations of antigens were tested in CD1
mice:
5 Bactericidal Antigens* Adjuvant FACS ELISA activity 919-his +
Orf4-his + Freund +++ + 8192 225-his + Orf40-his Orf4-L + Orf37-GST
+ Freund +++ + Bacteriostatic Orf40-his + 502-his + 8-his 919-untag
+ 791-his + Freund +++ + 4096 792-his 919-untag + 287-GST + Freund
+++ + 8192 953-his 919-untag + 287-GST Freund +++ + 32000 287-GST +
953-his Freund +++ + 8192 919-untag + 953-his Freund +++ + 8192
919-untag + Orf1-his + Al(OH).sub.3 +++ + 2048 287-GST 919-untag +
Orf1-his + Al(OH).sub.3 +++ + 2048 287-GST + MenC glycoconj.
Orf-46.1-his + 287-GST Al(OH).sub.3 n.d. + 128 *"his" indicates
expression and immunisation with a histidine-tagged protein;
"ORF4-L" is the lipidated form of ORF4; "GST" indicates expression
and immunisation with a GST fusion protein; "919-untag" is as
defined in Example 2; "MenC glycoconj" is the MenC glycoconjugate
described in Example 1.
[0066] Further combinations of antigens were tested in guinea
pigs:
6 Bactericidal Antigens Adjuvant FACS ELISA activity 919-his +
287-GST + Freund + + 4096 953-his + Orf46.1-his 919-untage +
287-GST + Freund + + 4096 953-his 287-GST + 953-his Al(OH).sub.3
n.d. + 256
[0067] Evidently the combinations give excellent immunological
results.
Example 4
[0068] NspA Combinations
[0069] NspA protein is disclosed in WO96/29412, and is represented
herein as SEQ ID NOS:7-14. The academic literature disclosure of
this protein [Martin et al. (1997) J. Exp. Med 185 1173-1183]
reported the protein to be highly conserved between Neisseria
strains (99% cross-reactivity of anti-NspA antibodies with 250
meningococcal A, B & C strains) and also efficient protection
against deadly challenge with live bacteria. There have also been
reports that NspA adsorbed on alum elicits serum meningococcal
bactericidal antibody responses in rabbits and monkeys [Martin et
al. (1998) Abstracts of 11th International pathogenic Neisseria
conference, page 198]. On the basis of these data, rNspA
(recombinant NspA) is being developed as a vaccine for the
prevention of meningococcal disease caused by all serogroups.
[0070] Despite sequence conservation, however, it has surprisingly
been discovered that rNspA cell surface epitopes are detected on
only 65% of the serogroup B strains tested below, and
susceptibility to anti-NspA bactericidal activity is also less than
that reported by Martin et al. These results contrast with Martin
et al., and suggest that a rNspA-based meningococcal B vaccines
will need to be supplemented with additional antigens in order to
be effective.
[0071] The N.meningitidis strains tested in this example were
isolated from patients residing in different countries over a
period of more than 30 years (see table on page 17). These strains
were selected to be representative of widely divergent `clonal`
groups, as defined by multilocus isoenzyme typing [Seiler et al.
(1996) Mol. Microbiol. 19:841-856] and/or multilocus sequence
typing [Maiden et al. (1998) PNAS USA 95:3140-45]. Strain M7, which
is derived from strain NMB, contains a transposon insertion that
blocks capsular polysaccharide biosynthesis [Stephens et al. (1991)
Infect. Immun. 59:4097-4102], but all the other strains are
encapsulated.
[0072] Based on the nucleotide sequence in Martin et al. (1997),
PCR primers were designed and the NspA gene from strain 8047 was
amplified. The sequence, including the promoter region, was cloned
into pSK+ plasmid (rNspA). A plasmid pTrc.NspA.1 encoding a protein
in which a portion of the signal sequence has been replaced with a
poly-histidine tag was also used. Both plasmids were expressed in
E.coli strain BL21(DE3) and the proteins were purified. In E.coli,
rNspA is secreted, rather than being associated with the outer
membrane. The protein was partially purified from the culture
medium by precipitation with 55% w/v ammonium sulphate, and had an
apparent MW of 18.6 kDa, confirmed by Western Blot.
[0073] The two forms of NspA (rNspA and denature His-tage NspA)
were injected into 6-week old female CD-1 mice to raise antisera.
The ability of these to bind to the surface of N.meningitidis
strain B was determined using flow cytometric detection of indirect
fluorescence assay [Granoff et al. (1998) J. Immunol. 160:5028-36].
The results for strains NMB and M7 (an acapsulated mutant of NMB)
are shown in FIG. 4. As expected, anti-group B polysaccharide mAb
SEAM-3 [Granoff et al.] only binds to the encapsulated strain,
whereas the positive anti-P1.2 (PorA) control mAb binds to both
strains. The antisera raised against rNspA is able to bind both
strains. Antisera against the His-tag NspA gave negative results,
however. These antisera were also negative for strains 8047, CU385
and M986 (FIGS. 5A-5B), but by Western Blot these antisera gave
positive results.
[0074] These data suggest that antibodies prepared using His-tag
NspA recognise epitopes that are present in denatured NspA, but not
native NspA as found on the cell-surface in vivo. In contrast,
antibodies prepared against rNspA seem to recognise conformational
NspA epitopes.
[0075] The flow cytometric assay was applied to the strains shown
in the table on page 17. FIGS. 5A-5B show that murine antibodies
raised against rNspA bind to the surface of strain 8047 (the strain
from which the nspA gene was cloned) and strain CU385, but not to
M986. FIGS. 5C-5E show similar negative results for strains BZ232,
MC58, NG3/88 and NGP165. In all of these negative cases, however,
the anticapsular mAb control was positive.
[0076] The table on page 17 summarises the flow cytometry results.
Although NspA is reported o be accessible at the surface of all
intact N.meningitidis strains tested [Martin et al. (1997) J. Exp.
Med 185 1173-1183; Plante et al. (1999) Infect. Immun. 67:2855-61],
only 11 of the 17 test strains (65%) reacted with the anti-rNspA
sera. There was no apparent relationship between cell-surface
expression in a given strain and classification (by serotype,
subtype, or electrophoretic type), or with year or country of
isolation.
[0077] In an attempt to explain the differences in reactivity with
the anti-rNspA sera, the nspA genes from five of the six negative
strains (BX232, NG3/88, NGP165, M136 & M986) and from three of
the positive strains (8047, CU385 & NG6/88) were sequenced. The
sequence for the sixth negative strain (MC58) was already available
from the complete genome sequence.
[0078] The nspA sequences for all ten strains were highly
conserved, varying at most by 5 nucleotides from the prototype
sequence of Martin et al. The most variant protein had only 3 amino
acid differences (see FIG. 3). With one exception, all of the amino
acid variants involved the same respective residues in discrete
segments of the protein. These include the signal peptide, which is
not present in the mature protein, and two short segments in the 50
C-terminal residues. These differences do not explain the antisera
results, as there are examples of identical variant sequences in
strains that were positive and those that were negative (compare
M136 & 8047; NGP165 & NG6/88; MC58 & CU385).
[0079] As neither lack of the gene nor polymorphism explained the
antiserum results, the amount of NspA protein in the outer
membranes of five strains (8047, CU385 & NG6/88--all positive
for anti-rNspA; M986 & M 136--both negative) were tested.
Bacterial cell pellets were extracted with lauryl sarcosinate, and
the insoluble outer membrane fractions were analysed. An 18.6 kDa
band was seen for all five strains, and this was cross-reactive
with anti-His-tag-NspA by Western Blot. Thus strain differences in
nspA expression also failed to explain the results.
[0080] The ability of anti-rNspA to bind to the bacterial cell
surface could be influenced by the amount of polysaccharide capsule
present. The quantity of capsular polysaccharide produced by the 17
test strains was therefore assessed by inhibition ELISA.
[0081] Extracts of capsular polysaccharide were prepared based on a
method described by Corn et al. [J. Infect. Dis. (1993)
167:356-64]. Individual bacterial clones were grown to an
OD.sub.620 0.5-0.7 in 7 mL of Mueller-Hinton broth. Bacteria were
collected by centrifugation at 5000 g for 15 min, washed in 0.6 mL
of 10 mM Hepes, pH 8.0, and then resuspended in 0.6 mL of the same
buffer containing 10 mM EDTA and incubated at 37.degree. C. for 1
hr. The cells were pelleted at 10,000 g for 1 minute and the
relative amount of meningococcal B polysaccharide antigen released
into the supernatant was determined by an inhibition ELISA,
performed as described by Azmi et al. [Infect. Immun. (1995)
63:1906-13]. The solid phase antigen in the ELISA was meningococcal
B polysaccharide-ADH-biotin absorbed to avidin-coated microtiter
plates [Granoff et al.]. The meningococcal B
polysaccharide-reactive human paraprotein LIP [Azmi et al.] was
used as the primary antibody (0.2 .mu.g/ml). In the absence of
inhibitor, this concentration of antibody was sufficient to given
an OD of 0.7 to 1.0 after 30 minutes incubation with substrate
[Azmi et al.]. The titre of polysaccharide released into the
supernatant was measured by determining the dilution of supernatant
that resulted in 50% inhibition of antibody binding. Controls in
this assay included an EDTA extract prepared from the strain M7,
which does not produce any capsular polysaccharide, and purified
meningococal B polysaccharide. To ensure that all of the capsular
polysaccharide was released by the EDTA treatment, the same
inhibition ELISA was performed using the cell pellet resuspended in
the same buffer and volume as the capsule extract. The observable
inhibitory activity from the cell pellet was between 0 and 10% of
the activity observed in the capsule extracts with the latter,
higher percentage coming from cell pellets of strains that produce
the largest amounts of capsule.
[0082] The results for each strain are shown in the table on page
17. On average, the six negative anti-rNspA strains produced
three-fold more capsular polysaccharide than the eleven positive
strains (respective reciprocal geometric mean dilutions of 676 vs.
224, p<0.05). This may explain the results obtained with the
antiserum--conceivably, the presence of larger amounts of capsule
could interfere with the ability of the anti-rNspA antibody to bind
to NspA epitopes which, in strains with lower amounts of capsule,
are accessible.
[0083] The complement-dependent bactericidal activity of the
anti-rNspA antisera were tested using an assay similar to that
described by Mandrell et al. [J. Infect. Dis. (1995) 172:1279-89].
The complement source was human serum from a healthy adult with no
detectable anti-capsular antibody to group B polysaccharide and no
intrinsic bactericidal activity against the test strain. Serum
bactericidal titres were defined as the serum dilution resulting in
a 50% decrease in CFU/ml after 60 minutes incubation of bacteria in
the reaction mixture, compared to the control CFU/ml at time
zero.
[0084] Typically, bacteria incubated with a negative control
antibody showed a 150-200% increase in CFU/ml during the 60 minutes
of incubation. The positive control antibody [anti-capsular IgG2a
mAb SEAM 12, Granoff et al. ] showed complement-mediated
bactericidal activity against all 17 strains. In contrast, the six
strains that were negative for anti-rNspA antisera binding by flow
assay were resistant, showing no bactericidal or bacteriostatic
effects. Ten of the other eleven positive strains were either
killed by complement and the antisera (SWZ107, J351, CU385, NG6/88,
BZ198, H44/76, NMB & 8047) or were inhibited (H355 &
S3446); strain 1000, however, was not affected.
[0085] The ability of the anti-rNspA antisera to confer passive
protection against meningococcal B bacteremia was tested in infant
rats using a method adapted from Saukkonen [J. Infect. Dis. (1988)
158:209-212]. Briefly, 6-7 day old rats were randomly distributed
to nursing mothers. Groups of 5-6 animals were challenged IP with
100 .mu.l of approximately 5000 CFU of N.meningitidis group B
bacteria. One strain negative for NspA surface epitopes (M986) and
one positive strain (8047) were tested, each of which having been
passaged three times in infant rats. Immediately before
administration, the bacterial suspension was mixed with different
dilutions of test or control antibody (positive control:
anticapsular mAb; negative control: anti-E.coli). 18 hours after
challenge, blood specimens were obtained from the heart. Aliquots
were plated onto chocolate agar, and CFU/ml was determined after
overnight incubation at 37.degree. C. in 5% CO.sub.2.
[0086] The protective activities of the various co-administered
antibodies were as follows:
7 Dose per rat Blood culture or Positive/ CFU/ml CFU/ml Antibody
treatment serum dilution Strain total (mean .times. 10.sup.-3) (%
of control) Anticapsular mAb 2 .mu.g M986 0/6 <1 <1
Anti-rNspA 1:5 M986 6/6 44.sup.a 45 Anti-rNspA 1:25 M986 6/6
93.sup.a 95 Anti-E. coli control 1:5 M986 6/6 98.sup.a --
Anticapsular mAb 2 .mu.g 8047 0/5 <1 <1 Anti-rNspA 1:5 8047
1/6 0.2.sup.b 2 Anti-rNspA 1:25 8047 1/5 0.4.sup.b 4 Anti-E. coli
control 1:5 8047 6/6 10.sup.b -- .sup.ap > 0.5, compared to
geometric mean CFU/ml of control rats .sup.bp < 0.001, compared
to geometric mean CFU/ml of control rats
[0087] As can be seen, a dose of 2 .mu.g per rat of the positive
anticapsular control was protective against both strains. A 1:5 or
1:25 dilution of anti-rNspA antiserum protected against bacteremia
caused by strain 8047. Neither dilution was effective in preventing
M986 bacteremia, however.
[0088] Despite the positive conclusions of Martin et al.,
therefore, NspA does not seem to be effective in preventing
meningococcal B infection. Approximately one third of strains have
decreased cell-surface expression of NspA epitopes when grown in
vitro, are resistant to anti-NspA induced complement-mediated
bacteriolysis, and are resistant to passive antiserum immunisation.
These strains produce large amounts of capsular polysaccharide, and
would thus be expected to have the greatest virulence. The ability
of a vaccine containing only NspA to confer broad protective
immunity against meningococcal B thus has to be doubted.
[0089] Compositions comprising NspA [FIG. 3] therefore
advantageously comprise further antigens. A preferred aspect of the
invention is thus a combination of NspA protein with one or more
further Neisserial antigens.
Example 5
[0090] NspA Fragments
[0091] A model of the secondary structure of NspA is shown in FIG.
6, containing eight transmembrane .beta.-strands and 4
surface-exposed connecting loops. This fits the pattern of
alternating hydrophobic and hydrophilic amino acids in NspA, which
is characteristic of many .beta.-barrel porins [Weiss et al. (1990)
FEBS Letts 267:268-272]
[0092] The grey shaded areas in the model indicate segments that
are >40% identical and >70% similar to encoded amino acid
sequences of opacity proteins (Opa) from N.meningitidis, N.
gonorrhoeae, N. flavius, N. sicca, and H. influenzae identified in
a BLAST search of the non-redundant GenBank CDS. The alternating
sequences are predicted amphiphilic .beta.-strands; vertical
segments correspond to transmembrane segments; the top of the
figure corresponds to surface exposed segments, labelled as loops 1
to 4.
[0093] According to Martin et al., the only significant homology
between the deduced amino acid sequence of NspA and those of other
proteins are weak homologies with the Neisseria opacity protein
(Opa) family in two small segments (.about.20 amino acids) near the
C-terminal end of the protein. However, separate comparisons of the
N- and C-termini of NspA with GenBank reveals a high degree of
homology (>40% identity and >70% similarity) between NspA and
Opa proteins from N.meningitidis, N. gonorrhoeae, N. flavius, N.
sicca, and H. influenzae. The Opa proteins are thought to be
integral membrane proteins that have eight transmembrane segments
and a .beta.-barrel topology in the membrane similar to that of
porin [Merker et al. (1997) Mol. Microbiol. 23:281-293]. The
presence of NspA in detergent-insoluble membrane preparations
indicate that NspA is located in the outer membrane, which would be
consistent with the Opa-like membrane topology shown in the model.
In addition, the segments of NspA that are most homologous to those
of the Opa proteins are the putative transmembrane segments
indicated in the shaded areas of FIG. 6.
[0094] The opacity proteins of Neisseria can, under certain
circumstances, elicit protective antibody. However, problems with
limited antibody accessibility of the opacity proteins in
encapsulated bacteria, variability of amino acid sequences in
exposed loop segments, and phase variation of protein expression
during clinical infection, have limited the ability of Opa to
elicit protective antibody consistently [Malorny et al. (1998) J.
Infect. Dis. 172:1279-89]. In contrast, there appears to be little
or no sequence variation in the surface exposed loops of NspA in
FIG. 6. However, it was recently reported that a panel of
anti-N.meningitidis NspA monoclonal antibodies that reacted with
all meningococcal strains tested reacted with only a limited number
of N.gonorrhoeae strains, even though the respective amino acid
sequences in the two species are 92% identical. When the respective
NspA sequences of the meningococcal and gonococcal strains are
compared (FIG. 3), all of the respective amino acid differences
result in changes in hydrophobicity or charge, and are located in
the putative surface exposed connecting loops (FIG. 6). This
finding suggests that the connecting loops in NspA, which are
highly conserved in N.meningitidis, may be important epitopes for
antibodies that bind to native NspA.
[0095] These segments of the molecule, therefore, would appear to
be of greatest interest with respect to interacting with protective
antibody. However, the putative surface loops of NspA are
relatively small (10-14 amino acids) compared to, for example, the
highly immunogenic external loops of PorA and Opc (24 to 45 amino
acids). The shorter length of the loops may limit the accessibility
of NspA surface epitopes for binding interactions with serum
antibody, especially in the presence of abundant capsular
polysaccharide.
[0096] Accordingly, the invention provides the fragments of NspA
that are exposed on the cell-surface in FIG. 6, namely SSSLGSAKG
(SEQ ID NO:15), NYKAPSTDFKLY (SEQ ID NO:16), NRASVDLGGSDSFSQT (SEQ
ID NO:17), and NYIGKVNTVKNVRSG (SEQ ID NO:18), and also provides
corresponding fragments from allelic variants of NspA. In addition,
the invention provides sub-sequences of these fragments, comprising
7 or more contiguous amino acids from the fragments. The invention
further provides proteins comprising these fragments. Nucleic acid
encoding these fragments and proteins is also provided.
[0097] These NspA fragments, proteins comprising the fragments, and
nucleic acid, may be used in the compositions of the invention, in
particular as substitutes for full-length NspA. In a further
aspect, these fragments, proteins and nucleic acids may be used as
products in isolation, that is to say they need not exclusively be
used in combination with other biological molecules.
[0098] It will be appreciated that the invention has been described
by means of example only, and that modifications may be made whilst
remaining within the spirit and scope of the invention.
8 Reactivity of anti-rNspA polyclonal antisera with native NspA
exposed on the surface of live, encapsulated, Neisseria
meningitidis B bacteria in relation to susceptibility to
bacteriolysis and capsular production. Polysaccharide Meningococcal
B Strains NspA Cell Anti-rNspA Capsule Serologic Surface
Bactericidal Production Strain Country Year Classification ET
Complex Reactivity.sup.c Activity.sup.d (l/titer) (l/titer .+-. SE)
SWZ107.sup.a Switzerland 1980 4: P1.2 104 Positive .gtoreq.64 28
.+-. 4 NG6/88.sup.a Norway 1988 NT: P1.1 173 Positive 4 115 .+-. 23
CU385.sup.b Cuba 1980 4: P1.15 5 Positive 4 116 .+-. 1 IH5341
Finland 1985 15: P1.7, 16 ND Positive 16 176 .+-. 61 BZ198
Netherlands 1986 NT: P- 154 Positive .gtoreq.64 362 .+-. 1 NMB US
1968 2b: P1.2, 5 ND Positive 16 244 .+-. 20 8047 US 1978 2b: P1.2
ND Positive 16 1125 .+-. 50 H44/76 Norway 1976 15: P1.7, 16 5
Positive 24 99 .+-. 16 1000.sup.a USSR 1989 NT: P1.5 61 Positive
<4 287 .+-. 12 S3446.sup.b US 1972 14: P1.23, 14 11 (A1 cluster)
Positive <4 (static = 16) 585 .+-. 151 H355.sup.b Norway 1973
15: P1.15 11 cluster Positive <4 (static = 4) 656 .+-. 141
BZ232.sup.a Netherlands 1964 NT: P1.2 76 Negative <4 1493 .+-.
18 NG3/88.sup.a Norway 1988 8: P1.1 A4 cluster Negative <4 498
.+-. 105 MC58 UK 1985 15: P1.7, 16b 5 Negative <4 627 .+-. 121
M136.sup.b US 1968 11: P1.15 D1 cluster Negative <4 1056 .+-. 81
M986.sup.b US 1963 2a: P1.5, 2 B2 cluster Negative <4 1442 .+-.
206 NGP165 Norway 1974 NT: P1.2 37 Negative <4 138 .+-. 6
.sup.aDenotes strains that have been characterized further by
multilocus sequence typing [Maiden, 1998]. .sup.bDenotes strains
obtained from the Frasch collection, US FDA. 8047 was obtained from
W. Zollinger, Walter Reed Army Institute of Research, Washington,
D.C. MC58 is the strain selected by TIGR for genomic sequencing.
J351 was obtained from M. Sarvas, National Public Health Institute,
Helsinki, Finland. # The remaining strains are from the collection
described by Seiler et al. [Seiler, 1996]. ET data are from
Caugnant et al. [j. Infect. Dis. (1990) 162: 867-874], and Seiler
et al. .sup.cBy indirect fluorescence flow cytometry with
anti-rNspA antisera. .sup.dDilution of anti-rNspA antisera that
when incubated for 60 min. with bacterial cells and 20% human
complement yielded .gtoreq.50% decrease in CFU/ml, compared to that
at time 0. "Static" refers to strains that were inhibited but not
killed in the assay (.gtoreq.50% but <100% survival at 60 mins).
.sup.eTitre defined as dilution of capsule extract giving 50%
inhibition of antibody binding to meningococcal B polysaccharide
antigen in an ELISA.
[0099]
Sequence CWU 1
1
18 1 488 PRT Artificial representative ORF 268 protein 1 Met Phe
Lys Arg Ser Val Ile Ala Met Ala Cys Ile Phe Ala Leu Ser 1 5 10 15
Ala Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Val Lys Ser Ala Asp 20
25 30 Thr Leu Ser Lys Pro Ala Ala Pro Val Val Ser Glu Lys Glu Thr
Glu 35 40 45 Ala Lys Glu Asp Ala Pro Gln Ala Gly Ser Gln Gly Gln
Gly Ala Pro 50 55 60 Ser Ala Gln Gly Ser Gln Asp Met Ala Ala Val
Ser Glu Glu Asn Thr 65 70 75 80 Gly Asn Gly Gly Ala Val Thr Ala Asp
Asn Pro Lys Asn Glu Asp Glu 85 90 95 Val Ala Gln Asn Asp Met Pro
Gln Asn Ala Ala Gly Thr Asp Ser Ser 100 105 110 Thr Pro Asn His Thr
Pro Asp Pro Asn Met Leu Ala Gly Asn Met Glu 115 120 125 Asn Gln Ala
Thr Asp Ala Gly Glu Ser Ser Gln Pro Ala Asn Gln Pro 130 135 140 Asp
Met Ala Asn Ala Ala Asp Gly Met Gln Gly Asp Asp Pro Ser Ala 145 150
155 160 Gly Gly Gln Asn Ala Gly Asn Thr Ala Ala Gln Gly Ala Asn Gln
Ala 165 170 175 Gly Asn Asn Gln Ala Ala Gly Ser Ser Asp Pro Ile Pro
Ala Ser Asn 180 185 190 Pro Ala Pro Ala Asn Gly Gly Ser Asn Phe Gly
Arg Val Asp Leu Ala 195 200 205 Asn Gly Val Leu Ile Asp Gly Pro Ser
Gln Asn Ile Thr Leu Thr His 210 215 220 Cys Lys Gly Asp Ser Cys Ser
Gly Asn Asn Phe Leu Asp Glu Glu Val 225 230 235 240 Gln Leu Lys Ser
Glu Phe Glu Lys Leu Ser Asp Ala Asp Lys Ile Ser 245 250 255 Asn Tyr
Lys Lys Asp Gly Lys Asn Asp Lys Phe Val Gly Leu Val Ala 260 265 270
Asp Ser Val Gln Met Lys Gly Ile Asn Gln Tyr Ile Ile Phe Tyr Lys 275
280 285 Pro Lys Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser
Arg 290 295 300 Arg Ser Leu Pro Ala Glu Met Pro Leu Ile Pro Val Asn
Gln Ala Asp 305 310 315 320 Thr Leu Ile Val Asp Gly Glu Ala Val Ser
Leu Thr Gly His Ser Gly 325 330 335 Asn Ile Phe Ala Pro Glu Gly Asn
Tyr Arg Tyr Leu Thr Tyr Gly Ala 340 345 350 Glu Lys Leu Pro Gly Gly
Ser Tyr Ala Leu Arg Val Gln Gly Glu Pro 355 360 365 Ala Lys Gly Glu
Met Leu Ala Gly Ala Ala Val Tyr Asn Gly Glu Val 370 375 380 Leu His
Phe His Thr Glu Asn Gly Arg Pro Tyr Pro Thr Arg Gly Arg 385 390 395
400 Phe Ala Ala Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile
405 410 415 Asp Ser Gly Asp Asp Leu His Met Gly Thr Gln Lys Phe Lys
Ala Ala 420 425 430 Ile Asp Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu
Asn Gly Ser Gly 435 440 445 Asp Val Ser Gly Lys Phe Tyr Gly Pro Ala
Gly Glu Glu Val Ala Gly 450 455 460 Lys Tyr Ser Tyr Arg Pro Thr Asp
Ala Glu Lys Gly Gly Phe Gly Val 465 470 475 480 Phe Ala Gly Lys Lys
Glu Gln Asp 485 2 429 PRT Artificial representative ORF 268 protein
2 Met Phe Lys Arg Ser Val Ile Ala Met Ala Cys Ile Phe Pro Leu Ser 1
5 10 15 Ala Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Val Lys Ser Ala
Asp 20 25 30 Thr Pro Ser Lys Pro Ala Ala Pro Val Val Ala Glu Asn
Ala Gly Glu 35 40 45 Gly Val Leu Pro Lys Glu Lys Lys Asp Glu Glu
Ala Ala Gly Gly Ala 50 55 60 Pro Gln Ala Asp Thr Gln Asp Ala Thr
Ala Gly Glu Gly Ser Gln Asp 65 70 75 80 Met Ala Ala Val Ser Ala Glu
Asn Thr Gly Asn Gly Gly Ala Ala Thr 85 90 95 Thr Asp Asn Pro Lys
Asn Glu Asp Ala Gly Ala Gln Asn Asp Met Pro 100 105 110 Gln Asn Ala
Ala Glu Ser Ala Asn Gln Thr Gly Asn Asn Gln Pro Ala 115 120 125 Gly
Ser Ser Asp Ser Ala Pro Ala Ser Asn Pro Ala Pro Ala Asn Gly 130 135
140 Gly Ser Asp Phe Gly Arg Thr Asn Val Gly Asn Ser Val Val Ile Asp
145 150 155 160 Gly Pro Ser Gln Asn Ile Thr Leu Thr His Cys Lys Gly
Asp Ser Cys 165 170 175 Asn Gly Asp Asn Leu Leu Asp Glu Glu Ala Pro
Ser Lys Ser Glu Phe 180 185 190 Glu Lys Leu Ser Asp Glu Glu Lys Ile
Lys Arg Tyr Lys Lys Asp Glu 195 200 205 Gln Arg Glu Asn Phe Val Gly
Leu Val Ala Asp Arg Val Lys Lys Asp 210 215 220 Gly Thr Asn Lys Tyr
Ile Ile Phe Tyr Thr Asp Lys Pro Pro Thr Arg 225 230 235 240 Ser Ala
Arg Ser Arg Arg Ser Leu Pro Ala Glu Ile Pro Leu Ile Pro 245 250 255
Val Asn Gln Ala Asp Thr Leu Ile Val Asp Gly Glu Ala Val Ser Leu 260
265 270 Thr Gly His Ser Gly Asn Ile Phe Ala Pro Glu Gly Asn Tyr Arg
Tyr 275 280 285 Leu Thr Tyr Gly Ala Glu Lys Leu Pro Gly Gly Ser Tyr
Ala Leu Arg 290 295 300 Val Gln Gly Glu Pro Ala Lys Gly Glu Met Leu
Val Gly Thr Ala Val 305 310 315 320 Tyr Asn Gly Glu Val Leu His Phe
His Met Glu Asn Gly Arg Pro Tyr 325 330 335 Pro Ser Gly Gly Arg Phe
Ala Ala Lys Val Asp Phe Gly Ser Lys Ser 340 345 350 Val Asp Gly Ile
Ile Asp Ser Gly Asp Asp Leu His Met Gly Thr Gln 355 360 365 Lys Phe
Lys Ala Ala Ile Asp Gly Asn Gly Phe Lys Gly Thr Trp Thr 370 375 380
Glu Asn Gly Gly Gly Asp Val Ser Gly Arg Phe Tyr Gly Pro Ala Gly 385
390 395 400 Glu Glu Val Ala Gly Lys Tyr Ser Tyr Arg Pro Thr Asp Ala
Glu Lys 405 410 415 Gly Gly Phe Gly Val Phe Ala Gly Lys Lys Asp Arg
Asp 420 425 3 497 PRT Artificial representative ORF 268 protein 3
Met Phe Lys Arg Ser Val Ile Ala Met Ala Cys Ile Val Ala Leu Ser 1 5
10 15 Ala Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Val Lys Ser Ala
Asp 20 25 30 Thr Leu Ser Lys Pro Ala Ala Pro Val Val Thr Glu Asp
Val Gly Glu 35 40 45 Glu Val Leu Pro Lys Glu Lys Lys Asp Glu Glu
Ala Val Ser Gly Ala 50 55 60 Pro Gln Ala Asp Thr Gln Asp Ala Thr
Ala Gly Lys Gly Gly Gln Asp 65 70 75 80 Met Ala Ala Val Ser Ala Glu
Asn Thr Gly Asn Gly Gly Ala Ala Thr 85 90 95 Thr Asp Asn Pro Glu
Asn Lys Asp Glu Gly Pro Gln Asn Asp Met Pro 100 105 110 Gln Asn Ala
Ala Asp Thr Asp Ser Ser Thr Pro Asn His Thr Pro Ala 115 120 125 Pro
Asn Met Pro Thr Arg Asp Met Gly Asn Gln Ala Pro Asp Ala Gly 130 135
140 Glu Ser Ala Gln Pro Ala Asn Gln Pro Asp Met Ala Asn Ala Ala Asp
145 150 155 160 Gly Met Gln Gly Asp Asp Pro Ser Ala Gly Glu Asn Ala
Gly Asn Thr 165 170 175 Ala Asp Gln Ala Ala Asn Gln Ala Glu Asn Asn
Gln Val Gly Gly Ser 180 185 190 Gln Asn Pro Ala Ser Ser Thr Asn Pro
Asn Ala Thr Asn Gly Gly Ser 195 200 205 Asp Phe Gly Arg Ile Asn Val
Ala Asn Gly Ile Lys Leu Asp Ser Gly 210 215 220 Ser Glu Asn Val Thr
Leu Thr His Cys Lys Asp Lys Val Cys Asp Arg 225 230 235 240 Asp Phe
Leu Asp Glu Glu Ala Pro Pro Lys Ser Glu Phe Glu Lys Leu 245 250 255
Ser Asp Glu Glu Lys Ile Asn Lys Tyr Lys Lys Asp Glu Gln Arg Glu 260
265 270 Asn Phe Val Gly Leu Val Ala Asp Arg Val Glu Lys Asn Gly Thr
Asn 275 280 285 Lys Tyr Val Ile Ile Tyr Lys Asp Lys Ser Ala Ser Ser
Ser Ser Ala 290 295 300 Arg Phe Arg Arg Ser Ala Arg Ser Arg Arg Ser
Leu Pro Ala Glu Met 305 310 315 320 Pro Leu Ile Pro Val Asn Gln Ala
Asp Thr Leu Ile Val Asp Gly Glu 325 330 335 Ala Val Ser Leu Thr Gly
His Ser Gly Asn Ile Phe Ala Pro Glu Gly 340 345 350 Asn Tyr Arg Tyr
Leu Thr Tyr Gly Ala Glu Lys Leu Ser Gly Gly Ser 355 360 365 Tyr Ala
Leu Ser Val Gln Gly Glu Pro Ala Lys Gly Glu Met Leu Ala 370 375 380
Gly Thr Ala Val Tyr Asn Gly Glu Val Leu His Phe His Met Glu Asn 385
390 395 400 Gly Arg Pro Ser Pro Ser Gly Gly Arg Phe Ala Ala Lys Val
Asp Phe 405 410 415 Gly Ser Lys Ser Val Asp Gly Ile Ile Asp Ser Gly
Asp Asp Leu His 420 425 430 Met Gly Thr Gln Lys Phe Lys Ala Val Ile
Asp Gly Asn Gly Phe Lys 435 440 445 Gly Thr Trp Thr Glu Asn Gly Gly
Gly Asp Val Ser Gly Arg Phe Tyr 450 455 460 Gly Pro Ala Gly Glu Glu
Val Ala Gly Lys Tyr Ser Tyr Arg Pro Thr 465 470 475 480 Asp Ala Glu
Lys Gly Gly Phe Gly Val Phe Ala Gly Lys Lys Glu Gln 485 490 495 Asp
4 488 PRT Artificial representative ORF 268 protein 4 Met Phe Lys
Arg Ser Val Ile Ala Met Ala Cys Ile Phe Ala Leu Ser 1 5 10 15 Ala
Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Val Lys Ser Ala Asp 20 25
30 Thr Leu Ser Lys Pro Ala Ala Pro Val Val Ser Glu Lys Glu Thr Glu
35 40 45 Ala Lys Glu Asp Ala Pro Gln Ala Gly Ser Gln Gly Gln Gly
Ala Pro 50 55 60 Ser Ala Gln Gly Ser Gln Asp Met Ala Ala Val Ser
Glu Glu Asn Thr 65 70 75 80 Gly Asn Gly Gly Ala Val Thr Ala Asp Asn
Pro Lys Asn Glu Asp Glu 85 90 95 Val Ala Gln Asn Asp Met Pro Gln
Asn Ala Ala Gly Thr Asp Ser Ser 100 105 110 Thr Pro Asn His Thr Pro
Asp Pro Asn Met Leu Ala Gly Asn Met Glu 115 120 125 Asn Gln Ala Thr
Asp Ala Gly Glu Ser Ser Gln Pro Ala Asn Gln Pro 130 135 140 Asp Met
Ala Asn Ala Ala Asp Gly Met Gln Gly Asp Asp Pro Ser Ala 145 150 155
160 Gly Gly Gln Asn Ala Gly Asn Thr Ala Ala Gln Gly Ala Asn Gln Ala
165 170 175 Gly Asn Asn Gln Ala Ala Gly Ser Ser Asp Pro Ile Pro Ala
Ser Asn 180 185 190 Pro Ala Pro Ala Asn Gly Gly Ser Asn Phe Gly Arg
Val Asp Leu Ala 195 200 205 Asn Gly Val Leu Ile Asp Gly Pro Ser Gln
Asn Ile Thr Leu Thr His 210 215 220 Cys Lys Gly Asp Ser Cys Ser Gly
Asn Asn Phe Leu Asp Glu Glu Val 225 230 235 240 Gln Leu Lys Ser Glu
Phe Glu Lys Leu Ser Asp Ala Asp Lys Ile Ser 245 250 255 Asn Tyr Lys
Lys Asp Gly Lys Asn Asp Lys Phe Val Gly Leu Val Ala 260 265 270 Asp
Ser Val Gln Met Lys Gly Ile Asn Gln Tyr Ile Ile Phe Tyr Lys 275 280
285 Pro Lys Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg
290 295 300 Arg Ser Leu Pro Ala Glu Met Pro Leu Ile Pro Val Asn Gln
Ala Asp 305 310 315 320 Thr Leu Ile Val Asp Gly Glu Ala Val Ser Leu
Thr Gly His Ser Gly 325 330 335 Asn Ile Phe Ala Pro Glu Gly Asn Tyr
Arg Tyr Leu Thr Tyr Gly Ala 340 345 350 Glu Lys Leu Pro Gly Gly Ser
Tyr Ala Leu Arg Val Gln Gly Glu Pro 355 360 365 Ala Lys Gly Glu Met
Leu Ala Gly Ala Ala Val Tyr Asn Gly Glu Val 370 375 380 Leu His Phe
His Thr Glu Asn Gly Arg Pro Tyr Pro Thr Arg Gly Arg 385 390 395 400
Phe Ala Ala Lys Val Asp Phe Gly Ser Lys Ser Val Asp Gly Ile Ile 405
410 415 Asp Ser Gly Asp Asp Leu His Met Gly Thr Gln Lys Phe Lys Ala
Ala 420 425 430 Ile Asp Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn
Gly Ser Gly 435 440 445 Asp Val Ser Gly Lys Phe Tyr Gly Pro Ala Gly
Glu Glu Val Ala Gly 450 455 460 Lys Tyr Ser Tyr Arg Pro Thr Asp Ala
Glu Lys Gly Gly Phe Gly Val 465 470 475 480 Phe Ala Gly Lys Lys Glu
Gln Asp 485 5 364 PRT Artificial representative ORF 961 protein 5
Met Ser Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu 1 5
10 15 Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp
Val 20 25 30 Lys Lys Ala Ala Thr Val Ala Ile Val Ala Ala Tyr Asn
Asn Gly Gln 35 40 45 Glu Ile Asn Gly Phe Lys Ala Gly Glu Thr Ile
Tyr Asp Ile Gly Glu 50 55 60 Asp Gly Thr Ile Thr Gln Lys Asp Ala
Thr Ala Ala Asp Val Glu Ala 65 70 75 80 Asp Asp Phe Lys Gly Leu Gly
Leu Lys Lys Val Val Thr Asn Leu Thr 85 90 95 Lys Thr Val Asn Glu
Asn Lys Gln Asn Val Asp Ala Lys Val Lys Ala 100 105 110 Ala Glu Ser
Glu Ile Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp 115 120 125 Ala
Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala 130 135
140 Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys
145 150 155 160 Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val
Ala Asp Thr 165 170 175 Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile
Ala Asp Ser Leu Asp 180 185 190 Glu Thr Asn Thr Lys Ala Asp Glu Ala
Val Lys Thr Ala Asn Glu Ala 195 200 205 Lys Gln Thr Ala Glu Glu Thr
Lys Gln Asn Val Asp Ala Lys Val Lys 210 215 220 Ala Ala Glu Thr Ala
Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala 225 230 235 240 Asn Thr
Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp 245 250 255
Ile Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser 260
265 270 Ala Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys
Glu 275 280 285 Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly
Leu Phe Gln 290 295 300 Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala
Ala Val Gly Gly Tyr 305 310 315 320 Lys Ser Glu Ser Ala Val Ala Ile
Gly Thr Gly Phe Arg Phe Thr Glu 325 330 335 Asn Phe Ala Ala Lys Ala
Gly Val Ala Val Gly Thr Ser Ser Gly Ser 340 345 350 Ser Ala Ala Tyr
His Val Gly Val Asn Tyr Glu Trp 355 360 6 364 PRT Artificial
representative ORF 961 protein 6 Met Ser Met Lys His Phe Pro Ala
Lys Val Leu Thr Thr Ala Ile Leu 1 5 10 15 Ala Thr Phe Cys Ser Gly
Ala Leu Ala Ala Thr Ser Asp Asp Asp Val 20 25 30 Lys Lys Ala Ala
Thr Val Ala Ile Val Ala Ala Tyr Asn Asn Gly Gln 35 40 45 Glu Ile
Asn Gly Phe Lys Ala Gly Glu Thr Ile Tyr Asp Ile Gly Glu 50 55 60
Asp Gly Thr Ile Thr Gln Lys Asp Ala Thr Ala Ala Asp Val Glu Ala 65
70 75 80 Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Val Val Thr Asn
Leu Thr 85 90 95 Lys Thr Val Asn Glu Asn Lys Gln Asn Val Asp Ala
Lys Val Lys Ala 100 105 110 Ala Glu Ser Glu Ile Glu Lys Leu Thr Thr
Lys Leu Ala Asp Thr Asp 115 120 125 Ala Ala Leu Ala Asp Thr Asp Ala
Ala Leu Asp Glu Thr Thr Asn Ala 130 135
140 Leu Asn Lys Leu Gly Glu Asn Ile Thr Thr Phe Ala Glu Glu Thr Lys
145 150 155 160 Thr Asn Ile Val Lys Ile Asp Glu Lys Leu Glu Ala Val
Ala Asp Thr 165 170 175 Val Asp Lys His Ala Glu Ala Phe Asn Asp Ile
Ala Asp Ser Leu Asp 180 185 190 Glu Thr Asn Thr Lys Ala Asp Glu Ala
Val Lys Thr Ala Asn Glu Ala 195 200 205 Lys Gln Thr Ala Glu Glu Thr
Lys Gln Asn Val Asp Ala Lys Val Lys 210 215 220 Ala Ala Glu Thr Ala
Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala 225 230 235 240 Asn Thr
Ala Ala Asp Lys Ala Glu Ala Val Ala Ala Lys Val Thr Asp 245 250 255
Ile Lys Ala Asp Ile Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Ser 260
265 270 Ala Arg Ile Asp Ser Leu Asp Lys Asn Val Ala Asn Leu Arg Lys
Glu 275 280 285 Thr Arg Gln Gly Leu Ala Glu Gln Ala Ala Leu Ser Gly
Leu Phe Gln 290 295 300 Pro Tyr Asn Val Gly Arg Phe Asn Val Thr Ala
Ala Val Gly Gly Tyr 305 310 315 320 Lys Ser Glu Ser Ala Val Ala Ile
Gly Thr Gly Phe Arg Phe Thr Glu 325 330 335 Asn Phe Ala Ala Lys Ala
Gly Val Ala Val Gly Thr Ser Ser Gly Ser 340 345 350 Ser Ala Ala Tyr
His Val Gly Val Asn Tyr Glu Trp 355 360 7 174 PRT Artificial NspA 7
Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5
10 15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala
Ala 20 25 30 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly
Phe Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu
Arg Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala
Pro Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala
Ile Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95 Val Lys Pro Tyr Leu
Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100 105 110 Asp Leu Gly
Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile Gly Leu Gly 115 120 125 Val
Leu Thr Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 130 135
140 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn
145 150 155 160 Val Arg Ser Gly Glu Leu Ser Val Gly Val Arg Val Lys
Phe 165 170 8 174 PRT Artificial NspA 8 Met Lys Lys Ala Leu Ala Thr
Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly
Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys
Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg
Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55
60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu
65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln
Ser Pro 85 90 95 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn
Arg Ala Ser Val 100 105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln
Thr Ser Ile Gly Leu Gly 115 120 125 Val Leu Thr Gly Val Ser Tyr Ala
Val Thr Pro Asn Val Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn
Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser
Gly Glu Leu Ser Val Ala Val Arg Val Lys Phe 165 170 9 174 PRT
Artificial NspA 9 Met Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala
Leu Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr
Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys Ala Ser Ser Ser Leu
Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr
Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr
Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser
Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95
Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100
105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile Thr Leu
Gly 115 120 125 Val Leu Thr Ala Val Ser Tyr Ala Val Thr Pro Asn Val
Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val
Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser Gly Glu Leu Ser Val
Gly Val Arg Val Lys Phe 165 170 10 174 PRT Artificial NspA 10 Met
Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala Ile Pro Ala Ala 1 5 10
15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala
20 25 30 His Ala Lys Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe
Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg
Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro
Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala Ile
Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95 Val Lys Pro Tyr Leu Gly
Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100 105 110 Asp Leu Gly Gly
Ser Asp Ser Phe Ser Gln Thr Ser Ile Thr Leu Gly 115 120 125 Val Leu
Thr Gly Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp 130 135 140
Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 145
150 155 160 Val Arg Ser Gly Glu Leu Ser Val Ala Val Arg Val Lys Phe
165 170 11 174 PRT Artificial NspA 11 Met Lys Lys Ala Leu Ala Thr
Leu Ile Ala Leu Ala Ile Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly
Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys
Ala Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg
Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55
60 Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu
65 70 75 80 Tyr Ser Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln
Ser Pro 85 90 95 Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn
Arg Ala Ser Val 100 105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln
Thr Ser Ile Thr Leu Gly 115 120 125 Val Leu Thr Ala Val Ser Tyr Ala
Val Thr Pro Asn Val Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn
Tyr Ile Gly Lys Val Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser
Gly Glu Leu Ser Val Gly Val Arg Val Lys Phe 165 170 12 174 PRT
Artificial NspA 12 Met Lys Lys Ala Leu Ala Thr Leu Ile Ala Leu Ala
Ile Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr
Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys Ala Ser Ser Ser Leu
Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg Ile Ser Ala Gly Tyr
Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55 60 Tyr Thr Arg Tyr
Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 65 70 75 80 Tyr Ser
Ile Gly Ala Ser Ala Ile Tyr Asp Phe Asp Thr Gln Ser Pro 85 90 95
Val Lys Pro Tyr Leu Gly Ala Arg Leu Ser Leu Asn Arg Ala Ser Val 100
105 110 Asp Leu Gly Gly Ser Asp Ser Phe Ser Gln Thr Ser Ile Thr Leu
Gly 115 120 125 Val Leu Thr Ala Val Ser Tyr Ala Val Thr Pro Asn Val
Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn Tyr Ile Gly Lys Val
Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser Gly Glu Leu Ser Val
Ala Val Arg Val Lys Phe 165 170 13 173 PRT Artificial NspA 13 Met
Lys Lys Ala Leu Ala Ala Leu Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10
15 Ala Leu Ala Glu Gly Ala Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala
20 25 30 His Ala Lys Ala Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser
Pro Arg 35 40 45 Ile Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe
Ala Val Asp Tyr 50 55 60 Thr Arg Tyr Lys Asn Tyr Lys Gln Pro Ser
Thr Asp Phe Lys Leu Tyr 65 70 75 80 Ser Ile Gly Ala Ser Ala Val Tyr
Asp Phe Asp Thr Gln Ser Pro Val 85 90 95 Lys Pro Tyr Leu Phe Ala
Arg Leu Ser Leu Asn Arg Ala Ser Val Ala 100 105 110 His Gly Gly Ser
Asp Ser Phe Ser Gln Lys Ser Ile Ala Leu Gly Val 115 120 125 Leu Thr
Ala Val Ser Tyr Ala Val Thr Pro Asn Val Asp Leu Asp Ala 130 135 140
Gly Tyr Arg Tyr Asn Tyr Ile Val Lys Val Asn Thr Val Lys Asn Val 145
150 155 160 Arg Ser Gly Glu Leu Ser Val Ala Val Arg Val Lys Phe 165
170 14 174 PRT Artificial NspA 14 Met Lys Lys Ala Leu Ala Ala Leu
Ile Ala Leu Ala Leu Pro Ala Ala 1 5 10 15 Ala Leu Ala Glu Gly Ala
Ser Gly Phe Tyr Val Gln Ala Asp Ala Ala 20 25 30 His Ala Lys Ala
Ser Ser Ser Leu Gly Ser Ala Lys Gly Phe Ser Pro 35 40 45 Arg Ile
Ser Ala Gly Tyr Arg Ile Asn Asp Leu Arg Phe Ala Val Asp 50 55 60
Tyr Thr Arg Tyr Lys Asn Tyr Lys Ala Pro Ser Thr Asp Phe Lys Leu 65
70 75 80 Tyr Ser Ile Gly Ala Ser Ala Val Tyr Asp Phe Asp Thr Gln
Ser Pro 85 90 95 Val Lys Pro Tyr Leu Phe Ala Arg Leu Ser Leu Asn
Arg Ala Ser Val 100 105 110 Ala His Gly Gly Ser Asp Ser Phe Ser Gln
Lys Ser Ile Ala Leu Gly 115 120 125 Val Leu Thr Ala Val Ser Tyr Ala
Val Thr Pro Asn Val Asp Leu Asp 130 135 140 Ala Gly Tyr Arg Tyr Asn
Tyr Ile Val Lys Val Asn Thr Val Lys Asn 145 150 155 160 Val Arg Ser
Gly Glu Leu Ser Val Ala Val Arg Val Lys Phe 165 170 15 9 PRT
Artificial fragment of NspA exposed on the cell-surface 15 Ser Ser
Ser Leu Gly Ser Ala Lys Gly 1 5 16 12 PRT Artificial fragment of
NspA exposed on the cell-surface 16 Asn Tyr Lys Ala Pro Ser Thr Asp
Phe Lys Leu Tyr 1 5 10 17 16 PRT Artificial fragment of NspA
exposed on the cell-surface 17 Asn Arg Ala Ser Val Asp Leu Gly Gly
Ser Asp Ser Phe Ser Gln Thr 1 5 10 15 18 15 PRT Artificial fragment
of NspA exposed on the cell-surface 18 Asn Tyr Ile Gly Lys Val Asn
Thr Val Lys Asn Val Arg Ser Gly 1 5 10 15
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