U.S. patent application number 12/201731 was filed with the patent office on 2009-06-18 for antigen arrays for treatment of allergic eosinophilic diseases.
This patent application is currently assigned to Cytos Biotechnology AG. Invention is credited to Martin F. BACHMANN, Gary Jennings, Ivo Sonderegger.
Application Number | 20090155302 12/201731 |
Document ID | / |
Family ID | 46281503 |
Filed Date | 2009-06-18 |
United States Patent
Application |
20090155302 |
Kind Code |
A1 |
BACHMANN; Martin F. ; et
al. |
June 18, 2009 |
Antigen Arrays for Treatment of Allergic Eosinophilic Diseases
Abstract
The present invention is related to the fields of molecular
biology, virology, immunology and medicine. The invention provides
a composition comprising an ordered and repetitive antigen or
antigenic determinant array, and in particular an array comprising
a protein or peptide of IL-5, IL-13 or eotaxin. More specifically,
the invention provides a composition comprising a virus-like
particle and at least one protein, or peptide of IL-5, IL-13 and/or
eotaxin bound thereto. The invention also provides a process for
producing the conjugates and the ordered and repetitive arrays,
respectively. The compositions of the invention are useful in the
production of vaccines for the treatment of allergic diseases with
an eosinophilic component and as a pharmaccine to prevent or cure
allergic diseases with an eosinophilic component and to efficiently
induce immune responses, in particular antibody responses.
Furthermore, the compositions of the invention are particularly
useful to efficiently induce self-specific immune responses within
the indicated context.
Inventors: |
BACHMANN; Martin F.;
(Seuzach, CH) ; Jennings; Gary; (Zurich, CH)
; Sonderegger; Ivo; (Zurich, CH) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Cytos Biotechnology AG
Zurich-Schlieren
CH
|
Family ID: |
46281503 |
Appl. No.: |
12/201731 |
Filed: |
August 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11499659 |
Aug 7, 2006 |
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12201731 |
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10289454 |
Nov 7, 2002 |
7094409 |
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11499659 |
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10050902 |
Jan 18, 2002 |
7264810 |
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10289454 |
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60331045 |
Nov 7, 2001 |
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60396636 |
Jul 19, 2002 |
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60262379 |
Jan 19, 2001 |
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60288549 |
May 4, 2001 |
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60326998 |
Oct 5, 2001 |
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60331045 |
Nov 7, 2001 |
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Current U.S.
Class: |
424/199.1 ;
424/204.1 |
Current CPC
Class: |
A61K 39/35 20130101;
A61K 38/00 20130101; C07K 16/22 20130101; A61K 39/385 20130101;
A61K 47/646 20170801; A61K 2039/5256 20130101; C07K 2319/00
20130101; A61P 11/06 20180101; C07K 14/5437 20130101; A61K 2039/627
20130101; C07K 14/523 20130101; C07K 16/00 20130101; C07K 16/40
20130101; C07K 2317/52 20130101; A61K 2039/6075 20130101; C07K
16/4291 20130101; C12N 2810/852 20130101; C12N 2795/18122 20130101;
C07K 16/2863 20130101; A61P 31/12 20180101; A61P 37/08 20180101;
C07K 14/005 20130101; C12N 2730/10122 20130101; C07K 2319/30
20130101; A61K 39/0005 20130101; A61K 39/0007 20130101; A61K 39/001
20130101; A61K 47/6901 20170801; C07K 2317/34 20130101; A61K
39/0008 20130101; A61K 39/39 20130101; A61K 2039/5258 20130101;
A61K 38/162 20130101; C07K 2317/55 20130101; A61P 35/00 20180101;
C07K 14/5409 20130101; C07K 16/082 20130101; C07K 16/2875 20130101;
C12N 2730/10123 20130101; C12N 2730/10142 20130101 |
Class at
Publication: |
424/199.1 ;
424/204.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61P 37/08 20060101 A61P037/08; A61P 31/12 20060101
A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 21, 2002 |
IB |
PCT/IB02/00166 |
Claims
1-21. (canceled)
22. A composition comprising: (a) a core particle with at least one
first attachment site, wherein said core particle is a virus-like
particle of an RNA-bacteriophage; and (b) at least one antigen or
antigenic determinant with at least one second attachment site,
wherein said antigen or antigenic determinant is a protein or
peptide of IL-5, and wherein said second attachment site is
selected from the group consisting of: (i) an attachment site not
naturally occurring with said antigen or antigenic determinant; and
(ii) an attachment site naturally occurring with said antigen or
antigenic determinant, wherein said second attachment site
associates with said first attachment site through at least one
non-peptide covalent bond; and wherein said antigen or antigenic
determinant and said core particle interact through said
association to form an ordered and repetitive antigen array.
23-25. (canceled)
26. The composition of claim 22, wherein said core particle (a) is
a recombinant virus-like particle.
27-28. (canceled)
29. The composition of claim 22, wherein said virus-like particle
comprises recombinant proteins, or fragments thereof, of a
RNA-bacteriophage.
30. The composition of claim 29, wherein said RNA-bacteriophage is
selected from the group consisting of: (a) bacteriophage Q.beta.;
(b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA;
(e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M
11; (h) bacteriophage MX1; (i) bacteriophage NL95; (j)
bacteriophage f2; (k) bacteriophage PP7; and (l) bacteriophage
AP205.
31. The composition of claim 22, wherein said virus-like particle
comprises recombinant proteins, or fragments thereof, of
RNA-bacteriophage Q.beta..
32-34. (canceled)
35. The composition of claim 22, wherein said antigen or antigenic
determinant is a peptide of IL-5.
36. The composition of claim 22, wherein said protein or peptide of
IL-5 comprises an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of SEQ ID NO:233; (b)
the amino acid sequence of SEQ ID NO:234; and (c) the amino acid
sequence of a fragment of any of SEQ ID NO:233 or 234.
37-66. (canceled)
67. A method of immunization comprising administering the
composition of claim 1 to an animal or human.
68-73. (canceled)
74. A composition comprising: (a) at least one first core particle
and at least one second core particle each comprising at least one
first attachment site, wherein said first and said second core
particle each is a virus-like particle of an RNA-bacteriophage; and
(b) at least one first antigen or antigenic determinant and at
least one second antigen or antigenic determinant each comprising
at least one second attachment site, wherein said at least one
first antigen or antigenic determinant and said at least one second
antigen or antigenic determinant is selected from a protein or
peptide of IL-5, IL-13 or eotaxin, and wherein said second
attachment site is selected from the group consisting of: (i) an
attachment site not naturally occurring with said antigen or
antigenic determinant; and (ii) an attachment site naturally
occurring with said antigen or antigenic determinant, wherein said
second attachment sites associate with said first attachment sites
through each at least one non-peptide covalent bond; and wherein
said first core particle interacts with said first antigen or
antigen determinant to form an ordered and repetitive antigen
array, and said second core particle interacts with said second
antigen or antigen determinant to form ordered and repetitive
antigen arrays.
75-94. (canceled)
95. The composition of claim 22, wherein said first attachment site
comprises an amino group.
96. The composition of claim 22, wherein said second attachment
site comprises a sulfhydryl group.
97. The composition of claim 22, wherein said first attachment site
comprises an amino group and wherein said second attachment site
comprises a sulfhydryl group.
98. The composition of claim 22, wherein said RNA-bacteriophage is
bacteriophage Q.beta., and wherein said virus-like particle of an
RNA bacteriophage Q.beta. comprises one or more coat proteins
having the amino acid sequence set forth in SEQ ID NO:10.
99. The composition of claim 22, wherein said first attachment site
is not a sulfhydryl group of a cysteine.
100. The composition of claim 97, wherein said at least one first
attachment site and said at least one second attachment site are
linked through a heterobifunctional cross-linker.
101. The composition of claim 100, wherein said heterobifunctional
cross-linker is SMPH.
102. The composition of claim 101, wherein said RNA-bacteriophage
is bacteriophage Q.beta., and wherein said virus-like particle of
RNA-bacteriophage Q.beta. comprises one or more proteins having the
amino acid sequence as set forth in SEQ ID NO:10.
103. The composition of claim 102, wherein said antigen or
antigenic determinant is a protein of IL-5 having the amino acid
sequence as set forth in SEQ ID NO:234.
104. The composition of claim 103 further comprising an amino acid
linker, wherein said amino acid linker is bound to said antigen or
said antigenic determinant by way of a peptide bond, wherein said
amino acid linker comprises said second attachment site, and
wherein said amino acid linker with said second attachment site is
bound to the C-terminus of said protein of IL-5 by way of a peptide
bond.
105. A method of treating an allergic eosinophilic disease
comprising administering the composition of claim 22 to a human or
an animal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 11/499,659, filed Aug. 7, 2006, now pending,
which is a continuation of U.S. application Ser. No. 10/289,454,
filed Nov. 7, 2002, now U.S. Pat. No. 7,094,409, which claims the
benefit of the filing dates of U.S. Provisional Appl. Nos.
60/331,045, filed Nov. 7, 2001, and 60/396,636, filed Jul. 19,
2002. U.S. application Ser. No. 10/289,454 is also a
continuation-in-part of, and claims priority to, U.S. application
Ser. No. 10/050,902, filed Jan. 18, 2002, now U.S. Pat. No.
7,264,810, and International Appl. No. PCT/IB02/00166, filed Jan.
21, 2002, the latter of which was published under PCT Article 21(2)
in the English language as WO 02/056905 on Jul. 25, 2002, both of
which applications claim the benefit of the filing dates of U.S.
Provisional Application Nos. 60/262,379, 60/288,549, 60/326,998 and
60/331,045, filed Jan. 19, 2001, May 4, 2001, Oct. 5, 2001, and
Nov. 7, 2001, respectively, now abandoned. The disclosures of all
of the above-referenced applications are incorporated by reference
herein in their entireties.
REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ELECTRONICALLY VIA EFS-WEB
[0002] The Sequence Listing in "SequenceListing.txt", 262,144
bytes, created on Aug. 28, 2008, and submitted electronically via
EFS-Web, is herein incorporated-by-reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention is related to the fields of molecular
biology, virology, immunology and medicine. The invention provides
a composition comprising an ordered and repetitive antigen or
antigenic determinant array, and in particular an array comprising
a protein or peptide of IL-5, IL-13 or eotaxin. More specifically,
the invention provides a composition comprising a virus-like
particle and at least one protein, or peptide of IL-5, IL-13 and/or
eotaxin bound thereto. The invention also provides a process for
producing the conjugates and the ordered and repetitive arrays,
respectively. The compositions of the invention are useful in the
production of vaccines for the treatment of allergic diseases with
an eosinophilic component and as a pharmaccine to prevent or cure
allergic diseases with an eosinophilic component and to efficiently
induce immune responses, in particular antibody responses.
Furthermore, the compositions of the invention are particularly
useful to efficiently induce self-specific immune responses within
the indicated context.
[0005] 2. Related Art
[0006] A number of allergic diseases including asthma, nasal
rhinitis, nasal polyps, eosinophilic syndromes and atopic
dermatitis have prominent inflammatory components characterized by
pronounced eosinophilic infiltration.
[0007] The most medically important group of these diseases, atopic
asthma is recognized as a chronic inflammatory disease of the
airways that is clinically characterized by episodic airflow
obstruction, inflammation of the airways, and enhanced bronchial
reactivity to nonspecific allergens. The degree of obstruction of
the airways and hyperreactivity often correlates with the level of
airway inflammation. These clinical features are indicative of
asthma severity (Kay, A. B., J Allergy Clin Immunol, 1991, 87:893;
De Monchy, J. G. et al., Am Rev Respir Dis, 1985, 131:373; Beasley,
R. et al., Am Rev Respir Dis, 1989, 139:806; Azzawi, M. et al., Am
Rev Respir Dis, 1990, 142:1407; Ohashi, Y. et al., Am Rev Respir
Dis, 1992, 145:1469; Nakajima, H. et al., Am Rev Respir Dis, 1992,
146:374; Broide, D. H. et al., J Allergy Clin Immunol, 1991,
88:637; Warlaw, A. J. et al., Am Rev Respir Dis, 1988, 137:62).
Cellular infiltration correlates with disease progression and
indicates inflammation of the airways that is a major contributing
factor to pathogenesis and pathobiology. The inflammatory
infiltrate in asthma is complex; however, it is now widely
recognized that CD4.sup.+ Th lymphocytes with a Th2 profile (Th2
cells) of cytokine expression play a pivotal role in the clinical
expression and pathogenesis of this disorder (Robinson, D. S. et
al., J Allergy Clin Immunol, 1993, 92:397; Walker, C. et al., J
Allergy Clin Immunol, 1991, 88:935). Th2 cells regulate disease
progression and airways hyperresponsiveness (AHR) by orchestrating
allergic inflammation of the airways through the release of a range
of cytokines such as IL-4, -5, -9, -10, -13 (Robinson, D. S. et
al., N Eng J Med, 1992, 326:298; Robinson, D. S. et al., J Allergy
Clin Immunol, 1993, 92:313; Walker, C. et al., Am Rev Respir Dis,
1992, 146:109; Drazen, J. M. et al., J Exp Med, 1996, 183:1). Like
Th2 cells, the levels of eosinophils and their inflammatory
products in the lung correlate with disease severity, and
accumulation of this leukocyte in the airways is a central feature
of bronchial dysfunction during the late-phase asthmatic response
(Bousquet, J. et al., N Eng J Med, 1990, 323:1033). Although Th2
cells orchestrate many facets of the allergic response, their role
in regulating eosinophilia through the secretion of IL-5 is thought
to be a major proinflammatory pathway in asthma.
[0008] Interleukin-5 (IL-5) is a proinflammatory cytokine expressed
at high levels in asthmatics. Moreover, IL-5 is a cytokine
primarily involved in the pathogenesis of atopic diseases. It
specifically controls the production, activation and localization
of eosinophils, the major cause of tissue damage in atopic
diseases. Furthermore, IL-5 is an inducible T-cell derived cytokine
with remarkable specificity for the eosinophil lineage. IL-5 is
controlled at the level of transcription and regulation of the gene
represents a promising target for therapy of eosinophil-dependent
allergic disorders such as asthma, eczema and rhinitis.
[0009] There is a large body of evidence that eosinophils are a key
component of the allergic response in asthma. IL-5 is uniquely
involved in the production of eosinophils, and with a variety of
other cytokines such as IL-13, chemokines such as Eotaxin and other
factors controls their activation, localization and survival. Thus,
IL-5 has become an important drug target for new anti-asthmatics
(Foster, P. S. et al., Pharmacol Ther, 2002, 94(3):253; Foster, P.
S. et al., Trends Mol Med, 2002, 8(4):162).
[0010] There is 71% homology between human and murine proteins
(Cytokine hand book). IL-5 exhibits no significant amino acid
sequence homology with other cytokines, except for short stretches
in the murine interleukin-3, murine GM-CSF, and murine
interferon-.gamma. proteins. The predicted molecular mass of both
the human and mouse protein sequences are 13.1 kDa. Biologically
active IL-5 is a disulfide-linked homodimer that is covalently
linked by highly conserved cysteine residues (44-86' and 86-44')
that orient the monomers in a head to tail configuration (Takahashi
T. et al Mol. Immunol. 27:911-920 1990). Although wild-type
monomeric IL-5 is biologically inactive a functional IL-5 monomer
has been engineered by insertional mutagenesis (Dickason R R, et al
J. Mol. Med 74: 535-46 1996) Analysis of the crystal structure of
human IL-5 demonstrated a novel two-domain configuration with each
domain requiring the participation of two chains, with a high
degree of similarity to the cytokine fold found in GM-CSF,
interleukin-3, and interleukin-4 (Milburn M. V et al Nature 363:
172-176). The C-terminal region of IL-5 appears to be important for
binding to the IL-5 receptor and for biological activity (Proudfoot
et al J. Protein Chem. 15(5):491-9.1996). Binding of IL-5 to its
receptor is thought occur in regions overlapping helices A and D
where helix A is principally involved in binding the
.alpha.-subunit of the receptor (Graber P. et al J. Biol Chem 270:
15762-15769 1995). Native human IL-5 has 2 potential glycosylation
sites and mouse IL-5 three. Human IL-5 is both N-glycosylated and
O-glycosylated at Thr 3. Recombinant IL-5 expressed in eukaryotic
systems exhibits a broad range of molecular masses from 45-60 kDa
due to differential glycosylation. Deglycosylated IL-5 and IL-5
expressed in prokaryotic cells retain full biologic activity
(Tominaga A. et al J. Immunol. 144: 1345-1352, 1990).
[0011] The routes to drug discovery are typically based on screens
for inhibitors of IL-5 production, ligand antagonists, control of
receptor expression and receptor activation. In particular,
inhibition of the action of IL-5 might provide a way of treatment
against asthma and other diseases associated with eosinophils.
Immunotherapy represents another and very attractive approach to
controlling IL-5 levels and disease conditions associated with
eosinophilia such as asthma.
[0012] Currently, the commonest treatment for prevention of the
symptoms of asthma is the use of inhaled corticosteroids. Generally
the use of these agents is fairly safe and cheap. However they
function by inducing a general immunosuppressive effect and there
are adverse side effects associated with their long term use
including high blood pressure, osteoporosis and development of
cataracts. Corticosteroids must be taken everyday and hence patient
compliance is another issue in the successful use of these
medicines. Furthermore there are asthmatic patients refractory to
the use of corticosteroids necessitating the use of alternative
therapies. Selective targeting of eosinophils using
immunotherapeutic agents directed against IL-5 may overcome the
adverse effects of using general immunosuppressive agents with
pleiotropic actions.
[0013] Possible future treatment of diseases such as asthma may
include passive immunization and, thus, the use of monoclonal
antibodies specific for IL-5. Clinical trials with humanized
monoclonal antibodies against IL-5 aimed at reducing eosinophilia
in asthmatic patients are ongoing. In particular, clinical trials
using SCH55700 (eslizumab, Schering Plough) which is a humanized
monoclonal antibody with activity against IL-5 from various species
[Egan, R. W. et al., Arzneimittel-Forschung, 1999, 49:779] and
SB240563 (mepolizumab, Glaxo Smith Kline) which is a humanized
antibody with specificity for human and primate interleukin-5
[Hart, T. K. et al., Am J Respir Crit. Care Med, 1998, 157:A744;
Zia-Amirhosseini, P. et al., J Pharmacol Exp Ther, 1999, 291:1060]
have been reported. Both monoclonal antibodies demonstrated
acceptable safety profiles in phase 1 trials and led to reduction
of eosinophil numbers but no reduction in airway hyperreactivity
was, observed. The deleterious action that eosinophils exert on the
airways of asthmatics is thought to be a chronic phenomena
involving tissue re-modelling. Studies designed to test efficacy of
anti IL-5 therapy in this context need to be assessed and are in
development.
[0014] The treatment with mAbs, however, entails several
disadvantages. Monoclonal antibodies are expensive therapeutic
agents which must be taken monthly or bimonthly. The issue of
patient non-compliance resulting form repeated medical visits for
administration of the injected drug is an important problem.
Furthermore, allotype variation between the patient and therapeutic
antibody may lead to the monoclonal antibody therapy eventually
becoming ineffective. The high dose of mAb and the possibility of
immune complex formation may also reduce the efficacy of passive
immunisation. An active vaccination strategy limits these
complications.
[0015] Another approach to provide therapeutic agents for chronic
asthma or other disease states with demonstrated eosinophilia or
other conditions associated with IL-5 has been described in WO
97/45448. Therein, the use of "modified and variant forms of IL5
molecules capable of antagonising the activity of IL5" in
ameliorating, abating or otherwise reducing the aberrant effects
caused by native or mutant forms of IL5 has been proposed. The
antagonizing effect is reported to be the result of the variant
forms of IL5 binding to the low affinity a chain of IL5R but not to
the high affinity receptors. By this way of action the variants
compete with IL5 for binding to its receptors without exerting the
physiological effects of IL5.
[0016] Eotaxin is a chemokine specific for Chemokine receptor 3,
present on eosinophils, basophils and Th2 cells. However, Eotaxin
has high specificity for eosinophils (Zimmerman et al., J. Immunol.
165: 5839-46 (2000)). Eosinophil migration is reduced by 70% in
eotaxin-1 knock-out mice, which however can still develop
eosinophilia (Rothenberg et al., J. Exp. Med. 185: 785-90 (1997)).
IL-5 seems to be responsible for the migration of eosinophils from
bone-marrow to blood, and eotaxin for the local migration in the
tissue (Humbles et al., J. Exp. Med. 186: 601-12 (1997). Thus
targeting eotaxin in addition to IL-5 may enhance immunotherapies
directed towards lowering eosinophilia.
[0017] The human genome contains 3 eotaxin genes, eotaxin 1-3 which
share 30% homology. To date 2 genes are known in the mouse: eotaxin
1 and eotaxin 2 (Zimmerman et al., J. Immunol. 165: 5839-46
(2000)). They share 38% homology. Murine eotaxin-2 shares 59%
homology with human eotaxin-2. In the mouse, eotaxin-1 seems to be
ubiquitously expressed in the gastro-intestinal tract, while
eotaxin-2 seems to be predominantly expressed in the jejunum
(Zimmerman et al., J. Immunol. 165: 5839-46 (2000)). Eotaxin-1 is
present in broncheo-alveolar fluid (Teixeira et al., J. Clin.
Invest. 100: 1657-66 (1997)). The sequence of human eotaxin-1 is
shown in SEQ ID No.: 242 (aa 1-23 corresponds to the signal
peptide), the sequence of human eotaxin-2 is shown in SEQ ID No.:
243 (aa 1-26 corresponds to the signal peptide), the sequence of
human eotaxin-3 is shown in SEQ ID No.: 244 (aa 1-23 corresponds to
the signal peptide), the sequence of mouse eotaxin-1 is shown in
SEQ ID No.: 245 (aa 1-23 corresponds to the signal peptide), and
the sequence of mouse eotaxin-2 is shown in SEQ ID No.: 246 (aa
1-23 corresponds to the signal peptide).
[0018] The monomer of eotaxin has a mass of 8.3 kDa and is in
equilibrium with dimeric eotaxin over a wide range of conditions.
The estimated Kd is 1.3 mM at 37.degree. C. however the monomer is
the predominant form (Crump et al., J. Biol. Chem. 273: 22471-9
(1998). The structure of Eotaxin has been elucidated by NMR
spectroscopy. The binding site to its receptor CCR3 is at the
N-terminus and the region preceding the first cysteine is crucial
(Crump et al., J. Biol. Chem. 273: 22471-9 1998). Peptides derived
from chemokine receptors bound to Eotaxin confirmed this finding.
Eotaxin has four cysteines forming two disulfide bridges and can be
chemically synthesized (Clark-Lewis et al., Biochemistry
30:3128-3135 1991). Eotaxin 1 is variably O-glycosylated on Thr71
(Noso, N. et al Eur J. Biochem. 253: 114-122). Expression of
Eotaxin 1 in E. coli cytosol has also been described (Crump et al.,
J. Biol. Chem. 273: 22471-9 (1998)). Expression in E. coli as
inclusion bodies with subsequent refolding (Mayer et al.,
Biochemistry 39: 8382-95 (2000)), and Insect cell expression
(Forssmann et al., J. Exp. Med. 185: 2171-6 (1997)) have been
reported for Eotaxin-2.
[0019] Interleukin 13 (IL-13) is secreted as a biologically active
monomeric Th2 cytokine. The mature form of IL-13 comprises 112
amino acids in humans and 111 amino acids in mice. The calculated
molecular mass of the protein is approximately 12.4 kDa. IL-13 can
be N-linked glycosylated (Fitzgerald K. A. et al The Cytokines Fact
Book 2.sup.nd edition Academic Press) IL-13 is produced by Th2
cells, mast cells, basophils and natural killer cells (Brombacher
F, 2000 Bioessays July; 22(7):646-56). The functional IL-13
receptor is a heterodimer composed of the Interleukin 4 receptor
.alpha. chain (IL-4R .alpha. chain) and one of the two IL-13
receptor .alpha. binding proteins (Brombacher F, 2000 Bioessays
July; 22(7):646-56).
[0020] IL 13 plays a significant role in the pathology of asthma.
It has been shown that IL 13 is involved in the central features of
this disease. It has direct effects on allergen-induced airway
hyperresponsiveness (AHR) and mucus production and has an
involvement in eosinophilia (Kuperman D. A. 2002 Nature Medicine
epub ahead of print). Selective neutralization of IL-13 in mice
significantly attenuated the asthma phenotype. Furthermore,
administration of IL-13 conferred an asthma-like phenotype to
nonsensitized T-cell deficient or naive mice, respectively (Grunig
G. et al., 1998 Science, 282(5397): 2261-3, Wills-Karp, M. et al,
1998 Science 282(5397): 2258-61). Mice with a targeted deletion of
IL-13 failed to develop allergen-induced AHR and showed a marked
decrease in mucus production (Walter, D. M. et al, 2001 J Immunol
167(8): 4668-75). Since IL-13 also influences eosinophilia in the
murine asthma model (Grunig G. et al., 1998 Science, 282(5397):
2261-3), it possible IL-13 is involved in many more allergic
diseases associated with eosinophilia and neutralizing its activity
may offers a promising treatment for patients.
[0021] Additionally, upregulation of IL-13 and IL-13 receptor has
been found in many tumor types (e.g. in all Hodgkin lymphoma
disease cell lines examined to date). Thus immunization against
IL-13 may provide a way of curing tumor patients overexpressing
IL-13.
[0022] One way to improve the efficiency of vaccination is to
increase the degree of repetitiveness of the antigen applied.
Unlike isolated proteins, viruses induce prompt and efficient
immune responses in the absence of any adjuvants both with and
without T-cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol:
15:235-270 (1991)). Although viruses often consist of few proteins,
they are able to trigger much stronger immune responses than their
isolated components. For B-cell responses, it is known that one
crucial factor for the immunogenicity of viruses is the
repetitiveness and order of surface epitopes. Many viruses exhibit
a quasi-crystalline surface that displays a regular array of
epitopes which efficiently crosslinks epitope-specific
immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol.
Today 17:553-558 (1996)). This crosslinking of surface
immunoglobulins on B cells is a strong activation signal that
directly induces cell-cycle progression and the production of IgM
antibodies. Further, such triggered B cells are able to activate T
helper cells, which in turn induce a switch from IgM to IgG
antibody production in B cells and the generation of long-lived B
cell memory--the goal of any vaccination (Bachmann and Zinkernagel,
Ann. Rev. Immunol. 15:235-270 (1997)). Viral structure is even
linked to the generation of anti-antibodies in autoimmune disease
and as a part of the natural response to pathogens (see Fehr, T.,
et al., J. Exp. Med. 185:1785-1792 (1997)). Thus, antibodies
presented by a highly organized viral surface are able to induce
strong anti-antibody responses.
[0023] As indicated, however, the immune system usually fails to
produce antibodies against self-derived structures. For soluble
antigens present at low concentrations, this is due to tolerance at
the Th cell level. Under these conditions, coupling the
self-antigen to a carrier that can deliver T help may break
tolerance. For soluble proteins present at high concentrations or
membrane proteins at low concentration, B and Th cells may be
tolerant. However, B cell tolerance may be reversible (anergy) and
can be broken by administration of the antigen in a highly
organized fashion coupled to a foreign carrier (Bachmann and
Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1997)).
BRIEF SUMMARY OF THE INVENTION
[0024] We have now found that a protein or peptide of IL-5, IL-13
or eotaxin bound to a core particle having a structure with an
inherent repetitive organization, and hereby in particular to
virus-like-particles (VLP's) and subunits of VLP's, respectively,
leading to highly ordered and repetitive conjugates represent
potent immunogens for the induction of antibodies specific for
IL-5, IL-13 or eotaxin. Furthermore these auto-reactive antibodies
inhibit eosinophilia in a mouse model of asthma. Therefore, the
present invention provides a therapeutic mean for the treatment of
allergic eosinophilic disease, which is based on an ordered and
repetitive protein or peptide of IL-5, IL-13 or eotaxin-core
particle array, and in particular a VLP-protein or peptide of IL-5,
IL-13 or eotaxin-conjugate and -array, respectively. This
therapeutic is able to induce high titers of anti-IL-5, IL-13 or
eotaxin antibodies in a vaccinated animal and inhibit eosinophilia
in a mouse model of asthma.
[0025] The present invention, thus, provides for a composition
comprising: (a) a core particle with at least one first attachment
site; and (b) at least one antigen or antigenic determinant with at
least one second attachment site, wherein said antigen or antigenic
determinant is a protein or peptide of IL-5, IL-13 or eotaxin and
wherein said second attachment site being selected from the group
consisting of (i) an attachment site not naturally occurring with
said antigen or antigenic determinant; and (ii) an attachment site
naturally occurring with said antigen or antigenic determinant,
wherein said second attachment site is capable of association to
said first attachment site; and wherein said antigen or antigenic
determinant and said core particle interact through said
association to form an ordered and repetitive antigen array.
Preferred embodiments of core particles suitable for use in the
present invention are a virus, a virus-like particle, a
bacteriophage, a bacterial pilus or flagella or any other core
particle having an inherent repetitive structure capable of forming
an ordered and repetitive antigen array in accordance with the
present invention.
[0026] More specifically, the invention provides a composition
comprising an ordered and repetitive antigen or antigenic
determinant array, and hereby in particular protein or peptide of
IL-5, IL-13 or eotaxin-VLP conjugates. More specifically, the
invention provides a composition comprising a virus-like particle
and at least one protein or peptide of IL-5, IL-13 or eotaxin bound
thereto. The invention also provides a process for producing the
conjugates and the ordered and repetitive arrays, respectively. The
compositions of the invention are useful in the production of
vaccines for the treatment of allergic diseases with an
eosinophilic component and as a pharmaccine to prevent or cure
allergic diseases with an eosinophilic component and to efficiently
induce immune responses, in particular antibody responses.
Furthermore, the compositions of the invention are particularly
useful to efficiently induce self-specific immune responses within
the indicated context.
[0027] In the present invention, a protein or peptide of IL-5,
IL-13 or eotaxin is bound to a core particle and VLP, respectively,
typically in an oriented manner, yielding an ordered and repetitive
protein or peptide of IL-5, IL-13 or eotaxin antigen array.
Furthermore, the highly repetitive and organized structure of the
core particles and VLPs, respectively, mediates the display of the
protein or peptide of IL-5, IL-13 or eotaxin in a highly ordered
and repetitive fashion leading to a highly organized and repetitive
antigen array. Furthermore, binding of the protein or peptide of
IL-5, IL-13 or eotaxin to the core particle and VLP, respectively,
provides T helper cell epitopes, since the core particle and VLP is
foreign to the host immunized with the core particle--protein or
peptide of IL-5, IL-13 or eotaxin array and VLP-protein or peptide
of IL-5, IL-13 or eotaxin array, respectively. Those arrays differ
from prior art conjugates in their highly organized structure,
dimensions, and in the repetitiveness of the antigen on the surface
of the array.
[0028] In one aspect of the invention, the protein or peptide of
IL-5, IL-13 or eotaxin is expressed in a suitable expression host
compatible with proper folding of the IL-5, IL-13 or eotaxin
protein or IL-5, IL-13 or eotaxin peptide, or synthesized, while
the core particle and the VLP, respectively, is expressed and
purified from an expression host suitable for the folding and
assembly of the core particle and the VLP, respectively. The
protein or peptide of IL-5, IL-13 or eotaxin may be chemically
synthesized. The protein or peptide of IL-5, IL-13 or eotaxin array
is then assembled by binding the protein or peptide of IL-5, IL-13
or eotaxin to the core particle and the VLP, respectively.
[0029] In another aspect, the present invention provides for a
composition comprising (a) a virus-like particle, and (b) at least
one antigen or antigenic determinant, wherein said antigen or said
antigenic determinant is a protein or peptide of IL-5, IL-13 or
eotaxin.
[0030] In still a further aspect, the present invention provides
for a vaccine composition comprising a composition comprising: (a)
a core particle with at least one first attachment site; and (b) at
least one antigen or antigenic determinant with at least one second
attachment site, wherein said antigen or antigenic determinant is a
protein or peptide of IL-5, IL-13 or eotaxin, and wherein said
second attachment site being selected from the group consisting of
(i) an attachment site not naturally occurring with said antigen or
antigenic determinant; and (ii) an attachment site naturally
occurring with said antigen or antigenic determinant, wherein said
second attachment site is capable of association to said first
attachment site; and wherein said antigen or antigenic determinant
and said core particle interact through said association to form an
ordered and repetitive antigen array.
[0031] In a further aspect, the present invention provides for a
vaccine composition comprising a composition, wherein said
composition comprising (a) a virus-like particle; and (b) at least
one antigen or antigenic determinant, wherein said antigen or said
antigenic determinant is a protein or peptide of IL-5, IL-13 or
eotaxin; and wherein said at least one antigen or antigenic
determinant is bound to said virus-like particle.
[0032] In still a further aspect, the present invention provides
for a process for producing a composition of the invention
comprising (a) providing a virus-like particle; and (b) providing
at least one antigen or protein or peptide of IL-5, IL-13 or
eotaxin; (c) combining said virus-like particle and said at least
one antigen or antigenic determinant so that said at least one
antigen or antigenic determinant is bound to said virus-like
particle.
[0033] In still a further aspect, the present invention provides a
process for producing a composition of the invention comprising:
(a) providing a core particle with at least one first attachment
site; (b) providing at least one antigen or antigenic determinant
with at least one second attachment site, wherein said antigen or
antigenic determinant is a protein or peptide of IL-5, IL-13 or
eotaxin, and wherein said second attachment site being selected
from the group consisting of (i) an attachment site not naturally
occurring with said antigen or antigenic determinant; and (ii) an
attachment site naturally occurring with said antigen or antigenic
determinant; and wherein said second attachment site is capable of
association to said first attachment site; and (c) combining said
core particle and said at least one antigen or antigenic
determinant, wherein said antigen or antigenic determinant and said
core particle interact through said association to form an ordered
and repetitive antigen array.
[0034] In another aspect, the present invention provides for a
method of immunization comprising administering the composition of
the invention to an animal or human.
[0035] In a further aspect, the present invention provides for a
use of the compositions of the invention for the manufacture of a
medicament for treatment of allergic diseases with an eosinophilic
component.
[0036] In a still further aspect, the present invention provides
for a use of the compositions of the invention for the preparation
of a medicament for the therapeutic or prophylactic treatment of
allergic diseases with an eosinophilic component, preferably of
asthma. Furthermore, in a still further aspect, the present
invention provides for a use of the compositions of the invention,
either in isolation or in combination with other agents, for the
manufacture of a composition, vaccine, drug or medicament for
therapy or prophylaxis of allergic diseases with an eosinophilic
component, in particular asthma.
[0037] Therefore, the invention provides, in particular, vaccine
compositions which are suitable for preventing and/or attenuating
allergic diseases with an eosinophilic component or conditions
related thereto. The invention further provides and immunization
and vaccination methods, respectively, for preventing and/or
attenuating allergic diseases with an eosinophilic component or
conditions related thereto, in animals, and in particular in cows,
sheep and cattle as well as in humans. The inventive compositions
may be used prophylactically or therapeutically.
[0038] In specific embodiments, the invention provides methods for
preventing and/or attenuating allergic diseases with an
eosinophilic component or conditions related thereto which are
caused or exacerbated by "self" gene products, i.e. "self antigens"
as used herein. In related embodiments, the invention provides
methods for inducing immunological responses in animals and
individuals, respectively, which lead to the production of
antibodies that prevent and/or attenuate allergic diseases with an
eosinophilic component or conditions related thereto, which are
caused or exacerbated by "self" gene products.
[0039] As would be understood by one of ordinary skill in the art,
when compositions of the invention are administered to an animal or
a human, they may be in a composition which contains salts,
buffers, adjuvants, or other substances which are desirable for
improving the efficacy of the composition. Examples of materials
suitable for use in preparing pharmaceutical compositions are
provided in numerous sources including Remington's Pharmaceutical
Sciences (Osol, A, ed., Mack Publishing Co. (1990)).
[0040] Compositions of the invention are said to be
"pharmacologically acceptable" if their administration can be
tolerated by a recipient individual. Further, the compositions of
the invention will be administered in a "therapeutically effective
amount" (i.e., an amount that produces a desired physiological
effect).
[0041] The compositions of the present invention may be
administered by various methods known in the art, but will normally
be administered by injection, infusion, inhalation, oral
administration, or other suitable physical methods. The
compositions may alternatively be administered intramuscularly,
intravenously, or subcutaneously. Components of compositions for
administration include sterile aqueous (e.g., physiological saline)
or non-aqueous solutions and suspensions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Carriers or occlusive dressings can be used to increase
skin permeability and enhance antigen absorption.
[0042] Other embodiments of the present invention will be apparent
to one of ordinary skill in light of what is known in the art, the
following drawings and description of the invention, and the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1. Expression of mouse His-C-IL5. Extracts from the
insoluble cellular fraction obtained after culturing
pMODC6-IL5/BL21-DE3, either with or without IPTG, were prepared as
described above. Equivalent amounts of extract were loaded onto a
16% SDS-polyacrylamide gel, electrophoresed and stained with
Coomassie Blue. Lane M, Size Marker (NEB, Broad range, pre-stained
marker), Lane 1, Extract from uninduced culture, Lane 2, extract
from culture induced for 4 h with IPTG.
[0044] FIG. 2. SDS-PAGE analysis of the purification of His-C-IL-5
with Ni-NTA. Samples from various stages of the purification were
applied to a 16% SDS-PAGE and run under reducing conditions.
Proteins were stained with Coomassie blue. M, Marker; 1:
Solubilised inclusion bodies; 2: Flow through (unbound material);
3: Wash 1 pH 6.5; 4: Wash 2 pH 6.5; 5: Wash 3 pH 5.9; 6-8: Eluate
pH 4.5; 9: pure recombinant mouse IL-5.
[0045] FIG. 3. SDS-PAGE showing purification of recombinant
mouse-His-C-IL5. Five .mu.g aliquots of purified mouse-His-C-IL5
were separated on a 16% SDS polyacrylamide gel either in the
presence (2nd lane from left) or absence (3.sup.rd Lane from left)
of dithiothreitol. The gel was stained for protein with Coomassie
Blue R-250. Lane M contains a size marker (NEB, Broad range,
pre-stained marker).
[0046] FIG. 4. Effect of His-C-IL-5 on the Proliferation of BCL1
cells. BCL1 cells were incubated with .sup.3H-Thymidine in the
presence of the following: Murine IL-5 (30 ng/ml) His-C-IL5, (30
ng/ml); Q.beta. (200 ng/ml); Q.beta.-chemically crosslinked with an
unrelated cytokine (200 ng/ml) or Q.beta.-His-C-IL5 (105 ng/ml).
Undiluted starting concentrations are indicated in parentheses and
five-fold serial dilutions were made from the indicated starting
concentrations. The incorporation of .sup.3H-Thymidine was
determined by liquid scintillation counting.
[0047] FIG. 5. Analysis of the coupling reaction by Coomassie blue
stained SDS-PAGE. Lane M: pre-stained molecular weight marker Lane
1, Purified His-C-IL-5, Lane 2, Q.beta. after derivitisation with
the chemical cross-linker SMPH. Lane 3, Coupling reaction, Lane 4,
Coupling reaction after dialysis. The identity of the different
molecular species in the coupling reaction is identified on the
right of the figure.
[0048] FIG. 6. Analysis of the coupling reaction by Western-blot.
Lane M: Molecular weight marker; Lane 1, Purified His-C-IL-5; Lane
2, Q.beta. after derivitisation with the chemical cross-linker
SMPH. Lane 3, Coupling reaction. The primary antibody for detecting
His-C-IL5 was a rat anti-His antibody subsequently incubated with
an anti-Rat antibody conjugated to HRP. Q.beta. was detected by
staining with rabbit polyclonal antiserum against Q.beta. followed
by an HRP-conjugated anti-rabbit antibody. Identical blots were
stained as indicated.
[0049] FIG. 7: Quadruple ELISA. A. Schematic representation of the
capture ELISA. The various components of the assay are 1, goat
anti-rabbit IgG; 2, rabbit anti-Q.beta. polyclonal antisera; 3,
either Q.beta.-His-C-IL5, Q.beta. or PBS; 4, anti-IL5 monoclonal
Ab, TRFK 4 or 5; 5, Anti mouse IgG-HRP. B. Results of the quadruple
ELISA. The ability of neutralizing monoclonal antibodies to
interact with His-C-IL5 covalently coupled to the ordered antigen
array was determined by ELISA.
[0050] FIG. 8. ELISA of sera against IL-5. ELISA plates were coated
with His-C-IL5 and incubated with either pre-immune or day 21
collected from mice vaccinated with Q.beta.-His-C-IL5 (4 mice) or
Q.beta. mixed with His-C-IL-5 (5 mice). The starting dilution of
the sera was 1:50 and five-fold dilutions were made. Binding of
IL-5 specific antibodies was detected with anti-mouse IgG
conjugated to HRP and the chromogenic substrate.
[0051] FIG. 9. Induction of recombinant GST-EK-IL13-C1-His
expression. in BL21. Coomassie blue stain of a 16% SDS-PAGE. Load
corresponds to 0.1 OD.sub.600 of the indicated bacterial lysates.
Expression of the IL-13-fusion protein was induced with 0.75 mM
IPTG and samples were analysed after 4 hrs by SDS-PAGE. Note, there
is strong expression of the IL-13-fusion protein in bacteria that
had been transformed with the corresponding plasmid
(pMod-GST-EK-IL13-C1-His) and induced with IPTG (see arrow).
[0052] FIG. 10. Purification of GST-EK-IL13-C1-His under
denaturing. Coomassie blue stain of two 16% SDS-PAGEs. Load
corresponds to 5 .mu.l of the indicated fraction. The IL-13-fusion
protein was obtained from inclusion bodies, solubilized in a
Guanidine-HCl denaturing buffer and loaded onto a Ni.sup.2+-agarose
column, equilibrated with the same buffer. Bound proteins were
eluted in two steps with different pH. The figure shows analysis of
TCA-precipitated aliquots of the indicated fractions (#5-#30)
eluted with the second buffer at pH 4.5. Note, due to the
C-terminal His-tag, the IL-13 fusion protein was efficiently bound
to the Ni.sup.2+-agarose column and eluted by lowering the pH.
[0053] FIG. 11. Analysis of soluble IL-13 fusion protein after
refolding. The GST-EK-IL13-C1-His fusion protein was refolded as
described in section 18D. After the refolding reaction was finished
an aliquot of the protein solution was analysed by SDS-PAGE
followed by Coomassie-stain (A) or by Western blot (B). The
indicated primary antibodies were purchased from R&D Systems
(.alpha.-IL13, AF-4,3-NA), by Qiagen (.alpha.-PentaHis, 34660) and
Amersham Biosciences (.alpha.-GST, 24-4577), respectively.
Antibodies were used in concentrations according to the
manufacturer's manuals.
[0054] FIG. 12. Expression of mouse eotaxin-C1. The supernatants
from cell lysates of BL21/DE3 cells transformed with pmEo-C1, after
9 hours of induction with 1 mM of IPTG were run on 16% PAGE gel,
blotted to nitrocellulose membrane and reacted with goat anti-mouse
eotaxin antibody (R & D system). Lane 1: Pre-stained protein
marker (New England Biolabs). Lane 2: the supernatant of the cell
lysates of BL21/DE3 cells transformed with pmEo-C1, after 9 hours
of induction with 1 mM of IPTG. Lane 3: Pre-stained protein marker
(New England Biolabs). Lane 4. Western blot of the same lysates as
lane 2 probed with anti-mouse eotaxin antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0055] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are hereinafter
described.
1. DEFINITIONS
[0056] Allergic diseases with an eosinophilic component: The term
allergic diseases with an eosinophilic component as used within
refers to disease states or conditions where there is an increase
in the number of eosinophils in the circulating blood or body
tissues and fluids. Diseases where eosinophils are elevated and
have either a direct or indirect effect on the disease state
include; asthma, hay fever, nasal rhinitis, nasal polyps,
idiopathic eosinophilic syndromes, atopic dermatitis, skin diseases
and rashes, lung diseases such as Loefflers syndrome, chronic
eosinophilic pneumonia, Churg-Strauss syndrome and
hyper-eosinophilic syndromes of unknown causes. Those skilled in
the art can recognize allergic diseases with an eosinophilic
component.
[0057] Amino acid linker: An "amino acid linker", or also just
termed "linker" within this specification, as used herein, either
associates the antigen or antigenic determinant with the second
attachment site, or more preferably, already comprises or contains
the second attachment site, typically--but not necessarily--as one
amino acid residue, preferably as a cysteine residue. The term
"amino acid linker" as used herein, however, does not intend to
imply that such an amino acid linker consists exclusively of amino
acid residues, even if an amino acid linker consisting of amino
acid residues is a preferred embodiment of the present invention.
The amino acid residues of the amino acid linker are, preferably,
composed of naturally occurring amino acids or unnatural amino
acids known in the art, all-L or all-D or mixtures thereof.
However, an amino acid linker comprising a molecule with a
sulfhydryl group or cysteine residue is also encompassed within the
invention. Such a molecule comprise preferably a C1-C6 alkyl-,
cycloalkyl (C5, C6), aryl or heteroaryl moiety. However, in
addition to an amino acid linker, a linker comprising preferably a
C1-C6 alkyl-, cycloalkyl- (C5, C6), aryl- or heteroaryl- moiety and
devoid of any amino acid(s) shall also be encompassed within the
scope of the invention. Association between the antigen or
antigenic determinant or optionally the second attachment site and
the amino acid linker is preferably by way of at least one covalent
bond, more preferably by way of at least one peptide bond.
[0058] Animal: As used herein, the term "animal" is meant to
include, for example, humans, sheep, elks, deer, mule deer, minks,
mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats,
mice, birds, chicken, reptiles, fish, insects and arachnids.
[0059] Antibody: As used herein, the term "antibody" refers to
molecules which are capable of binding an epitope or antigenic
determinant. The term is meant to include whole antibodies and
antigen-binding fragments thereof, including single-chain
antibodies. Most preferably the antibodies are human antigen
binding antibody fragments and include, but are not limited to,
Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain
antibodies, disulfide-linked Fvs (sdFv) and fragments comprising
either a V.sub.L or V.sub.H domain. The antibodies can be from any
animal origin including birds and mammals. Preferably, the
antibodies are human, murine, rabbit, goat, guinea pig, camel,
horse or chicken. As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human immunoglobulin
and include antibodies isolated from human immunoglobulin libraries
or from animals transgenic for one or more human immunoglobulins
and that do not express endogenous immunoglobulins, as described,
for example, in U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0060] Antigen: As used herein, the term "antigen" refers to a
molecule capable of being bound by an antibody or a T cell receptor
(TCR) if presented by MHC molecules. The term "antigen", as used
herein, also encompasses T-cell epitopes. An antigen is
additionally capable of being recognized by the immune system
and/or being capable of inducing a humoral immune response and/or
cellular immune response leading to the activation of B- and/or
T-lymphocytes. This may, however, require that, at least in certain
cases, the antigen contains or is linked to a Th cell epitope and
is given in adjuvant. An antigen can have one or more epitopes (B-
and T-epitopes). The specific reaction referred to above is meant
to indicate that the antigen will preferably react, typically in a
highly selective manner, with its corresponding antibody or TCR and
not with the multitude of other antibodies or TCRs which may be
evoked by other antigens. Antigens as used herein may also be
mixtures of several individual antigens.
[0061] Antigenic determinant: As used herein, the term "antigenic
determinant" is meant to refer to that portion of an antigen that
is specifically recognized by either B- or T-lymphocytes.
B-lymphocytes responding to antigenic determinants produce
antibodies, whereas T-lymphocytes respond to antigenic determinants
by proliferation and establishment of effector functions critical
for the mediation of cellular and/or humoral immunity.
[0062] Association: As used herein, the term "association" as it
applies to the first and second attachment sites, refers to the
binding of the first and second attachment sites that is preferably
by way of at least one non-peptide bond. The nature of the
association may be covalent, ionic, hydrophobic, polar or any
combination thereof, preferably the nature of the association is
covalent.
[0063] Attachment Site, First: As used herein, the phrase "first
attachment site" refers to an element of non-natural or natural
origin, to which the second attachment site located on the antigen
or antigenic determinant may associate. The first attachment site
may be a protein, a polypeptide, an amino acid, a peptide, a sugar,
a polynucleotide, a natural or synthetic polymer, a secondary
metabolite or compound (biotin, fluorescein, retinol, digoxigenin,
metal ions, phenylmethylsulfonylfluoride), or a combination
thereof, or a chemically reactive group thereof. The first
attachment site is located, typically and preferably on the
surface, of the core particle such as, preferably the virus-like
particle. Multiple first attachment sites are present on the
surface of the core and virus-like particle, respectively,
typically in a repetitive configuration.
[0064] Attachment Site, Second: As used herein, the phrase "second
attachment site" refers to an element associated with the antigen
or antigenic determinant to which the first attachment site located
on the surface of the core particle and virus-like particle,
respectively, may associate. The second attachment site of the
antigen or antigenic determinant may be a protein, a polypeptide, a
peptide, a sugar, a polynucleotide, a natural or synthetic polymer,
a secondary metabolite or compound (biotin, fluorescein, retinol,
digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a
combination thereof, or a chemically reactive group thereof. At
least one second attachment site is present on the antigen or
antigenic determinant. The term "antigen or antigenic determinant
with at least one second attachment site" refers, therefore, to an
antigen or antigenic construct comprising at least the antigen or
antigenic determinant and the second attachment site. However, in
particular for a second attachment site, which is of non-natural
origin, i.e. not naturally occurring within the antigen or
antigenic determinant, these antigen or antigenic constructs
comprise an "amino acid linker".
[0065] Bound: As used herein, the term "bound" refers to binding or
attachment that may be covalent, e.g., by chemically coupling, or
non-covalent, e.g., ionic interactions, hydrophobic interactions,
hydrogen bonds, etc. Covalent bonds can be, for example, ester,
ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds,
carbon-phosphorus bonds, and the like. The term "bound" is broader
than and includes terms such as "coupled," "fused" and
"attached".
[0066] Coat protein(s): As used herein, the term "coat protein(s)"
refers to the protein(s) of a bacteriophage or a RNA-phage capable
of being incorporated within the capsid assembly of the
bacteriophage or the RNA-phage. However, when referring to the
specific gene product of the coat protein gene of RNA-phages the
term "CP" is used. For example, the specific gene product of the
coat protein gene of RNA-phage Q.beta. is referred to as "Q.beta.
CP", whereas the "coat proteins" of bacteriophage Q.beta. comprise
the "Q.beta. CP" as well as the A1 protein. The capsid of
Bacteriophage Q.beta. is composed mainly of the Q.beta. CP, with a
minor content of the A1 protein. Likewise, the VLP Q.beta. coat
protein contains mainly Q.beta. CP, with a minor content of A1
protein.
[0067] Core particle: As used herein, the term "core particle"
refers to a rigid structure with an inherent repetitive
organization. A core particle as used herein may be the product of
a synthetic process or the product of a biological process.
[0068] Coupled: The term "coupled", as used herein, refers to
attachment by covalent bonds or by strong non-covalent
interactions, typically and preferably to attachment by covalent
bonds. Any method normally used by those skilled in the art for the
coupling of biologically active materials can be used in the
present invention.
[0069] Effective Amount: As used herein, the term "effective
amount" refers to an amount necessary or sufficient to realize a
desired biologic effect. An effective amount of the composition
would be the amount that achieves this selected result, and such an
amount could be determined as a matter of routine by a person
skilled in the art. For example, an effective amount for treating
an immune system deficiency could be that amount necessary to cause
activation of the immune system, resulting in the development of an
antigen specific immune response upon exposure to antigen. The term
is also synonymous with "sufficient amount."
[0070] The effective amount for any particular application can vary
depending on such factors as the disease or condition being
treated, the particular composition being administered, the size of
the subject, and/or the severity of the disease or condition. One
of ordinary skill in the art can empirically determine the
effective amount of a particular composition of the present
invention without necessitating undue experimentation.
[0071] Eotaxin protein: The term "eotaxin protein" as used herein
refers to a protein encoded by an eotaxin gene. Different variants
of the eotaxin protein may be caused by nucleotide point mutations
and polymorphisms, respectively, as well as insertions, deletions
and/or substitutions of one or more nucleotides, and shall be
explicitly encompassed within the scope of the present invention.
Further variability can be caused by post-translational
modifications, such as differentially glycosylated forms of eotaxin
as well as prototypically cleaved forms of eotaxin. The, term
"eotaxin protein", as used herein, shall also encompass eotaxin
protein variants, including but not limiting to the above indicated
preferred examples.
[0072] Eotaxin peptide: As used herein, the term "eotaxin peptide"
is broadly defined as any peptide which represents a fraction of an
eotaxin protein and containing at least two, preferably at least
three, more preferably at least four, more preferably at least
five, more preferably at least six consecutive amino acids of the
original eotaxin protein which represents part of a eotaxin
protein, most preferably representative of a folded part of eotaxin
containing a B cell epitope, and again more preferably of the part
of eotaxin containing a neutralizing epitope.
[0073] The term "eotaxin peptide" shall further preferably
encompass any fraction of said eotaxin peptide, wherein said
fraction may be, preferably, derived by deletion of one or more
amino acids at the N and/or C terminus of eotaxin protein. The
eotaxin peptide can be obtained by recombinant expression in
eukaryotic or prokaryotic expression systems as eotaxin peptide
alone or as a fusion with other amino acids or proteins, e.g. to
facilitate folding, expression or solubility of the eotaxin peptide
or to facilitate purification of the eotaxin peptide. To enable
coupling of eotaxin peptides and subunit proteins of VLP's or
capsids, at least one second attachment site may be preferably
added to the eotaxin peptide. Alternatively eotaxin peptides may be
synthesized using methods known to the art. The term eotaxin
peptide as used herein shall also preferably encompass a peptide
which simulates the three dimensional surface structure of eotaxin.
Such eotaxin peptide is not necessarily derived from a continuous
amino acid sequence of eotaxin, but may be formed by discontinuous
amino acid residues from eotaxin. Such peptides may even contain
amino acids which are not present in the corresponding eotaxin
protein.
[0074] Epitope: As used herein, the term "epitope" refers to
continuous or discontinuous portions of a polypeptide having
antigenic or immunogenic activity in an animal, preferably a
mammal, and most preferably in a human. An epitope is recognized by
an antibody or a T cell through its T cell receptor in the context
of an MHC molecule. An "immunogenic epitope," as used herein, is
defined as a portion of a polypeptide that elicits an antibody
response or induces a T-cell response in an animal, as determined
by any method known in the art. (See, for example, Geysen et al.,
Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term
"antigenic epitope," as used herein, is defined as a portion of a
protein to which an antibody can immunospecifically bind its
antigen as determined by any method well known in the art.
Immunospecific binding excludes non-specific binding but does not
necessarily exclude cross-reactivity with other antigens. Antigenic
epitopes need not necessarily be immunogenic. Antigenic epitopes
can also be T-cell epitopes, in which case they can be bound
immunospecifically by a T-cell receptor within the context of an
MHC molecule.
[0075] An epitope can comprise 3 amino acids in a spatial
conformation which is unique to the epitope. Generally, an epitope
consists of at least about 5 such amino acids, and more usually,
consists of at least about 8-10 such amino acids. If the epitope is
an organic molecule, it may be as small as Nitrophenyl.
[0076] Fusion: As used herein, the term "fusion" refers to the
combination of amino acid sequences of different origin in one
polypeptide chain by in-frame combination of their coding
nucleotide sequences. The term "fusion" explicitly encompasses
internal fusions, i.e., insertion of sequences of different origin
within a polypeptide chain, in addition to fusion to one of its
termini.
[0077] Immune response: As used herein, the term "immune response"
refers to a humoral immune response and/or cellular immune response
leading to the activation or proliferation of B- and/or
T-lymphocytes and/or and antigen presenting cells. In some
instances, however, the immune responses may be of low intensity
and become detectable only when using at least one substance in
accordance with the invention. "Immunogenic" refers to an agent
used to stimulate the immune system of a living organism, so that
one or more functions of the immune system are increased and
directed towards the immunogenic agent. An "immunogenic
polypeptide" is a polypeptide that elicits a cellular and/or
humoral immune response, whether alone or linked to a carrier in
the presence or absence of an adjuvant. Preferably, antigen
presenting cell may be activated.
[0078] A substance which "enhances" an immune response refers to a
substance in which an immune response is observed that is greater
or intensified or deviated in any way with the addition of the
substance when compared to the same immune response measured
without the addition of the substance. For example, the lytic
activity of cytotoxic T cells can be measured, e.g. using a
.sup.51Cr release assay, in samples obtained with and without the
use of the substance during immunization. The amount of the
substance at which the CTL lytic activity is enhanced as compared
to the CTL lytic activity without the substance is said to be an
amount sufficient to enhance the immune response of the animal to
the antigen. In a preferred embodiment, the immune response in
enhanced by a factor of at least about 2, more preferably by a
factor of about 3 or more. The amount or type of cytokines secreted
may also be altered. Alternatively, the amount of antibodies
induced or their subclasses may be altered.
[0079] Immunization: As used herein, the terms "immunize" or
"immunization" or related terms refer to conferring the ability to
mount a substantial immune response (comprising antibodies and/or
cellular immunity such as effector CTL) against a target antigen or
epitope. These terms do not require that complete immunity be
created, but rather that an immune response be produced which is
substantially greater than baseline. For example, a mammal may be
considered to be immunized against a target antigen if the cellular
and/or humoral immune response to the target antigen occurs
following the application of methods of the invention.
[0080] Natural origin: As used herein, the term "natural origin"
means that the whole or parts thereof are not synthetic and exist
or are produced in nature.
[0081] Non-natural: As used herein, the term generally means not
from nature, more specifically, the term means from the hand of
man.
[0082] Non-natural origin: As used herein, the term "non-natural
origin" generally means synthetic or not from nature; more
specifically, the term means from the hand of man.
[0083] Ordered and repetitive antigen or antigenic determinant
array: As used herein, the term "ordered and repetitive antigen or
antigenic determinant array" generally refers to a repeating
pattern of antigen or antigenic determinant, characterized by a
typically and preferably uniform spatial arrangement of the
antigens or antigenic determinants with respect to the core
particle and virus-like particle, respectively. In one embodiment
of the invention, the repeating pattern may be a geometric pattern.
Typical and preferred examples of suitable ordered and repetitive
antigen or antigenic determinant arrays are those which possess
strictly repetitive paracrystalline orders of antigens or antigenic
determinants, preferably with spacings of 1 to 30 nanometers,
preferably 5 to 15 nanometers.
[0084] Pili: As used herein, the term "pili" (singular being
"pilus") refers to extracellular structures of bacterial cells
composed of protein monomers (e.g., pilin monomers) which are
organized into ordered and repetitive patterns. Further, pili are
structures which are involved in processes such as the attachment
of bacterial cells to host cell surface receptors, inter-cellular
genetic exchanges, and cell-cell recognition. Examples of pili
include Type-1 pili, P-pili, F1C pili, S-pili, and 987P-pili.
Additional examples of pili are set out below.
[0085] Pilus-like structure: As used herein, the phrase "pilus-like
structure" refers to structures having characteristics similar to
that of pili and composed of protein monomers. One example of a
"pilus-like structure" is a structure formed by a bacterial cell
which expresses modified pilin proteins that do not form ordered
and repetitive arrays that are identical to those of natural
pili.
[0086] Polypeptide: As used herein, the term "polypeptide" refers
to a molecule composed of monomers (amino acids) linearly linked by
amide bonds (also known as peptide bonds). It indicates a molecular
chain of amino acids and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides and
proteins are included within the definition of polypeptide. This
term is also intended to refer to post-expression modifications of
the polypeptide, for example, glycosylations, acetylations,
phosphorylations, and the like. A recombinant or derived
polypeptide is not necessarily translated from a designated nucleic
acid sequence. It may also be generated in any manner, including
chemical synthesis.
[0087] IL-5 protein: The term "IL-5 protein" as used herein refers
to a protein encoded by an IL-5 gene. Different variants of the
IL-5 protein may be caused by nucleotide point mutations and
polymorphisms, respectively, as well as insertions, deletions
and/or substitutions of one or more nucleotides, and shall be
explicitly encompassed within the scope of the present invention.
Further variability can be caused by post-translational
modifications, such as differentially glycosylated forms of IL-5 as
well as proteolytically cleaved forms of IL-5. The, term "IL-5
protein", as used herein, shall also encompass IL-5 protein
variants, including but not limiting to the above indicated
preferred examples.
[0088] IL-5 peptide: As used herein, the term "IL-5 peptide" is
broadly defined as any peptide which represents a fraction of an
IL-5 protein and containing at least two, preferably at least
three, more preferably at least four, more preferably at least
five, more preferably at least six consecutive amino acids of the
original IL-5 protein which represents part of a IL-5 protein, most
preferably representative of a folded part of IL-5 containing a B
cell epitope, and again more preferably of the part of IL-5
containing a neutralizing epitope.
[0089] The term "IL-5 peptide" shall further preferably encompass
any fraction of said IL5 peptide, wherein said fraction may be,
preferably, derived by deletion of one or more amino acids at the N
and/or C terminus of IL-5 protein. The IL-5 peptide can be obtained
by recombinant expression in eukaryotic or prokaryotic expression
systems as IL5 peptide alone or as a fusion with other amino acids
or proteins, e.g. to facilitate folding, expression or solubility
of the IL-5 peptide or to facilitate purification of the IL-5
peptide. To enable coupling of IL-5 peptides and subunit proteins
of VLP's or capsids, at least one second attachment site may be
preferably added to the IL-5 peptide. Alternatively IL-5 peptides
may be synthesized using methods known to the art. The term IL-5
peptide as used herein shall also prefereably encompass a peptide
which simulates the three dimensional surface structure of IL5.
Such IL5 peptide is not necessarily derived from a continuous amino
acid sequence of IL5, but may be formed by discontinuous amino acid
residues from IL5. Such peptides may even contain amino acids which
are not present in the corresponding IL5 protein.
[0090] IL-13 protein: The term "IL-13 protein" as used herein
refers to a protein encoded by an IL-13 gene. Different variants of
the IL-13 protein may be caused by nucleotide point mutations and
polymorphisms, respectively, as well as insertions, deletions
and/or substitutions of one or more nucleotides, and shall be
explicitly encompassed within the scope of the present invention.
Further variability can be caused by post-translational
modifications, such as differentially glycosylated forms of IL-13
as well as proteolytically cleaved forms of IL-13. The, term "IL-13
protein", as used herein, shall also encompass IL-13 protein
variants, including but not limiting to the above indicated
preferred examples.
[0091] IL-13 peptide: As used herein, the term "IL-13 peptide" is
broadly defined as any peptide which represents a fraction of an
IL-13 protein and containing at least two, preferably at least
three, more prefereably at least four, more prefereably at least
five, more prefereably at least six consecutive amino acids of the
original IL-13 protein which represents part of a IL-13 protein,
most preferably representative of a folded part of IL-13 containing
a B cell epitope, and again more preferably of the part of IL-13
containing a neutralizing epitope.
[0092] The term "IL-13 peptide" shall further preferably encompass
any fraction of said IL-13 peptide, wherein said fraction may be,
preferably, derived by deletion of one or more amino acids at the N
and/or C terminus of IL-13 protein. The IL-13 peptide can be
obtained by recombinant expression in eucaryotic or prokaryotic
expression systems as IL-13 peptide alone or as a fusion with other
amino acids or proteins, e.g. to facilitate folding, expression or
solubility of the IL-13 peptide or to facilitate purification of
the IL-13 peptide. To enable coupling of IL-13 peptides and subunit
proteins of VLP's or capsids, at least one second attachment site
may be preferably added to the IL-13 peptide. Alternatively IL-13
peptides may be synthesized using methods known to the art. The
term IL-13 peptide as used herein shall also prefereably encompass
a peptide which simulates the three dimensional surface structure
of IL-13. Such IL-13 peptide is not necessarily derived from a
continuous amino acid sequence of IL-13, but may be formed by
discontinuous amino acid residues from IL-13. Such peptides may
even contain amino acids which are not present in the corresponding
IL-13 protein.
[0093] Residue: As used herein, the term "residue" is meant to mean
a specific amino acid in a polypeptide backbone or side chain.
[0094] Self antigen: As used herein, the term "self antigen" refers
to proteins encoded by the host's DNA and products generated by
proteins or RNA encoded by the host's DNA are defined as self. In
addition, proteins that result from a combination of two or several
self-molecules or that represent a fraction of a self-molecule and
proteins that have a high homology two self-molecules as defined
above (>95%, preferably >97%, more preferably >99%) may
also be considered self.
[0095] Treatment: As used herein, the terms "treatment", "treat",
"treated" or "treating" refer to prophylaxis and/or therapy. When
used with respect to an infectious disease, for example, the term
refers to a prophylactic treatment which increases the resistance
of a subject to infection with a pathogen or, in other words,
decreases the likelihood that the subject will become infected with
the pathogen or will show signs of illness attributable to the
infection, as well as a treatment after the subject has become
infected in order to fight the infection, e.g., reduce or eliminate
the infection or prevent it from becoming worse. When used with
respect to allergic diseases with an eosinophilic component, the
term "treatment" refers to a prophylactic or therapeutic treatment
which inhibits or reduces, inter alia and preferably, allergic
inflammatory components associated with allergic eosinophilic
diseases.
[0096] Vaccine: As used herein, the term "vaccine" refers to a
formulation which contains the composition of the present invention
and which is in a form that is capable of being administered to an
animal. Typically, the vaccine comprises a conventional saline or
buffered aqueous solution medium in which the composition of the
present invention is suspended or dissolved. In this form, the
composition of the present invention can be used conveniently to
prevent, ameliorate, or otherwise treat a condition. Upon
introduction into a host, the vaccine is able to provoke an immune
response including, but not limited to, the production of
antibodies and/or cytokines and/or the activation of cytotoxic T
cells, antigen presenting cells, helper T cells, dendritic cells
and/or other cellular responses.
[0097] Optionally, the vaccine of the present invention
additionally includes an adjuvant which can be present in either a
minor or major proportion relative to the compound of the present
invention. The term "adjuvant" as used herein refers to
non-specific stimulators of the immune response or substances that
allow generation of a depot in the host which when combined with
the vaccine of the present invention provide for an even more
enhanced immune response. A variety of adjuvants can be used.
Examples include complete and incomplete Freund's adjuvant,
aluminum hydroxide and modified muramyldipeptide.
[0098] Virus-like particle (VLP): As used herein, the term
"virus-like particle" refers to a structure resembling a virus
particle. Moreover, a virus-like particle in accordance with the
invention is non replicative and noninfectious since it lacks all
or part of the viral genome, in particular the replicative and
infectious components of the viral genome. A virus-like particle in
accordance with the invention may contain nucleic acid distinct
from their genome. A typical and preferred embodiment of a
virus-like particle in accordance with the present invention is a
viral capsid such as the viral capsid of the corresponding virus,
bacteriophage, or RNA-phage. The terms "viral capsid" or "capsid",
as interchangeably used herein, refer to a macromolecular assembly
composed of viral protein subunits. Typically and preferably, the
viral protein subunits assemble into a viral capsid and capsid,
respectively, having a structure with an inherent repetitive
organization, wherein said structure is, typically, spherical or
tubular. For example, the capsids of RNA-phages or HBcAg's have a
spherical form of icosahedral symmetry. The term "capsid-like
structure" as used herein, refers to a macromolecular assembly
composed of viral protein subunits resembling the capsid morphology
in the above defined sense but deviating from the typical
symmetrical assembly while maintaining a sufficient degree of order
and repetitiveness.
[0099] Virus-like particle of a bacteriophage: As used herein, the
term "virus-like particle of a bacteriophage" refers to a
virus-like particle resembling the structure of a bacteriophage,
being non replicative and noninfectious, and lacking at least the
gene or genes encoding for the replication machinery of the
bacteriophage, and typically also lacking the gene or genes
encoding the protein or proteins responsible for viral attachment
to or entry into the host. This definition should, however, also
encompass virus-like particles of bacteriophages, in which the
aforementioned gene or genes are still present but inactive, and,
therefore, also leading to non-replicative and noninfectious
virus-like particles of a bacteriophage.
[0100] VLP of RNA phage coat protein: The capsid structure formed
from the self-assembly of 180 subunits of RNA phage coat protein
and optionally containing host RNA is referred to as a "VLP of RNA
phage coat protein". A specific example is the VLP of Q.beta. coat
protein. In this particular case, the VLP of Q.beta. coat protein
may either be assembled exclusively from Q.beta. CP subunits
(generated by expression of a Q.beta. CP gene containing, for
example, a TAA stop codon precluding any expression of the longer
A1 protein through suppression, see Kozlovska, T. M., et al.,
Intervirology 39: 9-15 (1996)), or additionally contain A1 protein
subunits in the capsid assembly.
[0101] Virus particle: The term "virus particle" as used herein
refers to the morphological form of a virus. In some virus types it
comprises a genome surrounded by a protein capsid; others have
additional structures (e.g., envelopes, tails, etc.).
[0102] One, a, or an: When the terms "one," "a," or "an" are used
in this disclosure, they mean "at least one" or "one or more,"
unless otherwise indicated.
[0103] As will be clear to those skilled in the art, certain
embodiments of the invention involve the use of recombinant nucleic
acid technologies such as cloning, polymerase chain reaction, the
purification of DNA and RNA, the expression of recombinant proteins
in prokaryotic and eukaryotic cells, etc. Such methodologies are
well known to those skilled in the art and can be conveniently
found in published laboratory methods manuals (e.g., Sambrook, J.
et al., eds., Molecular Cloning, A Laboratory Manual, 2nd. edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989); Ausubel, F. et al., eds., Current Protocols in Molecular
Biology, John H. Wiley & Sons, Inc. (1997)). Fundamental
laboratory techniques for working with tissue culture cell lines
(Celis, J., ed., Cell Biology, Academic Press, 2.sup.nd edition,
(1998)) and antibody-based technologies (Harlow, E. and Lane, D.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y. (1988); Deutscher, M. P., "Guide to
Protein Purification," Meth. Enzymol. 128, Academic Press San Diego
(1990); Scopes, R. K., Protein Purification Principles and
Practice, 3rd ed., Springer-Verlag, New York (1994)) are also
adequately described in the literature, all of which are
incorporated herein by reference.
2. COMPOSITIONS AND METHODS FOR ENHANCING AN IMMUNE RESPONSE
[0104] The disclosed invention provides compositions and methods
for enhancing an immune response against protein or peptide of
IL-5, IL-13 or eotaxin in an animal. Compositions of the invention
comprise, or alternatively consist of (a) a core particle with at
least one first attachment site; and (b) at least one antigen or
antigenic determinant with at least one second attachment site,
wherein said antigen or antigenic determinant is a protein or
peptide of IL-5, IL-13 or eotaxin, and wherein said second
attachment site being selected from the group consisting of (i) an
attachment site not naturally occurring with said antigen or
antigenic determinant; and (ii) an attachment site naturally
occurring with said antigen or antigenic determinant, wherein said
second attachment site is capable of association to said first
attachment site; and wherein said antigen or antigenic determinant
and said core particle interact through said association to form an
ordered and repetitive antigen array. More specifically,
compositions of the invention comprise, or alternatively consist
of, a virus-like particle and at least one antigen or antigenic
determinant, wherein the antigen or antigenic determinant is a
protein or peptide of IL-5, IL-13 or eotaxin, and wherein the at
least one antigen or antigenic determinant is bound to the
virus-like particle so as to form an ordered and repetitive
antigen-VLP-array. Furthermore, the invention conveniently enables
the practitioner to construct such a composition, inter alia, for
treatment and/or prophylactic prevention of allergic diseases with
an eosinophilic component.
[0105] In one embodiment, the core particle comprises a virus, a
bacterial pilus, a structure formed from bacterial pilin, a
bacteriophage, a virus-like particle, a viral capsid particle or a
recombinant form thereof. Any virus known in the art having an
ordered and repetitive coat and/or core protein structure may be
selected as a core particle of the invention; examples of suitable
viruses include Sindbis and other alphaviruses, rhabdoviruses (e.g.
vesicular stomatitis virus), picornaviruses (e.g., human rhino
virus, Aichi virus), togaviruses (e.g., rubella virus),
orthomyxoviruses (e.g., Thogoto virus, Batken virus, fowl plague
virus), polyomaviruses (e.g., polyomavirus BK, polyomavirus JC,
avian polyomavirus BFDV), parvoviruses, rotaviruses, Norwalk virus,
foot and mouth disease virus, a retrovirus, Hepatitis B virus,
Tobacco mosaic virus, Flock House Virus, and human Papillomavirus,
and preferably a RNA phage, bacteriophage Q.beta., bacteriophage
R17, bacteriophage M11, bacteriophage MX1, bacteriophage NL95,
bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage
MS2, bacteriophage f2, bacteriophage PP7 (for example, see Table 1
in Bachmann, M. F. and Zinkernagel, R. M., Immunol. Today
17:553-558 (1996)).
[0106] In a further embodiment, the invention utilizes genetic
engineering of a virus to create a fusion between an ordered and
repetitive viral envelope protein and a first attachment site
comprising a heterologous protein, peptide, antigenic determinant
or a reactive amino acid residue of choice. Other genetic
manipulations known to those in the art may be included in the
construction of the inventive compositions; for example, it may be
desirable to restrict the replication ability of the recombinant
virus through genetic mutation. Furthermore, the virus used for the
present invention is replication incompetent due to chemical or
physical inactivation or, as indicated, due to lack of a
replication competent genome. The viral protein selected for fusion
to the first attachment site should have an organized and
repetitive structure. Such an organized and repetitive structure
includes paracrystalline organizations with a spacing of 5-30 nm,
preferably 5-15 nm, on the surface of the virus. The creation of
this type of fusion protein will result in multiple, ordered and
repetitive first attachment sites on the surface of the virus and
reflect the normal organization of the native viral protein. As
will be understood by those in the art, the first attachment site
may be or be a part of any suitable protein, polypeptide, sugar,
polynucleotide, peptide (amino acid), natural or synthetic polymer,
a secondary metabolite or combination thereof that may serve to
specifically attach the antigen or antigenic determinant leading an
ordered and repetitive antigen array.
[0107] In another embodiment of the invention, the core particle is
a recombinant alphavirus, and more specifically, a recombinant
Sindbis virus. Alphaviruses are positive stranded RNA viruses that
replicate their genomic RNA entirely in the cytoplasm of the
infected cell and without a DNA intermediate (Strauss, J. and
Strauss, E., Microbiol. Rev. 58:491-562 (1994)). Several members of
the alphavirus family, Sindbis (Xiong, C. et al., Science
243:1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11:18-22
(1993)), Semliki Forest Virus (SFV) (Liljestrom, P. & Garoff,
H., Bio/Technology 9:1356-1361 (1991)) and others (Davis, N. L. et
al., Virology 171:189-204 (1989)), have received considerable
attention for use as virus-based expression vectors for a variety
of different proteins (Lundstrom, K., Curr. Opin. Biotechnol.
8:578-582 (1997); Liljestrom, P., Curr. Opin. Biotechnol. 5:495-500
(1994)) and as candidates for vaccine development. Recently, a
number of patents have issued directed to the use of alphaviruses
for the expression of heterologous proteins and the development of
vaccines (see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026;
5,789,245 and 5,814,482). The construction of the alphaviral core
particles of the invention may be done by means generally known in
the art of recombinant DNA technology, as described by the
aforementioned articles, which are incorporated herein by
reference.
[0108] A variety of different recombinant host cells can be
utilized to produce a viral-based core particle for antigen or
antigenic determinant attachment. For example, alphaviruses are
known to have a wide host range; Sindbis virus infects cultured
mammalian, reptilian, and amphibian cells, as well as some insect
cells (Clark, H., J. Natl. Cancer Inst. 51:645 (1973); Leake, C.,
J. Gen. Virol. 35:335 (1977); Stollar, V. in THE TOGAVIRUSES, R. W.
Schlesinger, Ed., Academic Press, (1980), pp. 583-621). Thus,
numerous recombinant host cells can be used in the practice of the
invention. BHK, COS, Vero, HeLa and CHO cells are particularly
suitable for the production of heterologous proteins because they
have the potential to glycosylate heterologous proteins in a manner
similar to human cells (Watson, E. et al., Glycobiology 4:227,
(1994)) and can be selected (Zang, M. et al., Bio/Technology 13:389
(1995)) or genetically engineered (Renner W. et al., Biotech.
Bioeng. 4:476 (1995); Lee K. et al. Biotech. Bioeng. 50:336 (1996))
to grow in serum-free medium, as well as in suspension.
[0109] Introduction of the polynucleotide vectors into host cells
can be effected by methods described in standard laboratory manuals
(see, e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, A
LABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; Ausubel, F. et
al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley
& Sons, Inc. (1997), Chapter 16), including methods such as
electroporation, DEAE-dextran mediated transfection, transfection,
microinjection, cationic lipid-mediated transfection, transduction,
scrape loading, ballistic introduction, and infection. Methods for
the introduction of exogenous DNA sequences into host cells are
discussed in Felgner, P. et al., U.S. Pat. No. 5,580,859.
[0110] Packaged RNA sequences can also be used to infect host
cells. These packaged RNA sequences can be introduced to host cells
by adding them to the culture medium. For example, the preparation
of non-infective alphaviral particles is described in a number of
sources, including "Sindbis Expression System", Version C
(Invitrogen Catalog No. K750-1).
[0111] When mammalian cells are used as recombinant host cells for
the production of viral-based core particles, these cells will
generally be grown in tissue culture. Methods for growing cells in
culture are well known in the art (see, e.g., Celis, J., ed., CELL
BIOLOGY, Academic Press, 2.sup.nd edition, (1998); Sambrook, J. et
al., eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989); Ausubel, F. et al., eds., CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John H. Wiley & Sons, Inc. (1997); Freshney, R.,
CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc. (1983)).
[0112] Further examples of RNA viruses suitable for use as core
particle in the present invention include, but are not limited to,
the following: members of the family Reoviridae, including the
genus Orthoreovirus (multiple serotypes of both mammalian and avian
retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee
virus, Kemerovo virus, African horse sickness virus, and Colorado
Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska
calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or
ovine rotavirus, avian rotavirus); the family Picornaviridae,
including the genus Enterovirus (poliovirus, Coxsackie virus A and
B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A, C,
D, E and G viruses, Simian enteroviruses, Murine encephalomyelitis
(ME) viruses, Poliovirus muris, Bovine enteroviruses, Porcine
enteroviruses, the genus Cardiovirus (Encephalomyocarditis virus
(EMC), Mengovirus), the genus Rhinovirus (Human rhinoviruses
including at least 113 subtypes; other rhinoviruses), the genus
Apthovirus (Foot and Mouth disease (FMDV); the family
Caliciviridae, including Vesicular exanthema of swine virus, San
Miguel sea lion virus, Feline picornavirus and Norwalk virus; the
family Togaviridae, including the genus Alphavirus (Eastern equine
encephalitis virus, Semliki forest virus, Sindbis virus,
Chikungunya virus, O'Nyong-Nyong virus, Ross river virus,
Venezuelan equine encephalitis virus, Western equine encephalitis
virus), the genus Flavirius (Mosquito borne yellow fever virus,
Dengue virus, Japanese encephalitis virus, St. Louis encephalitis
virus, Murray Valley encephalitis virus, West Nile virus, Kunjin
virus, Central European tick borne virus, Far Eastern tick borne
virus, Kyasanur forest virus, Louping III virus, Powassan virus,
Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus),
the genus Pestivirus (Mucosal disease virus, Hog cholera virus,
Border disease virus); the family Bunyaviridae, including the genus
Bunyvirus (Bunyamwera and related viruses, California encephalitis
group viruses), the genus Phlebovirus (Sandfly fever Sicilian
virus, Rift Valley fever virus), the genus Nairovirus
(Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease
virus), and the genus Uukuvirus (Uukuniemi and related viruses);
the family Orthomyxoviridae, including the genus Influenza virus
(Influenza virus type A, many human subtypes); Swine influenza
virus, and Avian and Equine Influenza viruses; influenza type B
(many human subtypes), and influenza type C (possible separate
genus); the family paramyxoviridae, including the genus
Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute schlerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); forest virus, Sindbis virus, Chikungunya virus,
O'Nyong-Nyong virus, Ross river virus, Venezuelan equine
encephalitis virus, Western equine encephalitis virus), the genus
Flavirius (Mosquito borne yellow fever virus, Dengue virus,
Japanese encephalitis virus, St. Louis encephalitis virus, Murray
Valley encephalitis virus, West Nile virus, Kunjin virus, Central
European tick borne virus, Far Eastern tick borne virus, Kyasanur
forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic
fever virus), the genus Rubivirus (Rubella virus), the genus
Pestivirus (Mucosal disease virus, Hog cholera virus, Border
disease virus); the family Bunyaviridae, including the genus
Bunyvirus (Bunyamwera and related viruses, California encephalitis
group viruses), the genus Phlebovirus (Sandfly fever Sicilian
virus, Rift Valley fever virus), the genus Nairovirus
(Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease
virus), and the genus Uukuvirus (Uukuniemi and related viruses);
the family Orthomyxoviridae, including the genus Influenza virus
(Influenza virus type A, many human subtypes); Swine influenza
virus, and Avian and Equine Influenza viruses; influenza type B
(many human subtypes), and influenza type C (possible separate
genus); the family paramyxoviridae, including the genus
Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute schlerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the genus Lyssavirus (Rabies virus), fish Rhabdoviruses and,
filoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0113] Illustrative DNA viruses that may be used as core particles
include, but are not limited to: the family Poxyiridae, including
the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox
Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus
Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox,
other avian poxvirus), the genus Capripoxvirus (sheep pox, goat
pox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus
(contagious postular dermatitis virus, pseudocowpox, bovine papular
stomatitis virus); the family Iridoviridae (African swine fever
virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the
family Herpesviridae, including the alpha-Herpesviruses (Herpes
Simplex Types 1 and 2, Varicella-Zoster, Equine abortion virus,
Equine herpes virus 2 and 3, pseudorabies virus, infectious bovine
keratoconjunctivitis virus, infectious bovine rhinotracheitis
virus, feline rhinotracheitis virus, infectious laryngotracheitis
virus) the Beta-herpesviruses (Human cytomegalovirus and
cytomegaloviruses of swine, monkeys and rodents); the
gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease
virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus,
guinea pig herpes virus, Lucke tumor virus); the family
Adenoviridae, including the genus Mastadenovirus (Human subgroups
A, B, C, D and E and ungrouped; simian adenoviruses (at least 23
serotypes), infectious canine hepatitis, and adenoviruses of
cattle, pigs, sheep, frogs and many other species, the genus
Aviadenovirus (Avian adenoviruses); and non-cultivatable
adenoviruses; the family Papoviridae, including the genus
Papillomavirus (Human papilloma viruses, bovine papilloma viruses,
Shope rabbit papilloma virus, and various pathogenic papilloma
viruses of other species), the genus Polyomavirus (polyomavirus,
Simian vacuolating agent (SV-40), Rabbit vacuolating agent (RKV), K
virus, BK virus, JC virus, and other primate polyoma viruses such
as Lymphotrophic papilloma virus); the family Parvoviridae
including the genus Adeno-associated viruses, the genus Parvovirus
(Feline panleukopenia virus, bovine parvovirus, canine parvovirus,
Aleutian mink disease virus, etc.). Finally, DNA viruses may
include viruses such as chronic infectious neuropathic agents
(CHINA virus).
[0114] In other embodiments, a bacterial pilin, a subportion of a
bacterial pilin, or a fusion protein which contains either a
bacterial pilin or subportion thereof is used to prepare
compositions and vaccine compositions, respectively, of the
invention. Examples of pilin proteins include pilins produced by
Escherichia coli, Haemophilus influenzae, Neisseria meningitidis,
Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas
stutzeri, and Pseudomonas aeruginosa. The amino acid sequences of
pilin proteins suitable for use with the present invention include
those set out in GenBank reports AJ000636 (SEQ ID NO:1), AJ132364
(SEQ ID NO:2), AF229646 (SEQ ID NO:3), AF051814 (SEQ ID NO:4),
AF051815 (SEQ ID NO:5), and X00981 (SEQ ID NO:6), the entire
disclosures of which are incorporated herein by reference.
[0115] Bacterial pilin proteins are generally processed to remove
N-terminal leader sequences prior to export of the proteins into
the bacterial periplasm. Further, as one skilled in the art would
recognize, bacterial pilin proteins used to prepare compositions
and vaccine compositions, respectively, of the invention will
generally not have the naturally present leader sequence.
[0116] One specific example of a pilin protein suitable for use in
the present invention is the P-pilin of E. coli (GenBank report
AF237482 (SEQ ID NO:7)). An example of a Type-1 E. coli pilin
suitable for use with the invention is a pilin having the amino
acid sequence set out in GenBank report P04128 (SEQ ID NO:8), which
is encoded by nucleic acid having the nucleotide sequence set out
in GenBank report M27603 (SEQ ID NO:9). The entire disclosures of
these GenBank reports are incorporated herein by reference. Again,
the mature form of the above referenced protein would generally be
used to prepare compositions and vaccine compositions,
respectively, of the invention.
[0117] Bacterial pilins or pilin subportions suitable for use in
the practice of the present invention will generally be able to
associate to form ordered and repetitive antigen arrays.
[0118] Methods for preparing pili and pilus-like structures in
vitro are known in the art. Bullitt et al., Proc. Natl. Acad. Sci.
USA 93:12890-12895 (1996), for example, describe the in vitro
reconstitution of E. coli P-pili subunits. Furthermore, Eshdat et
al., J. Bacteriol. 148:308-314 (1981) describe methods suitable for
dissociating Type-1 pili of E. coli and the reconstitution of pili.
In brief, these methods are as follows: pili are dissociated by
incubation at 37.degree. C. in saturated guanidine hydrochloride.
Pilin proteins are then purified by chromatography, after which
pilin dimers are formed by dialysis against 5 mM
tris(hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et
al. also found that pilin dimers reassemble to form pili upon
dialysis against the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0)
containing 5 mM MgCl.sub.2.
[0119] Further, using, for example, conventional genetic
engineering and protein modification methods, pilin proteins may be
modified to contain a first attachment site to which an antigen or
antigenic determinant is linked through a second attachment site.
Alternatively, antigens or antigenic determinants can be directly
linked through a second attachment site to amino acid residues
which are naturally resident in these proteins. These modified
pilin proteins may then be used in vaccine compositions of the
invention.
[0120] Bacterial pilin proteins used to prepare compositions and
vaccine compositions, respectively, of the invention may be
modified in a manner similar to that described herein for HBcAg.
For example, cysteine and lysine residues may be either deleted or
substituted with other amino acid residues and first attachment
sites may be added to these proteins. Further, pilin proteins may
either be expressed in modified form or may be chemically modified
after expression. Similarly, intact pili may be harvested from
bacteria and then modified chemically.
[0121] In another embodiment, pili or pilus-like structures are
harvested from bacteria (e.g., E. coli) and used to form
compositions and vaccine compositions of the invention. One example
of pili suitable for preparing compositions and vaccine
compositions is the Type-1 pilus of E. coli, which is formed from
pilin monomers having the amino acid sequence set out in SEQ ID
NO:8.
[0122] A number of methods for harvesting bacterial pili are known
in the art. Bullitt and Makowski (Biophys. J. 74:623-632 (1998)),
for example, describe a pilus purification method for harvesting
P-pili from E. coli. According to this method, pili are sheared
from hyperpiliated E. coli containing a P-pilus plasmid and
purified by cycles of solubilization and MgCl.sub.2 (1.0 M)
precipitation.
[0123] Once harvested, pili or pilus-like structures may be
modified in a variety of ways. For example, a first attachment site
can be added to the pili to which antigens or antigen determinants
may be attached through a second attachment site. In other words,
bacterial pili or pilus-like structures can be harvested and
modified to lead to ordered and repetitive antigen arrays.
[0124] Antigens or antigenic determinants could be linked to
naturally occurring cysteine resides or lysine residues present in
Pili or pilus-like structures. In such instances, the high order
and repetitiveness of a naturally occurring amino acid residue
would guide the coupling of the antigens or antigenic determinants
to the pili or pilus-like structures. For example, the pili or
pilus-like structures could be linked to the second attachment
sites of the antigens or antigenic determinants using a
heterobifunctional cross-linking agent.
[0125] When structures which are naturally synthesized by organisms
(e.g., pili) are used to prepare compositions and vaccine
compositions of the invention, it will often be advantageous to
genetically engineer these organisms so that they produce
structures having desirable characteristics. For example, when
Type-1 pili of E. coli are used, the E. coli from which these pili
are harvested may be modified so as to produce structures with
specific characteristics. Examples of possible modifications of
pilin proteins include the insertion of one or more lysine
residues, the deletion or substitution of one or more of the
naturally resident lysine residues, and the deletion or
substitution of one or more naturally resident cysteine residues
(e.g., the cysteine residues at positions 44 and 84 in SEQ ID
NO:8).
[0126] Further, additional modifications can be made to pilin genes
which result in the expression products containing a first
attachment site other than a lysine residue (e.g., a FOS or JUN
domain). Of course, suitable first attachment sites will generally
be limited to those which do not prevent pilin proteins from
forming pili or pilus-like structures suitable for use in vaccine
compositions of the invention.
[0127] Pilin genes which naturally reside in bacterial cells can be
modified in vivo (e.g., by homologous recombination) or pilin genes
with particular characteristics can be inserted into these cells.
For examples, pilin genes could be introduced into bacterial cells
as a component of either a replicable cloning vector or a vector
which inserts into the bacterial chromosome. The inserted pilin
genes may also be linked to expression regulatory control sequences
(e.g., a lac operator).
[0128] In most instances, the pili or pilus-like structures used in
compositions and vaccine compositions, respectively, of the
invention will be composed of single type of a pilin subunit. Pili
or pilus-like structures composed of identical subunits will
generally be used because they are expected to form structures
which present highly ordered and repetitive antigen arrays.
[0129] However, the compositions of the invention also include
compositions and vaccines comprising pili or pilus-like structures
formed from heterogenous pilin subunits. The pilin subunits which
form these pili or pilus-like structures can be expressed from
genes naturally resident in the bacterial cell or may be introduced
into the cells. When a naturally resident pilin gene and an
introduced gene are both expressed in a cell which forms pili or
pilus-like structures, the result will generally be structures
formed from a mixture of these pilin proteins. Further, when two or
more pilin genes are expressed in a bacterial cell, the relative
expression of each pilin gene will typically be the factor which
determines the ratio of the different pilin subunits in the pili or
pilus-like structures.
[0130] When pili or pilus-like structures having a particular
composition of mixed pilin subunits is desired, the expression of
at least one of the pilin genes can be regulated by a heterologous,
inducible promoter. Such promoters, as well as other genetic
elements, can be used to regulate the relative amounts of different
pilin subunits produced in the bacterial cell and, hence, the
composition of the pili or pilus-like structures.
[0131] In additional, the antigen or antigenic determinant can be
linked to bacterial pili or pilus-like structures by a bond which
is not a peptide bond, bacterial cells which produce pili or
pilus-like structures used in the compositions of the invention can
be genetically engineered to generate pilin proteins which are
fused to an antigen or antigenic determinant. Such fusion proteins
which form pili or pilus-like structures are suitable for use in
vaccine compositions of the invention.
[0132] Virus-like particles in the context of the present
application refer to structures resembling a virus particle but
which are not pathogenic. In general, virus-like particles lack the
viral genome and, therefore, are noninfectious. Also, virus-like
particles can be produced in large quantities by heterologous
expression and can be easily purified.
[0133] In a preferred embodiment, the virus-like particle is a
recombinant virus-like particle. The skilled artisan can produce
VLPs using recombinant DNA technology and virus coding sequences
which are readily available to the public. For example, the coding
sequence of a virus envelope or core protein can be engineered for
expression in a baculovirus expression vector using a commercially
available baculovirus vector, under the regulatory control of a
virus promoter, with appropriate modifications of the sequence to
allow functional linkage of the coding sequence to the regulatory
sequence. The coding sequence of a virus envelope or core protein
can also be engineered for expression in a bacterial expression
vector, for example.
[0134] Examples of VLPs include, but are not limited to, the capsid
proteins of Hepatitis B virus (Ulrich, et al., Virus Res.
50:141-182 (1998)), measles virus (Warnes, et al., Gene 160:173-178
(1995)), Sindbis virus, rotavirus (U.S. Pat. No. 5,071,651 and U.S.
Pat. No. 5,374,426), foot-and-mouth-disease virus (Twomey, et al.,
Vaccine 13:1603-1610, (1995)), Norwalk virus (Jiang, X., et al.,
Science 250:1580-1583 (1990); Matsui, S. M., et al., J. Clin.
Invest. 87:1456-1461 (1991)), the retroviral GAG protein (WO
96/30523), the retrotransposon Ty protein p1, the surface protein
of Hepatitis B virus (WO 92/11291), human papilloma virus (WO
98/15631), RNA phages, Ty, fr-phage, GA-phage and
Q.beta.-phage.
[0135] As will be readily apparent to those skilled in the art, the
VLP of the invention is not limited to any specific form. The
particle can be synthesized chemically or through a biological
process, which can be natural or non-natural. By way of example,
this type of embodiment includes a virus-like particle or a
recombinant form thereof.
[0136] In a more specific embodiment, the VLP can comprise, or
alternatively essentially consist of, or alternatively consist of
recombinant polypeptides, or fragments thereof, being selected from
recombinant polypeptides of Rotavirus, recombinant polypeptides of
Norwalk virus, recombinant polypeptides of Alphavirus, recombinant
polypeptides of Foot and Mouth Disease virus, recombinant
polypeptides of measles virus, recombinant polypeptides of Sindbis
virus, recombinant polypeptides of Polyoma virus, recombinant
polypeptides of Retrovirus, recombinant polypeptides of Hepatitis B
virus (e.g., a HBcAg), recombinant polypeptides of Tobacco mosaic
virus, recombinant polypeptides of Flock House Virus, recombinant
polypeptides of human Papillomavirus, recombinant polypeptides of
bacteriophages, recombinant polypeptides of RNA phages, recombinant
polypeptides of Ty, recombinant polypeptides of fr-phage,
recombinant polypeptides of GA-phage and recombinant polypeptides
of Q.beta.-phage. The virus-like particle can further comprise, or
alternatively essentially consist of, or alternatively consist of,
one or more fragments of such polypeptides, as well as variants of
such polypeptides. Variants of polypeptides can share, for example,
at least 80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid
level with their wild-type counterparts.
[0137] In a preferred embodiment, the virus-like particle
comprises, consists essentially of, or alternatively consists of
recombinant proteins, or fragments thereof, of a RNA-phage.
Preferably, the RNA-phage is selected from the group consisting of
a) bacteriophage Q.beta.; b) bacteriophage R17; c) bacteriophage
fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2;
g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95;
k) bacteriophage f2; and 1) bacteriophage PP7.
[0138] In another preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of, or alternatively consists of recombinant proteins,
or fragments thereof, of the RNA-bacteriophage Q.beta. or of the
RNA-bacteriophage fr.
[0139] In a further preferred embodiment of the present invention,
the recombinant proteins comprise, or alternatively consist
essentially of, or alternatively consist of coat proteins of RNA
phages.
[0140] RNA-phage coat proteins forming capsids or VLP's, or
fragments of the bacteriophage coat proteins compatible with
self-assembly into a capsid or a VLP, are, therefore, further
preferred embodiments of the present invention. Bacteriophage
Q.beta. coat proteins, for example, can be expressed recombinantly
in E. coli. Further, upon such expression these proteins
spontaneously form capsids. Additionally, these capsids form a
structure with an inherent repetitive organization.
[0141] Specific preferred examples of bacteriophage coat proteins
which can be used to prepare compositions of the invention include
the coat proteins of RNA bacteriophages such as bacteriophage
Q.beta. (SEQ ID NO:10; PIR Database, Accession No. VCBPQ.beta.
referring to Q.beta. CP and SEQ ID NO: 11; Accession No. AAA16663
referring to Q.beta. A1 protein), bacteriophage R17 (SEQ ID NO:12;
PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO:13; PIR
Accession No. VCBPFR), bacteriophage GA (SEQ ID NO:14; GenBank
Accession No. NP-040754), bacteriophage SP (SEQ ID NO:15; GenBank
Accession No. CAA30374 referring to SP CP and SEQ ID NO: 16;
Accession No. referring to SP A1 protein), bacteriophage MS2 (SEQ
ID NO:17; PIR Accession No. VCBPM2), bacteriophage M11 (SEQ ID
NO:18; GenBank Accession No. AAC06250), bacteriophage Mx1 (SEQ ID
NO:19; GenBank Accession No. AAC14699), bacteriophage NL95 (SEQ ID
NO:20; GenBank Accession No. AAC14704), bacteriophage f2 (SEQ ID
NO: 21; GenBank Accession No. P03611), bacteriophage PP7 (SEQ ID
NO: 22). Furthermore, the A1 protein of bacteriophage Q.beta. or
C-terminal truncated forms missing as much as 100, 150 or 180 amino
acids from its C-terminus may be incorporated in a capsid assembly
of Q.beta. coat proteins. Generally, the percentage of Q.beta. A1
protein relative to Q.beta. CP in the capsid assembly will be
limited, in order to ensure capsid formation.
[0142] Q.beta. coat protein has also been found to self-assemble
into capsids when expressed in E. coli (Kozlovska T M. et al., GENE
137: 133-137 (1993)). The obtained capsids or virus-like particles
showed an icosahedral phage-like capsid structure with a diameter
of 25 nm and T=3 quasi symmetry. Further, the crystal structure of
phage Q.beta. has been solved. The capsid contains 180 copies of
the coat protein, which are linked in covalent pentamers and
hexamers by disulfide bridges (Golmohammadi, R. et al., Structure
4: 543-5554 (1996)) leading to a remarkable stability of the capsid
of Q.beta. coat protein. Capsids or VLP's made from recombinant
Q.beta. coat protein may contain, however, subunits not linked via
disulfide links to other subunits within the capsid, or
incompletely linked. Thus, upon loading recombinant Q.beta. capsid
on non-reducing SDS-PAGE, bands corresponding to monomeric Q.beta.
coat protein as well as bands corresponding to the hexamer or
pentamer of Q.beta. coat protein are visible. Incompletely
disulfide-linked subunits could appear as dimer, trimer or even
tetramer band in non-reducing SDS-PAGE. Q.beta. capsid protein also
shows unusual resistance to organic solvents and denaturing agents.
Surprisingly, we have observed that DMSO and acetonitrile
concentrations as high as 30%, and Guanidinium concentrations as
high as 1 M do not affect the stability of the capsid. The high
stability of the capsid of Q.beta. coat protein is an advantageous
feature, in particular, for its use in immunization and vaccination
of mammals and humans in accordance of the present invention.
[0143] Upon expression in E. coli, the N-terminal methionine of
Q.beta. coat protein is usually removed, as we observed by
N-terminal Edman sequencing as described in Stoll, E. et al. J.
Biol. Chem. 252:990-993 (1977). VLP composed from Q.beta. coat
proteins where the N-terminal methionine has not been removed, or
VLPs comprising a mixture of Q.beta. coat proteins where the
N-terminal methionine is either cleaved or present are also within
the scope of the present invention.
[0144] Further RNA phage coat proteins have also been shown to
self-assemble upon expression in a bacterial host (Kastelein, R A.
et al., Gene 23: 245-254 (1983), Kozlovskaya, T M. et al., Dokl.
Akad. Nauk SSSR 287: 452-455 (1986), Adhin, M R. et al., Virology
170: 238-242 (1989), Ni, C Z., et al., Protein Sci. 5: 2485-2493
(1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). The
Q.beta. phage capsid contains, in addition to the coat protein, the
so called read-through protein A1 and the maturation protein A2. A1
is generated by suppression at the UGA stop codon and has a length
of 329 aa. The capsid of phage Q.beta. recombinant coat protein
used in the invention is devoid of the A2 lysis protein, and
contains RNA from the host. The coat protein of RNA phages is an
RNA binding protein, and interacts with the stem loop of the
ribosomal binding site of the replicase gene acting as a
translational repressor during the life cycle of the virus. The
sequence and structural elements of the interaction are known
(Witherell, G W. & Uhlenbeck, O C. Biochemistry 28: 71-76
(1989); Lim F. et al., J. Biol. Chem. 271: 31839-31845 (1996)). The
stem loop and RNA in general are known to be involved in the virus
assembly (Golmohammadi, R. et al., Structure 4: 543-5554
(1996)).
[0145] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of, or alternatively consists of recombinant proteins,
or fragments thereof, of a RNA-phage, wherein the recombinant
proteins comprise, consist essentially of or alternatively consist
of mutant coat proteins of a RNA phage, preferably of mutant coat
proteins of the RNA phages mentioned above. In another preferred
embodiment, the mutant coat proteins of the RNA phage have been
modified by removal of at least one lysine residue by way of
substitution, or by addition of at least one lysine residue by way
of substitution; alternatively, the mutant coat proteins of the RNA
phage have been modified by deletion of at least one lysine
residue, or by addition of at least one lysine residue by way of
insertion.
[0146] In another preferred embodiment, the virus-like particle
comprises, or alternatively consists essentially of, or
alternatively consists of recombinant proteins, or fragments
thereof, of the RNA-bacteriophage Q.beta., wherein the recombinant
proteins comprise, or alternatively consist essentially of, or
alternatively consist of coat proteins having an amino acid
sequence of SEQ ID NO:10, or a mixture of coat proteins having
amino acid sequences of SEQ ID NO:10 and of SEQ ID NO: 11 or
mutants of SEQ ID NO: 11 and wherein the N-terminal methionine is
preferably cleaved.
[0147] In a further preferred embodiment of the present invention,
the virus-like particle comprises, consists essentially of or
alternatively consists of recombinant proteins of Q.beta., or
fragments thereof, wherein the recombinant proteins comprise, or
alternatively consist essentially of, or alternatively consist of
mutant Q.beta. coat proteins. In another preferred embodiment,
these mutant coat proteins have been modified by removal of at
least one lysine residue by way of substitution, or by addition of
at least one lysine residue by way of substitution. Alternatively,
these mutant coat proteins have been modified by deletion of at
least one lysine residue, or by addition of at least one lysine
residue by way of insertion.
[0148] Four lysine residues are exposed on the surface of the
capsid of Q.beta. coat protein. Q.beta. mutants, for which exposed
lysine residues are replaced by arginines can also be used for the
present invention. The following Q.beta. coat protein mutants and
mutant Q.beta. VLP's can, thus, be used in the practice of the
invention: "Q.beta.-240" (Lys13-Arg; SEQ ID NO:23), "Q.beta.-243"
(Asn 10-Lys; SEQ ID NO:24), "Q.beta.-250" (Lys 2-Arg, Lys13-Arg;
SEQ ID NO:25), "Q.beta.-251" (SEQ ID NO:26) and "Q.beta.-259" (Lys
2-Arg, Lys16-Arg; SEQ ID NO:27). Thus, in further preferred
embodiment of the present invention, the virus-like particle
comprises, consists essentially of or alternatively consists of
recombinant proteins of mutant Q.beta. coat proteins, which
comprise proteins having an amino acid sequence selected from the
group of a) the amino acid sequence of SEQ ID NO:23; b) the amino
acid sequence of SEQ ID NO:24; c) the amino acid sequence of SEQ ID
NO:25; d) the amino acid sequence of SEQ ID NO:26; and e) the amino
acid sequence of SEQ ID NO:27. The construction, expression and
purification of the above indicated Q.beta. coat proteins, mutant
Q.beta. coat protein VLP's and capsids, respectively, are disclosed
in pending U.S. application Ser. No. 10/050,902 filed by the
present assignee on Jan. 18, 2002. In particular is hereby referred
to Example 18 of above mentioned application.
[0149] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively consists
essentially of, or alternatively consists of recombinant proteins
of Q.beta., or fragments thereof, wherein the recombinant proteins
comprise, consist essentially of or alternatively consist of a
mixture of either one of the foregoing Q.beta. mutants and the
corresponding A1 protein.
[0150] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of recombinant proteins, or
fragments thereof, of RNA-phage AP205.
[0151] The AP205 genome consists of a maturation protein, a coat
protein, a replicase and two open reading frames not present in
related phages; a lysis gene and an open reading frame playing a
role in the translation of the maturation gene (Klovins, J., et
al., J. Gen. Virol. 83: 1523-33 (2002)). AP205 coat protein can be
expressed from plasmid pAP283-58 (SEQ ID NO: 79), which is a
derivative of pQb10 (Kozlovska, T. M. et al., Gene 137:133-37
(1993)), and which contains an AP205 ribosomal binding site.
Alternatively, AP205 coat protein may be cloned into pQb 185,
downstream of the ribosomal binding site present in the vector.
Both approaches lead to expression of the protein and formation of
capsids as described in the co-pending US provisional patent
application with the title "Molecular Antigen Arrays" and having
filed by the present assignee on Jul. 16, 2002, which is
incorporated by reference in its entirety. Vectors pQb10 and pQb185
are vectors derived from pGEM vector, and expression of the cloned
genes in these vectors is controlled by the trp promoter
(Kozlovska, T. M. et al., Gene 137:133-37 (1993)). Plasmid
pAP283-58 (SEQ ID NO:79) comprises a putative AP205 ribosomal
binding site in the following sequence, which is downstream of the
XbaI site, and immediately upstream of the ATG start codon of the
AP205 coat protein: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAA
GTGAGGAAAATCACatg (bases 77-133 of SEQ ID NO:94). The vector pQb185
comprises a Shine Delagarno sequence downstream from the XbaI site
and upstream of the start codon (tctagaTTAACCCAACGCGTAGGAG
TCAGGCCatg (SEQ ID NO:343), Shine Delagarno sequence
underlined).
[0152] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of recombinant coat
proteins, or fragments thereof, of the RNA-phage AP205.
[0153] This preferred embodiment of the present invention, thus,
comprises AP205 coat proteins that form capsids. Such proteins are
recombinantly expressed, or prepared from natural sources. AP205
coat proteins produced in bacteria spontaneously form capsids, as
evidenced by Electron Microscopy (EM) and immunodiffusion. The
structural properties of the capsid formed by the AP205 coat
protein (SEQ ID NO: 80) and those formed by the coat protein of the
AP205 RNA phage are nearly indistinguishable when seen in EM. AP205
VLPs are highly immunogenic, and can be linked with antigens and/or
antigenic determinants to generate vaccine constructs displaying
the antigens and/or antigenic determinants oriented in a repetitive
manner. High titers are elicited against the so displayed antigens
showing that bound antigens and/or antigenic determinants are
accessible for interacting with antibody molecules and are
immunogenic.
[0154] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of recombinant mutant coat
proteins, or fragments thereof, of the RNA-phage AP205.
[0155] Assembly-competent mutant forms of AP205 VLPs, including
AP205 coat protein with the substitution of proline at amino acid 5
to threonine (SEQ ID NO: 81), may also be used in the practice of
the invention and leads to a further preferred embodiment of the
invention. These VLPs, AP205 VLPs derived from natural sources, or
AP205 viral particles, may be bound to antigens to produce ordered
repetitive arrays of the antigens in accordance with the present
invention.
[0156] AP205 P5-T mutant coat protein can be expressed from plasmid
pAP281-32 (SEQ ID No. 82), which is derived directly from pQb185,
and which contains the mutant AP205 coat protein gene instead of
the Q.beta. coat protein gene. Vectors for expression of the AP205
coat protein are transfected into E. coli for expression of the
AP205 coat protein.
[0157] Methods for expression of the coat protein and the mutant
coat protein, respectively, leading to self-assembly into VLPs are
described in co-pending US provisional patent application with the
title "Molecular Antigen Arrays" and having filed by the present
assignee on Jul. 16, 2002, which is incorporated by reference in
its entirety. Suitable E. coli strains include, but are not limited
to, E. coli K802, JM 109, RR1. Suitable vectors and strains and
combinations thereof can be identified by testing expression of the
coat protein and mutant coat protein, respectively, by SDS-PAGE and
capsid formation and assembly by optionally first purifying the
capsids by gel filtration and subsequently testing them in an
immunodiffusion assay (Ouchterlony test) or Electron Microscopy
(Kozlovska, T. M. et al., Gene 137:133-37 (1993)).
[0158] AP205 coat proteins expressed from the vectors pAP283-58 and
pAP281-32 may be devoid of the initial Methionine amino-acid, due
to processing in the cytoplasm of E. coli. Cleaved, uncleaved forms
of AP205 VLP, or mixtures thereof are further preferred embodiments
of the invention.
[0159] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of a mixture of recombinant
coat proteins, or fragments thereof, of the RNA-phage AP205 and of
recombinant mutant coat proteins, or fragments thereof, of the
RNA-phage AP205.
[0160] In a further preferred embodiment of the present invention,
the virus-like particle comprises, or alternatively essentially
consists of, or alternatively consists of fragments of recombinant
coat proteins or recombinant mutant coat proteins of the RNA-phage
AP205.
[0161] Recombinant AP205 coat protein fragments capable of
assembling into a VLP and a capsid, respectively are also useful in
the practice of the invention. These fragments may be generated by
deletion, either internally or at the termini of the coat protein
and mutant coat protein, respectively. Insertions in the coat
protein and mutant coat protein sequence or fusions of antigen
sequences to the coat protein and mutant coat protein sequence, and
compatible with assembly into a VLP, are further embodiments of the
invention and lead to chimeric AP205 coat proteins, and particles,
respectively. The outcome of insertions, deletions and fusions to
the coat protein sequence and whether it is compatible with
assembly into a VLP can be determined by electron microscopy.
[0162] The particles formed by the AP205 coat protein, coat protein
fragments and chimeric coat proteins described above, can be
isolated in pure form by a combination of fractionation steps by
precipitation and of purification steps by gel filtration using
e.g. Sepharose CL-4B, Sepharose CL-2B, Sepharose CL-6B columns and
combinations thereof as described in the co-pending US provisional
patent application with the title "Molecular Antigen Arrays" and
having filed by the present assignee on Jul. 16, 2002, which is
incorporated by reference in its entirety. Other methods of
isolating virus-like particles are known in the art, and may be
used to isolate the virus-like particles (VLPs) of bacteriophage
AP205. For example, the use of ultracentrifugation to isolate VLPs
of the yeast retrotransposon Ty is described in U.S. Pat. No.
4,918,166, which is incorporated by reference herein in its
entirety.
[0163] The crystal structure of several RNA bacteriophages has been
determined (Golmohammadi, R. et al., Structure 4:543-554 (1996)).
Using such information, surface exposed residues can be identified
and, thus, RNA-phage coat proteins can be modified such that one or
more reactive amino acid residues can be inserted by way of
insertion or substitution. As a consequence, those modified forms
of bacteriophage coat proteins can also be used for the present
invention. Thus, variants of proteins which form capsids or
capsid-like structures (e.g., coat proteins of bacteriophage
Q.beta., bacteriophage R17, bacteriophage fr, bacteriophage GA,
bacteriophage SP, and bacteriophage MS2) can also be used to
prepare compositions of the present invention.
[0164] Although the sequence of the variants proteins discussed
above will differ from their wild-type counterparts, these variant
proteins will generally retain the ability to form capsids or
capsid-like structures. Thus, the invention further includes
compositions and vaccine compositions, respectively, which further
includes variants of proteins which form capsids or capsid-like
structures, as well as methods for preparing such compositions and
vaccine compositions, respectively, individual protein subunits
used to prepare such compositions, and nucleic acid molecules which
encode these protein subunits. Thus, included within the scope of
the invention are variant forms of wild-type proteins which form
capsids or capsid-like structures and retain the ability to
associate and form capsids or capsid-like structures.
[0165] As a result, the invention further includes compositions and
vaccine compositions, respectively, comprising proteins, which
comprise, or alternatively consist essentially of, or alternatively
consist of amino acid sequences which are at least 80%, 85%, 90%,
95%, 97%, or 99% identical to wild-type proteins which form ordered
arrays and having an inherent repetitive structure,
respectively.
[0166] Further included within the scope of the invention are
nucleic acid molecules which encode proteins used to prepare
compositions of the present invention.
[0167] In other embodiments, the invention further includes
compositions comprising proteins, which comprise, or alternatively
consist essentially of, or alternatively consist of amino acid
sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99%
identical to any of the amino acid sequences shown in SEQ ID NOs:
10-27.
[0168] Proteins suitable for use in the present invention also
include C-terminal truncation mutants of proteins which form
capsids or capsid-like structures, or VLP's. Specific examples of
such truncation mutants include proteins having an amino acid
sequence shown in any of SEQ ID NOs:10-27 where 1, 2, 5, 7, 9, 10,
12, 14, 15, or 17 amino acids have been removed from the
C-terminus. Typically, theses C-terminal truncation mutants will
retain the ability to form capsids or capsid-like structures.
[0169] Further proteins suitable for use in the present invention
also include N-terminal truncation mutants of proteins which form
capsids or capsid-like structures. Specific examples of such
truncation mutants include proteins having an amino acid sequence
shown in any of SEQ ID NOs:10-27 where 1, 2, 5, 7, 9, 10, 12, 14,
15, or 17 amino acids have been removed from the N-terminus.
Typically, these N-terminal truncation mutants will retain the
ability to form capsids or capsid-like structures.
[0170] Additional proteins suitable for use in the present
invention include N- and C-terminal truncation mutants which form
capsids or capsid-like structures. Suitable truncation mutants
include proteins having an amino acid sequence shown in any of SEQ
ID NOs:10-27 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids
have been removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12,
14, 15, or 17 amino acids have been removed from the C-terminus.
Typically, these N-terminal and C-terminal truncation mutants will
retain the ability to form capsids or capsid-like structures.
[0171] The invention further includes compositions comprising
proteins which comprise, or alternatively consist essentially of,
or alternatively consist of, amino acid sequences which are at
least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above
described truncation mutants.
[0172] The invention thus includes compositions and vaccine
compositions prepared from proteins which form capsids or VLP's,
methods for preparing these compositions from individual protein
subunits and VLP's or capsids, methods for preparing these
individual protein subunits, nucleic acid molecules which encode
these subunits, and methods for vaccinating and/or eliciting
immunological responses in individuals using these compositions of
the present invention.
[0173] As previously stated, the invention includes virus-like
particles or recombinant forms thereof. In one further preferred
embodiment, the particles used in compositions of the invention are
composed of a Hepatitis B core protein (HBcAg) or a fragment of an
HBcAg. In a further embodiment, the particles used in compositions
of the invention are composed of a Hepatitis B core protein (HBcAg)
or a fragment of a HBcAg protein, which has been modified to either
eliminate or reduce the number of free cysteine residues. Zhou et
al. (J. Virol. 66:5393-5398 (1992)) demonstrated that HBcAgs which
have been modified to remove the naturally resident cysteine
residues retain the ability to associate and form capsids. Thus,
VLP's suitable for use in compositions of the invention include
those comprising modified HBcAgs, or fragments thereof, in which
one or more of the naturally resident cysteine residues have been
either deleted or substituted with another amino acid residue
(e.g., a serine residue).
[0174] The HBcAg is a protein generated by the processing of a
Hepatitis B core antigen precursor protein. A number of isotypes of
the HBcAg have been identified and their amino acids sequences are
readily available to those skilled in the art. In most instances,
compositions and vaccine compositions, respectively, of the
invention will be prepared using the processed form of a HBcAg
(i.e., a HBcAg from which the N-terminal leader sequence of the
Hepatitis B core antigen precursor protein have been removed).
[0175] Further, when HBcAgs are produced under conditions where
processing will not occur, the HBcAgs will generally be expressed
in "processed" form. For example, when an E. coli expression system
directing expression of the protein to the cytoplasm is used to
produce HBcAgs of the invention, these proteins will generally be
expressed such that the N-terminal leader sequence of the Hepatitis
B core antigen precursor protein is not present.
[0176] The preparation of Hepatitis B virus-like particles, which
can be used for the present invention, is disclosed, for example,
in WO 00/32227, and hereby in particular in Examples 17 to 19 and
21 to 24, as well as in WO 01/85208, and hereby in particular in
Examples 17 to 19, 21 to 24, 31 and 41, and in pending U.S.
application Ser. No. 10/050,902 filed by the present assignee on
Jan. 18, 2002. For the latter application, it is in particular
referred to Example 23, 24, 31 and 51. All three documents are
explicitly incorporated herein by reference.
[0177] The present invention also includes HBcAg variants which
have been modified to delete or substitute one or more additional
cysteine residues. It is known in the art that free cysteine
residues can be involved in a number of chemical side reactions.
These side reactions include disulfide exchanges, reaction with
chemical substances or metabolites that are, for example, injected
or formed in a combination therapy with other substances, or direct
oxidation and reaction with nucleotides upon exposure to UV light.
Toxic adducts could thus be generated, especially considering the
fact that HBcAgs have a strong tendency to bind nucleic acids. The
toxic adducts would thus be distributed between a multiplicity of
species, which individually may each be present at low
concentration, but reach toxic levels when together.
[0178] In view of the above, one advantage to the use of HBcAgs in
vaccine compositions which have been modified to remove naturally
resident cysteine residues is that sites to which toxic species can
bind when antigens or antigenic determinants are attached would be
reduced in number or eliminated altogether.
[0179] A number of naturally occurring HBcAg variants suitable for
use in the practice of the present invention have been identified.
Yuan et al., (J. Virol. 73:10122-10128 (1999)), for example,
describe variants in which the isoleucine residue at position
corresponding to position 97 in SEQ ID NO:28 is replaced with
either a leucine residue or a phenylalanine residue. The amino acid
sequences of a number of HBcAg variants, as well as several
Hepatitis B core antigen precursor variants, are disclosed in
GenBank reports AAF121240 (SEQ ID NO:29), AF121239 (SEQ ID NO:30),
X85297 (SEQ ID NO:31), X02496 (SEQ ID NO:32), X85305 (SEQ ID
NO:33), X85303 (SEQ ID NO:34), AF151735 (SEQ ID NO:35), X85259 (SEQ
ID NO:36), X85286 (SEQ ID NO:37), X85260 (SEQ ID NO:38), X85317
(SEQ ID NO:39), X85298 (SEQ ID NO:40), AF043593 (SEQ ID NO:41),
M20706 (SEQ ID NO:42), X85295 (SEQ ID NO:43), X80925 (SEQ ID
NO:44), X85284 (SEQ ID NO:45), X85275 (SEQ ID NO:46), X72702 (SEQ
ID NO:47), X85291 (SEQ ID NO:48), X65258 (SEQ ID NO:49), X85302
(SEQ ID NO:50), M32138 (SEQ ID NO:51), X85293 (SEQ ID NO:52),
X85315 (SEQ ID NO:53), U95551 (SEQ ID NO:54), X85256 (SEQ ID
NO:55), X85316 (SEQ ID NO:56), X85296 (SEQ ID NO:57), AB033559 (SEQ
ID NO:58), X59795 (SEQ ID NO:59), X85299 (SEQ ID NO:60), X85307
(SEQ ID NO:61), X65257 (SEQ ID NO:62), X85311 (SEQ ID NO:63),
X85301 (SEQ ID NO:64), X85314 (SEQ ID NO:65), X85287 (SEQ ID
NO:66), X85272 (SEQ ID NO:67), X85319 (SEQ ID NO:68), AB010289 (SEQ
ID NO:69), X85285 (SEQ ID NO:70), AB010289 (SEQ ID NO:71), AF121242
(SEQ i) NO:72), M90520 (SEQ ID NO:73), PO.sub.3153 (SEQ ID NO:74),
AF1 10999 (SEQ ID NO:75), and M95589 (SEQ ID NO:76), the
disclosures of each of which are incorporated herein by reference.
These HBcAg variants differ in amino acid sequence at a number of
positions, including amino acid residues which corresponds to the
amino acid residues located at positions 12, 13, 21, 22, 24, 29,
32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69,
74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113,
116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and
183 in SEQ ID NO:77. Further HBcAg variants suitable for use in the
compositions of the invention, and which may be further modified
according to the disclosure of this specification are described in
WO 00/198333, WO 00/177158 and WO 00/214478.
[0180] As noted above, generally processed HBcAgs (i.e., those
which lack leader sequences) will be used in the compositions and
vaccine compositions, respectively, of the invention. The present
invention includes vaccine compositions, as well as methods for
using these compositions, which employ the above described variant
HBcAgs.
[0181] Whether the amino acid sequence of a polypeptide has an
amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99%
identical to one of the above wild-type amino acid sequences, or a
subportion thereof, can be determined conventionally using known
computer programs such the Bestfit program. When using Bestfit or
any other sequence alignment program to determine whether a
particular sequence is, for instance, 95% identical to a reference
amino acid sequence, the parameters are set such that the
percentage of identity is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5%
of the total number of amino acid residues in the reference
sequence are allowed.
[0182] The HBcAg variants and precursors having the amino acid
sequences set out in SEQ ID NOs: 29-72 and 73-77 are relatively
similar to each other. Thus, reference to an amino acid residue of
a HBcAg variant located at a position which corresponds to a
particular position in SEQ ID NO:77, refers to the amino acid
residue which is present at that position in the amino acid
sequence shown in SEQ ID NO:77. The homology between these HBcAg
variants is for the most part high enough among Hepatitis B viruses
that infect mammals so that one skilled in the art would have
little difficulty reviewing both the amino acid sequence shown in
SEQ ID NO:77 and that of a particular HBcAg variant and identifying
"corresponding" amino acid residues. Furthermore, the HBcAg amino
acid sequence shown in SEQ ID NO:73, which shows the amino acid
sequence of a HBcAg derived from a virus which infect woodchucks,
has enough homology to the HBcAg having the amino acid sequence
shown in SEQ ID NO:77 that it is readily apparent that a three
amino acid residue insert is present in SEQ ID NO:64 between amino
acid residues 155 and 156 of SEQ ID NO:77.
[0183] The invention also includes vaccine compositions which
comprise HBcAg variants of Hepatitis B viruses which infect birds,
as wells as vaccine compositions which comprise fragments of these
HBcAg variants. For these HBcAg variants one, two, three or more of
the cysteine residues naturally present in these polypeptides could
be either substituted with another amino acid residue or deleted
prior to their inclusion in vaccine compositions of the
invention.
[0184] As discussed above, the elimination of free cysteine
residues reduces the number of sites where toxic components can
bind to the HBcAg, and also eliminates sites where cross-linking of
lysine and cysteine residues of the same or of neighboring HBcAg
molecules can occur. Therefore, in another embodiment of the
present invention, one or more cysteine residues of the Hepatitis B
virus capsid protein have been either deleted or substituted with
another amino acid residue.
[0185] In other embodiments, compositions and vaccine compositions,
respectively, of the invention will contain HBcAgs from which the
C-terminal region (e.g., amino acid residues 145-185 or 150-185 of
SEQ ID NO:77) has been removed. Thus, additional modified HBcAgs
suitable for use in the practice of the present invention include
C-terminal truncation mutants. Suitable truncation mutants include
HBcAgs where 1, 5, 10, 15, 20, 25, 30, 34, 35, amino acids have
been removed from the C-terminus.
[0186] HBcAgs suitable for use in the practice of the present
invention also include N-terminal truncation mutants. Suitable
truncation mutants include modified HBcAgs where 1, 2, 5, 7, 9, 10,
12, 14, 15, or 17 amino acids have been removed from the
N-terminus.
[0187] Further HBcAgs suitable for use in the practice of the
present invention include N- and C-terminal truncation mutants.
Suitable truncation mutants include HBcAgs where 1, 2, 5, 7, 9, 10,
12, 14, 15, and 17 amino acids have been removed from the
N-terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids have
been removed from the C-terminus.
[0188] The invention further includes compositions and vaccine
compositions, respectively, comprising HBcAg polypeptides
comprising, or alternatively essentially consisting of, or
alternatively consisting of, amino acid sequences which are at
least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above
described truncation mutants.
[0189] In certain embodiments of the invention, a lysine residue is
introduced into a HBcAg polypeptide, to mediate the binding of the
protein or peptide of IL-5, IL-13 or eotaxin o the VLP of HBcAg. In
preferred embodiments, compositions of the invention are prepared
using a HBcAg comprising, or alternatively consisting of, amino
acids 1-144, or 1-149, 1-185 of SEQ ID NO:77, which is modified so
that the amino acids corresponding to positions 79 and 80 are
replaced with a peptide having the amino acid sequence of
Gly-Gly-Lys-Gly-Gly (SEQ ID NO: 387). In further preferred
embodiments, the cysteine residues at positions 48 and 107 of SEQ
ID NO:77 are mutated to serine. The invention further includes
compositions comprising the corresponding polypeptides having amino
acid sequences shown in any of SEQ ID NOs:29-74, which also have
above noted amino acid alterations. Further included within the
scope of the invention are additional HBcAg variants which are
capable of associating to form a capsid or VLP and have the above
noted amino acid alterations. Thus, the invention further includes
compositions and vaccine compositions, respectively, comprising
HBcAg polypeptides which comprise, or alternatively consist of,
amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or
99% identical to any of the wild-type amino acid sequences, and
forms of these proteins which have been processed, where
appropriate, to remove the N-terminal leader sequence and modified
with above noted alterations.
[0190] Compositions or vaccine compositions of the invention may
comprise mixtures of different HBcAgs. Thus, these vaccine
compositions may be composed of HBcAgs which differ in amino acid
sequence. For example, vaccine compositions could be prepared
comprising a "wild-type" HBcAg and a modified HBcAg in which one or
more amino acid residues have been altered (e.g., deleted, inserted
or substituted). Further, preferred vaccine compositions of the
invention are those which present highly ordered and repetitive
antigen array, wherein the antigen is a protein or peptide of IL-5,
IL-13 or eotaxin In a further preferred embodiment of the present
invention, the at least one protein or peptide of IL-5, IL-13 or
eotaxin is bound to said core particle and virus-like particle,
respectively, by at least one covalent bond. Preferably, the least
one protein or peptide of IL-5, IL-13 or eotaxin is bound to the
core particle and virus-like particle, respectively, by at least
one covalent bond, said covalent bond being a non-peptide bond
leading to a core particle--protein or peptide of IL-5, IL-13 or
eotaxin ordered and repetitive array and a protein or peptide of
IL-5, IL-13 or eotaxin -VLP-array or -conjugate, respectively. This
protein or peptide of IL-5, IL-13 or eotaxin-VLP array and
conjugate, respectively, has typically and preferably a repetitive
and ordered structure since the at least one, but usually more than
one, protein or peptide of IL-5, IL-13 or eotaxin is bound to the
VLP in an oriented manner. Preferably, more than 10, 20, 40, 80,
120 protein or peptide of IL-5, IL-13 or eotaxin are bound to the
VLP or VLP subunit. The formation of a repetitive and ordered
protein or peptide of IL-5, IL-13 or eotaxin array and conjugate,
respectively, is ensured by an oriented and directed as well as
defined binding and attachment, respectively, of the at least one
protein or peptide of IL-5, IL-13 or eotaxin to the VLP as will
become apparent in the following. Furthermore, the typical inherent
highly repetitive and organized structure of the VLP's
advantageously contributes to the display of the protein or peptide
of IL-5, IL-13 or eotaxin in a highly ordered and repetitive
fashion leading to a highly organized and repetitive protein or
peptide of IL-5, IL-13 or eotaxin array and conjugate,
respectively.
[0191] Therefore, the preferred inventive conjugates and arrays,
respectively, differ from prior art conjugates in their highly
organized structure, dimensions, and in the repetitiveness of the
antigen on the surface of the array. The preferred embodiment of
this invention, furthermore, allows expression of both the particle
and the antigen in an expression host guaranteeing proper folding
of the antigen, i.e. the at least one protein or peptide of IL-5,
IL-13 or eotaxin, and proper folding and assembly of the VLP.
[0192] The present invention discloses methods of binding of
protein or peptide of IL-5, IL-13 or eotaxin to core particles and
VLPs, respectively. As indicated, in one aspect of the invention,
the protein or peptide of IL-5, IL-13 or eotaxin is bound to the
core particle and VLP, respectively, by way of chemical
cross-linking, typically and preferably by using a
heterobifunctional cross-linker. Several hetero-bifunctional
cross-linkers are known to the art. In preferred embodiments, the
hetero-bifunctional cross-linker contains a functional group which
can react with preferred first attachment sites, i.e. with the
side-chain amino group of lysine residues of the core particle and
the VLP or at least one VLP subunit, respectively, and a further
functional group which can react with a preferred second attachment
site, i.e. a cysteine residue naturally present, made available for
reaction by reduction, or engineered on the protein or peptide of
IL-5, IL-13 or eotaxin, and optionally also made available for
reaction by reduction. The first step of the procedure, typically
called the derivitization, is the reaction of the core particle or
the VLP with the cross-linker. The product of this reaction is an
activated core particle or activated VLP, also called activated
carrier. In the second step, unreacted cross-linker is removed
using usual methods such as gel filtration or dialysis. In the
third step, the protein or peptide of IL-5, IL-13 or eotaxin is
reacted with the activated carrier, and this step is typically
called the coupling step. Unreacted protein or peptide of IL-5,
IL-13 or eotaxin may be optionally removed in a fourth step, for
example by dialysis. Several hetero-bifunctional cross-linkers are
known to the art. These include the preferred cross-linkers SMPH
(Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB,
Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available
for example from the Pierce Chemical Company (Rockford, Ill., USA),
and having one functional group reactive towards amino groups and
one functional group reactive towards cysteine residues. The above
mentioned cross-linkers all lead to formation of a thioether
linkage. Another class of cross-linkers suitable in the practice of
the invention is characterized by the introduction of a disulfide
linkage between the protein or peptide of IL-5, IL-13 or eotaxin
and the core particle or VLP upon coupling. Preferred cross-linkers
belonging to this class include for example SPDP and Sulfo-LC-SPDP
(Pierce). The extent of derivatization of the core particle and
VLP, respectively, with cross-linker can be influenced by varying
experimental conditions such as the concentration of each of the
reaction partners, the excess of one reagent over the other, the
pH, the temperature and the ionic strength. The degree of coupling,
i.e. the amount of protein or peptide of IL-5, IL-13 or eotaxin per
subunits of the core particle and VLP, respectively, can be
adjusted by varying the experimental conditions described above to
match the requirements of the vaccine. Solubility of the protein or
peptide of IL-5, IL-13 or eotaxin peptide may impose a limitation
on the amount of protein or peptide of IL-5, IL-13 or eotaxin that
can be coupled on each subunit, and in those cases where the
obtained vaccine would be insoluble, reducing the amount of protein
or peptide of IL-5, IL-13 or eotaxin per subunit is beneficial.
[0193] A particularly favored method of binding of protein or
peptide of IL-5, IL-13 or eotaxin to the core particle and the VLP,
respectively, is the linking of a lysine residue on the surface of
the core particle and the VLP, respectively, with a cysteine
residue on the protein or peptide of IL-5, IL-13 or eotaxin. Thus,
in a preferred embodiment of the present invention, the first
attachment site is a lysine residue and the second attachment site
is a cysteine residue. In some embodiments, engineering of an amino
acid linker containing a cysteine residue, as a second attachment
site or as a part thereof, to the protein or peptide of IL-5, IL-13
or eotaxin for coupling to the core particle and VLP, respectively,
may be required. Alternatively, a cysteine may be introduced either
by insertion or mutation within the protein or peptide of IL-5,
IL-13 or eotaxin. Alternatively, the cysteine residue or a thiol
group may be introduced by chemical coupling.
[0194] The selection of the amino acid linker will be dependent on
the nature of the antigen and self-antigen, respectively, i.e. on
the nature of the protein or peptide of IL-5, IL-13 or eotaxin, on
its biochemical properties, such as pI, charge distribution and
glycosylation. In general, flexible amino acid linkers are favored.
Preferred embodiments of the amino acid linker are selected from
the group consisting of: (a) CGG; (b) N-terminal gamma 1-linker;
(c) N-terminal gamma 3-linker; (d) Ig hinge regions; (e) N-terminal
glycine linkers; (f) (G).sub.kC(G).sub.n with n=0-12 and k=0-5; (g)
N-terminal glycine-serine linkers; (h)
(G).sub.kC(G).sub.m(S).sub.l(GGGGS).sub.n with n=0-3, k=0-5,
m=0-10, l=0-2 (SEQ ID NO:344); (i) GGC; (k) GGC-NH2; (1) C-terminal
gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminal
glycine linkers; (o) (G).sub.nC(G).sub.k with n=0-12 and k=0-5; (p)
C-terminal glycine-serine linkers; (q)
(G).sub.m(S).sub.l(GGGGS).sub.n(G).sub.oC(G).sub.k with n=0-3,
k=0-5, m=O-10, l=0-2, and o=0-8 (SEQ ID NO:345).
[0195] Further preferred examples of amino acid linkers are the
hinge region of Immunoglobulins, glycine serine linkers
(GGGGS).sub.n(SEQ ID NO:346), and glycine linkers (G).sub.n all
further containing a cysteine residue as second attachment site and
optionally further glycine residues. Typically preferred examples
of said amino acid linkers are N-terminal gamma1: CGDKTHTSPP (SEQ
ID NO:347); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:348);
N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO:349); C-terminal
gamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO:350); N-terminal glycine
linker: GCGGGG (SEQ ID NO:351); C-terminal glycine linker: GGGGCG
(SEQ ID NO:352); C-terminal glycine-lysine linker: GGKKGC (SEQ ID
NO:353); N-terminal glycine-lysine linker: CGKKGG (SEQ ID
NO:354).
[0196] In a further preferred embodiment of the present invention,
and in particular if the antigen is a IL-5, IL-13 or eotaxin
peptide, GGCG (SEQ ID NO:355), GGC or GGC-NH2 ("NH2" stands for
amidation) linkers at the C-terminus of the peptide or CGG at its
N-terminus are preferred as amino acid linkers. In general, glycine
residues will be inserted between bulky amino acids and the
cysteine to be used as second attachment site, to avoid potential
steric hindrance of the bulkier amino acid in the coupling
reaction.
[0197] The cysteine residue present on the protein or peptide of
IL-5, IL-13 or eotaxin has to be in its reduced state to react with
the hetero-bifunctional cross-linker on the activated VLP, that is
a free cysteine or a cysteine residue with a free sulfhydryl group
has to be available. In the instance where the cysteine residue to
function as binding site is in an oxidized form, for example if it
is forming a disulfide bridge, reduction of this disulfide bridge
with e.g. DTT, TCEP or .beta.-mercaptoethanol is required.
[0198] Binding of the protein or peptide of IL-5, IL-13 or eotaxin
to the core particle and VLP, respectively, by using a
hetero-bifunctional cross-linker according to the preferred methods
described above, allows coupling of the protein or peptide of IL-5,
IL-13 or eotaxin to the core particle and the VLP, respectively, in
an oriented fashion. Other methods of binding the protein or
peptide of IL-5, IL-13 or eotaxin to the core particle and the VLP,
respectively, include methods wherein the protein or peptide of
IL-5, IL-13 or eotaxin is cross-linked to the core particle and the
VLP, respectively, using the carbodiimide EDC, and NHS. The protein
or peptide of IL-5, IL-13 or eotaxin may also be first thiolated
through reaction, for example with SATA, SATP or iminothiolane. The
protein or peptide of IL-5, IL-13 or eotaxin, after deprotection if
required, may then be coupled to the core particle and the VLP,
respectively, as follows. After separation of the excess thiolation
reagent, the protein or peptide of IL-5, IL-13 or eotaxin is
reacted with the core particle and the VLP, respectively,
previously activated with a hetero-bifunctional cross-linker
comprising a cysteine reactive moiety, and therefore displaying at
least one or several functional groups reactive towards cysteine
residues, to which the thiolated protein or peptide of IL-5, IL-13
or eotaxin can react, such as described above. Optionally, low
amounts of a reducing agent are included in the reaction mixture.
In further methods, the protein or peptide of IL-5, IL-13 or
eotaxin is attached to the core particle and the VLP, respectively,
using a homo-bifunctional cross-linker such as glutaraldehyde, DSG,
BM[PEO].sub.4, BS.sup.3, (Pierce Chemical Company, Rockford, Ill.,
USA) or other known homo-bifunctional cross-linkers with functional
groups reactive towards amine groups or carboxyl groups of the core
particle and the VLP, respectively.
[0199] In a further embodiment, the protein or peptide of IL-5,
IL-13 or eotaxin is bound to the core particle and the VLP,
respectively, through modification of the carbohydrate moieties
present on glycosylated protein or peptide of IL-5, IL-13 or
eotaxin and subsequent reaction with the core particle and the VLP,
respectively. In one embodiment, the glycosylated protein or
peptide of IL-5, IL-13 or eotaxin is reacted with sodium periodate
in a mild oxidation reaction of the carbohydrate moiety, to yield
an activated protein or peptide of IL-5, IL-13 or eotaxin with one
or more aldehyde functional groups. The so activated protein or
peptide of IL-5, IL-13 or eotaxin is separated from excess sodium
periodate, and further reacted with the core particle and the VLP,
respectively, wherein lysine residues of the core particle and the
VLP, respectively, or of at least one VLP subunit are reacting with
the previously formed aldehyde functional group on the protein or
peptide of IL-5, IL-13 or eotaxin, for example as described by
Hermanson, G. T. in Bioconjugate Techniques, Academic Press Inc.,
San Diego, Calif., USA. Self polymerization of the activated
protein or peptide of IL-5, IL-13 or eotaxin may be controlled by
adjusting the pH as described in the aforementioned publication.
The formed Schiff base is preferably further reduced with sodium
cyanoborohydride, which is subsequently removed by gel filtration
or dialysis. Alternatively, the core particle and the VLP,
respectively, may be reacted with EDC at carboxyl groups of the
core particle and the VLP, respectively, or at least one VLP
subunit and a dihydrazide, such as adipic acid dihydrazide, to
yield a hydrazide moiety available for reaction with the one or
more aldehyde functional groups present on the activated protein or
peptide of IL-5, IL-13 or eotaxin. The so formed hydrazone may be
further reduced with sodium cyanoborohydride. Alternatively, the
activated protein or peptide of IL-5, IL-13 or eotaxin with one or
more aldehyde functional groups is reacted with cysteamine,
resulting in the introduction of a cysteine group in the protein or
peptide of IL-5, IL-13 or eotaxin. Additional cross-linking methods
and cross-linkers, suitable for protein or peptide of IL-5, IL-13
or eotaxin to a core particle and a VLP, respectively, as well as
guidance on performing the coupling reactions and on the use of
chemical cross-linkers and chemical cross-linking procedures can be
found in Hermanson, G. T. in Bioconjugate Techniques, Academic
Press Inc., San Diego, Calif., USA.
[0200] Other methods of binding the VLP to a protein or peptide of
IL-5, IL-13 or eotaxin include methods where the core particle and
the VLP, respectively, is biotinylated, and the protein or peptide
of IL-5, IL-13 or eotaxin expressed as a streptavidin-fusion
protein, or methods wherein both the protein or peptide of IL-5,
IL-13 or eotaxin and the core particle and the VLP, respectively,
are biotinylated, for example as described in WO 00/23955. In this
case, the protein or peptide of IL-5, IL-13 or eotaxin may be first
bound to streptavidin or avidin by adjusting the ratio of protein
or peptide of IL-5, IL-13 or eotaxin to streptavidin such that free
binding sites are still available for binding of the core particle
and the VLP, respectively, which is added in the next step.
Alternatively, all components may be mixed in a "one pot" reaction.
Other ligand-receptor pairs, where a soluble form of the receptor
and of the ligand is available, and are capable of being
cross-linked to the core particle and the VLP, respectively, or the
protein or peptide of IL-5, IL-13 or eotaxin, may be used as
binding agents for binding the protein or peptide of IL-5, IL-13 or
eotaxin to the core particle and the VLP, respectively.
Alternatively, either the ligand or the receptor may be fused to
the protein or peptide of IL-5, IL-13 or eotaxin and so mediate
binding to the core particle and the VLP, respectively, chemically
bound or fused either to the receptor, or the ligand respectively.
Fusion may also be effected by insertion or substitution.
[0201] As already indicated, in a favored embodiment of the present
invention, the VLP is the VLP of a RNA phage, and in a more
preferred embodiment, the VLP is the VLP of RNA phage Q.beta. coat
protein.
[0202] One or several antigen molecules, i.e. a protein or peptide
of IL-5, IL-13 or eotaxin, can be attached to one subunit of the
capsid or VLP of RNA phages coat proteins, preferably through the
exposed lysine residues of the VLP of RNA phages, if sterically
allowable. A specific feature of the VLP of the coat protein of RNA
phages and in particular of the Q.beta. coat protein VLP is thus
the possibility to couple several antigens per subunit. This allows
for the generation of a dense antigen array.
[0203] In a preferred embodiment of the invention, the binding and
attachment, respectively, of the at least protein or peptide of
IL-5, IL-13 or eotaxin to the core particle and the virus-like
particle, respectively, is by way of interaction and association,
respectively, between at least one first attachment site of the
virus-like particle and at least one second attachment of the
antigen or antigenic determinant.
[0204] VLPs or capsids of Q.beta. coat protein display a defined
number of lysine residues on their surface, with a defined topology
with three lysine residues pointing towards the interior of the
capsid and interacting with the RNA, and four other lysine residues
exposed to the exterior of the capsid. These defined properties
favor the attachment of antigens to the exterior of the particle,
rather than to the interior of the particle where the lysine
residues interact with RNA. VLPs of other RNA phage coat proteins
also have a defined number of lysine residues on their surface and
a defined topology of these lysine residues.
[0205] In further preferred embodiments of the present invention,
the first attachment site is a lysine residue and/or the second
attachment comprises sulfhydryl group or a cysteine residue. In a
very preferred embodiment of the present invention, the first
attachment site is a lysine residue and the second attachment is a
cysteine residue.
[0206] In very preferred embodiments of the invention, the protein
or peptide of IL-5, IL-13 or eotaxin is bound via a cysteine
residue, either naturally present on the protein or peptide of
IL-5, IL-13 or eotaxin or engineered, to lysine residues of the VLP
of RNA phage coat protein, and in particular to the VLP of Q.beta.
coat protein.
[0207] Another advantage of the VLPs derived from RNA phages is
their high expression yield in bacteria that allows production of
large quantities of material at affordable cost.
[0208] As indicated, the inventive conjugates and arrays,
respectively, differ from prior art conjugates in their highly
organized structure, dimensions, and in the repetitiveness of the
antigen on the surface of the array. Moreover, the use of the VLPs
as carriers allow the formation of robust antigen arrays and
conjugates, respectively, with variable antigen density. In
particular, the use of VLP's of RNA phages, and hereby in
particular the use of the VLP of RNA phage Q.beta. coat protein
allows to achieve very high epitope density. The preparation of
compositions of VLPs of RNA phage coat proteins with a high epitope
density can be effected by using the teaching of this
application.
[0209] The second attachment site, as defined herein, may be either
naturally or non-naturally present with the antigen or the
antigenic determinant. In the case of the absence of a suitable
natural occurring second attachment site on the antigen or
antigenic determinant, such a, then non-natural second attachment
has to be engineered to the antigen.
[0210] As described above, four lysine residues are exposed on the
surface of the VLP of Q.beta. coat protein. Typically these
residues are derivatized upon reaction with a cross-linker
molecule. In the instance where not all of the exposed lysine
residues can be coupled to an antigen, the lysine residues which
have reacted with the cross-linker are left with a cross-linker
molecule attached to the .epsilon.-amino group after the
derivatization step. This leads to disappearance of one or several
positive charges, which may be detrimental to the solubility and
stability of the VLP. By replacing some of the lysine residues with
arginines, as in the disclosed Q.beta. coat protein mutants
described below, we prevent the excessive disappearance of positive
charges since the arginine residues do not react with the
cross-linker. Moreover, replacement of lysine residues by arginines
may lead to more defined antigen arrays, as fewer sites are
available for reaction to the antigen.
[0211] Accordingly, exposed lysine residues were replaced by
arginines in the following Q.beta. coat protein mutants and mutant
Q.beta. VLPs disclosed in this application: Q.beta.-240 (Lys13-Arg;
SEQ ID NO:23), Q.beta.-250 (Lys 2-Arg, Lys13-Arg; SEQ ID NO:25) and
Q.beta.-259 (Lys 2-Arg, Lys16-Arg; SEQ ID NO:27). The constructs
were cloned, the proteins expressed, the VLPs purified and used for
coupling to peptide and protein antigens. Q.beta.-251; (SEQ ID
NO:26) was also constructed, and guidance on how to express, purify
and couple the VLP of Q.beta.-251 coat protein can be found
throughout the application.
[0212] In a further embodiment, we disclose a Q.beta. mutant coat
protein with one additional lysine residue, suitable for obtaining
even higher density arrays of antigens. This mutant Q.beta. coat
protein, Q.beta.-243 (Asn 10-Lys; SEQ ID NO:24), was cloned, the
protein expressed, and the capsid or VLP isolated and purified,
showing that introduction of the additional lysine residue is
compatible with self-assembly of the subunits to a capsid or VLP.
Thus, protein or peptide of IL-5, IL-13 or eotaxin and conjugates,
respectively, may be prepared using VLP of Q.beta. coat protein
mutants. A particularly favored method of attachment of antigens to
VLPs, and in particular to VLPs of RNA phage coat proteins is the
linking of a lysine residue present on the surface of the VLP of
RNA phage coat proteins with a cysteine residue naturally present
or engineered on the antigen, i.e. the protein or peptide of IL-5,
IL-13 or eotaxin. In order for a cysteine residue to be effective
as second attachment site, a sulfhydryl group must be available for
coupling. Thus, a cysteine residue has to be in its reduced state,
that is, a free cysteine or a cysteine residue with a free
sulfhydryl group has to be available. In the instant where the
cysteine residue to function as second attachment site is in an
oxidized form, for example if it is forming a disulfide bridge,
reduction of this disulfide bridge with e.g. DTT, TCEP or
.beta.-mercaptoethanol is required. The concentration of reductant
and the molar excess of reductant over antigen has to be adjusted
for each antigen. A titration range, starting from concentrations
as low as 10 .mu.M or lower, up to 10 to 20 mM or higher reductant
if required is tested, and coupling of the antigen to the carrier
assessed. Although low concentrations of reductant are compatible
with the coupling reaction as described in pending U.S. application
Ser. No. 10/050,902 filed by the present assignee on Jan. 18, 2002,
higher concentrations inhibit the coupling reaction, as a skilled
artisan would know, in which case the reductant has to be removed
by dialysis or gel filtration. Advantageously, the pH of the
dialysis or equilibration buffer is lower than 7, preferably 6. The
compatibility of the low pH buffer with antigen activity or
stability has to be tested.
[0213] Epitope density on the VLP of RNA phage coat proteins can be
modulated by the choice of cross-linker and other reaction
conditions. For example, the cross-linkers Sulfo-GMBS and SMPH
typically allow reaching high epitope density. Derivatization is
positively influenced by high concentration of reactants, and
manipulation of the reaction conditions can be used to control the
number of antigens coupled to VLPs of RNA phage coat proteins, and
in particular to VLPs of Q.beta. coat protein.
[0214] Prior to the design of a non-natural second attachment site
the position at which it should be fused, inserted or generally
engineered has to be chosen. The selection of the position of the
second attachment site may, by way of example, be based on a
crystal structure of the antigen. Such a crystal structure of the
antigen may provide information on the availability of the C- or
N-termini of the molecule (determined for example from their
accessibility to solvent), or on the exposure to solvent of
residues suitable for use as second attachment sites, such as
cysteine residues. Exposed disulfide bridges, as is the case for
Fab fragments, may also be a source of a second attachment site,
since they can be generally converted to single cysteine residues
through mild reduction. Mild reduction conditions not affecting the
immunogenicity of protein or peptide of IL-5, IL-13 or eotaxin will
be chosen. In general, in the case where immunization with a
self-antigen is aiming at inhibiting the interaction of this
self-antigen with its natural ligands, the second attachment site
will be added such that it allows generation of antibodies against
the site of interaction with the natural ligands. Thus, the
location of the second attachment site will be selected such that
steric hindrance from the second attachment site or any amino acid
linker containing the same is avoided. In further embodiments, an
antibody response directed at a site distinct from the interaction
site of the self-antigen with its natural ligand is desired. In
such embodiments, the second attachment site may be selected such
that it prevents generation of antibodies against the interaction
site of the self-antigen with its natural ligands.
[0215] Other criteria in selecting the position of the second
attachment site include the oligomerization state of the antigen,
the site of oligomerization, the presence of a cofactor, and the
availability of experimental evidence disclosing sites in the
antigen structure and sequence where modification of the antigen is
compatible with the function of the self-antigen, or with the
generation of antibodies recognizing the self-antigen.
[0216] In the most preferred embodiments, the protein or peptide of
IL-5, IL-13 or eotaxin comprises a single second attachment site or
a single reactive attachment site capable of association with the
first attachment sites on the core particle and the VLPs or VLP
subunits, respectively. This ensures a defined and uniform binding
and association, respectively, of the at least one, but typically
more than one, preferably more than 10, 20, 40, 80, 120 antigens to
the core particle and VLP, respectively. The provision of a single
second attachment site or a single reactive attachment site on the
antigen, thus, ensures a single and uniform type of binding and
association, respectively leading to a very highly ordered and
repetitive array. For example, if the binding and association,
respectively, is effected by way of a lysine--(as the first
attachment site) and cysteine--(as a second attachment site)
interaction, it is ensured, in accordance with this preferred
embodiment of the invention, that only one cysteine residue per
antigen, independent whether this cysteine residue is naturally or
non-naturally present on the antigen, is capable of binding and
associating, respectively, with the VLP and the first attachment
site of the core particle, respectively.
[0217] In some embodiments, engineering of a second attachment site
onto the antigen require the fusion of an amino acid linker
containing an amino acid suitable as second attachment site
according to the disclosures of this invention. Therefore, in a
preferred embodiment of the present invention, an amino acid linker
is bound to the antigen or the antigenic determinant by way of at
least one covalent bond. Preferably, the amino acid linker
comprises, or alternatively consists of, the second attachment
site. In a further preferred embodiment, the amino acid linker
comprises a sulfhydryl group or a cysteine residue. In another
preferred embodiment, the amino acid linker is cysteine. Some
criteria of selection of the amino acid linker as well as further
preferred embodiments of the amino acid linker according to the
invention have already mentioned above.
[0218] In a further preferred embodiment of the invention, the at
least one antigen or antigenic determinant, i.e. the protein or
peptide of IL-5, IL-13 or eotaxin is fused to the core particle and
the virus-like particle, respectively. As outlined above, a VLP is
typically composed of at least one subunit assembling into a VLP.
Thus, in again a further preferred embodiment of the invention, the
antigen or antigenic determinant, preferably the at least one
protein or peptide of IL-5, IL-13 or eotaxin, is fused to at least
one subunit of the virus-like particle or of a protein capable of
being incorporated into a VLP generating a chimeric
VLP-subunit-protein or peptide of IL-5, IL-13 or eotaxin
fusion.
[0219] Fusion of the protein or peptide of IL-5, IL-13 or eotaxin
can be effected by insertion into the VLP subunit sequence, or by
fusion to either the N- or C-terminus of the VLP-subunit or protein
capable of being incorporated into a VLP. Hereinafter, when
referring to fusion proteins of a peptide to a VLP subunit, the
fusion to either ends of the subunit sequence or internal insertion
of the peptide within the subunit sequence are encompassed.
[0220] Fusion may also be effected by inserting the protein or
peptide of IL-5, IL-13 or eotaxin sequences into a variant of a VLP
subunit where part of the subunit sequence has been deleted, that
are further referred to as truncation mutants. Truncation mutants
may have N- or C-terminal, or internal deletions of part of the
sequence of the VLP subunit. For example, the specific VLP HBcAg
with, for example, deletion of amino acid residues 79 to 81 is a
truncation mutant with an internal deletion. Fusion of protein or
peptide of IL-5, IL-13 or eotaxin to either the N- or C-terminus of
the truncation mutants VLP-subunits also lead to embodiments of the
invention. Likewise, fusion of an epitope into the sequence of the
VLP subunit may also be effected by substitution, where for example
for the specific VLP HBcAg, amino acids 79-81 are replaced with a
foreign epitope. Thus, fusion, as referred to hereinafter, may be
effected by insertion of the protein or peptide of IL-5, IL-13 or
eotaxin sequence in the sequence of a VLP subunit, by substitution
of part of the sequence of the VLP subunit with the protein or
peptide of IL-5, IL-13 or eotaxin sequence, or by a combination of
deletion, substitution or insertions.
[0221] The chimeric protein or peptide of IL-5, IL-13 or eotaxin
subunit will be in general capable of self-assembly into a VLP. VLP
displaying epitopes fused to their subunits are also herein
referred to as chimeric VLPs. As indicated, the virus-like particle
comprises or alternatively is composed of at least one VLP subunit.
In a further embodiment of the invention, the virus-like particle
comprises or alternatively is composed of a mixture of chimeric VLP
subunits and non-chimeric VLP subunits, i.e. VLP subunits not
having an antigen fused thereto, leading to so called mosaic
particles. This may be advantageous to ensure formation of and
assembly to a VLP. In those embodiments, the proportion of chimeric
VLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,
95% or higher.
[0222] Flanking amino acid residues may be added to either end of
the sequence of the peptide or epitope to be fused to either end of
the sequence of the subunit of a VLP, or for internal insertion of
such peptidic sequence into the sequence of the subunit of a VLP.
Glycine and serine residues are particularly favored amino acids to
be used in the flanking sequences added to the protein or peptide
of IL-5, IL-13 or eotaxin to be fused. Glycine residues confer
additional flexibility, which may diminish the potentially
destabilizing effect of fusing a foreign sequence into the sequence
of a VLP subunit.
[0223] In a specific embodiment of the invention, the VLP is a
Hepatitis B core antigen VLP. Fusion proteins to either the
N-terminus of a HBcAg (Neyrinck, S. et al., Nature Med. 5:1157-1163
(1999)) or insertions in the so called major immunodominant region
(MIR) have been described (Pumpens, P. and Grens, E., Intervirology
44:98-114 (2001)), WO 01/98333), and are preferred embodiments of
the invention. Naturally occurring variants of HBcAg with deletions
in the MIR have also been described (Pumpens, P. and Grens, E.,
Intervirology 44:98-114 (2001), which is expressly incorporated by
reference in their entirety), and fusions to the N- or C-terminus,
as well as insertions at the position of the MIR corresponding to
the site of deletion as compared to a wt HBcAg are further
embodiments of the invention. Fusions to the C-terminus have also
been described (Pumpens, P. and Grens, E., Intervirology 44:98-114
(2001)). One skilled in the art will easily find guidance on how to
construct fusion proteins using classical molecular biology
techniques (Sambrook, J. et al., eds., Molecular Cloning, A
Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51
(1989)). Vectors and plasmids encoding HBcAg and HBcAg fusion
proteins and useful for the expression of a HBcAg and HBcAg fusion
proteins have been described (Pumpens, P. & Grens, E.
Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med.
5:1157-1163 (1999)) and can be used in the practice of the
invention. We also describe by way of example (Example 6) the
insertion of an epitope into the MIR of HBcAg, resulting in a
chimeric self-assembling HBcAg. An important factor for the
optimization of the efficiency of self-assembly and of the display
of the epitope to be inserted in the MIR of HBcAg is the choice of
the insertion site, as well as the number of amino acids to be
deleted from the HBcAg sequence within the MIR (Pumpens, P. and
Grens, E., Intervirology 44:98-114 (2001); EP 421'635; U.S. Pat.
No. 6,231,864) upon insertion, or in other words, which amino acids
form HBcAg are to be substituted with the new epitope. For example,
substitution of HBcAg amino acids 76-80, 79-81, 79-80, 75-85 or
80-81 with foreign epitopes has been described (Pumpens, P. and
Grens, E., Intervirology 44:98-114 (2001); EP0421635; U.S. Pat. No.
6,231,864). HBcAg contains a long arginine tail (Pumpens, P. and
Grens, E., Intervirology 44:98-114 (2001)) which is dispensable for
capsid assembly and capable of binding nucleic acids (Pumpens, P.
and Grens, E., Intervirology 44:98-114 (2001)). HBcAg either
comprising or lacking this arginine tail are both embodiments of
the invention.
[0224] In a further preferred embodiment of the invention, the VLP
is a VLP of a RNA phage. The major coat proteins of RNA phages
spontaneously assemble into VLPs upon expression in bacteria, and
in particular in E. coli. Specific examples of bacteriophage coat
proteins which can be used to prepare compositions of the invention
include the coat proteins of RNA bacteriophages such as
bacteriophage Q.beta. (SEQ ID NO:10; PIR Database, Accession No.
VCBPQ.beta. referring to Q.beta. CP and SEQ ID NO: 11; Accession
No. AAA16663 referring to Q.beta. A1 protein) and bacteriophage fr
(SEQ ID NO:4; PIR Accession No. VCBPFR).
[0225] In a more preferred embodiment, the at least one protein or
peptide of IL-5, IL-13 or eotaxin is fused to a Q.beta. coat
protein. Fusion protein constructs wherein epitopes have been fused
to the C-terminus of a truncated form of the A1 protein of Q.beta.,
or inserted within the A1 protein have been described (Kozlovska,
T. M., et al., Intervirology, 39:9-15 (1996)). The A1 protein is
generated by suppression at the UGA stop codon and has a length of
329 aa, or 328 aa, if the cleavage of the N-terminal methionine is
taken into account. Cleavage of the N-terminal methionine before an
alanine (the second amino acid encoded by the Q.beta. CP gene)
usually takes place in E. coli, and such is the case for N-termini
of the Q.beta. coat proteins CP. The part of the A1 gene, 3' of the
UGA amber codon encodes the CP extension, which has a length of 195
amino acids. Insertion of the at least one protein or peptide of
IL-5, IL-13 or eotaxin between position 72 and 73 of the CP
extension leads to further embodiments of the invention (Kozlovska,
T. M., et al., Intervirology 39:9-15 (1996)). Fusion of a protein
or peptide of IL-5, IL-13 or eotaxin at the C-terminus of a
C-terminally truncated Q.beta. A1 protein leads to further
preferred embodiments of the invention. For example, Kozlovska et
al., (Intervirology, 39: 9-15 (1996)) describe Q.beta. A1 protein
fusions where the epitope is fused at the C-terminus of the Q.beta.
CP extension truncated at position 19.
[0226] As described by Kozlovska et al. (Intervirology, 39: 9-15
(1996)), assembly of the particles displaying the fused epitopes
typically requires the presence of both the A1 protein-protein or
peptide of IL-5, IL-13 or eotaxin fusion and the wt CP to form a
mosaic particle. However, embodiments comprising virus-like
particles, and hereby in particular the VLPs of the RNA phage
Q.beta. coat protein, which are exclusively composed of VLP
subunits having at least one protein or peptide of IL-5, IL-13 or
eotaxin fused thereto, are also within the scope of the present
invention.
[0227] The production of mosaic particles may be effected in a
number of ways. Kozlovska et al., Intervirology, 39:9-15 (1996),
describe two methods, which both can be used in the practice of the
invention. In the first approach, efficient display of the fused
epitope on the VLPs is mediated by the expression of the plasmid
encoding the Q.beta. A1 protein fusion having a UGA stop codon
between CP and CP extension in a E. coli strain harboring a plasmid
encoding a cloned UGA suppressor tRNA which leads to translation of
the UGA codon into Trp (pISM3001 plasmid (Smiley B. K., et al.,
Gene 134:33-40 (1993))). In another approach, the CP gene stop
codon is modified into UAA, and a second plasmid expressing the A1
protein-protein or peptide of IL-5, IL-13 or eotaxin fusion is
cotransformed. The second plasmid encodes a different antibiotic
resistance and the origin of replication is compatible with the
first plasmid (Kozlovska, T. M., et al., Intervirology 39:9-15
(1996)). In a third approach, CP and the A1 protein-protein or
peptide of IL-5, IL-13 or eotaxin fusion are encoded in a
bicistronic manner, operatively linked to a promoter such as the
Trp promoter, as described in FIG. 1 of Kozlovska et al.,
Intervirology, 39:9-15 (1996).
[0228] In a further embodiment, the protein or peptide of IL-5,
IL-13 or eotaxin is inserted between amino acid 2 and 3 (numbering
of the cleaved CP, that is wherein the N-terminal methionine is
cleaved) of the fr CP, thus leading to a protein or peptide of
IL-5, IL-13 or eotaxin-fr CP fusion protein. Vectors and expression
systems for construction and expression of fr CP fusion proteins
self-assembling to VLP and useful in the practice of the invention
have been described (Pushko P. et al., Prot. Eng. 6:883-891
(1993)). In a specific embodiment, the protein or peptide of IL-5,
IL-13 or eotaxin sequence is inserted into a deletion variant of
the fr CP after amino acid 2, wherein residues 3 and 4 of the fr CP
have been deleted (Pushko P. et al., Prot. Eng. 6:883-891
(1993)).
[0229] Fusion of epitopes in the N-terminal protuberant
.beta.-hairpin of the coat protein of RNA phage MS-2 and subsequent
presentation of the fused epitope on the self-assembled VLP of RNA
phage MS-2 has also been described (WO 92/13081), and fusion of
protein or peptide of IL-5, IL-13 or eotaxin by insertion or
substitution into the coat protein of MS-2 RNA phage is also
falling under the scope of the invention.
[0230] In another embodiment of the invention, the protein or
peptide of IL-5, IL-13 or eotaxin are fused to a capsid protein of
papillomavirus. In a more specific embodiment, the protein or
peptide of IL-5, IL-13 or eotaxin are fused to the major capsid
protein L1 of bovine papillomavirus type 1 (BPV-1). Vectors and
expression systems for construction and expression of BPV-1 fusion
proteins in a baculovirus/insect cells systems have been described
(Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96:2373-2378
(1999), WO 00/23955). Substitution of amino acids 130-136 of BPV-1
L1 with a protein or peptide of IL-5, IL-13 or eotaxin leads to a
BPV-1 L1-protein or peptide of IL-5, IL-13 or eotaxin fusion
protein, which is a preferred embodiment of the invention. Cloning
in a baculovirus vector and expression in baculovirus infected Sf9
cells has been described, and can be used in the practice of the
invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA
96:2373-2378 (1999), WO 00/23955). Purification of the assembled
particles displaying the fused protein or peptide of IL-5, IL-13 or
eotaxin can be performed in a number of ways, such as for example
gel filtration or sucrose gradient ultracentrifugation (Chackerian,
B. et al., Proc. Natl. Acad. Sci. USA 96:2373-2378 (1999), WO
00/23955).
[0231] In a further embodiment of the invention, the protein or
peptide of IL-5, IL-13 or eotaxin are fused to a Ty protein capable
of being incorporated into a Ty VLP. In a more specific embodiment,
the protein or peptide of IL-5, IL-13 or eotaxin are fused to the
p1 or capsid protein encoded by the TYA gene (Roth, J. F., Yeast
16:785-795 (2000)). The yeast retrotransposons Ty1, 2, 3 and 4 have
been isolated from Saccharomyces cerevisiae, while the
retrotransposon Tf1 has been isolated from Schizosaccharomyces
Pombae (Boeke, J. D. and Sandmeyer, S. B., "Yeast Transposable
elements," in The molecular and Cellular Biology of the Yeast
Saccharomyces: Genome dynamics, Protein Synthesis, and Energetics.,
p. 193, Cold Spring Harbor Laboratory Press (1991)). The
retrotransposons Ty1 and 2 are related to the copia class of plant
and animal elements, while Ty3 belongs to the gypsy family of
retrotransposons, which is related to plants and animal
retroviruses. In the Ty1 retrotransposon, the p1 protein also
referred to as Gag or capsid protein has a length of 440 amino
acids. P1 is cleaved during maturation of the VLP at position 408,
leading to the p2 protein, the essential component of the VLP.
[0232] Fusion proteins to p1 and vectors for the expression of said
fusion proteins in Yeast have been described (Adams, S. E., et al.,
Nature 329:68-70 (1987)). So, for example, a protein or peptide of
IL-5, IL-13 or eotaxin peptide may be fused to p1 by inserting a
sequence coding for the protein or peptide of IL-5, IL-13 or
eotaxin into the BamHI site of the pMA5620 plasmid (Adams, S. E.,
et al., Nature 329:68-70 (1987)). The cloning of sequences coding
for foreign epitopes into the pMA5620 vector leads to expression of
fusion proteins comprising amino acids 1-381 of p1 of Ty1-15, fused
C-terminally to the N-terminus of the foreign epitope. Likewise,
N-terminal fusion of protein or peptide of IL-5, IL-13 or eotaxin,
or internal insertion into the p1 sequence, or substitution of part
of the p1 sequence is also meant to fall within the scope of the
invention. In particular, insertion of protein or peptide of IL-5,
IL-13 or eotaxin into the Ty sequence between amino acids 30-31,
67-68, 113-114 and 132-133 of the Ty protein p1 (EP0677111) leads
to preferred embodiments of the invention.
[0233] Further VLPs suitable for fusion of protein or peptide of
IL-5, IL-13 or eotaxin are, for example, Retrovirus-like-particles
(WO9630523), HIV2 Gag (Kang, Y. C., et al, Biol. Chem. 380:353-364
(1999)), Cowpea Mosaic Virus (Taylor, K. M. et al., Biol. Chem.
380:387-392 (1999)), parvovirus VP2 VLP (Rueda, P. et al., Virology
263:89-99 (1999)), HBsAg (U.S. Pat. No. 4,722,840,
EP0020416B1).
[0234] Examples of chimeric VLPs suitable for the practice of the
invention are also those described in Intervirology 39:1 (1996).
Further examples of VLPs contemplated for use in the invention are:
HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV,
HIV GAG, Tobacco Mosaic Virus. Virus-like particles of SV-40,
Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus and
Norwalk virus have also been made, and chimeric VLPs of those VLPs
are also within the scope of the present invention.
[0235] In a further preferred embodiment of the present invention,
the antigen or antigenic determinant is protein or peptide of IL-5,
IL-13 or eotaxin
[0236] In a further preferred embodiment of the invention, the
antigen or antigenic determinant is a protein or peptide of IL-5,
IL-13 or eotaxin variant, e.g. containing amino acid substitutions
or peptide insertions or polymorphisms. As already indicated,
compositions and vaccine compositions, respectively, comprising
protein or peptide of IL-5, IL-13 or eotaxin variants are included
within the scope of the present invention.
[0237] Protein or peptide of IL-5, IL-13 or eotaxin can be produced
by expression of the IL-5, IL-13 or eotaxin cDNA in procaryotic or
eucaryotic expression systems. Various examples hereto have been
described in the literature and can be used, possibly after
modifications, to express any protein or peptide of IL-5, IL-13 or
eotaxin of any desired species. Disclosures how to produce protein
or peptide of IL-5, is also given in WO 900/65058 and references
provided within
[0238] In a further preferred embodiment of the invention, the
antigen or antigenic determinant is an IL-5, IL-13 or eotaxin
peptide. Such IL-5, IL-13 or eotaxin peptides or fragments thereof
can be produced using standard molecular biological technologies
where the nucleotide sequence coding for the fragment of interest
is amplified by PCR and is cloned as a fusion to a polypeptide tag,
such as the GST tag, MBP tag, histdine tag, the Flag tag, myc tag
or the constant region of an antibody (Fc region). By introducing a
protease cleavage site between the IL-5, IL-13 or eotaxin fragment
and the tag, the IL-5, IL-13 or eotaxin peptide can be separated
from the tag after purification by digestion with corresponding
protease. In another approach the protein or peptide of IL-5, IL-13
or eotaxin peptide can be synthesized in vitro using standard
peptide synthesis reactions known to a person skilled in the art.
In a further approach, peptides of IL-5, IL-13 or eotaxin can be
produced by protease digestion or chemical cleavage of the full
length protein of IL-5, IL-13 or eotaxin, both methods of which are
well known to people trained in the art.
[0239] In a still further preferred embodiment of the present
invention, the antigen or antigenic determinant further comprise at
least one second attachment site being selected from the group
consisting of: (i) an attachment site not naturally occurring with
said antigen or antigenic determinant; and (ii) an attachment site
naturally occurring with said antigen or antigenic determinant.
Guidance on how to modify protein or peptide of IL-5, IL-13 or
eotaxin for binding to the virus-like particle is given throughout
the application. Preferred second attachment sites contain a
cysteine residue for binding to the derivatized VLP and examples
are given in the above description and in Example 12 and 13.
[0240] We have performed an analysis of the model for the
3-dimensional structure of IL-5 to determine accessibility of the
chosen second attachment (NH.sub.2 terminus) to permit coupling to
the first attachment site on the VLP in accordance with the present
invention. The N-terminus is preferred for attaching a second
attachment site comprising an amino acid linker with an additional
cysteine residue. However, an amino-acid linker containing a
cysteine residue as second attachment site and being fused at the
C-terminus of the IL-5 construct leads to a further preferred
embodiment of the invention. A human IL-5 construct with an
N-terminal amino acid linker containing a cysteine residue fused L
is a very preferred embodiment of the invention.
[0241] Similar procedures could be used by a person skilled in the
art to model the accessibility of attachment sites on IL-13 and
eotaxin to optimize coupling to the first attachment site of the
VLP.
[0242] Mouse protein or peptide of IL-5, IL-13 or eotaxin
constructs are disclosed, and preferred human protein or peptide of
IL-5, IL-13 or eotaxin fragment constructs can also be generated.
Further preferred constructs comprise the whole human protein of
IL-5, IL-13 or eotaxin protein, a human peptide of IL-5, IL-13 or
eotaxin. Immunization against protein or peptide of IL-5, IL-13 or
eotaxin using the inventive compositions comprising, preferably a
protein or peptide of IL-5, IL-13 or eotaxin bound to a VLP may
provide a way of treatment or prevention of allergic diseases with
an eosinophilic component.
[0243] In a further preferred embodiment of the present invention,
the protein or peptide of IL-5, IL-13 or eotaxin comprises at least
one antigenic site of a protein of IL-5, IL-13 or eotaxin. The
skilled person in the art knows how to identify the corresponding
peptides and amino acid sequences, respectively.
[0244] In a further preferred embodiment of the present invention
non-contiguous or contiguous peptides of IL-5, IL-13 or eotaxin
such as those defined by neutralizing monoclonal antibodies
(Dickason, R. R. et al J. Immunol. 156(3):1030-71996) are
included.
[0245] In a further preferred embodiment of the present invention
non-contiguous or contiguous peptides of IL-5, IL-13 or eotaxin
predicted to be involved in receptor interaction and crucial for
interaction with the receptor such as those from the COO-terminal
of IL-5, are included.
[0246] Further peptides of IL-5, IL-13 or eotaxin suitable for use
in the present invention can be experimentally determined by their
intrinsic property to induce a T cell or an antibody response. This
is generally achieved by immunizing an experimental animal
separately with selected peptides in an immunologically suitable
formulation and by measuring T cell and B cell, i.e. antibody
responses, using methods known to a person trained in the art. In
the case where the antigen is a protein or a peptide, this region
can be formed by a continuous amino acid sequence. Alternatively,
the antibody epitope can be formed by a discontinuous amino acid
sequence in which, after three dimensional folding of the protein,
polypeptide or peptide, the aminoacids are arranged in such a
manner that they spatially come close together and form the
epitope. Continuous peptide fragments of interest can identified by
immunization experiments as described above.
[0247] Further preferred peptides of IL-5, IL-13 or eotaxin
suitable for use for the present invention can be identified by
using existing or future monoclonal or polyclonal antibodies, the
procedures hereto are know to those skilled in the art.
[0248] Further peptides of IL-5, IL-13 or eotaxin suitable for use
for the present invention may be identified by screening phage
display peptide libraries with antibodies specific for protein or
peptide of IL-5, IL-13 or eotaxin, a method well known to a person
trained in the art.
[0249] In a further preferred embodiment of the invention, the
antigen or antigenic determinant is isolated protein IL-5, IL-13 or
eotaxin of any animal as well as any antigenic peptides of IL-5,
IL-13 or eotaxin of any animal. Those skilled in the art know how
to produce peptides from those isolated proteins or peptides of
IL-5, IL-13 or eotaxin.
[0250] In another preferred embodiment of the invention the
antigenic determinant is Interleukin-13 (IL-13). IL-13 is a
cytokine that is secreted by activated T lymphocytes and primarily
impacts monocytes, macrophages, and B cells. The amino acid
sequence of precursor human IL-13 is shown in SEQ ID No: 230 and
the amino acid sequence of processed human IL-13 is shown in SEQ ID
No: 231. The first 20 amino acids of the precursor protein
correspond to the signal peptide, and are absent of the processed
protein. The mouse sequence has also been described, and the
processed amino acid sequence is shown in SEQ ID No: 232 (Brown K.
D. et al., J. Immunol. 142:679-687 (1989)). Depending on the
expression host, the IL-13 construct will comprise the sequence of
the precursor protein, e.g. for expression and secretion in
eukaryotic hosts, or consist of the mature protein, e.g. for
cytoplasmic expression in E. coli. For expression in the periplasm
of E. coli, the signal peptide of IL-13 is replaced by a bacterial
signal peptide.
[0251] IL-13 is a T helper 2-derived cytokine (like IL-4, IL-5)
that has recently been implicated in allergic airway responses
(asthma). Upregulation of IL-13 and IL-13 receptor has been found
in many tumour types (e.g. Hodgkin lymphoma). Interleukin 13 is
secreted by and stimulates the growth of Hodgkin and Reed-Sternberg
cells (Kapp U et al., J Exp Med. 189:1939-46 (1999)). Thus,
immunization against IL-13 provides a way of treating among others
the conditions described above, such as Asthma or Hodgkins
Lymphoma.
[0252] Preferably, the composition comprises an amino acid linker
containing a free cysteine residue and being fused to the N or
C-terminus of the sequence of mature IL-13 to introduce a second
attachment site within the protein. In further preferred
embodiments, an amino acid linker containing a free cysteine is
added to the N-terminus of the mature form of IL-13, since it is
freely accessible according to the NMR structure of IL-13
(Eisenmesser, E. Z. et al., J. Mol. Biol. 310: 231 (2001)). In
again further preferred embodiments, the amino acid linker
containing a free cysteine is fused to the N-terminus of the
sequence corresponding to the sequence of the processed protein, or
inserted at the N-terminus of the sequence of the mature form of
the protein, C-terminally of the signal peptide. In still further
preferred embodiments, an amino acid linker containing a free
cysteine residue is added to the C-terminus of the protein.
[0253] IL-13 may be expressed in E. coli (Eisenmesser E. Z. et al.,
Protein Expr. Purif 20:186-95 (2000)), or in NS-0 cells (eukaryotic
cell line) (Cannon-Carlson S. et al., Protein Expr. Purif.
12:239-48 (1998)). EXAMPLE 8 demonstrates cloning, and expression
of constructs and purification of murine IL-13, fused to an amino
acid linker containing a cysteine residue, in bacteria. It also
describes production and testing of an Eotaxin-VLP vaccine. Human
IL-13 constructs can be generated according to the teachings of
EXAMPLE 8 and yielding the proteins human C-IL-13-F (SEQ ID NO:
330) and human C-IL-13-S (SEQ ID NO:331) after expression of the
fusion proteins and cleavage with Factor Xa, and enterokinase
respectively. The so generated proteins can be coupled to VLPs and
Pili, leading to preferred embodiments of the invention.
[0254] In yet another embodiment of the invention, the antigenic
determinant is Interleukin-5 (IL-5). IL-5 is a lineage-specific
cytokine for eosinophilopoiesis and plays an important part in
diseases associated with increased number of eosinophils, such as
asthma. The sequence of precursor and processed human IL-5 is
provided in SEQ ID No: 233 and in SEQ ID No: 234, respectively, and
the processed mouse amino acid sequence is shown in SEQ ID No:
235.
[0255] The biological function of IL-5 has been shown in several
studies (Coffman R. L. et al., Science 245: 308-10 (1989); Kopf et
al., Immunity 4:15-24 (1996)), which point to a beneficial effect
of inhibiting IL-5 function in diseases mediated through
eosinophils. Inhibition of the action of IL-5 provides thus a way
of treatment against asthma and other diseases associated with
eosinophils.
[0256] IL-5 forms a dimer, covalently linked by a disulfide bridge.
A single chain (sc) construct has been reported wherein two
monomers of IL-5 are linked by a peptide linker.
[0257] In preferred embodiments of the invention, a peptide linker
containing a free cysteine is added at the N-terminus of the
sequence of the processed form of IL-5. Addition of a linker
containing a free cysteine is also, preferably, envisaged at the
N-terminus of the sequence of the processed form of a scIL-5. In
further preferred embodiments, the amino acid linker containing a
free cysteine is fused to the N-terminus of the sequence
corresponding to the sequence of the processed protein, or inserted
at the N-terminus of the sequence of the mature form of the
protein, C-terminally of the signal peptide.
[0258] In again further preferred embodiments, a linker containing
a free cysteine is fused to the C-terminus of the sequence of IL-5,
or to the C-terminus of a scIL-5 sequence.
[0259] A number of expression systems have been described for IL-5
and can be used in preparing the compositions of the invention. A
bacterial expression system using E. coli has been described by
Proudfoot et al., (Biochem J 270:357-61 (1990)). In the case where
IL-5 is expressed in the cytoplasm of E. coli, the IL-5 construct
is devoid of a signal peptide. Insect cells may also be used for
producing IL-5 constructs for making the compositions of the
invention (Pierrot C. et al., Biochem. Biophys. Res. Commun.
253:756-60 (1998)). Likewise, Baculovirus expression systems (sf9
cells; Ingley E. et al., Eur. J. Biochem. 196:623-9 (1991) and
Brown P. M. et al., Protein Expr. Purif. 6: 63-71 (1995)) can also
be used. Finally, mammalian expression systems have also been
reported (CHO cells) and can be used in preparing these
compositions of the invention (Kodama S et al., J. Biochem. (Tokyo)
110:693-701 (1991)).
[0260] Baculovirus expression systems (Mitchell et al., Biochem.
Soc. Trans. 21:332 S (1993); Kunimoto D Y et al., Cytokine 3:224-30
(1991)) and a mammalian cell expression system using CHO cells
(Kodama S et al., Glycobiology 2:419-27 (1992)) have also been
described for mouse IL-5.
[0261] EXAMPLE 7 and 10 describes the expression, purification,
coupling to VLP, immunisation and testing in a murine model of
experimental asthma of a murine IL-5 construct wherein the IL-5
sequence is fused at its N-terminus to amino acid linkers
containing a cysteine residue for coupling to VLPs and Pili. Human
constructs can be generated according to the teaching of EXAMPLE 7
and 10 and yield the proteins human C-IL-5-E (SEQ ID NO:335), human
C-IL-5-F (SEQ ID NO:336) and human C-IL-5-S: (SEQ ID NO:337)
suitable for coupling to VLPs and Pili and leading to preferred
embodiments of the invention.
[0262] In another specific embodiment, the antigenic determinant is
Eotaxin. Eotaxin is a chemokine specific for Chemokine receptor 3,
present on eosinophils, basophils and Th2 cells. Eotaxin seems
however to be highly specific for Eosinophils (Zimmerman et al., J.
Immunol. 165: 5839-46 (2000)). Eosinophil migration is reduced by
70% in the eotaxin-1 knock-out mouse, which however can still
develop eosinophilia (Rothenberg et al., J. Exp. Med. 185: 785-90
(1997)). IL-5 seems to be responsible for the migration of
eosinophils from bone-marrow to blood, and eotaxin for the local
migration in the tissue (Humbles et al., J. Exp. Med. 186: 601-12
(1997).
[0263] Therefore, in a preferred embodiment, the inventive
composition comprises an amino-acid linker containing a cysteine
residue as second attachment site and being, preferably, fused to
the C-terminus of the Eotaxin sequence. In other preferred
embodiments, an amino acid linker containing a free cysteine is
fused to the N-terminus of the sequence corresponding to the
sequence of the processed protein, or inserted at the N-terminus of
the sequence of the mature form of the protein, C-terminally of the
signal peptide. The genes coding for these specific constructs are
cloned in a suitable expression vector.
[0264] EXAMPLE 9 and 11 describes the cloning and expression of a
murine eotaxin construct wherein the eotaxin sequence is fused at
its C-terminus to amino acid linkers containing a cysteine residue
for coupling to VLPs and Pili. Human constructs can be generated
according to the teaching of EXAMPLE 9 and yield proteins suitable
for coupling to VLPs and Pili and leading to preferred embodiments
of the invention. Eotaxin can be chemically synthesized
(Clark-Lewis et al., Biochemistry 30:3128-3135 (1991)). Expression
in E. coli has also been described for Eotaxin-1, in the cytoplasm
(Crump et al., J. Biol. Chem. 273: 22471-9 (1998)). Expression in
E. coli as inclusion bodies with subsequent refolding (Mayer et
al., Biochemistry 39: 8382-95 (2000)), and Insect cell expression
(Forssmann et al., J. Exp. Med. 185: 2171-6 (1997)) have been
described for Eotaxin-2, and may, moreover, be used to arrive at
the specific embodiments of the invention.
[0265] It will be understood by one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are readily apparent
and may be made without departing from the scope of the invention
or any embodiment thereof. Having now described the present
invention in detail, the same will be more clearly understood by
reference to the following examples, which are included herewith
for purposes of illustration only and are not intended to be
limiting of the invention.
EXAMPLES
Example 1
Construction and Expression of Mutant Q.beta. Coat Proteins, and
Purification of Mutant Q.beta. Coat Protein VLPS or Capsids
Plasmid Construction and Cloning of Mutant Coat Proteins
[0266] Construction of pQ.beta.-240:
[0267] The plasmid pQ.beta.10 (Kozlovska, T M, et al., Gene
137:133-137) was used as an initial plasmid for the construction of
pQ.beta.-240. The mutation Lys13.fwdarw.Arg was created by inverse
PCR. The inverse primers were designed in inverted tail-to-tail
directions:
TABLE-US-00001 (SEQ ID NO:356)
5'-GGTAACATCGGTCGAGATGGAAAACAAACTCTGGTCC-3' and (SEQ ID NO:357)
5'-GGACCAGAGTTTGTTTTCCATCTCGACCGATGTTACC-3'.
The products of the first PCR were used as templates for the second
PCR reaction, in which an upstream primer
TABLE-US-00002 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO:358)
and a downstream primer
TABLE-US-00003 (SEQ ID NO:356)
5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3'
were used. The product of the second PCR was digested with XbaI and
Mph1103I and cloned into the pQ.beta.10 expression vector, which
was cleaved by the same restriction enzymes. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentas, Vilnius, Lithuania).
[0268] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-240 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles.
Resulting Amino Acid Sequence:
TABLE-US-00004 [0269] (SEQ ID NO:23)
AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTV
SVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYS
TDEERAFVRTELAALLASPLLIDAIDQLNPAY
Construction of pQ.beta.-243:
[0270] The plasmid pQ.beta.10 was used as an initial plasmid for
the construction of pQ.beta.-243. The mutation Asn10.fwdarw.Lys was
created by inverse PCR. The inverse primers were designed in
inverted tail-to-tail directions:
TABLE-US-00005 (SEQ ID NO:360)
5'-GGCAAAATTAGAGACTGTTACTTTAGGTAAGATCGG-3' and (SEQ ID NO:361)
5'-CCGATCTTACCTAAAGTAACAGTCTCTAATTTTGCC-3'.
The products of the first PCR were used as templates for the second
PCR reaction, in which an upstream primer
TABLE-US-00006 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO:358)
and a downstream primer
TABLE-US-00007 (SEQ ID NO:359)
5'-CGATGCATTTCATCCTTAGTTATCAATACGCT-GGGTTCAG-3'
were used. The product of the second PCR was digested with XbaI and
Mph1103I and cloned into the pQ.beta.10 expression vector, which
was cleaved by the same restriction enzymes. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentas, Vilnius, Lithuania).
[0271] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-243 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles.
Resulting Amino Acid Sequence:
TABLE-US-00008 [0272] (SEQ ID NO:24)
AKLETVTLGKIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTV
SVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYS
TDEERAFVRTELAALLASPLLIDAIDQLNPAY
Construction of pQ.beta.-250:
[0273] The plasmid pQ.beta.-240 was used as an initial plasmid for
the construction of pQ.beta.-250. The mutation Lys2.fwdarw.Arg was
created by site-directed mutagenesis. An upstream primer
TABLE-US-00009 (SEQ ID NO:362)
5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3'
and a downstream primer
TABLE-US-00010 5'-GATTTAGGTGACACTATAG-3' (SEQ ID NO:363)
were used for the synthesis of the mutant PCR-fragment, which was
introduced into the pQ.beta.-185 expression vector at the unique
restriction sites NcoI and HindIII The PCR reactions were performed
with PCR kit reagents and according to producer protocol (MBI
Fermentas, Vilnius, Lithuania).
[0274] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-250 supported efficient synthesis of 14-kD protein co
migrating upon PAGE with control Q.beta. coat protein isolated from
Q.beta. phage particles.
Resulting Amino Acid Sequence:
TABLE-US-00011 [0275] (SEQ ID NO:25)
ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTV
SVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYS
TDEEPAFVRTELAALLASPLLIDAIDQLNPAY
Construction of pQ.beta.-251:
[0276] The plasmid pQ.beta.10 was used as an initial plasmid for
the construction of pQ.beta.-251. The mutation Lys16.fwdarw.Arg was
created by inverse PCR. The inverse primers were designed in
inverted tail-to-tail directions:
TABLE-US-00012 (SEQ ID NO:364)
5'-GATGGACGTCAAACTCTGGTCCTCAATCCGCGTGGGG-3' and (SEQ ID NO:365)
5'-CCCCACGCGGATTGAGGACCAGAGTTTGACGTCCATC-3'.
The products of the first PCR were used as templates for the second
PCR reaction, in which an upstream primer
TABLE-US-00013 5'-AGCTCGCCCGGGGATCCTCTAG-3' (SEQ ID NO:358)
and a downstream primer
TABLE-US-00014 (SEQ ID NO:359)
5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3'
were used. The product of the second PCR was digested with XbaI and
Mph1103I and cloned into the pQ.beta.10 expression vector, which
was cleaved by the same restriction enzymes. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentas, Vilnius, Lithuania).
[0277] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-251 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles. The resulting amino acid sequence
encoded by this construct is shown in SEQ. ID NO: 26.
Construction of pQ.beta.-259:
[0278] The plasmid pQ.beta.-251 was used as an initial plasmid for
the construction of pQ.beta.-259. The mutation Lys2.fwdarw.Arg was
created by site-directed mutagenesis. An upstream primer
TABLE-US-00015 (SEQ ID NO:362)
5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3'
and a downstream primer
TABLE-US-00016 5'-GATTTAGGTGACACTATAG-3' (SEQ D NO:263)
were used for the synthesis of the mutant PCR-fragment, which was
introduced into the pQ.beta.-185 expression vector at the unique
restriction sites NcoI and HindIII. The PCR reactions were
performed with PCR kit reagents and according to producer protocol
(MBI Fermentas, Vilnius, Lithuania).
[0279] Sequencing using the direct label incorporation method
verified the desired mutations. E. coli cells harbouring
pQ.beta.-259 supported efficient synthesis of 14-kD protein co
migrating upon SDS-PAGE with control Q.beta. coat protein isolated
from Q.beta. phage particles.
Resulting amino acid sequence:
TABLE-US-00017 (SEQ ID NO: 27)
AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTV
SVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQKYADVTFSFTQYS
TDEERAFVRTELAALLASPLLIDAIDQLNPAY
General Procedures for Expression and Purification of Q.beta. and
Q.beta. Mutants
Expression
[0280] E. coli JM109 was transformed with Q.mu. coat protein
expression plasmids. 5 ml of LB liquid medium containing 20
.mu.g/ml ampicillin were inoculated with clones transformed with
Q.beta. coat protein expression plasmids. The inoculated culture
was incubated at 37.degree. C. for 16-24 h without shaking. The
prepared inoculum was subsequently diluted 1:100 in 100-300 ml of
fresh LB medium, containing 20 .mu.g/ml ampicillin. and incubated
at 37.degree. C. overnight without shaking. The resulting second
inoculum was diluted 1:50 in M9 medium containing 1% Casamino acids
and 0.2% glucose in flasks, and incubated at 37.degree. C.
overnight under shaking.
Purification
[0281] Solutions and buffers for the purification procedure:
1. Lysis buffer LB 50 mM Tris-HCl pH8.0 with 5 mM EDTA, 0.1%
[0282] tritonX100 and freshly prepared PMSF at a concentration of 5
micrograms per ml. Without lysozyme and DNAse.
2. SAS
[0283] Saturated ammonium sulphate in water
3. Buffer NET.
[0284] 20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and
[0285] 150 mM NaCl.
4. PEG
[0286] 40% (w/v) polyethyleneglycol 6000 in NET
Disruption and Lysis
[0287] Frozen cells were resuspended in LB at 2 ml/g cells. The
mixture was sonicated with 22 kH five times for 15 seconds, with
intervals of 1 min to cool the solution on ice. The lysate was then
centrifuged at 14 000 rpm, for 1 h using a Janecki K 60 rotor. The
centrifugation steps described below were all performed using the
same rotor, except otherwise stated. The supernatant was stored at
4.degree. C., while cell debris were washed twice with LB. After
centrifugation, the supernatants of the lysate and wash fractions
were pooled.
Fractionation
[0288] A saturated ammonium sulphate solution was added dropwise
under stirring to the above pooled lysate. The volume of the SAS
was adjusted to be one fifth of total volume, to obtain 20% of
saturation. The solution was left standing overnight, and was
centrifuged the next day at 14 000 rpm, for 20 min. The pellet was
washed with a small amount of 20% ammonium sulphate, and
centrifuged again. The obtained supernatants were pooled, and SAS
was added dropwise to obtain 40% of saturation. The solution was
left standing overnight, and was centrifuged the next day at 14 000
rpm, for 20 min. The obtained pellet was solubilised in NET
buffer.
Chromatography
[0289] The capsid or VLP protein resolubilized in NET buffer was
loaded on a Sepharose CL-4B column. Three peaks eluted during
chromatography. The first one mainly contained membranes and
membrane fragments, and was not collected. Capsids were contained
in the second peak, while the third one contained other E. coli
proteins.
[0290] The peak fractions were pooled, and the NaCl concentration
was adjusted to a final concentration of 0.65 M. A volume of PEG
solution corresponding to one half of the pooled peak fraction was
added dropwise under stirring. The solution was left to stand
overnight without stirring. The capsid protein was sedimented by
centrifugation at 14 000 rpm for 20 min. It was then solubilized in
a minimal volume of NET and loaded again on the Sepharose CL-4B
column. The peak fractions were pooled, and precipitated with
ammonium sulphate at 60% of saturation (w/v). After centrifugation
and resolubilization in NET buffer, capsid protein was loaded on a
Sepharose CL-6B column for rechromatography.
Dialysis and Drying
[0291] The peak fractions obtained above were pooled and
extensively dialysed against sterile water, and lyophilized for
storage.
Expression and Purification Q.beta.-240
[0292] Cells (E. coli JM 109, transformed with the plasmid
pQ.beta.-240) were resuspended in LB, sonicated five times for 15
seconds (water ice jacket) and centrifuged at 13000 rpm for one
hour. The supernatant was stored at 4.degree. C. until further
processing, while the debris were washed 2 times with 9 ml of LB,
and finally with 9 ml of 0.7 M urea in LB. All supernatants were
pooled, and loaded on the Sepharose CL-4B column. The pooled peak
fractions were precipitated with ammonium sulphate and centrifuged.
The resolubilized protein was then purified further on a Sepharose
2B column and finally on a Sepharose 6B column. The capsid peak was
finally extensively dialyzed against water and lyophilized as
described above. The assembly of the coat protein into a capsid was
confirmed by electron microscopy.
Expression and Purification Q.beta.-243
[0293] Cells (E. coli RR1) were resuspended in LB and processed as
described in the general procedure. The protein was purified by two
successive gel filtration steps on the sepharose CL-4B column and
finally on a sepharose CL-2B column. Peak fractions were pooled and
lyophilized as described above. The assembly of the coat protein
into a capsid was confirmed by electron microscopy.
Expression and Purification of Q.beta.-250
[0294] Cells (E. coli JM 109, transformed with pQ.beta.-250) were
resuspended in LB and processed as described above. The protein was
purified by gel filtration on a Sepharose CL-4B and finally on a
Sepharose CL-2B column, and lyophilized as described above. The
assembly of the coat protein into a capsid was confirmed by
electron microscopy.
Expression and Purification of Q.beta.-259
[0295] Cells (E. coli JM 109, transformed with pQ.beta.-259) were
resuspended in LB and sonicated. The debris were washed once with
10 ml of LB and a second time with 10 ml of 0.7 M urea in LB. The
protein was purified by two gel-filtration chromatography steps, on
a Sepharose CL-4 B column. The protein was dialyzed and
lyophilized, as described above. The assembly of the coat protein
into a capsid was confirmed by electron microscopy.
Example 2
[0296] Insertion of a Peptide Containing a Lysine Residue into the
c/e1 Epitope of HBcAg(1-149).
[0297] The c/e1 epitope (residues 72 to 88) of HBcAg is located in
the tip region on the surface of the Hepatitis B virus capsid
(HBcAg). A part of this region (Proline 79 and Alanine 80) was
genetically replaced by the peptide Gly-Gly-Lys-Gly-Gly (SEQ ID NO:
387) (HBcAg-Lys construct). The introduced Lysine residue contains
a reactive amino group in its side chain that can be used for
intermolecular chemical crosslinking of HBcAg particles with any
antigen containing a free cysteine group.
[0298] HBcAg-Lys DNA, having the amino acid sequence shown in SEQ
ID NO:78, was generated by PCRs: The two fragments encoding HBcAg
fragments (amino acid residues 1 to 78 and 81 to 149) were
amplified separately by PCR. The primers used for these PCRs also
introduced a DNA sequence encoding the Gly-Gly-Lys-Gly-Gly peptide
(SEQ ID NO: 387). The HBcAg (1 to 78) fragment was amplified from
pEco63 using primers EcoRIHBcAg(s) and Lys-HBcAg(as). The HBcAg (81
to 149) fragment was amplified from pEco63 using primers
Lys-HBcAg(s) and HBcAg(1-149)Hind(as). Primers Lys-HBcAg(as) and
Lys-HBcAg(s) introduced complementary DNA sequences at the ends of
the two PCR products allowing fusion of the two PCR products in a
subsequent assembly PCR. The assembled fragments were amplified by
PCR using primers EcoRIHBcAg(s) and HbcAg(1-149)Hind(as).
[0299] For the PCRs, 100 .mu.mol of each oligo and 50 ng of the
template DNAs were used in the 50 ml reaction mixtures with 2 units
of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO4. For both reactions,
temperature cycling was carried out as follows: 94.degree. C. for 2
minutes; 30 cycles of 94.degree. C. (1 minute), 50.degree. C. (1
minute), 72.degree. C. (2 minutes).
Primer Sequences:
TABLE-US-00018 [0300] EcoRIHBcAg(s): (SEQ ID NO:366)
(5'-CCGGAATTCATGGACATTGACCCTTATAAAG-3'); Lys-HBcAg(as): (SEQ ID
NO:367) (5'-CCTAGAGCCACCTTTGCCACCATCTTCTAAATTA-GTACCCACCCA
GGTAGC-3'); Lys-HBcAg(s): (SEQ ID NO:368)
(5'-GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTA-GTAGTCAGTTAT GTC-3');
HBcAg(1-149)Hind(as): (SEQ ID NO:369)
(5'-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3').
[0301] For fusion of the two PCR fragments by PCR 100 pmol of
primers EcoRIHBcAg(s) and HBcAg(1-149)Hind(as) were used with 100
ng of the two purified PCR fragments in a 50 ml reaction mixture
containing 2 units of Pwo polymerase, 0.1 mM dNTPs and 2 mM
MgSO.sub.4. PCR cycling conditions were: 94.degree. C. for 2
minutes; 30 cycles of 94.degree. C. (1 minute), 50.degree. C. (1
minute), 72.degree. C. (2 minutes). The assembled PCR product was
analyzed by agarose gel electrophoresis, purified and digested for
19 hours in an appropriate buffer with EcoRI and HindIII
restriction enzymes. The digested DNA fragment was ligated into
EcoRI/HindIII-digested pKK vector to generate pKK-HBcAg-Lys
expression vector. Insertion of the PCR product into the vector was
analyzed by EcoRI/HindIII restriction analysis and DNA sequencing
of the insert.
Example 3
Expression and Purification of HBcAg-Lys
[0302] E. coli strains K802 or JM109 were transformed with
pKK-HBcAg-Lys. 1 ml of an overnight culture of bacteria was used to
innoculate 100 ml of LB medium containing 100 .mu.g/ml ampicillin.
This culture was grown for 4 hours at 37.degree. C. until an OD at
600 nm of approximately 0.8 was reached. Induction of the synthesis
of HBcAg-Lys was performed by addition of IPTG to a final
concentration of 1 mM. After induction, bacteria were further
shaken at 37.degree. C. for 4 hours. Bacteria were harvested by
centrifugation at 5000.times.g for 15 minutes. The pellet was
frozen at -80.degree. C. The pellet was thawed and resuspended in
bacteria lysis buffer (10 mM Na.sub.2HPO.sub.4, pH 7.0, 30 mM NaCl,
0.25% Tween-20, 10 mM EDTA) supplemented with 200 .mu.g/ml lysozyme
and 10 .mu.l of Benzonase (Merck). Cells were incubated for 30
minutes at room temperature and disrupted by sonication. E. coli
cells harboring pKK-HBcAg-Lys expression plasmid or a control
plasmid were used for induction of HBcAg-Lys expression with IPTG.
Prior to the addition of IPTG, a sample was removed from the
bacteria culture carrying the pKK-HBcAg-Lys plasmid and from a
culture carrying the control plasmid. Four hours after addition of
IPTG, samples were again removed from the culture containing
pKK-HBcAg-Lys and from the control culture. Protein expression was
monitored by SDS-PAGE followed by Coomassie staining.
[0303] The lysate was then centrifuged for 30 minutes at
12,000.times.g in order to remove insoluble cell debris. The
supernatant and the pellet were analyzed by Western blotting using
a monoclonal antibody against HBcAg (YVS1841, purchased from
Accurate Chemical and Scientific Corp., Westbury, N.Y., USA),
indicating that a significant amount of HBcAg-Lys protein was
soluble. Briefly, lysates from E. coli cells expressing HBcAg-Lys
and from control cells were centrifuged at 14,000.times.g for 30
minutes. Supernatant (=soluble fraction) and pellet (=insoluble
fraction) were separated and diluted with SDS sample buffer to
equal volumes. Samples were analyzed by SDS-PAGE followed by
Western blotting with anti-HBcAg monoclonal antibody YVS1841.
[0304] The cleared cell lysate was used for step-gradient
centrifugation using a sucrose step gradient consisting of a 4 ml
65% sucrose solution overlaid with 3 ml 15% sucrose solution
followed by 4 ml of bacterial lysate. The sample was centrifuged
for 3 hrs with 100,000.times.g at 4.degree. C. After
centrifugation, 1 ml fractions from the top of the gradient were
collected and analyzed by SDS-PAGE followed by Coomassie staining.
The HBcAg-Lys protein was detected by Coomassie staining.
[0305] The HBcAg-Lys protein was enriched at the interface between
15 and 65% sucrose indicating that it had formed a capsid particle.
Most of the bacterial proteins remained in the sucrose-free upper
layer of the gradient, therefore step-gradient centrifugation of
the HBcAg-Lys particles led both to enrichment and to a partial
purification of the particles.
[0306] Expression and purification of HBcAg-Lys in large scale was
performed as follows. An overnight culture was prepared by
inoculating a single colony in 100 ml LB, 100 .mu.g/ml Ampicillin
and growing the culture overnight at 37.degree. C. 25 ml of the
preculture were diluted in 800 ml LB Ampicillin medium the next
day, and the culture gorwn to an optical density OD.sup.600 of
0.6-0.8. The culture was then induced with 1 mM IPTG, and left to
grow for another 4 hours. The cells were harvested and lysed
essentially as described above.
[0307] BBcAg-Lys was then purified by first precipitating the
protein with ammonium sulphate (30% saturation) from the cleared
cell lysate, then loading the resolubilized pellet on a gel
filtration column (Sephacryl S-400, Pharmacia). The pooled
fractions were precipitated again with ammonium sulphate, the
pellet resolubilized and loaded a second time on the same gel
filtration column. The fractions were finally pooled and
concentrated, and the concentration assessed using a Bradford test
(BioRad).
Example 4
Construction of a HBcAg Devoid of Free Cysteine Residues and
Containing an Inserted Lysine Residue
[0308] A Hepatitis core Antigen (HBcAg), referred to herein as
HBcAg-lys-2cys-Mut, devoid of cysteine residues at positions
corresponding to 48 and 107 in SEQ ID NO:77 and containing an
inserted lysine residue was constructed using the following
methods.
[0309] The two mutations were introduced by first separately
amplifying three fragments of the HBcAg-Lys gene prepared as
described above in Example 2 with the following PCR primer
combinations. PCR methods and conventional cloning techniques were
used to prepare the HBcAg-lys-2cys-Mut gene.
[0310] In brief, the following primers were used to prepare
fragment 1:
TABLE-US-00019 Primer 1: EcoRIHBcAg(s) (SEQ ID NO:366)
CCGGAATTCATGGACATTGACCCTTATAAAG Primer 2: 48as (SEQ ID NO:370)
GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC
[0311] The following primers were used to prepare fragment 2:
TABLE-US-00020 Primer 3: 48s (SEQ ID NO:371)
GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC Primer 4: 107as (SEQ ID
NO:372) CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC
[0312] The following primers were used to prepare fragment 3:
TABLE-US-00021 Primer 5: HBcAg149hind-as (SEQ ID NO:369)
CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGA-AGCGTTGATAG
TABLE-US-00022 Primer 6: 107s GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG
(SEQ ID NO:373)
[0313] Fragments 1 and 2 were then combined with PCR primers
EcoRIHBcAg(s) and 107 as to give fragment 4. Fragment 4 and
fragment 3 were then combined with primers EcoRIHBcAg(s) and
HBcAg149hind-as to produce the full length gene. The full length
gene was then digested with the EcoRI (GAATTC) and HindIII (AAGCTT)
enzymes and cloned into the pKK vector (Pharmacia) cut at the same
restriction sites. Expression and purification of
HBcAg-lys-2cys-Mut were performed as set out in Example 3.
Example 5
Construction of HBcAg1-185-Lys
[0314] Hepatitis core Antigen (HBcAg) 1-185 was modified as
described in Example 2. A part of the c/e1 epitope (residues 72 to
88) region (Proline 79 and Alanine 80) was genetically replaced by
the peptide Gly-Gly-Lys-Gly-Gly (HBcAg1-185-Lys construct, SEQ ID
NO: 78). The introduced Lysine residue contains a reactive amino
group in its side chain that can be used for intermolecular
chemical crosslinking of HBcAg particles with any antigen
containing a free cysteine group. PCR methods and conventional
cloning techniques were used to prepare the HBcAg1-185-Lys
gene.
[0315] The Gly-Gly-Lys-Gly-Gly sequence (SEQ ID NO: 387) was
inserted by amplifying two separate fragments of the HBcAg gene
from pEco63, as described above in Example 2 and subsequently
fusing the two fragments by PCR to assemble the full length gene.
The following PCR primer combinations were used:
[0316] Fragment 1:
Primer 1: EcoRIHBcAg(s) (see Example 2)
Primer 2: Lys-HBcAg(as) (see Example 2)
[0317] Fragment 2:
Primer 3: Lys-HBcAg(s) (see Example 2)
TABLE-US-00023 [0318] Primer 4: HBcAgwtHindIIII (SEQ ID NO:374)
CGCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG
[0319] Assembly:
Primer 1: EcoRIHBcAg(s) (see example 2)
Primer 2: HBcAgwtHindIII
[0320] The assembled full length gene was then digested with the
EcoRI (GAATTC) (4-9 of SEQ ID NO:366) and HindIII (AAGCTT) (9-14 of
SEQ ID NO:374) enzymes and cloned into the pKK vector (Pharmacia)
cut at the same restriction sites.
Example 6
Fusion of a Peptide Epitope in the MIR Region of HbcAg
[0321] The residues 79 and 80 of HBcAg1-185 were substituted with
the epitope C.epsilon.H3 of sequence VNLTWSRASG. The C.epsilon.H3
sequence stems from the sequence of the third constant domain of
the heavy chain of human IgE. The epitope was inserted in the
HBcAg1-185 sequence using an assembly PCR method. In the first PCR
step, the HBcAg1-185 gene originating from ATCC clone pEco63 and
amplified with primers HBcAg-wt EcoRI fwd and HBcAg-wt Hind III rev
was used as template in two separate reactions to amplify two
fragments containing sequence elements coding for the C.epsilon.H3
sequence. These two fragments were then assembled in a second PCR
step, in an assembly PCR reaction.
[0322] Primer combinations in the first PCR step: C.epsilon.H3fwd
with HBcAg-wt Hind III rev, and HBcAg-wt EcoRI fwd with
C.epsilon.H3rev. In the assembly PCR reaction, the two fragments
isolated in the first PCR step were first assembled during 3 PCR
cycles without outer primers, which were added afterwards to the
reaction mixture for the next 25 cycles. Outer primers: HBcAg-wt
EcoRI fwd and HBcAg-wt Hind III rev.
[0323] The PCR product was cloned in the pKK223.3 using the EcoRI
and HindIII sites, for expression in E. coli (see Example 2). The
chimeric VLP was expressed in E. coli and purified as described in
Example 2. The elution volume at which the HBcAg1-185-C.epsilon.H3
eluted from the gel filtration showed assembly of the fusion
proteins to a chimeric VLP.
Primer Sequences:
C.epsilon.H3fwd:
TABLE-US-00024 [0324] (SEQ ID NO:375) 5'GTT AAC TTG ACC TGG TCT CGT
GCT TCT GGT GCA TCC AGG GAT CTA GTA GTC 3' (SEQ ID NO:376) V N L T
W S R A S G A80 S R D L V V86
C.epsilon.H3rev:
TABLE-US-00025 [0325] (SEQ ID NO:377) 5' ACC AGA AGC ACG AGA CCA
GGT CAA GTT AAC ATC TTC CAA ATT ATT ACC CAC 3' (SEQ ID NO:378) D78
E L N N G V72
HBcAg-wt EcoRI fwd:
TABLE-US-00026 [0326] 5' CCGgaattcATGGACATTGACCCTTATAAAG (SEQ ID
NO:366)
HBcAg-wt Hind III rev:
TABLE-US-00027 [0327] (SEQ ID NO:374) 5'
CGCGTCCCaagcttCTAACATTGAGATTCCCGAGATTG
Example 7
[0328] Cloning, expression and purification of IL-5 with an
N-terminal amino acid linker containing a cysteine residue.
Coupling to VLP, immunization and demonstration of efficacy in an
experimental model of allergic asthma with an eosinophilic
component.
A. Cloning of Mouse His-C-IL-5 and Expression as Inclusion Bodies
in E. Coli
[0329] IL-5 was amplified from an ATCC clone (pmIL5-4G; ATCC
number: 37562) by PCR using the following two primers:
Spelinker3-F1 (SEQ ID NO:340) and I15StopXho-R (SEQ ID NO:342). The
product of this PCR was used as template for a second PCR with the
primers SpeNlinker3-F2 (SEQ ID NO: 341) and 115StopXho-R. The
insert was digested with SpeI and NotI. This insert was ligated
into a pET vector derivative (pMODEC.sub.3-8 vector), previously
digested with Nhe I and Not I, and transformed into E. coli TG1
cells. The construct generated by cloning IL5 into pMODEC3-8
comprises, from its N-terminus, a hexa-histidine tag (to facilitate
purification), an Enterokinase cleavage site, a gamma 3 derived
amino acid linker (flanked N-terminally by the amino acids ALV and
C-terminally by AS) containing a cysteine residue and the DNA
encoding the mature form of IL-5 protein. Fidelity of the cloning
procedure was confirmed by DNA sequencing.
[0330] The construct containing IL-5 described above was termed
pMODC6-IL5.2 (also referred to as pMODC6-IL5) and transformed into
E. coli strain BL21-DE3. The recombinant protein expressed in E.
coli is termed His-C-IL5.
[0331] Clonal BL21-DE3 cells harboring pMODC6-IL5 were grown over
night in 5 ml of LB containing 1 mg/L Ampicillin. A 2.0 ml aliquot
of this culture was diluted into 100 ml terrific broth (TB)
containing 1 mg/L Ampicillin. The culture was grown to an optical
density, OD.sub.600nm, of 0.7-1.0 and expression induced for 4
hours by adding 0.1 ml of a 1.0 M stock of Ispropyl
.beta.-D-Thiogalactopyranoside (IPTG). Recombinant His-C-IL5 was
expressed in an insoluble form and located in the inclusion body
fraction of induced cells. Expression of His-C-IL5 was confirmed in
the flowing manner. A 10 ml sample of culture was taken 4 hours
after induction and centrifuged for 10 min at 4000.times.g. The
pellet was suspended in 0.5 ml lysis buffer consisting of 50 mM
Tris-HCl, 2 mM EDTA, 0.1% triton X-100 (pH 8.0). To the suspension
was added 20 .mu.l of Lysozyme (40 mg/ml) and after 30 min at
4.degree. C. sonicated for 2 min. A 1.0 ml aliquot of benzonase and
100 .mu.l aliquot of 50 mM MgCl.sub.2 were added and incubated for
30 min at room temperature. After centrifugation for 15 min at
13000.times.g the supernatant was discarded and the pellet heated
for 5 min at 98.degree. C. in 100 .mu.l of SDS loading buffer.
Aliquots of 10 .mu.l were then analyzed by SDS-PAGE under reducing
conditions (FIG. 17A). SDS-PAGE analysis demonstrated a protein
band of 17 kDa corresponding to the mass of IL-5. As control,
BL21-DE2 cells containing pMODC6-IL5 were grown in the absence of
IPTG and extracts prepared from the insoluble cell fraction as
described above.
B. Purification and Refolding of Mouse-His-C-IL5.
[0332] A larger scale expression of IL-5 from clone pMODC6-IL5 in
BL21-DE3 cells was performed in order to obtain sufficient
quantities of pure IL-5 for vaccine production. Overnight cultures
were grown and diluted into either 100 ml or IL volumes of TB
medium containing 1.0 mg/L Ampicillin. A total of 3 liters of
culture was thus prepared and grown at 37.degree. C. until
OD.sub.600nm reached 0.7 at which time IPTG was added to give a
final concentration of 1.0 mM. After 4 h incubation cells were
harvested by centrifugation for 30 min at 10 000.times.g. After
harvesting the pellet was resuspended in PBS (5.0 ml/g wet weight)
and centrifuged for 15 minutes at 10 000.times.g. The washed pellet
was stored at -20.degree. C. until further use.
[0333] The bacterial pellet was suspended in PBS (2.0 ml/g cell wet
weight) using a Dounce homogenizer. Lysozyme (0.8 mg/ml) was added
to the suspension and incubated for 30 minutes at room temperature.
The suspension was sonicated for 1 minute, 3 times on ice then
benzonase and MgCl.sub.2 (10 mM final concentration) were added and
incubated for 30 minutes at room temperature. Triton X-100 was
added to a final concentration of 1% (w/v) the mixture gently
stirred at room temperature for 30 minutes. The solution was
centrifuged for 20 minutes at 20 000.times.g (SS34 tubes) and the
supernatant discarded. The pellet harbouring the inclusion bodies
was suspended (5.0 ml/g wet weight) in washing buffer (PBS
containing 2M Urea and 1% (w/v) Triton X-100) using a Dounce
homogenizer and agitated for 5 minutes. The solution was
centrifuged for 20 minutes at 20 000.times.g and the supernatant
discarded. The pellet was washed and centrifuged as above 2 more
times. A final wash of the inclusion bodies was performed with
washing buffer in the absence of Triton X-100.
[0334] The His-C-IL-5 present in inclusion bodies of the pellet was
solubilized in (5.0 ml/g cell wet weight) denaturing buffer (100 mM
NaH.sub.2PO.sub.4, 10 mM Tris-HCl, 6.0 M Guanidine-hydrochloride,
pH 8.0) and gently stirred for 1 h at 25.degree. C. The suspension
was centrifuged for 20 min. at 20 000.times.g and the supernatant
mixed with Ni-NTA resin (QIAgen, equilibrated with solubilization
buffer). After 3 hours of gentle agitation at 4.degree. C. the
slurry was poured into a glass column (C10/10) and the resin washed
with 100 ml of 100 mM NaH.sub.2PO.sub.4, 10 mM Tris, 6.0 M
Guanidine-hydrochloride (pH 6.3). An additional washing step was
performed with 15 ml of 100 mM NaH.sub.2PO.sub.4, 10 mM Tris, 6.0 M
Guanidine-hydrochloride (pH 5.9). Mouse His-C-IL5 was eluted from
the resin by applying 20 ml of 100 mM NaH.sub.2PO.sub.4, 10 mM
Tris, 6.0 M Guanidine-hydrochloride (pH 4.5). Purification was
analyzed by SDS-PAGE.
[0335] Fractions from the elution step containing His-C-IL-5 were
pooled and dialysed against buffer comprising 8.0 M Urea 100 mM
NaH.sub.2PO.sub.4, 10 mM Tris-HCl (pH 8.0) at 4.degree. C. using a
10 kDa cut-off membrane. Following dialysis, the protein
concentration was determined spectrophotometrically using the
following formula; Protein
(mg/ml)=(1.55.times.A.sub.280nm)-(0.76.times.A.sub.260nm). The
concentration of the protein was diluted with dialysis buffer to
0.2 mg/ml. The solution was then dialysed with a 3.5 kDa membrane
for 24 hours at 4.degree. C. against refolding buffer 1 comprising
2.0 M urea, 50 mM NaH.sub.2PO.sub.4, 5 mM reduced Glutathione, 0.5
mM oxidized Glutathione, 0.5 M Arginine, 10% (v/v) glycerol (pH
8.5) and for a further 24 h against another refolding buffer 2
comprising 50 mM NaH.sub.2PO.sub.4, 5 mM reduced Glutathione, 0.5
mM oxidized Glutathione, 0.5 M Arginine, 10% (v/v) glycerol, (pH
8.5). At the end the protein was dialysed for 24 h at 4.degree. C.
against PBS pH 8.0 then centrifuged at 10 000.times.g for 30 min.
The protein content of the supernatant was estimated by Bradford
assay.
[0336] In order to further purify His-C-IL5, anion exchange with
Hitrap Q resin (Amersham Pharmacia, Uppsala Sweden) was performed.
His-C-IL5 was concentrated to 1 mg/ml using Centrifugal Filters
(Ultrafree-15 Millipore, 10 kDa cut-off) and dialyzed for 14 h
against 50 mM Phosphate buffer pH 8.4. The solution was loaded onto
a Hitrap Q column and washed with 50 mM Phosphate pH 8.4 buffer.
His-C-IL-5 was eluted from the column by applying a NaCl gradient
from 0-1 M. His-C-IL5 eluted from the column at 100 mM NaCl.
Analysis of the purification was performed by SDS-PAGE and
concentration measured by Bradford assay. Quaternary structure of
the protein was assessed by SDS-PAGE performed under non-reducing
conditions.
C. Vaccine Production: Coupling His-C-IL5 to Q.beta.
[0337] A variety of conditions were investigated to optimize the
efficiency of the coupling reaction. These included the addition of
reducing agent, (TCEP) to His-C-IL5 and varying the molar ratios of
Q.beta. monomer and His-C-IL5 in the coupling reaction and are
summarized in Table 1. The vaccine for the efficacy study was
produced in the following way. Purified His-C-IL-5 (40 .mu.M) was
reduced for 1 h with an equimolar amount of TCEP in PBS pH 8.0.
Reduced IL-5 (80 .mu.M) was incubated for 4 hours at 22.degree. C.
with 40 .mu.M Q.beta. derivatized with SMPH in a total volume of
700 .mu.l. The reaction was dialysed 12 hours against PBS pH 8.0
using a 300 kDa cut-off dialysis membrane. The coupling reaction
was analysed by SDS-PAGE and Western-Blot with anti-His and
anti-Q.beta. antibodies. Protein concentration was measured by
Bradford. The coupling efficiency [i.e. mol Q.beta.-IL5/mol Q.beta.
monomer (total)] was estimated, by densitometric analysis of the
Coomassie blue stained SDS-PAGE.
TABLE-US-00028 TABLE 1 Different coupling conditions used to
optimize the chemical cross-linking of His-C-IL5 to Q.beta..
Concentration of Concentration of TCEP/IL5 ratio derivatized
Q.beta. (.mu.M) His-C-IL5 (.mu.M) (.mu.M) 70 40 No TCEP 70 40 1:2
70 40 1:1 70 40 1.5:1 70 40 2:1 70 40 16.6:1 20 30 No TCEP 20 30
1:2 20 30 1:1 20 30 1.5:1 20 30 2:1 20 30 16.6:1
D. ELISA to Assess Vaccine
[0338] The coupling of mouse His-C-IL5 to Q.beta., was assessed
using a "quadruple" ELISA which is represented in FIG. 4. A 96 well
ELISA plate was coated over-night with 100 ul of 1 mg/L goat
anti-rabbit IgG per well. The plate was washed four times with
PBS-Tween 0.1% (v/v) (PBST) then blocked for 2 h at 37.degree. C.
with 2% (w/v) Bovine serum albumin (BSA) in PBST. After washing
with PBST polyclonal, anti-Q.beta. serum from rabbit (diluted
1:5000) was added and incubated for 1 h. The plate was washed twice
with PBST and either varying amounts of Q.beta.-His-C--IL5 or
control were added (FIG. 5) and incubated for 1 h at 25.degree. C.
Two different tertiary antibodies were used in the assay; rat
anti-mouse IL5 (TRFK4) or rat anti-mouse IL5 (TRFK5), both are
neutralizing monoclonal antibodies. All were used at concentrations
of 1 .mu.g/ml. The detecting antibodies were conjugated with Horse
Radish Peroxidase (HRP) and specific for the particular Fc-fragment
of the tertiary antibody. Binding in the sandwich assay was
measured by a chemiluminescence (ECL) at 450 nm.
F. Assay of IL-5 Activity
[0339] The ability of the B cell lymphoma line BCL1 to proliferate
in response to murine IL-5 was used to check the bioactivity of the
re-folded recombinant His-C-IL-5 (Harriman G. R. (1991) Current
Protocols in Immunology 6.5.1-6.5.5 John Wiley and Sons Inc). The
proliferative activity of His-C-IL5 covalently coupled to Q.beta.
was also assessed. Recombinant murine IL-5 (R&D systems,
Minneapolis USA) was used as a control. The various forms of
recombinant IL-5 were incubated in flat bottom 96 well plates with
2.times.10.sup.4 BCL1 cells per well and incubated for 24 h at
37.degree. C., 5% CO.sub.2.1 .mu.Ci of .sup.3H-Thymidine (Hartmann
Analytic, Switzerland) was added to each well and the plates
incubated for another 6 h at 37.degree. C. 5% CO.sub.2. The cells
are harvested, washed and the incorporation of Thymidine determined
by counting the .beta.-emission with a liquid scintillation
counter.
G. Immunization Protocol
[0340] In order to generate self reactive antibodies to mouse IL-5,
four BalbC mice were injected subcutaneously a day 0 and day 14
with 25 .mu.g of Q.beta.-His-C-IL5 vaccine in 200 .mu.l of PBS. To
serve as a negative control, five mice were immunized at day 0 and
14 with a simple mixture of 6.4 .mu.g Q.beta. and 16 .mu.g IL5 i.e.
not covalently coupled (Q.beta.+His-C-IL-5) in PBS. Mice were bled
prior to immunization and at day 21 of the immunisation protocol.
Sera were analysed by ELISA.
[0341] H. Sera Analysis
[0342] ELISA. Maxisorp ELISA plates (Nunc) were coated with 50
.mu.l of purified His-C-IL-5 (3 .mu.g/ml) for 14 h at 4.degree. C.
The plates were washed 3 times with PBS and blocked with 2% BSA in
PBS for 2 h at 37.degree. C. then washed twice with PBS. Five-fold
dilutions of sera were added in 2% BSA, 0.1% FCS in PBS and
incubated at room temperature for 1 hour. The plates were
subsequently washed 3 times with PBS and incubated with anti-mouse
IgG conjugated with HRP (dilution 1:1000) at room temperature for 1
h. The plates were again washed 3 times with PBS and 100 .mu.l/well
developing solution (0.066 M Na2HPO4, 0.035 M citric acid, 0.032%
H.sub.2O.sub.2, 0.4% 1,2-Phenylenediamine dihydrochloride) were
added. After 2 minutes of reaction at room temperature the ELISA
was stopped with 50 .mu.l per well 5% H.sub.2SO.sub.4. Absorbance
was measured at 450 nm on a Spectramax spectrophotometer (Molecular
Devices).
[0343] Western blot staining with serum of mice immunized with
Q.beta.-IL5. His-C-IL5, Q.beta. and controls were separated by
SDS-PAGE and electroblotted onto a nitrocellulose membrane. The
membrane was blocked for 1 h with 5% (w/v) milk powder in PBS, then
incubated with 20 .mu.l of day 21 serum from vaccinated mice in 10
ml 1% (w/v) milk powder in PBS. The membrane was washed with PBS
for 15 minutes and then incubated for 1 h with 10 ml 1% (w/v) milk
powder in PBS containing anti-mouse IgG antibody conjugated with
horse radish peroxidase (HRP) at a dilution of 1:1000. The membrane
was washed for 15 minutes in PBS and developed with ECL (Amersham
Pharmacia, Sweden) and exposed to Photographic film.
I. Eosinophilia Model.
[0344] An experimental asthma model of allergic airway inflammation
was used to assess the effects of vaccination on eosinophilia.
Balb/c mice (4 per group) were immunised with either
Q.beta.-His-C-IL-5 as described above. At day 23 of the vaccination
program mice were injected intraperitoneally with 50 .mu.g
Ovalbumin (OVA) in Alumn (Alu-Gel-S) A third group of 4 mice which
received no immunisation, were also injected. After 10 days (i.e.
day 33) the mice received 100 .mu.g OVA in PBS administered
intranasally each day for 4 days. 24 hours after the last challenge
the mice were sacrificed and the lungs washed with PBS. The cells
contained in the broncheo alveolar lavage (BAL) were stained with
Maigrunwald-Giemsa and counted (Trifilieff A, et al. Clin Exp
Allergy. 2001 June; 31 (6):934-42.
RESULTS AND DISCUSSION
[0345] Expression. Expression of the construct pMODC6-IL5 in
BL21-DE2 cells was analysed by SDS-PAGE (FIG. 1). The Coomassie
Blue stained gel demonstrated the IPTG-induced expression of a 17
kDa protein corresponding to the mass of IL-5. As control, BL21-DE2
cells containing pMODC6-IL5 were grown in the absence of IPTG and
extracts prepared from the insoluble cell fraction as described
above. As expected there was no induction of a 17 kDa under these
conditions. His-C-IL5 was localized in the insoluble inclusion body
fraction.
[0346] Extraction purification and refolding. Insoluble His-C-IL5
was extracted from detergent washed inclusion bodies with 6M
guanidine hydrochloride. The solubilised protein was purified by
metal chelate affinity chromatography and analysed by SDS-PAGE
(FIG. 2). Recombinant His-C-IL5 was found to be highly enriched by
this procedure. The denatured protein was subjected to a refolding
procedure in urea as described above and further purified by anion
exchange chromatography. These steps yielded soluble, highly pure
His-C-IL5 as judged by SDS-PAGE (FIG. 5, lane 1) with a recovery of
23% and yield of 6.9 mg.
[0347] Since biologically active native IL-5 is a disulfide-linked
homodimer, the ability of purified recombinant His-C-IL5 to form
dimmers was assessed by SDS-PAGE performed under non-reducing
conditions (FIG. 3). As judged by the molecular mass of 37 kDa,
His-C-IL5 was demonstrated be dimeric in nature indicating
conservation of the native quaternary structure.
[0348] The biological activity of recombinant His-C-IL5 was
assessed by determining its ability to stimulate proliferation of a
murine B cell line (FIG. 4). BCL1 cells cultured in the presence of
His-C-IL5 were shown to have enhanced proliferative rates when
compared to culture medium alone or other proteins. Furthermore the
enhanced proliferation was similar to that observed for a
commercially obtained murine IL-5. The ability of His-C-IL5 to
stimulate B cell proliferation, presumably by interacting with it's
cognate receptor, and to adopt a dimeric structure both indicate
the recombinant protein has adopted native conformation.
[0349] Vaccine production and analytics. The covalent chemical
coupling of His-C-IL5 to the virus-like particle Q.beta. was
assessed by SDS-PAGE and Western blot analyses. Coomassie blue
stained gels of the coupling reaction demonstrated the appearance
of bands with molecular weights corresponding to those predicted
for His-C-IL5 covalently linked to Q.beta. (FIG. 5). Moreover,
Western analyses showed co-localisation of these bands when stained
with either anti-His or anti-Q.beta. antibodies (FIG. 6). The
coupling efficiency [i.e. mol Q.beta.-IL5/mol Q.beta. monomer
(total)] was estimated, by densitometric analysis of the Coomassie
blue stained SDS-PAGE, to be of 40.6%.
[0350] The ability of His-C-IL-5 covalently cross-linked to Q.beta.
to stimulate B cell proliferation was assessed as described
previously. FIG. 5 shows that Q.beta.-His-C-IL5 was able to cause
enhanced proliferation compared to Q.beta. coupled to an unrelated
cytokine.
[0351] The conformation of His-C-IL5 coupled to Q.beta. was further
analysed using a quadruple ELISA. (FIG. 7a). FIG. 7b, demonstrates
that His-C-IL5 is recognised by the IL-5 neutralising monoclonal
antibodies TRFK 4 and TRFK 5. When the reaction was performed with
Q.beta. rather than Q.beta.-His-C-IL-5 no signal was detected. The
monoclonal antibody TRFK4 recognises a neutralising epitope within
IL-5. The ability of the IL-5 specific monoclonal antibodies to
recognise covalently linked His-C-IL-5 indicates the neutralising
epitopes are conserved within the vaccine preparation.
[0352] Analysis of sera. Preimmune sera and day 21 sera from mice
vaccinated with Q.beta.-His-C-IL5 were collected and analysed by
ELISA (FIG. 8). The result shows that immunological tolerance
towards the self-antigen 1L-5 was overcome in the absence of
adjuvant and after only in 4/4 vaccinated mice. Half maximal titres
were calculated to be in the range of 1:2000 to 1:6000. In the
control group that received Q.beta. mixed with His-C-IL5 no
significant anti-IL-5 titres were detected. However, 3 of the 5
mice produced a low antibody titre<1:50. Immune sera from mice
vaccinated with Q.beta.-His-C-IL5 were further tested by Western
blot analysis. In all cases the immune sera specifically recognized
murine IL-5.
[0353] Vaccine efficacy in an animal model of experimental asthma.
The effect of vaccination with Q.beta.-His-C-IL-5 on eosinophilia
was assessed in a murine model of allergic airway inflammation that
mimics key pathological events in asthma. This experiment tested
the ability of the anti-IL5 antibodies generated by vaccination
with Q.beta.-His-C-IL-5 to down-regulate the in vivo action of
endogenous IL-5. In the control experiment mice were vaccinated
with PBS before OVA sensitisation and challenge. In this case high
numbers of eosinophils were counted in the BAL. The mean number of
eosinophils/200 cells counted was 96.+-.14 S. D. In contrast mean
value of the BAL eosinophils from the four mice vaccinated with
Q.beta.-His-C-IL-5 was 27.5+11 S.D./200 cells counted. This is a
reduction of 71.4% and is evidence the autoantibodies generated by
immunisation with His-C-IL-5 presented as a highly ordered immune
array recognise the endogenous target molecule and thereby down
regulate eosinophilia in an experimental model of asthma.
Example 8
Molecular Cloning, Expression, Refolding and Purification of Mouse
mIL-13 with a C-Terminal Amino Acid Linker Containing a Cysteine
Residue for Coupling to VLPs and Pili. Coupling of Mouse
Interleukin 13 to VLPs and Pili
A. Cloning IL-13 for Prokaryotic Expression.
[0354] The DNA for cloning IL-13 was isolated by RT-PCR from in
vitro activated splenocytes, which were obtained as following: CD4+
T cells were isolated from mouse spleen cells and incubated 3 days
in IMDM (+5% FCS+10 ng/ml IL4) in 6 well plates previously coated
with anti-CD3 and anti-CD28 antibodies. RNA from these cells was
used to amplify cDNA encoding IL13 by one-step RT-PCR (Qiagen
one-step PCR kit). Primer XhoIL13-R was used for reverse
transcription of the RNA and the primers NheIL13-F (SEQ ID NO:338)
and XhoIL13-R (SEQ ID NO:339) were used for the PCR amplification
of the IL13 cDNA. Amplified IL13 cDNA was ligated in a pMOD vector
using the NheI/XhoI restriction sites (giving the vector
pMODB1-IL13). The identity of the resulting cDNA sequence was
determined by nucleotide sequencing.
[0355] Using the same primer, NheIL13-F (SEQ ID NO:338) and
XhoIL13-R (SEQ ID NO:339), the IL-13 cDNA was amplified from the
pModB1-IL13 plasmid and ligated into the pMODGST-EK-C1 vector
resulting in the plasmid pModGST-EK-IL13-C1. The cDNA sequence of
this plasmid was determined by nucleotide sequencing. A cDNA
comprising the coding sequence for the glutathione S transferase
fused to an enterokinase cleavage site followed by the IL-13
sequence with the C-terminal linker 1 was amplified by PCR with the
primer GST-BamHI ss and C1-BsmBI/XhoI using the plasmid
pModGST-EK-IL13-C1 as template. This cDNA was digested with
restriction enzymes BamHI and BsmBI and ligated into the pModB-N1
vector using the BamHI/XhoI restriction site. The resulting plasmid
pMod-GST-EK-IL13-C1-His encodes a fusion protein consisting of
glutathione S transferase, an enterokinase cleavage site, IL-13, a
cysteine containing linker and a polyhistidine-tag
(GST-EK-IL13-C1-His). The identity of the cDNA encoding this fusion
protein was confirmed by nucleotide sequencing.
Sequence of Oligonucleotides:
GST-BamHI ss:
TABLE-US-00029 [0356] 5'-CGCCGGATCCTATACTAGGTTATTGG-3' (SEQ ID
NO:379)
C1-BsmBI/XhoI as:
TABLE-US-00030 [0357] (SEQ ID NO:380)
5'-GGGCGCGTCTCCTCGAGACCGCAACCACCACCA-3'
B. Expression of IL-13 in E. coli.
[0358] The plasmid pMod-GST-EK-IL13-C1-His was transformed into the
bacterial host strain BL 21 (DE3). After 90 minutes of recovery in
LB-Media containing 2% Glucose (preculture), 250 ml MOPS-buffered
SB-Media containing 0.2% Glucose and 100 .mu.g Ampicillin/1 was
inoculated with 250 .mu.l preculture and incubated on a shaking
platform at 37.degree. C. over night. The next morning the seed
culture was diluted with 750 ml prewarmed MOPS-buffered SB-Media
containing 100 .mu.g Ampicillin/I and incubated on a shaking
platform with 125 rpm at 37.degree. C. for another 90 min until an
OD.sub.600 of 4.5 was reached. The 1000 ml culture was diluted with
500 ml MOPS-buffered SB-Media containing 100 .mu.g Ampicillin/I and
shifted to a 24.degree. C. incubator where it was incubated with
shaking platform for 30 min until an OD.sub.600 of 3.75 was
reached. Expression of the GST-EK-IL13-C1-His fusion protein was
induced by adding 0.75 mM IPTG. After 4 hrs bacteria were harvested
by centrifugation and disrupted by sonication.
C. Purification of IL-13 from Inclusion Bodies Under Denaturing
Conditions:
[0359] After lysis the inclusion bodies were sedimented by low
speed centrifugation (10 000 g, 60 min., at 4.degree. C.). The
supernatant was collected and centrifuged again under the same
conditions. Pellets were kept as crude inclusion bodies fraction.
The inclusion bodies were washed 4 times with the following
wash-buffer: 50 mM TrisHCl, pH 7.6, 250 mM NaCl, 5 mM MgCl.sub.2, 2
M Urea, 2% Triton X-100 and 10 U Benzonase/ml. Inclusion bodies
were collected by centrifugation and resuspended in denaturing
buffer containing 100 mM NaH.sub.2PO.sub.4, 10 mM TrisHCl and 6 M
Guanidine-HC pH 8.0. Inclusion bodies were sonicated in the
presence of 10 U Benzonase/ml and incubated for 2 hrs on a rotating
wheel at room temperature. After centrifugation the supernatant
were retained and the pellets resuspended again in denaturing
buffer and treated as described above. Supernatants were pooled and
loaded onto Ni.sup.2+-agarose column equilibrated with the
denaturing buffer. Bound protein was eluted in two steps with
denaturing buffer pH 6.3 and pH 4.5. Aliquots of the fractions were
analysed by Amidoblack staining and after TCA-precipitation by
SDS-PAGE (FIG. 10).
D. Refolding GST-EK-IL13-C1-His.
[0360] P-Mercaptoethanol was added to the eluted protein to a final
concentration of 10 mM and dialysed overnight against 2 liters of
buffer containing 8.0 M Urea, 100 mM NaH.sub.2PO.sub.4, 10 mM
TrisHCl, 10 mM .beta.-Mercaptoethanol (pH 8.0) at 4.degree. C.
using a 10 kDa cut-off membrane. Following dialysis, the protein
concentration was determined and the concentration of the protein
diluted with dialysis buffer to 0.2 mg/ml. The solution was
dialysed for 24 hrs at 4.degree. C. against refolding-buffer 1
comprising 2.0 M Urea, 50 mM NaH.sub.2PO.sub.4, 5 mM reduced
Glutathione, 0.5 mM oxidized Glutathione, 0.5 M Arginine, 10% (v/v)
glycerol (pH 8.5). The next day the refolding buffer 1 was
exchanged against refolding buffer 2 containing 50 mM
NaH.sub.2PO.sub.4, 2.5 mM reduced Glutathione, 0.25 mM oxidized
Glutathione, 0.25 M Arginine, 10% (v/v) glycerol (pH 8.5) and
dialysed at 4.degree. C. for another 24 hrs. Finally the solution
was dialysed at 4.degree. C. against refolding buffer 3 comprising
20 mM ethanolamine, 150 mM NaCl and 10% (v/v) glycerol (pH 9.0).
Refolding buffer 3 was exchanged once after 2 hrs and dialysis
proceeded for another 14 hrs. The dialysate was centrifuged at
4.degree. C. and 20 000 g for 15 min. The supernatant was retained
and the protein concentrated by centrifugation in "biomax
centrifugal filter devices" with a 5 kDa molecular weight cut-off
(Millipore) to a final protein concentration of 2 mg/ml. Protein
was analysed by SDS-PAGE and Western blot with monospecific
antibodies against GST, mouse IL-13 and the His-tag,
respectively.
E. Cleavage of GST-EK-IL13-C1-His Fusion Protein with
Enterokinase:
[0361] The GST-EK-IL13-C1-His fusion protein is incubated with
1.times. enterokinase buffer (50 mM TrisHCl pH 8.0, 10 mM
CaCl.sub.2 and 1% Tween-20) and 1 U Enterokinase (Invitrogene) per
12.5 ug fusion protein for 24 hrs at 4.degree. C.
F. Purification of IL13-C1-His:
[0362] After the enterokinase treatment, cleaved GST is separated
by a combination of ion-exchange chromatography, gelfiltration and
affinity chromatography. The IL-13-C1-His protein is concentrated
to a final protein concentration of 2 mg/ml.
G. Preparing the IL-13-C1-His Protein for the Coupling
Reaction:
[0363] In order to determine optimal conditions for coupling the
IL-13-C1-His protein is treated under mild reducing conditions with
various concentrations (0 .mu.M to 500 .mu.M) of a reducing reagent
(DTT or TCEP). The reduced IL-13-C1-His protein is tested for
efficient coupling to derivatized VLPs and Pilis.
H. Coupling of IL-13-C1-His to Q.beta. Capsids:
[0364] A solution of 120 .mu.M Q.beta. capsid in 20 mM Hepes, 150
mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar
excess of a heterobifunctional crosslinker like SMPH (Pierce),
diluted from a stock solution in DMSO, at 25.degree. C. on a
rocking shaker. The reaction solution is subsequently dialyzed
twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2
at 4.degree. C. The dialyzed, derivatized Q.beta. reaction mixture
is then mixed with the prepared IL-13-C1-His protein. In the
coupling reaction the IL-13-C1-His protein is in twofold molar
excess over the derivatized Q.beta. capsid. The coupling reaction
proceeds for four hours at 25.degree. C. on a rocking shaker.
Coupling products are analysed by SDS-PAGE and in addition by
Westernblot.
Coupling of IL-13-C1-His to fr Capsid Protein
[0365] A solution of 120 .mu.M fr capsid in 20 mM Hepes, 150 mM
NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess
of SMPH (Pierce), diluted from a stock solution in DMSO, at
25.degree. C. on a rocking shaker. The reaction solution is
subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes,
150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed fr capsid protein
reaction mixture is then reacted with the prepared IL-13-C1-His
protein. In the coupling reaction the IL-13-C1-His protein is in
twofold molar excess over the derivatized fr capsid. The coupling
reaction proceeds for four hours at 25.degree. C. on a rocking
shaker. Coupling products are analysed by SDS-PAGE and in addition
by Westernblot.
Coupling IL-13-C1-His to HBcAg-Lys-2cys-Mut
[0366] A solution of 120 .mu.M HBcAg-Lys-2cys-Mut capsid in 20 mM
Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold
molar excess of SMPH (Pierce), diluted from a stock solution in
DMSO, at 25.degree. C. on a rocking shaker. The reaction solution
is subsequently dialyzed twice for 2 hours against 1 L of 20 mM
Hepes, 150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed
HBcAg-Lys-2cys-Mut reaction mixture is then reacted with the
prepared IL-13-C1-His protein. In the coupling reaction the
IL-13-C1-His protein is in twofold molar excess over the
derivatized HBcAg-Lys-2cys-Mut capsid. The coupling reaction
proceeds for four hours at 25.degree. C. on a rocking shaker.
Coupling products are analysed by SDS-PAGE and in addition by
Westernblot.
Coupling of IL-13-C1-His protein to Pili
[0367] A solution of 125 .mu.M Type-1 pili of E. coli in 20 mM
Hepes, pH 7.4, is reacted for 60 minutes with a 50-fold molar
excess of cross-linker SMPH, diluted from a stock solution in DMSO,
at RT on a rocking shaker. The reaction mixture is desalted on a
PD-10 column (Amersham-Pharmacia Biotech). The protein-containing
fractions eluating from the column are pooled, and the desalted
derivatized pili protein is reacted with the prepared IL-13-C1-His
protein. In the coupling reaction the IL-13-C1-His protein is in
twofold molar excess over the derivatized Type-1 pili of E. coli.
The coupling reaction proceeds for four hours at 25.degree. C. on a
rocking shaker. Coupling products are analysed by SDS-PAGE and in
addition by Westernblot.
Immunization of Mice with IL-13-C1-His Coupled to Q.beta. Capsid
Protein
[0368] Female Balb/c mice are vaccinated with IL-13-C1-His coupled
to a VLP without the addition of adjuvants. 25 .mu.g of total
protein of each sample is diluted in PBS to 200 ul and injected
subcutaneously (100 .mu.l on two ventral sides) on day 0 and day
14. Mice are bleed retroorbitally on day 31 and their serum is
analyzed using a IL-13-specific ELISA.
Example 9
Cloning, Expression, Purification and Coupling of Eotaxin with a
Cys-Containing Amino Acid Linker Sequence
[0369] Mouse eotaxin was recombinantly expressed with an amino acid
linker C1 fused at its C-terminus. This linker contained one
cysteine for coupling to VLP.
Construction of pmEo-C1 and pmHisEo-C1
[0370] The MCS of pET22b(+) (Novagen, Inc.) was changed to
GTTTAACTTTAAGAAGGAGATATACATATGGATCCGGCTAGCGCTCGAGGGTT
TAAACGGCGGCCGCATGCACC (SEQ ID NO:381) by replacing the original
sequence from the NdeI site to XhoI site with annealed oligos
primerMCS-1F and primerMCS-1R (annealing in 15 mM TrisHCl pH 8
buffer). The resulting plasmid was termed pMod00, which had NdeI,
BamHI, NheI, XhoI, PmeI and NotI restriction sites in its MCS. The
annealed pair of oligos Bamhis6-EK-Nhe-F and Bamhis6-EKNhe-R and
the annealed pair of oligo1F-C-glycine-linker and
oligo1R-C-glycine-linker were together ligated into BamHI-NotI
digested pMod00 plasmid to get pModEC1, which had an N terminal
hexahistidine tag, an enterokinase cleavage site and a C-terminal
amino acid glycine linker containing one cysteine residue. Mouse
eotaxin was amplified from an ATCC clone (ATCC number 3148394) by
PCR using the following primers: mEotaxin-F, Nhe-mEotaxin-F, and
mEotaxin-Xho-R. mEotaxin-F had an internal NdeI site,
Nhe-mEotaxin-F had an internal NheI site and mEotaxin-Xho-R had an
internal XhoI site. The PCR product from primer pair mEotaxin-F and
mEotaxin-Xho-R was digested with NdeI and XhoI and ligated into
pModEC1 digested with the same enzymes. The resulting plasmid was
named pmEo-C1, which encodes a fusing protein consisting of eotaxin
and a cysteine containing linker at its C-terminus. The PCR product
from primer pair Nhe-mEotaxin-F and mEotaxin-Xho-R was digested
with NheI and XhoI and ligated into pModEC1 digested with the same
enzymes. The resulting plasmid was named pHismEo-C1, which encodes
a fusing protein consisting of an N-terminal His-tag followed by an
enterokinase cleavage site, eotaxin and a cysteine linker.
[0371] For the PCR reaction, 15 pmol of each oligo and 1 ng of the
template DNA was used in the 50 .mu.l reaction mixture (2 units of
PFX polymerase, 0.3 mM dNTPs and 2 mM MgSO.sub.4). The temperature
cycles were as following: 94.degree. C. for 2 minutes, followed by
30 cycles of 94.degree. C. (30 seconds), 60.degree. C. (30
seconds), 68.degree. C. (30 seconds) and followed by 68.degree. C.
for 2 minutes. All other steps were performed by standard molecular
biology protocols.
[0372] Sequence of the oligonucleotides:
TABLE-US-00031 mEotaxin-F: (SEQ ID NO:382)
5'GGAATTCCATATGCACCCAGGCTCCATCCCAAC3' Nhe-mEotaxin-F: (SEQ ID
NO:383) 5'CCTAGCTAGCGCACCCAGGCTCCATCCCAAC3' mEotaxin-Xho-R: (SEQ ID
NO:384) 5'CCCGCTCGAGTGGTTTTGGAGTTTGGAGTT3'
Expression of pmEo-C1
[0373] Competent E. coli BL21 (DE3) cells were transformed with
plasmid pmEo-C1. Single colonies from ampicillin (Amp)-containing
agar plates were expanded in liquid culture (SB with 150 mM MOPS,
pH 7.0, 100 ug/ml Amp, 0.5% glucose) and incubated at 30.degree. C.
with 220 rpm shaking overnight. 1 l of SB (150 mM MOPS, pH 7.0, 100
ug/ml Amp) was then inoculated 1:50 v/v with the overnight culture
and grown to OD600=1.7 at 30.degree. C. with 150 rpm shaking.
Expression was induced with 1 mM IPTG. Cells were harvested after 9
hours' induction by centrifuging at 6000 rpm for 5 minutes. Cell
pellet was suspended in lysis buffer (10mM Na.sub.2HPO.sub.4, 30 mM
NaCl, 10 mM EDTA and 0.25% Tween-20) with 0.8 mg/ml lysozyme,
sonicated and treated with benzonase. After centrifugation with
48000 RCF for 20 minutes, the supernatant was resolved on 16% PAGE
gel and the mouse eotaxin expression was confirmed by anti-mouse
eotaxin antibody (R & D system) on Western blot (FIG. 12). This
clearly demonstrated the expression of eotaxin-C1 which ran at the
expected molecular weight of 8.8 KD.
[0374] The protein sequences of the mouse eotaxin-C1 and mouse
His-eotaxin-C1 were translated from the cDNA sequences.
TABLE-US-00032 Mouse eotaxin-C1: (SEQ ID NO:385)
MHPGSIPTSCCFIMTSKKIPNTLLKSYKRITNNRCTLKAIVFKTRLGKEI
CADPKKKWVQDATKHLDQKLQTPKPLRGGGGGCG Mouse His-eotaxin-C1: (SEQ ID
NO:386) MDPHHHHHHGSGDDDDKALAHPGSIPTSCCFIMTSKKIPNTLLKSYKRIT
NNRCTLKAIVFKTRLGKEICADPKKKWVQDATKHLDQKLQTPKPLRGGGG GCG
Coupling of Mouse Eotaxin-C1 to Q.beta. Capsid Protein
[0375] A solution of 1.48 ml of 6 mg/ml Q.beta. capsid protein in
20 mM Hepes, 150 mM NaCl pH 7.2 is reacted for 60 minutes with 14.8
.mu.l of a SMPH (Pierce) (from a 100 mM stock solution dissolved in
DMSO) at 25.degree. C. The reaction solution is subsequently
dialyzed twice for 3 hours against 2 1 of 20 mM Hepes, 150 mM NaCl,
pH 7.0 at 4.degree. C. A solution of 1.3 ml of 3.6 mg/ml mouse
eotaxin-C1 protein in 20 mM Hepes, 150 mM NaCl pH 7.2 is reacted
for 1 hour with 9.6 .mu.l of a TCEP (Pierce) (from a 36 mM stock
solution dissolved in H.sub.2O) at 25.degree. C. 130 .mu.l of the
derivatized and dialyzed Q.beta. is then react with 129 .mu.l of
reduced eotaxin-C1 in 241 .mu.l of 20 mM Hepes, 150 mM NaCl, pH 7.0
over night at 25.degree. C. Western blot analyses with an
anti-Q.beta. and an anti eotaxin antibody demonstrate covalent
coupling of eotaxin to Q.beta..
B. Immunization of Mice with Mouse Eotaxin-C1 Coupled to Q.beta.
Capsid Protein
[0376] Female Balb/c mice are vaccinated with mouse eotaxin-C1
coupled to Q.beta. capsid protein without the addition of
adjuvants. 25 .mu.g of total protein of each sample is diluted in
PBS to 200 ul and injected subcutaneously (100 .mu.l on two ventral
sides) on day 0 and day 14. Mice are bled retroorbitally on day 31
and their serum is analyzed using an eotaxin-specific ELISA.
C. ELISA
[0377] ELISA plates are coated with mouse eotaxin-C1 at a
concentration of 5 .mu.g/ml. The plates are blocked and then
incubated with serially diluted mouse sera. Bound antibodies are
detected with enzymatically labeled anti-mouse IgG antibody. As a
control, preimmune serum from the same mice are also tested.
Example 10
Cloning and Expression of Interleukin 5 (IL-5) with an N-Terminal
Amino Acid Linker Containing a Cysteine Residue for Coupling to
VLPs and Pili
[0378] A. Cloning of IL-5 for Expression as Inclusion Bodies in E.
coli
[0379] IL-5 was amplified from an ATCC clone (pmIL5-4G; ATCC
number: 37562) by PCR using the following two primers:
Spelinker3-F1 (SEQ ID NO:340) and 115StopXho-R (SEQ ID NO:342). The
product of this PCR was used as template for a second PCR with the
primers SpeNlinker3-F2 (SEQ ID NO:341) and 115StopXho-R. The insert
was digested with SpeI and NotI. This insert was ligated into a pET
vector derivative (pMODEC3-8 vector), previously digested with NheI
and NotI (not dephosphorylated), and transformed into E. coli TG1
cells. The IL5 construct generated by cloning into pMODEC3-8 vector
contains at its N-terminus a hexa-histidine tag, followed by an
enterokinase site, an N-terminal gamma 3 amino acid linker
containing a cysteine residue, flanked C-terminally by the sequence
AS and N-terminally by the sequence ALV, and the mature form of the
IL 5 gene. The protein released by cleavage with enterokinase is
called "mouse C-IL-5-E" (SEQ ID NO:332). Plasmid DNA of resulting
clone pMODC6-IL5.2 (also called pMODC6-IL5), whose sequence had
been confirmed by DNA sequencing, was transformed into E. coli
strain BL21.
[0380] Clone pMODC6-IL5/BL21 was grown over night in 5 ml LB
containing 1 mg/L Ampicillin. 2 ml of this culture were diluted in
100 ml terrific broth (TB) containing 1 mg/L Ampicillin. The
culture was induced by adding 0.1 ml of a 1M solution of Ispropyl
.beta.-D-Thiogalactopyranoside (IPTG) when the culture reached an
optical density OD600=0.7. 10 ml samples were taken every 2 h. The
samples were centrifuged 10 min at 4000.times.g. The pellet was
resuspended in 0.5 ml Lysis buffer containing 50 mM Tris-HCl, 2 mM
EDTA, 0.1% triton X-100 (pH8). After having added 20 .mu.l of
Lysozyme (40 mg/ml) and having incubated the tube 30 min at
4.degree. C., the cells were sonicated for 2 min. 100 .mu.l of a 50
mM MgCl.sub.2 solution and 1 ml of benzonase were added. The cells
were then incubated 30 min at room temperature and centrifuged 15
min at 13000.times.g.
[0381] The supernatant was discarded and the pellet was boiled 5
min at 98.degree. C. in 100 .mu.l of SDS loading buffer. 10 .mu.l
of the samples in loading buffer were analyzed by SDS-PAGE under
reducing conditions (FIG. 17 A). The gel of FIG. 17 A clearly
demonstrates expression of the IL-5 construct. The samples loaded
on the gel of FIG. 17 A were the following:
[0382] Lane M: Marker (NEB, Broad range prestained marker). Lane 1:
cell extract of 1 ml culture before induction. Lane 2: cell extract
of 1 ml culture 4 h after induction.
B. Cloning of IL-5 for Expression in Mammalian Cells (HEK-293T)
[0383] a) IL-5 fused at its N-terminus to an amino acid linker
containing a cysteine residue and fused at its C-terminus to the Fc
fragment
[0384] The template described under (A) (ATCC clone 37562) was used
for the cloning of the following construct. The plasmid pMODB1-IL5
(a pET derivative) was digested with BamHI/XhoI to yield a small
fragment encoding IL5 fused to an N terminal amino acid linker
containing a cysteine. This fragment was ligated in the vector
pCEP-SP-XA-Fc*(.DELTA.Xho) which had previously been digested with
BamHI and XhoI. The ligation was electroporated into E. coli strain
TG1 and plasmid DNA of resulting clone pCEP-SP-IL5-Fc.2, whose
sequence had been confirmed by DNA sequencing, was used to
transfect HEK-293T cells. The resulting IL-5 construct encoded by
this plasmid had the amino acid sequence ADPGCGGGGGLA (1-12 of SEQ
ID NO:333) fused at the N-terminus of the IL-5 mature sequence.
This sequence comprises the amino acid linker sequence GCGGGGG
(4-10 of SEQ ID NO:333) containing a cysteine and flanked by
additional amino acids introduced during the cloning procedure. The
IL-5 protein released by cleavage of the fusion protein with
Factor-Xa is named hereinafter "mouse C-IL-5-F" (SEQ ID
NO:333).
[0385] After transfection and selection on Puromycin the culture
supernatant was analyzed by Western-Blot (FIG. 17 B) using an
anti-His (mouse) and an anti-mouse IgG antibody conjugated to Horse
radish peroxidase. The anti-mouse IgG antibody conjugated to Horse
radish peroxidase also detects Fc-fusion proteins. Purification of
the protein was performed by affinity chromatography on Protein-A
resin. The result of FIG. 17 B clearly demonstrates expression of
the IL-5 construct.
[0386] The samples loaded on the Western-Blot of FIG. 17 B were the
following:
[0387] Lane 1: supernatant of HEK culture expressing IL5-Fc (20
.mu.l). SDS-PAGE was performed under reducing conditions. Lane 2:
supernatant of HEK culture expressing IL13-Fc (20 .mu.l). SDS-PAGE
was performed under non reducing conditions. Lane 3: supernatant of
HEK culture expressing IL5-Fc (20 .mu.l). SDS-PAGE was performed
under non reducing conditions. [0388] b) IL-5 cloned with GST
(Glutathione-S-transferase) and an amino acid linker containing a
cysteine residue fused at its N-terminus
[0389] IL-5 (ATCC 37562) was amplified with the primers
Nhe-link1-IL13-F and IL5StopXho-R. After digestion with NheI and
XhoI the insert was ligated into pCEP-SP-GST-EK which had been
previously digested with NheI and XhoI. The resulting plasmid
pCEP-SP-GST-IL5 was sequenced and used for transfection of HEK-293T
cells. The resulting IL-5 construct encoded by this plasmid had the
amino acid sequence LACGGGGG (1-80 of SEQ ID NO:334) fused at the
N-terminus of the IL-5 mature sequence. This sequence comprises the
amino acid linker sequence ACGGGGG (2-8 of SEQ ID NO:334)
containing a cysteine residue and flanked by additional amino acids
introduced during the cloning procedure. The protein released by
cleavage with enterokinase was named hereinafter "mouse C-IL-5-S"
(SEQ ID NO:334). The purification of the resulting protein was
performed by affinity chromatography on Glutathione affinity
resin.
C. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S to Q.beta. Capsid
Protein
[0390] A solution of 120 .mu.M Q.beta. capsid protein in 20 mM
Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold
molar excess of SMPH (Pierce), diluted from a stock solution in
DMSO, at 25.degree. C. on a rocking shaker. The reaction solution
is subsequently dialyzed twice for 2 hours against 1 L of 20 mM
Hepes, 150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed Q.beta.
reaction mixture is then reacted with the mouse C-IL-5-F or mouse
C-IL-5-S solution (end concentrations: 60 .mu.M Q.beta. capsid
protein, 60 .mu.M mouse C-IL-5-F or mouse C-IL-5-S) for four hours
at 25.degree. C. on a rocking shaker. Coupling products are
analysed by SDS-PAGE.
D. Coupling of Mouse Mouse C-IL-5-F or Mouse C-IL-5-S to fr Capsid
Protein
[0391] A solution of 120 .mu.M fr capsid protein in 20 mM Hepes,
150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar
excess of SMPH (Pierce), diluted from a stock solution in DMSO, at
25.degree. C. on a rocking shaker. The reaction solution is
subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes,
150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed fr reaction
mixture is then reacted with the mouse C-IL-5-F or mouse C-IL-5-S
solution (end concentrations: 60 .mu.M fr capsid protein, 60 .mu.M
mouse C-IL-5-F or mouse C-IL-5-S) for four hours at 25.degree. C.
on a rocking shaker. Coupling products are analysed by
SDS-PAGE.
E. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S Solution to
HBcAg-Lys-2cys-Mut
[0392] A solution of 120 .mu.M HBcAg-Lys-2cys-Mut capsid in 20 mM
Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold
molar excess of SMPH (Pierce), diluted from a stock solution in
DMSO, at 25.degree. C. on a rocking shaker. The reaction solution
is subsequently dialyzed twice for 2 hours against 1 L of 20 mM
Hepes, 150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed
HBcAg-Lys-2cys-Mut reaction mixture is then reacted with the mouse
C-IL-5-F or mouse C-IL-5-S solution (end concentrations: 60 .mu.M
HBcAg-Lys-2cys-Mut, 60 .mu.M mouse C-IL-5-F or mouse C-IL-5-S) for
four hours at 25.degree. C. on a rocking shaker. Coupling products
are analysed by SDS-PAGE.
F. Coupling of Mouse C-IL-5-F or Mouse C-IL-5-S Solution to
Pili
[0393] A solution of 125 .mu.M Type-1 pili of E. coli in 20 mM
Hepes, pH 7.4, is reacted for 60 minutes with a 50-fold molar
excess of cross-linker SMPH, diluted from a stock solution in DMSO,
at RT on a rocking shaker. The reaction mixture is desalted on a
PD-10 column (Amersham-Pharmacia Biotech). The protein-containing
fractions eluating from the column are pooled, and the desalted
derivatized pili protein is reacted with the mouse C-IL-5-F or
mouse C-IL-5-S solution (end concentrations: 60 .mu.M pili, 60
.mu.M mouse C-IL-5-F or mouse C-IL-5-S) for four hours at
25.degree. C. on a rocking shaker. Coupling products are analysed
by SDS-PAGE.
Example 11
Cloning, Expression and Purification of IL-13 to VLPs and Pili
[0394] A. Cloning and expression of Interleukin 13 (IL-13) with an
N-terminal amino acid linker containing a cysteine residue for
coupling to VLPs and Pili [0395] a) Cloning of mouse IL-13
(HEK-293T) for expression in mammalian cells as Fc fusion
protein
[0396] The DNA for the cloning of IL-13 was isolated by RT-PCR from
in vitro activated splenocytes, which were obtained as following:
CD4+ T cells were isolated from mouse spleen cells and incubated 3
days in IMDM (+5% FCS+10 ng/ml IL4) in 6 well plates which have
been previously coated with anti-CD3 and anti-CD28 antibodies. The
RNA from these cells was used to amplify IL13 by one-step RT-PCR
(Qiagen one-step PCR kit). Primer XhoIL13-R was used for the
reverse transcription of the RNA and the primers NheIL13-F (SEQ ID
NO:338) and XhoIL13-R (SEQ ID NO:339) were used for the PCR
amplification of the IL13 cDNA. Amplified IL13 cDNA was ligated in
a pMOD vector using the Nhe/XhoI restriction sites (giving the
vector pMODB1-IL13). pMODB1-1113 was digested BamHI/XhoI and the
fragment containing IL13 was ligated in the
pCEP-SP-XA-Fc*(.DELTA.xho) vector, an analogue of pCEP-SP-XA-Fc*
where a XhoI site at the end of the Fc sequence has been removed,
which had been previously digested with BamHI/XhoI. The plasmid
resulting from this ligation (pCEP-SP-IL13-Fc) was sequenced and
used to transfect HEK-293T cells. The resulting IL 13 construct
encoded by this plasmid had the amino acid sequence ADPGCGGGGGLA
(1-12 of SEQ ID NO:328) fused at the N-terminus of the IL-13 mature
sequence. This sequence comprises the amino acid linker sequence
GCGGGGG (4-10 of SEQ ID NO:328) flanked by additional amino acids
introduced during the cloning procedure. IL13-Fc could be purified
with Protein-A resin from the supernatant of the cells transfected
with pCEP-SP-IL13-Fc. The result of the expression is shown on FIG.
17 B (see EXAMPLE 10 for description of the samples). Mature IL-13
fused at its N-terminus with the aforementioned amino acid sequence
is released upon cleavage of the fusion protein with Factor-Xa,
leading to a protein called hereinafter "mouse C-IL-13-F" and
having a sequence of SEQ ID NO:328. The result of FIG. 17 B clearly
demonstrates expression of the IL-13 construct. [0397] b) Cloning
of mouse IL-13 (HEK-293T) for expression in mammalian cells with
GST (Glutathione-S-transferase) fused at its N-terminus
[0398] The cDNA used for cloning IL-13 with an N-terminal GST
originated from the cDNA of TH2 activated T-cells as described
above (a.). IL-13 was amplified from this cDNA using the primers
Nhelink1IL13-F and IL13StopXhoNot-R. The PCR product was digested
with NheI and XhoI and ligated in the pCEP-SP-GST-EK vector
previously digested with NheI/XhoI. The plasmid which could be
isolated from the ligation (pCEP-SP-GST-IL13) was used to transfect
HEK-293T cells. The resulting IL 13 construct encoded by this
plasmid had the amino acid sequence LACGGGGG (1-8 of SEQ ID NO:329)
fused at the N-terminus of the IL-13 mature sequence. This sequence
comprises the amino acid linker sequence ACGGGGG (2-8 of SEQ ID
NO:329) flanked by an additional amino acid introduced during the
cloning procedure. The culture supernatant of the cells transfected
with pCEP-SP-GST-IL13 contained the fusion protein GST-IL13 which
could be purified by Glutathione affinity chromatography according
to standard protocols. Mature IL-13 fused at its N-terminus with
aforementioned amino acid sequence is released upon cleavage of the
fusion protein with enterokinase, leading to a protein called
hereinafter "mouse C-IL-13-S" and having a sequence of SEQ ID
NO:329.
B. Coupling of Mouse C-IL-13-F, Mouse C-IL-13-S to Q.beta. Capsid
Protein
[0399] A solution of 120 .mu.M Q.beta. capsid in 20 mM Hepes, 150
mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar
excess of SMPH (Pierce), diluted from a stock solution in DMSO, at
25.degree. C. on a rocking shaker. The reaction solution is
subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes,
150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed Q.beta. reaction
mixture is then reacted with the mouse C-IL-13-F or mouse C-IL-13-S
solution (end concentrations: 60 .mu.M Q.beta. capsid protein, 60
.mu.M mouse C-IL-13-F or mouse C-IL-13-S) for four hours at
25.degree. C. on a rocking shaker. Coupling products are analysed
by SDS-PAGE.
C. Coupling of Mouse C-IL-13-F, Mouse C-IL-13-S to fr Capsid
Protein
[0400] A solution of 120 .mu.M fr capsid protein in 20 mM Hepes,
150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar
excess of SMPH (Pierce), diluted from a stock solution in DMSO, at
25.degree. C. on a rocking shaker. The reaction solution is
subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes,
150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed fr reaction
mixture is then reacted with the mouse C-IL-13-F or mouse C-IL-13-S
solution (end concentrations: 60 .mu.M fr capsid protein, 60 .mu.M
mouse C-IL-13-F or mouse C-IL-13-S) for four hours at 25.degree. C.
on a rocking shaker. Coupling products are analysed by
SDS-PAGE.
D. Coupling of Mouse C-IL-13-F or Mouse C-IL-13-S Solution to
HBcAg-Lys-2cys-Mut
[0401] A solution of 120 .mu.M HBcAg-Lys-2cys-Mut capsid in 20 mM
Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold
molar excess of SMPH (Pierce), diluted from a stock solution in
DMSO, at 25.degree. C. on a rocking shaker. The reaction solution
is subsequently dialyzed twice for 2 hours against 1 L of 20 mM
Hepes, 150 mM NaCl, pH 7.2 at 4.degree. C. The dialyzed
HBcAg-Lys-2cys-Mut reaction mixture is then reacted with the mouse
C-IL-13-F or mouse C-IL-13-S solution (end concentrations: 60 .mu.M
HBcAg-Lys-2cys-Mut, 60 .mu.M mouse C-IL-13-F or mouse C-IL-13-S)
for four hours at 25.degree. C. on a rocking shaker. Coupling
products are analysed by SDS-PAGE.
E. Coupling of Mouse C-IL-13-F or Mouse C-IL-13-S Solution to
Pili
[0402] A solution of 125 .mu.M Type-1 pili of E. coli in 20 mM
Hepes, pH 7.4, is reacted for 60 minutes with a 50-fold molar
excess of cross-linker SMPH, diluted from a stock solution in DMSO,
at RT on a rocking shaker. The reaction mixture is desalted on a
PD-10 column (Amersham-Pharmacia Biotech). The protein-containing
fractions eluating from the column are pooled, and the desalted
derivatized pili protein is reacted with the mouse C-IL-13-F or
mouse C-IL-13-S solution (end concentrations: 60 .mu.M pili, 60
.mu.M mouse C-IL-13-F or mouse C-IL-13-S) for four hours at
25.degree. C. on a rocking shaker. Coupling products are analysed
by SDS-PAGE.
[0403] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0404] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 387 <210> SEQ ID NO 1 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Haemophilus influenzae
<400> SEQUENCE: 1 Met Lys Lys Thr Leu Leu Gly Ser Leu Ile Leu
Leu Ala Phe Ala Gly 1 5 10 15 Asn Val Gln Ala Ala Ala Asn Ala Asp
Thr Ser Gly Thr Val Thr Phe 20 25 30 Phe Gly Lys Val Val Glu Asn
Thr Cys Gln Val Asn Gln Asp Ser Glu 35 40 45 Tyr Glu Cys Asn Leu
Asn Asp Val Gly Lys Asn His Leu Ser Gln Gln 50 55 60 Gly Tyr Thr
Ala Met Gln Thr Pro Phe Thr Ile Thr Leu Glu Asn Cys 65 70 75 80 Asn
Val Thr Thr Thr Asn Asn Lys Pro Lys Ala Thr Lys Val Gly Val 85 90
95 Tyr Phe Tyr Ser Trp Glu Ile Ala Asp Lys Asp Asn Lys Tyr Thr Leu
100 105 110 Lys Asn Ile Lys Glu Asn Thr Gly Thr Asn Asp Ser Ala Asn
Lys Val 115 120 125 Asn Ile Gln Leu Leu Glu Asp Asn Gly Thr Ala Glu
Ile Lys Val Val 130 135 140 Gly Lys Thr Thr Thr Asp Phe Thr Ser Glu
Asn His Asn Gly Ala Gly 145 150 155 160 Ala Asp Pro Val Ala Thr Asn
Lys His Ile Ser Ser Leu Thr Pro Leu 165 170 175 Asn Asn Gln Asn Ser
Ile Asn Leu His Tyr Ile Ala Gln Tyr Tyr Ala 180 185 190 Thr Gly Val
Ala Glu Ala Gly Lys Val Pro Ser Ser Val Asn Ser Gln 195 200 205 Ile
Ala Tyr Glu 210 <210> SEQ ID NO 2 <211> LENGTH: 139
<212> TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri
<400> SEQUENCE: 2 Met Lys Ala Gln Met Gln Lys Gly Phe Thr Leu
Ile Glu Leu Met Ile 1 5 10 15 Val Val Ala Ile Ile Gly Ile Leu Ala
Ala Ile Ala Leu Pro Ala Tyr 20 25 30 Gln Asp Tyr Thr Val Arg Ser
Asn Ala Ala Ala Ala Leu Ala Glu Ile 35 40 45 Thr Pro Gly Lys Ile
Gly Phe Glu Gln Ala Ile Asn Glu Gly Lys Thr 50 55 60 Pro Ser Leu
Thr Ser Thr Asp Glu Gly Tyr Ile Gly Ile Thr Asp Ser 65 70 75 80 Thr
Ser Tyr Cys Asp Val Asp Leu Asp Thr Ala Ala Asp Gly His Ile 85 90
95 Glu Cys Thr Ala Lys Gly Gly Asn Ala Gly Lys Phe Asp Gly Lys Thr
100 105 110 Ile Thr Leu Asn Arg Thr Ala Asp Gly Glu Trp Ser Cys Ala
Ser Thr 115 120 125 Leu Asp Ala Lys Tyr Lys Pro Gly Lys Cys Ser 130
135 <210> SEQ ID NO 3 <211> LENGTH: 59 <212>
TYPE: PRT <213> ORGANISM: Caulobacter crescentus <400>
SEQUENCE: 3 Met Thr Lys Phe Val Thr Arg Phe Leu Lys Asp Glu Ser Gly
Ala Thr 1 5 10 15 Ala Ile Glu Tyr Gly Leu Ile Val Ala Leu Ile Ala
Val Val Ile Val 20 25 30 Thr Ala Val Thr Thr Leu Gly Thr Asn Leu
Arg Thr Ala Phe Thr Lys 35 40 45 Ala Gly Ala Ala Val Ser Thr Ala
Ala Gly Thr 50 55 <210> SEQ ID NO 4 <211> LENGTH: 173
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 4 Met Ala Val Val Ser Phe Gly Val Asn Ala Ala
Pro Thr Ile Pro Gln 1 5 10 15 Gly Gln Gly Lys Val Thr Phe Asn Gly
Thr Val Val Asp Ala Pro Cys 20 25 30 Ser Ile Ser Gln Lys Ser Ala
Asp Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45 Ser Lys Ser Phe Leu
Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu 50 55 60 Asp Ile Glu
Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn 65 70 75 80 Gly
Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile Val 85 90
95 Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr Gly Thr Ala
100 105 110 Ile Val Val Gln Gly Ala Gly Lys Asn Val Val Phe Asp Gly
Ser Glu 115 120 125 Gly Asp Ala Asn Thr Leu Lys Asp Gly Glu Asn Val
Leu His Tyr Thr 130 135 140 Ala Val Val Lys Lys Ser Ser Ala Val Gly
Ala Ala Val Thr Glu Gly 145 150 155 160 Ala Phe Ser Ala Val Ala Asn
Phe Asn Leu Thr Tyr Gln 165 170 <210> SEQ ID NO 5 <211>
LENGTH: 173 <212> TYPE: PRT <213> ORGANISM: Escherichia
coli <400> SEQUENCE: 5 Met Ala Val Val Ser Phe Gly Val Asn
Ala Ala Pro Thr Ile Pro Gln 1 5 10 15 Gly Gln Gly Lys Val Thr Phe
Asn Gly Thr Val Val Asp Ala Pro Cys 20 25 30 Ser Ile Ser Gln Lys
Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45 Ser Lys Ser
Phe Leu Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu 50 55 60 Asp
Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn 65 70
75 80 Gly Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile
Val 85 90 95 Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr
Gly Thr Ala 100 105 110 Ile Val Val Gln Gly Ala Gly Lys Asn Val Val
Phe Asp Gly Ser Glu 115 120 125 Gly Asp Ala Asn Thr Leu Lys Asp Gly
Glu Asn Val Leu His Tyr Thr 130 135 140 Ala Val Val Lys Lys Ser Ser
Ala Val Gly Ala Ala Val Thr Glu Gly 145 150 155 160 Ala Phe Ser Ala
Val Ala Asn Phe Asn Leu Thr Tyr Gln 165 170 <210> SEQ ID NO 6
<211> LENGTH: 181 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 6 Met Lys Ile Lys Thr Leu
Ala Ile Val Val Leu Ser Ala Leu Ser Leu 1 5 10 15 Ser Ser Thr Ala
Ala Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr 20 25 30 Val His
Phe Lys Gly Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala 35 40 45
Gly Ser Val Asp Gln Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser 50
55 60 Leu Ala Gln Glu Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile
Gln 65 70 75 80 Leu Asn Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala
Val Ala Phe 85 90 95 Leu Gly Thr Ala Ile Asp Ala Gly His Thr Asn
Val Leu Ala Leu Gln 100 105 110 Ser Ser Ala Ala Gly Ser Ala Thr Asn
Val Gly Val Gln Ile Leu Asp 115 120 125 Arg Thr Gly Ala Ala Leu Thr
Leu Asp Gly Ala Thr Phe Ser Ser Glu 130 135 140 Thr Thr Leu Asn Asn
Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr 145 150 155 160 Phe Ala
Gly Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr Phe 165 170 175
Lys Val Gln Tyr Gln 180 <210> SEQ ID NO 7 <211> LENGTH:
172 <212> TYPE: PRT <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 7 Met Ala Val Val Ser Phe Gly Val Asn Ala Ala
Pro Thr Thr Pro Gln 1 5 10 15 Gly Gln Gly Arg Val Thr Phe Asn Gly
Thr Val Val Asp Ala Pro Cys 20 25 30 Ser Ile Ser Gln Lys Ser Ala
Asp Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45 Ser Lys Ser Phe Leu
Ala Asn Asp Gly Gln Ser Lys Pro Met Asn Leu 50 55 60 Asp Ile Glu
Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Asn Gly Asn 65 70 75 80 Ala
Lys Thr Gly Ser Val Lys Leu Ala Phe Thr Gly Pro Thr Val Ser 85 90
95 Gly His Pro Ser Glu Leu Ala Thr Asn Gly Gly Pro Gly Thr Ala Ile
100 105 110 Met Ile Gln Ala Ala Gly Lys Asn Val Pro Phe Asp Gly Thr
Glu Gly 115 120 125 Asp Pro Asn Leu Leu Lys Asp Gly Asp Asn Val Leu
His Tyr Thr Thr 130 135 140 Val Gly Lys Lys Ser Ser Asp Gly Asn Ala
Gln Ile Thr Glu Gly Ala 145 150 155 160 Phe Ser Gly Val Ala Thr Phe
Asn Leu Ser Tyr Gln 165 170 <210> SEQ ID NO 8 <211>
LENGTH: 182 <212> TYPE: PRT <213> ORGANISM: Escherichia
coli <400> SEQUENCE: 8 Met Lys Ile Lys Thr Leu Ala Ile Val
Val Leu Ser Ala Leu Ser Leu 1 5 10 15 Ser Ser Thr Thr Ala Leu Ala
Ala Ala Thr Thr Val Asn Gly Gly Thr 20 25 30 Val His Phe Lys Gly
Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala 35 40 45 Gly Ser Val
Asp Gln Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser 50 55 60 Leu
Ala Gln Glu Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile Gln 65 70
75 80 Leu Asn Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala Val Ala
Phe 85 90 95 Leu Gly Thr Ala Ile Asp Ala Gly His Thr Asn Val Leu
Ala Leu Gln 100 105 110 Ser Ser Ala Ala Gly Ser Ala Thr Asn Val Gly
Val Gln Ile Leu Asp 115 120 125 Arg Thr Gly Ala Ala Leu Thr Leu Asp
Gly Ala Thr Phe Ser Ser Glu 130 135 140 Thr Thr Leu Asn Asn Gly Thr
Asn Thr Ile Pro Phe Gln Ala Arg Tyr 145 150 155 160 Phe Ala Thr Gly
Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr 165 170 175 Phe Lys
Val Gln Tyr Gln 180 <210> SEQ ID NO 9 <211> LENGTH: 853
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 9 acgtttctgt ggctcgacgc atcttcctca ttcttctctc
caaaaaccac ctcatgcaat 60 ataaacatct ataaataaag ataacaaata
gaatattaag ccaacaaata aactgaaaaa 120 gtttgtccgc gatgctttac
ctctatgagt caaaatggcc ccaatgtttc atcttttggg 180 ggaaactgtg
cagtgttggc agtcaaactc gttgacaaac aaagtgtaca gaacgactgc 240
ccatgtcgat ttagaaatag ttttttgaaa ggaaagcagc atgaaaatta aaactctggc
300 aatcgttgtt ctgtcggctc tgtccctcag ttctacgacg gctctggccg
ctgccacgac 360 ggttaatggt gggaccgttc actttaaagg ggaagttgtt
aacgccgctt gcgcagttga 420 tgcaggctct gttgatcaaa ccgttcagtt
aggacaggtt cgtaccgcat cgctggcaca 480 ggaaggagca accagttctg
ctgtcggttt taacattcag ctgaatgatt gcgataccaa 540 tgttgcatct
aaagccgctg ttgccttttt aggtacggcg attgatgcgg gtcataccaa 600
cgttctggct ctgcagagtt cagctgcggg tagcgcaaca aacgttggtg tgcagatcct
660 ggacagaacg ggtgctgcgc tgacgctgga tggtgcgaca tttagttcag
aaacaaccct 720 gaataacgga accaatacca ttccgttcca ggcgcgttat
tttgcaaccg gggccgcaac 780 cccgggtgct gctaatgcgg atgcgacctt
caaggttcag tatcaataac ctacctaggt 840 tcagggacgt tca 853 <210>
SEQ ID NO 10 <211> LENGTH: 132 <212> TYPE: PRT
<213> ORGANISM: Bacteriophage Q beta <400> SEQUENCE: 10
Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Lys 1 5
10 15 Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly
Val 20 25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu
Lys Arg Val 35 40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg
Lys Asn Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala
Cys Thr Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln
Ala Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr
Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu
Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn
Pro Ala Tyr 130 <210> SEQ ID NO 11 <211> LENGTH: 329
<212> TYPE: PRT <213> ORGANISM: Bacteriophage Q beta CP
<400> SEQUENCE: 11 Met Ala Lys Leu Glu Thr Val Thr Leu Gly
Asn Ile Gly Lys Asp Gly 1 5 10 15 Lys Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30 Val Ala Ser Leu Ser Gln
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45 Val Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60 Val Gln
Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser 65 70 75 80
Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser 85
90 95 Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr
Glu 100 105 110 Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala
Ile Asp Gln 115 120 125 Leu Asn Pro Ala Tyr Trp Thr Leu Leu Ile Ala
Gly Gly Gly Ser Gly 130 135 140 Ser Lys Pro Asp Pro Val Ile Pro Asp
Pro Pro Ile Asp Pro Pro Pro 145 150 155 160 Gly Thr Gly Lys Tyr Thr
Cys Pro Phe Ala Ile Trp Ser Leu Glu Glu 165 170 175 Val Tyr Glu Pro
Pro Thr Lys Asn Arg Pro Trp Pro Ile Tyr Asn Ala 180 185 190 Val Glu
Leu Gln Pro Arg Glu Phe Asp Val Ala Leu Lys Asp Leu Leu 195 200 205
Gly Asn Thr Lys Trp Arg Asp Trp Asp Ser Arg Leu Ser Tyr Thr Thr 210
215 220 Phe Arg Gly Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp Ala Thr
Tyr 225 230 235 240 Leu Ala Thr Asp Gln Ala Met Arg Asp Gln Lys Tyr
Asp Ile Arg Glu 245 250 255 Gly Lys Lys Pro Gly Ala Phe Gly Asn Ile
Glu Arg Phe Ile Tyr Leu 260 265 270 Lys Ser Ile Asn Ala Tyr Cys Ser
Leu Ser Asp Ile Ala Ala Tyr His 275 280 285 Ala Asp Gly Val Ile Val
Gly Phe Trp Arg Asp Pro Ser Ser Gly Gly 290 295 300 Ala Ile Pro Phe
Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys Pro Ile 305 310 315 320 Gln
Ala Val Ile Val Val Pro Arg Ala 325 <210> SEQ ID NO 12
<211> LENGTH: 129 <212> TYPE: PRT <213> ORGANISM:
Bacteriophage R17 <400> SEQUENCE: 12 Ala Ser Asn Phe Thr Gln
Phe Val Leu Val Asn Asp Gly Gly Thr Gly 1 5 10 15 Asn Val Thr Val
Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30 Ile Ser
Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35 40 45
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val 50
55 60 Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
Ala 65 70 75 80 Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
Ile Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
Met Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro Ser Ala
Ile Ala Ala Asn Ser Gly Ile 115 120 125 Tyr <210> SEQ ID NO
13 <211> LENGTH: 130 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage fr <400> SEQUENCE: 13 Met Ala Ser Asn
Phe Glu Glu Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp
Val Lys Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35
40 45 Val Arg Gln Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val
Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu
Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Met Asn Met Glu Leu
Thr Ile Pro Val Phe 85 90 95 Ala Thr Asn Asp Asp Cys Ala Leu Ile
Val Lys Ala Leu Gln Gly Thr 100 105 110 Phe Lys Thr Gly Asn Pro Ile
Ala Thr Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130
<210> SEQ ID NO 14 <211> LENGTH: 130 <212> TYPE:
PRT <213> ORGANISM: Bacteriophage GA <400> SEQUENCE: 14
Met Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly 1 5
10 15 Asn Val Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu
Trp 20 25 30 Leu Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr
Ala Ser Tyr 35 40 45 Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala
Ile Lys Leu Glu Val 50 55 60 Pro Lys Ile Val Thr Gln Val Val Asn
Gly Val Glu Leu Pro Gly Ser 65 70 75 80 Ala Trp Lys Ala Tyr Ala Ser
Ile Asp Leu Thr Ile Pro Ile Phe Ala 85 90 95 Ala Thr Asp Asp Val
Thr Val Ile Ser Lys Ser Leu Ala Gly Leu Phe 100 105 110 Lys Val Gly
Asn Pro Ile Ala Glu Ala Ile Ser Ser Gln Ser Gly Phe 115 120 125 Tyr
Ala 130 <210> SEQ ID NO 15 <211> LENGTH: 132
<212> TYPE: PRT <213> ORGANISM: Bacteriophage SP
<400> SEQUENCE: 15 Met Ala Lys Leu Asn Gln Val Thr Leu Ser
Lys Ile Gly Lys Asn Gly 1 5 10 15 Asp Gln Thr Leu Thr Leu Thr Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30 Val Ala Ser Leu Ser Glu
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45 Val Thr Val Ser
Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe Lys 50 55 60 Val Gln
Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys 65 70 75 80
Asp Pro Ser Val Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe 85
90 95 Thr Ser Tyr Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu
Leu 100 105 110 Ala Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile
Asp Asn Leu 115 120 125 Asn Pro Ala Tyr 130 <210> SEQ ID NO
16 <211> LENGTH: 329 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage SP CP <400> SEQUENCE: 16 Ala Lys Leu
Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly Asp 1 5 10 15 Gln
Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25
30 Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val
35 40 45 Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe
Lys Val 50 55 60 Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg
Asp Ala Cys Asp 65 70 75 80 Pro Ser Val Thr Arg Ser Ala Phe Ala Asp
Val Thr Leu Ser Phe Thr 85 90 95 Ser Tyr Ser Thr Asp Glu Glu Arg
Ala Leu Ile Arg Thr Glu Leu Ala 100 105 110 Ala Leu Leu Ala Asp Pro
Leu Ile Val Asp Ala Ile Asp Asn Leu Asn 115 120 125 Pro Ala Tyr Trp
Ala Ala Leu Leu Val Ala Ser Ser Gly Gly Gly Asp 130 135 140 Asn Pro
Ser Asp Pro Asp Val Pro Val Val Pro Asp Val Lys Pro Pro 145 150 155
160 Asp Gly Thr Gly Arg Tyr Lys Cys Pro Phe Ala Cys Tyr Arg Leu Gly
165 170 175 Ser Ile Tyr Glu Val Gly Lys Glu Gly Ser Pro Asp Ile Tyr
Glu Arg 180 185 190 Gly Asp Glu Val Ser Val Thr Phe Asp Tyr Ala Leu
Glu Asp Phe Leu 195 200 205 Gly Asn Thr Asn Trp Arg Asn Trp Asp Gln
Arg Leu Ser Asp Tyr Asp 210 215 220 Ile Ala Asn Arg Arg Arg Cys Arg
Gly Asn Gly Tyr Ile Asp Leu Asp 225 230 235 240 Ala Thr Ala Met Gln
Ser Asp Asp Phe Val Leu Ser Gly Arg Tyr Gly 245 250 255 Val Arg Lys
Val Lys Phe Pro Gly Ala Phe Gly Ser Ile Lys Tyr Leu 260 265 270 Leu
Asn Ile Gln Gly Asp Ala Trp Leu Asp Leu Ser Glu Val Thr Ala 275 280
285 Tyr Arg Ser Tyr Gly Met Val Ile Gly Phe Trp Thr Asp Ser Lys Ser
290 295 300 Pro Gln Leu Pro Thr Asp Phe Thr Gln Phe Asn Ser Ala Asn
Cys Pro 305 310 315 320 Val Gln Thr Val Ile Ile Ile Pro Ser 325
<210> SEQ ID NO 17 <211> LENGTH: 130 <212> TYPE:
PRT <213> ORGANISM: Bacteriophage MS2 <400> SEQUENCE:
17 Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15 Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val
Ala Glu 20 25 30 Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys
Val Thr Cys Ser 35 40 45 Val Arg Gln Ser Ser Ala Gln Asn Arg Lys
Tyr Thr Ile Lys Val Glu 50 55 60 Val Pro Lys Val Ala Thr Gln Thr
Val Gly Gly Val Glu Leu Pro Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr
Leu Asn Met Glu Leu Thr Ile Pro Ile Phe 85 90 95 Ala Thr Asn Ser
Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu 100 105 110 Leu Lys
Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly 115 120 125
Ile Tyr 130 <210> SEQ ID NO 18 <211> LENGTH: 133
<212> TYPE: PRT <213> ORGANISM: Bacteriophage M11
<400> SEQUENCE: 18 Met Ala Lys Leu Gln Ala Ile Thr Leu Ser
Gly Ile Gly Lys Lys Gly 1 5 10 15 Asp Val Thr Leu Asp Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30 Val Ala Ala Leu Ser Glu
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45 Val Thr Ile Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60 Val Gln
Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr 65 70 75 80
Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ser Asp Val Thr Phe Ser 85
90 95 Phe Thr Gln Tyr Ser Thr Val Glu Glu Arg Ala Leu Val Arg Thr
Glu 100 105 110 Leu Gln Ala Leu Leu Ala Asp Pro Met Leu Val Asn Ala
Ile Asp Asn 115 120 125 Leu Asn Pro Ala Tyr 130 <210> SEQ ID
NO 19 <211> LENGTH: 133 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage MX1 <400> SEQUENCE: 19 Met Ala Lys
Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Asn Gly 1 5 10 15 Asp
Val Thr Leu Asn Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25
30 Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
35 40 45 Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys 50 55 60 Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr
Ala Ser Gly Thr 65 70 75 80 Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr
Ala Asp Val Thr Phe Ser 85 90 95 Phe Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Leu Val Arg Thr Glu 100 105 110 Leu Lys Ala Leu Leu Ala
Asp Pro Met Leu Ile Asp Ala Ile Asp Asn 115 120 125 Leu Asn Pro Ala
Tyr 130 <210> SEQ ID NO 20 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Bacteriophage NL95
<400> SEQUENCE: 20 Met Ala Lys Leu Asn Lys Val Thr Leu Thr
Gly Ile Gly Lys Ala Gly 1 5 10 15 Asn Gln Thr Leu Thr Leu Thr Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30 Val Ala Ser Leu Ser Glu
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45 Val Thr Val Ser
Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60 Val Gln
Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys Asp Ala Cys 65 70 75 80
Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp Val Thr Leu Ser Phe 85
90 95 Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala Leu Ile Arg Thr Glu
Leu 100 105 110 Ala Ala Leu Leu Lys Asp Asp Leu Ile Val Asp Ala Ile
Asp Asn Leu 115 120 125 Asn Pro Ala Tyr Trp Ala Ala Leu Leu Ala Ala
Ser Pro Gly Gly Gly 130 135 140 Asn Asn Pro Tyr Pro Gly Val Pro Asp
Ser Pro Asn Val Lys Pro Pro 145 150 155 160 Gly Gly Thr Gly Thr Tyr
Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly 165 170 175 Glu Leu Ile Thr
Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys 180 185 190 Gly Ser
Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp Phe Leu 195 200 205
Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu Ser Lys Tyr Asp 210
215 220 Ile Glu Thr His Arg Arg Cys Arg Gly Asn Gly Tyr Val Asp Leu
Asp 225 230 235 240 Ala Ser Val Met Gln Ser Asp Glu Tyr Val Leu Ser
Gly Ala Tyr Asp 245 250 255 Val Val Lys Met Gln Pro Pro Gly Thr Phe
Asp Ser Pro Arg Tyr Tyr 260 265 270 Leu His Leu Met Asp Gly Ile Tyr
Val Asp Leu Ala Glu Val Thr Ala 275 280 285 Tyr Arg Ser Tyr Gly Met
Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300 Pro Gln Leu Pro
Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys Pro 305 310 315 320 Val
Gln Thr Val Ile Val Ile Pro Ser Leu 325 330 <210> SEQ ID NO
21 <211> LENGTH: 129 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage f2 <400> SEQUENCE: 21 Ala Ser Asn Phe
Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly 1 5 10 15 Asn Val
Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30
Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35
40 45 Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu
Val 50 55 60 Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu
Pro Val Ala 65 70 75 80 Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr
Ile Pro Ile Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val
Lys Ala Met Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro
Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125 Tyr <210> SEQ
ID NO 22 <211> LENGTH: 128 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage PP7 <400> SEQUENCE: 22 Met Ser Lys
Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu 1 5 10 15 Thr
Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val 20 25
30 Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn
35 40 45 Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln
Ala Asp 50 55 60 Val Val Asp Cys Ser Thr Ser Val Cys Gly Glu Leu
Pro Lys Val Arg 65 70 75 80 Tyr Thr Gln Val Trp Ser His Asp Val Thr
Ile Val Ala Asn Ser Thr 85 90 95 Glu Ala Ser Arg Lys Ser Leu Tyr
Asp Leu Thr Lys Ser Leu Val Ala 100 105 110 Thr Ser Gln Val Glu Asp
Leu Val Val Asn Leu Val Pro Leu Gly Arg 115 120 125 <210> SEQ
ID NO 23 <211> LENGTH: 132 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta 240 <400> SEQUENCE: 23 Ala Lys
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys 1 5 10 15
Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20
25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr
Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr
Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala
Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala
Tyr 130 <210> SEQ ID NO 24 <211> LENGTH: 132
<212> TYPE: PRT <213> ORGANISM: Bacteriophage Q beta
243 <400> SEQUENCE: 24 Ala Lys Leu Glu Thr Val Thr Leu Gly
Lys Ile Gly Lys Asp Gly Lys 1 5 10 15 Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Gln
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60 Gln
Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys 65 70
75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser
Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg
Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp
Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130 <210> SEQ
ID NO 25 <211> LENGTH: 132 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta 250 <400> SEQUENCE: 25 Ala Arg
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys 1 5 10 15
Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20
25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr
Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr
Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala
Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala
Tyr 130 <210> SEQ ID NO 26 <211> LENGTH: 132
<212> TYPE: PRT <213> ORGANISM: Bacteriophage Q beta
251 <400> SEQUENCE: 26 Ala Lys Leu Glu Thr Val Thr Leu Gly
Asn Ile Gly Lys Asp Gly Arg 1 5 10 15 Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Gln
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60 Gln
Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys 65 70
75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser
Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg
Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp
Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130 <210> SEQ
ID NO 27 <211> LENGTH: 132 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta 259 <400> SEQUENCE: 27 Ala Arg
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg 1 5 10 15
Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20
25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr
Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr
Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala
Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala
Tyr 130 <210> SEQ ID NO 28 <211> LENGTH: 185
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 28 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr
Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met
Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80
Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85
90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Asp
Arg Gly Arg Ser Pro Arg Arg 145 150 155 160 Arg Thr Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg 165 170 175 Arg Ser Gln Ser
Arg Glu Ser Gln Cys 180 185 <210> SEQ ID NO 29 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 29 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Gly Ser Gln Cys 180 <210> SEQ ID NO 30 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 30 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Thr Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Cys Val Ile Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Gly Ser Gln Cys 180 <210> SEQ ID NO 31 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 31 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 32 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 32 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Asn Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 33 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 33 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Thr Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Cys Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 34 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 34 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 35 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 35 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Asp Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 36 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 36 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro Gln 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 37 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 37 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Lys Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Gly Ser Gln Cys 210 <210> SEQ ID NO 38 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 38 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Phe Arg Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Asp Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Ser Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Cys Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 39 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 39 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 40 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 40 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg His Ala Ile Leu Cys Trp Gly Asp Leu Arg
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 41 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 41 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Asp Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Phe Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Ala Asn Leu Glu Asp Pro Ala
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Gln Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Cys 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 42 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 42 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 43 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 43 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met
Ser 85 90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 44 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 44 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 45 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 45 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 46 <211>
LENGTH: 183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 46 Met Asp Ile Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu
Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu
Met Thr Leu Ala Thr Trp Val Gly Ala Asn Leu Glu Asp Pro Ala 65 70
75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr
Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly
Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg
Arg Gly Arg Thr Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser
Arg Glu Ser Gln Cys 180 <210> SEQ ID NO 47 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 47 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 48 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 48 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 49 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 49 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Thr Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ala Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 50 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <400> SEQUENCE: 50 Met Gln Leu Phe His Leu Cys Leu Ile
Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu
Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp
Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala
Leu Tyr Arg Glu Ala Phe Glu Cys Ser Glu His Cys Ser Pro His 65 70
75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met
Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile
Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly
Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val
Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195
200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO 51 <211>
LENGTH: 212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B
virus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (28)..(28) <223> OTHER INFORMATION: Xaa
can represent any amino acid <400> SEQUENCE: 51 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Xaa Asp Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Ile Thr 85 90 95 Leu Ser Thr Trp Val Gly Gly Asn
Leu Glu Asp Pro Thr Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Thr Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 52 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 52 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Asn
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 53 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 53 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Cys Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 54 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 54 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Pro Gln Cys 210 <210> SEQ ID
NO 55 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 55 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Ser Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 56 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 56 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Leu Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 57 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 57 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Lys Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 58 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 58 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ala 50 55 60 Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Thr Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Leu Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 59 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 59 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Ser Met Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Tyr Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Thr Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu
Gln Asp Pro Thr 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Val Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Val Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Gln Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
60 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 60 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg His Val Phe Leu Cys Trp
Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu
Glu Asp Pro Thr 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
61 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 61 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Thr Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 62 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 62 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Ile Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 63 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 63 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu
Glu Asp Pro Val 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Val Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Ala Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
64 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 64 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Met Asn 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn
Leu Glu Asp Pro Val Ser Arg Asp 100 105 110 Leu Val Val Gly Tyr Val
Asn Thr Thr Val Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 65 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 65 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu
Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Thr Val Val Arg Arg Arg Gly Arg Thr Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
66 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 66 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Ala Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Ile Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 67 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 67 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Thr Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Val Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 68 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 68 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Arg Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Thr Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 69 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 69 Met Gln Leu
Phe His Leu Cys Leu Val Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ala 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Asn Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Asn Tyr Val
Asn Thr Asn Met Gly Leu Lys Ile Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Leu Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 70 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 70 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Ala Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Asp Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 71 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 71 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ala Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu
Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
72 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 72 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu
Glu Asp Pro Ile 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Gly Ser Gln Cys 180 <210> SEQ ID NO
73 <211> LENGTH: 188 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 73 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu 1 5 10 15 Asn
Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25
30 Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys
35 40 45 Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp
Asp Glu 50 55 60 Leu Thr Lys Leu Ile Ala Trp Met Ser Ser Asn Ile
Thr Ser Glu Gln 65 70 75 80 Val Arg Thr Ile Ile Val Asn His Val Asn
Asp Thr Trp Gly Leu Lys 85 90 95 Val Arg Gln Ser Leu Trp Phe His
Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110 His Thr Val Gln Glu Phe
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Ala Pro Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu His
Thr Val Ile Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser 145 150 155
160 Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
165 170 175 Arg Arg Arg Arg Ser Gln Ser Pro Ser Thr Asn Cys 180 185
<210> SEQ ID NO 74 <211> LENGTH: 217 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
74 Met Tyr Leu Phe His Leu Cys Leu Val Phe Ala Cys Val Pro Cys Pro
1 5 10 15 Thr Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp
Met Asp 20 25 30 Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln
Leu Leu Asn Phe 35 40 45 Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn
Ala Leu Val Asp Thr Ala 50 55 60 Ala Ala Leu Tyr Glu Glu Glu Leu
Thr Gly Arg Glu His Cys Ser Pro 65 70 75 80 His His Thr Ala Ile Arg
Gln Ala Leu Val Cys Trp Glu Glu Leu Thr 85 90 95 Arg Leu Ile Thr
Trp Met Ser Glu Asn Thr Thr Glu Glu Val Arg Arg 100 105 110 Ile Ile
Val Asp His Val Asn Asn Thr Trp Gly Leu Lys Val Arg Gln 115 120 125
Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln His Thr Val 130
135 140 Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Ala
Pro 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu His Thr 165 170 175 Val Ile Arg Arg Arg Gly Gly Ser Arg Ala
Ala Arg Ser Pro Arg Arg 180 185 190 Arg Thr Pro Ser Pro Arg Arg Arg
Arg Ser Gln Ser Pro Arg Arg Arg 195 200 205 Arg Ser Gln Ser Pro Ala
Ser Asn Cys 210 215 <210> SEQ ID NO 75 <211> LENGTH:
262 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 75 Met Asp Val Asn Ala Ser Arg Ala Leu Ala
Asn Val Tyr Asp Leu Pro 1 5 10 15 Asp Asp Phe Phe Pro Lys Ile Glu
Asp Leu Val Arg Asp Ala Lys Asp 20 25 30 Ala Leu Glu Pro Tyr Trp
Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45 Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60 Gln Gly
Met His Glu Ile Ala Glu Ala Ile Arg Ala Val Ile Pro Pro 65 70 75 80
Thr Thr Ala Pro Val Pro Ser Gly Tyr Leu Ile Gln His Asp Glu Ala 85
90 95 Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys Glu Gln Glu Glu Arg
Ile 100 105 110 Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile
His Ala His 115 120 125 Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser
Leu Asp Arg Ala Arg 130 135 140 Arg Leu Leu Trp Trp His Tyr Asn Cys
Leu Leu Trp Gly Glu Ala Thr 145 150 155 160 Val Thr Asn Tyr Ile Ser
Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175 Lys Tyr Arg Gly
Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190 Ile Gln
Val Ala Gln Gly Gly Arg Lys Thr Ser Thr Ala Thr Arg Lys 195 200 205
Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val 210
215 220 Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu Arg Arg Ala Ser Ser
Pro 225 230 235 240 Gln Arg Ala Gly Ser Pro Leu Pro Arg Ser Ser Ser
Ser His His Arg 245 250 255 Ser Pro Ser Pro Arg Lys 260 <210>
SEQ ID NO 76 <211> LENGTH: 305 <212> TYPE: PRT
<213> ORGANISM: Hepatitis B virus <400> SEQUENCE: 76
Met Trp Asp Leu Arg Leu His Pro Ser Pro Phe Gly Ala Ala Cys Gln 1 5
10 15 Gly Ile Phe Thr Ser Ser Leu Leu Leu Phe Leu Val Thr Val Pro
Leu 20 25 30 Val Cys Thr Ile Val Tyr Asp Ser Cys Leu Cys Met Asp
Ile Asn Ala 35 40 45 Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro
Asp Asp Phe Phe Pro 50 55 60 Lys Ile Asp Asp Leu Val Arg Asp Ala
Lys Asp Ala Leu Glu Pro Tyr 65 70 75 80 Trp Arg Asn Asp Ser Ile Lys
Lys His Val Leu Ile Ala Thr His Phe 85 90 95 Val Asp Leu Ile Glu
Asp Phe Trp Gln Thr Thr Gln Gly Met His Glu 100 105 110 Ile Ala Glu
Ala Leu Arg Ala Ile Ile Pro Ala Thr Thr Ala Pro Val 115 120 125 Pro
Gln Gly Phe Leu Val Gln His Glu Glu Ala Glu Glu Ile Pro Leu 130 135
140 Gly Glu Leu Phe Arg Tyr Gln Glu Glu Arg Leu Thr Asn Phe Gln Pro
145 150 155 160 Asp Tyr Pro Val Thr Ala Arg Ile His Ala His Leu Lys
Ala Tyr Ala 165 170 175 Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg
Arg Leu Leu Trp Trp 180 185 190 His Tyr Asn Cys Leu Leu Trp Gly Glu
Pro Asn Val Thr Asn Tyr Ile 195 200 205 Ser Arg Leu Arg Thr Trp Leu
Ser Thr Pro Glu Lys Tyr Arg Gly Lys 210 215 220 Asp Ala Pro Thr Ile
Glu Ala Ile Thr Arg Pro Ile Gln Val Ala Gln 225 230 235 240 Gly Gly
Arg Asn Lys Thr Gln Gly Val Arg Lys Ser Arg Gly Leu Glu 245 250 255
Pro Arg Arg Arg Arg Val Lys Thr Thr Ile Val Tyr Gly Arg Arg Arg 260
265 270 Ser Lys Ser Arg Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala Gly
Ser 275 280 285 Pro Leu Pro Arg Thr Ser Arg Asp His His Arg Ser Pro
Ser Pro Arg 290 295 300 Glu 305 <210> SEQ ID NO 77
<211> LENGTH: 185 <212> TYPE: PRT <213> ORGANISM:
Hepatitis B virus <400> SEQUENCE: 77 Met Asp Ile Asp Pro Tyr
Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro
Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala
Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45
Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50
55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro
Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met
Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys
Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser
Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val
Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg 145 150 155 160 Arg Thr
Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg 165 170 175
Arg Ser Gln Ser Arg Glu Ser Gln Cys 180 185 <210> SEQ ID NO
78 <211> LENGTH: 152 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 78 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ala Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Thr Asn Leu
Glu Asp Gly Gly 65 70 75 80 Lys Gly Gly Ser Arg Asp Leu Val Val Ser
Tyr Val Asn Thr Asn Val 85 90 95 Gly Leu Lys Phe Arg Gln Leu Leu
Trp Phe His Ile Ser Cys Leu Thr 100 105 110 Phe Gly Arg Glu Thr Val
Leu Glu Tyr Leu Val Ser Phe Gly Val Trp 115 120 125 Ile Arg Thr Pro
Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser 130 135 140 Thr Leu
Pro Glu Thr Thr Val Val 145 150 <210> SEQ ID NO 79
<211> LENGTH: 3635 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Plasmid pAP283 58 <400> SEQUENCE: 79
cgagctcgcc cctggcttat cgaaattaat acgactcact atagggagac cggaattcga
60 gctcgcccgg ggatcctcta gaattttctg cgcacccatc ccgggtggcg
cccaaagtga 120 ggaaaatcac atggcaaata agccaatgca accgatcaca
tctacagcaa ataaaattgt 180 gtggtcggat ccaactcgtt tatcaactac
attttcagca agtctgttac gccaacgtgt 240 taaagttggt atagccgaac
tgaataatgt ttcaggtcaa tatgtatctg tttataagcg 300 tcctgcacct
aaaccggaag gttgtgcaga tgcctgtgtc attatgccga atgaaaacca 360
atccattcgc acagtgattt cagggtcagc cgaaaacttg gctaccttaa aagcagaatg
420 ggaaactcac aaacgtaacg ttgacacact cttcgcgagc ggcaacgccg
gtttgggttt 480 ccttgaccct actgcggcta tcgtatcgtc tgatactact
gcttaagctt gtattctata 540 gtgtcaccta aatcgtatgt gtatgataca
taaggttatg tattaattgt agccgcgttc 600 taacgacaat atgtacaagc
ctaattgtgt agcatctggc ttactgaagc agaccctatc 660 atctctctcg
taaactgccg tcagagtcgg tttggttgga cgaaccttct gagtttctgg 720
taacgccgtt ccgcaccccg gaaatggtca ccgaaccaat cagcagggtc atcgctagcc
780 agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
gcggttgctg 840 gcgcctatat cgccgacatc accgatgggg aagatcgggc
tcgccacttc gggctcatga 900 gcgcttgttt cggcgtgggt atggtggcag
gccccgtggc cgggggactg ttgggcgcca 960 tctccttgca tgcaccattc
cttgcggcgg cggtgctcaa cggcctcaac ctactactgg 1020 gctgcttcct
aatgcaggag tcgcataagg gagagcgtcg atatggtgca ctctcagtac 1080
aatctgctct gatgccgcat agttaagcca actccgctat cgctacgtga ctgggtcatg
1140 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
tctgctcccg 1200 gcatccgctt acagacaagc tgtgaccgtc tccgggagct
gcatgtgtca gaggttttca 1260 ccgtcatcac cgaaacgcgc gaggcagctt
gaagacgaaa gggcctcgtg atacgcctat 1320 ttttataggt taatgtcatg
ataataatgg tttcttagac gtcaggtggc acttttcggg 1380 gaaatgtgcg
cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 1440
tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
1500 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
cctgtttttg 1560 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
tcagttgggt gcacgagtgg 1620 gttacatcga actggatctc aacagcggta
agatccttga gagttttcgc cccgaagaac 1680 gttttccaat gatgagcact
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 1740 acgccgggca
agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1800
actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
1860 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
atcggaggac 1920 cgaaggagct aaccgctttt ttgcacaaca tgggggatca
tgtaactcgc cttgatcgtt 1980 gggaaccgga gctgaatgaa gccataccaa
acgacgagcg tgacaccacg atgcctgtag 2040 caatggcaac aacgttgcgc
aaactattaa ctggcgaact acttactcta gcttcccggc 2100 aacaattaat
agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 2160
ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
2220 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
tacacgacgg 2280 ggagtcaggc aactatggat gaacgaaata gacagatcgc
tgagataggt gcctcactga 2340 ttaagcattg gtaactgtca gaccaagttt
actcatatat actttagatt gatttaaaac 2400 ttcattttta atttaaaagg
atctaggtga agatcctttt tgataatctc atgaccaaaa 2460 tcccttaacg
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2520
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
2580 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
aaggtaactg 2640 gcttcagcag agcgcagata ccaaatactg tccttctagt
gtagccgtag ttaggccacc 2700 acttcaagaa ctctgtagca ccgcctacat
acctcgctct gctaatcctg ttaccagtgg 2760 ctgctgccag tggcgataag
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2820 ataaggcgca
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2880
cgacctacac cgaactgaga tacctacagc gcgagcattg agaaagcgcc acgcttcccg
2940 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
gagcgcacga 3000 gggagcttcc agggggaaac gcctggtatc tttatagtcc
tgtcgggttt cgccacctct 3060 gacttgagcg tcgatttttg tgatgctcgt
caggggggcg gagcctatgg aaaaacgcca 3120 gcaacgcggc ctttttacgg
ttcctggcct tttgctggcc ttttgctcac atgttctttc 3180 ctgcgttatc
ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 3240
ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
3300 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
tgtggtgtca 3360 tggtcggtga tcgccagggt gccgacgcgc atctcgactg
catggtgcac caatgcttct 3420 ggcgtcaggc agccatcgga agctgtggta
tggccgtgca ggtcgtaaat cactgcataa 3480 ttcgtgtcgc tcaaggcgca
ctcccgttct ggataatgtt ttttgcgccg acatcataac 3540 ggttctggca
aatattctga aatgagctgt tgacaattaa tcatcgaact agttaactag 3600
tacgcaagtt cacgtaaaaa gggtatcgcg gaatt 3635 <210> SEQ ID NO
80 <211> LENGTH: 131 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Ap205 coat protein <400> SEQUENCE: 80 Met
Ala Asn Lys Pro Met Gln Pro Ile Thr Ser Thr Ala Asn Lys Ile 1 5 10
15 Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu
20 25 30 Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn
Val Ser 35 40 45 Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro
Lys Pro Glu Gly 50 55 60 Cys Ala Asp Ala Cys Val Ile Met Pro Asn
Glu Asn Gln Ser Ile Arg 65 70 75 80 Thr Val Ile Ser Gly Ser Ala Glu
Asn Leu Ala Thr Leu Lys Ala Glu 85 90 95 Trp Glu Thr His Lys Arg
Asn Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110 Ala Gly Leu Gly
Phe Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125 Thr Thr
Ala 130 <210> SEQ ID NO 81 <211> LENGTH: 131
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: AP205 coat
protein <400> SEQUENCE: 81 Met Ala Asn Lys Thr Met Gln Pro
Ile Thr Ser Thr Ala Asn Lys Ile 1 5 10 15 Val Trp Ser Asp Pro Thr
Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30 Leu Arg Gln Arg
Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45 Gly Gln
Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60
Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg 65
70 75 80 Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys
Ala Glu 85 90 95 Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe
Ala Ser Gly Asn 100 105 110 Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala
Ala Ile Val Ser Ser Asp 115 120 125 Thr Thr Ala 130 <210> SEQ
ID NO 82 <211> LENGTH: 3613 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Plasmid pAP281 32 <400> SEQUENCE: 82
cgagctcgcc cctggcttat cgaaattaat acgactcact atagggagac cggaattcga
60 gctcgcccgg ggatcctcta gattaaccca acgcgtagga gtcaggccat
ggcaaataag 120 acaatgcaac cgatcacatc tacagcaaat aaaattgtgt
ggtcggatcc aactcgttta 180 tcaactacat tttcagcaag tctgttacgc
caacgtgtta aagttggtat agccgaactg 240 aataatgttt caggtcaata
tgtatctgtt tataagcgtc ctgcacctaa accggaaggt 300 tgtgcagatg
cctgtgtcat tatgccgaat gaaaaccaat ccattcgcac agtgatttca 360
gggtcagccg aaaacttggc taccttaaaa gcagaatggg aaactcacaa acgtaacgtt
420 gacacactct tcgcgagcgg caacgccggt ttgggtttcc ttgaccctac
tgcggctatc 480 gtatcgtctg atactactgc ttaagcttgt attctatagt
gtcacctaaa tcgtatgtgt 540 atgatacata aggttatgta ttaattgtag
ccgcgttcta acgacaatat gtacaagcct 600 aattgtgtag catctggctt
actgaagcag accctatcat ctctctcgta aactgccgtc 660 agagtcggtt
tggttggacg aaccttctga gtttctggta acgccgttcc gcaccccgga 720
aatggtcacc gaaccaatca gcagggtcat cgctagccag atcctctacg ccggacgcat
780 cgtggccggc atcaccggcg ccacaggtgc ggttgctggc gcctatatcg
ccgacatcac 840 cgatggggaa gatcgggctc gccacttcgg gctcatgagc
gcttgtttcg gcgtgggtat 900 ggtggcaggc cccgtggccg ggggactgtt
gggcgccatc tccttgcatg caccattcct 960 tgcggcggcg gtgctcaacg
gcctcaacct actactgggc tgcttcctaa tgcaggagtc 1020 gcataaggga
gagcgtcgat atggtgcact ctcagtacaa tctgctctga tgccgcatag 1080
ttaagccaac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
1140 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
agacaagctg 1200 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
gtcatcaccg aaacgcgcga 1260 ggcagcttga agacgaaagg gcctcgtgat
acgcctattt ttataggtta atgtcatgat 1320 aataatggtt tcttagacgt
caggtggcac ttttcgggga aatgtgcgcg gaacccctat 1380 ttgtttattt
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 1440
aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
1500 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
cgctggtgaa 1560 agtaaaagat gctgaagatc agttgggtgc acgagtgggt
tacatcgaac tggatctcaa 1620 cagcggtaag atccttgaga gttttcgccc
cgaagaacgt tttccaatga tgagcacttt 1680 taaagttctg ctatgtggcg
cggtattatc ccgtattgac gccgggcaag agcaactcgg 1740 tcgccgcata
cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 1800
tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
1860 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
ccgctttttt 1920 gcacaacatg ggggatcatg taactcgcct tgatcgttgg
gaaccggagc tgaatgaagc 1980 cataccaaac gacgagcgtg acaccacgat
gcctgtagca atggcaacaa cgttgcgcaa 2040 actattaact ggcgaactac
ttactctagc ttcccggcaa caattaatag actggatgga 2100 ggcggataaa
gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 2160
tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
2220 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
ctatggatga 2280 acgaaataga cagatcgctg agataggtgc ctcactgatt
aagcattggt aactgtcaga 2340 ccaagtttac tcatatatac tttagattga
tttaaaactt catttttaat ttaaaaggat 2400 ctaggtgaag atcctttttg
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 2460 ccactgagcg
tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 2520
gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
2580 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
cgcagatacc 2640 aaatactgtc cttctagtgt agccgtagtt aggccaccac
ttcaagaact ctgtagcacc 2700 gcctacatac ctcgctctgc taatcctgtt
accagtggct gctgccagtg gcgataagtc 2760 gtgtcttacc gggttggact
caagacgata gttaccggat aaggcgcagc ggtcgggctg 2820 aacggggggt
tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 2880
cctacagcgc gagcattgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
2940 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc 3000 ctggtatctt tatagtcctg tcgggtttcg ccacctctga
cttgagcgtc gatttttgtg 3060 atgctcgtca ggggggcgga gcctatggaa
aaacgccagc aacgcggcct ttttacggtt 3120 cctggccttt tgctggcctt
ttgctcacat gttctttcct gcgttatccc ctgattctgt 3180 ggataaccgt
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 3240
gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
3300 cgcgcgttgg ccgattcatt aatgcagctg tggtgtcatg gtcggtgatc
gccagggtgc 3360 cgacgcgcat ctcgactgca tggtgcacca atgcttctgg
cgtcaggcag ccatcggaag 3420 ctgtggtatg gccgtgcagg tcgtaaatca
ctgcataatt cgtgtcgctc aaggcgcact 3480 cccgttctgg ataatgtttt
ttgcgccgac atcataacgg ttctggcaaa tattctgaaa 3540 tgagctgttg
acaattaatc atcgaactag ttaactagta cgcaagttca cgtaaaaagg 3600
gtatcgcgga att 3613 <210> SEQ ID NO 83 <400> SEQUENCE:
83 000 3 <210> SEQ ID NO 84 <400> SEQUENCE: 84 000 3
<210> SEQ ID NO 85 <400> SEQUENCE: 85 000 3 <210>
SEQ ID NO 86 <400> SEQUENCE: 86 000 3 <210> SEQ ID NO
87 <400> SEQUENCE: 87 000 3 <210> SEQ ID NO 88
<400> SEQUENCE: 88 000 3 <210> SEQ ID NO 89 <400>
SEQUENCE: 89 000 3 <210> SEQ ID NO 90 <400> SEQUENCE:
90 000 3 <210> SEQ ID NO 91 <400> SEQUENCE: 91 000 3
<210> SEQ ID NO 92 <400> SEQUENCE: 92 000 3 <210>
SEQ ID NO 93 <400> SEQUENCE: 93 000 3 <210> SEQ ID NO
94 <400> SEQUENCE: 94 000 3 <210> SEQ ID NO 95
<400> SEQUENCE: 95 000 3 <210> SEQ ID NO 96 <400>
SEQUENCE: 96 000 3 <210> SEQ ID NO 97 <400> SEQUENCE:
97 000 3 <210> SEQ ID NO 98 <400> SEQUENCE: 98 000 3
<210> SEQ ID NO 99 <400> SEQUENCE: 99 000 3 <210>
SEQ ID NO 100 <400> SEQUENCE: 100 000 3 <210> SEQ ID NO
101 <400> SEQUENCE: 101 000 3 <210> SEQ ID NO 102
<400> SEQUENCE: 102 000 3 <210> SEQ ID NO 103
<400> SEQUENCE: 103 000 3 <210> SEQ ID NO 104
<400> SEQUENCE: 104 000 3 <210> SEQ ID NO 105
<400> SEQUENCE: 105 000 3 <210> SEQ ID NO 106
<400> SEQUENCE: 106 000 3 <210> SEQ ID NO 107
<400> SEQUENCE: 107 000 3 <210> SEQ ID NO 108
<400> SEQUENCE: 108 000 3 <210> SEQ ID NO 109
<400> SEQUENCE: 109 000 3 <210> SEQ ID NO 110
<400> SEQUENCE: 110 000 3 <210> SEQ ID NO 111
<400> SEQUENCE: 111 000 3 <210> SEQ ID NO 112
<400> SEQUENCE: 112 000 3 <210> SEQ ID NO 113
<400> SEQUENCE: 113 000 3 <210> SEQ ID NO 114
<400> SEQUENCE: 114 000 3 <210> SEQ ID NO 115
<400> SEQUENCE: 115 000 3 <210> SEQ ID NO 116
<400> SEQUENCE: 116 000 3 <210> SEQ ID NO 117
<400> SEQUENCE: 117 000 3 <210> SEQ ID NO 118
<400> SEQUENCE: 118 000 3 <210> SEQ ID NO 119
<400> SEQUENCE: 119 000 3 <210> SEQ ID NO 120
<400> SEQUENCE: 120 000 3 <210> SEQ ID NO 121
<400> SEQUENCE: 121 000 3 <210> SEQ ID NO 122
<400> SEQUENCE: 122 000 3 <210> SEQ ID NO 123
<400> SEQUENCE: 123 000 3 <210> SEQ ID NO 124
<400> SEQUENCE: 124 000 3 <210> SEQ ID NO 125
<400> SEQUENCE: 125 000 3 <210> SEQ ID NO 126
<400> SEQUENCE: 126 000 3 <210> SEQ ID NO 127
<400> SEQUENCE: 127 000 3 <210> SEQ ID NO 128
<400> SEQUENCE: 128 000 3 <210> SEQ ID NO 129
<400> SEQUENCE: 129 000 3 <210> SEQ ID NO 130
<400> SEQUENCE: 130 000 3 <210> SEQ ID NO 131
<400> SEQUENCE: 131 000 3 <210> SEQ ID NO 132
<400> SEQUENCE: 132 000 3 <210> SEQ ID NO 133
<400> SEQUENCE: 133 000 3 <210> SEQ ID NO 134
<400> SEQUENCE: 134 000 3 <210> SEQ ID NO 135
<400> SEQUENCE: 135 000 3 <210> SEQ ID NO 136
<400> SEQUENCE: 136 000 3 <210> SEQ ID NO 137
<400> SEQUENCE: 137 000 3 <210> SEQ ID NO 138
<400> SEQUENCE: 138 000 3 <210> SEQ ID NO 139
<400> SEQUENCE: 139 000 3 <210> SEQ ID NO 140
<400> SEQUENCE: 140 000 3 <210> SEQ ID NO 141
<400> SEQUENCE: 141 000 3 <210> SEQ ID NO 142
<400> SEQUENCE: 142 000 3 <210> SEQ ID NO 143
<400> SEQUENCE: 143 000 3 <210> SEQ ID NO 144
<400> SEQUENCE: 144 000 3 <210> SEQ ID NO 145
<400> SEQUENCE: 145 000 3 <210> SEQ ID NO 146
<400> SEQUENCE: 146 000 3 <210> SEQ ID NO 147
<400> SEQUENCE: 147 000 3 <210> SEQ ID NO 148
<400> SEQUENCE: 148 000 3 <210> SEQ ID NO 149
<400> SEQUENCE: 149 000 3 <210> SEQ ID NO 150
<400> SEQUENCE: 150 000 3 <210> SEQ ID NO 151
<400> SEQUENCE: 151 000 3 <210> SEQ ID NO 152
<400> SEQUENCE: 152 000 3 <210> SEQ ID NO 153
<400> SEQUENCE: 153 000 3 <210> SEQ ID NO 154
<400> SEQUENCE: 154 000 3 <210> SEQ ID NO 155
<400> SEQUENCE: 155 000 3 <210> SEQ ID NO 156
<400> SEQUENCE: 156 000 3 <210> SEQ ID NO 157
<400> SEQUENCE: 157 000 3 <210> SEQ ID NO 158
<400> SEQUENCE: 158 000 3 <210> SEQ ID NO 159
<400> SEQUENCE: 159 000 3 <210> SEQ ID NO 160
<400> SEQUENCE: 160 000 3 <210> SEQ ID NO 161
<400> SEQUENCE: 161 000 3 <210> SEQ ID NO 162
<400> SEQUENCE: 162 000 3 <210> SEQ ID NO 163
<400> SEQUENCE: 163 000 3 <210> SEQ ID NO 164
<400> SEQUENCE: 164 000 3 <210> SEQ ID NO 165
<400> SEQUENCE: 165 000 3 <210> SEQ ID NO 166
<400> SEQUENCE: 166 000 3 <210> SEQ ID NO 167
<400> SEQUENCE: 167 000 3 <210> SEQ ID NO 168
<400> SEQUENCE: 168 000 3 <210> SEQ ID NO 169
<400> SEQUENCE: 169 000 3 <210> SEQ ID NO 170
<400> SEQUENCE: 170 000 3 <210> SEQ ID NO 171
<400> SEQUENCE: 171 000 3 <210> SEQ ID NO 172
<400> SEQUENCE: 172 000 3 <210> SEQ ID NO 173
<400> SEQUENCE: 173 000 3 <210> SEQ ID NO 174
<400> SEQUENCE: 174 000 3 <210> SEQ ID NO 175
<400> SEQUENCE: 175 000 3 <210> SEQ ID NO 176
<400> SEQUENCE: 176 000 3 <210> SEQ ID NO 177
<400> SEQUENCE: 177 000 3 <210> SEQ ID NO 178
<400> SEQUENCE: 178 000 3 <210> SEQ ID NO 179
<400> SEQUENCE: 179 000 3 <210> SEQ ID NO 180
<400> SEQUENCE: 180 000 3 <210> SEQ ID NO 181
<400> SEQUENCE: 181 000 3 <210> SEQ ID NO 182
<400> SEQUENCE: 182 000 3 <210> SEQ ID NO 183
<400> SEQUENCE: 183 000 3 <210> SEQ ID NO 184
<400> SEQUENCE: 184 000 3 <210> SEQ ID NO 185
<400> SEQUENCE: 185 000 3 <210> SEQ ID NO 186
<400> SEQUENCE: 186 000 3 <210> SEQ ID NO 187
<400> SEQUENCE: 187 000 3 <210> SEQ ID NO 188
<400> SEQUENCE: 188 000 3 <210> SEQ ID NO 189
<400> SEQUENCE: 189 000 3 <210> SEQ ID NO 190
<400> SEQUENCE: 190 000 3 <210> SEQ ID NO 191
<400> SEQUENCE: 191 000 3 <210> SEQ ID NO 192
<400> SEQUENCE: 192 000 3 <210> SEQ ID NO 193
<400> SEQUENCE: 193 000 3 <210> SEQ ID NO 194
<400> SEQUENCE: 194 000 3 <210> SEQ ID NO 195
<400> SEQUENCE: 195 000 3 <210> SEQ ID NO 196
<400> SEQUENCE: 196 000 3 <210> SEQ ID NO 197
<400> SEQUENCE: 197 000 3 <210> SEQ ID NO 198
<400> SEQUENCE: 198 000 3 <210> SEQ ID NO 199
<400> SEQUENCE: 199 000 3 <210> SEQ ID NO 200
<400> SEQUENCE: 200 000 3 <210> SEQ ID NO 201
<400> SEQUENCE: 201 000 3 <210> SEQ ID NO 202
<400> SEQUENCE: 202 000 3 <210> SEQ ID NO 203
<400> SEQUENCE: 203 000 3 <210> SEQ ID NO 204
<400> SEQUENCE: 204 000 3 <210> SEQ ID NO 205
<400> SEQUENCE: 205 000 3 <210> SEQ ID NO 206
<400> SEQUENCE: 206 000 3 <210> SEQ ID NO 207
<400> SEQUENCE: 207 000 3 <210> SEQ ID NO 208
<400> SEQUENCE: 208 000 3 <210> SEQ ID NO 209
<400> SEQUENCE: 209 000 3 <210> SEQ ID NO 210
<400> SEQUENCE: 210 000 3 <210> SEQ ID NO 211
<400> SEQUENCE: 211 000 3 <210> SEQ ID NO 212
<400> SEQUENCE: 212 000 3 <210> SEQ ID NO 213
<400> SEQUENCE: 213 000 3 <210> SEQ ID NO 214
<400> SEQUENCE: 214 000 3 <210> SEQ ID NO 215
<400> SEQUENCE: 215 000 3 <210> SEQ ID NO 216
<400> SEQUENCE: 216 000 3 <210> SEQ ID NO 217
<400> SEQUENCE: 217 000 3 <210> SEQ ID NO 218
<400> SEQUENCE: 218 000 3 <210> SEQ ID NO 219
<400> SEQUENCE: 219 000 3 <210> SEQ ID NO 220
<400> SEQUENCE: 220 000 3 <210> SEQ ID NO 221
<400> SEQUENCE: 221 000 3 <210> SEQ ID NO 222
<400> SEQUENCE: 222 000 3 <210> SEQ ID NO 223
<400> SEQUENCE: 223 000 3 <210> SEQ ID NO 224
<400> SEQUENCE: 224 000 3 <210> SEQ ID NO 225
<400> SEQUENCE: 225 000 3 <210> SEQ ID NO 226
<400> SEQUENCE: 226 000 3 <210> SEQ ID NO 227
<400> SEQUENCE: 227 000 3 <210> SEQ ID NO 228
<400> SEQUENCE: 228 000 3 <210> SEQ ID NO 229
<400> SEQUENCE: 229 000 3 <210> SEQ ID NO 230
<211> LENGTH: 132 <212> TYPE: PRT <213> ORGANISM:
precursor human IL 13 <400> SEQUENCE: 230 Met Ala Leu Leu Leu
Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly 1 5 10 15 Phe Ala Ser
Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu 20 25 30 Ile
Glu Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys 35 40
45 Asn Gly Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys
50 55 60 Ala Ala Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala
Ile Glu 65 70 75 80 Lys Thr Gln Arg Met Leu Ser Gly Phe Cys Pro His
Lys Val Ser Ala 85 90 95 Gly Gln Phe Ser Ser Leu His Val Arg Asp
Thr Lys Ile Glu Val Ala 100 105 110 Gln Phe Val Lys Asp Leu Leu Leu
His Leu Lys Lys Leu Phe Arg Glu 115 120 125 Gly Arg Phe Asn 130
<210> SEQ ID NO 231 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: processed human IL 13 <400>
SEQUENCE: 231 Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile
Glu Glu Leu 1 5 10 15 Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu
Cys Asn Gly Ser Met 20 25 30 Val Trp Ser Ile Asn Leu Thr Ala Gly
Met Tyr Cys Ala Ala Leu Glu 35 40 45 Ser Leu Ile Asn Val Ser Gly
Cys Ser Ala Ile Glu Lys Thr Gln Arg 50 55 60 Met Leu Ser Gly Phe
Cys Pro His Lys Val Ser Ala Gly Gln Phe Ser 65 70 75 80 Ser Leu His
Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val Lys 85 90 95 Asp
Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe Asn 100 105
110 <210> SEQ ID NO 232 <211> LENGTH: 111 <212>
TYPE: PRT <213> ORGANISM: processed mouse IL 13 <400>
SEQUENCE: 232 Gly Pro Val Pro Arg Ser Val Ser Leu Pro Leu Thr Leu
Lys Glu Leu 1 5 10 15 Ile Glu Glu Leu Ser Asn Ile Thr Gln Asp Gln
Thr Pro Leu Cys Asn 20 25 30 Gly Ser Met Val Trp Ser Val Asp Leu
Ala Ala Gly Gly Phe Cys Val 35 40 45 Ala Leu Asp Ser Leu Thr Asn
Ile Ser Asn Cys Asn Ala Ile Tyr Arg 50 55 60 Thr Gln Arg Ile Leu
His Gly Leu Cys Asn Arg Lys Ala Pro Thr Thr 65 70 75 80 Val Ser Ser
Leu Pro Asp Thr Lys Ile Glu Val Ala His Phe Ile Thr 85 90 95 Lys
Leu Leu Ser Tyr Thr Lys Gln Leu Phe Arg His Gly Pro Phe 100 105 110
<210> SEQ ID NO 233 <211> LENGTH: 134 <212> TYPE:
PRT <213> ORGANISM: precursor human IL 5 <400>
SEQUENCE: 233 Met Arg Met Leu Leu His Leu Ser Leu Leu Ala Leu Gly
Ala Ala Tyr 1 5 10 15 Val Tyr Ala Ile Pro Thr Glu Ile Pro Thr Ser
Ala Leu Val Lys Glu 20 25 30 Thr Leu Ala Leu Leu Ser Thr His Arg
Thr Leu Leu Ile Ala Asn Glu 35 40 45 Thr Leu Arg Ile Pro Val Pro
Val His Lys Asn His Gln Leu Cys Thr 50 55 60 Glu Glu Ile Phe Gln
Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln 65 70 75 80 Gly Gly Thr
Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys 85 90 95 Tyr
Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val 100 105
110 Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr
115 120 125 Glu Trp Ile Ile Glu Ser 130 <210> SEQ ID NO 234
<211> LENGTH: 115 <212> TYPE: PRT <213> ORGANISM:
processed human IL 5 <400> SEQUENCE: 234 Ile Pro Thr Glu Ile
Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser
Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile
Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40
45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr
50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr
Ile Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg
Val Asn Gln Phe 85 90 95 Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val
Met Asn Thr Glu Trp Ile 100 105 110 Ile Glu Ser 115 <210> SEQ
ID NO 235 <211> LENGTH: 113 <212> TYPE: PRT <213>
ORGANISM: processed mouse IL 5 <400> SEQUENCE: 235 Met Glu
Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15
Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20
25 30 Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe
Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly
Thr Val Glu 50 55 60 Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys
Tyr Ile Asp Arg Gln 65 70 75 80 Lys Glu Lys Cys Gly Glu Glu Arg Arg
Arg Thr Arg Gln Phe Leu Asp 85 90 95 Tyr Leu Gln Glu Phe Leu Gly
Val Met Ser Thr Glu Trp Ala Met Glu 100 105 110 Gly <210> SEQ
ID NO 236 <400> SEQUENCE: 236 000 3 <210> SEQ ID NO 237
<400> SEQUENCE: 237 000 3 <210> SEQ ID NO 238
<400> SEQUENCE: 238 000 3 <210> SEQ ID NO 239
<400> SEQUENCE: 239 000 3 <210> SEQ ID NO 240
<400> SEQUENCE: 240 000 3 <210> SEQ ID NO 241
<400> SEQUENCE: 241 000 3 <210> SEQ ID NO 242
<211> LENGTH: 97 <212> TYPE: PRT <213> ORGANISM:
Human Eotaxin 1 <400> SEQUENCE: 242 Met Lys Val Ser Ala Ala
Leu Leu Trp Leu Leu Leu Ile Ala Ala Ala 1 5 10 15 Phe Ser Pro Gln
Gly Leu Ala Gly Pro Ala Ser Val Pro Thr Thr Cys 20 25 30 Cys Phe
Asn Leu Ala Asn Arg Lys Ile Pro Leu Gln Arg Leu Glu Ser 35 40 45
Tyr Arg Arg Ile Thr Ser Gly Lys Cys Pro Gln Lys Ala Val Ile Phe 50
55 60 Lys Thr Lys Leu Ala Lys Asp Ile Cys Ala Asp Pro Lys Lys Lys
Trp 65 70 75 80 Val Gln Asp Ser Met Lys Tyr Leu Asp Gln Lys Ser Pro
Thr Pro Lys 85 90 95 Pro <210> SEQ ID NO 243 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Human
Eotaxin 2 <400> SEQUENCE: 243 Met Ala Gly Leu Met Thr Ile Val
Thr Ser Leu Leu Phe Leu Gly Val 1 5 10 15 Cys Ala His His Ile Ile
Pro Thr Gly Ser Val Val Ile Pro Ser Pro 20 25 30 Cys Cys Met Phe
Phe Val Ser Lys Arg Ile Pro Glu Asn Arg Val Val 35 40 45 Ser Tyr
Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala Gly Val Ile 50 55 60
Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro Lys Gln Glu 65
70 75 80 Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala Lys Gln Lys
Lys Ala 85 90 95 Ser Pro Arg Ala Arg Ala Val Ala Val Lys Gly Pro
Val Gln Arg Tyr 100 105 110 Pro Gly Asn Gln Thr Thr Cys 115
<210> SEQ ID NO 244 <211> LENGTH: 94 <212> TYPE:
PRT <213> ORGANISM: Human Eotaxin 3 <400> SEQUENCE: 244
Met Met Gly Leu Ser Leu Ala Ser Ala Val Leu Leu Ala Ser Leu Leu 1 5
10 15 Ser Leu His Leu Gly Thr Ala Thr Arg Gly Ser Asp Ile Ser Lys
Thr 20 25 30 Cys Cys Phe Gln Tyr Ser His Lys Pro Leu Pro Trp Thr
Trp Val Arg 35 40 45 Ser Tyr Glu Phe Thr Ser Asn Ser Cys Ser Gln
Arg Ala Val Ile Phe 50 55 60 Thr Thr Lys Arg Gly Lys Lys Val Cys
Thr His Pro Arg Lys Lys Trp 65 70 75 80 Val Gln Lys Tyr Ile Ser Leu
Leu Lys Thr Pro Lys Gln Leu 85 90 <210> SEQ ID NO 245
<211> LENGTH: 97 <212> TYPE: PRT <213> ORGANISM:
Mouse Eotaxin 1 <400> SEQUENCE: 245 Met Gln Ser Ser Thr Ala
Leu Leu Phe Leu Leu Leu Thr Val Thr Ser 1 5 10 15 Phe Thr Ser Gln
Val Leu Ala His Pro Gly Ser Ile Pro Thr Ser Cys 20 25 30 Cys Phe
Ile Met Thr Ser Lys Lys Ile Pro Asn Thr Leu Leu Lys Ser 35 40 45
Tyr Lys Arg Ile Thr Asn Asn Arg Cys Thr Leu Lys Ala Ile Val Phe 50
55 60 Lys Thr Arg Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys Lys Lys
Trp 65 70 75 80 Val Gln Asp Ala Thr Lys His Leu Asp Gln Lys Leu Gln
Thr Pro Lys 85 90 95 Pro <210> SEQ ID NO 246 <211>
LENGTH: 119 <212> TYPE: PRT <213> ORGANISM: Mouse
Eotaxin 2 <400> SEQUENCE: 246 Met Ala Gly Ser Ala Thr Ile Val
Ala Gly Leu Leu Leu Leu Val Ala 1 5 10 15 Cys Ala Cys Cys Ile Phe
Pro Ile Asp Ser Val Thr Ile Pro Ser Ser 20 25 30 Cys Cys Thr Ser
Phe Ile Ser Lys Lys Ile Pro Glu Asn Arg Val Val 35 40 45 Ser Tyr
Gln Leu Ala Asn Gly Ser Ile Cys Pro Lys Ala Gly Val Ile 50 55 60
Phe Ile Thr Lys Lys Gly His Lys Ile Cys Thr Asp Pro Lys Leu Leu 65
70 75 80 Trp Val Gln Arg His Ile Gln Lys Leu Asp Ala Lys Lys Asn
Gln Pro 85 90 95 Ser Lys Gly Ala Lys Ala Val Arg Thr Lys Phe Ala
Val Gln Arg Arg 100 105 110 Arg Gly Asn Ser Thr Glu Val 115
<210> SEQ ID NO 247 <400> SEQUENCE: 247 000 3
<210> SEQ ID NO 248 <400> SEQUENCE: 248 000 3
<210> SEQ ID NO 249 <400> SEQUENCE: 249 000 3
<210> SEQ ID NO 250 <400> SEQUENCE: 250 000 3
<210> SEQ ID NO 251 <400> SEQUENCE: 251 000 3
<210> SEQ ID NO 252 <400> SEQUENCE: 252 000 3
<210> SEQ ID NO 253 <400> SEQUENCE: 253 000 3
<210> SEQ ID NO 254 <400> SEQUENCE: 254 000 3
<210> SEQ ID NO 255 <400> SEQUENCE: 255 000 3
<210> SEQ ID NO 256 <400> SEQUENCE: 256 000 3
<210> SEQ ID NO 257 <400> SEQUENCE: 257 000 3
<210> SEQ ID NO 258 <400> SEQUENCE: 258 000 3
<210> SEQ ID NO 259 <400> SEQUENCE: 259 000 3
<210> SEQ ID NO 260 <400> SEQUENCE: 260 000 3
<210> SEQ ID NO 261 <400> SEQUENCE: 261 000 3
<210> SEQ ID NO 262 <400> SEQUENCE: 262 000 3
<210> SEQ ID NO 263 <400> SEQUENCE: 263 000 3
<210> SEQ ID NO 264 <400> SEQUENCE: 264 000 3
<210> SEQ ID NO 265 <400> SEQUENCE: 265 000 3
<210> SEQ ID NO 266 <400> SEQUENCE: 266 000 3
<210> SEQ ID NO 267 <400> SEQUENCE: 267 000 3
<210> SEQ ID NO 268 <400> SEQUENCE: 268 000 3
<210> SEQ ID NO 269 <400> SEQUENCE: 269 000 3
<210> SEQ ID NO 270 <400> SEQUENCE: 270 000 3
<210> SEQ ID NO 271 <400> SEQUENCE: 271 000 3
<210> SEQ ID NO 272 <400> SEQUENCE: 272 000 3
<210> SEQ ID NO 273 <400> SEQUENCE: 273 000 3
<210> SEQ ID NO 274 <400> SEQUENCE: 274 000 3
<210> SEQ ID NO 275 <400> SEQUENCE: 275 000 3
<210> SEQ ID NO 276 <400> SEQUENCE: 276 000 3
<210> SEQ ID NO 277 <400> SEQUENCE: 277 000 3
<210> SEQ ID NO 278 <400> SEQUENCE: 278 000 3
<210> SEQ ID NO 279 <400> SEQUENCE: 279 000 3
<210> SEQ ID NO 280 <400> SEQUENCE: 280 000 3
<210> SEQ ID NO 281 <400> SEQUENCE: 281 000 3
<210> SEQ ID NO 282 <400> SEQUENCE: 282 000 3
<210> SEQ ID NO 283 <400> SEQUENCE: 283 000 3
<210> SEQ ID NO 284 <400> SEQUENCE: 284 000 3
<210> SEQ ID NO 285 <400> SEQUENCE: 285 000 3
<210> SEQ ID NO 286 <400> SEQUENCE: 286 000 3
<210> SEQ ID NO 287 <400> SEQUENCE: 287 000 3
<210> SEQ ID NO 288 <400> SEQUENCE: 288 000 3
<210> SEQ ID NO 289 <400> SEQUENCE: 289 000 3
<210> SEQ ID NO 290 <400> SEQUENCE: 290 000 3
<210> SEQ ID NO 291 <400> SEQUENCE: 291 000 3
<210> SEQ ID NO 292 <400> SEQUENCE: 292 000 3
<210> SEQ ID NO 293 <400> SEQUENCE: 293 000 3
<210> SEQ ID NO 294 <400> SEQUENCE: 294 000 3
<210> SEQ ID NO 295 <400> SEQUENCE: 295 000 3
<210> SEQ ID NO 296 <400> SEQUENCE: 296 000 3
<210> SEQ ID NO 297 <400> SEQUENCE: 297 000 3
<210> SEQ ID NO 298 <400> SEQUENCE: 298 000 3
<210> SEQ ID NO 299 <400> SEQUENCE: 299 000 3
<210> SEQ ID NO 300 <400> SEQUENCE: 300 000 3
<210> SEQ ID NO 301 <400> SEQUENCE: 301 000 3
<210> SEQ ID NO 302 <400> SEQUENCE: 302 000 3
<210> SEQ ID NO 303 <400> SEQUENCE: 303 000 3
<210> SEQ ID NO 304 <400> SEQUENCE: 304 000 3
<210> SEQ ID NO 305 <400> SEQUENCE: 305 000 3
<210> SEQ ID NO 306 <400> SEQUENCE: 306 000 3
<210> SEQ ID NO 307 <400> SEQUENCE: 307 000 3
<210> SEQ ID NO 308 <400> SEQUENCE: 308 000 3
<210> SEQ ID NO 309 <400> SEQUENCE: 309 000 3
<210> SEQ ID NO 310 <400> SEQUENCE: 310 000 3
<210> SEQ ID NO 311 <400> SEQUENCE: 311 000 3
<210> SEQ ID NO 312 <400> SEQUENCE: 312 000 3
<210> SEQ ID NO 313 <400> SEQUENCE: 313 000 3
<210> SEQ ID NO 314 <400> SEQUENCE: 314 000 3
<210> SEQ ID NO 315 <400> SEQUENCE: 315 000 3
<210> SEQ ID NO 316 <400> SEQUENCE: 316 000 3
<210> SEQ ID NO 317 <400> SEQUENCE: 317 000 3
<210> SEQ ID NO 318 <400> SEQUENCE: 318 000 3
<210> SEQ ID NO 319 <400> SEQUENCE: 319 000 3
<210> SEQ ID NO 320 <400> SEQUENCE: 320 000 3
<210> SEQ ID NO 321 <400> SEQUENCE: 321 000 3
<210> SEQ ID NO 322 <400> SEQUENCE: 322 000 3
<210> SEQ ID NO 323 <400> SEQUENCE: 323 000 3
<210> SEQ ID NO 324 <400> SEQUENCE: 324 000 3
<210> SEQ ID NO 325 <400> SEQUENCE: 325 000 3
<210> SEQ ID NO 326 <400> SEQUENCE: 326 000 3
<210> SEQ ID NO 327 <400> SEQUENCE: 327 000 3
<210> SEQ ID NO 328 <211> LENGTH: 132 <212> TYPE:
PRT <213> ORGANISM: Mouse C IL 13 F <400> SEQUENCE: 328
Ala Asp Pro Gly Cys Gly Gly Gly Gly Gly Leu Ala Gly Pro Val Pro 1 5
10 15 Arg Ser Val Ser Leu Pro Leu Thr Leu Lys Glu Leu Ile Glu Glu
Leu 20 25 30 Ser Asn Ile Thr Gln Asp Gln Thr Pro Leu Cys Asn Gly
Ser Met Val 35 40 45 Trp Ser Val Asp Leu Ala Ala Gly Gly Phe Cys
Val Ala Leu Asp Ser 50 55 60 Leu Thr Asn Ile Ser Asn Cys Asn Ala
Ile Tyr Arg Thr Gln Arg Ile 65 70 75 80 Leu His Gly Leu Cys Asn Arg
Lys Ala Pro Thr Thr Val Ser Ser Leu 85 90 95 Pro Asp Thr Lys Ile
Glu Val Ala His Phe Ile Thr Lys Leu Leu Ser 100 105 110 Tyr Thr Lys
Gln Leu Phe Arg His Gly Pro Phe Leu Glu Val Leu Ala 115 120 125 Ile
Glu Gly Arg 130 <210> SEQ ID NO 329 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Mouse C IL 13 S
<400> SEQUENCE: 329 Leu Ala Cys Gly Gly Gly Gly Gly Gly Pro
Val Pro Arg Ser Val Ser 1 5 10 15 Leu Pro Leu Thr Leu Lys Glu Leu
Ile Glu Glu Leu Ser Asn Ile Thr 20 25 30 Gln Asp Gln Thr Pro Leu
Cys Asn Gly Ser Met Val Trp Ser Val Asp 35 40 45 Leu Ala Ala Gly
Gly Phe Cys Val Ala Leu Asp Ser Leu Thr Asn Ile 50 55 60 Ser Asn
Cys Asn Ala Ile Tyr Arg Thr Gln Arg Ile Leu His Gly Leu 65 70 75 80
Cys Asn Arg Lys Ala Pro Thr Thr Val Ser Ser Leu Pro Asp Thr Lys 85
90 95 Ile Glu Val Ala His Phe Ile Thr Lys Leu Leu Ser Tyr Thr Lys
Gln 100 105 110 Leu Phe Arg His Gly Pro Phe 115 <210> SEQ ID
NO 330 <211> LENGTH: 133 <212> TYPE: PRT <213>
ORGANISM: Human C IL 13 F <400> SEQUENCE: 330 Ala Asp Pro Gly
Cys Gly Gly Gly Gly Gly Leu Ala Gly Pro Val Pro 1 5 10 15 Pro Ser
Thr Ala Leu Arg Glu Leu Ile Glu Glu Leu Val Asn Ile Thr 20 25 30
Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser Met Val Trp Ser Ile 35
40 45 Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu Glu Ser Leu Ile
Asn 50 55 60 Val Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg Met
Leu Ser Gly 65 70 75 80 Phe Cys Pro His Lys Val Ser Ala Gly Gln Phe
Ser Ser Leu His Val 85 90 95 Arg Asp Thr Lys Ile Glu Val Ala Gln
Phe Val Lys Asp Leu Leu Leu 100 105 110 His Leu Lys Lys Leu Phe Arg
Glu Gly Arg Phe Asn Leu Glu Val Leu 115 120 125 Ala Ile Glu Gly Arg
130 <210> SEQ ID NO 331 <211> LENGTH: 120 <212>
TYPE: PRT <213> ORGANISM: Human C IL 13 S <400>
SEQUENCE: 331 Leu Ala Cys Gly Gly Gly Gly Gly Gly Pro Val Pro Pro
Ser Thr Ala 1 5 10 15 Leu Arg Glu Leu Ile Glu Glu Leu Val Asn Ile
Thr Gln Asn Gln Lys 20 25 30 Ala Pro Leu Cys Asn Gly Ser Met Val
Trp Ser Ile Asn Leu Thr Ala 35 40 45 Gly Met Tyr Cys Ala Ala Leu
Glu Ser Leu Ile Asn Val Ser Gly Cys 50 55 60 Ser Ala Ile Glu Lys
Thr Gln Arg Met Leu Ser Gly Phe Cys Pro His 65 70 75 80 Lys Val Ser
Ala Gly Gln Phe Ser Ser Leu His Val Arg Asp Thr Lys 85 90 95 Ile
Glu Val Ala Gln Phe Val Lys Asp Leu Leu Leu His Leu Lys Lys 100 105
110 Leu Phe Arg Glu Gly Arg Phe Asn 115 120 <210> SEQ ID NO
332 <211> LENGTH: 136 <212> TYPE: PRT <213>
ORGANISM: Mouse C IL 5 E <400> SEQUENCE: 332 Ala Leu Val Gly
Cys Gly Gly Pro Lys Pro Ser Thr Pro Pro Gly Ser 1 5 10 15 Ser Gly
Gly Ala Pro Ala Ser Met Glu Ile Pro Met Ser Thr Val Val 20 25 30
Lys Glu Thr Leu Thr Gln Leu Ser Ala His Arg Ala Leu Leu Thr Ser 35
40 45 Asn Glu Thr Met Arg Leu Pro Val Pro Thr His Lys Asn His Gln
Leu 50 55 60 Cys Ile Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys
Asn Gln Thr 65 70 75 80 Val Arg Gly Gly Thr Val Glu Met Leu Phe Gln
Asn Leu Ser Leu Ile 85 90 95 Lys Lys Tyr Ile Asp Arg Gln Lys Glu
Lys Cys Gly Glu Glu Arg Arg 100 105 110 Arg Thr Arg Gln Phe Leu Asp
Tyr Leu Gln Glu Phe Leu Gly Val Met 115 120 125 Ser Thr Glu Trp Ala
Met Glu Gly 130 135 <210> SEQ ID NO 333 <211> LENGTH:
134 <212> TYPE: PRT <213> ORGANISM: Mouse C IL 5 F
<400> SEQUENCE: 333 Ala Asp Pro Gly Cys Gly Gly Gly Gly Gly
Leu Ala Met Glu Ile Pro 1 5 10 15 Met Ser Thr Val Val Lys Glu Thr
Leu Thr Gln Leu Ser Ala His Arg 20 25 30 Ala Leu Leu Thr Ser Asn
Glu Thr Met Arg Leu Pro Val Pro Thr His 35 40 45 Lys Asn His Gln
Leu Cys Ile Gly Glu Ile Phe Gln Gly Leu Asp Ile 50 55 60 Leu Lys
Asn Gln Thr Val Arg Gly Gly Thr Val Glu Met Leu Phe Gln 65 70 75 80
Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln Lys Glu Lys Cys 85
90 95 Gly Glu Glu Arg Arg Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln
Glu 100 105 110 Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu Gly
Leu Glu Val 115 120 125 Leu Ala Ile Glu Gly Arg 130 <210> SEQ
ID NO 334 <211> LENGTH: 121 <212> TYPE: PRT <213>
ORGANISM: Mouse C IL 5 S <400> SEQUENCE: 334 Leu Ala Cys Gly
Gly Gly Gly Gly Met Glu Ile Pro Met Ser Thr Val 1 5 10 15 Val Lys
Glu Thr Leu Thr Gln Leu Ser Ala His Arg Ala Leu Leu Thr 20 25 30
Ser Asn Glu Thr Met Arg Leu Pro Val Pro Thr His Lys Asn His Gln 35
40 45 Leu Cys Ile Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys Asn
Gln 50 55 60 Thr Val Arg Gly Gly Thr Val Glu Met Leu Phe Gln Asn
Leu Ser Leu 65 70 75 80 Ile Lys Lys Tyr Ile Asp Arg Gln Lys Glu Lys
Cys Gly Glu Glu Arg 85 90 95 Arg Arg Thr Arg Gln Phe Leu Asp Tyr
Leu Gln Glu Phe Leu Gly Val 100 105 110 Met Ser Thr Glu Trp Ala Met
Glu Gly 115 120 <210> SEQ ID NO 335 <211> LENGTH: 138
<212> TYPE: PRT <213> ORGANISM: Human C IL 5 E
<400> SEQUENCE: 335 Ala Leu Val Gly Cys Gly Gly Pro Lys Pro
Ser Thr Pro Pro Gly Ser 1 5 10 15 Ser Gly Gly Ala Pro Ala Ser Ile
Pro Thr Glu Ile Pro Thr Ser Ala 20 25 30 Leu Val Lys Glu Thr Leu
Ala Leu Leu Ser Thr His Arg Thr Leu Leu 35 40 45 Ile Ala Asn Glu
Thr Leu Arg Ile Pro Val Pro Val His Lys Asn His 50 55 60 Gln Leu
Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser 65 70 75 80
Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser 85
90 95 Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu
Glu 100 105 110 Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu
Phe Leu Gly 115 120 125 Val Met Asn Thr Glu Trp Ile Ile Glu Ser 130
135 <210> SEQ ID NO 336 <211> LENGTH: 136 <212>
TYPE: PRT <213> ORGANISM: Human C IL 5 F <400>
SEQUENCE: 336 Ala Asp Pro Gly Cys Gly Gly Gly Gly Gly Leu Ala Ile
Pro Thr Glu 1 5 10 15 Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu
Ala Leu Leu Ser Thr 20 25 30 His Arg Thr Leu Leu Ile Ala Asn Glu
Thr Leu Arg Ile Pro Val Pro 35 40 45 Val His Lys Asn His Gln Leu
Cys Thr Glu Glu Ile Phe Gln Gly Ile 50 55 60 Gly Thr Leu Glu Ser
Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu 65 70 75 80 Phe Lys Asn
Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys 85 90 95 Lys
Cys Gly Glu Glu Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu 100 105
110 Gln Glu Phe Leu Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser Leu
115 120 125 Glu Val Leu Ala Ile Glu Gly Arg 130 135 <210> SEQ
ID NO 337 <211> LENGTH: 123 <212> TYPE: PRT <213>
ORGANISM: Human C IL 5 S <400> SEQUENCE: 337 Leu Ala Cys Gly
Gly Gly Gly Gly Ile Pro Thr Glu Ile Pro Thr Ser 1 5 10 15 Ala Leu
Val Lys Glu Thr Leu Ala Leu Leu Ser Thr His Arg Thr Leu 20 25 30
Leu Ile Ala Asn Glu Thr Leu Arg Ile Pro Val Pro Val His Lys Asn 35
40 45 His Gln Leu Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu
Glu 50 55 60 Ser Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe
Lys Asn Leu 65 70 75 80 Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys
Lys Lys Cys Gly Glu 85 90 95 Glu Arg Arg Arg Val Asn Gln Phe Leu
Asp Tyr Leu Gln Glu Phe Leu 100 105 110 Gly Val Met Asn Thr Glu Trp
Ile Ile Glu Ser 115 120 <210> SEQ ID NO 338 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic primer NheIL13 F <400> SEQUENCE: 338 ctagctagcc
gggccggtgc caagatc 27 <210> SEQ ID NO 339 <211> LENGTH:
26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
primer NheIL13 R <400> SEQUENCE: 339 tttctcgagg aaggggccgt
ggcgaa 26 <210> SEQ ID NO 340 <211> LENGTH: 55
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
primer Spelinker3 F1 <400> SEQUENCE: 340 ccccgccggg
ttcttctggc ggtgctccgg ctagcatgga gattcccatg agcac 55 <210>
SEQ ID NO 341 <211> LENGTH: 52 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic primer SpeNlinker3 F2
<400> SEQUENCE: 341 ttttactagt tggttgcggc ggcccgaaac
cgagcacccc gccgggttct tc 52 <210> SEQ ID NO 342 <211>
LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic primer IL5StopXho R <400> SEQUENCE: 342 ttttgcggcc
gcgtttaaac tcgagttatt agccttccat tgcccactc 49 <210> SEQ ID NO
343 <211> LENGTH: 35 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Shine Delagarno sequence of vector
pQb185 <400> SEQUENCE: 343 tctagattaa cccaacgcgt aggagtcagg
ccatg 35 <210> SEQ ID NO 344 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic Amino
acid linker <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (1)..(1) <223> OTHER INFORMATION:
Glycine can be repeated from zero to five times <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(3)..(3) <223> OTHER INFORMATION: Glycine can be repeated
from zero to ten times <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Serine can be repeated from zero to two times
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (5)..(9) <223> OTHER INFORMATION: These amino acids
can be repeated from zero to three times as a group <400>
SEQUENCE: 344 Gly Cys Gly Ser Gly Gly Gly Gly Ser 1 5 <210>
SEQ ID NO 345 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Amino Acid Linker
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: Glycine can be
repeated from zero to ten times <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Serine can be repeated from zero to two times
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (3)..(7) <223> OTHER INFORMATION: These amino acids
can be repeated from zero to three times as a group <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: Glycine can be repeated
from zero to eight times <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (10)..(10) <223> OTHER
INFORMATION: Glycine can be repeated from zero to five times
<400> SEQUENCE: 345 Gly Ser Gly Gly Gly Gly Ser Gly Cys Gly 1
5 10 <210> SEQ ID NO 346 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic Glycine serine
linkers <400> SEQUENCE: 346 Gly Gly Gly Gly Ser 1 5
<210> SEQ ID NO 347 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic N terminal gamma 1
<400> SEQUENCE: 347 Cys Gly Asp Lys Thr His Thr Ser Pro Pro 1
5 10 <210> SEQ ID NO 348 <211> LENGTH: 10 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: C terminal gamma 1
<400> SEQUENCE: 348 Asp Lys Thr His Thr Ser Pro Pro Cys Gly 1
5 10 <210> SEQ ID NO 349 <211> LENGTH: 17 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: N terminal gamma 3
<400> SEQUENCE: 349 Cys Gly Gly Pro Lys Pro Ser Thr Pro Pro
Gly Ser Ser Gly Gly Ala 1 5 10 15 Pro <210> SEQ ID NO 350
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic C terminal gamma 3 <400> SEQUENCE: 350
Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala Pro Gly Gly 1 5
10 15 Cys Gly <210> SEQ ID NO 351 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic N
terminal glycine linker <400> SEQUENCE: 351 Gly Cys Gly Gly
Gly Gly 1 5 <210> SEQ ID NO 352 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: ARtificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic C
terminal glycine linker <400> SEQUENCE: 352 Gly Gly Gly Gly
Cys Gly 1 5 <210> SEQ ID NO 353 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic C
terminal glycine lysine linker <400> SEQUENCE: 353 Gly Gly
Lys Lys Gly Cys 1 5 <210> SEQ ID NO 354 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic N
terminal glycine lysine linker <400> SEQUENCE: 354 Cys Gly
Lys Lys Gly Gly 1 5 <210> SEQ ID NO 355 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic C
terminal linker <400> SEQUENCE: 355 Gly Gly Cys Gly 1
<210> SEQ ID NO 356 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 356 ggtaacatcg gtcgagatgg aaaacaaact ctggtcc
37 <210> SEQ ID NO 357 <211> LENGTH: 37 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer <400> SEQUENCE: 357 ggaccagagt ttgttttcca tctcgaccga
tgttacc 37 <210> SEQ ID NO 358 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer <400> SEQUENCE: 358 agctcgcccg
gggatcctct ag 22 <210> SEQ ID NO 359 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer <400> SEQUENCE: 359 cgatgcattt
catccttagt tatcaatacg ctgggttcag 40 <210> SEQ ID NO 360
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer <400> SEQUENCE:
360 ggcaaaatta gagactgtta ctttaggtaa gatcgg 36 <210> SEQ ID
NO 361 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer <400>
SEQUENCE: 361 ccgatcttac ctaaagtaac agtctctaat tttgcc 36
<210> SEQ ID NO 362 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 362 ggccatggca cgactcgaga ctgttacttt agg 33
<210> SEQ ID NO 363 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 363 gatttaggtg acactatag 19 <210> SEQ
ID NO 364 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucloetide primer <400>
SEQUENCE: 364 gatggacgtc aaactctggt cctcaatccg cgtgggg 37
<210> SEQ ID NO 365 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 365 ccccacgcgg attgaggacc agagtttgac gtccatc
37 <210> SEQ ID NO 366 <211> LENGTH: 31 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic EcoRIHBcAg(s)
<400> SEQUENCE: 366 ccggaattca tggacattga cccttataaa g 31
<210> SEQ ID NO 367 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Lys HBcAg(as) <400>
SEQUENCE: 367 cctagagcca cctttgccac catcttctaa attagtaccc
acccaggtag c 51 <210> SEQ ID NO 368 <211> LENGTH: 48
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic Lys
HBcAg(s) <400> SEQUENCE: 368 gaagatggtg gcaaaggtgg ctctagggac
ctagtagtca gttatgtc 48 <210> SEQ ID NO 369 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic HbcAg(1 149)Hind(as) <400> SEQUENCE: 369 cgcgtcccaa
gcttctaaac aacagtagtc tccggaag 38 <210> SEQ ID NO 370
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic 48as primer <400> SEQUENCE: 370
gtgcagtatg gtgaggtgag gaatgctcag gagactc 37 <210> SEQ ID NO
371 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic 48s primer <400> SEQUENCE: 371
gsgtctcctg agcattcctc acctcaccat actgcac 37 <210> SEQ ID NO
372 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic 107as primer <400> SEQUENCE: 372
cttccaaaag tgagggaaga aatgtgaaac cac 33 <210> SEQ ID NO 373
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic 107s primer <400> SEQUENCE: 373
gtggtttcac atttcttccc tcacttttgg aag 33 <210> SEQ ID NO 374
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic HBcAgwtHindIIII <400> SEQUENCE: 374
cgcgtcccaa gcttctaaca ttgagattcc cgagattg 38 <210> SEQ ID NO
375 <211> LENGTH: 51 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic CepsilonH3 foreward <400>
SEQUENCE: 375 gttaacttga cctggtctcg tgcttctggt gcatccaggg
atctagtagt c 51 <210> SEQ ID NO 376 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
CepsilonH3 foreward <400> SEQUENCE: 376 Val Asn Leu Thr Trp
Ser Arg Ala Ser Gly Ala Ser Arg Asp Leu Val 1 5 10 15 Val
<210> SEQ ID NO 377 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic CepsilonH3 reversed
<400> SEQUENCE: 377 accagaagca cgagaccagg tcaagttaac
atcttccaaa ttattaccca c 51 <210> SEQ ID NO 378 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic CepsilonH3 reversed <400> SEQUENCE: 378 Asp Glu Leu
Asn Asn Gly Val 1 5 <210> SEQ ID NO 379 <211> LENGTH:
26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic GST
BamHI ss <400> SEQUENCE: 379 cgccggatcc tatactaggt tattgg 26
<210> SEQ ID NO 380 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic C1 BsmBI/XhoI as
<400> SEQUENCE: 380 gggcgcgtct cctcgagacc gcaaccacca cca 33
<210> SEQ ID NO 381 <211> LENGTH: 74 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic MCS of pET22b(+)
<400> SEQUENCE: 381 gtttaacttt aagaaggaga tatacatatg
gatccggcta gcgctcgagg gtttaaacgg 60 cggccgcatg cacc 74 <210>
SEQ ID NO 382 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: mEotaxin F <400> SEQUENCE: 382
ggaattccat atgcacccag gctccatccc aac 33 <210> SEQ ID NO 383
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Nhe mEotaxin F <400> SEQUENCE: 383 cctagctagc gcacccaggc
tccatcccaa c 31 <210> SEQ ID NO 384 <211> LENGTH: 30
<212> TYPE: DNA <213> ORGANISM: mEotaxin Xho R
<400> SEQUENCE: 384 cccgctcgag tggttttgga gtttggagtt 30
<210> SEQ ID NO 385 <211> LENGTH: 84 <212> TYPE:
PRT <213> ORGANISM: Mouse eotaxin C1 <400> SEQUENCE:
385 Met His Pro Gly Ser Ile Pro Thr Ser Cys Cys Phe Ile Met Thr Ser
1 5 10 15 Lys Lys Ile Pro Asn Thr Leu Leu Lys Ser Tyr Lys Arg Ile
Thr Asn 20 25 30 Asn Arg Cys Thr Leu Lys Ala Ile Val Phe Lys Thr
Arg Leu Gly Lys 35 40 45 Glu Ile Cys Ala Asp Pro Lys Lys Lys Trp
Val Gln Asp Ala Thr Lys 50 55 60 His Leu Asp Gln Lys Leu Gln Thr
Pro Lys Pro Leu Arg Gly Gly Gly 65 70 75 80 Gly Gly Cys Gly
<210> SEQ ID NO 386 <211> LENGTH: 103 <212> TYPE:
PRT <213> ORGANISM: Mouse His eotaxin C1 <400>
SEQUENCE: 386 Met Asp Pro His His His His His His Gly Ser Gly Asp
Asp Asp Asp 1 5 10 15 Lys Ala Leu Ala His Pro Gly Ser Ile Pro Thr
Ser Cys Cys Phe Ile 20 25 30 Met Thr Ser Lys Lys Ile Pro Asn Thr
Leu Leu Lys Ser Tyr Lys Arg 35 40 45 Ile Thr Asn Asn Arg Cys Thr
Leu Lys Ala Ile Val Phe Lys Thr Arg 50 55 60 Leu Gly Lys Glu Ile
Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp 65 70 75 80 Ala Thr Lys
His Leu Asp Gln Lys Leu Gln Thr Pro Lys Pro Leu Arg 85 90 95 Gly
Gly Gly Gly Gly Cys Gly 100 <210> SEQ ID NO 387 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic peptide sequence designed to modify HBcAg <400>
SEQUENCE: 387 Gly Gly Lys Gly Gly 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 387
<210> SEQ ID NO 1 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Haemophilus influenzae <400>
SEQUENCE: 1 Met Lys Lys Thr Leu Leu Gly Ser Leu Ile Leu Leu Ala Phe
Ala Gly 1 5 10 15 Asn Val Gln Ala Ala Ala Asn Ala Asp Thr Ser Gly
Thr Val Thr Phe 20 25 30 Phe Gly Lys Val Val Glu Asn Thr Cys Gln
Val Asn Gln Asp Ser Glu 35 40 45 Tyr Glu Cys Asn Leu Asn Asp Val
Gly Lys Asn His Leu Ser Gln Gln 50 55 60 Gly Tyr Thr Ala Met Gln
Thr Pro Phe Thr Ile Thr Leu Glu Asn Cys 65 70 75 80 Asn Val Thr Thr
Thr Asn Asn Lys Pro Lys Ala Thr Lys Val Gly Val 85 90 95 Tyr Phe
Tyr Ser Trp Glu Ile Ala Asp Lys Asp Asn Lys Tyr Thr Leu 100 105 110
Lys Asn Ile Lys Glu Asn Thr Gly Thr Asn Asp Ser Ala Asn Lys Val 115
120 125 Asn Ile Gln Leu Leu Glu Asp Asn Gly Thr Ala Glu Ile Lys Val
Val 130 135 140 Gly Lys Thr Thr Thr Asp Phe Thr Ser Glu Asn His Asn
Gly Ala Gly 145 150 155 160 Ala Asp Pro Val Ala Thr Asn Lys His Ile
Ser Ser Leu Thr Pro Leu 165 170 175 Asn Asn Gln Asn Ser Ile Asn Leu
His Tyr Ile Ala Gln Tyr Tyr Ala 180 185 190 Thr Gly Val Ala Glu Ala
Gly Lys Val Pro Ser Ser Val Asn Ser Gln 195 200 205 Ile Ala Tyr Glu
210 <210> SEQ ID NO 2 <211> LENGTH: 139 <212>
TYPE: PRT <213> ORGANISM: Pseudomonas stutzeri <400>
SEQUENCE: 2 Met Lys Ala Gln Met Gln Lys Gly Phe Thr Leu Ile Glu Leu
Met Ile 1 5 10 15 Val Val Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala
Leu Pro Ala Tyr 20 25 30 Gln Asp Tyr Thr Val Arg Ser Asn Ala Ala
Ala Ala Leu Ala Glu Ile 35 40 45 Thr Pro Gly Lys Ile Gly Phe Glu
Gln Ala Ile Asn Glu Gly Lys Thr 50 55 60 Pro Ser Leu Thr Ser Thr
Asp Glu Gly Tyr Ile Gly Ile Thr Asp Ser 65 70 75 80 Thr Ser Tyr Cys
Asp Val Asp Leu Asp Thr Ala Ala Asp Gly His Ile 85 90 95 Glu Cys
Thr Ala Lys Gly Gly Asn Ala Gly Lys Phe Asp Gly Lys Thr 100 105 110
Ile Thr Leu Asn Arg Thr Ala Asp Gly Glu Trp Ser Cys Ala Ser Thr 115
120 125 Leu Asp Ala Lys Tyr Lys Pro Gly Lys Cys Ser 130 135
<210> SEQ ID NO 3 <211> LENGTH: 59 <212> TYPE:
PRT <213> ORGANISM: Caulobacter crescentus <400>
SEQUENCE: 3 Met Thr Lys Phe Val Thr Arg Phe Leu Lys Asp Glu Ser Gly
Ala Thr 1 5 10 15 Ala Ile Glu Tyr Gly Leu Ile Val Ala Leu Ile Ala
Val Val Ile Val 20 25 30 Thr Ala Val Thr Thr Leu Gly Thr Asn Leu
Arg Thr Ala Phe Thr Lys 35 40 45 Ala Gly Ala Ala Val Ser Thr Ala
Ala Gly Thr 50 55 <210> SEQ ID NO 4 <211> LENGTH: 173
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 4 Met Ala Val Val Ser Phe Gly Val Asn Ala Ala
Pro Thr Ile Pro Gln 1 5 10 15 Gly Gln Gly Lys Val Thr Phe Asn Gly
Thr Val Val Asp Ala Pro Cys 20 25 30 Ser Ile Ser Gln Lys Ser Ala
Asp Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45 Ser Lys Ser Phe Leu
Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu 50 55 60 Asp Ile Glu
Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn 65 70 75 80 Gly
Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile Val 85 90
95 Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr Gly Thr Ala
100 105 110 Ile Val Val Gln Gly Ala Gly Lys Asn Val Val Phe Asp Gly
Ser Glu 115 120 125 Gly Asp Ala Asn Thr Leu Lys Asp Gly Glu Asn Val
Leu His Tyr Thr 130 135 140 Ala Val Val Lys Lys Ser Ser Ala Val Gly
Ala Ala Val Thr Glu Gly 145 150 155 160 Ala Phe Ser Ala Val Ala Asn
Phe Asn Leu Thr Tyr Gln 165 170 <210> SEQ ID NO 5 <211>
LENGTH: 173 <212> TYPE: PRT <213> ORGANISM: Escherichia
coli <400> SEQUENCE: 5 Met Ala Val Val Ser Phe Gly Val Asn
Ala Ala Pro Thr Ile Pro Gln 1 5 10 15 Gly Gln Gly Lys Val Thr Phe
Asn Gly Thr Val Val Asp Ala Pro Cys 20 25 30 Ser Ile Ser Gln Lys
Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu 35 40 45 Ser Lys Ser
Phe Leu Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu 50 55 60 Asp
Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn 65 70
75 80 Gly Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile
Val 85 90 95 Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr
Gly Thr Ala 100 105 110 Ile Val Val Gln Gly Ala Gly Lys Asn Val Val
Phe Asp Gly Ser Glu 115 120 125 Gly Asp Ala Asn Thr Leu Lys Asp Gly
Glu Asn Val Leu His Tyr Thr 130 135 140 Ala Val Val Lys Lys Ser Ser
Ala Val Gly Ala Ala Val Thr Glu Gly 145 150 155 160 Ala Phe Ser Ala
Val Ala Asn Phe Asn Leu Thr Tyr Gln 165 170 <210> SEQ ID NO 6
<211> LENGTH: 181 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 6 Met Lys Ile Lys Thr Leu
Ala Ile Val Val Leu Ser Ala Leu Ser Leu 1 5 10 15 Ser Ser Thr Ala
Ala Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr 20 25 30 Val His
Phe Lys Gly Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala 35 40 45
Gly Ser Val Asp Gln Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser 50
55 60 Leu Ala Gln Glu Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile
Gln 65 70 75 80 Leu Asn Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala
Val Ala Phe 85 90 95 Leu Gly Thr Ala Ile Asp Ala Gly His Thr Asn
Val Leu Ala Leu Gln 100 105 110 Ser Ser Ala Ala Gly Ser Ala Thr Asn
Val Gly Val Gln Ile Leu Asp 115 120 125 Arg Thr Gly Ala Ala Leu Thr
Leu Asp Gly Ala Thr Phe Ser Ser Glu 130 135 140 Thr Thr Leu Asn Asn
Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr 145 150 155 160 Phe Ala
Gly Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr Phe 165 170 175
Lys Val Gln Tyr Gln 180 <210> SEQ ID NO 7 <211> LENGTH:
172 <212> TYPE: PRT <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 7 Met Ala Val Val Ser Phe Gly Val Asn Ala Ala
Pro Thr Thr Pro Gln 1 5 10 15 Gly Gln Gly Arg Val Thr Phe Asn Gly
Thr Val Val Asp Ala Pro Cys 20 25 30
Ser Ile Ser Gln Lys Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu 35
40 45 Ser Lys Ser Phe Leu Ala Asn Asp Gly Gln Ser Lys Pro Met Asn
Leu 50 55 60 Asp Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys
Asn Gly Asn 65 70 75 80 Ala Lys Thr Gly Ser Val Lys Leu Ala Phe Thr
Gly Pro Thr Val Ser 85 90 95 Gly His Pro Ser Glu Leu Ala Thr Asn
Gly Gly Pro Gly Thr Ala Ile 100 105 110 Met Ile Gln Ala Ala Gly Lys
Asn Val Pro Phe Asp Gly Thr Glu Gly 115 120 125 Asp Pro Asn Leu Leu
Lys Asp Gly Asp Asn Val Leu His Tyr Thr Thr 130 135 140 Val Gly Lys
Lys Ser Ser Asp Gly Asn Ala Gln Ile Thr Glu Gly Ala 145 150 155 160
Phe Ser Gly Val Ala Thr Phe Asn Leu Ser Tyr Gln 165 170 <210>
SEQ ID NO 8 <211> LENGTH: 182 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <400> SEQUENCE: 8 Met
Lys Ile Lys Thr Leu Ala Ile Val Val Leu Ser Ala Leu Ser Leu 1 5 10
15 Ser Ser Thr Thr Ala Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr
20 25 30 Val His Phe Lys Gly Glu Val Val Asn Ala Ala Cys Ala Val
Asp Ala 35 40 45 Gly Ser Val Asp Gln Thr Val Gln Leu Gly Gln Val
Arg Thr Ala Ser 50 55 60 Leu Ala Gln Glu Gly Ala Thr Ser Ser Ala
Val Gly Phe Asn Ile Gln 65 70 75 80 Leu Asn Asp Cys Asp Thr Asn Val
Ala Ser Lys Ala Ala Val Ala Phe 85 90 95 Leu Gly Thr Ala Ile Asp
Ala Gly His Thr Asn Val Leu Ala Leu Gln 100 105 110 Ser Ser Ala Ala
Gly Ser Ala Thr Asn Val Gly Val Gln Ile Leu Asp 115 120 125 Arg Thr
Gly Ala Ala Leu Thr Leu Asp Gly Ala Thr Phe Ser Ser Glu 130 135 140
Thr Thr Leu Asn Asn Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr 145
150 155 160 Phe Ala Thr Gly Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp
Ala Thr 165 170 175 Phe Lys Val Gln Tyr Gln 180 <210> SEQ ID
NO 9 <211> LENGTH: 853 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <400> SEQUENCE: 9 acgtttctgt
ggctcgacgc atcttcctca ttcttctctc caaaaaccac ctcatgcaat 60
ataaacatct ataaataaag ataacaaata gaatattaag ccaacaaata aactgaaaaa
120 gtttgtccgc gatgctttac ctctatgagt caaaatggcc ccaatgtttc
atcttttggg 180 ggaaactgtg cagtgttggc agtcaaactc gttgacaaac
aaagtgtaca gaacgactgc 240 ccatgtcgat ttagaaatag ttttttgaaa
ggaaagcagc atgaaaatta aaactctggc 300 aatcgttgtt ctgtcggctc
tgtccctcag ttctacgacg gctctggccg ctgccacgac 360 ggttaatggt
gggaccgttc actttaaagg ggaagttgtt aacgccgctt gcgcagttga 420
tgcaggctct gttgatcaaa ccgttcagtt aggacaggtt cgtaccgcat cgctggcaca
480 ggaaggagca accagttctg ctgtcggttt taacattcag ctgaatgatt
gcgataccaa 540 tgttgcatct aaagccgctg ttgccttttt aggtacggcg
attgatgcgg gtcataccaa 600 cgttctggct ctgcagagtt cagctgcggg
tagcgcaaca aacgttggtg tgcagatcct 660 ggacagaacg ggtgctgcgc
tgacgctgga tggtgcgaca tttagttcag aaacaaccct 720 gaataacgga
accaatacca ttccgttcca ggcgcgttat tttgcaaccg gggccgcaac 780
cccgggtgct gctaatgcgg atgcgacctt caaggttcag tatcaataac ctacctaggt
840 tcagggacgt tca 853 <210> SEQ ID NO 10 <211> LENGTH:
132 <212> TYPE: PRT <213> ORGANISM: Bacteriophage Q
beta <400> SEQUENCE: 10 Ala Lys Leu Glu Thr Val Thr Leu Gly
Asn Ile Gly Lys Asp Gly Lys 1 5 10 15 Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Gln
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60 Gln
Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys 65 70
75 80 Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser
Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg
Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp
Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130 <210> SEQ
ID NO 11 <211> LENGTH: 329 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta CP <400> SEQUENCE: 11 Met Ala
Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly 1 5 10 15
Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20
25 30 Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys
Arg 35 40 45 Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys
Asn Tyr Lys 50 55 60 Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys
Thr Ala Asn Gly Ser 65 70 75 80 Cys Asp Pro Ser Val Thr Arg Gln Ala
Tyr Ala Asp Val Thr Phe Ser 85 90 95 Phe Thr Gln Tyr Ser Thr Asp
Glu Glu Arg Ala Phe Val Arg Thr Glu 100 105 110 Leu Ala Ala Leu Leu
Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln 115 120 125 Leu Asn Pro
Ala Tyr Trp Thr Leu Leu Ile Ala Gly Gly Gly Ser Gly 130 135 140 Ser
Lys Pro Asp Pro Val Ile Pro Asp Pro Pro Ile Asp Pro Pro Pro 145 150
155 160 Gly Thr Gly Lys Tyr Thr Cys Pro Phe Ala Ile Trp Ser Leu Glu
Glu 165 170 175 Val Tyr Glu Pro Pro Thr Lys Asn Arg Pro Trp Pro Ile
Tyr Asn Ala 180 185 190 Val Glu Leu Gln Pro Arg Glu Phe Asp Val Ala
Leu Lys Asp Leu Leu 195 200 205 Gly Asn Thr Lys Trp Arg Asp Trp Asp
Ser Arg Leu Ser Tyr Thr Thr 210 215 220 Phe Arg Gly Cys Arg Gly Asn
Gly Tyr Ile Asp Leu Asp Ala Thr Tyr 225 230 235 240 Leu Ala Thr Asp
Gln Ala Met Arg Asp Gln Lys Tyr Asp Ile Arg Glu 245 250 255 Gly Lys
Lys Pro Gly Ala Phe Gly Asn Ile Glu Arg Phe Ile Tyr Leu 260 265 270
Lys Ser Ile Asn Ala Tyr Cys Ser Leu Ser Asp Ile Ala Ala Tyr His 275
280 285 Ala Asp Gly Val Ile Val Gly Phe Trp Arg Asp Pro Ser Ser Gly
Gly 290 295 300 Ala Ile Pro Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys
Cys Pro Ile 305 310 315 320 Gln Ala Val Ile Val Val Pro Arg Ala 325
<210> SEQ ID NO 12 <211> LENGTH: 129 <212> TYPE:
PRT <213> ORGANISM: Bacteriophage R17 <400> SEQUENCE:
12 Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly
1 5 10 15 Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala
Glu Trp 20 25 30 Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val
Thr Cys Ser Val 35 40 45 Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr
Thr Ile Lys Val Glu Val 50 55 60 Pro Lys Val Ala Thr Gln Thr Val
Gly Gly Val Glu Leu Pro Val Ala 65 70 75 80 Ala Trp Arg Ser Tyr Leu
Asn Met Glu Leu Thr Ile Pro Ile Phe Ala 85 90 95 Thr Asn Ser Asp
Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu 100 105 110 Lys Asp
Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125
Tyr <210> SEQ ID NO 13
<211> LENGTH: 130 <212> TYPE: PRT <213> ORGANISM:
Bacteriophage fr <400> SEQUENCE: 13 Met Ala Ser Asn Phe Glu
Glu Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Lys
Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile
Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45
Val Arg Gln Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu 50
55 60 Val Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro
Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile
Pro Val Phe 85 90 95 Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys
Ala Leu Gln Gly Thr 100 105 110 Phe Lys Thr Gly Asn Pro Ile Ala Thr
Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130 <210> SEQ
ID NO 14 <211> LENGTH: 130 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage GA <400> SEQUENCE: 14 Met Ala Thr Leu
Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly 1 5 10 15 Asn Val
Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp 20 25 30
Leu Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr 35
40 45 Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu
Val 50 55 60 Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu
Pro Gly Ser 65 70 75 80 Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr
Ile Pro Ile Phe Ala 85 90 95 Ala Thr Asp Asp Val Thr Val Ile Ser
Lys Ser Leu Ala Gly Leu Phe 100 105 110 Lys Val Gly Asn Pro Ile Ala
Glu Ala Ile Ser Ser Gln Ser Gly Phe 115 120 125 Tyr Ala 130
<210> SEQ ID NO 15 <211> LENGTH: 132 <212> TYPE:
PRT <213> ORGANISM: Bacteriophage SP <400> SEQUENCE: 15
Met Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly 1 5
10 15 Asp Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn
Gly 20 25 30 Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu
Glu Lys Arg 35 40 45 Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn
Arg Lys Asn Phe Lys 50 55 60 Val Gln Ile Lys Leu Gln Asn Pro Thr
Ala Cys Thr Arg Asp Ala Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Ser
Ala Phe Ala Asp Val Thr Leu Ser Phe 85 90 95 Thr Ser Tyr Ser Thr
Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu 100 105 110 Ala Ala Leu
Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu 115 120 125 Asn
Pro Ala Tyr 130 <210> SEQ ID NO 16 <211> LENGTH: 329
<212> TYPE: PRT <213> ORGANISM: Bacteriophage SP CP
<400> SEQUENCE: 16 Ala Lys Leu Asn Gln Val Thr Leu Ser Lys
Ile Gly Lys Asn Gly Asp 1 5 10 15 Gln Thr Leu Thr Leu Thr Pro Arg
Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Glu Ala
Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser Val
Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe Lys Val 50 55 60 Gln Ile
Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys Asp 65 70 75 80
Pro Ser Val Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe Thr 85
90 95 Ser Tyr Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu
Ala 100 105 110 Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp
Asn Leu Asn 115 120 125 Pro Ala Tyr Trp Ala Ala Leu Leu Val Ala Ser
Ser Gly Gly Gly Asp 130 135 140 Asn Pro Ser Asp Pro Asp Val Pro Val
Val Pro Asp Val Lys Pro Pro 145 150 155 160 Asp Gly Thr Gly Arg Tyr
Lys Cys Pro Phe Ala Cys Tyr Arg Leu Gly 165 170 175 Ser Ile Tyr Glu
Val Gly Lys Glu Gly Ser Pro Asp Ile Tyr Glu Arg 180 185 190 Gly Asp
Glu Val Ser Val Thr Phe Asp Tyr Ala Leu Glu Asp Phe Leu 195 200 205
Gly Asn Thr Asn Trp Arg Asn Trp Asp Gln Arg Leu Ser Asp Tyr Asp 210
215 220 Ile Ala Asn Arg Arg Arg Cys Arg Gly Asn Gly Tyr Ile Asp Leu
Asp 225 230 235 240 Ala Thr Ala Met Gln Ser Asp Asp Phe Val Leu Ser
Gly Arg Tyr Gly 245 250 255 Val Arg Lys Val Lys Phe Pro Gly Ala Phe
Gly Ser Ile Lys Tyr Leu 260 265 270 Leu Asn Ile Gln Gly Asp Ala Trp
Leu Asp Leu Ser Glu Val Thr Ala 275 280 285 Tyr Arg Ser Tyr Gly Met
Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300 Pro Gln Leu Pro
Thr Asp Phe Thr Gln Phe Asn Ser Ala Asn Cys Pro 305 310 315 320 Val
Gln Thr Val Ile Ile Ile Pro Ser 325 <210> SEQ ID NO 17
<211> LENGTH: 130 <212> TYPE: PRT <213> ORGANISM:
Bacteriophage MS2 <400> SEQUENCE: 17 Met Ala Ser Asn Phe Thr
Gln Phe Val Leu Val Asp Asn Gly Gly Thr 1 5 10 15 Gly Asp Val Thr
Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu 20 25 30 Trp Ile
Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser 35 40 45
Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu 50
55 60 Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro
Val 65 70 75 80 Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile
Pro Ile Phe 85 90 95 Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys
Ala Met Gln Gly Leu 100 105 110 Leu Lys Asp Gly Asn Pro Ile Pro Ser
Ala Ile Ala Ala Asn Ser Gly 115 120 125 Ile Tyr 130 <210> SEQ
ID NO 18 <211> LENGTH: 133 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage M11 <400> SEQUENCE: 18 Met Ala Lys
Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Lys Gly 1 5 10 15 Asp
Val Thr Leu Asp Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly 20 25
30 Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
35 40 45 Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys 50 55 60 Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr
Ala Ser Gly Thr 65 70 75 80 Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr
Ser Asp Val Thr Phe Ser 85 90 95 Phe Thr Gln Tyr Ser Thr Val Glu
Glu Arg Ala Leu Val Arg Thr Glu 100 105 110 Leu Gln Ala Leu Leu Ala
Asp Pro Met Leu Val Asn Ala Ile Asp Asn 115 120 125 Leu Asn Pro Ala
Tyr 130 <210> SEQ ID NO 19 <211> LENGTH: 133
<212> TYPE: PRT <213> ORGANISM: Bacteriophage MX1
<400> SEQUENCE: 19
Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Asn Gly 1 5
10 15 Asp Val Thr Leu Asn Leu Asn Pro Arg Gly Val Asn Pro Thr Asn
Gly 20 25 30 Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu
Glu Lys Arg 35 40 45 Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn
Arg Lys Asn Tyr Lys 50 55 60 Val Gln Val Lys Ile Gln Asn Pro Thr
Ser Cys Thr Ala Ser Gly Thr 65 70 75 80 Cys Asp Pro Ser Val Thr Arg
Ser Ala Tyr Ala Asp Val Thr Phe Ser 85 90 95 Phe Thr Gln Tyr Ser
Thr Asp Glu Glu Arg Ala Leu Val Arg Thr Glu 100 105 110 Leu Lys Ala
Leu Leu Ala Asp Pro Met Leu Ile Asp Ala Ile Asp Asn 115 120 125 Leu
Asn Pro Ala Tyr 130 <210> SEQ ID NO 20 <211> LENGTH:
330 <212> TYPE: PRT <213> ORGANISM: Bacteriophage NL95
<400> SEQUENCE: 20 Met Ala Lys Leu Asn Lys Val Thr Leu Thr
Gly Ile Gly Lys Ala Gly 1 5 10 15 Asn Gln Thr Leu Thr Leu Thr Pro
Arg Gly Val Asn Pro Thr Asn Gly 20 25 30 Val Ala Ser Leu Ser Glu
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg 35 40 45 Val Thr Val Ser
Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys 50 55 60 Val Gln
Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys Asp Ala Cys 65 70 75 80
Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp Val Thr Leu Ser Phe 85
90 95 Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala Leu Ile Arg Thr Glu
Leu 100 105 110 Ala Ala Leu Leu Lys Asp Asp Leu Ile Val Asp Ala Ile
Asp Asn Leu 115 120 125 Asn Pro Ala Tyr Trp Ala Ala Leu Leu Ala Ala
Ser Pro Gly Gly Gly 130 135 140 Asn Asn Pro Tyr Pro Gly Val Pro Asp
Ser Pro Asn Val Lys Pro Pro 145 150 155 160 Gly Gly Thr Gly Thr Tyr
Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly 165 170 175 Glu Leu Ile Thr
Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys 180 185 190 Gly Ser
Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp Phe Leu 195 200 205
Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu Ser Lys Tyr Asp 210
215 220 Ile Glu Thr His Arg Arg Cys Arg Gly Asn Gly Tyr Val Asp Leu
Asp 225 230 235 240 Ala Ser Val Met Gln Ser Asp Glu Tyr Val Leu Ser
Gly Ala Tyr Asp 245 250 255 Val Val Lys Met Gln Pro Pro Gly Thr Phe
Asp Ser Pro Arg Tyr Tyr 260 265 270 Leu His Leu Met Asp Gly Ile Tyr
Val Asp Leu Ala Glu Val Thr Ala 275 280 285 Tyr Arg Ser Tyr Gly Met
Val Ile Gly Phe Trp Thr Asp Ser Lys Ser 290 295 300 Pro Gln Leu Pro
Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys Pro 305 310 315 320 Val
Gln Thr Val Ile Val Ile Pro Ser Leu 325 330 <210> SEQ ID NO
21 <211> LENGTH: 129 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage f2 <400> SEQUENCE: 21 Ala Ser Asn Phe
Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly 1 5 10 15 Asn Val
Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp 20 25 30
Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val 35
40 45 Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu
Val 50 55 60 Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu
Pro Val Ala 65 70 75 80 Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr
Ile Pro Ile Phe Ala 85 90 95 Thr Asn Ser Asp Cys Glu Leu Ile Val
Lys Ala Met Gln Gly Leu Leu 100 105 110 Lys Asp Gly Asn Pro Ile Pro
Ser Ala Ile Ala Ala Asn Ser Gly Ile 115 120 125 Tyr <210> SEQ
ID NO 22 <211> LENGTH: 128 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage PP7 <400> SEQUENCE: 22 Met Ser Lys
Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu 1 5 10 15 Thr
Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val 20 25
30 Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn
35 40 45 Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln
Ala Asp 50 55 60 Val Val Asp Cys Ser Thr Ser Val Cys Gly Glu Leu
Pro Lys Val Arg 65 70 75 80 Tyr Thr Gln Val Trp Ser His Asp Val Thr
Ile Val Ala Asn Ser Thr 85 90 95 Glu Ala Ser Arg Lys Ser Leu Tyr
Asp Leu Thr Lys Ser Leu Val Ala 100 105 110 Thr Ser Gln Val Glu Asp
Leu Val Val Asn Leu Val Pro Leu Gly Arg 115 120 125 <210> SEQ
ID NO 23 <211> LENGTH: 132 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta 240 <400> SEQUENCE: 23 Ala Lys
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys 1 5 10 15
Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20
25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn
Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr
Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr
Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu
Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala
Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala
Tyr 130 <210> SEQ ID NO 24 <211> LENGTH: 132
<212> TYPE: PRT <213> ORGANISM: Bacteriophage Q beta
243 <400> SEQUENCE: 24 Ala Lys Leu Glu Thr Val Thr Leu Gly
Lys Ile Gly Lys Asp Gly Lys 1 5 10 15 Gln Thr Leu Val Leu Asn Pro
Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Gln
Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser
Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60 Gln
Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys 65 70
75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser
Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg
Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp
Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130 <210> SEQ
ID NO 25 <211> LENGTH: 132 <212> TYPE: PRT <213>
ORGANISM: Bacteriophage Q beta 250 <400> SEQUENCE: 25 Ala Arg
Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Arg Asp Gly Lys 1 5 10 15
Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20
25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg
Val 35 40 45
Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val 50
55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser
Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp Val Thr
Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe
Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro Leu Leu
Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130
<210> SEQ ID NO 26 <211> LENGTH: 132 <212> TYPE:
PRT <213> ORGANISM: Bacteriophage Q beta 251 <400>
SEQUENCE: 26 Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys
Asp Gly Arg 1 5 10 15 Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn
Pro Thr Asn Gly Val 20 25 30 Ala Ser Leu Ser Gln Ala Gly Ala Val
Pro Ala Leu Glu Lys Arg Val 35 40 45 Thr Val Ser Val Ser Gln Pro
Ser Arg Asn Arg Lys Asn Tyr Lys Val 50 55 60 Gln Val Lys Ile Gln
Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys 65 70 75 80 Asp Pro Ser
Val Thr Arg Gln Lys Tyr Ala Asp Val Thr Phe Ser Phe 85 90 95 Thr
Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu 100 105
110 Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu
115 120 125 Asn Pro Ala Tyr 130 <210> SEQ ID NO 27
<211> LENGTH: 132 <212> TYPE: PRT <213> ORGANISM:
Bacteriophage Q beta 259 <400> SEQUENCE: 27 Ala Arg Leu Glu
Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Arg 1 5 10 15 Gln Thr
Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val 20 25 30
Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val 35
40 45 Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys
Val 50 55 60 Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn
Gly Ser Cys 65 70 75 80 Asp Pro Ser Val Thr Arg Gln Lys Tyr Ala Asp
Val Thr Phe Ser Phe 85 90 95 Thr Gln Tyr Ser Thr Asp Glu Glu Arg
Ala Phe Val Arg Thr Glu Leu 100 105 110 Ala Ala Leu Leu Ala Ser Pro
Leu Leu Ile Asp Ala Ile Asp Gln Leu 115 120 125 Asn Pro Ala Tyr 130
<210> SEQ ID NO 28 <211> LENGTH: 185 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
28 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg
Arg 145 150 155 160 Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro Arg Arg Arg 165 170 175 Arg Ser Gln Ser Arg Glu Ser Gln Cys 180
185 <210> SEQ ID NO 29 <211> LENGTH: 183 <212>
TYPE: PRT <213> ORGANISM: Hepatitis B virus <400>
SEQUENCE: 29 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val
Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val
Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala
Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu
Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala
Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile 65 70 75 80 Ser Arg Asp
Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe
Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105
110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr
115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr
Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro
Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln
Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Gly Ser Gln Cys
180 <210> SEQ ID NO 30 <211> LENGTH: 183 <212>
TYPE: PRT <213> ORGANISM: Hepatitis B virus <400>
SEQUENCE: 30 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val
Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val
Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala
Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu
Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala
Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr 65 70 75 80 Ser Arg Asp
Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe
Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105
110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr
115 120 125 Pro Pro Ala Tyr Arg Pro Thr Asn Ala Pro Ile Leu Ser Thr
Leu Pro 130 135 140 Glu Thr Cys Val Ile Arg Arg Arg Gly Arg Ser Pro
Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln
Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Gly Ser Gln Cys
180 <210> SEQ ID NO 31 <211> LENGTH: 212 <212>
TYPE: PRT <213> ORGANISM: Hepatitis B virus <400>
SEQUENCE: 31 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser
Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu
Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr
Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser
Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu
Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala
Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85 90 95 Leu
Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp 100 105
110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg
Gln
115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu
Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg
Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu
Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg
Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser
Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln
Cys 210 <210> SEQ ID NO 32 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 32 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Asn Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 33 <211> LENGTH:
183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 33 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Thr Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr
Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met
Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80
Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85
90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140 Glu Thr Cys Val Val Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg
Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu
Ser Gln Cys 180 <210> SEQ ID NO 34 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 34 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 35 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 35 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Asp Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 36 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 36 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro Gln 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165
170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser
Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO
37 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 37 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Val Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Lys Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Gly Ser Gln Cys 210 <210> SEQ ID
NO 38 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 38 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Phe Arg Asp Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu
Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Asp Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Ser Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
39 <211> LENGTH: 183 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 39 Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser
Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25
30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu
Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr
Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155
160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> SEQ ID NO
40 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 40 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg His Ala Ile Leu Cys
Trp Gly Asp Leu Arg Thr 85 90 95 Leu Ala Thr Trp Val Gly Gly Asn
Leu Glu Asp Pro Ile Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID
NO 41 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 41 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr
Ala Ser 50 55 60 Ala Leu Phe Arg Asp Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp Val Gly Ala Asn
Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val
Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Gln Ala 145 150 155
160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Cys
165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro
Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 42 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
42 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 43 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
43 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Asp Leu Met Ser 85 90 95 Leu Ala Thr Trp
Val Gly Val Asn Leu Glu Asp Pro Ile Ser Arg Asp 100 105 110 Leu Val
Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 44 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
44 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 45 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
45 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 46 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
46 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Ala Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Thr Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 47 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
47 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Asp Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85 90 95
Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp 100
105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg
Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg
Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile
Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg
Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser
Gln Cys 210 <210> SEQ ID NO 48 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 48 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 49 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 49 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Thr Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ala Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 50 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 50 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Phe Glu Cys Ser Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 51 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (28)..(28) <223> OTHER INFORMATION: Xaa can
represent any amino acid <400> SEQUENCE: 51 Met Gln Leu Phe
His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln
Ala Ser Lys Leu Cys Leu Gly Trp Leu Xaa Asp Met Asp Ile 20 25 30
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35
40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala
Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp
Gly Asp Leu Ile Thr 85 90 95 Leu Ser Thr Trp Val Gly Gly Asn Leu
Glu Asp Pro Thr Ser Arg Asp 100 105 110 Leu Val Val Ser Tyr Val Asn
Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125 Leu Leu Trp Phe His
Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 145 150 155 160
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165
170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser
Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Thr
Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210 <210> SEQ ID NO
52 <211> LENGTH: 212 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 52 Met Gln Leu
Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr 1 5 10 15 Val
Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile 20 25
30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu
35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Asn
Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His
Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 53 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 53 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Cys Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 54 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 54 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Pro Gln Cys 210 <210> SEQ ID NO 55 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 55 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Ser Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 56 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 56 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Leu Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 57 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 57 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75
80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Lys Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 58 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 58 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ala 50 55 60 Ala Leu
Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Leu Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 59 <211> LENGTH:
183 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 59 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ala Ser Met Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Tyr
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr
Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Thr Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met
Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Gln Asp Pro Thr 65 70 75 80
Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85
90 95 Phe Arg Gln Leu Leu Trp Phe His Val Ser Cys Leu Thr Phe Gly
Arg 100 105 110 Glu Thr Val Val Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125 Pro Gln Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140 Glu Thr Cys Val Val Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg
Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu
Ser Gln Cys 180 <210> SEQ ID NO 60 <211> LENGTH: 183
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 60 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr
Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His
Thr Ala Leu Arg His Val Phe Leu Cys Trp Gly Asp 50 55 60 Leu Met
Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr 65 70 75 80
Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85
90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg
Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu
Ser Gln Cys 180 <210> SEQ ID NO 61 <211> LENGTH: 212
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 61 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Thr Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 165 170 175 Val Val Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205
Glu Ser Gln Cys 210 <210> SEQ ID NO 62 <211> LENGTH:
212 <212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 62 Met Gln Leu Phe His Leu Cys Leu Ile Ile
Ser Cys Ser Cys Pro Thr 1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu
Gly Trp Leu Trp Gly Met Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe
Gly Ala Thr Val Glu Leu Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe
Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu
Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 65 70 75 80
His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85
90 95 Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg
Asp 100 105 110 Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
Phe Arg Gln 115 120 125 Leu Leu Trp Phe His Ile Ser Cys Leu Ile Phe
Gly Arg Glu Thr Val 130 135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala
145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro
Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg
Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 63 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
63 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Val 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Val Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Ala Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 64 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
64 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Asp Leu Met Asn 85 90 95 Leu Ala Thr Trp
Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg Asp 100 105 110 Leu Val
Val Gly Tyr Val Asn Thr Thr Val Gly Leu Lys Phe Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 65 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
65 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Asp 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Thr Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 66 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
66 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Ala
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp
Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val
Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125
Ile Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 67 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
67 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85 90 95 Leu Ala Thr Trp
Val Gly Val Asn Leu Glu Asp Pro Ala Thr Arg Asp 100 105 110 Leu Val
Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys Phe Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 68 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
68 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Arg Ile Leu Cys Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp
Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val
Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Thr Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 69 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
69 Met Gln Leu Phe His Leu Cys Leu Val Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ala 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 85 90 95 Leu Ala Thr Trp
Val Gly Asn Asn Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val
Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 70 <211> LENGTH: 212 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
70 Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr
1 5 10 15 Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met
Asp Ile 20 25 30 Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu Ser Phe Leu 35 40 45 Pro Ser Ala Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp Thr Ala Ser 50 55 60 Ala Leu Tyr Arg Glu Ala Leu Glu
Ser Pro Glu His Cys Ser Pro His 65 70 75 80 His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Asp Leu Met Thr 85 90 95 Leu Ala Thr Trp
Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp 100 105 110 Leu Val
Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln 115 120 125
Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 130
135 140 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala 145 150 155 160 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu
Pro Glu Thr Thr 165 170 175 Val Val Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro 180 185 190 Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser Gln Ser Arg 195 200 205 Glu Ser Gln Cys 210
<210> SEQ ID NO 71 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
71 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ala Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180
<210> SEQ ID NO 72 <211> LENGTH: 183 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
72 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Gly Asn Leu Glu Asp Pro Ile 65 70 75 80 Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175 Gln Ser Arg Gly Ser Gln Cys 180
<210> SEQ ID NO 73 <211> LENGTH: 188 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
73 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu
1 5 10 15 Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu
Val Asp
20 25 30 Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu
His Cys 35 40 45 Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val
Cys Trp Asp Glu 50 55 60 Leu Thr Lys Leu Ile Ala Trp Met Ser Ser
Asn Ile Thr Ser Glu Gln 65 70 75 80 Val Arg Thr Ile Ile Val Asn His
Val Asn Asp Thr Trp Gly Leu Lys 85 90 95 Val Arg Gln Ser Leu Trp
Phe His Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110 His Thr Val Gln
Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Ala
Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140
Glu His Thr Val Ile Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser 145
150 155 160 Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln
Ser Pro 165 170 175 Arg Arg Arg Arg Ser Gln Ser Pro Ser Thr Asn Cys
180 185 <210> SEQ ID NO 74 <211> LENGTH: 217
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 74 Met Tyr Leu Phe His Leu Cys Leu Val Phe
Ala Cys Val Pro Cys Pro 1 5 10 15 Thr Val Gln Ala Ser Lys Leu Cys
Leu Gly Trp Leu Trp Asp Met Asp 20 25 30 Ile Asp Pro Tyr Lys Glu
Phe Gly Ser Ser Tyr Gln Leu Leu Asn Phe 35 40 45 Leu Pro Leu Asp
Phe Phe Pro Asp Leu Asn Ala Leu Val Asp Thr Ala 50 55 60 Ala Ala
Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys Ser Pro 65 70 75 80
His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Glu Glu Leu Thr 85
90 95 Arg Leu Ile Thr Trp Met Ser Glu Asn Thr Thr Glu Glu Val Arg
Arg 100 105 110 Ile Ile Val Asp His Val Asn Asn Thr Trp Gly Leu Lys
Val Arg Gln 115 120 125 Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe
Gly Gln His Thr Val 130 135 140 Gln Glu Phe Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Ala Pro 145 150 155 160 Tyr Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu His Thr 165 170 175 Val Ile Arg Arg
Arg Gly Gly Ser Arg Ala Ala Arg Ser Pro Arg Arg 180 185 190 Arg Thr
Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg 195 200 205
Arg Ser Gln Ser Pro Ala Ser Asn Cys 210 215 <210> SEQ ID NO
75 <211> LENGTH: 262 <212> TYPE: PRT <213>
ORGANISM: Hepatitis B virus <400> SEQUENCE: 75 Met Asp Val
Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro 1 5 10 15 Asp
Asp Phe Phe Pro Lys Ile Glu Asp Leu Val Arg Asp Ala Lys Asp 20 25
30 Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu
35 40 45 Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln
Thr Thr 50 55 60 Gln Gly Met His Glu Ile Ala Glu Ala Ile Arg Ala
Val Ile Pro Pro 65 70 75 80 Thr Thr Ala Pro Val Pro Ser Gly Tyr Leu
Ile Gln His Asp Glu Ala 85 90 95 Glu Glu Ile Pro Leu Gly Asp Leu
Phe Lys Glu Gln Glu Glu Arg Ile 100 105 110 Val Ser Phe Gln Pro Asp
Tyr Pro Ile Thr Ala Arg Ile His Ala His 115 120 125 Leu Lys Ala Tyr
Ala Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135 140 Arg Leu
Leu Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Thr 145 150 155
160 Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu
165 170 175 Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr
Arg Pro 180 185 190 Ile Gln Val Ala Gln Gly Gly Arg Lys Thr Ser Thr
Ala Thr Arg Lys 195 200 205 Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val 210 215 220 Tyr Gly Arg Arg Arg Ser Lys Ser
Arg Glu Arg Arg Ala Ser Ser Pro 225 230 235 240 Gln Arg Ala Gly Ser
Pro Leu Pro Arg Ser Ser Ser Ser His His Arg 245 250 255 Ser Pro Ser
Pro Arg Lys 260 <210> SEQ ID NO 76 <211> LENGTH: 305
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 76 Met Trp Asp Leu Arg Leu His Pro Ser Pro
Phe Gly Ala Ala Cys Gln 1 5 10 15 Gly Ile Phe Thr Ser Ser Leu Leu
Leu Phe Leu Val Thr Val Pro Leu 20 25 30 Val Cys Thr Ile Val Tyr
Asp Ser Cys Leu Cys Met Asp Ile Asn Ala 35 40 45 Ser Arg Ala Leu
Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro 50 55 60 Lys Ile
Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr 65 70 75 80
Trp Arg Asn Asp Ser Ile Lys Lys His Val Leu Ile Ala Thr His Phe 85
90 95 Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr Gln Gly Met His
Glu 100 105 110 Ile Ala Glu Ala Leu Arg Ala Ile Ile Pro Ala Thr Thr
Ala Pro Val 115 120 125 Pro Gln Gly Phe Leu Val Gln His Glu Glu Ala
Glu Glu Ile Pro Leu 130 135 140 Gly Glu Leu Phe Arg Tyr Gln Glu Glu
Arg Leu Thr Asn Phe Gln Pro 145 150 155 160 Asp Tyr Pro Val Thr Ala
Arg Ile His Ala His Leu Lys Ala Tyr Ala 165 170 175 Lys Ile Asn Glu
Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp 180 185 190 His Tyr
Asn Cys Leu Leu Trp Gly Glu Pro Asn Val Thr Asn Tyr Ile 195 200 205
Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Lys 210
215 220 Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro Ile Gln Val Ala
Gln 225 230 235 240 Gly Gly Arg Asn Lys Thr Gln Gly Val Arg Lys Ser
Arg Gly Leu Glu 245 250 255 Pro Arg Arg Arg Arg Val Lys Thr Thr Ile
Val Tyr Gly Arg Arg Arg 260 265 270 Ser Lys Ser Arg Glu Arg Arg Ala
Pro Thr Pro Gln Arg Ala Gly Ser 275 280 285 Pro Leu Pro Arg Thr Ser
Arg Asp His His Arg Ser Pro Ser Pro Arg 290 295 300 Glu 305
<210> SEQ ID NO 77 <211> LENGTH: 185 <212> TYPE:
PRT <213> ORGANISM: Hepatitis B virus <400> SEQUENCE:
77 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu
1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu
Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val
Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val
Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr
Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125
Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130
135 140 Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg
Arg 145 150 155 160 Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro Arg Arg Arg 165 170 175 Arg Ser Gln Ser Arg Glu Ser Gln Cys
180 185 <210> SEQ ID NO 78 <211> LENGTH: 152
<212> TYPE: PRT <213> ORGANISM: Hepatitis B virus
<400> SEQUENCE: 78 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe
Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ala Ala Leu Tyr
Arg Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp 50 55 60 Leu Met
Thr Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Gly Gly 65 70 75 80
Lys Gly Gly Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val 85
90 95 Gly Leu Lys Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu
Thr 100 105 110 Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe
Gly Val Trp 115 120 125 Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn
Ala Pro Ile Leu Ser 130 135 140 Thr Leu Pro Glu Thr Thr Val Val 145
150 <210> SEQ ID NO 79 <211> LENGTH: 3635 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Plasmid pAP283 58
<400> SEQUENCE: 79 cgagctcgcc cctggcttat cgaaattaat
acgactcact atagggagac cggaattcga 60 gctcgcccgg ggatcctcta
gaattttctg cgcacccatc ccgggtggcg cccaaagtga 120 ggaaaatcac
atggcaaata agccaatgca accgatcaca tctacagcaa ataaaattgt 180
gtggtcggat ccaactcgtt tatcaactac attttcagca agtctgttac gccaacgtgt
240 taaagttggt atagccgaac tgaataatgt ttcaggtcaa tatgtatctg
tttataagcg 300 tcctgcacct aaaccggaag gttgtgcaga tgcctgtgtc
attatgccga atgaaaacca 360 atccattcgc acagtgattt cagggtcagc
cgaaaacttg gctaccttaa aagcagaatg 420 ggaaactcac aaacgtaacg
ttgacacact cttcgcgagc ggcaacgccg gtttgggttt 480 ccttgaccct
actgcggcta tcgtatcgtc tgatactact gcttaagctt gtattctata 540
gtgtcaccta aatcgtatgt gtatgataca taaggttatg tattaattgt agccgcgttc
600 taacgacaat atgtacaagc ctaattgtgt agcatctggc ttactgaagc
agaccctatc 660 atctctctcg taaactgccg tcagagtcgg tttggttgga
cgaaccttct gagtttctgg 720 taacgccgtt ccgcaccccg gaaatggtca
ccgaaccaat cagcagggtc atcgctagcc 780 agatcctcta cgccggacgc
atcgtggccg gcatcaccgg cgccacaggt gcggttgctg 840 gcgcctatat
cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga 900
gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
960 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
ctactactgg 1020 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
atatggtgca ctctcagtac 1080 aatctgctct gatgccgcat agttaagcca
actccgctat cgctacgtga ctgggtcatg 1140 gctgcgcccc gacacccgcc
aacacccgct gacgcgccct gacgggcttg tctgctcccg 1200 gcatccgctt
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca 1260
ccgtcatcac cgaaacgcgc gaggcagctt gaagacgaaa gggcctcgtg atacgcctat
1320 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
acttttcggg 1380 gaaatgtgcg cggaacccct atttgtttat ttttctaaat
acattcaaat atgtatccgc 1440 tcatgagaca ataaccctga taaatgcttc
aataatattg aaaaaggaag agtatgagta 1500 ttcaacattt ccgtgtcgcc
cttattccct tttttgcggc attttgcctt cctgtttttg 1560 ctcacccaga
aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 1620
gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
1680 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
tcccgtattg 1740 acgccgggca agagcaactc ggtcgccgca tacactattc
tcagaatgac ttggttgagt 1800 actcaccagt cacagaaaag catcttacgg
atggcatgac agtaagagaa ttatgcagtg 1860 ctgccataac catgagtgat
aacactgcgg ccaacttact tctgacaacg atcggaggac 1920 cgaaggagct
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1980
gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
2040 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
gcttcccggc 2100 aacaattaat agactggatg gaggcggata aagttgcagg
accacttctg cgctcggccc 2160 ttccggctgg ctggtttatt gctgataaat
ctggagccgg tgagcgtggg tctcgcggta 2220 tcattgcagc actggggcca
gatggtaagc cctcccgtat cgtagttatc tacacgacgg 2280 ggagtcaggc
aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 2340
ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
2400 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
atgaccaaaa 2460 tcccttaacg tgagttttcg ttccactgag cgtcagaccc
cgtagaaaag atcaaaggat 2520 cttcttgaga tccttttttt ctgcgcgtaa
tctgctgctt gcaaacaaaa aaaccaccgc 2580 taccagcggt ggtttgtttg
ccggatcaag agctaccaac tctttttccg aaggtaactg 2640 gcttcagcag
agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 2700
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
2760 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
tagttaccgg 2820 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
acagcccagc ttggagcgaa 2880 cgacctacac cgaactgaga tacctacagc
gcgagcattg agaaagcgcc acgcttcccg 2940 aagggagaaa ggcggacagg
tatccggtaa gcggcagggt cggaacagga gagcgcacga 3000 gggagcttcc
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 3060
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
3120 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
atgttctttc 3180 ctgcgttatc ccctgattct gtggataacc gtattaccgc
ctttgagtga gctgataccg 3240 ctcgccgcag ccgaacgacc gagcgcagcg
agtcagtgag cgaggaagcg gaagagcgcc 3300 caatacgcaa accgcctctc
cccgcgcgtt ggccgattca ttaatgcagc tgtggtgtca 3360 tggtcggtga
tcgccagggt gccgacgcgc atctcgactg catggtgcac caatgcttct 3420
ggcgtcaggc agccatcgga agctgtggta tggccgtgca ggtcgtaaat cactgcataa
3480 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
acatcataac 3540 ggttctggca aatattctga aatgagctgt tgacaattaa
tcatcgaact agttaactag 3600 tacgcaagtt cacgtaaaaa gggtatcgcg gaatt
3635 <210> SEQ ID NO 80 <211> LENGTH: 131 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Ap205 coat protein
<400> SEQUENCE: 80 Met Ala Asn Lys Pro Met Gln Pro Ile Thr
Ser Thr Ala Asn Lys Ile 1 5 10 15 Val Trp Ser Asp Pro Thr Arg Leu
Ser Thr Thr Phe Ser Ala Ser Leu 20 25 30 Leu Arg Gln Arg Val Lys
Val Gly Ile Ala Glu Leu Asn Asn Val Ser 35 40 45 Gly Gln Tyr Val
Ser Val Tyr Lys Arg Pro Ala Pro Lys Pro Glu Gly 50 55 60 Cys Ala
Asp Ala Cys Val Ile Met Pro Asn Glu Asn Gln Ser Ile Arg 65 70 75 80
Thr Val Ile Ser Gly Ser Ala Glu Asn Leu Ala Thr Leu Lys Ala Glu 85
90 95 Trp Glu Thr His Lys Arg Asn Val Asp Thr Leu Phe Ala Ser Gly
Asn 100 105 110 Ala Gly Leu Gly Phe Leu Asp Pro Thr Ala Ala Ile Val
Ser Ser Asp 115 120 125 Thr Thr Ala 130 <210> SEQ ID NO 81
<211> LENGTH: 131 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: AP205 coat protein <400> SEQUENCE: 81 Met Ala
Asn Lys Thr Met Gln Pro Ile Thr Ser Thr Ala Asn Lys Ile 1 5 10 15
Val Trp Ser Asp Pro Thr Arg Leu Ser Thr Thr Phe Ser Ala Ser Leu 20
25 30 Leu Arg Gln Arg Val Lys Val Gly Ile Ala Glu Leu Asn Asn Val
Ser 35 40 45 Gly Gln Tyr Val Ser Val Tyr Lys Arg Pro Ala Pro Lys
Pro Glu Gly 50 55 60 Cys Ala Asp Ala Cys Val Ile Met Pro Asn Glu
Asn Gln Ser Ile Arg 65 70 75 80 Thr Val Ile Ser Gly Ser Ala Glu Asn
Leu Ala Thr Leu Lys Ala Glu 85 90 95 Trp Glu Thr His Lys Arg Asn
Val Asp Thr Leu Phe Ala Ser Gly Asn 100 105 110 Ala Gly Leu Gly Phe
Leu Asp Pro Thr Ala Ala Ile Val Ser Ser Asp 115 120 125 Thr Thr Ala
130 <210> SEQ ID NO 82 <211> LENGTH: 3613 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Plasmid pAP281
32 <400> SEQUENCE: 82 cgagctcgcc cctggcttat cgaaattaat
acgactcact atagggagac cggaattcga 60 gctcgcccgg ggatcctcta
gattaaccca acgcgtagga gtcaggccat ggcaaataag 120 acaatgcaac
cgatcacatc tacagcaaat aaaattgtgt ggtcggatcc aactcgttta 180
tcaactacat tttcagcaag tctgttacgc caacgtgtta aagttggtat agccgaactg
240 aataatgttt caggtcaata tgtatctgtt tataagcgtc ctgcacctaa
accggaaggt 300 tgtgcagatg cctgtgtcat tatgccgaat gaaaaccaat
ccattcgcac agtgatttca 360 gggtcagccg aaaacttggc taccttaaaa
gcagaatggg aaactcacaa acgtaacgtt 420 gacacactct tcgcgagcgg
caacgccggt ttgggtttcc ttgaccctac tgcggctatc 480 gtatcgtctg
atactactgc ttaagcttgt attctatagt gtcacctaaa tcgtatgtgt 540
atgatacata aggttatgta ttaattgtag ccgcgttcta acgacaatat gtacaagcct
600 aattgtgtag catctggctt actgaagcag accctatcat ctctctcgta
aactgccgtc 660 agagtcggtt tggttggacg aaccttctga gtttctggta
acgccgttcc gcaccccgga 720 aatggtcacc gaaccaatca gcagggtcat
cgctagccag atcctctacg ccggacgcat 780 cgtggccggc atcaccggcg
ccacaggtgc ggttgctggc gcctatatcg ccgacatcac 840 cgatggggaa
gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat 900
ggtggcaggc cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct
960 tgcggcggcg gtgctcaacg gcctcaacct actactgggc tgcttcctaa
tgcaggagtc 1020 gcataaggga gagcgtcgat atggtgcact ctcagtacaa
tctgctctga tgccgcatag 1080 ttaagccaac tccgctatcg ctacgtgact
gggtcatggc tgcgccccga cacccgccaa 1140 cacccgctga cgcgccctga
cgggcttgtc tgctcccggc atccgcttac agacaagctg 1200 tgaccgtctc
cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 1260
ggcagcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
1320 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
gaacccctat 1380 ttgtttattt ttctaaatac attcaaatat gtatccgctc
atgagacaat aaccctgata 1440 aatgcttcaa taatattgaa aaaggaagag
tatgagtatt caacatttcc gtgtcgccct 1500 tattcccttt tttgcggcat
tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 1560 agtaaaagat
gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 1620
cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
1680 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
agcaactcgg 1740 tcgccgcata cactattctc agaatgactt ggttgagtac
tcaccagtca cagaaaagca 1800 tcttacggat ggcatgacag taagagaatt
atgcagtgct gccataacca tgagtgataa 1860 cactgcggcc aacttacttc
tgacaacgat cggaggaccg aaggagctaa ccgctttttt 1920 gcacaacatg
ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 1980
cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
2040 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
actggatgga 2100 ggcggataaa gttgcaggac cacttctgcg ctcggccctt
ccggctggct ggtttattgc 2160 tgataaatct ggagccggtg agcgtgggtc
tcgcggtatc attgcagcac tggggccaga 2220 tggtaagccc tcccgtatcg
tagttatcta cacgacgggg agtcaggcaa ctatggatga 2280 acgaaataga
cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 2340
ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
2400 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
agttttcgtt 2460 ccactgagcg tcagaccccg tagaaaagat caaaggatct
tcttgagatc ctttttttct 2520 gcgcgtaatc tgctgcttgc aaacaaaaaa
accaccgcta ccagcggtgg tttgtttgcc 2580 ggatcaagag ctaccaactc
tttttccgaa ggtaactggc ttcagcagag cgcagatacc 2640 aaatactgtc
cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 2700
gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
2760 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
ggtcgggctg 2820 aacggggggt tcgtgcacac agcccagctt ggagcgaacg
acctacaccg aactgagata 2880 cctacagcgc gagcattgag aaagcgccac
gcttcccgaa gggagaaagg cggacaggta 2940 tccggtaagc ggcagggtcg
gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 3000 ctggtatctt
tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 3060
atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
3120 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
ctgattctgt 3180 ggataaccgt attaccgcct ttgagtgagc tgataccgct
cgccgcagcc gaacgaccga 3240 gcgcagcgag tcagtgagcg aggaagcgga
agagcgccca atacgcaaac cgcctctccc 3300 cgcgcgttgg ccgattcatt
aatgcagctg tggtgtcatg gtcggtgatc gccagggtgc 3360 cgacgcgcat
ctcgactgca tggtgcacca atgcttctgg cgtcaggcag ccatcggaag 3420
ctgtggtatg gccgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
3480 cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
tattctgaaa 3540 tgagctgttg acaattaatc atcgaactag ttaactagta
cgcaagttca cgtaaaaagg 3600 gtatcgcgga att 3613 <210> SEQ ID
NO 83 <400> SEQUENCE: 83 000 3 <210> SEQ ID NO 84
<400> SEQUENCE: 84 000 3 <210> SEQ ID NO 85 <400>
SEQUENCE: 85 000 3 <210> SEQ ID NO 86 <400> SEQUENCE:
86 000 3 <210> SEQ ID NO 87 <400> SEQUENCE: 87 000 3
<210> SEQ ID NO 88 <400> SEQUENCE: 88 000 3 <210>
SEQ ID NO 89 <400> SEQUENCE: 89 000 3 <210> SEQ ID NO
90 <400> SEQUENCE: 90 000 3 <210> SEQ ID NO 91
<400> SEQUENCE: 91 000 3 <210> SEQ ID NO 92 <400>
SEQUENCE: 92 000 3 <210> SEQ ID NO 93 <400> SEQUENCE:
93 000 3 <210> SEQ ID NO 94 <400> SEQUENCE: 94 000 3
<210> SEQ ID NO 95 <400> SEQUENCE: 95 000 3 <210>
SEQ ID NO 96 <400> SEQUENCE: 96 000 3 <210> SEQ ID NO
97 <400> SEQUENCE: 97 000 3 <210> SEQ ID NO 98
<400> SEQUENCE: 98 000 3 <210> SEQ ID NO 99 <400>
SEQUENCE: 99 000 3 <210> SEQ ID NO 100 <400> SEQUENCE:
100
000 3 <210> SEQ ID NO 101 <400> SEQUENCE: 101 000 3
<210> SEQ ID NO 102 <400> SEQUENCE: 102 000 3
<210> SEQ ID NO 103 <400> SEQUENCE: 103 000 3
<210> SEQ ID NO 104 <400> SEQUENCE: 104 000 3
<210> SEQ ID NO 105 <400> SEQUENCE: 105 000 3
<210> SEQ ID NO 106 <400> SEQUENCE: 106 000 3
<210> SEQ ID NO 107 <400> SEQUENCE: 107 000 3
<210> SEQ ID NO 108 <400> SEQUENCE: 108 000 3
<210> SEQ ID NO 109 <400> SEQUENCE: 109 000 3
<210> SEQ ID NO 110 <400> SEQUENCE: 110 000 3
<210> SEQ ID NO 111 <400> SEQUENCE: 111 000 3
<210> SEQ ID NO 112 <400> SEQUENCE: 112 000 3
<210> SEQ ID NO 113 <400> SEQUENCE: 113 000 3
<210> SEQ ID NO 114 <400> SEQUENCE: 114 000 3
<210> SEQ ID NO 115 <400> SEQUENCE: 115 000 3
<210> SEQ ID NO 116 <400> SEQUENCE: 116 000 3
<210> SEQ ID NO 117 <400> SEQUENCE: 117 000 3
<210> SEQ ID NO 118 <400> SEQUENCE: 118 000 3
<210> SEQ ID NO 119 <400> SEQUENCE: 119 000 3
<210> SEQ ID NO 120 <400> SEQUENCE: 120 000 3
<210> SEQ ID NO 121 <400> SEQUENCE: 121 000 3
<210> SEQ ID NO 122 <400> SEQUENCE: 122 000 3
<210> SEQ ID NO 123 <400> SEQUENCE: 123 000 3
<210> SEQ ID NO 124 <400> SEQUENCE: 124 000 3
<210> SEQ ID NO 125 <400> SEQUENCE: 125 000 3
<210> SEQ ID NO 126 <400> SEQUENCE: 126 000 3
<210> SEQ ID NO 127 <400> SEQUENCE: 127 000 3
<210> SEQ ID NO 128 <400> SEQUENCE: 128 000 3
<210> SEQ ID NO 129 <400> SEQUENCE: 129 000 3
<210> SEQ ID NO 130 <400> SEQUENCE: 130 000 3
<210> SEQ ID NO 131 <400> SEQUENCE: 131 000 3
<210> SEQ ID NO 132 <400> SEQUENCE: 132 000 3
<210> SEQ ID NO 133 <400> SEQUENCE: 133 000 3
<210> SEQ ID NO 134 <400> SEQUENCE: 134 000 3
<210> SEQ ID NO 135 <400> SEQUENCE: 135 000 3
<210> SEQ ID NO 136 <400> SEQUENCE: 136
000 3 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000 3
<210> SEQ ID NO 138 <400> SEQUENCE: 138 000 3
<210> SEQ ID NO 139 <400> SEQUENCE: 139 000 3
<210> SEQ ID NO 140 <400> SEQUENCE: 140 000 3
<210> SEQ ID NO 141 <400> SEQUENCE: 141 000 3
<210> SEQ ID NO 142 <400> SEQUENCE: 142 000 3
<210> SEQ ID NO 143 <400> SEQUENCE: 143 000 3
<210> SEQ ID NO 144 <400> SEQUENCE: 144 000 3
<210> SEQ ID NO 145 <400> SEQUENCE: 145 000 3
<210> SEQ ID NO 146 <400> SEQUENCE: 146 000 3
<210> SEQ ID NO 147 <400> SEQUENCE: 147 000 3
<210> SEQ ID NO 148 <400> SEQUENCE: 148 000 3
<210> SEQ ID NO 149 <400> SEQUENCE: 149 000 3
<210> SEQ ID NO 150 <400> SEQUENCE: 150 000 3
<210> SEQ ID NO 151 <400> SEQUENCE: 151 000 3
<210> SEQ ID NO 152 <400> SEQUENCE: 152 000 3
<210> SEQ ID NO 153 <400> SEQUENCE: 153 000 3
<210> SEQ ID NO 154 <400> SEQUENCE: 154 000 3
<210> SEQ ID NO 155 <400> SEQUENCE: 155 000 3
<210> SEQ ID NO 156 <400> SEQUENCE: 156 000 3
<210> SEQ ID NO 157 <400> SEQUENCE: 157 000 3
<210> SEQ ID NO 158 <400> SEQUENCE: 158 000 3
<210> SEQ ID NO 159 <400> SEQUENCE: 159 000 3
<210> SEQ ID NO 160 <400> SEQUENCE: 160 000 3
<210> SEQ ID NO 161 <400> SEQUENCE: 161 000 3
<210> SEQ ID NO 162 <400> SEQUENCE: 162 000 3
<210> SEQ ID NO 163 <400> SEQUENCE: 163 000 3
<210> SEQ ID NO 164 <400> SEQUENCE: 164 000 3
<210> SEQ ID NO 165 <400> SEQUENCE: 165 000 3
<210> SEQ ID NO 166 <400> SEQUENCE: 166 000 3
<210> SEQ ID NO 167 <400> SEQUENCE: 167 000 3
<210> SEQ ID NO 168 <400> SEQUENCE: 168 000 3
<210> SEQ ID NO 169 <400> SEQUENCE: 169 000 3
<210> SEQ ID NO 170 <400> SEQUENCE: 170 000 3
<210> SEQ ID NO 171 <400> SEQUENCE: 171 000 3
<210> SEQ ID NO 172
<400> SEQUENCE: 172 000 3 <210> SEQ ID NO 173
<400> SEQUENCE: 173 000 3 <210> SEQ ID NO 174
<400> SEQUENCE: 174 000 3 <210> SEQ ID NO 175
<400> SEQUENCE: 175 000 3 <210> SEQ ID NO 176
<400> SEQUENCE: 176 000 3 <210> SEQ ID NO 177
<400> SEQUENCE: 177 000 3 <210> SEQ ID NO 178
<400> SEQUENCE: 178 000 3 <210> SEQ ID NO 179
<400> SEQUENCE: 179 000 3 <210> SEQ ID NO 180
<400> SEQUENCE: 180 000 3 <210> SEQ ID NO 181
<400> SEQUENCE: 181 000 3 <210> SEQ ID NO 182
<400> SEQUENCE: 182 000 3 <210> SEQ ID NO 183
<400> SEQUENCE: 183 000 3 <210> SEQ ID NO 184
<400> SEQUENCE: 184 000 3 <210> SEQ ID NO 185
<400> SEQUENCE: 185 000 3 <210> SEQ ID NO 186
<400> SEQUENCE: 186 000 3 <210> SEQ ID NO 187
<400> SEQUENCE: 187 000 3 <210> SEQ ID NO 188
<400> SEQUENCE: 188 000 3 <210> SEQ ID NO 189
<400> SEQUENCE: 189 000 3 <210> SEQ ID NO 190
<400> SEQUENCE: 190 000 3 <210> SEQ ID NO 191
<400> SEQUENCE: 191 000 3 <210> SEQ ID NO 192
<400> SEQUENCE: 192 000 3 <210> SEQ ID NO 193
<400> SEQUENCE: 193 000 3 <210> SEQ ID NO 194
<400> SEQUENCE: 194 000 3 <210> SEQ ID NO 195
<400> SEQUENCE: 195 000 3 <210> SEQ ID NO 196
<400> SEQUENCE: 196 000 3 <210> SEQ ID NO 197
<400> SEQUENCE: 197 000 3 <210> SEQ ID NO 198
<400> SEQUENCE: 198 000 3 <210> SEQ ID NO 199
<400> SEQUENCE: 199 000 3 <210> SEQ ID NO 200
<400> SEQUENCE: 200 000 3 <210> SEQ ID NO 201
<400> SEQUENCE: 201 000 3 <210> SEQ ID NO 202
<400> SEQUENCE: 202 000 3 <210> SEQ ID NO 203
<400> SEQUENCE: 203 000 3 <210> SEQ ID NO 204
<400> SEQUENCE: 204 000 3 <210> SEQ ID NO 205
<400> SEQUENCE: 205 000 3 <210> SEQ ID NO 206
<400> SEQUENCE: 206 000 3 <210> SEQ ID NO 207
<400> SEQUENCE: 207 000 3 <210> SEQ ID NO 208
<400> SEQUENCE: 208 000 3 <210> SEQ ID NO 209
<400> SEQUENCE: 209 000 3 <210> SEQ ID NO 210
<400> SEQUENCE: 210 000 3 <210> SEQ ID NO 211
<400> SEQUENCE: 211 000 3 <210> SEQ ID NO 212
<400> SEQUENCE: 212 000 3 <210> SEQ ID NO 213
<400> SEQUENCE: 213 000 3 <210> SEQ ID NO 214
<400> SEQUENCE: 214 000 3 <210> SEQ ID NO 215
<400> SEQUENCE: 215 000 3 <210> SEQ ID NO 216
<400> SEQUENCE: 216 000 3 <210> SEQ ID NO 217
<400> SEQUENCE: 217 000 3 <210> SEQ ID NO 218
<400> SEQUENCE: 218 000 3 <210> SEQ ID NO 219
<400> SEQUENCE: 219 000 3 <210> SEQ ID NO 220
<400> SEQUENCE: 220 000 3 <210> SEQ ID NO 221
<400> SEQUENCE: 221 000 3 <210> SEQ ID NO 222
<400> SEQUENCE: 222 000 3 <210> SEQ ID NO 223
<400> SEQUENCE: 223 000 3 <210> SEQ ID NO 224
<400> SEQUENCE: 224 000 3 <210> SEQ ID NO 225
<400> SEQUENCE: 225 000 3 <210> SEQ ID NO 226
<400> SEQUENCE: 226 000 3 <210> SEQ ID NO 227
<400> SEQUENCE: 227 000 3 <210> SEQ ID NO 228
<400> SEQUENCE: 228 000 3 <210> SEQ ID NO 229
<400> SEQUENCE: 229 000 3 <210> SEQ ID NO 230
<211> LENGTH: 132 <212> TYPE: PRT <213> ORGANISM:
precursor human IL 13 <400> SEQUENCE: 230 Met Ala Leu Leu Leu
Thr Thr Val Ile Ala Leu Thr Cys Leu Gly Gly 1 5 10 15 Phe Ala Ser
Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu 20 25 30 Ile
Glu Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys 35 40
45 Asn Gly Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys
50 55 60 Ala Ala Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala
Ile Glu 65 70 75 80 Lys Thr Gln Arg Met Leu Ser Gly Phe Cys Pro His
Lys Val Ser Ala 85 90 95 Gly Gln Phe Ser Ser Leu His Val Arg Asp
Thr Lys Ile Glu Val Ala 100 105 110 Gln Phe Val Lys Asp Leu Leu Leu
His Leu Lys Lys Leu Phe Arg Glu 115 120 125 Gly Arg Phe Asn 130
<210> SEQ ID NO 231 <211> LENGTH: 112 <212> TYPE:
PRT <213> ORGANISM: processed human IL 13 <400>
SEQUENCE: 231 Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile
Glu Glu Leu 1 5 10 15 Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu
Cys Asn Gly Ser Met 20 25 30 Val Trp Ser Ile Asn Leu Thr Ala Gly
Met Tyr Cys Ala Ala Leu Glu 35 40 45 Ser Leu Ile Asn Val Ser Gly
Cys Ser Ala Ile Glu Lys Thr Gln Arg 50 55 60 Met Leu Ser Gly Phe
Cys Pro His Lys Val Ser Ala Gly Gln Phe Ser 65 70 75 80 Ser Leu His
Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val Lys 85 90 95 Asp
Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe Asn 100 105
110 <210> SEQ ID NO 232 <211> LENGTH: 111 <212>
TYPE: PRT <213> ORGANISM: processed mouse IL 13 <400>
SEQUENCE: 232 Gly Pro Val Pro Arg Ser Val Ser Leu Pro Leu Thr Leu
Lys Glu Leu 1 5 10 15 Ile Glu Glu Leu Ser Asn Ile Thr Gln Asp Gln
Thr Pro Leu Cys Asn 20 25 30 Gly Ser Met Val Trp Ser Val Asp Leu
Ala Ala Gly Gly Phe Cys Val 35 40 45 Ala Leu Asp Ser Leu Thr Asn
Ile Ser Asn Cys Asn Ala Ile Tyr Arg 50 55 60 Thr Gln Arg Ile Leu
His Gly Leu Cys Asn Arg Lys Ala Pro Thr Thr 65 70 75 80 Val Ser Ser
Leu Pro Asp Thr Lys Ile Glu Val Ala His Phe Ile Thr 85 90 95 Lys
Leu Leu Ser Tyr Thr Lys Gln Leu Phe Arg His Gly Pro Phe 100 105 110
<210> SEQ ID NO 233 <211> LENGTH: 134 <212> TYPE:
PRT <213> ORGANISM: precursor human IL 5
<400> SEQUENCE: 233 Met Arg Met Leu Leu His Leu Ser Leu Leu
Ala Leu Gly Ala Ala Tyr 1 5 10 15 Val Tyr Ala Ile Pro Thr Glu Ile
Pro Thr Ser Ala Leu Val Lys Glu 20 25 30 Thr Leu Ala Leu Leu Ser
Thr His Arg Thr Leu Leu Ile Ala Asn Glu 35 40 45 Thr Leu Arg Ile
Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr 50 55 60 Glu Glu
Ile Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln 65 70 75 80
Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys 85
90 95 Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg
Val 100 105 110 Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val
Met Asn Thr 115 120 125 Glu Trp Ile Ile Glu Ser 130 <210> SEQ
ID NO 234 <211> LENGTH: 115 <212> TYPE: PRT <213>
ORGANISM: processed human IL 5 <400> SEQUENCE: 234 Ile Pro
Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15
Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20
25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu Glu
Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln
Gly Gly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile
Lys Lys Tyr Ile Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Glu Glu
Arg Arg Arg Val Asn Gln Phe 85 90 95 Leu Asp Tyr Leu Gln Glu Phe
Leu Gly Val Met Asn Thr Glu Trp Ile 100 105 110 Ile Glu Ser 115
<210> SEQ ID NO 235 <211> LENGTH: 113 <212> TYPE:
PRT <213> ORGANISM: processed mouse IL 5 <400>
SEQUENCE: 235 Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu
Thr Gln Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu
Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln Leu
Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asn
Gln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 Met Leu Phe Gln Asn
Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Lys Glu Lys
Cys Gly Glu Glu Arg Arg Arg Thr Arg Gln Phe Leu Asp 85 90 95 Tyr
Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu 100 105
110 Gly <210> SEQ ID NO 236 <400> SEQUENCE: 236 000 3
<210> SEQ ID NO 237 <400> SEQUENCE: 237 000 3
<210> SEQ ID NO 238 <400> SEQUENCE: 238 000 3
<210> SEQ ID NO 239 <400> SEQUENCE: 239 000 3
<210> SEQ ID NO 240 <400> SEQUENCE: 240 000 3
<210> SEQ ID NO 241 <400> SEQUENCE: 241 000 3
<210> SEQ ID NO 242 <211> LENGTH: 97 <212> TYPE:
PRT <213> ORGANISM: Human Eotaxin 1 <400> SEQUENCE: 242
Met Lys Val Ser Ala Ala Leu Leu Trp Leu Leu Leu Ile Ala Ala Ala 1 5
10 15 Phe Ser Pro Gln Gly Leu Ala Gly Pro Ala Ser Val Pro Thr Thr
Cys 20 25 30 Cys Phe Asn Leu Ala Asn Arg Lys Ile Pro Leu Gln Arg
Leu Glu Ser 35 40 45 Tyr Arg Arg Ile Thr Ser Gly Lys Cys Pro Gln
Lys Ala Val Ile Phe 50 55 60 Lys Thr Lys Leu Ala Lys Asp Ile Cys
Ala Asp Pro Lys Lys Lys Trp 65 70 75 80 Val Gln Asp Ser Met Lys Tyr
Leu Asp Gln Lys Ser Pro Thr Pro Lys 85 90 95 Pro <210> SEQ ID
NO 243 <211> LENGTH: 119 <212> TYPE: PRT <213>
ORGANISM: Human Eotaxin 2 <400> SEQUENCE: 243 Met Ala Gly Leu
Met Thr Ile Val Thr Ser Leu Leu Phe Leu Gly Val 1 5 10 15 Cys Ala
His His Ile Ile Pro Thr Gly Ser Val Val Ile Pro Ser Pro 20 25 30
Cys Cys Met Phe Phe Val Ser Lys Arg Ile Pro Glu Asn Arg Val Val 35
40 45 Ser Tyr Gln Leu Ser Ser Arg Ser Thr Cys Leu Lys Ala Gly Val
Ile 50 55 60 Phe Thr Thr Lys Lys Gly Gln Gln Phe Cys Gly Asp Pro
Lys Gln Glu 65 70 75 80 Trp Val Gln Arg Tyr Met Lys Asn Leu Asp Ala
Lys Gln Lys Lys Ala 85 90 95 Ser Pro Arg Ala Arg Ala Val Ala Val
Lys Gly Pro Val Gln Arg Tyr 100 105 110 Pro Gly Asn Gln Thr Thr Cys
115 <210> SEQ ID NO 244 <211> LENGTH: 94 <212>
TYPE: PRT <213> ORGANISM: Human Eotaxin 3 <400>
SEQUENCE: 244 Met Met Gly Leu Ser Leu Ala Ser Ala Val Leu Leu Ala
Ser Leu Leu 1 5 10 15 Ser Leu His Leu Gly Thr Ala Thr Arg Gly Ser
Asp Ile Ser Lys Thr 20 25 30 Cys Cys Phe Gln Tyr Ser His Lys Pro
Leu Pro Trp Thr Trp Val Arg 35 40 45 Ser Tyr Glu Phe Thr Ser Asn
Ser Cys Ser Gln Arg Ala Val Ile Phe 50 55 60 Thr Thr Lys Arg Gly
Lys Lys Val Cys Thr His Pro Arg Lys Lys Trp 65 70 75 80 Val Gln Lys
Tyr Ile Ser Leu Leu Lys Thr Pro Lys Gln Leu 85 90 <210> SEQ
ID NO 245 <211> LENGTH: 97 <212> TYPE: PRT <213>
ORGANISM: Mouse Eotaxin 1 <400> SEQUENCE: 245 Met Gln Ser Ser
Thr Ala Leu Leu Phe Leu Leu Leu Thr Val Thr Ser 1 5 10 15 Phe Thr
Ser Gln Val Leu Ala His Pro Gly Ser Ile Pro Thr Ser Cys 20 25 30
Cys Phe Ile Met Thr Ser Lys Lys Ile Pro Asn Thr Leu Leu Lys Ser 35
40 45 Tyr Lys Arg Ile Thr Asn Asn Arg Cys Thr Leu Lys Ala Ile Val
Phe 50 55 60 Lys Thr Arg Leu Gly Lys Glu Ile Cys Ala Asp Pro Lys
Lys Lys Trp 65 70 75 80 Val Gln Asp Ala Thr Lys His Leu Asp Gln Lys
Leu Gln Thr Pro Lys 85 90 95 Pro <210> SEQ ID NO 246
<211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Mouse Eotaxin 2
<400> SEQUENCE: 246 Met Ala Gly Ser Ala Thr Ile Val Ala Gly
Leu Leu Leu Leu Val Ala 1 5 10 15 Cys Ala Cys Cys Ile Phe Pro Ile
Asp Ser Val Thr Ile Pro Ser Ser 20 25 30 Cys Cys Thr Ser Phe Ile
Ser Lys Lys Ile Pro Glu Asn Arg Val Val 35 40 45 Ser Tyr Gln Leu
Ala Asn Gly Ser Ile Cys Pro Lys Ala Gly Val Ile 50 55 60 Phe Ile
Thr Lys Lys Gly His Lys Ile Cys Thr Asp Pro Lys Leu Leu 65 70 75 80
Trp Val Gln Arg His Ile Gln Lys Leu Asp Ala Lys Lys Asn Gln Pro 85
90 95 Ser Lys Gly Ala Lys Ala Val Arg Thr Lys Phe Ala Val Gln Arg
Arg 100 105 110 Arg Gly Asn Ser Thr Glu Val 115 <210> SEQ ID
NO 247 <400> SEQUENCE: 247 000 3 <210> SEQ ID NO 248
<400> SEQUENCE: 248 000 3 <210> SEQ ID NO 249
<400> SEQUENCE: 249 000 3 <210> SEQ ID NO 250
<400> SEQUENCE: 250 000 3 <210> SEQ ID NO 251
<400> SEQUENCE: 251 000 3 <210> SEQ ID NO 252
<400> SEQUENCE: 252 000 3 <210> SEQ ID NO 253
<400> SEQUENCE: 253 000 3 <210> SEQ ID NO 254
<400> SEQUENCE: 254 000 3 <210> SEQ ID NO 255
<400> SEQUENCE: 255 000 3 <210> SEQ ID NO 256
<400> SEQUENCE: 256 000 3 <210> SEQ ID NO 257
<400> SEQUENCE: 257 000 3 <210> SEQ ID NO 258
<400> SEQUENCE: 258 000 3 <210> SEQ ID NO 259
<400> SEQUENCE: 259 000 3 <210> SEQ ID NO 260
<400> SEQUENCE: 260 000 3 <210> SEQ ID NO 261
<400> SEQUENCE: 261 000 3 <210> SEQ ID NO 262
<400> SEQUENCE: 262 000 3 <210> SEQ ID NO 263
<400> SEQUENCE: 263 000 3 <210> SEQ ID NO 264
<400> SEQUENCE: 264 000 3 <210> SEQ ID NO 265
<400> SEQUENCE: 265 000 3 <210> SEQ ID NO 266
<400> SEQUENCE: 266 000 3 <210> SEQ ID NO 267
<400> SEQUENCE: 267 000 3 <210> SEQ ID NO 268
<400> SEQUENCE: 268 000 3 <210> SEQ ID NO 269
<400> SEQUENCE: 269 000 3 <210> SEQ ID NO 270
<400> SEQUENCE: 270 000 3 <210> SEQ ID NO 271
<400> SEQUENCE: 271 000 3 <210> SEQ ID NO 272
<400> SEQUENCE: 272 000 3 <210> SEQ ID NO 273
<400> SEQUENCE: 273 000 3 <210> SEQ ID NO 274
<400> SEQUENCE: 274 000 3 <210> SEQ ID NO 275
<400> SEQUENCE: 275 000 3 <210> SEQ ID NO 276
<400> SEQUENCE: 276 000 3 <210> SEQ ID NO 277
<400> SEQUENCE: 277 000 3 <210> SEQ ID NO 278
<400> SEQUENCE: 278
000 3 <210> SEQ ID NO 279 <400> SEQUENCE: 279 000 3
<210> SEQ ID NO 280 <400> SEQUENCE: 280 000 3
<210> SEQ ID NO 281 <400> SEQUENCE: 281 000 3
<210> SEQ ID NO 282 <400> SEQUENCE: 282 000 3
<210> SEQ ID NO 283 <400> SEQUENCE: 283 000 3
<210> SEQ ID NO 284 <400> SEQUENCE: 284 000 3
<210> SEQ ID NO 285 <400> SEQUENCE: 285 000 3
<210> SEQ ID NO 286 <400> SEQUENCE: 286 000 3
<210> SEQ ID NO 287 <400> SEQUENCE: 287 000 3
<210> SEQ ID NO 288 <400> SEQUENCE: 288 000 3
<210> SEQ ID NO 289 <400> SEQUENCE: 289 000 3
<210> SEQ ID NO 290 <400> SEQUENCE: 290 000 3
<210> SEQ ID NO 291 <400> SEQUENCE: 291 000 3
<210> SEQ ID NO 292 <400> SEQUENCE: 292 000 3
<210> SEQ ID NO 293 <400> SEQUENCE: 293 000 3
<210> SEQ ID NO 294 <400> SEQUENCE: 294 000 3
<210> SEQ ID NO 295 <400> SEQUENCE: 295 000 3
<210> SEQ ID NO 296 <400> SEQUENCE: 296 000 3
<210> SEQ ID NO 297 <400> SEQUENCE: 297 000 3
<210> SEQ ID NO 298 <400> SEQUENCE: 298 000 3
<210> SEQ ID NO 299 <400> SEQUENCE: 299 000 3
<210> SEQ ID NO 300 <400> SEQUENCE: 300 000 3
<210> SEQ ID NO 301 <400> SEQUENCE: 301 000 3
<210> SEQ ID NO 302 <400> SEQUENCE: 302 000 3
<210> SEQ ID NO 303 <400> SEQUENCE: 303 000 3
<210> SEQ ID NO 304 <400> SEQUENCE: 304 000 3
<210> SEQ ID NO 305 <400> SEQUENCE: 305 000 3
<210> SEQ ID NO 306 <400> SEQUENCE: 306 000 3
<210> SEQ ID NO 307 <400> SEQUENCE: 307 000 3
<210> SEQ ID NO 308 <400> SEQUENCE: 308 000 3
<210> SEQ ID NO 309 <400> SEQUENCE: 309 000 3
<210> SEQ ID NO 310 <400> SEQUENCE: 310 000 3
<210> SEQ ID NO 311 <400> SEQUENCE: 311 000 3
<210> SEQ ID NO 312 <400> SEQUENCE: 312 000 3
<210> SEQ ID NO 313 <400> SEQUENCE: 313 000 3
<210> SEQ ID NO 314 <400> SEQUENCE: 314
000 3 <210> SEQ ID NO 315 <400> SEQUENCE: 315 000 3
<210> SEQ ID NO 316 <400> SEQUENCE: 316 000 3
<210> SEQ ID NO 317 <400> SEQUENCE: 317 000 3
<210> SEQ ID NO 318 <400> SEQUENCE: 318 000 3
<210> SEQ ID NO 319 <400> SEQUENCE: 319 000 3
<210> SEQ ID NO 320 <400> SEQUENCE: 320 000 3
<210> SEQ ID NO 321 <400> SEQUENCE: 321 000 3
<210> SEQ ID NO 322 <400> SEQUENCE: 322 000 3
<210> SEQ ID NO 323 <400> SEQUENCE: 323 000 3
<210> SEQ ID NO 324 <400> SEQUENCE: 324 000 3
<210> SEQ ID NO 325 <400> SEQUENCE: 325 000 3
<210> SEQ ID NO 326 <400> SEQUENCE: 326 000 3
<210> SEQ ID NO 327 <400> SEQUENCE: 327 000 3
<210> SEQ ID NO 328 <211> LENGTH: 132 <212> TYPE:
PRT <213> ORGANISM: Mouse C IL 13 F <400> SEQUENCE: 328
Ala Asp Pro Gly Cys Gly Gly Gly Gly Gly Leu Ala Gly Pro Val Pro 1 5
10 15 Arg Ser Val Ser Leu Pro Leu Thr Leu Lys Glu Leu Ile Glu Glu
Leu 20 25 30 Ser Asn Ile Thr Gln Asp Gln Thr Pro Leu Cys Asn Gly
Ser Met Val 35 40 45 Trp Ser Val Asp Leu Ala Ala Gly Gly Phe Cys
Val Ala Leu Asp Ser 50 55 60 Leu Thr Asn Ile Ser Asn Cys Asn Ala
Ile Tyr Arg Thr Gln Arg Ile 65 70 75 80 Leu His Gly Leu Cys Asn Arg
Lys Ala Pro Thr Thr Val Ser Ser Leu 85 90 95 Pro Asp Thr Lys Ile
Glu Val Ala His Phe Ile Thr Lys Leu Leu Ser 100 105 110 Tyr Thr Lys
Gln Leu Phe Arg His Gly Pro Phe Leu Glu Val Leu Ala 115 120 125 Ile
Glu Gly Arg 130 <210> SEQ ID NO 329 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Mouse C IL 13 S
<400> SEQUENCE: 329 Leu Ala Cys Gly Gly Gly Gly Gly Gly Pro
Val Pro Arg Ser Val Ser 1 5 10 15 Leu Pro Leu Thr Leu Lys Glu Leu
Ile Glu Glu Leu Ser Asn Ile Thr 20 25 30 Gln Asp Gln Thr Pro Leu
Cys Asn Gly Ser Met Val Trp Ser Val Asp 35 40 45 Leu Ala Ala Gly
Gly Phe Cys Val Ala Leu Asp Ser Leu Thr Asn Ile 50 55 60 Ser Asn
Cys Asn Ala Ile Tyr Arg Thr Gln Arg Ile Leu His Gly Leu 65 70 75 80
Cys Asn Arg Lys Ala Pro Thr Thr Val Ser Ser Leu Pro Asp Thr Lys 85
90 95 Ile Glu Val Ala His Phe Ile Thr Lys Leu Leu Ser Tyr Thr Lys
Gln 100 105 110 Leu Phe Arg His Gly Pro Phe 115 <210> SEQ ID
NO 330 <211> LENGTH: 133 <212> TYPE: PRT <213>
ORGANISM: Human C IL 13 F <400> SEQUENCE: 330 Ala Asp Pro Gly
Cys Gly Gly Gly Gly Gly Leu Ala Gly Pro Val Pro 1 5 10 15 Pro Ser
Thr Ala Leu Arg Glu Leu Ile Glu Glu Leu Val Asn Ile Thr 20 25 30
Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser Met Val Trp Ser Ile 35
40 45 Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu Glu Ser Leu Ile
Asn 50 55 60 Val Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg Met
Leu Ser Gly 65 70 75 80 Phe Cys Pro His Lys Val Ser Ala Gly Gln Phe
Ser Ser Leu His Val 85 90 95 Arg Asp Thr Lys Ile Glu Val Ala Gln
Phe Val Lys Asp Leu Leu Leu 100 105 110 His Leu Lys Lys Leu Phe Arg
Glu Gly Arg Phe Asn Leu Glu Val Leu 115 120 125 Ala Ile Glu Gly Arg
130 <210> SEQ ID NO 331 <211> LENGTH: 120 <212>
TYPE: PRT <213> ORGANISM: Human C IL 13 S <400>
SEQUENCE: 331 Leu Ala Cys Gly Gly Gly Gly Gly Gly Pro Val Pro Pro
Ser Thr Ala 1 5 10 15 Leu Arg Glu Leu Ile Glu Glu Leu Val Asn Ile
Thr Gln Asn Gln Lys 20 25 30 Ala Pro Leu Cys Asn Gly Ser Met Val
Trp Ser Ile Asn Leu Thr Ala 35 40 45 Gly Met Tyr Cys Ala Ala Leu
Glu Ser Leu Ile Asn Val Ser Gly Cys 50 55 60 Ser Ala Ile Glu Lys
Thr Gln Arg Met Leu Ser Gly Phe Cys Pro His 65 70 75 80 Lys Val Ser
Ala Gly Gln Phe Ser Ser Leu His Val Arg Asp Thr Lys 85 90 95 Ile
Glu Val Ala Gln Phe Val Lys Asp Leu Leu Leu His Leu Lys Lys 100 105
110 Leu Phe Arg Glu Gly Arg Phe Asn 115 120 <210> SEQ ID NO
332 <211> LENGTH: 136 <212> TYPE: PRT <213>
ORGANISM: Mouse C IL 5 E <400> SEQUENCE: 332 Ala Leu Val Gly
Cys Gly Gly Pro Lys Pro Ser Thr Pro Pro Gly Ser 1 5 10 15 Ser Gly
Gly Ala Pro Ala Ser Met Glu Ile Pro Met Ser Thr Val Val 20 25 30
Lys Glu Thr Leu Thr Gln Leu Ser Ala His Arg Ala Leu Leu Thr Ser 35
40 45 Asn Glu Thr Met Arg Leu Pro Val Pro Thr His Lys Asn His Gln
Leu 50 55 60 Cys Ile Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys
Asn Gln Thr 65 70 75 80
Val Arg Gly Gly Thr Val Glu Met Leu Phe Gln Asn Leu Ser Leu Ile 85
90 95 Lys Lys Tyr Ile Asp Arg Gln Lys Glu Lys Cys Gly Glu Glu Arg
Arg 100 105 110 Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu
Gly Val Met 115 120 125 Ser Thr Glu Trp Ala Met Glu Gly 130 135
<210> SEQ ID NO 333 <211> LENGTH: 134 <212> TYPE:
PRT <213> ORGANISM: Mouse C IL 5 F <400> SEQUENCE: 333
Ala Asp Pro Gly Cys Gly Gly Gly Gly Gly Leu Ala Met Glu Ile Pro 1 5
10 15 Met Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu Ser Ala His
Arg 20 25 30 Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro Val
Pro Thr His 35 40 45 Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe
Gln Gly Leu Asp Ile 50 55 60 Leu Lys Asn Gln Thr Val Arg Gly Gly
Thr Val Glu Met Leu Phe Gln 65 70 75 80 Asn Leu Ser Leu Ile Lys Lys
Tyr Ile Asp Arg Gln Lys Glu Lys Cys 85 90 95 Gly Glu Glu Arg Arg
Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu 100 105 110 Phe Leu Gly
Val Met Ser Thr Glu Trp Ala Met Glu Gly Leu Glu Val 115 120 125 Leu
Ala Ile Glu Gly Arg 130 <210> SEQ ID NO 334 <211>
LENGTH: 121 <212> TYPE: PRT <213> ORGANISM: Mouse C IL
5 S <400> SEQUENCE: 334 Leu Ala Cys Gly Gly Gly Gly Gly Met
Glu Ile Pro Met Ser Thr Val 1 5 10 15 Val Lys Glu Thr Leu Thr Gln
Leu Ser Ala His Arg Ala Leu Leu Thr 20 25 30 Ser Asn Glu Thr Met
Arg Leu Pro Val Pro Thr His Lys Asn His Gln 35 40 45 Leu Cys Ile
Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys Asn Gln 50 55 60 Thr
Val Arg Gly Gly Thr Val Glu Met Leu Phe Gln Asn Leu Ser Leu 65 70
75 80 Ile Lys Lys Tyr Ile Asp Arg Gln Lys Glu Lys Cys Gly Glu Glu
Arg 85 90 95 Arg Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe
Leu Gly Val 100 105 110 Met Ser Thr Glu Trp Ala Met Glu Gly 115 120
<210> SEQ ID NO 335 <211> LENGTH: 138 <212> TYPE:
PRT <213> ORGANISM: Human C IL 5 E <400> SEQUENCE: 335
Ala Leu Val Gly Cys Gly Gly Pro Lys Pro Ser Thr Pro Pro Gly Ser 1 5
10 15 Ser Gly Gly Ala Pro Ala Ser Ile Pro Thr Glu Ile Pro Thr Ser
Ala 20 25 30 Leu Val Lys Glu Thr Leu Ala Leu Leu Ser Thr His Arg
Thr Leu Leu 35 40 45 Ile Ala Asn Glu Thr Leu Arg Ile Pro Val Pro
Val His Lys Asn His 50 55 60 Gln Leu Cys Thr Glu Glu Ile Phe Gln
Gly Ile Gly Thr Leu Glu Ser 65 70 75 80 Gln Thr Val Gln Gly Gly Thr
Val Glu Arg Leu Phe Lys Asn Leu Ser 85 90 95 Leu Ile Lys Lys Tyr
Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu Glu 100 105 110 Arg Arg Arg
Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 115 120 125 Val
Met Asn Thr Glu Trp Ile Ile Glu Ser 130 135 <210> SEQ ID NO
336 <211> LENGTH: 136 <212> TYPE: PRT <213>
ORGANISM: Human C IL 5 F <400> SEQUENCE: 336 Ala Asp Pro Gly
Cys Gly Gly Gly Gly Gly Leu Ala Ile Pro Thr Glu 1 5 10 15 Ile Pro
Thr Ser Ala Leu Val Lys Glu Thr Leu Ala Leu Leu Ser Thr 20 25 30
His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg Ile Pro Val Pro 35
40 45 Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile Phe Gln Gly
Ile 50 55 60 Gly Thr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr Val
Glu Arg Leu 65 70 75 80 Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr Ile
Asp Gly Gln Lys Lys 85 90 95 Lys Cys Gly Glu Glu Arg Arg Arg Val
Asn Gln Phe Leu Asp Tyr Leu 100 105 110 Gln Glu Phe Leu Gly Val Met
Asn Thr Glu Trp Ile Ile Glu Ser Leu 115 120 125 Glu Val Leu Ala Ile
Glu Gly Arg 130 135 <210> SEQ ID NO 337 <211> LENGTH:
123 <212> TYPE: PRT <213> ORGANISM: Human C IL 5 S
<400> SEQUENCE: 337 Leu Ala Cys Gly Gly Gly Gly Gly Ile Pro
Thr Glu Ile Pro Thr Ser 1 5 10 15 Ala Leu Val Lys Glu Thr Leu Ala
Leu Leu Ser Thr His Arg Thr Leu 20 25 30 Leu Ile Ala Asn Glu Thr
Leu Arg Ile Pro Val Pro Val His Lys Asn 35 40 45 His Gln Leu Cys
Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu Glu 50 55 60 Ser Gln
Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn Leu 65 70 75 80
Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly Glu 85
90 95 Glu Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe
Leu 100 105 110 Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120
<210> SEQ ID NO 338 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic primer NheIL13 F
<400> SEQUENCE: 338 ctagctagcc gggccggtgc caagatc 27
<210> SEQ ID NO 339 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic primer NheIL13 R
<400> SEQUENCE: 339 tttctcgagg aaggggccgt ggcgaa 26
<210> SEQ ID NO 340 <211> LENGTH: 55 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic primer Spelinker3 F1
<400> SEQUENCE: 340 ccccgccggg ttcttctggc ggtgctccgg
ctagcatgga gattcccatg agcac 55 <210> SEQ ID NO 341
<211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic primer SpeNlinker3 F2 <400> SEQUENCE:
341 ttttactagt tggttgcggc ggcccgaaac cgagcacccc gccgggttct tc 52
<210> SEQ ID NO 342 <211> LENGTH: 49 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic primer IL5StopXho R
<400> SEQUENCE: 342 ttttgcggcc gcgtttaaac tcgagttatt
agccttccat tgcccactc 49 <210> SEQ ID NO 343 <211>
LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Shine Delagarno sequence of vector pQb185
<400> SEQUENCE: 343 tctagattaa cccaacgcgt aggagtcagg ccatg 35
<210> SEQ ID NO 344 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Amino acid linker
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: Glycine can be
repeated from zero to five times <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3) <223>
OTHER INFORMATION: Glycine can be repeated from zero to ten times
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Serine can be
repeated from zero to two times <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(9) <223>
OTHER INFORMATION: These amino acids can be repeated from zero to
three times as a group <400> SEQUENCE: 344 Gly Cys Gly Ser
Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 345 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Amino Acid Linker <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: Glycine can be repeated from zero to ten times
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Serine can be
repeated from zero to two times <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(7) <223>
OTHER INFORMATION: These amino acids can be repeated from zero to
three times as a group <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (8)..(8) <223> OTHER
INFORMATION: Glycine can be repeated from zero to eight times
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (10)..(10) <223> OTHER INFORMATION: Glycine can be
repeated from zero to five times <400> SEQUENCE: 345 Gly Ser
Gly Gly Gly Gly Ser Gly Cys Gly 1 5 10 <210> SEQ ID NO 346
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Glycine serine linkers <400> SEQUENCE:
346 Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 347 <211>
LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic N terminal gamma 1 <400> SEQUENCE: 347 Cys Gly Asp
Lys Thr His Thr Ser Pro Pro 1 5 10 <210> SEQ ID NO 348
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: C terminal gamma 1 <400> SEQUENCE: 348 Asp Lys
Thr His Thr Ser Pro Pro Cys Gly 1 5 10 <210> SEQ ID NO 349
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: N terminal gamma 3 <400> SEQUENCE: 349 Cys Gly
Gly Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser Gly Gly Ala 1 5 10 15
Pro <210> SEQ ID NO 350 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic C terminal gamma
3 <400> SEQUENCE: 350 Pro Lys Pro Ser Thr Pro Pro Gly Ser Ser
Gly Gly Ala Pro Gly Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 351
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic N terminal glycine linker <400>
SEQUENCE: 351 Gly Cys Gly Gly Gly Gly 1 5 <210> SEQ ID NO 352
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
ARtificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic C terminal glycine linker <400>
SEQUENCE: 352 Gly Gly Gly Gly Cys Gly 1 5 <210> SEQ ID NO 353
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic C terminal glycine lysine linker <400>
SEQUENCE: 353 Gly Gly Lys Lys Gly Cys 1 5 <210> SEQ ID NO 354
<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic N terminal glycine lysine linker <400>
SEQUENCE: 354 Cys Gly Lys Lys Gly Gly 1 5 <210> SEQ ID NO 355
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic C terminal linker <400> SEQUENCE: 355
Gly Gly Cys Gly 1 <210> SEQ ID NO 356 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer <400> SEQUENCE: 356 ggtaacatcg
gtcgagatgg aaaacaaact ctggtcc 37 <210> SEQ ID NO 357
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer <400> SEQUENCE:
357 ggaccagagt ttgttttcca tctcgaccga tgttacc 37 <210> SEQ ID
NO 358 <211> LENGTH: 22 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer <400>
SEQUENCE: 358 agctcgcccg gggatcctct ag 22 <210> SEQ ID NO 359
<211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer <400> SEQUENCE: 359 cgatgcattt
catccttagt tatcaatacg ctgggttcag 40 <210> SEQ ID NO 360
<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer <400> SEQUENCE:
360 ggcaaaatta gagactgtta ctttaggtaa gatcgg 36 <210> SEQ ID
NO 361 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer <400>
SEQUENCE: 361 ccgatcttac ctaaagtaac agtctctaat tttgcc 36
<210> SEQ ID NO 362 <211> LENGTH: 33 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 362 ggccatggca cgactcgaga ctgttacttt agg 33
<210> SEQ ID NO 363 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 363 gatttaggtg acactatag 19 <210> SEQ
ID NO 364 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucloetide primer <400>
SEQUENCE: 364 gatggacgtc aaactctggt cctcaatccg cgtgggg 37
<210> SEQ ID NO 365 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
<400> SEQUENCE: 365 ccccacgcgg attgaggacc agagtttgac gtccatc
37 <210> SEQ ID NO 366 <211> LENGTH: 31 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic EcoRIHBcAg(s)
<400> SEQUENCE: 366 ccggaattca tggacattga cccttataaa g 31
<210> SEQ ID NO 367 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Lys HBcAg(as) <400>
SEQUENCE: 367 cctagagcca cctttgccac catcttctaa attagtaccc
acccaggtag c 51 <210> SEQ ID NO 368 <211> LENGTH: 48
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic Lys
HBcAg(s) <400> SEQUENCE: 368 gaagatggtg gcaaaggtgg ctctagggac
ctagtagtca gttatgtc 48 <210> SEQ ID NO 369 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic HbcAg(1 149)Hind(as) <400> SEQUENCE: 369 cgcgtcccaa
gcttctaaac aacagtagtc tccggaag 38 <210> SEQ ID NO 370
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic 48as primer <400> SEQUENCE: 370
gtgcagtatg gtgaggtgag gaatgctcag gagactc 37 <210> SEQ ID NO
371 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic 48s primer <400> SEQUENCE: 371
gsgtctcctg agcattcctc acctcaccat actgcac 37 <210> SEQ ID NO
372 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic 107as primer <400> SEQUENCE: 372
cttccaaaag tgagggaaga aatgtgaaac cac 33 <210> SEQ ID NO 373
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic 107s primer <400> SEQUENCE: 373
gtggtttcac atttcttccc tcacttttgg aag 33 <210> SEQ ID NO 374
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic HBcAgwtHindIIII <400> SEQUENCE: 374
cgcgtcccaa gcttctaaca ttgagattcc cgagattg 38 <210> SEQ ID NO
375 <211> LENGTH: 51 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic CepsilonH3 foreward <400>
SEQUENCE: 375 gttaacttga cctggtctcg tgcttctggt gcatccaggg
atctagtagt c 51 <210> SEQ ID NO 376 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
CepsilonH3 foreward <400> SEQUENCE: 376 Val Asn Leu Thr Trp
Ser Arg Ala Ser Gly Ala Ser Arg Asp Leu Val 1 5 10 15 Val
<210> SEQ ID NO 377 <211> LENGTH: 51 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic CepsilonH3 reversed
<400> SEQUENCE: 377 accagaagca cgagaccagg tcaagttaac
atcttccaaa ttattaccca c 51 <210> SEQ ID NO 378 <211>
LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic CepsilonH3 reversed <400> SEQUENCE: 378 Asp Glu Leu
Asn Asn Gly Val 1 5 <210> SEQ ID NO 379 <211> LENGTH:
26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic GST
BamHI ss <400> SEQUENCE: 379
cgccggatcc tatactaggt tattgg 26 <210> SEQ ID NO 380
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic C1 BsmBI/XhoI as <400> SEQUENCE: 380
gggcgcgtct cctcgagacc gcaaccacca cca 33 <210> SEQ ID NO 381
<211> LENGTH: 74 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic MCS of pET22b(+) <400> SEQUENCE: 381
gtttaacttt aagaaggaga tatacatatg gatccggcta gcgctcgagg gtttaaacgg
60 cggccgcatg cacc 74 <210> SEQ ID NO 382 <211> LENGTH:
33 <212> TYPE: DNA <213> ORGANISM: mEotaxin F
<400> SEQUENCE: 382 ggaattccat atgcacccag gctccatccc aac 33
<210> SEQ ID NO 383 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Nhe mEotaxin F <400> SEQUENCE: 383
cctagctagc gcacccaggc tccatcccaa c 31 <210> SEQ ID NO 384
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
mEotaxin Xho R <400> SEQUENCE: 384 cccgctcgag tggttttgga
gtttggagtt 30 <210> SEQ ID NO 385 <211> LENGTH: 84
<212> TYPE: PRT <213> ORGANISM: Mouse eotaxin C1
<400> SEQUENCE: 385 Met His Pro Gly Ser Ile Pro Thr Ser Cys
Cys Phe Ile Met Thr Ser 1 5 10 15 Lys Lys Ile Pro Asn Thr Leu Leu
Lys Ser Tyr Lys Arg Ile Thr Asn 20 25 30 Asn Arg Cys Thr Leu Lys
Ala Ile Val Phe Lys Thr Arg Leu Gly Lys 35 40 45 Glu Ile Cys Ala
Asp Pro Lys Lys Lys Trp Val Gln Asp Ala Thr Lys 50 55 60 His Leu
Asp Gln Lys Leu Gln Thr Pro Lys Pro Leu Arg Gly Gly Gly 65 70 75 80
Gly Gly Cys Gly <210> SEQ ID NO 386 <211> LENGTH: 103
<212> TYPE: PRT <213> ORGANISM: Mouse His eotaxin C1
<400> SEQUENCE: 386 Met Asp Pro His His His His His His Gly
Ser Gly Asp Asp Asp Asp 1 5 10 15 Lys Ala Leu Ala His Pro Gly Ser
Ile Pro Thr Ser Cys Cys Phe Ile 20 25 30 Met Thr Ser Lys Lys Ile
Pro Asn Thr Leu Leu Lys Ser Tyr Lys Arg 35 40 45 Ile Thr Asn Asn
Arg Cys Thr Leu Lys Ala Ile Val Phe Lys Thr Arg 50 55 60 Leu Gly
Lys Glu Ile Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp 65 70 75 80
Ala Thr Lys His Leu Asp Gln Lys Leu Gln Thr Pro Lys Pro Leu Arg 85
90 95 Gly Gly Gly Gly Gly Cys Gly 100 <210> SEQ ID NO 387
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic peptide sequence designed to modify HBcAg
<400> SEQUENCE: 387 Gly Gly Lys Gly Gly 1 5
* * * * *