U.S. patent application number 12/978277 was filed with the patent office on 2011-08-25 for hepatitis virus core proteins as vaccine platforms and methods of use thereof.
This patent application is currently assigned to Vaccine Research Institute of San Diego. Invention is credited to Jean-Noel Billaud, David R. MILICH.
Application Number | 20110206724 12/978277 |
Document ID | / |
Family ID | 34119216 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110206724 |
Kind Code |
A1 |
MILICH; David R. ; et
al. |
August 25, 2011 |
HEPATITIS VIRUS CORE PROTEINS AS VACCINE PLATFORMS AND METHODS OF
USE THEREOF
Abstract
The present invention relates to hepatitis virus core proteins
and nucleic acids. In particular, the present invention provides
compositions and methods comprising recombinant hepatitis virus
core proteins or nucleic acids for use in vaccine formulations.
Inventors: |
MILICH; David R.;
(Escondido, CA) ; Billaud; Jean-Noel; (San Diego,
CA) |
Assignee: |
Vaccine Research Institute of San
Diego
San Diego
CA
|
Family ID: |
34119216 |
Appl. No.: |
12/978277 |
Filed: |
December 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10566322 |
Oct 18, 2007 |
7883843 |
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PCT/US04/23391 |
Jul 19, 2004 |
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12978277 |
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10630074 |
Jul 30, 2003 |
7144712 |
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10566322 |
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10630070 |
Jul 30, 2003 |
7320795 |
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10630074 |
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Current U.S.
Class: |
424/196.11 ;
435/243; 435/320.1; 435/325; 530/350; 536/23.72 |
Current CPC
Class: |
A61K 2039/6075 20130101;
C12N 2730/10022 20130101; Y02A 50/412 20180101; Y02A 50/30
20180101; C12N 2730/10122 20130101; A61K 39/385 20130101; C12N
2730/10023 20130101; C12N 2730/10123 20130101; C07K 14/005
20130101; A61K 39/00 20130101; A61P 43/00 20180101; A61P 37/00
20180101 |
Class at
Publication: |
424/196.11 ;
530/350; 536/23.72; 435/320.1; 435/325; 435/243 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C07K 14/02 20060101 C07K014/02; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12N 1/00 20060101 C12N001/00; A61P 37/00 20060101
A61P037/00 |
Goverment Interests
[0002] The invention was made in part with Government support by
the National Institutes of Health, Grants RO1 AI020720 and RO1
AI049730. As such, the Government has certain rights in the
invention.
Claims
1. An antigenic composition comprising a heterologous antigen
inserted within the amino acid sequence of a hepadnavirus core
antigen, wherein said heterologous antigen is 50 or fewer amino
acids in length, and wherein said heterologous antigen and said
amino acid sequence assemble as a hybrid particle.
2. The composition of claim 1, wherein said heterologous antigen is
inserted within an immunodominant loop or within an alpha-helix
adjacent to said immunodominant loop of said hepadnavirus core
antigen.
3. The composition of claim 1, wherein said heterologous antigen is
inserted at a position chosen from N-terminal or C-terminal.
4. The composition of claim 1, wherein said heterologous antigen
comprises at least one B cell epitope.
5. The composition of claim 1, wherein said heterologous antigen
comprises at least one T helper cell epitope.
6. The composition of claim 1, wherein C-terminal sequence of the
hepadnavirus core antigen is replaced by from 1 to 100 amino
acids.
7. The composition of claim 6, wherein said 1 to 100 amino acids
does not consist of cysteine or of wild type C-terminal sequence of
said hepadnavirus core antigen.
8. The composition of claim 1, wherein said hepadnavirus core
antigen is selected from the group consisting of a rodent
hepadnavirus core antigen, an avihepadnavirus core antigen, and a
primate hepadnavirus core antigen.
9. The composition of claim 8, wherein said rodent hepadnavirus
core antigen is selected from the group consisting of wherein a
woodchuck hepadnavirus core antigen, a ground squirrel hepadnavirus
core antigen, and an arctic ground squirrel hepadnavirus core
antigen.
10. The composition of claim 8, wherein said avihepadnavirus core
antigen is selected from the group consisting of a duck
hepadnavirus core antigen, a Ross' goose hepadnavirus core antigen,
a hereon hepadnavirus core antigen, a Sheldgoose hepadnavirus core
antigen, and a stork hepadnavirus core antigen.
11. The composition of claim 8, wherein said primate hepadnavirus
core antigen is selected from the group consisting of a human
hepatitis B virus core antigen and a nonhuman primate hepadnavirus
core antigen.
12. The composition of claim 11, wherein said nonhuman primate
hepadnavirus core antigen is selected from the group consisting of
a chimpanzee hepatitis B virus core antigen, a gibbon hepatitis B
virus core antigen, an orangutan hepatitis virus core antigen, and
a woolly monkey hepatitis virus core antigen.
13. The composition of claim 1, wherein said amino acid sequence
further comprises at least one immune enhancer sequence.
14. The composition of claim 1, wherein said heterologous antigen
further comprises flanking glutamic acid residues.
15. The composition of claim 1, wherein said heterologous antigen
further comprises flanking aspartic acid residues.
16. A vaccine comprising the antigenic composition of claim 1 and
an adjuvant.
17. The vaccine of claim 16, formulated for human
administration.
18. A nucleic acid sequence encoding a hybrid hepadnavirus core
antigen comprising a heterologous antigen inserted within the amino
acid sequence of a hepadnavirus core antigen, wherein said
heterologous antigen is 50 or fewer amino acids in length, and
wherein said heterologous antigen and said amino acid sequence
assemble as a hybrid particle.
19. An expression vector comprising the nucleic acid sequence of
claim 18.
20. A host cell comprising the expression vector of claim 19.
Description
[0001] This application is a continuation-in-part of, and claims
priority to, application Ser. No. 10/630,070, filed Jul. 30, 2003,
and application Ser. No. 10/630,074, filed Jul. 30, 2003.
FIELD OF THE INVENTION
[0003] The present invention relates to hepatitis virus core
proteins and nucleic acids. In particular, the present invention
provides compositions and method's comprising recombinant hepatitis
virus core proteins or nucleic acids for use in vaccine
formulations.
BACKGROUND OF THE INVENTION
[0004] The ability to map neutralizing B cell epitopes on protein
and carbohydrate antigens has created much interest in the
potential use of these hapten-like antigens in vaccine development.
There are a number of advantages to the use of peptides and small
well-defined oligosaccharides (OS) for subunit vaccine design,
including for example, chemical purity and safety, ease of
production, cost, stability, defined and targeted B and/or T cell
epitopes and mutability. The promise of the hapten-like technology
hasn't been fully realized because efficient and reproducible
methods for the delivery of these small epitopes to the immune
system are lacking. Peptidic and OS antigens often require
conjugation to an immunogenic carrier in order to provide efficient
T cell help for antibody producing B cells, as peptide antigens
often do not contain helper T (Th) cell epitopes and carbohydrate
antigens are not recognized by T cells.
[0005] The particulate human hepatitis B virus (HBV) core protein
(HBcAg) has been utilized as a carrier platform as it possesses
many of the characteristics uniquely required for the delivery of
weak immunogens to the immune system (See, Pumpens and Grens,
Intervirology, 44:98-114, 2001). Although the HBcAg is highly
immunogenic, the existing HBcAg-based platform technology has a
number of serious theoretical and practical limitations. For
example, less than 50% of foreign epitopes can be accommodated by
the HBcAg platform because of adverse effects on particle assembly
(Jegerlehner et al., Vaccine, 20:3104, 2002 and PCT/US01/25625);
use of the HBcAg compromises the use of the anti-HBc diagnostic
assay; pre-existing anti-HBc antibody is present in all HBV
chronically infected patients and in most previously infected and
recovered patients, which may limit the effectiveness of the HBcAg
as a vaccine carrier; and immune tolerance to HBcAg in individuals
chronically infected with HBV (300-400 million worldwide) limits
immunogenicity in this population. Thus, there is a profound need
in the art for particulate carrier platforms capable of delivering
a wide variety of heterologous peptide and oligosaccharide epitopes
in an immunogenic form. This need is particularly acute in the
event the vaccine recipient is chronically infected with or
suspected to be infected with HBV.
SUMMARY OF THE INVENTION
[0006] The present invention relates to hepatitis virus core
proteins and nucleic acids. In particular, the present invention
provides compositions and methods comprising recombinant hepatitis
virus core proteins or nucleic acids for use in raising antibodies
in vivo and in vitro against antigens of interest, including use in
vaccine formulations.
[0007] The invention provides a composition comprising a
heterologous antigen linked to one or more non-primate hepadnavirus
core antigen sequence that comprises a loop region. Without
limiting the particular location of the insertion, in one
embodiment, the heterologous antigen is inserted in the
hepadnavirus core antigen (e.g., Tables 7-2 and 9), such as inside
the loop region and/or outside the loop region. Without limiting
the particular type of C-terminal modification, in one embodiment,
the C-terminal sequence of the hepadnavirus core antigen sequence
is replaced by from 1 to 100 amino acids (e.g., Tables 7-2 and 9).
In an alternative embodiment, the heterologous antigen and/or the
hepadnavirus core antigen comprises one or more of 1) substitution
of an amino acid that is not an acidic amino acid with at least one
acidic amino acid, and 2) insertion of at least one acidic amino
acid compared to the wild type hepadnavirus core antigen sequence
(e.g., Tables 7-2, 17, 18). While not intending to limit the type
or source of heterologous antigen, in one embodiment, the
heterologous antigen comprises at least one B cell epitope, at
least one T cell epitope, and/or at least one CD4+ T cell epitope.
In a preferred embodiment, the CD4+ T cell epitope comprises a
sequence chosen from one or more of SEQ ID NOs:239-244 (derived
from Tetanus Toxin), SEQ ID NOs:245-250 (derived from Diphtheria
toxin), SEQ ID NOs:251-252 (derived from Plasmodium falciparum
circumsporozoite), SEQ ID NO:253 (derived from hepatitis B virus
antigen (HbsAg)), SEQ ID No:254 (derived from Influenza
hemagglutinin), SEQ ID NO:255 (derived from Influenza matrix), and
SEQ ID NO:256 (derived from measles virus fusion protein) (see FIG.
43). In one embodiment, the composition further comprises at least
one immune enhancer sequence linked to one or more of the
heterologous antigen and to the hepadnavirus core antigen sequence.
In an alternative embodiment, the composition further comprises one
or more of 1) wild type non-primate hepadnavirus core antigen, and
2) modified non-primate hepadnavirus core antigen lacking a
heterologous antigen.
[0008] In a further embodiment, the non-primate hepadnavirus core
antigen sequence is a rodent hepadnavirus core antigen sequence,
such as one or more of woodchuck hepatitis virus core antigen
(e.g., SEQ ID NO:1, 103-107), arctic ground squirrel hepatitis
virus core antigen (e.g., SEQ ID NO:102), and ground squirrel
hepatitis virus core antigen (e.g., SEQ ID NO:21 and 108). In one
embodiment, the heterologous antigen is inserted inside the loop
region. (e.g., Tables 7-2 and 9), such as amino acid residues 76,
77, 78, 81, and/or 82. In another embodiment, the heterologous
antigen is inserted at a position outside the loop region, such as
amino acid residues 44, 71, 72, 73, 74, 75, 83, 84, 85, 92,
N-terminal and/or C-terminal. In a further embodiment, the
heterologous antigen is inserted at a position inside the loop
region and in a position outside the loop region. In one
embodiment, the C-terminal sequence of the rodent hepadnavirus core
antigen sequence is replaced by from 1 to 100 amino acids (e.g.,
Tables 7-2 and 9). In a preferred embodiment, the 1 to 100 amino
acids is chosen from R, C, K, A, RRC, and SEQ ID NOs:2-20, 22-36,
42-56, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,
177, 179, 181, 183-238 (i.e., C-terminal modifications from all
rodent hepadnaviruses (Tables 1, 3-1, 3-2), all primate
hepadnaviruses (Tables 4-1, 4-2, 4-3), and all avihepadnaviruses
(Table 3-3)). More preferably, the hepadnavirus core antigen
sequence is a woodchuck hepadnavirus core antigen sequence, and the
1 to 100 amino acids does not consist of the wild type C-terminal
sequence of the woodchuck hepadnavirus core antigen (e.g., SEQ ID
NO:2).
[0009] In one embodiment, the hepadnavirus core antigen sequence is
a ground squirrel hepadnavirus core antigen sequence, and the 1 to
100 amino acids does not consist of the wild type C-terminal
sequence of the ground squirrel hepadnavirus core antigen (e.g.,
SEQ ID NO:22). In another embodiment, the hepadnavirus core antigen
sequence is arctic ground squirrel hepadnavirus core antigen
sequence, and the 1 to 100 amino acids does not consist of the wild
type C-terminal sequence of the arctic ground squirrel hepadnavirus
core antigen (e.g., SEQ ID NO:153).
[0010] In a further embodiment, the 1 to 100 amino acids is chosen
from R, C, K, A, RRC, SEQ ID NOS:2-20 (i.e., woodchuck core antigen
modification on any core antigen, Table 1), SEQ ID NOS:22-36 (i.e.,
ground squirrel core antigen modification on any core antigen,
Table 3-1), SEQ ID NOS:153, 183-196 (i.e., arctic ground squirrel
core antigen modification on any core antigen, Table 3-2), SEQ ID
NOS:42-56 (i.e., human core antigen modification on any core
antigen, Table 4-1), SEQ ID NOS:157, 159, 161, 211-224 (i.e.,
orangutan/gibbon/chimpanzee core antigen modification on any core
antigen, Table 4-3), SEQ ID NO:155, 197-210 (i.e., woolly monkey
core antigen modification on any core antigen, Table 4-2), SEQ ID
NOS:163, 165, 167, 169, 171, 173, 175, 177, 179, 181, and 230-238
(i.e., avian core antigen modification on any core antigen, Table
3-3). In an alternative embodiment, the heterologous antigen and/or
the hepadnavirus core antigen comprises one or more of 1)
substitution of an amino acid that is not an acidic amino acid with
at least one acidic amino acid, and 2) insertion of at least one
acidic amino acid compared to the wild type hepadnavirus core
antigen sequence (e.g., Tables 7-2, 17, 18). Alternatively, the
heterologous antigen comprises at least one B cell epitope, at
least one T cell epitope, and/or at least one CD4+ T cell epitope.
In a further embodiment, the composition further comprises at least
one immune enhancer sequence linked to one or more of the
heterologous antigen and to the hepadnavirus core antigen sequence.
In further embodiment, the composition further comprises one or
more of 1) wild type rodent hepadnavirus core antigen, and 2)
modified rodent hepadnavirus core antigen lacking a heterologous
antigen.
[0011] In yet another embodiment, the non-primate hepadnavirus core
antigen sequence is an avihepadnavirus core antigen sequence. In
one embodiment, the avihepadnavirus core antigen sequence is chosen
from one or more of duck avihepadnavirus core antigen sequence,
Ross' goose avihepadnavirus core antigen sequence, heron
avihepadnavirus core antigen sequence, Sheldgoose avihepadnavirus
core antigen sequence, and stork avihepadnavirus core antigen
sequence. In a further embodiment, the heterologous antigen is
inserted at a position within the loop region (e.g., Table 7-2),
such as amino acid residues 91, 92, 93, 96, and/or 97.
Alternatively, the heterologous antigen is inserted at a position
outside of the loop region, such as amino acid residues 40, 86, 87,
88, 89, 90, 98, 99, 131, 138, N-terminal and/or C-terminal. In one
embodiment the heterologous antigen is inserted at a position
within the loop region and in a position outside the loop region.
In a further embodiment, the C-terminal sequence of the
avihepadnavirus core antigen sequence is replaced by from 1 to 100
amino acids (e.g., Tables 7-2), such as R, C, K, A, RRC, and SEQ ID
NOs:2-20, 22-36, 42-56, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183-238 (Tables 1, 3-1, 3-2,
3-3, 4-1, 4-2, 4-3). In one preferred embodiment, the
avihepadnavirus core antigen sequence is a duck avihepadnavirus
core antigen sequence, and the 1 to 100 amino acids does not
consist of the wild type C-terminal sequence of the duck
avihepadnavirus core antigen. In another preferred embodiment, the
avihepadnavirus core antigen sequence is a Ross' goose
avihepadnavirus core antigen sequence, and the 1 to 100 amino acids
does not consist of the wild type C-terminal sequence of the Ross'
goose avihepadnavirus core antigen (e.g., SEQ ID NOS:175). In a
further preferred embodiment, the avihepadnavirus core antigen
sequence is a heron avihepadnavirus core antigen sequence, and the
1 to 100 amino acids does not consist of the wild type C-terminal
sequence of the heron avihepadnavirus core antigen (e.g., SEQ ID
NO:179). In yet another preferred embodiment, the avihepadnavirus
core antigen sequence is a Sheldgoose avihepadnavirus core antigen
sequence, and the 1 to 100 amino acids does not consist of the wild
type C-terminal sequence of the Sheldgoose avihepadnavirus core
antigen. (e.g., SEQ ID NO:177). In another preferred embodiment,
the avihepadnavirus core antigen sequence is a stork
avihepadnavirus core antigen sequence, and the 1 to 100 amino acids
does not consist of the wild type C-terminal sequence of the stork
avihepadnavirus core antigen. (such as SEQ ID NO:181). In a
particular embodiment, the 1 to 100 amino acids is chosen from R,
C, K, A, RRC, SEQ ID NOS:2-20 (i.e., woodchuck core antigen
modification on any core antigen, Table 1), SEQ ID NOS:22-36 (i.e.,
ground squirrel core antigen modification on any core antigen,
Table 3-1), SEQ ID NOS:153, 183-196 (i.e., arctic ground squirrel
core antigen modification on any core antigen, Table 3-2), SEQ ID
NOS:42-56 (i.e., human core antigen modification on any core
antigen, Table 4-1), SEQ ID NOS:157, 159, 161, 211-224 (i.e.,
orangutan/gibbon/chimpanzee core antigen modification on any core
antigen, Table 4-3), SEQ ID NO:155, 197-210 (i.e., woolly monkey
core antigen modification on any core antigen, Table 4-2), SEQ ID
NOS:163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 225-238
(i.e., avian core antigen modification on any core antigen, Table
3-3). In a further embodiment, the heterologous antigen and/or the
hepadnavirus core antigen comprises one or more of 1) substitution
of an amino acid that is not an acidic amino acid with at least one
acidic amino acid, and 2) insertion of at least one acidic amino
acid compared to the wild type hepadnavirus core antigen sequence
(e.g., Tables 7-2, 17, 18). In another embodiment, the heterologous
antigen comprises at least one B cell epitope, at least one T cell
epitope, and/or at least one CD4+ T cell epitope. In a further
embodiment, the composition further comprises at least one immune
enhancer sequence linked to one or more of the heterologous antigen
and to the hepadnavirus core antigen sequence. Alternatively, the
composition further comprises one or more of 1) wild type
avihepadnavirus core antigen, and 2) modified avihepadnavirus core
antigen lacking a heterologous antigen. In another embodiment, the
avihepadnavirus core antigen sequence comprises a deletion of the
loop region or of a portion thereof, such as deletion of from 1 to
40 amino acids of the loop region.
[0012] The invention further provides a composition comprising a
heterologous antigen linked to one or more primate hepadnavirus
core antigen sequence that comprises a loop region, wherein the
C-terminal sequence of the hepadnavirus core antigen sequence is
replaced by from 1 to 100 amino acids, and wherein the 1 to 100
amino acids does not consist of cysteine or of the wild type
C-terminal sequence of the hepadnavirus core antigen. In one
embodiment, the 1 to 100 amino acids is chosen from R, K, A, RRC,
and SEQ ID NOs:2-20, 22-36, 43-56, 153, 163, 165, 167, 169, 171,
173, 175, 177, 179, 181, 183-238 (Tables 1, 3-1, 3-2, 3-3, 4-1,
4-2, 4-3, 9 and 7-2). In one embodiment, the 1 to 100 amino acids
is chosen from R, C, K, A, RRC, SEQ ID NOS:2-20 (i.e., woodchuck
core antigen modification on any core antigen, Table 1), SEQ ID
NOS:22-36 (i.e., ground squirrel core antigen modification on any
core antigen, Table 3-1), SEQ ID NOS:153, 183-196 (i.e., arctic
ground squirrel core antigen modification on any core antigen,
Table 3-2), SEQ ID NOS:43-56 (i.e., human core antigen modification
on any core antigen, Table 4-1), SEQ ID NOS:211-224 (i.e.,
orangutan/gibbon/chimpanzee core antigen modification on any core
antigen, Table 4-3), SEQ ID NO:197-210 (i.e., woolly monkey core
antigen modification on any core antigen, Table 4-2), SEQ ID
NOS:163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 225-238
(i.e., avian hepadnavirus core antigen modification on any core
antigen, Table 3-3). In a further embodiment, the heterologous
antigen is inserted in the hepadnavirus core antigen inside and/or
outside the loop region. In an alternative embodiment, the
heterologous antigen and/or the hepadnavirus core antigen comprises
one or more of 1) substitution of an amino acid that is not an
acidic amino acid with at least one acidic amino acid, and 2)
insertion of at least one acidic amino acid compared to the wild
type hepadnavirus core antigen sequence (e.g., Tables 7-2, 17, 18).
In a further embodiment, the heterologous antigen comprises at
least one B cell epitope, at least one T cell epitope, and/or at
least one CD4+ T cell epitope. In another embodiment, the
composition further comprises at least one immune enhancer sequence
linked to one or more of the heterologous antigen and to the
hepadnavirus core antigen sequence. In a further embodiment, the
composition further comprises one or more of 1) wild type primate
hepadnavirus core antigen, and 2) modified primate hepadnavirus
core antigen lacking a heterologous antigen.
[0013] In a preferred embodiment, the primate hepadnavirus core
antigen sequence is a human hepatitis B virus core antigen
sequence. In a more preferred embodiment, the human hepatitis B
virus core antigen sequence is chosen from one or more of SEQ ID
NOS:41, and 109-114. Alternatively, the heterologous antigen is
inserted inside the loop region such as at amino acid residues 76,
77, 78, 81, and 82 (e.g., Tables 7-2 and 9). In another
alternative, the heterologous antigen is inserted at a position
outside the loop region, such as at amino acid residues 44, 71, 72,
73, 74, 75, 83, 84, 85, 92, N-terminal and C-terminal. In a further
embodiment, the heterologous antigen is inserted at a position
inside and outside the loop region. In a further embodiment, the
heterologous antigen and/or the hepadnavirus core antigen comprises
one or more of 1) substitution of an amino acid that is not an
acidic amino acid with at least one acidic amino acid, and 2)
insertion of at least one acidic amino acid compared to the wild
type hepadnavirus core antigen sequence (e.g., Tables 7-2, 17, 18).
In a further embodiment, the heterologous antigen comprises at
least one B cell epitope, at least one T cell epitope, and/or at
least one CD4+ T cell epitope. In preferred embodiment, the
composition further comprises at least one immune enhancer sequence
linked to one or more of the heterologous antigen and to the
hepadnavirus core antigen sequence. In yet another embodiment, the
composition further comprises one or more of 1) wild type human
hepatitis B virus core antigen, and 2) modified human hepatitis B
virus core antigen lacking a heterologous antigen.
[0014] In another preferred embodiment, the primate hepadnavirus
core antigen sequence is a non-human primate hepadnavirus core
antigen sequence, such as chimpanzee hepatitis B virus (e.g., SEQ
ID NO:115), gibbon hepatitis B virus (e.g., SEQ ID NO:116),
orangutan hepatitis virus (e.g., SEQ ID NO:117), and woolly monkey
hepatitis virus (e.g., SEQ ID NO:118). In one embodiment, the
heterologous antigen is inserted at a position within the loop
region such as amino acid residues 76, 77, 78, 81, and/or 82 (e.g.,
Tables 7-2 and 9). In another embodiment, the heterologous antigen
is inserted at a position outside of the loop region, e.g., at
amino acid residues 44, 71, 72, 73, 74, 75, 83, 84, 85, 92,
N-terminal and/or C-terminal. In yet a further embodiment, the
heterologous antigen is inserted at a position within and outside
the loop region. In another embodiment, the C-terminal sequence of
the non-human primate hepadnavirus core antigen sequence is
replaced by from 1 to 100 amino acids (e.g., Tables 7-2 and 9),
such as R, K, A, RRC, and SEQ ID NOs:2-20, 22-36, 43-56, 153, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183-238 (Tables 1,
3-1, 3-2, 3-3, 4-1, 4-2, 4-3). In a further embodiment, the 1 to
100 amino acids is chosen from R, C, K, A, RRC, SEQ ID NOS:2-20
(i.e., woodchuck core antigen modification on any core antigen,
Table 1), SEQ ID NOS:22-36 (i.e., ground squirrel core antigen
modification on any core antigen, Table 3-1), SEQ ID NOS:153,
183-196 (i.e., arctic ground squirrel core antigen modification on
any core antigen, Table 3-2), SEQ ID NOS:43-56 (i.e., human core
antigen modification on any core antigen, Table 4-1), SEQ ID
NOS:211-224 (i.e., orangutan/gibbon/chimpanzee core antigen
modification on any core antigen, Table 4-3), SEQ ID NO:197-210
(i.e., woolly monkey core antigen modification on any core antigen,
Table 4-2), SEQ ID NOS:163, 165, 167, 169, 171, 173, 175, 177, 179,
181, and 225-238 (i.e., avian core antigen modification on any core
antigen, Table 3-3). In a particular embodiment, the heterologous
antigen and/or hepadnavirus core antigen sequence comprises one or
more of 1) substitution of an amino acid that is not an acidic
amino acid with at least one acidic amino acid, and 2) insertion of
at least one acidic amino acid compared to the wild type
hepadnavirus core antigen sequence (e.g., Tables 7-2, 17, 18). In
another embodiment, the heterologous antigen comprises at least one
B cell epitope, at least one T cell epitope, and/or at least one
CD4+ T cell epitope. In a further embodiment, the composition
further comprises at least one immune enhancer sequence linked to
one or more of the heterologous antigen and to the hepadnavirus
core antigen sequence. In yet another embodiment, the composition
further comprises one or more of 1) wild type non-human primate
hepadnavirus core antigen, and 2) modified non-human primate
hepadnavirus core antigen lacking a heterologous antigen.
[0015] Also provided by the invention is a composition comprising
one or more non-primate hepadnavirus core antigen sequence that
comprises a loop region, wherein the C-terminal sequence of the
hepadnavirus core antigen sequence is replaced by from 1 to 100
amino acids (e.g., Tables 7-2 and 9).
[0016] The invention also provides a composition comprising one or
more primate hepadnavirus core antigen sequence that comprises a
loop region, wherein the C-terminal sequence of the hepadnavirus
core antigen sequence is replaced by from 1 to 100 amino acids, and
wherein the 1 to 100 amino acids does not consist of cysteine or of
the wild type C-terminal sequence of the hepadnavirus core antigen
(e.g., Tables 7-2 and 9).
[0017] Also provided herein is a method for modifying a non-primate
hepadnavirus core antigen, comprising: a) providing: i) a
non-primate hepadnavirus core antigen comprising a loop region; and
ii) antigen that is heterologous to the non-primate hepadnavirus;
and b) inserting the antigen in the non-primate hepadnavirus core
antigen (e.g., Tables 7-2 and 9). In one embodiment, the method
further comprises determining antigenicity, in vitro and/or in vivo
in an animal, of the non-primate hepadnavirus core antigen produced
by the method, wherein the mammal is chosen from mouse, non-human
primate, and human. In another embodiment, the method further
comprises expressing the modified antigen in any organism,
including plant. This may be useful in producing food based
vaccines, testing antigenicity, immunogenicity, etc. In one
embodiment, the non-primate hepadnavirus is a rodent hepadnavirus,
such as arctic ground squirrel hepatitis virus (AGSHV), ground
squirrel hepatitis virus (GSHV), and woodchuck hepatitis virus
(WHV). In one embodiment, the arctic ground squirrel hepatitis
virus (AGSHV) core antigen comprises SEQ ID NO:102 (Genbank
#NC.sub.--001719), or is encoded by a nucleic acid sequence
comprising SEQ ID NO:127 (Genbank #U29144). In another embodiment,
the ground squirrel hepatitis virus (GSHV) core antigen comprises
one or more of SEQ ID NO:21 and 108 or is encoded by a nucleic acid
sequence comprising one or more of SEQ ID NO:39 and 128 (Genbank
#NP.sub.--040993, #K02715). In a further embodiment, the woodchuck
hepatitis virus (WHV) core antigen comprises one or more of SEQ ID
NO:1, and 103-107 and/or is encoded by a nucleic acid sequence
comprising one or more of SEQ ID NOs:37 and 129-133 (Genbank
#NKVLC2, #M90520, #M18752, #M11082, #J04514, and #J02442).
[0018] In a further embodiment, the non-primate hepadnavirus is an
avihepadnavirus, such as Ross' goose hepatitis virus, heron
hepatitis virus, duck hepatitis virus, sheldgoose hepatitis virus,
and stork hepatitis virus. In one embodiment, the Ross' goose
hepatitis virus core antigen comprises SEQ ID NO:125 (Genbank
#NC.sub.--005888) and/or is encoded by a nucleic acid sequence
comprising SEQ ID NO:143 (Genbank #NC.sub.--005888). In a further
embodiment, the heron hepatitis virus core antigen comprises SEQ ID
NO:126 and/or is encoded by a nucleic acid sequence comprising SEQ
ID NO:144 (Genbank #M22056). In yet another embodiment, the duck
hepatitis virus core antigen comprises one or more of SEQ ID
NO:119-124 and/or is encoded by a nucleic acid sequence comprising
one or more of SEQ ID NOs:145-150 (Genbank #M32991, #M60677,
#M32990, #M21953, #NC.sub.--001344, #X60213). In a further
embodiment, the sheldgoose hepatitis virus core antigen comprises
SEQ ID NO:151 and/or is encoded by a nucleic acid sequence
comprising SEQ ID NO:124 (Genbank #AY494853). In another
embodiment, the stork hepatitis virus core antigen comprises SEQ ID
NO:152, and/or is encoded by a nucleic acid sequence comprising SEQ
ID NO:126 (Genbank #AJ251934). In one embodiment, the inserting of
the antigen is inside and/or outside the loop region. In a further
embodiment, the method further comprises c) replacing the
C-terminal sequence of the hepadnavirus core antigen sequence with
from 1 to 100 amino acids (e.g., Tables 7-2 and 9), wherein steps
b) and c) are carried out in any order or are concomitant. In a
further embodiment, the antigen comprises a polypeptide, and
wherein the method further comprises c) modifying one or more of
the non-primate hepadnavirus core antigen and the heterologous
antigen, by one or more of (i) inserting at least one acidic amino
acid, and (ii) substituting at least one amino acid that is not an
acidic amino acid with one or more acidic amino acid (e.g., Tables
7-2, 17, 18), wherein steps b) and c) are carried out in any order
or are concomitant. In a preferred embodiment, the modified
heterologous antigen comprises a sequence chosen from one or more
of SEQ ID no: 73, 74, 75, 77, 78, 79, 80, 81, 83, 98 (Table 17),
99, 100, and 101 (Table 18) (see also Table 7-2). In an alternative
embodiment, the method further comprises c) linking at least one
immune enhancer sequence to one or more of the heterologous antigen
and to the non-primate hepadnavirus core antigen sequence, wherein
steps b) and c) are carried out in any order or are concomitant. In
yet another embodiment, the antigen comprises one or more of SEQ ID
NOs:70-92 (Table 10). In one embodiment, the hepadnavirus is an
avihepadnavirus, and the method further comprises c) deleting at
least a portion of the loop region in the avihepadnavirus core
antigen, wherein steps b) and c) are carried out in any order or
are concomitant, and wherein the deleting comprises deleting the
loop region or a portion thereof.
[0019] The invention additionally provides a method for modifying a
primate hepadnavirus core antigen, comprising: a) providing: i) a
primate hepadnavirus core antigen comprising a loop region; and ii)
antigen that is heterologous to the primate hepadnavirus; b)
inserting the antigen in the primate hepadnavirus core antigen
(e.g., Tables 7-2 and 9); and c) replacing the C-terminal sequence
of the hepadnavirus core antigen sequence with from 1 to 100 amino
acids, wherein the 1 to 100 amino acids does not consist of
cysteine or of the wild type C-terminal sequence of the
hepadnavirus core antigen (e.g., Tables 7-2 and 9), wherein steps
b) and c) are carried out in any order or are concomitant. In one
embodiment, the method further comprises determining antigenicity
in vitro and/or in vivo in an animal of the primate hepadnavirus
core antigen produced by the method. In one embodiment, the mammal
is chosen from mouse, non-human primate, and human. In a preferred
embodiment, the primate hepadnavirus is a human hepatitis B virus,
such as one that comprises one or more of SEQ ID NOs:41, 109-114 or
that is encoded by a nucleic acid sequence comprising one or more
of SEQ ID NOs:138-142 (Genbank #X65257, #.times.02763,
#.times.01587, #J02202, #AY123041). In an alternative embodiment,
the primate hepadnavirus is a non-human primate hepadnavirus, such
as one chosen from orangutan hepatitis virus, woolly monkey
hepatitis virus, gibbon hepatitis B virus, and chimpanzee hepatitis
B virus. In one embodiment, the orangutan hepatitis virus core
antigen comprises SEQ ID NO:117 or is encoded by a nucleic acid
sequence comprising SEQ ID NO:134 (Genbank #NC.sub.--002168). In a
further embodiment, the woolly monkey hepatitis virus core antigen
comprises SEQ ID NO:118 or is encoded by a nucleic acid sequence
comprising SEQ ID NO:135 (Genbank #AF046996). In yet another
embodiment, the gibbon hepatitis B virus core antigen comprises SEQ
ID NO:116 or is encoded by a nucleic acid sequence comprising SEQ
ID NO:136 (Genbank #AY077735). In one embodiment, the chimpanzee
hepatitis B virus core antigen comprises SEQ ID NO:115 or is
encoded by a nucleic acid sequence comprising SEQ ID NO:137
(Genbank #AF222323). In a further embodiment, the inserting of the
antigen is inside and/or outside the loop region. In yet another
embodiment, the antigen comprises a polypeptide, and wherein the
method further comprises c) modifying one or more of the primate
hepadnavirus core antigen and the heterologous antigen, by one or
more of (i) inserting at least one acidic amino acid, and (ii)
substituting at least one amino acid that is not an acidic amino
acid with one or more acidic amino acid (e.g., Tables 7-2, 17, 18),
wherein steps b) and c) are carried out in any order or are
concomitant. In a further embodiment, the modified heterologous
antigen comprises a sequence chosen from one or more of SEQ ID NOs:
73, 74, 75, 77, 78, 79, 80, 81, 83, 98 (e.g., Table 17), 99, 100,
and 101 (e.g., Tables 7-2, 17, 18). In yet a further embodiment,
the method further comprises c) linking at least one immune
enhancer sequence to one or more of the heterologous antigen and to
the primate hepadnavirus core antigen sequence, wherein steps b)
and c) are carried out in any order or are concomitant. In one
embodiment, the antigen comprises one or more of SEQ ID NOs:70-92
(Table 10).
[0020] Also provided herein is a method for producing an
immunogenic composition, comprising: a) providing: i) a non-primate
hepadnavirus core antigen sequence comprising a loop region; and
ii) an antigen that is heterologous to the hepadnavirus core
antigen; b) altering at least one of the heterologous antigen and
the hepadnavirus core antigen with a modification chosen from one
or more of: i) insertion of at least one acidic amino acid; and ii)
substitution of an amino acid that is not an acidic amino acid with
at least one acidic amino acid; e) producing a modified
hepadnavirus core antigen by inserting one or more of: i) the
altered heterologous antigen of step b into the hepadnavirus core
antigen of step a; ii) the heterologous antigen of step a into the
altered hepadnavirus core antigen of step b; and iii) the altered
heterologous antigen of step b into the altered hepadnavirus core
antigen of step b; and d) expressing the modified hepadnavirus core
antigen under conditions suitable for producing hepadnavirus
particles having a diameter of 25 to 35 nm, wherein steps b) and c)
are in any order or are concomitant. In one embodiment, in the
absence of the altering, expression of the modified hepadnavirus
core antigen yields 25 fold less hepadnavirus particles than does
expression of a wild type hepadnavirus core antigen. In a further
embodiment, the at least one acidic amino acid residue comprises
one or more of at least one aspartic acid residue, and/or at least
one glutamic acid residue. Alternatively, the insertion of acidic
amino acid is in at least one position chosen from the N-terminus
and the C-terminus of the heterologous antigen. In another
alternative, the substitution with acidic amino acid comprises
replacement of at least one amino acid that is not an acidic amino
acid of the heterologous antigen with at least one acidic amino
acid residue. In yet a further embodiment, the altering produces a
modified heterologous antigen having an isoelectric point in the
range of 3.0 to 6.0.
[0021] The invention also provides a method for producing an immune
response, comprising: a) providing: i) an animal (e.g., mammalian
subject); and ii) a composition comprising one or more of: 1) a
polypeptide comprising a non-primate hepadnavirus core antigen
amino acid sequence linked to a heterologous antigen, wherein the
hepadnavirus core antigen comprises a loop region, and 2) an
expression vector encoding the polypeptide; and b) administering
the composition to the animal under conditions such that an immune
response is generated to the heterologous antigen. In one
embodiment, the immune response comprises one or more of lymphocyte
proliferative response, cytokine response and antibody response.
Preferably, the antibody response comprises production of IgG
antibodies, and more preferably the IgG antibodies comprise an
autoantibody. In another embodiment, the non-primate hepadnavirus
core antigen sequence is chosen from a rodent hepadnavirus core
antigen sequence and an avihepadnavirus core antigen sequence.
[0022] Also provided by the invention is a method for producing an
immune response, comprising: a) providing: i) an animal (e.g.,
mammalian subject); and ii) a composition comprising one or more
of: 1) a polypeptide comprising a heterologous antigen linked to
one or more primate hepadnavirus core antigen sequence that
comprises a loop region, wherein the C-terminal sequence of the
hepadnavirus core antigen sequence is replaced by from 1 to 100
amino acids, and wherein the 1 to 100 amino acids does not consist
of cysteine or of the wild type C-terminal sequence of the
hepadnavirus core antigen; and 2) an expression vector encoding the
polypeptide; and b) administering the composition to the animal
under conditions such that an immune response is generated to the
heterologous antigen. In one embodiment, the immune response
comprises one or more of lymphocyte proliferative response,
cytokine response and antibody response. Preferably, the antibody
response comprises production of IgG antibodies. More preferably,
the IgG antibodies comprise an autoantibody. In one embodiment, the
primate hepadnavirus core antigen sequence is chosen from human
hepatitis B virus core antigen sequence and a non-human primate
hepadnavirus core antigen sequence.
[0023] The invention also provides a method for producing an
immunogenic composition, comprising: a) providing: i) a primate
hepadnavirus core antigen sequence comprising a loop region; and
ii) an antigen that is heterologous to the hepadnavirus core
antigen; b) altering at least one of the heterologous antigen and
the hepadnavirus core antigen with a modification chosen from one
or more of i) insertion of at least one acidic amino acid; and ii)
substitution of an amino acid that is not an acidic amino acid with
at least one acidic amino acid; c) producing a modified
hepadnavirus core antigen by inserting one or more of: i) the
altered heterologous antigen of step b into the hepadnavirus core
antigen of step a; ii) the heterologous antigen of step a into the
altered hepadnavirus core antigen of step b; and iii) the altered
heterologous antigen of step b into the altered hepadnavirus core
antigen of step b; and d) expressing the modified hepadnavirus core
antigen under conditions suitable for producing hepadnavirus
particles having a diameter of 25 to 35 nm, wherein steps b) and c)
are in any order or are concomitant. In one embodiment, in the
absence of the altering, expression of the modified hepadnavirus
core antigen yields 25 fold less hepadnavirus particles than does
expression of a wild type hepadnavirus core antigen. In another
embodiment, the at least one acidic amino acid residue comprises
one or more of at least one aspartic acid residue, and/or at least
one glutamic acid residue. In an alternative embodiment, the
insertion of acidic amino acid is in at least one position chosen
from the N-terminus and the C-terminus of the heterologous antigen.
In yet a further embodiment, the substitution with acidic amino
acid comprises replacement of at least one amino acid that is not
an acidic amino acid of the heterologous antigen with at least one
acidic amino acid residue. In an alternative embodiment, the
altering produces a modified heterologous antigen having an
isoelectric point in the range of 3.0 to 6.0.
[0024] The invention also provides a nucleic acid sequence encoding
any one or more of the herein disclosed heterologous antigen linked
to a hepadnavirus core antigen sequence. Also provided is an
expression vector comprising the herein described nucleic acid
sequences. The invention additionally provides a cell comprising
the herein described nucleic acid sequences. The invention also
provides a vaccine comprising any one or more of the polypeptide
and/or nucleic acid sequences disclosed herein.
[0025] In particular, the present invention provides compositions
comprising a heterologous antigen linked to the amino acid sequence
set forth in SEQ ID NO:38, the amino acid sequence comprising a
loop region. In some embodiments, the heterologous antigen is
inserted at a position within the loop region. In preferred
embodiments, the position within the loop region is chosen from
amino acid residues 77, 78, 81, and 82. In another embodiment, the
position within the loop region is at amino acid residue 76. In
further embodiments, the heterologous antigen is inserted at a
position outside of the loop region. In preferred embodiments, the
position outside the loop region is chosen from amino acid residues
71, 72, 73, 74, 75, 83, 84, 85, 92, N-terminal and C-terminal. In
another embodiment, the position outside the loop region is at
amino acid residue 44. In still further embodiments, the
heterologous antigen is inserted at a position within the loop
region, and in a position outside the loop region. The present
invention also provides composition in which the heterologous
antigen is conjugated to the amino acid sequence. In preferred
embodiments, the heterologous antigen comprises at least one B cell
epitope. In further preferred embodiments, the heterologous antigen
comprises at least one T helper cell epitope. In exemplary
embodiments, the heterologous antigen is chosen from but not
limited to human immunodeficiency virus antigen, feline
immunodeficiency virus antigen, Plasmodium parasite antigen,
influenza virus antigen, Staphylococcus bacterium antigen,
cholesteryl ester transfer protein antigen, major
histocompatibility complex antigen, cytokine antigen, amyloid
.beta.-peptide antigen, peanut allergen antigen, latex allergen
hevein antigen, brown shrimp allergen antigen and major grass
pollen allergen antigen.
[0026] The present invention also provides compositions comprising
a heterologous antigen linked to the amino acid sequence set forth
in SEQ ID NO:38, the amino acid sequence comprising a loop region
and further comprising from 1 to 100 amino acids at the carboxy end
of residue I.sup.149. In some preferred embodiments, the 1 to 100
amino acids is chosen from R.sup.150, C.sup.150, K.sup.150,
A.sup.150, R.sup.150R.sup.151C.sup.152, and SEQ ID NOS:2-20. In
other preferred embodiments, the 1 to 100 amino acids is chosen
from SEQ ID NOS:22-36. In still further preferred embodiments, the
1 to 100 amino acids is chosen from SEQ ID NOS:42-56. Additionally,
in particularly preferred embodiments, the heterologous antigen
linked to the amino acid sequence set forth in SEQ ID NO:38,
comprises a particle having a diameter of 25 to 35 nm. In some
embodiments, the amino acid sequence further comprises at least one
immune enhancer sequence. The immune enhancer sequence is operably
linked to the C-terminus of the amino acid sequence in some
embodiments. In exemplary embodiments, the immune enhancer sequence
is chosen from but not limited to unmethylated CpG dinucleotides,
CD40 ligand, complement C3d fragment, B cell activating factor, and
soluble gene product of lymphocyte activation gene-3 and universal
human CD4+ T cell epitopes to which the human population has been
frequently exposed (e.g., tetanus toxoid epitopes). Also provided
are embodiments further comprising woodchuck hepatitis virus core
antigen chosen from wild type woodchuck hepatitis virus core
antigen and modified woodchuck hepatitis virus core antigen lacking
a heterologous antigen. In some embodiments, the composition
further comprises one or more compounds chosen from adjuvant,
diluent and carrier. In related embodiments, the adjuvant is chosen
from but not limited to aluminum hydroxide, aluminum phosphate,
squalene and mineral oil. Moreover, the present invention provides
a nucleic acid sequence encoding a heterologous antigen linked to
the amino acid sequence set forth in SEQ ID NO:38. Related
embodiments provide an expression vector comprising a nucleic acid
sequence encoding a heterologous antigen linked to the amino acid
sequence set forth in SEQ ID NO:38.
[0027] Additionally, the present invention provides compositions
comprising a heterologous antigen linked to an amino acid sequence
which is at least 95% identical to the sequence set forth in SEQ ID
NO:38, the amino acid sequence comprising a loop region and forming
a particle having a diameter of 25 to 35 nm. In some embodiments,
the heterologous antigen comprises at least one modification chosen
from insertion of at least one acidic amino acid residue, and
substitution of at least one acidic amino acid residue. In some
preferred embodiments, the at least one acidic amino acid residue
comprises from 1 to 10 residues of one or both of aspartic acid and
glutamic acid. In other preferred embodiments, the amino acid
sequence comprises at least one modification chosen from insertion
of at least one acidic amino acid residue, and substitution of at
least one acidic amino acid residue. In some preferred embodiments,
the at least one acidic amino acid residue comprises from 1 to 10
residues of one or both of aspartic acid and glutamic acid.
[0028] Also provided by the present invention are compositions
comprising the amino acid sequence set forth in SEQ ID NO:38, the
amino acid sequence comprising a loop region. In some embodiments,
the amino acid sequence further comprises from 1 to 100 amino acids
(excluding the wild type C-terminus set forth in SEQ ID NO:2) at
the carboxy end of residue I.sup.149. In some preferred
embodiments, the 1 to 100 amino acids is chosen from R.sup.150,
C.sup.150, K.sup.150, A.sup.150, R.sup.150R.sup.151C.sup.152, and
SEQ ID NOS:3-20. In other preferred embodiments, the 1 to 100 amino
acids is chosen from SEQ ID NOS:22-36. In still further preferred
embodiments, the 1 to 100 amino acids is chosen from SEQ ID
NOS:42-56. Additionally, in some embodiments, the amino acid
sequence further comprises at least one immune enhancer sequence.
In preferred embodiments, the immune enhancer sequence is operably
linked to the C-terminus of the amino acid sequence. In exemplary
embodiments, the immune enhancer sequence is chosen from but not
limited to unmethylated CpG dinucleotides, CD40 ligand, complement
C3d fragment, B cell activating factor, and soluble gene product of
lymphocyte activation gene-3 and universal human CD4+ T cell
epitopes to which the human population has been frequently exposed
(e.g., tetanus toxoid epitopes). Also provided is a nucleic acid
sequence encoding the amino acid sequence set forth in SEQ ID
NO:38, the amino acid sequence comprising a loop region. Related
embodiments provide an expression vector comprising the nucleic
acid sequence encoding the amino acid sequence set forth in SEQ ID
NO:38. Additionally, compositions further comprising a modified
woodchuck hepatitis virus core antigen comprising a heterologous
antigen are provided. In some particularly preferred embodiments,
compositions are provided comprising an amino acid sequence which
is at least 95% identical to SEQ ID NO:38, the amino acid sequence
comprising a loop region and forming a particle having a diameter
of 25 to 35 nm.
[0029] Importantly, the present invention provides methods,
comprising: providing: an animal (e.g., mammalian subject); and a
composition comprising one or more of a polypeptide comprising a
heterologous antigen linked to the amino acid sequence set forth in
SEQ ID NO:38, the amino acid sequence comprising a loop region, and
an expression vector encoding the polypeptide; and administering
the composition to the animal under conditions such that an immune
response is generated. In some embodiments, the immune response
comprises one or more of lymphocyte proliferative response,
cytokine response and antibody response. In some preferred
embodiments, the cytokine response comprises IL-2 production. In
further embodiments, the antibody response comprises at least three
fold higher levels of antibody than that observed before
administration of the at least one composition. In particularly
preferred embodiments, the antibody response comprises production
of IgG antibodies. In related embodiments, the IgG antibodies
comprise an autoantibody. In some preferred embodiments, the
composition further comprises one or more compounds chosen from
adjuvant, diluent and carrier. In related embodiments, the adjuvant
is chosen from but not limited to aluminum hydroxide, aluminum
phosphate, squalene and mineral oil.
[0030] Also provided by the present invention are methods for
producing an immunogenic composition, comprising: providing: a
heterologous antigen; and a hepatitis virus core antigen; altering
at least one of the heterologous antigen and the hepatitis virus
core antigen, with a modification chosen from insertion of at least
one acidic amino acid residue and substitution of at least one
amino acid residue; and inserting the heterologous antigen of step
b within the hepatitis virus core antigen of step b to produce a
modified hepatitis virus core antigen; expressing the modified
hepatitis virus core antigen under conditions suitable for
producing particles having a diameter of 25 to 35 nm. In some
embodiments, in the absence of the altering, expression of the
modified hepatitis virus core antigen yields aggregates rather than
particles. In other embodiments, in the absence of the altering,
expression of the modified hepatitis virus core antigen yields 25
fold less particles than does expression of a wild type hepatitis
virus core antigen. In some preferred embodiments, the at least one
acidic amino acid residue comprises from 1 to 10 aspartic acid
residues. In other preferred embodiments, the at least one acidic
amino acid residue comprises from 1 to 10 glutamic acid residues.
In related embodiments, the at least one acidic amino acid residue
comprises at least one aspartic acid residue and at least one
glutamic acid residue. In some embodiments, the insertion is in at
least one position chosen from the N-terminus and the C-terminus of
the heterologous antigen. In other embodiments, the substitution
comprises a replacement of at least one non-acidic amino acid
residue within the heterologous antigen, with the at least one
acidic amino acid residue. In preferred embodiments, the altering
produces a modified heterologous antigen with an isoelectric point
in the range of 2.0 to 7.0. In a subset of these embodiments, the
altering produces a modified heterologous antigen with an
isoelectric point more preferably in the range of 3.0 to 6.0, and
most preferably in the range of 4.0 to 5.0. In a preferred
embodiment, the hepatitis virus core antigen is a woodchuck
hepatitis virus core antigen.
[0031] Additionally, the present invention provides compositions
comprising a heterologous antigen linked to the amino acid sequence
set forth in SEQ ID NO:40, the amino acid sequence comprising a
loop region. In some embodiments, the heterologous antigen is
inserted at a position within the loop region. In preferred
embodiments, the position within the loop region is chosen from
amino acid residues 77, 80, and 81. In another embodiment, the
position within the loop region is at amino acid residue 76. In
further embodiments, the heterologous antigen is inserted at a
position outside of the loop region. In preferred embodiments, the
position outside the loop region is chosen from amino acid residues
71, 72, 73, 74, 75, 82, 83, 84, 91, N-terminal and C-terminal. In
another embodiment, the position outside the loop region is at
amino acid residue 44. In still further embodiments, the
heterologous antigen is inserted at a position within the loop
region, and in a position outside the loop region. The present
invention also provides composition in which the heterologous
antigen is conjugated to the amino acid sequence. In preferred
embodiments, the heterologous antigen comprises at least one B cell
epitope. In further preferred embodiments, the heterologous antigen
comprises at least one T helper cell epitope. In exemplary
embodiments, the heterologous antigen is chosen from but not
limited to human immunodeficiency virus antigen, feline
immunodeficiency virus antigen, Plasmodium parasite antigen,
influenza virus antigen, Staphylococcus bacterium antigen,
cholesteryl ester transfer protein antigen, major
histocompatibility complex antigen, cytokine antigen, amyloid
.beta.-peptide antigen, peanut allergen antigen, latex allergen
hevein antigen, brown shrimp allergen antigen and major grass
pollen allergen antigen.
[0032] The present invention also provides compositions comprising
a heterologous antigen linked to the amino acid sequence set forth
in SEQ ID NO:40, the amino acid sequence comprising a loop region
and further comprising from 1 to 100 amino acids at the carboxy end
of residue I.sup.148. In some preferred embodiments, the 1 to 100
amino acids is chosen from R.sup.149, C.sup.149, K.sup.149,
A.sup.149, R.sup.149R.sup.150C.sup.151, and SEQ ID NOS:3-6, 22-36,
153, and 183-196. In other preferred embodiments, the 1 to 100
amino acids is chosen from SEQ ID NOS:2, 7-20. In still further
preferred embodiments, the 1 to 100 amino acids is chosen from SEQ
ID NOS:42-56. Additionally, in particularly preferred embodiments,
the heterologous antigen linked to the amino acid sequence set
forth in SEQ ID NO:40, comprises a particle having a diameter of 25
to 35 nm. In some embodiments, the amino acid sequence further
comprises at least one immune enhancer sequence. The immune
enhancer sequence is operably linked to the C-terminus of the amino
acid sequence in some embodiments. In exemplary embodiments, the
immune enhancer sequence is chosen from but not limited to
unmethylated CpG dinucleotides, CD40 ligand, complement C3d
fragment, B cell activating factor, and soluble gene product of
lymphocyte activation gene-3 and universal human CD4+ T cell
epitopes to which the human population has been frequently exposed
(e.g., tetanus toxoid epitopes). Also provided are embodiments
further comprising ground squirrel hepatitis virus core antigen
chosen from wild type ground squirrel hepatitis virus core antigen
and modified ground squirrel hepatitis virus core antigen lacking a
heterologous antigen. In some embodiments, the composition further
comprises one or more compounds chosen from adjuvant, diluent and
carrier. In related embodiments, the adjuvant is chosen from but
not limited to aluminum hydroxide, aluminum phosphate, squalene and
mineral oil. Moreover, the present invention provides a nucleic
acid sequence encoding a heterologous antigen linked to the amino
acid sequence set forth in SEQ ID NO:40. Related embodiments
provide an expression vector comprising a nucleic acid sequence
encoding a heterologous antigen linked to the amino acid sequence
set forth in SEQ ID NO:40.
[0033] Additionally, the present invention provides compositions
comprising a heterologous antigen linked to an amino acid sequence
which is at least 95% identical to the sequence set forth in SEQ ID
NO:40, the amino acid sequence comprising a loop region and forming
a particle having a diameter of 25 to 35 nm. In some embodiments,
the heterologous antigen comprises at least one modification chosen
from insertion of at least one acidic amino acid residue, and
substitution of at least one acidic amino acid residue. In some
preferred embodiments, the at least one acidic amino acid residue
comprises from 1 to 10 residues of one or both of aspartic acid and
glutamic acid. In other preferred embodiments, the amino acid
sequence comprises at least one modification chosen from insertion
of at least one acidic amino acid residue, and substitution of at
least one acidic amino acid residue. In some preferred embodiments,
the at least one acidic amino acid residue comprises from 1 to 10
residues of one or both of aspartic acid and glutamic acid.
[0034] Also provided by the present invention are compositions
comprising the amino acid sequence set forth in SEQ ID NO:40, the
amino acid sequence comprising a loop region. In some embodiments,
the amino acid sequence further comprises from 1 to 100 amino acids
(excluding the wild type C-terminus set forth in SEQ ID NO:22) at
the carboxy end of residue I.sup.148. In some preferred
embodiments, the 1 to 100 amino acids is chosen from R.sup.149,
C.sup.149, K.sup.149, A.sup.149, R.sup.149R.sup.150C.sup.151, and
SEQ ID NOS:3-6, 23-36. In other preferred embodiments, the 1 to 100
amino acids is chosen from SEQ ID NOS:2-20. In still further
preferred embodiments, the 1 to 100 amino acids is chosen from SEQ
ID NOS:42-56. Additionally, in some embodiments, the amino acid
sequence further comprises at least one immune enhancer sequence.
In preferred embodiments, the immune enhancer sequence is operably
linked to the C-terminus of the amino acid sequence. In exemplary
embodiments, the immune enhancer sequence is chosen from
unmethylated CpG dinucleotides, CD40 ligand, complement C3d
fragment, B cell activating factor, and soluble gene product of
lymphocyte activation gene-3 and universal human CD4+ T cell
epitopes to which the human population has been frequently exposed
(e.g., tetanus toxoid epitopes). Also provided is a nucleic acid
sequence encoding the amino acid sequence set forth in SEQ ID
NO:40, the amino acid sequence comprising a loop region. Related
embodiments provide an expression vector comprising the nucleic
acid sequence encoding the amino acid sequence set forth in SEQ ID
NO:40. Additionally, compositions further comprising a modified
ground squirrel hepatitis virus core antigen comprising a
heterologous antigen are provided. In some particularly preferred
embodiments, compositions are provided comprising an amino acid
sequence which is at least 95% identical to SEQ ID NO:40, the amino
acid sequence comprising a loop region and forming a particle
having a diameter of 25 to 35 nm.
[0035] Importantly, the present invention provides methods,
comprising: providing: an animal (e.g., mammalian subject); and a
composition comprising one or more of a polypeptide comprising a
heterologous antigen linked to the amino acid sequence set forth in
SEQ ID NO:40, the amino acid sequence comprising a loop region, and
an expression vector encoding the polypeptide; and administering
the composition to the animal under conditions such that an immune
response is generated. In some embodiments, the immune response
comprises one or more of lymphocyte proliferative response,
cytokine response and antibody response. In some preferred
embodiments, the cytokine response comprises IL-2 production. In
further embodiments, the antibody response comprises at least three
fold higher levels of antibody than that observed before
administration of the at least one composition. In particularly
preferred embodiments, the antibody response comprises production
of IgG antibodies. In related embodiments, the IgG antibodies
comprise an autoantibody. In some preferred embodiments, the
composition further comprises one or more compounds chosen from
adjuvant, diluent and carrier. In related embodiments, the adjuvant
is chosen from but not limited to aluminum hydroxide, aluminum
phosphate, squalene and mineral oil.
[0036] Also provided by the present invention are methods for
producing an immunogenic composition, comprising: providing: a
heterologous antigen; and a hepatitis virus core antigen; altering
at least one of the heterologous antigen and the hepatitis virus
core antigen, with a modification chosen from insertion of at least
one acidic amino acid residue and substitution of at least one
acidic amino acid residue; and inserting the heterologous antigen
of step b within the hepatitis virus core antigen of step b to
produce a modified hepatitis virus core antigen; expressing the
modified hepatitis virus core antigen under conditions suitable for
producing particles having a diameter of 25 to 35 nm. In some
embodiments, in the absence of the altering, expression of the
modified hepatitis virus core antigen yields aggregates rather than
particles. In other embodiments, in the absence of the altering,
expression of the modified hepatitis virus core antigen yields 25
fold less particles than does expression of a wild type hepatitis
virus core antigen. In some preferred embodiments, the at least one
acidic amino acid residue comprises from 1 to 10 aspartic acid
residues. In other preferred embodiments, the at least one acidic
amino acid residue comprises from 1 to 10 glutamic acid residues.
In related embodiments, the at least one acidic amino acid residue
comprises at least one aspartic acid residue and at least one
glutamic acid residue. In some embodiments, the insertion is in at
least one position chosen from the N-terminus and the C-terminus of
the heterologous antigen. In other embodiments, the substitution
comprises a replacement of at least one non-acidic amino acid
residue within the heterologous antigen, with the at least one
acidic amino acid residue. In preferred embodiments, the altering
produces a modified heterologous antigen with an isoelectric point
in the range of 2.0 to 7.0. In a subset of these embodiments, the
altering produces a modified heterologous antigen with an
isoelectric point more preferably in the range of 3.0 to 6.0, and
most preferably in the range of 4.0 to 5.0. In a preferred
embodiment, the hepatitis virus core antigen is a ground squirrel
hepatitis virus core antigen.
[0037] Additionally, the present invention provides compositions
comprising a heterologous antigen linked to the amino acid sequence
set forth in SEQ ID NO:58, the amino acid sequence comprising a
loop region and further comprising from 1 to 100 amino acids at the
carboxy end of residue V.sup.149, and wherein the 1 to 100 amino
acids does not comprise C.sup.150 or the sequence set forth in SEQ
ID NO:42 (i.e., excluding C.sup.150, and the wild type C-terminus).
In some preferred embodiments, the 1 to 100 amino acids is chosen
from R.sup.150, K.sup.150, A.sup.150, R.sup.150R.sup.151C.sup.152,
and SEQ ID NOS:3-6, 43-56. In other preferred embodiments, the 1 to
100 amino acids is chosen from SEQ ID NOS:2, 7-20. In still further
preferred embodiments, the 1 to 100 amino acids is chosen from SEQ
ID NOS:22-36. Additionally, in particularly preferred embodiments,
the heterologous antigen linked to the amino acid sequence set
forth in SEQ ID NO:58, comprises a particle having a diameter of 25
to 35 nm.
[0038] In some embodiments, the heterologous antigen is inserted at
a position within the loop region. In preferred embodiments, the
position within the loop region is chosen from amino acid residues
77, 78, 81, and 82. In another embodiment, the position within the
loop region is at amino acid residue 76. In further embodiments,
the heterologous antigen is inserted at a position outside of the
loop region. In preferred embodiments, the position outside the
loop region is chosen from amino acid residues 71, 72, 73, 74, 75,
83, 84, 85, 92, N-terminal and C-terminal. In another embodiment,
the position outside the loop region is at amino acid residue 44.
In still further embodiments, the heterologous antigen is inserted
at a position within the loop region, and in a position outside the
loop region. The present invention also provides composition in
which the heterologous antigen is conjugated to the amino acid
sequence. In preferred embodiments, the heterologous antigen
comprises at least one B cell epitope. In further preferred
embodiments, the heterologous antigen comprises at least one T
helper cell epitope. In exemplary embodiments, the heterologous
antigen is chosen from but not limited to human immunodeficiency
virus antigen, feline immunodeficiency virus antigen, Plasmodium
parasite antigen, influenza virus antigen, Staphylococcus bacterium
antigen, cholesteryl ester transfer protein antigen, major
histocompatibility complex antigen, cytokine antigen, amyloid
.beta.-peptide antigen, peanut allergen antigen, latex allergen
hevein antigen, brown shrimp allergen antigen and major grass
pollen allergen antigen.
[0039] In some embodiments, the amino acid sequence further
comprises at least one immune enhancer sequence. The immune
enhancer sequence is operably linked to the C-terminus of the amino
acid sequence in some embodiments. In exemplary embodiments, the
immune enhancer sequence is chosen from but not limited to
unmethylated CpG dinucleotides, CD40 ligand, complement C3d
fragment, B cell activating factor, and soluble gene product of
lymphocyte activation gene-3 and universal human CD4+ T cell
epitopes to which the human population has been frequently exposed
(i.e., tetanus toxoid epitopes). Also provided are embodiments
further comprising human hepatitis B virus core antigen chosen from
wild type human hepatitis B virus core antigen and modified human
hepatitis B virus core antigen lacking a heterologous antigen. In
some embodiments, the composition further comprises one or more
compounds chosen from adjuvant, diluent and carrier. In related
embodiments, the adjuvant is chosen from but not limited to
aluminum hydroxide, aluminum phosphate, squalene and mineral oil.
Moreover, the present invention provides a nucleic acid sequence
encoding a heterologous antigen linked to the amino acid sequence
set forth in SEQ ID NO:58. Related embodiments provide an
expression vector comprising a nucleic acid sequence encoding a
heterologous antigen linked to the amino acid sequence set forth in
SEQ ID NO:58.
[0040] Additionally, the present invention provides compositions
comprising a heterologous antigen linked to an amino acid sequence
which is at least 95% identical to the sequence set forth in SEQ ID
NO:58, the amino acid sequence comprising a loop region and forming
a particle having a diameter of 25 to 35 nm. In some embodiments,
the heterologous antigen comprises at least one modification chosen
from insertion of at least one acidic amino acid residue, and
substitution of at least one acidic amino acid residue. In some
preferred embodiments, the at least one acidic amino acid residue
comprises from 1 to 10 residues of one or both of aspartic acid and
glutamic acid. In other preferred embodiments, the amino acid
sequence comprises at least one modification chosen from insertion
of at least one acidic amino acid residue, and substitution of at
least one acidic amino acid residue. In some preferred embodiments,
the at least one acidic amino acid residue comprises from 1 to 10
residues of one or both of aspartic acid and glutamic acid.
[0041] Also provided by the present invention are compositions
comprising the amino acid sequence set forth in SEQ ID NO:58, the
amino acid sequence comprising a loop region and further comprising
from 1 to 100 amino acids at the carboxy end of residue V.sup.149.
In some preferred embodiments, the 1 to 100 amino acids is chosen
from R.sup.150K.sup.150A.sup.150, R.sup.150R.sup.151C.sup.152, and
SEQ ID NOS:3-6, 43-56 (excluding C.sup.150, and the wild type
C-terminus set forth in SEQ ID NO:42). In other preferred
embodiments, the 1 to 100 amino acids is chosen from SEQ ID NOS:2,
7-20. In still further preferred embodiments, the 1 to 100 amino
acids is chosen from SEQ ID NOS:22-36. Additionally, in
particularly preferred embodiments, the heterologous antigen linked
to the amino acid sequence set forth in SEQ ID NO:58, comprises a
particle having a diameter of 25 to 35 nm.
[0042] Additionally, in some embodiments, the amino acid sequence
further comprises at least one immune enhancer sequence. In
preferred embodiments, the immune enhancer sequence is operably
linked to the C-terminus of the amino acid sequence. In exemplary
embodiments, the immune enhancer sequence is chosen from but not
limited to unmethylated CpG dinucleotides, CD40 ligand, complement
C3d fragment, B cell activating factor, and soluble gene product of
lymphocyte activation gene-3 and universal human CD4+ T cell
epitopes to which the human population has been frequently exposed
(i.e., tetanus toxoid epitopes). Also provided is a nucleic acid
sequence encoding the amino acid sequence set forth in SEQ ID NO:
58, the amino acid sequence comprising a loop region. Related
embodiments provide an expression vector comprising the nucleic
acid sequence encoding the amino acid sequence set forth in SEQ ID
NO:58. Additionally, compositions further comprising a modified
human hepatitis B virus core antigen comprising a heterologous
antigen are provided. In some particularly preferred embodiments,
compositions are provided comprising an amino acid sequence which
is at least 95% identical to SEQ ID NO:58, the amino acid sequence
comprising a loop region and forming a particle having a diameter
of 25 to 35 nm.
[0043] Importantly, the present invention provides methods,
comprising: providing: an animal (e.g., mammalian subject); and a
composition comprising one or more of a polypeptide comprising a
heterologous antigen linked to the amino acid sequence set forth in
SEQ ID NO:58, the amino acid sequence comprising a loop region, and
an expression vector encoding the polypeptide; and administering
the composition to the animal under conditions such that an immune
response is generated. In some embodiments, the immune response
comprises one or more of lymphocyte proliferative response,
cytokine response and antibody response. In some preferred
embodiments, the cytokine response comprises IL-2 production. In
further embodiments, the antibody response comprises at least three
fold higher levels of antibody than that observed before
administration of the at least one composition. In particularly
preferred embodiments, the antibody response comprises production
of IgG antibodies. In related embodiments, the IgG antibodies
comprise an autoantibody. In some preferred embodiments, the
composition further comprises one or more compounds chosen from
adjuvant, diluent and carrier. In related embodiments, the adjuvant
is chosen from but not limited to aluminum hydroxide, aluminum
phosphate, squalene and mineral oil.
[0044] Also provided by the present invention are methods for
producing an immunogenic composition, comprising: providing: a
heterologous antigen; and a hepatitis virus core antigen; altering
at least one of the heterologous antigen and the hepatitis virus
core antigen, with a modification chosen from insertion of at least
one acidic amino acid residue and substitution of at least one
acidic amino acid residue; and inserting the heterologous antigen
of step b within the hepatitis virus core antigen of step b to
produce a modified hepatitis virus core antigen; expressing the
modified hepatitis virus core antigen under conditions suitable for
producing particles having a diameter of 25 to 35 nm. In some
embodiments, in the absence of the altering, expression of the
modified hepatitis virus core antigen yields aggregates rather than
particles. In other embodiments, in the absence of the altering,
expression of the modified hepatitis virus core antigen yields 25
fold less particles than does expression of a wild type hepatitis
virus core antigen. In some preferred embodiments, the at least one
acidic amino acid residue comprises from 1 to 10 aspartic acid
residues. In other preferred embodiments, the at least one acidic
amino acid residue comprises from 1 to 10 glutamic acid residues.
In related embodiments, the at least one acidic amino acid residue
comprises at least one aspartic acid residue and at least one
glutamic acid residue. In some embodiments, the insertion is in at
least one position chosen from the N-terminus and the C-terminus of
the heterologous antigen. In other embodiments, the substitution
comprises a replacement of at least one non-acidic amino acid
residue within the heterologous antigen, with the at least one
acidic amino acid residue. In preferred embodiments, the altering
produces a modified heterologous antigen with an isoelectric point
in the range of 2.0 to 7.0. In a subset of these embodiments, the
altering produces a modified heterologous antigen with an
isoelectric point more preferably in the range of 3.0 to 6.0, and
most preferably in the range of 4.0 to 5.0. In a particularly
preferred embodiment, the hepatitis virus core antigen is a human
hepatitis B virus core antigen.
DESCRIPTION OF THE FIGURES
[0045] FIG. 1 depicts the structure of the HBc particle determined
at 7.4 angstrom resolution from cryoelectron micrographs. The
immunodominant loop from amino acid residues 76 to 82 is shown.
[0046] FIG. 2 provides a graph showing the comparative
immunogenicity of HBcAg-based (HBc-M) and WHcAg-based (WHc-M)
(NANP).sub.n vaccines. Groups of three mice were immunized with a
single dose of 20 .mu.g of the indicated particles in IFA and sera
were collected at the indicated time points for determination of
anti-NANP titer by ELISA.
[0047] FIG. 3 illustrates that the WHcAg accommodates insertion of
foreign epitopes at many positions, with insertion sites indicated
as numbered symbols. Tolerant (+) insertions sites are shown with
an oval, intermediate (+/-) insertion sites are shown with a
triangle, and non-tolerant (-) insertion sites are shown with a
square.
[0048] FIG. 4 provides an image of a 1% agarose gel indicating that
wild type HBcAg and WHcAg particles migrate differently under
non-denaturing conditions.
[0049] FIG. 5 graphically depicts the magnitude of the antibody
response elicited by immunizing mice of the indicated H-2 congenic
strains with 7.0 .mu.g of either WHcAg or HBcAg or GSHcAg in IFA.
Six weeks post immunization (1.degree., 6 wk), sera were collected
and analyzed for anti-WHc (solid) or anti-HBc (hatched) or
anti-GSHc (dots) antibody by ELISA. End-point serum titers are
shown.
[0050] FIG. 6 illustrates that there is a low level of
cross-reactivity between the WHcAg/GSHcAg and the HBcAg and a high
level of cross-reactivity between the WHcAg and the GSHcAg at the
antibody level. The indicated panel of H-2 congenic strains was
immunized with 7.0 .mu.g of either WHcAg (solid) or HBcAg (hatched)
or GSHcAg (dots) in IFA. Six weeks later sera were collected and
tested for binding to WHcAg, GSHcAg and HBcAg. End-point serum
titers are shown.
[0051] FIG. 7 illustrates that there is a low level of
cross-reactivity between the WHcAg and the HBcAg at the T cell
level. Balb/c mice were immunized with WHcAg (5.0 .mu.g) in
complete Freund's adjuvant. Ten days later draining lymph node (LN)
cells were harvested and cultured with varying concentrations of
WHcAg and HBcAg in vitro. T cell proliferation was measured by
[.sup.3H]TdR uptake and corrected for background.
[0052] FIG. 8 shows the proliferative response of cells from mice
of the indicated strains and H-2 haplotypes after immunization with
WHcAg (5.0 .mu.g) in CFA. Ten days later draining LN cells were
cultured with WHcAg, HBcAg and the indicated peptides in vitro, and
T cell proliferation was measured by [.sup.3H]TdR uptake. Panel A
shows the proliferative response of B10.S mice to WHc, HBc and the
Wp120-131 (VSFGVWIRTPAP, set forth as SEQ ID NO:59; while the
corresponding HBV sequence is VSFGVWIRTPPA, set forth as SEQ ID
NO:60). Panel B shows the proliferative response of cells of B10.D2
mice to WHc, HBc, and the Wp60-80 peptide (VCWDELTKLIAWMSSNITSEQ,
set forth as SEQ ID NO:61; while the corresponding HBV sequence is
LCWGELMTLATWVGGNLEDPI, set forth as SEQ ID NO:62). Panel C shows
the proliferative response of cells from B10.M mice to WHc, HBc,
and the Wp60-80 peptide. The peptide T cell site recognized after
HBcAg immunization of B10.S mice is Hp120-131, of B10.D2 mice is
Hp85-100, and of B10.M mice is Hp100-120.
[0053] FIG. 9 provides the results of a CD4+ T cell epitope mapping
analysis in the indicated strains of mice. Briefly, mice were
immunized (7.0 .mu.g) and boosted (3.5 .mu.g) with either WHcAg or
HBcAg in IFA, and ten days later spleen cells were cultured with
WHcAg, HBcAg and a panel of WHcAg-derived or HBcAg-derived peptides
in vitro. After 4 days, tissue culture supernatants (SN) were
collected and IFN.gamma. (.mu.g/ml) was measured by ELISA. The
relative strengths of the peptide T cell sites are indicated by the
minimum peptide concentrations required to recall IFN.gamma.
production in vitro.
[0054] FIG. 10 provides the results of a CD4+ T cell epitope
mapping analysis in the indicated strains of mice. Briefly, mice
were immunized (7.0 .mu.g) and boosted (3.5 .mu.g) with either
WHcAg or HBcAg in IFA, and ten days later spleen cells were
cultured with WHcAg, HBcAg and a panel of WHcAg-derived or
HBcAg-derived peptides in vitro. After 4 days, tissue culture
supernatants (SN) were collected and IFN.gamma. (.mu.g/ml) was
measured by ELISA. The relative strengths of the peptide T cell
sites are indicated by the minimum peptide concentrations required
to recall IFN.gamma. production in vitro.
[0055] FIG. 11 illustrates that anti-WHc antibodies do not
interfere with the commercial anti-HBc diagnostic assay. The
commercial anti-HBc assay was performed as recommended by the
supplier with the exception that 1:10 dilutions of murine sera were
used.
[0056] FIG. 12, panels A-E, illustrate that WHc is an effective
immunogen in both wild type and transgenic mice. Wild type (+/+)
and HBeAg-expressing transgenic (HBe-Tg) mice were immunized with
20 .mu.g of either HBcAg, an HBc-based malaria vaccine candidate
(HBc-M, V12.PF3.1) or WHc-based malaria vaccine candidate (WHc-M,
HyW-M78) in IFA. Sera were collected at 2, 4 and 8 weeks after
immunization and analyzed for anti-core antibody (left panels A, B,
and C) and anti-NANP antibody (right panels D and E) by ELISA.
[0057] FIG. 13, panels A-F, depict the recall IFN.gamma.-response
of wild type and HBe-Tg mice after immunizing with 10 .mu.g of
HBcAg (panels A and D), HBc-M (panels B and E) or WHc-M (panels C
and F) as determined by ELISA of four day culture SNs. Two weeks
later spleen cells were collected and cultured with HBc, WHc and
p120-140 peptide, conserved between WHc and HBc (W/H), or the
WHc-derived peptides p60-80 (W) and p80-95 (W), which are not
conserved.
[0058] FIGS. 14 and 15 show the immunogenicity of hybrid core
particles. The indicated hybrid particles were injected (20 .mu.g,
1.degree.) and boosted (10 .mu.g, 2.degree.) in IFA. Eight weeks
after the primary and 6 weeks after the secondary immunization,
sera were collected, pooled, and anti-insert and anti-core antibody
titers were determined by ELISA. The hybrid particles are
designated by the C-terminus, the inserted epitope (e.g., M=malaria
P. falciparum) and the position of the insert (e.g., 74). Full
descriptions of the C-termini and the inserted epitopes are listed
in the accompanying tables.
[0059] FIG. 16 shows the effect of the position of the inserted
epitope (NANP), on the immunogenicity of the WHcAg hybrid particle.
Groups of 4 mice were primed with 20 .mu.g of the indicated
particles in IFA. Sera were collected, pooled, and analyzed for
anti-NANP by ELISA. The hybrid particles are identical except for
the position of the insert: NH.sub.2-terminus, COOH-terminus, or
internal (e.g., amino acid 78 or 74).
[0060] FIG. 17 illustrates the effect on immunogenicity of varying
the C-termini of hybrid WHcAg particles with the same (M)
heterologous sequence inserted at amino acid 74. In this study,
mice were immunized with a single dose of 20 .mu.g of the hybrid
particles in IFA.
[0061] FIG. 18 graphically depicts the lack of genetic
nonresponders and the magnitude of the antibody response elicited
by immunizing mice of the indicated H-2 congenic strains with 10
.mu.g of the WHc-based malaria vaccine candidate (HyW-M78) in IFA
and boosting with 5.0 .mu.g of HyW-M78 in IFA. Six weeks after the
primary (1.degree.) and the secondary (2.degree.) immunizations,
sera were collected and anti-WHc and anti-NANP antibodies were
determined by ELISA.
[0062] FIG. 19 provides the results of a CD4+ T cell epitope
mapping analysis in the indicated strains of mice. Briefly, mice of
the indicated strains and H-2 haplotypes were immunized and boosted
either with WHcAg (7.0 .mu.g) or a WHc-based malaria vaccine
candidate (HyW-M78) (10 .mu.g) both in IFA. Two weeks after the
boost spleen cells were harvested and cultured with a panel of
WHcAg-derived peptides in vitro. Four day culture SNs were
collected and IFN.gamma. was measured by ELISA. The minimum
concentration of peptide required to recall IFN.gamma. production
is indicated by the shaded boxes representing weak (light) to very
strong (dark) T cell sites.
[0063] FIG. 20 provides a schematic representation of the steps
involved in construction of the modified WHcAg vaccine
platform.
[0064] FIG. 21 depicts the results of capture ELISAs designed to
detect either WHcAg polypeptide as a marker for expression or WHcAg
particles as a marker for assembly in E. coli lysates. In panel A,
a polyclonal antibody that recognizes assembled particles
(anti-nWHc) is used to determine relative assembly competence,
while in panel B, a mAb specific for a peptidic epitope on WHcAg
(anti-pWHc) is used as the detecting antibody to determine relative
expression levels. In addition, a malaria (M) epitope-specific mAb
was used to detect the malaria repeat epitope (dashed line). The
capture antibody does not compete with either detecting
antibody.
[0065] FIG. 22 provides a list of WHcAg-IM2 or WHcAg-IM2 mutant(-)
hybrid constructs expressed in E. coli and analyzed for relative
expression level and assembly competence by capture ELISA. Lysates
were sequentially screened with mAbs that preferentially recognize
denatured WHcAg or assembled WHcAg particles and given relative
scores accordingly.
[0066] FIG. 23 shows an analysis of a M2e peptide analog panel for
binding to mAb 14C2 and to a polyclonal murine anti-HyW-M(-)78
antisera. The wild type M2e sequence is set forth herein as SEQ ID
NO:64.
[0067] FIG. 24 illustrates that particulate HBcAg is preferentially
presented by naive B cells to naive T cells. Either splenic
adherent cells including macrophage and dendritic cells (MO/DC) or
B cells from naive mice were fractionated and used as APC for
fractionated CD4.sup.+ T cells derived from naive TCR-Tg
(core-specific) mice. Purified APC plus CD4.sup.+ T cells were
cultured in the presence of HBcAg or peptide for 48 hrs before the
level of IL-2 in the SN was determined by ELISA.
[0068] FIG. 25 illustrates that particulate WHcAg and hybrid WHcAg
particles are preferentially presented by naive B cells to naive T
cells. Either naive spleen cells from wild type mice or from B cell
knockout (KO) mice were used as APCs for CD4.sup.+ T cells derived
from core-specific TCR-Tg mice. Purified APC plus CD4.sup.+ T cells
were cultured in the presence of the indicated antigen for 48 hrs
before the level of IL-2 in the SN was determined by ELISA.
[0069] FIG. 26 shows the magnitude of the in vitro primary antibody
response elicited by HBc, HBe, and WHc. Briefly, spleen cells
derived from core-specific TCR-Tg mice were cultured for 5 days in
the presence of the indicated antigen before SNs were collected and
analyzed for the respective IgM antibodies by ELISA.
[0070] FIG. 27 illustrates the correlation observed between in vivo
anti-insert IgG antibody production and primary in vitro IgM
antibody production. In vitro IgM was determined by ELISA, using
the respective hybrid particles as solid phase ligands, while the
in vivo anti-insert IgG level was measured on solid phase
peptides.
[0071] FIG. 28 provides a schematic representation of one of the
methods of the present invention used to obtain mosaic WHcAg
particles by the read-through mechanism.
[0072] FIG. 29 provides a schematic representation of one of the
methods of the present invention used to obtain mosaic WHcAg
particles by utilization of differentially induced plasmids.
[0073] FIG. 30 shows the effect adjuvant usage on the level of
insert-specific antibody production. Groups of mice were immunized
with C-longM78 hybrid WHcAg particles in saline (1.degree.; primary
and 2.degree., secondary) or formulated in the indicated adjuvants.
Sera were collected at 6 weeks post immunization; pooled and
analyzed for anti-NANP antibody by ELISA, as shown in Panel A.
Panel B depicts the IgG isotype distribution pattern of anti-NANP
antibodies elicited by C-longM78 particles administered in saline
or the indicated adjuvants.
[0074] FIG. 31 shows that antibodies are raised to a protein which
is chemically-coupled to WHcAg. Balb/c mice were immunized three
times with a WHcAg-PGA chemical conjugate (10 .mu.g) adsorbed on
alum. Two weeks after each immunization sera were collected and IgM
and IgG anti-PGA antibodies and anti-WHc antibody was determined by
ELISA.
[0075] FIG. 32 depicts the antibody response over the course of
four months after immunization of mice with an Influenza A
M2e-WHcAg hybrid particle (HyW-IM2(-)78). Five mice were immunized
with 20 .mu.g (1.degree.) and boosted with 10 .mu.g (2.degree.) of
M2e-WHcAg hybrid particles in IFA, and sera was collected, pooled
and analyzed for anti-WHc and anti-M2e antibodies by ELISA.
[0076] FIG. 33 shows the IgG isotype distribution of primary
(1.degree.) and secondary (2.degree.) sera reactive with M2e (panel
A) and WHc (panel B) of mice immunized with M2e-WHcAg hybrid
particles as described in FIG. 33.
[0077] FIG. 34 illustrates that sera from mice immunized with
WHcAg-M2e reacts with influenza A virus infected cells. Sera from
WHcAg immunized (panel A) or WHcAg-M2e (panel B) immunized mice
were incubated with mock (open histograms) or influenza A-infected
(filled histograms) 293T cells. After incubation with a goat
anti-mouse IgG conjugated to FITC, the cells were analyzed by flow
cytometry.
[0078] FIG. 35 depicts the inhibition of rWSN M-Udorn replication
by sera from WHcAg-M2e immunized mice. MDCK cells were infected at
an MOI of 0.1 for 1 hour, with either rWSN (anti-M2e insensitive)
(open bars) or the anti-M2e sensitive, rWSN M-Udorn (hatched bars)
strain. Cells were washed extensively then incubated in DMEM
containing trypsin and 1% sera from WHcAg-M2e immunized mice. At
the indicated times post infection supernatants were collected and
infectious virus particle concentration determined by plaque
assay.
[0079] FIG. 36 depicts the antibody response obtained upon
immunization (20 .mu.g) and boosting (10 .mu.g)
(B10.times.B10.S).sub.F1 mice with hybrid WHcAg particles
containing a CETP.sub.461-476 insert (HyW-CE.sub.74) in IFA. Sera
was collected at the indicated times and anti-WHc and
anti-CETP.sub.461-476 was determined by ELISA. The 14 week sera was
tested for the ability to inhibit human CETP enzymatic activity in
vitro (inset). The human CETP was obtained from hCETP-Tg mouse
sera.
[0080] FIG. 37 illustrates the in vivo persistence of induced
autoantibody. Double-Tg mice expressing an HBc/HBe-specific TCR
(7/16-5) and either HBeAg or HBcAg were injected with the TCR
target peptide (aa 129-140) at day O, Sera were collected at the
indicated times and anti-HBe (panel A) and anti-HBc autoantibody
(panel B) was determined by ELISA.
[0081] FIG. 38 depicts the migration patterns of WHcAg and
WHcAg-CD40L particles in a polyacrylamide gel following denaturing
and reducing conditions (SDS plus beta-mercaptoethanol). The amino
acid sequence of the WHcAg-CD40L is set forth herein as SEQ ID
NO:69. The predicted molecular weights of the two core proteins are
shown: WHc travels as a 20.5 kDa monomer and a 41.1 kDa dimer; and
WHc-CD40L travels as a 37.4 kDa monomer.
[0082] FIG. 39 illustrates that the addition of the molecular
adjuvant CD40L enhances the immunogenicity of WHcAg. Mice were
immunized with 20 .mu.g of WHcAg particles or WHcAg-CD40L hybrid
particles in IFA. At one week (Panel A) and two weeks (Panel B)
post-immunization, sera were collected and analyzed for anti-WHc
antibody by ELISA.
[0083] FIG. 40 provides the wild type WHcAg nucleic acid sequence
(Panel A), and the amino acid sequences of both wild type WHcAg
(Panel B) and truncated WHcAg (Panel C), as set forth in SEQ ID
NO:37, SEQ ID NO:1, and SEQ ID NO:38 respectively.
[0084] FIG. 41 provides the wild type GSHcAg nucleic acid sequence
(Panel A), and the amino acid sequences of both wild type GSHcAg
(Panel B) and truncated GSHcAg (Panel C), as set forth in SEQ ID
NO:39, SEQ ID NO:21, and SEQ ID NO:40 respectively.
[0085] FIG. 42 provides the wild type HBcAg nucleic acid sequence
(Panel A), and the amino acid sequences of both wild type HBcAg
(Panel B) and truncated HBcAg (Panel C), as set forth in SEQ ID
NO:57, SEQ ID NO:41, and SEQ ID NO:58 respectively.
[0086] FIG. 43 depicts the results of a CD4.sup.+ T cell epitope
mapping analysis of GSHcAg-primed mice of the eight indicated
strains of H-2 congenic mice. Briefly, mice were immunized with
GSHcAg (10 .mu.g in IFA) and 4 weeks later spleen cells were
cultured with panels of GSHcAg-derived, WHcAg-derived or
HBcAg-derived peptides in vitro. After 2 or 4 days, culture
supernatants (SN) were collected and IL-2 or IFN.gamma.,
respectively, were measured by ELISA. The relative strengths of the
peptide T cell sites depicted are indicated by the minimum peptide
concentrations required to recall either IL-2 or IFN.gamma.
production in vitro.
[0087] FIG. 44 depicts the ability of hybrid-WHcAg particles
carrying an immune enhancer, the tetanus toxin (TT) universal
CD4.sup.+ T cell epitope TT950-969, to "redirect" the TT-specific T
cell help to the WHcAg for anti-WHc antibody production. Mice of
the indicated strains were first primed with the tetanus toxin
fragment C(TTFc) (20 .mu.g, WA) to mimic TT immunization in humans.
Two months later either TTFc-primed or unprimed mice were injected
with hybrid WHc-TT950-969 particles (10 .mu.g, in saline). At 2, 6
and 28 weeks later serum anti-WHc antibody levels were determined
by ELISA. B10 and B10.S strain T cells recognize the TT950-969
peptide and Balb/c T cells do not.
[0088] FIG. 45 depicts splenic T cell recognition of the TT950-969
peptide in B10, B10.S and Balb/c mice either unprimed or primed
with TTFc (20 .mu.g, IFA) and then all groups were injected with
hybrid WHc-TT950-969 particles (10 .mu.g, in saline). Six months
later spleen cells were harvested and cultured with the TT950-969
peptide and IL-2 produced in the culture media was determined by
ELISA and quantitated (U/ml) by comparison to an IL-2 standard in
the same ELISA.
[0089] FIG. 46 A-C shows a ClustalW alignment of viral core
sequences for primates (human, chimpanzee, gibbon, orangutan,
etc.); rodents (woodchuck, grounds quirrel, artic grounds quirrel,
etc.); and birds/avians (duck, goose, heron, etc.).
GENERAL DESCRIPTION OF THE INVENTION
[0090] The present invention is directed to exploitation of hepadna
virus nucleocapsids/core antigens as multivalent carrier platforms
for enhancing the immune response to weak haptenic-like antigens.
During development of the present invention, theoretical and
practical limitations inherent to the original human hepatitis B
virus (HBV) to nucleocapsid/core antigen (HBcAg) platform
technology were addressed. In one embodiment, a new combinatorial
platform technology, which may be applied to any hepadnavirus, was
developed by modification of the exemplary woodchuck hepadna virus
(WHV) core antigen (WHcAg). To begin, three variables were
identified as considerations in designing WHcAg-hybrid particles:
insert position, C-terminal sequence and epitope sequence. A rapid
screening method to examine WHcAg-hybrid particle assembly within
bacterial lysates was developed as part of a combinatorial approach
involving shuffling of the insert position, and C-terminal
modifications for each epitope of interest. In another embodiment,
a second new combinatorial platform technology was developed by
modification of the exemplary ground squirrel hepadna virus (GHV)
nucleocapsid/core antigen (GSHcAg). While in a further embodiment,
the exemplary human hepatitis B virus core antigen platform was
improved through introduction of various modifications
[0091] As disclosed herein, one advantage of the invention's
combinatorial modification that includes epitope insertion at
preferred locations in a hepadnavirus core antigen as well as
preferred C-terminal modifications of the core antigen is that
inclusion of the C-terminal modification rescues assembly of the
resulting virus particles that contain the inserted epitope (e.g.,
Tables 12-14). Conversely, non-permissive C-terminal modifications
to hepadnavirus core antigens may be rescued by altering the
insertion position of the epitope into the hepadnavirus core
antigen (e.g., Tables 13, 14). Moreover, substitution of amino
acids with acidic amino acids and/or the use of acidic amino acid
linkers in the hepadnavirus core antigen rescue assembly of the
inserted epitope (e.g., Table 18).
DEFINITIONS
[0092] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor or RNA (e.g., tRNA, siRNA,
rRNA, etc.). The polypeptide can be encoded by a full length coding
sequence or by any portion of the coding sequence so long as the
desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends, such that
the gene corresponds to the length of the full-length mRNA. The
sequences that are located 5' of the coding region and which are
present on the mRNA are referred to as 5' untranslated sequences.
The sequences that are located 3' or downstream of the coding
region and that are present on the mRNA are referred to as 3'
untranslated sequences. The term "gene" encompasses both cDNA and
genomic forms of a gene. A genomic form or clone of a gene contains
the coding region, which may be interrupted with non-coding
sequences termed "introns" or "intervening regions" or "intervening
sequences." Introns are removed or "spliced out" from the nuclear
or primary transcript, and are therefore absent in the messenger
RNA (mRNA) transcript. The mRNA functions during translation to
specify the sequence or order of amino acids in a nascent
polypeptide.
[0093] In particular, the term "WHcAg gene" refers to the
full-length WHcAg nucleotide sequence (e.g., contained in SEQ ID
NO:37). However, it is also intended that the term encompass
fragments of the WHcAg sequence, and/or other domains within the
full-length WHcAg nucleotide sequence. Furthermore, the terms
"WHcAg nucleotide sequence" or "WHcAg polynucleotide sequence"
encompasses DNA, cDNA, and RNA (e.g., mRNA) sequences.
[0094] The term "plasmid" as used herein, refers to a small,
independently replicating, piece of DNA. Similarly, the term "naked
plasmid" refers to plasmid DNA devoid of extraneous material
typically used to affect transfection. As used herein, a "naked
plasmid" refers to a plasmid substantially free of
calcium-phosphate, DEAE-dextran, liposomes, and/or polyamines. As
used herein, the term "purified" refers to molecules
(polynucleotides or polypeptides) that are removed from their
natural environment, isolated or separated. "Purified" molecules
are at least 50% free, preferably at least 75% free, and more
preferably at least 90% free from other components with which they
are naturally associated.
[0095] The term "recombinant DNA" refers to a DNA molecule that is
comprised of segments of DNA joined together by means of molecular
biology techniques. Similarly, the term "recombinant protein"
refers to a protein molecule that is expressed from recombinant
DNA. The term "fusion protein" as used herein refers to a protein
formed by expression of a hybrid gene made by combining two gene
sequences. Typically this is accomplished by cloning a cDNA into an
expression vector in frame with an existing gene. The fusion
partner may act as a reporter (e.g., .beta.gal) or may provide a
tool for isolation purposes (e.g., GST).
[0096] Suitable systems for production of recombinant proteins
include but are not limited to prokaryotic (e.g., Escherichia
coli), yeast (e.g., Saccaromyces cerevisiae), insect (e.g.,
baculovirus), mammalian (e.g., Chinese hamster ovary), plant (e.g.,
safflower), and cell-free systems (e.g., rabbit reticulocyte).
[0097] As used herein, the term "coding region" refers to the
nucleotide sequences that encode the amino acid sequences found in
the nascent polypeptide as a result of translation of an mRNA
molecule. The coding region is bounded in eukaryotes, on the 5'
side by the nucleotide triplet "ATG" that encodes the initiator
methionine and on the 3' side by one of the three triplets which
specify stop codons (i.e., TAA, TAG, and TGA).
[0098] Where amino acid sequence is recited herein to refer to an
amino acid sequence of a protein molecule, "amino acid sequence"
and like terms, such as "polypeptide" or "protein" are not meant to
limit the amino acid sequence to the complete, native amino acid
sequence associated with the recited protein molecule. Rather the
terms "amino acid sequence" and "protein" encompass partial
sequences, and modified sequences.
[0099] The term "wild type" when in reference to a gene or gene
product refers to a gene or gene product that has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild type gene or gene product is
that which is most frequently observed in a population and is thus
arbitrarily designed the "normal" or "wild-type" form of the gene
or gene product.
[0100] In contrast, the terms "modified," "mutant," and "variant"
refer to a gene or gene product that displays changes in sequence
and or functional properties (i.e., altered characteristics) when
compared to the wild-type gene or gene product. In some
embodiments, the modification comprises at least one insertion,
deletion, or substitution. In preferred embodiments, the insertion
comprises introduction of a heterologous antigen sequence into a
hepatitis B virus antigen sequence (e.g., fusion protein).
[0101] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid and is referred to using the
functional term "substantially homologous." The term "inhibition of
binding," when used in reference to nucleic acid binding, refers to
reduction in binding caused by competition of homologous sequences
for binding to a target sequence. The inhibition of hybridization
of the completely complementary sequence to the target sequence may
be examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence to a target under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
that lacks even a partial degree of complementarity (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target.
[0102] The art knows well that numerous equivalent conditions may
be employed to comprise low stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.).
[0103] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0104] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0105] As used herein, the term "competes for binding" when used in
reference to a first and a second polypeptide means that the first
polypeptide with an activity binds to the same substrate as does
the second polypeptide with an activity. In one embodiment, the
second polypeptide is a variant of the first polypeptide (e.g.,
encoded by a different allele) or a related (e.g., encoded by a
homolog) or dissimilar (e.g., encoded by a second gene having no
apparent relationship to the first gene) polypeptide. The
efficiency (e.g., kinetics or thermodynamics) of binding by the
first polypeptide may be the same as or greater than or less than
the efficiency of substrate binding by the second polypeptide. For
example, the equilibrium binding constant (K.sub.D) for binding to
the substrate may be different for the two polypeptides. The term
"K.sub.m" as used herein refers to the Michaelis-Menton constant
for an enzyme and is defined as the concentration of the specific
substrate at which a given enzyme yields one-half its maximum
velocity in an enzyme catalyzed reaction.
[0106] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids.
[0107] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations that take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0108] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Those skilled in the art will
recognize that "stringency" conditions may be altered by varying
the parameters just described either individually or in concert.
With "high stringency" conditions, nucleic acid base pairing will
occur between nucleic acid fragments that have a high frequency of
complementary base sequences (e.g., hybridization under "high
stringency" conditions may occur between homologs with 85-100%
identity, preferably 70-100% identity). With medium stringency
conditions, nucleic acid base pairing will occur between nucleic
acids with an intermediate frequency of complementary base
sequences (e.g., hybridization under "medium stringency" conditions
may occur between homologs with 50-70% identity). Thus, conditions
of "weak" or "low" stringency are often required with nucleic acids
that are derived from organisms that are genetically diverse, as
the frequency of complementary sequences is usually less.
[0109] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution comprising
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 100 to about 1000
nucleotides in length is employed.
[0110] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution comprising
5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 100 to about 1000
nucleotides in length is employed.
[0111] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42.degree. C. in a solution
comprising 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.1% SDS, 5.times.Denhardt's reagent
[50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 g/ml denatured
salmon sperm DNA followed by washing in a 120 solution comprising
5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 100
to about 1000 nucleotides in length is employed.
[0112] The term "equivalent" when made in reference to a
hybridization condition as it relates to a hybridization condition
of interest means that the hybridization condition and the
hybridization condition of interest result in hybridization of
nucleic acid sequences which have the same range of percent (%)
homology. For example, if a hybridization condition of interest
results in hybridization of a first nucleic acid sequence with
other nucleic acid sequences that have from 85% to 95% homology to
the first nucleic acid sequence, then another hybridization
condition is said to be equivalent to the hybridization condition
of interest if this other hybridization condition also results in
hybridization of the first nucleic acid sequence with the other
nucleic acid sequences that have from 85% to 95% homology to the
first nucleic acid sequence.
[0113] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence", "sequence identity", "percentage of sequence identity",
and "substantial identity". A "reference sequence" is a defined
sequence used as a basis for a sequence comparison; a reference
sequence may be a subset of a larger sequence, for example, as a
segment of a full-length cDNA sequence given in a sequence listing
or may comprise a complete gene sequence. Generally, a reference
sequence is at least 20 nucleotides in length, frequently at least
25 nucleotides in length, and often at least 50 nucleotides in
length. Since two polynucleotides may each (1) comprise a sequence
(i.e., a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) may further
comprise a sequence that is divergent between the two
polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison
window", as used herein, refers to a conceptual segment of at least
20 contiguous nucleotide positions wherein a polynucleotide
sequence may be compared to a reference sequence of at least 20
contiguous nucleotides and wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(Smith and Waterman, Adv. Appl. Math., 2: 482, 1981) by the
homology alignment algorithm of Needleman and Wunsch (Needleman and
Wunsch, J. Mol. Biol., 48:443, 1970), by the search for similarity
method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad.
Sci., U.S.A., 85:2444, 1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group,
Madison, Wis.), or by inspection, and the best alignment (i.e.,
resulting in the highest percentage of homology over the comparison
window) generated by the various methods is selected. The term
"sequence identity" means that two polynucleotide sequences are
identical (i.e., on a nucleotide-by-nucleotide basis) over the
window of comparison. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. The terms "substantial identity" as used herein denotes a
characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 85 percent
sequence identity, preferably at least 90 to 95 percent sequence
identity, more usually at least 99 percent sequence identity as
compared to a reference sequence over a comparison window of at
least 20 nucleotide positions, frequently over a window of at least
25-50 nucleotides, wherein the percentage of sequence identity is
calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison. The reference sequence may be a subset of a
larger sequence, for example, as a segment of the full-length
sequences of the compositions claimed in the present invention
(e.g., WHcAg)
[0114] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity or more (e.g., 99 percent sequence identity). Preferably,
residue positions which are not identical differ by conservative
amino acid substitutions. Conservative amino acid substitutions
refer to the interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having acidic side
chains is glutamic acid and aspartic acid; a group of amino acids
having basic side chains is lysine, arginine, and histidine; and a
group of amino acids having sulfur-containing side chains is
cysteine and methionine. Preferred conservative amino acids
substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0115] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques have been designed primarily for this sorting out.
[0116] Template specificity is achieved in most amplification
techniques by the choice of enzyme. Amplification enzymes are
enzymes that, under conditions they are used, will process specific
sequences of nucleic acid in a heterogeneous mixture of nucleic
acid. In particular, Tth, Taq and Pfu polymerases, by virtue of
their ability to function at high temperature, are found to display
high specificity for the sequences bounded and thus defined by the
primers; the high temperature results in thermodynamic conditions
that favor primer hybridization with the target sequences and not
hybridization with non-target sequences.
[0117] As used herein, the term "sample template" refers to nucleic
acid originating from a sample that is analyzed for the presence of
"target" (defined below). In contrast, "background template" is
used in reference to nucleic acid other than sample template that
may or may not be present in a sample. Background template is most
often inadvertent. It may be the result of carryover, or it may be
due to the presence of nucleic acid contaminants sought to be
purified away from the sample. For example, nucleic acids from
organisms other than those to be detected may be present as
background in a test sample.
[0118] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0119] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular WHcAg
sequences. It is contemplated that any probe used in the present
invention will be labelled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0120] As used herein, the term "target," when used in reference to
the polymerase chain reaction, refers to the region of nucleic acid
bounded by the primers used for polymerase chain reaction. Thus,
the "target" is sought to be sorted out from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0121] As used herein, the term "polymerase chain reaction" ("PCR")
refers to the method of Mullis (U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,965,188, hereby incorporated by reference), that
describe a method for increasing the concentration of a segment of
a target sequence in a mixture of genomic DNA without cloning or
purification. This process for amplifying the target sequence
consists of introducing a large excess of two oligonucleotide
primers to the DNA mixture containing the desired target sequence,
followed by a precise sequence of thermal cycling in the presence
of a DNA polymerase. The two primers are complementary to their
respective strands of the double stranded target sequence. To
effect amplification, the mixture is denatured and the primers then
annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a
polymerase so as to form a new pair of complementary strands. The
steps of denaturation, primer annealing, and polymerase extension
can be repeated many times (i.e., denaturation, annealing and
extension constitute one "cycle"; there can be numerous "cycles")
to obtain a high concentration of an amplified segment of the
desired target sequence. The length of the amplified segment of the
desired target sequence is determined by the relative positions of
the primers with respect to each other, and therefore, this length
is a controllable parameter. By virtue of the repeating aspect of
the process, the method is referred to as the "polymerase chain
reaction" (hereinafter "PCR"). Because the desired amplified
segments of the target sequence become the predominant sequences
(in terms of concentration) in the mixture, they are said to be
"PCR amplified."
[0122] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0123] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one contaminant nucleic acid with which it is
ordinarily associated in its natural source. Isolated nucleic acid
is present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids are nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous
other mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding gene includes, by way of example, such
nucleic acid in cells ordinarily expressing gene where the nucleic
acid is in a chromosomal location different from that of natural
cells, or is otherwise flanked by a different nucleic acid sequence
than that found in nature. The isolated nucleic acid,
oligonucleotide, or polynucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be utilized to
express a protein, the oligonucleotide or polynucleotide will
contain at a minimum the sense or coding strand (i.e., the
oligonucleotide or polynucleotide may single-stranded), but may
contain both the sense and anti-sense strands (i.e., the
oligonucleotide or polynucleotide may be double-stranded).
[0124] The terms "fragment" and "portion" when used in reference to
a nucleotide sequence (as in "a portion of a given nucleotide
sequence") refers to partial segments of that sequence. The
fragments may range in size from four nucleotides to the entire
nucleotide sequence minus one nucleotide (10 nucleotides, 20, 30,
40, 50, 100, 200, etc.).
[0125] Similarly, the terms "fragment" and "portion" when used in
reference to a polypeptide sequence refers to partial segments of
that sequence. In some embodiments, the portion has an
amino-terminal and/or carboxy-terminal deletion as compared to the
native protein, but where the remaining amino acid sequence is
identical to the corresponding positions in the amino acid sequence
deduced from a full-length cDNA sequence. The fragments may range
in size from four consecutive amino acid residues to the entire
amino acid sequence minus one amino acid. In one embodiment,
fragments are preferably at least 4 amino acids long, more
preferably at least 50 amino acids long, and most preferably at
least 50 amino acids long or longer (the entire amino acid sequence
minus on amino acid). In particularly preferred embodiments, the
portion comprises the amino acid residues required for
intermolecular binding of the compositions of the present invention
with its various ligands and/or substrates.
[0126] Thus, "at least a portion of" a nucleotide sequence and of
an amino acid sequence refers to a sequence that comprises from
four (4) contiguous nucleotides and from four (4) contiguous amino
acid residues, respectively, of the nucleotide sequence and of the
amino acid sequence to the entire nucleotide sequence and amino
acid sequence.
[0127] As used herein the term "coding region" when used in
reference to structural gene refers to the nucleotide sequences
that encode the amino acids found in the nascent polypeptide as a
result of translation of a mRNA molecule. The coding region is
bounded, in eukaryotes, on the 5' side by the nucleotide triplet
"ATG" that encodes the initiator methionine and on the 3' side by
one of the three triplets which specify stop codons (i.e., TAA,
TAG, TGA).
[0128] As used herein, the term "purified" refers to molecules
(polynucleotides or polypeptides) that are separated from other
components with which they are naturally associated. "To purify"
refers to a reduction (preferably by at least 10%, more preferably
by at least 50%, and most preferably by at least 90%) of one or
more contaminants from a sample. For example, WHcAg antibodies are
purified by removal of contaminating non-immunoglobulin proteins;
they are also purified by the removal of immunoglobulin that does
not bind WHcAg. The removal of non-immunoglobulin proteins and/or
the removal of immunoglobulins that do not bind WHcAg results in an
increase in the percent of WHcAg-reactive immunoglobulins in the
sample. In another example, recombinant WHcAg polypeptides are
expressed in bacterial host cells and the polypeptides are purified
by the removal of host cell proteins; the percent of recombinant
WHcAg polypeptides is thereby increased in the sample.
[0129] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule that is comprised of segments of DNA joined together
by means of molecular biological techniques. Similarly, the term
"recombinant protein" or "recombinant polypeptide" as used herein
refers to a protein molecule that is expressed from a recombinant
DNA molecule.
[0130] The term "native protein" as used herein to indicate that a
protein does not contain amino acid residues encoded by vector
sequences, that is the native protein contains those amino acids
found in the protein as it occurs in nature. A native protein may
be produced by recombinant means or may be isolated from a
naturally occurring source.
[0131] The term "Southern blot," refers to the analysis of DNA on
agarose or acrylamide gels to fractionate the DNA according to size
followed by transfer of the DNA from the gel to a solid support,
such as nitrocellulose or a nylon membrane. The immobilized DNA is
then probed with a labeled probe to detect DNA species
complementary to the probe used. The DNA may be cleaved with
restriction enzymes prior to electrophoresis. Following
electrophoresis, the DNA may be partially depurinated and denatured
prior to or during transfer to the solid support. Southern blots
are a standard tool of molecular biologists (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
NY, pp 9.31-9.58, 1989).
[0132] The term "Northern blot," as used herein refers to the
analysis of RNA by electrophoresis of RNA on agarose gels to
fractionate the RNA according to size followed by transfer of the
RNA from the gel to a solid support, such as nitrocellulose or a
nylon membrane. The immobilized RNA is then probed with a labeled
probe to detect RNA species complementary to the probe used.
Northern blots are a standard tool of molecular biologists
(Sambrook, et al., supra, pp 7.39-7.52, 1989).
[0133] The term "Western blot" refers to the analysis of protein(s)
(or polypeptides) immobilized onto a support such as nitrocellulose
or a membrane. The proteins are run on acrylamide gels to separate
the proteins, followed by transfer of the protein from the gel to a
solid support, such as nitrocellulose or a nylon membrane. The
immobilized proteins are then exposed to antibodies with reactivity
against an antigen of interest. The binding of the antibodies may
be detected by various methods, including the use of radiolabelled
antibodies.
[0134] The terms "antigenic determinant" and "epitope" as used
herein refer to that portion of an antigen that makes contact with
a particular antibody and/or T cell receptor. When a protein or
fragment of a protein is used to immunize a host animal, numerous
regions of the protein may induce the production of antibodies that
bind specifically to a given region or three-dimensional structure
on the protein; these regions or structures are referred to as
antigenic determinants. An antigenic determinant may compete with
the intact antigen (i.e., the "immunogen" used to elicit the immune
response) for binding to an antibody.
[0135] The term "vaccine" as used herein refers to an antigen which
induces immunity against the antigen in an animal to which the
antigen is administered. Vaccines include, but are not limited to,
antigenic recombinant polypeptides, glycoproteins, and/or nucleic
sequences (e.g., plasmids) encoding them. Such plasmids may be
inoculated directly into the host, the antigen is expressed in the
host and antibody and/or cell-mediated immunity can then be induced
to the recombinant antigen. Vaccines may be used for immunizing an
animal against the antigen of interest, by administering the
vaccine to generate an immune response in animal against the
antigen. Vaccines and/or antibodies against the antigen may be used
therapeutically and/or prophylactically.
[0136] The term "transgene" as used herein refers to a foreign gene
that is placed into an organism by introducing the foreign gene
into newly fertilized eggs or early embryos. The term "foreign
gene" refers to any nucleic acid (e.g., gene sequence) that is
introduced into the genome of an animal by experimental
manipulations and may include gene sequences found in that animal
so long as the introduced gene does not reside in the same location
as does the naturally-occurring gene.
[0137] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector."
[0138] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
usually include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eukaryotic cells
are known to utilize promoters, enhancers, and termination and
polyadenylation signals.
[0139] As used herein, the term host cell refers to any eukaryotic
or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast
cells, mammalian cells, avian cells, amphibian cells, plant cells,
fish cells, and insect cells), whether located in vitro or in vivo.
For example, host cells may be located in a transgenic animal.
[0140] The term "transfection" as used herein refers to the
introduction of foreign DNA into eukaryotic cells. Transfection may
be accomplished by a variety of means known to the art including
calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, and biolistics.
[0141] The term "stable transfection" or "stably transfected"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell that has stably integrated foreign DNA into the
genomic DNA.
[0142] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells that have taken up foreign DNA but
have failed to integrate this DNA.
[0143] The term "calcium phosphate co-precipitation" refers to a
technique for the introduction of nucleic acids into a cell. The
uptake of nucleic acids by cells is enhanced when the nucleic acid
is presented as a calcium phosphate-nucleic acid co-precipitate.
The original technique of is modified to optimize conditions for
particular types of cells. The art is well aware of these numerous
modifications.
[0144] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise an
aqueous solution. Compositions comprising polynucleotide sequences
encoding WHcAg or fragments thereof may be employed as
hybridization probes. In this case, the WHcAg encoding
polynucleotide sequences are typically employed in an aqueous
solution containing salts (e.g., NaCl), detergents (e.g., SDS), and
other components (e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.).
[0145] As used herein, the terms "hepadnavirus" and "hepatitis
virus" refer to any one of a family of DNA-containing viruses that
cause hepatitis (inflammation of the liver) in a wide range of
vertebrate species. "Hepadnavirus" includes orthohepadnavirus such
as from rodents (e.g. from woodchuck, ground squirrel, arctic
ground squirrel, etc.) and from primates (e.g., chimpanzee, gibbon,
orangutan, woolly monkey, human, etc.) and avihepadnavirus (e.g.,
from duck, Ross' goose, heron, stork, etc.). The terms "human
hepatitis B virus" and "HBV" refer to a species of the genus
orthohepadnavirus which causes human hepatitis B and which is also
a causal agent in human hepatocellular carcinoma. Viruses similar
to HBV also infect animals (e.g., woodchuck, ground squirrel,
duck), and are encompassed by some embodiments of the present
invention.
[0146] The terms "WHcAg," "woodchuck hepadnavirus core antigen,"
and "woodchuck hepatitis virus core antigen" as used herein refer
to the core antigen of the woodchuck hepadna virus exemplified by
SEQ ID NO:1, 103-107, while the WHcAg coding region is exemplified
by SEQ ID NO:37, 129-133. The term WHcAg also encompasses the core
antigens of other woodchuck hepatitis viruses, such as the
woodchuck hepatitis virus clone 2 corresponding to GenBank
Accession No. NKVLC2, M90520, M18752, M11082, J04514, and
J02442.
[0147] As used herein, the terms "GSHcAg," "ground squirrel
hepadnavirus core antigen," and ground squirrel hepatitis virus
core antigen" refer to the core antigen of the ground squirrel
hepadna virus exemplified by SEQ ID NOs:21 and 108, while the
GSHcAg coding region is exemplified by SEQ ID NOs:39 and 128. The
term GSHcAg also encompasses the core antigens of other ground
squirrel hepadna viruses, such as the arctic ground squirrel
hepatitis B virus corresponding to GenBank Accession No.
NP.sub.--040993 and K02715.
[0148] The terms "HBcAg" and "human hepatitis B core antigen" refer
to the core antigen of the human hepatitis B virus exemplified by
SEQ ID NO:41, 109-114, while the HBcAg coding region is exemplified
by SEQ ID NO:57, 138-142 (Genbank No. X65257, X02763, X01587,
J02202, AY123041). The term HBcAg also encompasses the core
antigens of other HBV isolates, including but not limited to the
ADW subtypes (e.g., subtype ADW4, strain brazil/isolate w4b;
subtype ADW, strain okinawa/podw282; subtype ADW, strain
indonesia/pidw420; etc.), and the ADR subtypes. The term
"hepadnavirus core antigen" herein refers to a sequence of a
hepadnavirus that corresponds (e.g., by sequence alignment, see
FIG. 46) to the core antigen sequence of one or more of the
hepadnavirus core antigens disclosed herein. In one embodiment, the
"hepadnavirus core antigen" contains a region towards the carboxyl
terminus which is homologous with that found in protamines (DNA
binding proteins) and which interacts with DNA during assembly of
core particles (Pasek et al, Nature, 282, 575-579, 1979). In one
embodiment, the "hepadnavirus core antigen" has greater than 70%,
preferably greater than 80%, and most preferably greater than 90%,
homology to the core antigen sequence of one or more of the
hepadnavirus core antigens disclosed herein, such the woodchuck
hepatitis virus core antigen (e.g., SEQ ID NO:1, 103-107), arctic
ground squirrel hepatitis virus core antigen (e.g., SEQ ID NO:102),
ground squirrel hepatitis virus core antigen (e.g., SEQ ID NO:21
and 108), chimpanzee hepatitis B virus core antigen (e.g., SEQ ID
NO:115), gibbon hepatitis B virus core antigen (e.g., SEQ ID
NO:116), orangutan hepatitis virus core antigen (e.g., SEQ ID
NO:117), woolly monkey hepatitis virus core antigen (e.g., SEQ ID
NO:118), human hepatitis B virus core antigen (e.g. SEQ ID NOS:41,
and 109-114), duck hepatitis virus core antigen (SEQ ID
NO:119-124), Ross' goose hepatitis virus core antigen (SEQ ID
NO:125), heron hepatitis virus core antigen (SEQ ID NO:126),
sheldgoose hepatitis virus core antigen (SEQ ID NO:151), and stork
hepatitis virus core antigen (SEQ ID NO:152).
[0149] The terms "C-terminal sequence," "C-terminal portion,"
"COOH-terminal portion," "carboxy terminal portion," "C-terminal
domain," "COOH-terminal domain," and "carboxy terminal domain,"
when used in reference to an amino acid sequence of interest (such
as a hepadnavirus core antigen as exemplified by WHcAg) refer to
the amino acid sequence (and portions thereof) that is located from
approximately the middle of the amino acid sequence of interest to
the C-terminal-most amino acid residue of the sequence of interest.
In one embodiment, the "C-terminal sequence" of a hepadnavirus core
antigen as used herein also refers to that portion of the
hepadnavirus core antigen, the deletion of which from the core
antigen does not result in a complete absence of assembly (although
it may result in a reduced level of assembly) of a virus particle.
Thus, in one embodiment, the "C-terminal sequence" of a
hepadnavirus core antigen as used herein refers to a sequence of
the hepadnavirus core antigen that corresponds (e.g., by sequence
alignment, see FIG. 46) to the C-terminal sequence of one or more
of the hepadnavirus core antigens disclosed herein, such as amino
acids 150-188 of the woodchuck hepatitis virus core antigen (e.g.,
SEQ ID NO:1, 103-107), amino acids 150-187 of the arctic ground
squirrel hepatitis virus core antigen (e.g., SEQ ID NO:102), amino
acids 149-187 of the ground squirrel hepatitis virus core antigen
(e.g., SEQ ID NO:21 and 108), amino acids 150-183 of any one of the
chimpanzee hepatitis B virus core antigen (e.g., SEQ ID NO:115),
gibbon hepatitis B virus core antigen (e.g., SEQ ID NO:116), and
orangutan hepatitis virus core antigen (e.g., SEQ ID NO:117), amino
acids 150-182 of the woolly monkey hepatitis virus core antigen
(e.g., SEQ ID NO:118), amino acids 150-183 of the human hepatitis B
virus core antigen (e.g. SEQ ID NOS:41, and 109-114), amino acids
196-282 of any one of the duck hepatitis virus core antigen (SEQ ID
NO:119-124), Ross' goose hepatitis virus core antigen (SEQ ID
NO:125), heron hepatitis virus core antigen (SEQ ID NO:126),
sheldgoose hepatitis virus core antigen (SEQ ID NO:151), and stork
hepatitis virus core antigen (SEQ ID NO:152).
[0150] Alignment of sequences for comparison may be conducted by
computerized implementations of known algorithms (e.g., GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis., or BlastN and BlastX available from the National Center for
Biotechnology Information), by using the algorithm of E. Myers and
W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into
the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4, or by
inspection. Sequences are typically compared using either BlastN or
BlastX with default parameters. In one preferred embodiment,
alignment is conducted using MacVector.TM. software.
[0151] Exemplary C-terminal sequences of hepadnavirus core antigens
are illustrated by those from woodchuck hepatitis virus (SEQ ID
NO:2), ground squirrel hepatitis virus (SEQ ID NO:22), arctic
ground squirrel hepatitis virus (SEQ ID NO:153), chimpanzee
hepatitis B virus (SEQ ID NO:161), gibbon hepatitis B virus (SEQ ID
NO:159), orangutan hepatitis virus (SEQ ID NO:157), woolly monkey
hepatitis virus (SEQ ID NO:155), duck hepatitis virus (SEQ ID
NO:163, 165, 167, 169, 171, and/or 173), Ross' goose hepatitis
virus (SEQ ID NO:175), heron hepatitis virus (SEQ ID NO:179),
sheldgoose hepatitis virus (SEQ ID NO:177), stork hepatitis virus
(SEQ ID NO:181), and from human hepatitis B virus (SEQ ID NO:42).
Thus, in one embodiment, the C-terminal portion of WHcAg includes
the amino acid sequence from position 150 to 188 of WHcAg (SEQ ID
NO:2); the C-terminal portion of GSHcAg includes the amino acid
sequence from position 149 to 187 of GSHcAg (SEQ ID NO:22); the
C-terminal portion of HBcAg includes the amino acid sequence from
position 150 to 183 of HBcAg (SEQ ID NO:42).
[0152] The terms "truncated hepadnavirus core antigen" and
"N-terminal sequence of a hepadnavirus core antigen" refer to a
hepadnavirus core antigen from which the C-terminal sequence has
been deleted.
[0153] The term "hybrid" as used in reference to a hepadna virus
core antigen, refers to a fusion protein of the hepadna virus core
antigen and an unrelated antigen (e.g., Plasmodium antigen). For
instance, in some preferred embodiments of the present invention,
the term "hybrid WHcAg" refers to a fusion protein comprising both
a WHcAg component (full length, or partial) and a heterologous
antigen (e.g., non-WHcAg and/or modified WHcAg) component. In
particularly preferred embodiments, the heterologous antigen
comprises at least one B cell epitope and/or at least one T cell
epitope which may be conjugated (e.g., covalently linked) to a
residue of the WHcAg and/or which is inserted within the WHcAg via
expression as a fusion protein. In contrast, the term "nonhybrid"
refers to an antigen of a single origin (e.g., WHcAg in the absence
of a heterologous antigen insert or conjugate).
[0154] The term "modified antigen" refers to an antigen, any part
of which (such as the nucleic acid sequence and/or proteins) has
been modified by chemical, biochemical, and/or molecular biological
techniques compared to the wild-type antigen. In one embodiment,
the antigen is modified by means of molecular biological
techniques. In one embodiment, the modification may include one or
more of a deletion, an insertion, and a substitution. A "deletion"
is defined as a change in a nucleic acid sequence in which one or
more nucleotides is absent. An "insertion" or "addition" is that
change in a nucleic acid sequence which has resulted in the
addition of one or more nucleotides. The term "substitution of an
amino acid" and "substitution of a nucleic acid" as used herein
refer to the replacement of one or more amino acids and one or more
nucleic acids, respectively, by one or more amino acids and by one
or more nucleic acids. Similarly, in some preferred embodiments,
the term "modified WHcAg" refers to a woodchuck hepadna virus core
protein with a C-terminal truncation. In particularly preferred
embodiments, the modified WHcAg comprises both carboxy-terminal
amino acid deletions, and insertions within the loop and/or outside
the loop. In addition the term "modified WHcAg" refers to a
woodchuck virus core protein comprising a heterologous antigen in
the form of a conjugate or a fusion protein. Thus as used herein,
the terms "modified hepatitis virus core antigen" and grammatical
equivalents encompass hybrid core antigens, as well as mutant core
antigens.
[0155] The term insertion of a first amino acid (e.g., alanine) or
amino acid sequence (e.g., heterologous antigen) "at amino acid
position x" or "in amino acid position x" of a second amino acid
sequence (e.g., woodchuck hepadna virus core antigen) means
introduction of a first amino acid or sequence into a second amino
acid sequence, such that the first amino acid or sequence is placed
C-temiinal to amino acid x.
[0156] The term "conjugating" when made in reference to two
molecules (such as a to heterologous antigen and hepadna virus core
antigen) as used herein means covalently linking the two molecules.
In one embodiment, where one of the molecules is a viral core or
will be assembled into a viral nucleocapsid, it may be desirable to
modify the nature and size of the second molecule and the site at
which it is covalently linked to the core antigen such that it does
not interfere with the capacity of the modified core to assemble in
vitro and/or in vivo. In some embodiments, the heterologous antigen
is conjugated to a functional group on the hepadna virus core
antigen, chosen from but not limited to a carboxyl group, a primary
amine, and a sulfhydryl. In some preferred embodiments, a
heterobifunctional cross-linker is used to attach the heterologous
antigen to the hepadna virus core antigen. Exemplary cross-linkers
include but are not limited to MBS, EDC/Sulfo-NHS and ABH obtained
from Pierce (Rockford, Ill.).
[0157] As used herein in reference to a hepadna virus core antigen,
the term "loop" refers to a portion of the hepadna virus core
antigen which links the second and third alpha-helices and which
contains an immunodominant B cell epitope. Specifically, in
reference to HBcAG, the term "within the loop" refers to residues
at positions 76 to 82 of the wild type sequence, while the term
"outside the loop" refers to residues amino-terminal to residue 76
and carboxy-terminal to residue 82. Likewise, in reference to
WHcAg, the term "within the loop" refers to residues at positions
76 to 82 of the wild type sequence, while the term "outside the
loop" refers to residues amino-terminal to residue 76 and
carboxy-terminal to residue 82. In contrast, in reference to
GSHcAg, the term "within the loop" refers to residues at positions
76 to 81, while the term "outside the loop" refers to residues
amino-terminal to residue 76 and carboxy-terminal to residue
81.
[0158] The terms "N-terminus" "NH.sub.2-terminus" and
"amino-terminus" refer to the amino acid residue corresponding to
the methionine encoded by the start codon (e.g., position or
residue 1). In contrast the terms "C-terminus" "COOH-terminus" and
"carboxy terminus" refer to the amino acid residue encoded by the
final codon (e.g., last or final residue prior to the stop
codon).
[0159] The term "antibody" refers to polyclonal and monoclonal
antibodies. Polyclonal antibodies which are formed in the animal as
the result of an immunological reaction against a protein of
interest or a fragment thereof, can then be readily isolated from
the blood using well-known methods and purified by column
chromatography, for example. Monoclonal antibodies can also be
prepared using known methods (See, Winter and Milstein, Nature,
349, 293-299, 1991). As used herein, the term "antibody"
encompasses recombinantly prepared, and modified antibodies and
antigen-binding fragments thereof, such as chimeric antibodies,
humanized antibodies, multifunctional antibodies, bispecific or
oligo-specific antibodies, single-stranded antibodies and F(ab) or
F(ab).sub.2 fragments. The term "reactive" when used in reference
to an antibody indicates that the antibody is capable of binding an
antigen of interest. For example, a WHcAg-reactive antibody is an
antibody which binds to WHcAg or to a fragment of WHcAg.
[0160] The terms "auto-antibody" or "auto-antibodies" refer to any
immunoglobulin that binds specifically to an antigen that is native
to the host organism that produced the antibody (i.e., the antigen
is not synthetic and/or has not been artificially supplied to the
host organism). However, the term encompasses antibodies originally
produced in response to the administration or presence of a foreign
and/or synthetic substance in the host, but also cross-react with
"self" antigens. Exemplary auto-antibodies include, without
limitation, anti-cholesterol ester transfer protein (CETP)
antibody, anti-major histocompatibility complex class II antibody,
anti-cytokine antibody, and anti amyloid-.beta.-peptide antibody.
The presence of auto-antibodies is termed "autoimmunity."
[0161] The term "cytokine" refers to a molecule, such a protein or
glycoprotein, involved in the regulation of cellular proliferation
and function. Cytokines are exemplified by lymphokines (e.g., tumor
necrosis factor-.alpha., tumor necrosis factor-.beta.,
interferon-.gamma., etc.), growth-factors (e.g., erythropoietin,
insulin, G-CSF, M-CSF, GM-CSF, EGF, PDGF, FGF, etc.), and
interleukins (e.g., IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13,
etc.).
[0162] The term "B cell epitope" as used herein refers to as
antigenic determinant (protein or carbohydrate) to which a single
antibody molecule binds. B cell epitopes may comprise linear
epitopes (amino acids adjacent to each other in the primary
sequence) or conformational epitopes (moities distant from each
other in the primary sequence, but which are brought in proximity
to one another during folding of the antigen) of at least four
amino acid residues.
[0163] The term "T cell epitope" as used herein refers to an
antigenic determinant presented by a MHC class I or class II
molecule for binding to a single T cell receptor. T cell epitopes
are linear epitopes comprising at least seven amino acid residues.
In some embodiments of the present invention, the term T cell
epitope comprises a T helper cell epitope which is an antigen
fragment presented by an MHC class II molecule for binding to T
cell receptor on the surface of a helper T cell (e.g., generally
CD4.sup.+).
[0164] The term "conservative substitution" as used herein refers
to a change that takes place within a family of amino acids that
are related in their side chains. Genetically encoded amino acids
can be divided into four families: (1) acidic (aspartate,
glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar
(alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan); and (4) uncharged polar (glycine,
asparagine, glutamine, cysteine, serine, threonine, tyrosine).
Phenylalanine, tryptophan, and tyrosine are sometimes classified
jointly as aromatic amino acids. In similar fashion, the amino acid
repertoire can be grouped as (1) acidic (aspartate, glutamate); (2)
basic (lysine, arginine, histidine), (3) aliphatic (glycine,
alanine, valine, leucine, isoleucine, serine, threonine), with
serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,
tryptophan); (5) amide (asparagine, glutamine); and (6)
sulfur-containing (cysteine and methionine).
[0165] The terms "non-acidic amino acid" and "not an acidic amino
acid" refer to any amino acid other than glutamic acid and aspartic
acid, such as lysine, arginine, histidine, alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan, glycine, asparagine, glutamine, cysteine, serine,
threonine, and tyrosine.
[0166] Whether a change in the amino acid sequence of a peptide
results in a functional homolog can be readily determined by
assessing the ability of the variant peptide to function in a
fashion similar to the wild-type protein. Peptides having more than
one replacement can readily be tested in the same manner. In
contrast, the term "nonconservative substitution" refers to a
change in which an amino acid from one family is replaced with an
amino acid from another family (e.g., replacement of a glycine with
a tryptophan). Guidance in determining which amino acid residues
can be substituted, inserted, or deleted without abolishing
biological activity can be found using computer programs (e.g.,
LASERGENE software, DNASTAR Inc., Madison, Wis.).
[0167] The terms "antigen," "immunogen," "antigenic,"
"immunogenic," "antigenically active," and "immunologically active"
refer to any substance that is capable of inducing a specific
humoral and/or cell-mediated immune response. An immunogen
generally contains at least one epitope. Immunogens are exemplified
by, but not restricted to molecules which contain a peptide,
polysaccharide, nucleic acid sequence, and/or lipid. Complexes of
peptides with lipids, polysaccharides, or with nucleic acid
sequences are also contemplated, including (without limitation)
glycopeptide, lipopeptide, glycolipid, etc. These complexes are
particularly useful immunogens where smaller molecules with few
epitopes do not stimulate a satisfactory immune response by
themselves.
[0168] A peptide sequence and nucleotide sequence may be
"endogenous" or "heterologous" (i.e., "foreign"). The term
"endogenous" refers to a sequence which is naturally found in the
cell or virus into which it is introduced so long as it does not
contain some modification relative to the naturally-occurring
sequence. The term "heterologous" refers to a sequence which is not
endogenous to the cell or virus into which it is introduced. For
example, heterologous DNA includes a nucleotide sequence which is
ligated to, or is manipulated to become ligated to, a nucleic acid
sequence to which it is not ligated in nature, or to which it is
ligated at a different location in nature. Heterologous DNA also
includes a nucleotide sequence which is naturally found in the cell
or virus into which it is introduced and which contains some
modification relative to the naturally-occurring sequence.
Generally, although not necessarily, heterologous DNA encodes
heterologous RNA and heterologous proteins that are not normally
produced by the cell or virus into which it is introduced. Examples
of heterologous DNA include reporter genes, transcriptional and
translational regulatory sequences, DNA sequences which encode
selectable marker proteins (e.g., proteins which confer drug
resistance), etc. In preferred embodiments, the terms "heterologous
antigen" and "heterologous sequence" refer to a non-hepadna virus
antigen or amino acid sequence including but not limited to
microbial antigens, mammalian antigens and allergen antigens.
[0169] The terms "peptide," "peptide sequence," "amino acid
sequence," "polypeptide," and "polypeptide sequence" are used
interchangeably herein to refer to at least two amino acids or
amino acid analogs which are covalently linked by a peptide bond or
an analog of a peptide bond. The term peptide includes oligomers
and polymers of amino acids or amino acid analogs. The term peptide
also includes molecules which are commonly referred to as peptides,
which generally contain from about two (2) to about twenty (20)
amino acids. The term peptide also includes molecules which are
commonly referred to as polypeptides, which generally contain from
about twenty (20) to about fifty amino acids (50). The term peptide
also includes molecules which are commonly referred to as proteins,
which generally contain from about fifty (50) to about three
thousand (3000) amino acids. The amino acids of the peptide may be
L-amino acids or D-amino acids. A peptide, polypeptide or protein
may be synthetic, recombinant or naturally occurring. A synthetic
peptide is a peptide which is produced by artificial means in
vitro.
[0170] The terms "oligosaccharide" and "OS" antigen refer to a
carbohydrate comprising up to ten component sugars, either O or N
linked to the next sugar. Likewise, the terms "polysaccharide" and
"PS" antigen refer to polymers of more than ten monosaccharide
residues linked glycosidically in branched or unbranched
chains.
[0171] The terms "microbial sequence" and "sequence of a microbe"
refers to synthetic, recombinant or purified sequences (preferably
sequence fragments comprising at least one B cell epitope) of a
virus, a bacterium, a fungus, and a parasite. Exemplary microbial
sequences include those of Influenza A, Staphylococcus sp., Candida
sp., and Plasmodium sp.
[0172] As used herein, the term "mammalian sequence" refers to
synthetic, recombinant or purified sequences (preferably sequence
fragments comprising at least one B cell epitope) of a mammal.
Exemplary mammalian sequences include cytokine sequence, MHC class
I heavy chain sequences, MHC class II alpha and beta chain
sequences, and amyloid .beta.-peptide sequences.
[0173] The term "allergen" as used herein, refers to an antigenic
substance capable of producing an immediate type hypersensitivity
reaction (allergy) in a animal. Exemplary allergens include food
allergens such as peanut allergen, grass pollen allergen and dust
mite allergen.
[0174] The term "particle" as used herein refers to a virus-like
protein structure of approximately 25-35 nm in diameter, into which
hepadnavirus core polypeptides spontaneously assemble. Particle
formation is measured by the exemplary methods for assessing
hepadnavirus core antigen expression and assembly disclosed
herein.
[0175] The term "aggregate" as used herein refers to a cluster,
clump, or mass of individual polypeptides and/or particles.
[0176] As used herein, the terms "immune enhancer" and "molecular
adjuvant" refer to molecules or antigens which provide a stimulus
to T cells or a co-stimulus to B cells or other antigen presenting
cells, thereby increasing the level of the immune response by the
cells to an antigen. Exemplary immune enhancers include but are not
limited to unmethylated CpG dinucleotides, CD40 ligand, complement
C3d fragment, BAFF, and LAG-3 and TT950-969.
[0177] An "animal" as used herein refers to any multicellular
animal, including mammals, birds (e.g., chickens, ducks, geese,
herons, storks, etc.), amphibians (e.g. Xenopus), reptiles, and
insects (e.g. mosquito, Drosophila, etc.). The terms "mammals" and
"mammalian" refer to members of the class mammalia which nourish
their young by fluid secreted from mammary glands of the mother,
including human beings. The class "mammalian" includes placental
animals, marsupial animals, and monotrematal animals. An exemplary
"mammal" may be a rodent, primate (including simian and human)
ovine, bovine, ruminant, lagomorph, porcine, caprine, equine,
canine, feline, ave, etc. Preferred non-human animals are selected
from the order RodentiaPreferred embodiments of the present
invention are primarily directed to vertebrate (backbone or
notochord) members of the animal kingdom.
[0178] The terms "patient" and "subject" refer to an animal (such
as a mammal) that may be treated using the methods of the present
invention.
[0179] The term "control" refers to animals or samples which
provide a basis for comparison for experimental animals or samples.
For instance, the use of control animals or samples permits
determinations to be made regarding the efficacy of experimental
procedures. In some embodiments, the term "control animal" refers
to a animal that which receives a mock treatment (e.g., saline
alone or WHcAg without a heterologous antigen insert or
conjugate).
[0180] As used herein, the term "immune response" refers to the
reactivity of an organism's immune system in response to an
antigen. In vertebrates, this may involve antibody production,
induction of cell-mediated immunity, and/or complement activation
(e.g., phenomena associated with the vertebrate immune system's
prevention and resolution of infection by microorganisms). In
preferred embodiments, the term immune response encompasses but is
not limited to one or more of a "lymphocyte proliferative
response," a "cytokine response," and an "antibody response."
[0181] In particularly preferred embodiments, the immune response
is largely reactive with an antigen of interest. For instance, when
used in reference to administration of a hybrid WHcAg-NANP vaccine
to an animal (e.g., mammalian subject), the term refers to the
immune response produced in the animal, which reacts with either
the WHcAg core or the NANP insert/conjugate of the vaccine. Immune
responses reactive with an antigen of interest are measured in
vitro using various methods disclosed herein.
[0182] The term "reactive with an antigen of interest" when made in
reference to an immune response refers to an increased level of the
immune response to the antigen of interest as compared to the level
of the immune response to control antigen. (e.g., unrelated
antigen).
[0183] The term "lymphocyte proliferative response" refers to
antigen-induced lymphocyte (e.g., PBL) increase in cell number.
Alternatively, or in addition, the term "proliferation" refers to
the physiological and morphological progression of changes that
cells undergo when dividing, for instance including DNA replication
as measured by tritiated thymidine incorporation.
[0184] The term "cytokine response" refers to antigen-induced
cytokine secretion by lymphocytes as measured for instance by
assaying culture supernatants for cytokine content (e.g., IL-2,
IFN.gamma., TNF.alpha., IL-4, etc) by ELISA.
[0185] The term "antibody response" refers to the production of
antibodies (e.g., IgM, IgA, IgG) which bind to an antigen of
interest, this response is measured for instance by assaying sera
by antigen ELISA.
[0186] The term "adjuvant" as used herein refers to any compound
which, when injected together with an antigen, non-specifically
enhances the immune response to that antigen.
[0187] Exemplary adjuvants include but are not limited to
incomplete Freunds adjuvant (IFA), aluminum-based adjuvants (e.g.,
AlOH, AIPO4, etc), and Montanide ISA 720.
[0188] The terms "diluent" and "diluting agent" as used herein
refer to agents used to diminish the strength of an admixture.
Exemplary diluents include water, physiological saline solution,
human serum albumin, oils, polyethylene glycols, glycerine,
propylene glycol or other synthetic solvents, antibacterial agents
such as benzyl alcohol, antioxidants such as ascorbic acid or
sodium bisulphite, chelating agents such as ethylene
diamine-tetra-acetic acid, buffers such as acetates, citrates or
phosphates and agents for adjusting the osmolarity, such as sodium
chloride or dextrose.
[0189] The terms "carrier" and "vehicle" as used herein refer to
usually inactive accessory substances into which a pharmaceutical
substance (e.g., WHcAg vaccine) is suspended. Exemplary carriers
include liquid carriers (such as water, saline, culture medium,
saline, aqueous dextrose, and glycols) and solid carriers (such as
carbohydrates exemplified by starch, glucose, lactose, sucrose, and
dextrans, anti-oxidants exemplified by ascorbic acid and
glutathione, and hydrolyzed proteins.
[0190] The term "derived" when in reference to a peptide derived
from a source (such as a microbe, cell, etc.) as used herein is
intended to refer to a peptide which has been obtained (e.g.,
isolated, purified, etc.) from the source. Alternatively, or in
addition, the peptide may be genetically engineered and/or
chemically synthesized.
[0191] The terms "operably linked," "in operable combination," and
"in operable order" as used herein refer to the linkage of nucleic
acid sequences such that they perform their intended function. For
example, operably linking a promoter sequence to a nucleotide
sequence of interest refers to linking the promoter sequence and
the nucleotide sequence of interest in a manner such that the
promoter sequence is capable of directing the transcription of the
nucleotide sequence of interest and/or the synthesis of a
polypeptide encoded by the nucleotide sequence of interest.
Similarly, operably linking a nucleic acid sequence encoding a
protein of interest means linking the nucleic acid sequence to
regulatory and other sequences in a manner such that the protein of
interest is expressed. The term also refers to the linkage of amino
acid sequences in such a manner so that a functional protein is
produced.
[0192] The terms "specific binding," "binding specificity," and
grammatical equivalents thereof when made in reference to the
binding of a first molecule (such as a polypeptide, glycoprotein,
nucleic acid sequence, etc.) to a second molecule (such as a
polypeptide, glycoprotein, nucleic acid sequence, etc.) refer to
the preferential interaction between the first molecule with the
second molecule as compared to the interaction between the second
molecule with a third molecule. Specific binding is a relative term
that does not require absolute specificity of binding; in other
words, the term "specific binding" does not require that the second
molecule interact with the first molecule in the absence of an
interaction between the second molecule and the third molecule.
Rather, it is sufficient that the level of interaction between the
first molecule and the second molecule is higher than the level of
interaction between the second molecule with the third molecule.
"Specific binding" of a first molecule with a second molecule also
means that the interaction between the first molecule and the
second molecule is dependent upon the presence of a particular
structure on or within the first molecule; in other words the
second molecule is recognizing and binding to a specific structure
on or within the first molecule rather than to nucleic acids or to
molecules in general. For example, if a second molecule is specific
for structure "A" that is on or within a first molecule, the
presence of a third nucleic acid sequence containing structure A
will reduce the amount of the second molecule which is bound to the
first molecule.
[0193] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" includes both singular and
plural references unless the content clearly dictates otherwise.
For example, the term "inserted at a position" as used herein in
reference to a polypeptide sequence refers to insertion at one or
more (such as one, two, three, etc.) amino acid positions in the
polypeptide sequence. In one preferred embodiment, insertion is at
one amino acid position as exemplified herein.
[0194] The phrase "chosen from A, B, and C" as used herein, means
selecting one or more of A, B, C.
[0195] As used herein, the term "or" when used in the expression "A
or B," where A and B refer to a composition, disease, product,
etc., means one, or the other, or both.
[0196] The term "on" when in reference to the location of a first
article with respect to a second article means that the first
article is on top and/or into the second article, including, for
example, where the first article permeates into the second article
after initially being placed on it.
[0197] As used herein, the term "comprising" when placed before the
recitation of steps in a method means that the method encompasses
one or more steps that are additional to those expressly recited,
and that the additional one or more steps may be performed before,
between, and/or after the recited steps. For example, a method
comprising steps a, b, and c encompasses a method of steps a, b, x,
and c, a method of steps a, b, c, and x, as well as a method of
steps x, a, b, and c. Furthermore, the term "comprising" when
placed before the recitation of steps in a method does not
(although it may) require sequential performance of the listed
steps, unless the content clearly dictates otherwise. For example,
a method comprising steps a, b, and c encompasses, for example, a
method of performing steps in the order of steps a, c, and b, the
order of steps c, b, and a, and the order of steps c, a, and b,
etc.
[0198] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth as used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters in the specification and claims are
approximations that may vary depending upon the desired properties
sought to be obtained by the present invention. At the very least,
and without limiting the application of the doctrine of equivalents
to the scope of the claims, each numerical parameter should at
least be construed in light of the number of reported significant
digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters describing
the broad scope of the invention are approximation, the numerical
values in the specific examples are reported as precisely as
possible. Any numerical value, however, inherently contains
standard deviations that necessarily result from the errors found
in the numerical value's testing measurements.
[0199] The term "not" when preceding, and made in reference to, any
particularly named molecule (mRNA, etc.) or phenomenon (such as
biological activity, biochemical activity, etc.) means that the
particularly named molecule or phenomenon is excluded.
[0200] The term "altering" and grammatical equivalents as used
herein in reference to the level of any substance and/or phenomenon
refers to an increase and/or decrease in the quantity of the
substance and/or phenomenon, regardless of whether the quantity is
determined objectively, and/or subjectively.
[0201] The term "increase," "elevate," "raise," and grammatical
equivalents when in reference to the level of a substance and/or
phenomenon in a first sample relative to a second sample, mean that
the quantity of the substance and/or phenomenon in the first sample
is higher than in the second sample by any amount that is
statistically significant using any art-accepted statistical method
of analysis. In one embodiment, the increase may be determined
subjectively, for example when a patient refers to their subjective
perception of disease symptoms, such as pain, clarity of vision,
etc. In another embodiment, the quantity of the substance and/or
phenomenon in the first sample is at least 10% greater than the
quantity of the same substance and/or phenomenon in a second
sample. In another embodiment, the quantity of the substance and/or
phenomenon in the first sample is at least 25% greater than the
quantity of the same substance and/or phenomenon in a second
sample. In yet another embodiment, the quantity of the substance
and/or phenomenon in the first sample is at least 50% greater than
the quantity of the same substance and/or phenomenon in a second
sample. In a further embodiment, the quantity of the substance
and/or phenomenon in the first sample is at least 75% greater than
the quantity of the same substance and/or phenomenon in a second
sample. In yet another embodiment, the quantity of the substance
and/or phenomenon in the first sample is at least 90% greater than
the quantity of the same substance and/or phenomenon in a second
sample.
[0202] The terms "reduce," "inhibit," "diminish," "suppress,"
"decrease," and grammatical equivalents when in reference to the
level of a substance and/or phenomenon in a first sample relative
to a second sample, mean that the quantity of substance and/or
phenomenon in the first sample is lower than in the second sample
by any amount that is statistically significant using any
art-accepted statistical method of analysis. In one embodiment, the
reduction may be determined subjectively, for example when a
patient refers to their subjective perception of disease symptoms,
such as pain, clarity of vision, etc. In another embodiment, the
quantity of substance and/or phenomenon in the first sample is at
least 10% lower than the quantity of the same substance and/or
phenomenon in a second sample. In another embodiment, the quantity
of the substance and/or phenomenon in the first sample is at least
25% lower than the quantity of the same substance and/or phenomenon
in a second sample. In yet another embodiment, the quantity of the
substance and/or phenomenon in the first sample is at least 50%
lower than the quantity of the same substance and/or phenomenon in
a second sample. In a further embodiment, the quantity of the
substance and/or phenomenon in the first sample is at least 75%
lower than the quantity of the same substance and/or phenomenon in
a second sample. In yet another embodiment, the quantity of the
substance and/or phenomenon in the first sample is at least 90%
lower than the quantity of the same substance and/or phenomenon in
a second sample.
[0203] Reference herein to any specifically named protein (such as
"WHcAg," "GSHcAg," and "HBcAg," etc.) refers to a polypeptide
having at least one of the biological activities of the
specifically named protein, wherein the biological activity is
detectable by any method. In addition, reference herein to any
specifically named protein (such as "WHcAg," "GSHcAg," and "HBcAg,"
etc.) includes within its scope fragments, fusion proteins, and
variants of the specifically named protein. The term "variant" of a
protein as used herein is defined as an amino acid sequence which
differs by insertion, deletion, and/or conservative substitution of
one or more amino acids from the protein. In one embodiment, the
sequence of the variant has at least 99% identity, preferably at
least 95% identity, and more preferably at least 90% identity with
the sequence of the protein in issue.
[0204] For example, the term "has the biological activity of a
specifically named protein" (such as "WHcAg," "GSHcAg," and
"HBcAg," etc.) when made in reference to the biological activity of
a variant of the specifically named protein refers, for example, to
a quantity of binding of an antibody that is specific for the
specifically named protein to the variant which is preferably
greater than 50% (preferably from 50% to 500%, more preferably from
50% to 200%, most preferably from 50% to 100%), as compared to the
quantity of binding of the same antibody to the specifically named
protein.
[0205] Reference herein to any specifically named nucleotide
sequence (such as a sequence encoding WHcAg, a sequence encoding
GSHcAg, and a sequence encoding HBcAg, etc.) includes within its
scope fragments, homologs, and sequences that hybridize under
stringent condition to the specifically named nucleotide sequence.
The term "homolog" of a specifically named nucleotide sequence
refers to an oligonucleotide sequence which exhibits greater than
or equal to 50% identity to the sequence of interest.
Alternatively, or in addition, a homolog of any specifically named
nucleotide sequence (such as a sequence encoding WHcAg, a sequence
encoding GSHcAg, and a sequence encoding HBcAg, etc.) is defined as
an oligonucleotide sequence which has at least 95% identity with
the sequence of the nucleotide sequence in issue. In another
embodiment, the sequence of the homolog has at least 90% identity,
and preferably at least 85% identity with the sequence of the
nucleotide sequence in issue.
DETAILED DESCRIPTION OF THE INVENTION
[0206] The present invention relates to modified hepatitis virus
core proteins and nucleic acids. In particular, the present
invention provides compositions and methods comprising recombinant
modified hepatitis virus core proteins or nucleic acids for use in,
for example, vaccine formulations.
I. Hepatitis B Virus Core Antigen (HBcAg)
[0207] The human hepatitis B virus core antigen is a 21 kDa
polypeptide. Two kinds of HBcAg core particles are spontaneously
assembled during the course of virion assembly and during
heterologous expression in both prokaryotic and eukaryotic systems:
particles with T=4 symmetry containing 240 polypeptides, and
particles with T=3 symmetry containing 180 polypeptides. Recent
cryoelectron microscopy studies have revealed the structure of
HBcAg particles to a resolution of 7.4 angstroms (Botcher et al.,
Nature, 386:88-91, 1997; and Conway et al., Nature, 386:91-94,
1997). Dimer clustering of HBcAg subunits produces spikes on the
surface of the core shell, which consist of radial bundles of four
long .alpha.-helices. The immunodominant B cell epitope on HBcAg is
localized around amino acids 76-82 (Salfeld et al., J Virol,
63:798-808, 1989; and Schodel et al., J Virol, 66:106-114, 1992),
apparently forming a loop connecting adjacent helices (See, FIG.
1). The spacing of the spikes on the core shell is optimal for B
cell mug receptor cross-linking. In addition, the inherent
immunogenicity of the native HBcAg B cell epitopes suggested the
desirability of substituting heterologous epitopes in the same
position (i.e., at the tip of the spike). In fact, a number of
pathogen-specific B cell epitopes have been chemically linked or
fused by recombinant methods to HBcAg in order to increase their
immunogenicity (See, Milich et al., Ann NY Acad Sci, 754:187-201,
1995; and Pumpens et al., Intervirology, 38:63-74, 1995, for
reviews). These studies, conducted by a number of independent
laboratories, have met with significant success including complete
protection against foot and mouth disease virus (Clarke et al.,
Nature, 330; 381-384, 1987), Plasmodium berghei (Schodel et al., J
Exp Med, 180:1037-1046, 1994), and Plasmodium yoelii (Schodel et
al., Behring Inst Mitt, 114-119, 1997).
[0208] Adjuvants are broadly separated into two classes based upon
their primary mechanism of action: vaccine delivery systems (e.g,
emulsions, microparticles, iscoms, liposomes, etc.) that target
associated antigens to antigen presenting cells (APC); and
immunostimulatory adjuvants (e.g., LPS, MLP, CpG, etc.) that
directly activate innate immune responses. The HBcAg platform
provides a delivery system that targets antigen-specific B cells
and other primary APC, as well as efficient T cell help for
antigen-specific B cells. Additionally, the core platform functions
as an immunostimulatory adjuvant by directly activating
antigen-specific B cells by virtue of cross-linking membrane
immunoglobulin (mIg) receptors for induction of B7.1 and B7.2
costimulatory molecule expression on naive resting B cells (Milich
et al., Proc Natl Acad Sci USA, 94:14648-14653, 1997).
II. Non-Primate (e.g., Rodent and Avian) Hepatitis Virus Core
Platform
[0209] A. Woodchuck Hepatitis Virus (WHV)
[0210] During development of the present invention, comparative
studies between the HBcAg and the WHcAg were undertaken. The WHcAg
is 67% identical at the amino acid level to the HBcAg, and the
comparative studies herein revealed that, the HBcAg and WHcAg
proteins do not significantly crossreact at the antibody level and
only partially at the CD4.sup.+ T cell level. However, these two
particulate antigens share a number of characteristics including:
enhanced immunogenicity of T cell-independent, as well as T
cell-dependent antibody production; the absence of nonresponder
MHC-haplotypes; efficient antigen-specific B cell activation; the
ability of naive WHcAg or HBcAg-specific B cells to act as primary
APC for naive core-specific Th cells; and the ability to act as a
carrier moiety for foreign epitopes. In fact, in a direct
comparison of an HBcAg-CS vaccine candidate (V12.PF3.1) and a
WHcAg-based hybrid particle containing the same CS repeat sequence
(188-M74), a single 20 .mu.g dose of the WHcAg hybrid particle in
IFA elicited significantly higher levels of anti-NANP antibodies
with a better persistence profile than the same dose of the HBcAg
hybrid vaccine (See, FIG. 2). Furthermore, the WHcAg appears to
tolerate insertions of foreign epitopes at a greater number of
positions than the HBcAg, as illustrated in FIG. 3. A number of
internal insertions inside the loop region (positions 76-82), as
well as internal insertions outside the loop region were tolerated
by WHcAg. This is in sharp contrast to the rather limited number of
efficient insertion sites described for the HBcAg, including those
in loop positions 77, 78, 81, 82 (Pumpens and Grens, Intervirology,
44:98-114, 2001). Importantly, the identification of an expanded
number of insertion sites was dependent on additional modifications
to the C-terminus that stabilize the internal insertions. Indeed,
21 separate C-terminal modifications (See, Table 1) have been
generated for use in combination with 17 insertion sites, to ensure
efficient hybrid WHcAg particle assembly. Additionally, the insert
sequence was found to effect hybrid WHcAg assembly competence. For
example, highly positively-charged epitope inserts tended to
destabilize hybrid particle assembly. Thus, three variables
relevant to the design of hybrid hepadnavirus core particles have
been identified including insert position, C-terminus and epitope
sequence.
TABLE-US-00001 TABLE 1 Sequences of the C-Termini of the Woodchuck
Vaccine Platform.sup.1 Designation WHcAg C-Terminal Sequence
Identifier wild type RRRGGARASRSPRRRTPSPRRRRSQS SEQ ID NO: 2
PRRRRSQSPSANC 150R R N/A 150C C N/A 150-2RC RRC N/A 150-3RC RRRC
SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC SEQ ID NO: 5
150-3AC AAAC SEQ ID NO: 6 WT-R AAGGARASRSPSQSPSQSPSANC SEQ ID NO: 7
WT-R1 AAGGARASRSQSPSQSPSANC SEQ ID NO: 8 WT-R2 AAGGARASRSQSSQSPSANC
SEQ ID NO: 9 WT-R3 AAGGARASRSQSSQSSANC SEQ ID NO: 10 C-Long
RRGGARASQSANC SEQ ID NO: 11 C-Long(M1) ARGGARASQSANC SEQ ID NO: 12
C-Long(M2) RAGGARASQSANC SEQ ID NO: 13 C-Long(M3) AAGGARASQSANC SEQ
ID NO: 14 HyW AAGRSPSQSPSQSRESQC SEQ ID NO: 15 HyW-1
AAGRSPSQSPSQSPSANC SEQ ID NO: 16 HyW-2 AAGRSPSQSPSQSSANC SEQ ID NO:
17 HyW-3 AAGRSQSPSQSSANC SEQ ID NO: 18 HyW-4 AAGRSPSQSSQSSANC SEQ
ID NO: 19 HyW-5 AAGRSQSSQSSANC SEQ ID NO: 20 .sup.1The wild type
C-terminal protein sequence corresponds to positions 150-188. The
full length protein sequence of WHcAg is set forth herein as SEQ ID
NO: 1, while the full length DNA sequence is set forth as SEQ ID
NO: 37. Additionally, the wild type N-terminal protein sequence
(corresponding to positions 1-149) is set forth as SEQ ID NO:
38.
[0211] A combinatorial approach was made feasible by development of
an ELISA-based screening system to detect core protein expression
level, insert antigenicity and particle assembly in the lysates of
transformed bacteria, prior to purification. Although a Plasmodium
circumsporozoite (CS) repeat was used as a model epitope, this
technology is not confined to a limited set of epitopes. In fact,
insertion of 22 out of 24 different epitopes into the WHcAg
platform has been successfully accomplished during development of
the present invention. Another bottleneck that had existed in the
characterization of hybrid core particles was the necessity for in
vivo immunogenicity testing requiring 4-6 weeks for the analysis of
a primary response. This bottleneck has been widened during
development of the present invention by utilizing in vitro antibody
production as a correlate of in vivo immunogenicity. The in vitro
antibody production assay requires just 5 days of tissue culture.
Establishment of in vitro antibody production as a predictor of in
vivo immunogenicity is a powerful screening tool dramatically
shortening the time necessary for the development of hybrid WHcAg
particle vaccine candidates. Subsequently, once a vaccine candidate
is shown to induce antibody production in vitro, then in vivo
studies of dose, route and formulation are completed.
[0212] Three exemplary categories of model antigens are
contemplated to be successfully accommodated by the WHcAg platform
system including: i) peptidic epitopes inserted into WHcAg by
recombinant methods; ii) polysaccharide (PS) antigens chemically
conjugated to directly to or lysine-modified WHcAg particles; and
iii) larger, non-linear protein/polypeptide antigens incorporated
into WHcAg by recombinant or chemical methods. In some embodiments,
the incorporation of larger protein sequences is accomplished by
production of mosaic WHcAg particles comprised of an optimal
mixture of wild-type WHcAg and WHcAg-fusion proteins containing the
desired inserted sequence. This mosaic approach is also suitable
for utilization of so-called molecular adjuvants through linkage to
the C-terminal amino acid residue of WHcAg particles. A number of
useful molecular adjuvants, which bridge the gap between innate and
adaptive immunity, have in common the ability to provide a
co-stimulus targeting immune cells (typically B cells or other
APCs). Linkage of a molecular adjuvant to a hybrid WHcAg particle
is contemplated to be advantageous in that the antigen-specific B
cell or APC taking up the particle become activated, as opposed to
the non-specific activation induced by merely mixing adjuvant and
antigen.
[0213] Additionally in other embodiments, the hepadna virus core
platforms are utilized in non-infectious disease situations, such
as those requiring high level in vivo antibody production (as an
alternative to monoclonal antibody therapy). For example, active
immunization to elicit anti-TNF.alpha. therapeutic autoantibodies
is contemplated to have a number of advantages over monoclonal
anti-TNF.alpha. therapy for the treatment of arthritis and other
inflammatory diseases.
[0214] Without limiting the invention, advantages of using modified
WHcAg particle vaccine provided by the present invention include:
i) WHcAg is equally or more immunogenic than the HBcAg at the T and
B cell levels; ii) WHcAg will not substantially compromise the use
of the anti-HBc diagnostic assay because the WHcAg and HBcAg are
not substantially crossreactive at the antibody level; iii)
pre-existing anti-HBe antibodies in HBV chronically infected
patients or in previously infected and recovered persons may limit
the efficacy of the HBcAg platform, whereas, the WHcAg and GSHcAg
platforms do not bind pre-existing anti-HBc antibodies; (iv) immune
tolerance in HBV chronic carriers can be circumvented by the use of
the WHcAg, GSHcAg, and/or ArGSHcAg platform because the HBcAg is
only partially crossreactive at the T cell level with WhcAg,
GSHcAg, and/or ArGSHcAg; and v) the WhcAg, GSHcAg, and/or ArGSHcAg
combinatorial technologies are more versatile than the HBcAg in
terms of accommodating the insertion of a greater variety of
foreign epitopes (Also see Table 2).
TABLE-US-00002 TABLE 2 Summary of Some Advantages Of Using WHcAg,
GSHcAg, and/or ArGSHcAg as Vaccine Platforms No. Advantage 1
Efficient self-assembly into 25-35 nm particles allowing for
multivalency of inserted epitopes and combination vaccines. 2
Highly immunogenic during natural infection and vaccination. 3 1-2
doses required in animal models. 4 A library comprising 17
insertion positions and 21 C-terminal modifications is provided by
the current invention. 5 A combinatorial technology involving
insert position, C-terminus and foreign sequence is provided by the
current invention. 6 Linker residues permitting assembly of core
particles containing destabilizing foreign sequences is provided by
the current invention. 7 Th cell as well as B cell epitopes are
accommodated on hybrid particles. 8 Hybrid particles elicit a broad
spectrum of IgG isotypes. 9 Hybrid particles do not require an
adjuvant, although immunogenicity can be enhanced by a
metabolizable oil/alum depot effect. 10 Core particles can
accommodate incorporation of a molecular adjuvant and/or immune
enhancer. 11 Core particles can accommodate linkage of carbohydrate
antigens and large non- linear protein antigens. 12 Hybrid
particles are very stable (e.g., a cold chain is not necessarily
required). 13 Use of core particles does not compromise the
anti-HBc diagnostic assay. 14 Use of core particles avoids the
problem of immune tolerance in HBV-infected individuals. 15
Recombinant core particles can be produced in E. coli, which is
cost effective and scaleable. 16 Use of core particles avoids the
problem of pre-existing anti-HBc antibodies.
[0215] B. Ground Squirrel Hepatitis Virus (GSHV) and Arctic Ground
Squirrel Hepatitis Virus (ArGHV)
[0216] In another embodiment, a second new combinatorial platform
technology is developed by modification of the ground squirrel
hepadna virus (GHV) core protein (GSHcAg) and the arctic ground
squirrel hepadna virus (ArGHV) core protein (ArGHcAg). The GSHcAg
is 91% identical at the amino acid level to the WHcAg.
Modifications to the C-terminus of the of the GSHcAg and ArGHcAg
proteins, similar to those described above for WHcAg, are made as
shown in Tables 3-1 and 3-2.
[0217] The GSHcAg is 67% identical at the amino acid level to the
HBcAg and the comparative studies revealed that, the HBcAg and
GSHcAg proteins do not significantly crossreact at the antibody
level (FIG. 6 and Table 19) and only partially at the CD4.sup.+ T
cell level (Table 20 and FIG. 43). However, the GSHcAg and the
WHcAg do show significant crossreactivity at both the antibody
level (FIG. 6 and Table 19) and at the CD4.sup.+ T cell level
(Table 20 and FIG. 43). It is notable that the B cell
crossreactivity between the GSHcAg and the WHcAg occurs outside the
loop regions, which are variable between the GSHcAg and the WHcAg.
For example, polyclonal anti-WHc antibodies recognize WHcAg
25-times better than GSHcAg, whereas, these same antibodies
recognize WHcAg with a disrupted loop region (WHc Aloop)
equivalently to GSHcAg and GSHcAg (A loop). The reciprocal is also
true, polyclonal anti-GSHc antibodies recognize GSHcAg 125-times
better than WHcAg yet recognize WHcAg, WHc (A loop) or GSHc (A
loop) equivalently (Table 19). Both anti-WHc and anti-GSHc
antibodies demonstrate a significant degree of crossreactivity for
Arctic Ground Squirrel Core particles (ArGSHc). Additionally, as
shown in FIG. 5, the GSHcAg is an efficient immunogen. Whereas the
WHcAg is more immunogenic than the HBcAg in 5 of 8 H-2 congenic
strains and equivalent in 3 other strains, the GSHcAg is equally or
more immunogenic than the WHcAg in the 8 H-2 congenic strains and
more immunogenic than the HBcAg in all 8 strains.
[0218] In terms of T cell crossreactivity, the GSHcAg and the WHcAg
demonstrate crossreactivity in all 8 H-2 congenic strains, whereas
the GSHcAg and the HBcAg demonstrate CD4+ T cell crossreactivity in
2 of 8 strains (Table 20). The basis for T cell crossreactivity
between GSHcAg, WHcAg and HBcAg is demonstrated by T cell epitope
mapping studies (FIG. 43). The GSHcAg and the WHcAg share at least
1 overlapping T cell epitope in all 8 strains examined, whereas the
GSHcAg and HBcAg share overlapping T cell sites in 2 of 8
strains.
TABLE-US-00003 TABLE 3-1 Sequences of the C-Termini of the Ground
Squirrel Vaccine Platform.sup.1 Designation GSHcAg C-Terminal
Sequence Identifier wild type RRRGGSRAARSPRRRTPSPRRRRSQS SEQ ID NO:
22 PRRRRSQSPASNC 150R R N/A 150C C N/A 150-2RC RRC N/A 150-3RC RRRC
SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC SEQ ID NO: 5
150-3AC AAAC SEQ ID NO: 6 WT-R AAGGSRAARSPSQSPSQSPASNC SEQ ID NO:
23 WT-R1 AAGGSRAARSQSPSQSPASNC SEQ ID NO: 24 WT-R2
AAGGSRAARSQSSQSPASNC SEQ ID NO: 25 WT-R3 AAGGSRAARSQSSQSASNC SEQ ID
NO: 26 C-Long RRGGSRAASQASNC SEQ ID NO: 27 C-Long(M1)
ARGGSRAASQASNC SEQ ID NO: 28 C-Long(M2) RAGGSRAASQASNC SEQ ID NO:
29 C-Long(M3) AAGGSRAASQASNC SEQ ID NO: 30 HyW AAGRSPSQSPSQSRESQC
SEQ ID NO: 31 HyW-1 AAGRSPSQSPSQSPASNC SEQ ID NO: 32 HyW-2
AAGRSPSQSPSQSASNC SEQ ID NO: 33 HyW-3 AAGRSQSPSQSASNC SEQ ID NO: 34
HyW-4 AAGRSPSQSSQSASNC SEQ ID NO: 35 HyW-5 AAGRSQSSQSASNC SEQ ID
NO: 36 .sup.1The wild type C-terminal sequence corresponds to
positions 149-187. The full length protein sequence of GSHcAg is
set forth herein as SEQ ID NO: 21, while the full length DNA
sequence is set forth as SEQ ID NO: 39. Additionally, the wild type
N-terminal protein sequence (corresponding to positions 1-148) is
set forth as SEQ ID NO: 40.
TABLE-US-00004 TABLE 3-2 Sequences of the C-Termini of the Arctic
Ground Squirrel Vaccine Platform.sup.1 ArGSHcAg Designation
C-Terminal Sequence Identifier wild type RRRGSARVVRSPRRRTPSPRRRRS
SEQ ID NO: 153 QSPRRRPQSPASNC 150R R N/A 150C C N/A 150-2RC RRC N/A
150-3RC RRRC SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC
SEQ ID NO: 5 150-3AC AAAC SEQ ID NO: 6 WT-R AAGSARVVRSPSQSPQSPASNC
SEQ ID NO: 183 WT-R1 AAGSARVVRSSQSPQSPASNC SEQ ID NO: 184 WT-R2
AAGSARVVRSSQSQSPASNC SEQ ID NO: 185 WT-R3 AAGSARVVRSSQSQSASNC SEQ
ID NO: 186 C-Long RRGSARVVSQASNC SEQ ID NO: 187 C-Long(M1)
ARGSARVVSQASNC SEQ ID NO: 188 C-Long(M2) RAGSARVVSQASNC SEQ ID NO:
189 C-Long(M3) AAGSARVVSQASNC SEQ ID NO: 190 HyW AAGRSPSQSPQSRESQC
SEQ ID NO: 191 HyW-1 AAGRSPSQSPQSPASNC SEQ ID NO: 192 HyW-2
AAGRSPSQSPQSASNC SEQ ID NO: 193 HyW-3 AAGRSQSPQSASNC SEQ ID NO: 194
HyW-4 AAGRSPSQSQSASNC SEQ ID NO: 195 HyW-5 AAGRSQSQSASNC SEQ ID NO:
196 .sup.1The wild type C-terminal protein sequence corresponds to
positions 150-187. The full length protein sequence of ArGSHcAg is
set forth herein as SEQ ID NO: 102, while the full length DNA
sequence is set forth as SEQ ID NO: 127. Additionally, the wild
type N-terminal protein sequence (corresponding to positions 1-149)
is set forth as SEQ ID NO: 154.
[0219] C. Avian Hepatitis Virus
[0220] In another embodiment, avian hepatitis virus core antigens
(such as from duck, Ross' goose, Sheldgoose, heron, stork, etc.)
may be modified as shown in Table 3-3.
TABLE-US-00005 TABLE 3-3 Sequences of the C-Termini of the Avian
Vaccine Platform.sup.1 Designation Avian HcAg C-Terminal Sequence
Identifier wild type AQGGRKTSSGTRKPRGLEPRRRKVKTTFVYGRRRSK SEQ ID
NO: 163 duck SRERRAPSPQRAGSPLPRSSSSHHRSPSPRK wild type
AQGGRKTTTGTRKPRGLEPRRRKVKTTVVYGRRRS SEQ ID NO: 165 duck
KSRERRAPTPQRAGSPLPRSSSSHHRSPSPRK wild type
AQGGRKTSSGTRKPRGLEPRRRKVKTTVVYGRRRS SEQ ID NO: 167 duck
KSRDRRAPSPQRAGSPLPRSSSSHHRSPSPRK wild type
AQGGRKTSSGTRKPRGLEPRRRKVKTTVVYGRRRS SEQ ID duck
KSRERRAPSPQRAGSPLPRSSSSHHRSPSPRK NOs: 169, 171, 173 wild type
AQGGRNKTQGVRKSRGLEPRRRRVKTTIVYGRRRS SEQ ID NO: 175 Ross' goose
KSRERRAPTPQRAGSPLPRTSRDHHRSPSPRE wild type
AQGGRNKTQGSRKPRGLQPRRRKVKTTVVYGRRRS SEQ ID NO: 177 Sheldgoose
KSRDRRAPSPQRAGSPLPRPSTSHHRSPSPRK wild type
AQGGRNQTKGTRKPRGLEPRRRKVKTTVVYGRRRS SEQ ID NO: 179 heron
KSRGRRSSPSQRAGSPLPRNRGNQTRSPSPRE wild type
AQGSRNQTKGVRKPRGLEPRRRKVKTTVVYGRRRS SEQ ID NO: 181 stork
KSRGRRSSPSQRAGSPEPRNRENQSRSSSPRE 150R R N/A 150C C N/A 150-2RC RRC
N/A 150-3RC RRRC SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC
KKKC SEQ ID NO: 5 150-3AC AAAC SEQ ID NO: 6 WT-R
AAGGERGVRSPSQSPSRSPSPRK SEQ ID NO: 225 WT-R1 AAGGERGVRSQSPSRSPSPRK
SEQ ID NO: 226 WT-R2 AAGGERGVRSQSSRSPSPRK SEQ ID NO: 227 WT-R3
AAGGERGVRSQSSRSSPRK SEQ ID NO: 228 C-Long RRGGARASRSPSPRK SEQ ID
NO: 229 C-Long(M1) ARGGARASRSPSPRK SEQ ID NO: 230 C-Long(M2)
RAGGARASRSPSPRK SEQ ID NO: 231 C-Long(M3) AAGGARASRSPSPRK SEQ ID
NO: 232 HyW AAGRSPSQSPSSRSPSPRK SEQ ID NO: 233 HyW-1
AAGRSPSQSPSSRSPSPRE SEQ ID NO: 234 HyW-2 AAGRSPSQSPSSRSSPRE SEQ ID
NO: 235 HyW-3 AAGRSQSPSSRSSPRE SEQ ID NO: 236 HyW-4
AAGRSPSQSSRSSPRE SEQ ID NO: 237 HyW-5 AAGRSQSSRSSPRE SEQ ID NO: 238
.sup.1The wild type C-terminal protein sequence corresponds to
positions 196-282. The full length protein sequence of Avian HcAg
(duck, goose, heron, etc.) are set forth herein as SEQ ID NOs:
119-126, 151 and 152, while the full length DNA sequence is set
forth as SEQ ID NOs: 143-150. Additionally, the wild type
N-terminal protein sequence (corresponding to positions 1-195) is
set forth as SEQ ID NOs: 164, 166, 168, 170, 172, 174, 176, 178,
180, and 182.
III. Primate Hepatitis B Virus (HBV) Core Platform
[0221] In a further embodiment, primate hepatitis virus core
antigens are provided by the invention. In one embodiment, the
human hepatitis B virus (HBV) core antigen (HBcAg) platform as well
as the hepatitis B virus core antigens for woolly monkey,
orangutan, gibbon, and chimpanzee are improved through introduction
of various modifications to the C-terminus as shown in Tables 4-1,
4-2, and 4-3.
TABLE-US-00006 TABLE 4-1 Sequences of the C-Termini of the Human
HBcAg Vaccine Platform Designation HBcAg C-Terminal Sequence
Identifier wild type RRRGRSPRRRTPSPRRRRSQS SEQ ID NO: 42
PRRRRSQSRESQC 150R R N/A 150C C N/A 150-2RC RRC N/A 150-3RC RRRC
SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC SEQ ID NO: 5
150-3AC AAAC SEQ ID NO: 6 WT-R AAGRSPSQSPSQSRESQC SEQ ID NO: 43
WT-R1 AAGRSQSPSQSRESQC SEQ ID NO: 44 WT-R2 AAGRSQSSQSRESQC SEQ ID
NO: 45 WT-R3 AAGRSQSSQSESQC SEQ ID NO: 46 C-Long RRGSQSRESQC SEQ ID
NO: 47 C-Long(M1) ARGSQSRESQC SEQ ID NO: 48 C-Long(M2) RAGSQSRESQC
SEQ ID NO: 49 C-Long(M3) AAGSQSRESQC SEQ ID NO: 50 HyW
AAGRSPSQSPSQSPSANC SEQ ID NO: 51 HyW-1 AAGRSPSQSPSQSRESQC SEQ ID
NO: 52 HyW-2 AAGRSPSQSPSQSESQC SEQ ID NO: 53 HyW-3 AAGRSQSPSQSESQC
SEQ ID NO: 54 HyW-4 AAGRSPSQSSQSESQC SEQ ID NO: 55 HyW-5
AAGRSQSSQSESQC SEQ ID NO: 56 .sup.1The wild type C-terminal
sequence corresponds to positions 150-183. The full length protein
sequence of HBcAg is set forth herein as SEQ ID NO: 41, while the
full length DNA sequence is set forth as SEQ ID NO: 57.
Additionally, the wild type N-terminal protein sequence
(corresponding to positions 1-149) is set forth as SEQ ID NO:
58.
TABLE-US-00007 TABLE 4-2 Sequences of the C-Termini of the Woolly
Monkey Vaccine Platform.sup.1 WMHcAg Designation C-Terminal
Sequence Identifier wild type RRRRPSGRRTPSPRRRRSQSPRR SEQ ID NO:
155 RRSQSPASSC 150R R N/A 150C C N/A 150-2RC RRC N/A 150-3RC RRRC
SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC SEQ ID NO: 5
150-3AC AAAC SEQ ID NO: 6 WT-R AARPSPSQSPSQSPASSC SEQ ID NO: 197
WT-R1 AARPSQSPSQSPASSC SEQ ID NO: 198 WT-R2 AARPSQSSQSPASSC SEQ ID
NO: 199 WT-R3 AARPSQSSQSASSC SEQ ID NO: 200 C-Long RRGSQSRRSQSSC
SEQ ID NO: 201 C-Long(M1) ARGSQSRRSQSSC SEQ ID NO: 202 C-Long(M2)
RAGSQSRRSQSSC SEQ ID NO: 203 C-Long(M3) AAGSQSRRSQSSC SEQ ID NO:
204 HyW AARRRPSQSPSQSPASSC SEQ ID NO: 205 HyW-1 AARRRPSQSPSQSASSC
SEQ ID NO: 206 HyW-2 AARRRPSQSPSQSSSC SEQ ID NO: 207 HyW-3
AARRSQSPSQSSSC SEQ ID NO: 208 HyW-4 AARRSPSQSSQSSSC SEQ ID NO: 209
HyW-5 AARRSQSSQSSSC SEQ ID NO: 210 .sup.1The wild type C-terminal
protein sequence corresponds to positions 150-182. The full length
protein sequence of WMHcAg is set forth herein as SEQ ID NO: 118,
while the full length DNA sequence is set forth as SEQ ID NO: 135.
Additionally, the wild type N-terminal protein sequence
(corresponding to positions 1-149) is set forth as SEQ ID NO:
156.
TABLE-US-00008 TABLE 4-3 Sequences of the C-Termini of the
Orangutan, Gibbon, and Chimpanzee Vaccine Platform.sup.1 Orangutan,
Gibbon, and Chimpanzee Ag Designation C-Terminal Sequence
Identifier wild type RRRGRSPRRRTPSPRRRRS SEQ ID NO: 157, 159
QSPRRRRSQSPASQC and 161 150R R N/A 150C C N/A 150-2RC RRC N/A
150-3RC RRRC SEQ ID NO: 3 150-4RC RRRRC SEQ ID NO: 4 150-3KC KKKC
SEQ ID NO: 5 150-3AC AAAC SEQ ID NO: 6 WT-R AAGRSPSQSPSQSPASQC SEQ
ID NO: 211 WT-R1 AAGRSQSPSQSPASQC SEQ ID NO: 212 WT-R2
AAGRSQSSQSPASQC SEQ ID NO: 213 WT-R3 AAGRSQSSQSASQC SEQ ID NO: 214
C-Long RRGSQSPASQC SEQ ID NO: 215 C-Long(M1) ARGSQSPASQC SEQ ID NO:
216 C-Long(M2) RAGSQSPASQC SEQ ID NO: 217 C-Long(M3) AAGSQSPASQC
SEQ ID NO: 218 HyW AAGRSPSQSPSQSPASQC SEQ ID NO: 219 HyW-1
AAGRSPSQSPSQSASQC SEQ ID NO: 220 HyW-2 AAGRSPSQSPSQSASQC SEQ ID NO:
221 HyW-3 AAGRSQSPSQSASQC SEQ ID NO: 222 HyW-4 AAGRSPSQSSQSASQC SEQ
ID NO: 223 HyW-5 AAGRSQSSQSASQC SEQ ID NO: 224 .sup.1The wild type
C-terminal protein sequence corresponds to positions 150-183. The
full length protein sequence of Orangutan HcAg, Gibbon HcAg, and
Chimpanzee HcAg, are set forth herein as SEQ ID NO: 117, 116, 115,
respectively, while the full length DNA sequences are set forth as
SEQ ID NOs: 134, 136, 137, respectively. Additionally, the wild
type N-terminal protein sequences (corresponding to positions
1-149) are set forth as SEQ ID NOs: 158, 160, and 162,
respectively.
IV. Additional Modifications to the WHcAg Vaccine Platform
[0222] In some embodiments, the 188 amino acid wild-type (WT) WHcAg
is further modified by recombinant technology to increase the
number of potential insertion sites. The WT WHcAg core gene is
modified by creating and inserting unique cloning restriction
nucleic acid sequences flanked by the conserved ends. The number of
sites per core gene is varied to obtain bi or multivalent
particles. In particular additional insertion sites are created in
the following locations: i) in the alpha-helical core regions
forming the stem of the spike, and ii) in the N-terminal and
non-helical regions of the protein. Different cloning restriction
sites are used at different positions, and different linkers are
used with different heterologous inserted sequences.
[0223] The pUCWHc vector expressing the WHcAg sequence under the
control of the Lac Operon promoter is inserted between NcoI-BamH1
sites for subcloning convenience. The foreign inserted sequences
are designed as follows: i) for small linear peptidic epitopes,
direct synthesis of the corresponding nucleotide sequences is done
in order to flank the foreign sequence with the desired unique
restriction site(s) created on the plasmid; ii) for larger protein
fragments, the foreign sequence is first obtained by PCR from
plasmids encoding the complete corresponding gene, and modified
accordingly with unique flanking restriction sites.
[0224] A. Creation of New Insertion Sites
[0225] All insertions are accomplished by either using the
EcoR1-Xho1 sites or Sad sites, with the position of the insertion
differing between contructs (See, FIG. 20). Accordingly, new
primers/oligonucleotides are designed in order to encompass either
one or two restriction sites present on the wild-type WHcAg gene
and to code for 5'EcoR1-3'Xho1 sites or Sad sites.
[0226] 1. Direct Synthesis of Sense and Antisense
Oligonucleotides
[0227] The oligonucleotides span two other WHcAg gene restriction
sites (e.g., RS1, RS2) and do not exceed 100 nucleotides in length
(limit for direct synthesis). Both the plasmid pUC-WT and the
oligonucleotides are then digested by the RS1 and RS2 enzymes and
purified from a low-melting point agarose gel. The
RS1-EcoR1-Xho1-RS2 fragment replaces the corresponding RS1-RS2
sequence of the pUC-WT plasmid via ligation to produce
pUC-WT.DELTA. insertion site (e.g., pUC WT.DELTA. 98-99 corresponds
to WT WHcAg gene with insertion between amino acids 98 and 99).
[0228] 2. Design of PCR Primers
[0229] In some instances, the direct synthesis of oligonucleotides
cannot be realized because the desired insertion site is distanced
from one of two usable restriction sites of the WHcAg gene by more
than 100 nucleotides. In these cases, one of the primers (forward
or reverse depending on the desired orientation) is designed to
match the exact sequence of the WT WHcAg gene and to include a
unique restriction site (RS3). The other primer is designed to
create a mismatch (PCR mutagenesis) from the WT sequence and to
introduce EcoR1-Xho1 sites, plus an RS4 site belonging to the WT
gene. PCR with the forward-RS3, and the reverse-EcoR1-Xho1-RS4
primers is performed using the pUC-WT plasmid as a template. The
resulting PCR product and the pUC-WT plasmid are then digested by
RS3-RS4 and ligated to create the new pUC-WT.DELTA. insertion site.
Linkers are used when necessary to accommodate foreign sequences
such as for insertion of large fragments (Kratz et al., Proc Natl
Acad Sci USA, 96:1915-1920, 1999). By creating 5' EcoR1-Xho13' as
insertion sites, and keeping the same reading frame as that of the
WT WHcAg gene, each foreign sequence is flanked by the same linker,
Gly-Ile-Leu on their N-terminus, and Leu-Glu on their C-terminus.
Similarly by creating 5' SacI-SacI 3' as insertion sites, and
keeping the same reading frame as that of the WT WHcAg gene, each
foreign sequence is flanked by the same linker, Ser-Ser, on both
their N- and C-termini. The following primer sequence containing
both EcoR1 and Xho1 restriction sites, GGAAATTCTTCTCCTCGAG (SEQ ID
NO:63) is used for this purpose. Similarly, others sequences are
introduced to code for new linkers (e.g., Gly4-S-Gly4) on each side
of the foreign sequence.
[0230] B. Modifications of the C-Terminus
[0231] The library of C-termini is expanded to eliminate certain
motifs (e.g., RNA/DNA binding motifs) and to accommodate the
addition of other linker/spacer sequences. As described below in
the examples, modifications of the C-tenninus that enhance
expression/assembly and/or antigenicity/immunogenicity of various
hybrid core constructs have been characterized. The new C-termini
are modified by designing oligonucleotides encoding the sequence of
interest and flanked by 5' BseA1 and 3' BamH1 sites as a general
pattern, and then using the oligonucleotides to replace the
corresponding native fragment on the pUC-WT plasmid. All the WHcAg
constructs (insert sites, C-termini, +/-foreign sequences) are
sequenced in both directions at an automated sequencing facility.
The hybrid WHcAg constructs (pUC vectors) are then used to
transform chemically-competent Top 10 E. coli by heat shock. The
transformed Top10 grow overnight at low temperature 28.degree. C.
to avoid inclusion body formation, before the expression of the
protein is induced by addition of IPTG (1 mM for 4 h). The bacteria
are lysed in a lysozyme-salt solution containing proteolysis
inhibitors. The resulting supernatant is precipitated overnight in
the cold with 50% ammonium sulfate. The proteins are then purified
by chromatography on hydroxylapatite and Sepharose 4B columns. In
some embodiments, for better and tighter control of the expression,
each hybrid WHcAg construct is subcloned into another expression
vector, pET11d, at the Nco1-BamH1 sites. The pET11d vector allows
expression of the corresponding protein under an inducible T7/Lac
Operon promoter. These hybrid constructs are then transformed in
the BL21 (DE3) E. coli strain.
[0232] C. Rapid Screening Technology
[0233] The approach of combining the optimal C-terminus from a
selection of 21 termini and the optimal insert position from a
choice of 17 positions, in the context of a given epitope requires
a rapid screening technology that can be applied early in the
manufacturing process. Therefore, an antibody-based method for
detecting expression of core polypeptide, assembly of polypeptide
into core hybrid particles and for assessing antigenicity of the
inserted heterologous epitope has been developed. This rapid
screening technique is applied to lysates of the transformed E.
coli to assess the desirability of any given hybrid core before a
significant investment in vaccine production is made. As described
in the examples, capture ELISAs were designed either to detect the
WHcAg polypeptide as a marker of expression or to detect the WHcAg
particle as a marker for assembly, while insert-specific mAbs were
used to assess the expression level and antigenicity of the insert
(See, FIG. 21). Lysates were sequentially screened with mAbs that
preferentially recognize denatured WHcAg (anti-p WHc), assembled
WHcAg particles (anti-nWHc), and proper display of insert sequence
(insert-specific mAbs). Based on relative assembly scores of the
lysates, optimal hybrid particle gene constructs were selected for
further purification. The assembly score was based on the dilution
of detecting antibody that binds the hybrid particle relative to
its binding to wild-type WHcAg. A strong correlation between the
relative lysate assembly scores and the ability to purify hybrid
core particles in high yield has been observed during development
of the present invention. Every hybrid particle construct with an
assembly score of three or greater in the transformed bacterial
lysate has yielded easily-purifiable particles. In contrast,
constructs with assembly scores of two or less have been
problematic to purify (Table 11).
V. Antigenic and Immunogenic Characterization of WHcAg-Hybrid
Particles
[0234] A. Epitope Selection
[0235] A group of model epitopes/antigens has been selected for use
to further develop the WHcAg platform technology. Three categories
of antigens are examined: (1) peptidic epitopes are inserted using
recombinant methods; (2) polysaccharide (PS) antigens are
chemically conjugated to lysine-modified core particles; and (3)
larger or non-linear protein antigens are incorporated onto core
particles by recombinant or chemical methods. Selected peptidic
epitopes include those shown in Table 9. Selected
protein/polypeptide antigens include but are not limited to the
Bacillus anthracis capsular polypeptide poly-gamma-D-glutamic acid,
which has been chemically conjugated to WHcAg (See, FIG. 31).
[0236] B. Epitope Optimization on Hybrid Core Particles
[0237] Because the inserted epitope sequence can effect hybrid core
assembly or stability, it is useful to perform mutational analysis
of the epitope in order to map the necessary antibody contact
residues. Non-essential residues are subsequently substituted with
other less disruptive residues as needed. This strategy is also
useful for identifying analogs with improved antibody binding. The
M2e epitope serves as an example of this strategy. A list of
M2-WHcAg hybrid constructs (-IM2 series) with different C-termini
and two different insert sites were produced and the relative
expression levels and assembly competence scores are shown in FIG.
22. Note that all constructs harboring the wild-type M2e sequence
either assembled poorly or were purified as aggregates instead of
core particles. It is contemplated that the two cysteines in the
wild-type M2e sequence result in inappropriate inter- or
intra-particle disulfide bridges. Therefore, an M2e-specific mAb
(14C2, which inhibits Influenza A growth of most strains) was
tested for binding to a peptide analog panel, including
cysteine-substituted peptides as shown in FIG. 23. Because
substitution of either or both cysteine residues did not effect the
binding of the 14C2 mAb, hybrid core constructs carrying the
mutated M2e sequence, IM2(-), inserted at position 78 were
produced. The IM2(-) sequence expressed in the context of the 150C
C-terminus still resulted in aggregates during purification.
However, the IM2(-) sequence inserted at position 78 in the context
of the HyW-C-terminus allowed assembly and was easily purified
(See, FIG. 22). Subsequently, other combinations of C-termini and
insert positions have been found to accommodate the IM2(-)
sequence, such as HyW2-IM2(-)81. These other M2e-WHcAg hybrid
particles have also been tested for immunogenicity. Additional
modifications to the M2e epitope are also done to optimize the
hybrid WHcAg expression and particle assembly. In some embodiments,
substitution of single cysteine residues (C16 and C18) have been
made, and multiple copies of a M2e(-) truncated sequence were
inserted. Note that P4 (a 15-mer) also bound mAb 14C2 efficiently,
as did the polyclonal anti-HyW-IM2(-)78 antisera (See, FIG.
23).
[0238] C. Antigenicity
[0239] Prior to in vivo immunogenicity testing all purified hybrid
WHcAg particles are characterized for antigen expression at the B
cell level by measuring the ability to bind polyclonal or mAbs
specific for the WHcAg carrier and the peptidic, protein or PS
insert. The same capture ELISA system used to detect hybrid WHcAg
particles in bacterial lysates is used for purified particles. T
cell antigenicity is determined by assessing the ability of the
hybrid WHcAg particles to activate core-specific T cells in vitro.
For this purpose naive splenic T cells from T cell receptor (TCR)
transgenic (Tg) (7/16-5-TCR) mice which have a high frequency of
HBcAg-specific CD4.sup.+ T cells (.about.50%) are used. The 7/16-5
TCR crossreacts with HBcAg and WHcAg as it recognizes
HBcAg.sub.129-140 presented by IA.sup.b and this sequence is very
similar between HBcAg and WHcAg. After a 2 day culture of
7/16-5-TCR spleen cells with HBcAg/WHcAg, the IL-2 that is secreted
into the supernatant (SN) is measured by ELISA (See, FIGS. 24 and
25). This is a convenient and rapid screen to check if any of the
modifications to the WHcAg protein have disrupted core-specific T
cell recognition. Additional WHcAg-specific TCR-Tg lineages that
recognize different T cell sites are similarly employed (Chen et
al., J. Virol. 74: 7587-7599, 2000). In cases when a
pathogen-specific heterologous CD4.sup.+ T cell epitope is inserted
into the hybrid core particle, mice of the appropriate H-2
haplotype (e.g., high responder) are immunized with the hybrid
particle (10 .mu.g, subcutaneously in IFA) and draining lymph node
cells are harvested 7-10 days later for culture with the
heterologous peptide, as well as a WHcAg-derived peptide panel. T
cell activation and specificity is determined by cytokine
production (IL-2, IL-4, IFN.gamma.) recalled by the peptide antigen
panel. Cytokines are measured in 2 day (IL-2) or 4 day (IL-4,
IFN.gamma.) SNs by ELISA.
[0240] D. Immunogenicity
[0241] The immune response to hybrid-WHcAg particles and WHcAg-PS
conjugates is examined in detail. In addition to anti-insert or
anti-PS and anti-WHcAg antibody end-point titers, antibody
specificity, isotype distribution, antibody persistence and
antibody avidity are monitored. Examples of these assays are
provided below. In vivo immune responses to PS-WHcAg conjugates are
compared to free PS and to the same PS linked to other protein
carriers (e.g., tetanus toxoid). In vivo antibody production is
studied in inbred murine strains, in athymic mice, in H-2 congenic
mice, and in core-specific TCR-Tg mice. The use of these strains
permits the evaluation of non-H-2 and H-2 dependent genetic
influences on immune responsiveness, as well as T cell independence
(athymic mice). In addition, the TCR-Tg mice permit the screening
of a number of hybrid particles/conjugates rapidly in vivo (e.g.,
2-4 weeks), because the kinetics of antibody production to the
WHcAg carrier and inserted epitopes is accelerated in these
mice.
[0242] Carrier-specific and insert-specific Th cell immunogenicity
is monitored by assessing T cell activation, fine-specificity and
cytokine production. For B cell peptidic epitopes inserted into
WHcAg, or PS antigens linked to WHcAg, the source of T cell help is
predictably WHcAg-specific Th cells. However, if peptidic T cell
sites are inserted into WHcAg or larger protein fragments are
incorporated into WHcAg particles, then the source of functional T
cell help is not readily apparent. To determine if exogenous T cell
sites are functional, mutant core particles with a single
substitution at residue 132 (Y132A) have been produced. The
tyrosine at position 132 represents a dominant aggretopic
(MHC-binding) residue in H-2.sup.b mice, and this alanine
substitution converts H-2.sup.b mice into WHcAg-nonresponders at
the Th cell level. Therefore, production of hybrid WHcAg particles
containing the Y132A mutation in WHcAg allow the T cell helper
function of the exogenous T cell site to be measured as a function
of in vivo antibody production to the inserted B cell epitope.
[0243] E. In Vitro Correlates of the Immune Response to Hybrid Core
Particles
[0244] The hepatitis core proteins are extremely immunogenic in
vivo during natural infection and as immunogens. It is contemplated
that in vitro correlates of immunogenicity can serve as rapid
screening methods to circumvent long term in vivo studies.
[0245] 1. Induction of Costimulatory Molecules and T Cell
Activation
[0246] In vitro culture of naive resting murine B cells with native
HBcAg or WHcAg sufficiently crosslinks mIg receptors on
core-specific B cells for induction of the costimulatory B7.2 (24
hrs.) and B7.1 (72 hrs.) molecules (Milich et al., Proc Natl Acad
Sci, USA, 94:14648-14653, 1997). The HBV envelope particulate
antigen (HBsAg) does not demonstrate this property nor do many
non-particulate experimental antigens (e.g., hen egg lysozyme,
pigeon cytochrome C, etc.). This property is important because it
allows naive, resting B cells to become competent APC for primary T
cells. FIG. 24 illustrates that naive B cells more efficiently
present the HBcAg to naive CD4.sup.+ cells derived from 7/16-5-TCR
Tg mice than do the more classic APCs, splenic adherent cells
(MO/DC). In contrast, MO/DC APC present peptide HBcAg.sub.120-140
more efficiently than do B cells. Previously it has been shown that
HBcAg-specific B cells are the primary APC in murine spleen
cultures (Milich et al., supra, 1997), and that there is a high
frequency (8%) of HBcAg-binding B cells among naive murine spleen
cells (Lazdina et al., J Virol, 75:6367-6374, 2001), as well as in
naive human PBL (Cao et al., J Virol, 75:6359-6366, 2001).
Preliminary studies illustrate that the WHcAg and hybrid-WHcAg
particles containing malaria inserts in the loop are also
preferentially presented to T cells by naive splenic B cells (See,
FIG. 25). To confiim that this B cell APC function is dependent
upon induction of B7.1 and B7.2 costimulatory molecules a variety
of WHcAg-hybrid particles are cultured with naive resting, splenic
B cells over a 72 hour period. Induction of B7.1 and B7.2 mRNA is
then measured by RT-PCR, and expression of B7.1 and B7.2 protein is
measured by FACs analysis. WHcAg hybrid particles differing in
number of inserts, position of those inserts, and C-termini are
compared to identify correlations between structure and induction
of B7.2 and/or B7.1. Inhibition of B7.2/B7.1 induction with
anti-mIg and anti-insert monoclonal antibodies is contemplated to
confirm that B7.2/B7.1 induction is mediated through crosslinking
of the antigen-specific mIg receptor.
[0247] 2. In Vitro Primary Antibody Production as an In Vitro Model
of Immunogenicity
[0248] It is contemplated that like T cell activation, that
anti-core antibody production is mediated through core
antigen-specific B cells acting as the primary APC source. Indeed,
after five days in culture with either HBcAg or WHcAg, spleen cells
from 7/16-5 TCR-Tg mice were shown to produce high levels of IgM
anti-HBc or anti-WHc (See, FIG. 26). Strikingly, the WHcAg induced
higher levels of IgM anti-core antibody than did the HBcAg at all
antigen concentrations. During development of the present
invention, In vitro IgM anti-core production was found to: (1)
require core-specific T cells since this response does not occur in
non-TCR.sup.+ control splenic cultures; (2) require particulate
core antigens since non-particulate HBeAg or WHeAg elicit low
levels of antibody; and (3) be antigen-specific since the IgM
anti-HBc and anti-WHc antibodies produced are non-crossreactive. To
determine the generality of this finding, a variety of hybrid WHcAg
particles containing different inserted epitopes at different
positions with varying C-termini were tested in the in vitro IgM
antibody production assay using 7/16-5-TCR spleen cells. Indeed, as
shown in FIG. 27, hybrid core particles did elicit in vitro IgM
antibody production variably from high levels to no antibody.
Importantly, the level of in vitro IgM antibody production
positively correlated with anti-insert IgG antibody production in
vivo after primary immunization with hybrid WHcAg particles. Thus,
substitution of primary in vitro IgM production for in vivo
immunization is contemplated to be an efficient method for
screening large numbers of hybrid WHcAg particles relatively
quickly. Interestingly, the IgM antibodies present in the SNs bind
to their respective hybrid WHcAg particles better than to native
WHcAg, and better than to their respective peptide inserts. Thus,
the IgM antibodies are contemplated to recognize a combined
structural component on the surface of the hybrid particles
[0249] F. Functional Characteristics of Immune Responses to Hybrid
Core Particles
[0250] As many of the heterologous epitopes utilized are engineered
or weak peptidic and PS epitopes, it is important to determine the
extent to which antibodies induced by hybrid WHcAg particle or
core-glycoconjugate immunization recognize the native antigen
(preferably in the context of the pathogen). For instance, anti-M2e
antibodies elicited by immunization with HyW-IM2(-)78 particles
recognize native M2 in Influenza A infected cell cultures (See,
FIG. 34). In some embodiments when it is not practical to test
antibody binding on the pathogen, at least a purified native
protein is also tested. For example anti-SEB.sub.140-151 and
anti-SEB.sub.152-161 antisera are tested by ELISA on a panel of
SEs. Similarly IgG antibody avidity for the native protein is
determined. For this purpose, Na SCN is used as a chaotropic agent
as previously described (Anttila et al., J Infect Dis,
177:1614-1621, 1998). The basic ELISA is performed with one
exception, before the detecting antibody is introduced, 100 .mu.l
of 0.5M Na SCN in PBS or PBS alone is added to each microtiter well
for 15 minutes at RT. The wells are then washed four times and the
ELISA is completed as usual. The results are expressed as avidity
indices (e.g., titer with Na SCN/titer without Na
SCN).times.100.
[0251] 1. Antibody Function
[0252] For example, anti-M2e antisera is assayed for in vitro
plaque size reduction (See, FIG. 35), as a measure of antibody
function. Another example involves an analysis of antibodies
reactive with SEB (a super antigen for V.beta.8.sup.+ T cells).
Both the SEB.sub.140-151 and SEB.sub.152-161 peptides and
antibodies have been shown to inhibit human V.beta.8+ T cell
activation in vitro (Arad et al., Nat Med, 6:414-421, 2000; and
Visvanathan et al., Infect Immunol, 69:875-884, 2001). Similarly,
passive transfer of anti-SEB.sub.152-161 and anti-SEB.sub.140-151
antisera have been shown to protect mice against a lethal
intra-peritoneal challenge with SEB and LPS. Although mice are not
very sensitive to SE-induced toxic shock, Balb/c mice primed with
D-galactosamine (20 mg) followed by low dose LPS (1-10 ng) become
extremely sensitive to SEB (20 ng) and exhibit 100% lethality
(Visvanathan et al., supra, 2001). Antisera generated by
immunization with HyW2-SE75 is tested in these two functional
assays. The host defense against many bacterial pathogens depends
on the opsonic activity of anti-PS antibodies, the complement
pathway, and phagocytosis by macrophages. Opsonic activity of mouse
antisera to WHcAg-PS conjugates is determined in an
opsonophagocytosis assay as described (DeVelasco et al., Vaccine,
12:1419-1422, 1994). Fluorescein-labelled bacteria are opsonized
with dilutions (0-20%) of heat-inactivated mouse antisera in the
presence or absence of complement (2%). Anti-PS antibodies are
neutralized prior to performing the assay. The mouse macrophage
cell line RAW-264 (10.sup.7 cells/ml) is used for phagocytosis of
bacteria at a bacteria/macrophage ratio of 10:1. Macrophages are
washed and analyzed by flow cytometry. The mean of the
FITC-intensity of the cells in each sample is used to estimate the
opsonic capacity of the antisera.
[0253] 2. Immunization and Challenge Studies
[0254] Once hybrid WHcAg particles or WHcAg-PS conjugates have been
optimized for immunogenicity and antibody function in vitro or via
passive transfer of immune sera, immunization/challenge experiments
are performed, dependent upon a number of factors. For example, the
availability of an infectious model system, the biosafety level of
the pathogen, and the appropriate collaborations with disease model
experts. In one embodiment, challenge experiments are done using
the Influenza A system.
[0255] VI. Expansion of the WHcAg Platform to Accommodate
Non-Linear, Large Protein and Carbohydrate Epitopes
[0256] A. Construct Design for Expression of Mosaic Core
Particles
[0257] The WHcAg platform technology is also applicable to
non-linear larger domains or protein fragments. Insertion of larger
amino acid sequences is advantageous not for presentation of a
larger number of epitopes, but also to allow appropriate folding of
conformational epitopes. Attempts to obtain stable large protein
hybrid HBcAg particles have failed (Koletzki et al., J Gen Virol,
78:2049-2053, 1997), with the single exception of the entire green
fluorescence protein inserted into the loop of the HBcAg (Kratz et
al., Proc Natl Acad Sci USA, 96:1915-1920). In addition to the
molecular adjuvants described below, the non-toxic C fragment of
tetanus toxoid (TTFC) is used as a model protein antigen. Since
challenge with tetanus toxoid (TT) is well established in the mouse
model, expression of TTFC is used to evaluate the ability of a
vaccine vector to elicit a protective antibody response.
Recombinant particles with TTFC inserted at the N- or C-terminus or
into the loop region of WHcAg are produced. In some embodiments,
the incorporation of TTFC into core particles is accomplished by
production of mosaic TTFC-WHcAg particles. Specifically, decreasing
the number of large foreign sequences per hybrid WHcAg particle to
be co-incorporated with unmodified WHcAg protein subunits is
contemplated to overcome any steric hindrance. The following
approach has been shown to be effective (Smiley and Minion, Gene,
134:33-40, 1993) and is compatible with the cloning and expression
vectors (pUC, and pET11d) described herein, although other
approaches are also suitable.
[0258] 1. Co-Expression of Wild-Type and WHcAg Fusion Proteins
Mediated by a Suppressor tRNA-Readthrough of a Stop Codon
[0259] As shown in FIG. 28, oligonucleotides are generated to
possess a TGA stop codon, as well as the coding information for an
additional five amino acids predicted to form a coil secondary
structure (Gly5). The oligonucleotides are annealed and the
resulting duplex is inserted between the wild-type WHcAg and the
fused protein (e.g., WHcAg-TTFC). The derivative plasmid (coding
for the fusion WHcAg protein) is used to transform the E. coli K12
K802 strain or others bacterial strains that possesses an opal
TGA-Trp suppressor tRNA under lac repressor control as one example
(Smiley and Minion, supra 1993). This approach results in the
co-expression of both the wild-type WHcAg protein (HyW) and the
fusion-WHcAg protein (HyW-TTFC) in the same bacterial cell. To
design the fusion-WHcAg core protein, several of the C-terminal
modifications are tested to identify those that favor the
expression/assembly of a mosaic core particle.
[0260] 2. Co-Expression of Wild-Type and WHcAg Fusion Proteins by
Using Differentially Inducible Plasmids
[0261] As shown in FIG. 29, a second approach is taken which
utilizes two plasmids differentially-induced to express the
wild-type and the fusion-WHcAg proteins. In some embodiments, the
constructs have been made in pUC18 as the cloning and expression
vector, permitting IPTG-inducible expression due to the presence of
the Lac promoter. For convenience, the gene coding for the
wild-type WHcAg is subcloned into the pLEX expression vector. The
pLex expression vector contains the strong P.sub.L promoter to
drive the expression of the gene of interest (e.g., wild-type WHc
gene). The P.sub.L promoter is controlled by the lambda cI
repressor protein, which is expressed in the E. coli host (G1698
strain). The cI repressor was engineered into the bacterial
chromosome under control of the tightly regulated trp promoter. The
expression of the gene is induced by addition of tryptophan thereby
suppressing the synthesis of the cI repressor. Therefore, the same
E. coli (G1698) is co-transformed with pUC encoding the
fusion-WHcAg protein and with pLEX encoding the wild-type WHcAg
protein. The induction of expression of the proteins is then
induced differentially by using IPTG and Tryptophan.
[0262] B. Traditional and Molecular Adjuvants
[0263] Although adjuvants are not required when using the WHcAg
delivery system, some embodiments of the present invention employ
traditional and/or molecular adjuvants. Specifically, immunization
in saline effectively elicits anti-insert antibody production.
However, formulation in non-inflammatory agents such as WA (mineral
oil), Montanide ISA 720 (squalene), and aluminum phosphate
(AlPO.sub.4), enhance immunogenicity (See, FIG. 30, Panel A).
Additionally, administration of WHcAg results in the production of
all four IgG isotypes, regardless of which if any adjuvant is
employed (See, FIG. 30, Panel B). Inclusion of a CpG motif also
enhances the primary response. Moreover, use of an inflammatory
adjuvant such as the Ribi formulation is not more beneficial than
is the use of non-inflammatory adjuvants, indicating that the
benefits of the adjuvants result from a depot effect rather than
from non-specific inflammation. Thus, the core platform is used
with no adjuvant or with non-inflammatory adjuvants depending upon
the application and the quantity of antibody desired. In some
embodiments of the present invention, IFA is used in murine
studies, whereas alum or squalene is used in human studies.
[0264] In instances where it is desirable to deliver hybrid WHcAg
particles in a single dose in saline (e.g., a nasal influenza A
M2e-core post-exposure vaccine), a molecular adjuvant is employed.
A number of molecular adjuvants are employed to bridge the gap
between innate and adaptive immunity by providing a co-stimulus to
target B cells or other APCs. For this purpose in some embodiments,
the complement C3d fragment (GenBank Accession No. NM 009778) is
employed, as two or three copies of C3d linked to the experimental
antigen hen egg lysozyme (HEL) was shown to be three to four orders
of magnitude more immunogenic than HEL alone (Dempsey et al.,
Science, 271:348-350, 1996), even in the absence of a traditional
adjuvant. C3d targets antigen to B cell and follicular dendritic
cells via binding to CD21, thereby costimulating B cells through
its association with CD19, a B cell membrane protein that amplifies
B cell activation (Tedder et al., Immunol Today, 15:437-442,
1994).
[0265] Similarly, soluble dimeric or trimeric forms of CD40L
(GenBank Accession No. X65453) have been shown to bind and
cross-link membrane CD40 sufficiently to induce B cell
proliferation, costimulate Ig class switching, suppress B cell
apoptosis and activate APC (Morris et al., J Biol Chem,
274:418-423, 1999). Additional potential molecular adjuvants
include but are not limited to: i) soluble BAFF (B cell activating
factor belonging to the TNF family; GenBank Accession No.
AF119383), which exclusively binds to B cells and functions as a
potent B cell growth factor (Mackay and Browning, Nature Reviews
Immunology, 2:465-475, 2002), ii) soluble LAG-3 (lymphocyte
activation gene-3; GenBank Accession No. NM 008479), which binds
MHC class II molecules with high avidity and elicits
activation/maturation of dendritic cells (ElMir and Triebel, J
Immunol, 164:5583-5589, 2000), and iii) immunostimulatory CpG
oligodeoxynucleotides, which costimulate a variety of immune cells
(Krieg et al., Nature, 374:546-549, 1995). In some embodiments,
these molecules are linked to the C-terminus of hybrid core
particles to activate the antigen-specific B cell or APC that takes
up the particle, as opposed to the non-specific activation induced
by merely mixing the adjuvants with antigen. Less than 100%
substitution is desirable because of potential negative effects on
particle assembly and/or overstimulation of the targeted cell.
Therefore, in some embodiments, mosaic hybrid core particles
carrying fewer adjuvant molecules are produced.
[0266] 1. Inclusion of CpG Dinucleotides in Hybrid Core
Particles
[0267] Unmethylated CpG dinucleotides have been shown to be potent
immune activators of B cells and macrophages (Krieg et al., supra,
1995; and Davis et al., J Immunol, 160:870-876, 1998).
Additionally, co-immunization of antigen and CpG dinucleotides (DN)
enhances the immune response similar to traditional adjuvants. Two
characteristics of the CpG effect include: i) B cell uptake is
required for activation; and ii) the CpG motif preferentially
activates B cells that simultaneously encounter their specific
antigen. Given the non-specificity of the effects (e.g., all B
cells will internalize CpG DN), large quantities of CpG DN are
necessary in vivo.
[0268] Hybrid core-CS(NANP) particles are a very efficient method
for delivery of CpG DN to the interior of antigen-specific B cells.
A core-CpG DN complex directly binds to core-specific B cells, and
B cell activation occurs due to crosslinking of the mIg receptor
and simultaneous delivery of the CpG DN to the B cell interior.
This permits efficient, selective delivery of CpG DN to activated,
antigen-specific B cells (e.g., NANP-specific B cells in the case
of core-CS hybrid particles). Indeed, preliminary data indicate
that HBcAg or HBcAg-CS hybrid particles carrying bacterial RNA/DNA
are more immunogenic than particles devoid of E. coli-derived
RNA/DNA. Full-length HBcAg possesses a RNA/DNA binding sequence at
the C-terminus, which is lost upon truncation at residue 149.
Unmethylated CpG DNs are much more frequently found in bacterial
DNA than in vertebrate DNA. As shown in Table 5, full-length
HBcAg.sub.183 is significantly more immunogenic than truncated
HBcAg.sub.149 when limiting doses (0.2 .mu.g) are injected in
saline in the absence of a traditional adjuvant. Unexpectedly, this
difference in immunogenicity is abolished when an adjuvant is used
(e.g., CFA).
TABLE-US-00009 TABLE 5 Bacterial Nucleic Acid Augments the
Immunogenicity of HBcAg Particles.sup.1 Dose RNA Anti-HBc Titer
Anti-NANP Titer Immunogen (.mu.g) DNA G.sub.1 G.sub.2a G.sub.2b
G.sub.3 G.sub.1 G.sub.2a G.sub.2b G.sub.3 HBcAg183 0.2 + 0 40,960
40,960 650 HBcAg149 0.2 - 0 0 640 0 HBcAg183- 1.0 + 10,240 2,560
10,240 640 160 160 10,240 640 NANP HBcAg149- 1.0 - 0 160 640 0 40
40 160 160 NANP .sup.1Balb/c mice were immunized with the indicated
dose of full-length HBcAg (183 amino acids), truncated HBcAg (149
amino acids), or full-length or trucated HBcAg containing
(NANP).sub.4 inserts in the loop region suspended in saline. Serum
was collected four weeks after primary immunization and analyzed by
IgG isotype-specific ELISA.
[0269] The positive effect of bacterial RNA/DNA was also observed
when full-length versus truncated HBcAg-CS hybrid particles were
used, resulting in significantly higher anti-NANP antibody
production. Synthetic CpG DNs are first chemically coupled to WHcAg
or WHcAg-CS hybrid particles. For this purpose, CpG DNA is modified
to contain 5' amino groups, which are subsequently used to
conjugate the oligonucleotides to WHcAg particles. In other
embodiments, recombinant methods are used to incorporate CpG motifs
into the interior of WHcAg using nucleic acid-binding motifs.
Internalization of the CpG sequence is expected to reduce its
sensitivity to nucleases. The effects of inclusion of CpG DN into
hybrid WHcAg particles is determined by immunization of hybrid
particles with and without CpG DN, by comparing the anti-insert and
anti-WHcAg humoral responses and the WHcAg-specific Th cell
responses in various in vivo assays. CpG DN-coupled hybrid
particles are also used in various TCR-Tg splenic in vitro assays
of cytokine production and IgM antibody production.
[0270] 2. Other Molecular Adjuvants
[0271] Genes encoding the murine CD40L (both 655 and 470 nucleic
acid versions) have been used successfully to express these ligands
at the C-terminus of WHcAg (See, FIG. 38). Moreover, immunization
of mice with hybrid WHcAg-CD40L particles results in the production
of higher anti-core antibody titers than does the immunization of
mice with WHcAg particles (See, FIG. 39). However, lower than
desirable yields of purified particles have been obtained.
Therefore, mosaic particles containing less than 100% CD40L-fused
polypeptides are produced to overcome this problem.
[0272] The other molecular adjuvants inserted within the WHcAg,
including the C3d fragment, BAFF and LAG-3, have a tendency to
become internalized when inserted at the C-terminus. Therefore
tandem repeats of molecular adjuvants are used to resist
internalization. Alternatively, various mutations within the
so-called hinge region of WHcAg, between the assembly domain and
the DNA/RNA-binding region of the core particle are made to prevent
internalization of C-terminal sequences. However, internalization
represents a problem for those molecular adjuvants such as CD40L,
C3d, BAFF and LAG-3, which function at the APC/B cell membrane. In
contrast, internalization of molecular adjuvants such as CpG DN is
not an issue as these types of adjuvants function at the level of
cytosolic receptors.
[0273] Another type of molecular adjuvant or immune enhancer is the
inclusion within hybrid core particles of a CD4.sup.+ T cell
epitope, preferably a "universal" CD4.sup.+ T cell epitope that is
recognized by a large proportion of CD4.sup.+ T cells (such as by
more than 50%, preferably more than 60%, more preferably more than
70%, most preferably greater than 80%), of CD4.sup.+ T cells. In
one embodiment, universal CD4.sup.+ T cell epitopes bind to a
variety of human MHC class II molecules and are able to stimulate T
helper cells. In another embodiment, universal CD4.sup.+ T cell
epitopes are preferably derived from antigens to which the human
population is frequently exposed either by natural infection or
vaccination (Falugi, et al., Eur. J. Immunol., 31:3816-3824, 2001).
A number of such universal CD4.sup.+ T cell epitopes have been
described including, but not limited to: Tetanus Toxin (TT)
residues 632-651 (SEQ ID NO:239); TT residues 950-969 (SEQ ID
NO:240); TT residues 947-967 (SEQ ID NO:241), TT residues 830-843
(SEQ ID NO:242), TT residues 1084-1099 (SEQ ID NO:243), TT residues
1174-1189 (SEQ ID NO:244) (Demotz, et al., Eur. J. Immunol.,
23:425-432, 1993); Diphtheria Toxin (DT) residues 271-290 (SEQ ID
NO:245); DT residues 321-340 (SEQ ID NO:246); DT residues 331-350
(SEQ ID NO:247); DT residues 411-430 (SEQ ID NO:248); DT residues
351-370 (SEQ ID NO:249); DT residues 431-450 (SEQ ID NO:250)
(Diethelm-Okita, et al., J. Infect. Dis., 1818:1001-1009, 2000);
Plasmodium falciparum circumsporozoite (CSP) residues 321-345 (SEQ
ID NO:251) and CSP residues 378-395 (SEQ ID NO:252) (Hammer, et
al., Cell, 74:197-203, 1993); Hepatitis B antigen (HbsAg)
residues19-33 (SEQ ID NO:253) (Greenstein.TM., et al., J. Immunol.,
148:3970-3977, 1992); Influenza hemagglutinin residues 307-319 (SEQ
ID NO:254); Influenza matrix residues 17-31 (SEQ ID NO:255)
(Alexander, et al., J. Immunol., 164:1625-1633, 2000); and measles
virus fusion protein (MVF) residues 288-302 (SEQ ID NO:256)
(Dakappagari, et al., J. Immunol., 170:4242-4253, 2003). One
purpose of including a universal CD4.sup.+ T cell epitope into
hybrid core particles is to take advantage of pre-existing memory
CD4.sup.+ T cells primed by previous infection or vaccination and
redirecting the T cell helper function to the B cell epitope
present on the core particle carrier. For example, we chose to
insert the TT950-969 sequence on the C-terminus of hybrid WHcAg
particles. The TT950-969 sequence is recognized by 86% of humans at
the T cell level due to TT vaccination according to a report by
Diethelm-Okita, et al., (J. Inf. Dis., 181:1001-1007, 2000).
Furthermore, this CD4+ T cell epitope is recognized by B10 and
B10.S murine strains but not by Balb/c mice. A single injection in
B10 and B10.S TT-immune mice with 10 .mu.g of WHc-TT950-969 hybrid
particles in saline, without the use of an adjuvant elicited
extremely high levels of anti-WHc antibodies, which persisted
beyond 7 months. Non-TT-immune B10 and B10.S mice produced
significantly less anti-WHc antibodies, which persisted poorly
(FIG. 44). While not intending to limit the invention to any
mechanism, in one embodiment, the mechanism responsible for
enhanced anti-WHc antibody production was demonstrated to be due to
TT950-969-specific T helper cell function (FIG. 45). Therefore,
pre-existing TT950-969-specific memory T helper cell function can
be "redirected" to provide T cell help for antibody production to B
cell epitopes presented on the WHcAg platform and this procedure
can obviate the need for an adjuvant and reduce the number of
vaccine doses required.
[0274] C. Chemical Coupling of Protein and Carbohydrate
Antigens
[0275] In those instances when it is not possible to incorporate
large protein epitopes or molecular adjuvants into the WHcAg by
recombinant methods, chemical conjugation is used. Similarly, the
WHcAg also serves as a new type of carrier platform for
polysaccharide or oligosaccharides (PS/OS) antigens upon chemically
coupling PS/OS epitopes to the WHcAg core.
[0276] 1. Modification of Core Genes for Subsequent Chemical
Conjugation
[0277] The wild type WHcAg is not efficiently chemically
derivatized. Therefore, reactive amine groups are added by the
insertion of one to several lysines via recombinant technologies.
The position and number of the added lysines is varied (e.g, N- and
C-termini, and within or outside the exposed loop region).
[0278] 2. Model Protein Antigen
[0279] One protein and two carbohydrate model antigens are used for
testing the feasibility of chemical conjugation to lysine-modified
WHcAg. The model protein antigen, Poly-gamma-D-glutamic acid (PGA),
is the capsular polypeptide of Bacillus sp. including B. anthracis
(Fouet et al., J Appl Microbiol, 87:251-255, 1999). The capsular
PGA of anthrax pathogens is very similar to bacterial cell surface
PS antigens in that they are both poorly immunogenic, repetitive
polymers require coupling to a carrier moiety. To produce WHcAg-PGA
conjugates, a carbodiime-mediated coupling reaction is used because
PGA molecules contain abundant carboxylate groups. The cores
initially employed for this purpose include WHcAg-HyW2 and
WHcAg-HyW2 modified with a lysine insert within the loop (K.sup.75)
or at the C-terminus. In preliminary studies using WHcAg-HyW2 and a
saturation coupling approach, equal amounts of PGA and WHcAg-HyW2
(1.3 mg) and 5.0 mg of carbodiimide were mixed and after a four
hour reaction time, SDS-PAGE analysis revealed that most of the PGA
was coupled to WHcAg. As shown in FIG. 31, immunization of Balb/c
mice with 10 .mu.g of the PGA-WHcAg-HyW2 conjugate formulated in
alum resulted in production of significant IgM and IgG anti-PGA
antibodies, whereas the uncoupled PGA in alum was non-immunogenic.
Note that IgM anti-PGA decreased from the first immunization to the
third and IgG anti-PGA antibodies increased from the first
immunization to the third. In other embodiments, the
lysine-modified WHcAg particles are used with various PGA polymer
sizes and WHcAg/PGA ratios, to optimize conjugate production and
immunogenicity. The WHcAg carrier is expected by the inventors to
compare favorably with common toxoid carriers (e.g., tetanus toxoid
and diptheria toxoid). In vitro opsonophagocytic assays (DeVelasco
et al., Vaccine, 12:1419-1422, 1994) are used to test the function
of anti-PGA antibodies. As a surrogate for B. anthracis, B.
licheniformis 9945A (not a human pathogen) which has the same PGA
capsule is used. In some embodiments, the ability of the PGA-WHcAg
conjugate to protect immunized mice against lethal B. anthracis
challenge is assessed.
[0280] 3. Model Carbohydrate Antigens
[0281] The O-antigenic PSs are both essential virulence factors and
protective Shigella antigens. Moreover, serum IgG specific for O-PS
has been demonstrated to confer immunity against shigellosis.
Despite these findings, to date no licensed Shigella vaccines
exist. To meet this need in the art, a lysine-modified WHcAg is
used as a carrier platform for the O-PSs of Shigella, with a
particular focus on S. dysenteriae 1 and S. flexneri 2a. A recent
study using recombinant core protein derived from the duck hepadna
virus coupled to purified type III capsular PS from group B
streptococcus (GBS) demonstrated 97% survival after GBS type III
challenge in newborn pups born to vaccinated mouse dams (Paoletti
et al., Vaccine, 20:370-376, 2002). A method is used that permits
the chemical synthesis of an array of glycoconjugates containing
saccharide antigens of desired molecular sizes and that employs
chemically controlled site-specific coupling (Wang et al., Vaccine,
21:1112-1117, 2003). Utilizing these chemical methods, particulate
PS-WHcAg conjugates are prepared incorporating PS epitopes of known
molecular size and orientation which are linked at specified sites
to core particles. The size of OS antigens deserves particular
attention in the context of using WHcAg as the carrier. The spacing
of natural WHcAg B-cell epitopes and of peptidic antigens inserted
in the tips of the spikes is contemplated to be an important
determining factor for immunogenicity. Therefore, it is important
to test a range of sizes of OS antigens beginning with sizes
comparable to peptidic antigens (e.g., 2,000-3,000 kDa), which
exhibit high levels of immunogenicity. In addition to coupling
large numbers of a single PS epitope to a single particle, the
multivalency of the particles provides the opportunity to couple PS
epitopes from many different serotypes to the same particle
yielding a multivalent vaccine. Alternatively, particles conjugated
with one PS serotype are mixed with other conjugated particles
carrying a different serotype PS. The WHcAg is contemplated to be
superior to commonly used carrier proteins for delivery of OS/PS
antigens.
VII. Applications of the Hepadnavirus Core AntigenCombinatorial
Technology
[0282] A. Infectious Diseases
[0283] Historically the use of the HBcAg as a platform has been
confined to use as a T cell carrier for neutralizing epitopes of
infectious disease pathogens. Subunit vaccine development for
infectious diseases remains an important application for the WHcAg
platform technology. In one embodiment, the P. falciparum CS repeat
epitope NANPNVDP(NANP).sub.3 (SEQ ID NO:75) was inserted in many
positions within the WHcAg, and complemented with diverse C-termini
as a model system to further develop the WHcAg as a vaccine
platform (See, FIGS. 2, 14 and 15).
[0284] 1. Plasmodium Vivax
[0285] This P. vivax malaria species is predominant in South and
Central America and is also found in Southeast Asia. A bivalent
WHcAg hybrid particle was produced containing the P. falciparum CS
repeat at the N-terminus, and the Type I variants of the P.
vivax-CS repeat as an insertion at position 78. Preliminary data
indicate that the vaccine candidate is effective and that
antibodies to both inserts were produced. Thus, the present
invention also provides bivalent vaccines based upon the WHcAg
technology (See, FIG. 15). Since the P. vivax system is more
complex (because several genotypes exist), more than one vaccine
particle is produced and tested. Alternatively, several
genotype-specific B cell epitopes are inserted into the same
particle at different sites to produce a bivalent or trivalent
vaccine particle. For instance, the Type II and Type III variants
of the P. vivax CS repeat epitopes (See, Table 6) are inserted into
the WHcAg platform in addition to the Type I variant:
TABLE-US-00010 TABLE 6 Plasmodium vivax Circumsporozoite (CS)
Sequences Type Sequence.sup.1 Identifier I DRAAGQPAGDRADGQPAG SEQ
ID NO: 74 II ANGAGNQPGANGAGDQPG SEQ ID NO: 65 II ANGADNQPGANGADDQPG
SEQ ID NO: 66 III APGANQEGGAAAPGANQEGGAA SEQ ID NO: 67 .sup.1Bold
type denotes variant residues.
[0286] 2. Foot and Mouth Disease (FMDV)
[0287] One of the first examples of the use of the HBcAg as a
vaccine carrier was for the major immunogenic B cell epitope of the
FMDV.sub.141-160. Previously, a hybrid HBcAg-FMDV particle was
shown to elicit protective antibodies (Bittle et al., Nature,
298:30-35, 1982), although there were problems eliciting
sufficiently high levels of anti-FMDV antibodies. The WHcAg
combinatorial technology described herein is contemplated to
provide a more effective vaccine candidate. Two protective linear
epitopes derived from the VP1 protein have been defined:
VP1.sub.141-160 and VP1.sub.200-213 (Van Lierop et al., Immunol,
75:406-413, 1992). Recently a DNA vaccine encoding VP1.sub.141-160
and VP1.sub.200-213 was shown to protect swine from a FMDV
challenge (Wong et al., Virol, 278:27-35, 2000). Thus, both
neutralizing epitopes are selected for incorporation into hybrid
WHcAg particles.
[0288] 3. Influenza A Virus
[0289] The extracellular domain of the matrix 2 (M2e) sequence of
influenza A has also been chosen as a model neutralizing B cell
epitope for insertion within WHcAg, as this sequence has a number
of features in common with the malaria CS repeat. The M2e sequence
is a linear protective epitope that is poorly immunogenic during
natural infection and in the context of various vaccine
formulations. Additionally, the M2e sequence permits the comparison
of the WHcAg and HBcAg vaccine platforms (Jegerlehner et al.,
Vaccine, 3104, 2002; Neirynck et al., Nat Med, 5:1157-1163, 1999;
and Heinen et al., J Gen Virol, 83:1851-1859, 2002). The kinetics
of the antibody response elicited by a M2e-WHcAg hybrid particle
(HyW-IM2(-)78) is shown in FIG. 32. Anti-WHc and anti-M2e
antibodies are detected within 2 weeks of primary immunization,
with serum titers reaching a plateau four weeks after the primary
immunization, and rising approximately 10-fold after boosting.
Although, an anti-M2e titer of 3.times.10.sup.6 after two doses is
two orders of magnitude higher than the levels previously obtained
using the HBcAg platform, improvements on the immunogenicity of the
HyW-IM2(-)78 particle are contemplated. The sera from mice
immunized with HyW-IM2(-)78 were analyzed to determine the isotype
distribution of anti-2Me and anti-WHc antibodies. As shown in FIG.
33, the response was well represented by all the IgG isotypes.
After the second immunization, anti-M2e serum titers greater than
1:100,000 were obtained for all four IgG isotypes. A similar
pattern was observed in the anti-WHc response with the exception of
a relatively low IgG.sub.3 response to the carrier. The broad
spectrum IgG isotype profile specific for the M2e epitope is a
positive characteristic, which guarantees a full spectrum of
biologic effector functions (complement fixation, ADCC, etc.).
Importantly, the HyW-IM2(-)78 antisera also binds to viral M2 and
inhibits influenza virus growth.
[0290] Quantitating the reactivity of sera from WHcAg-M2e immunized
mice against authentic M2 protein is the first step in
characterizing WHcAg-M2e as a vaccine candidate. The use of flow
cytometry against virus-infected cells is performed on live,
unfixed cells thereby ensuring the sera can recognize M2 in its
native conformation in the plasma membrane (Pekosz and Lamb, J
Virol, 73:8808-8812, 1999). Sera from WHcAg-M2e immunized mice
(diluted 1:100) were incubated at 4.degree. C. with cells infected
with influenza A virus for 12 hours. The samples were washed,
incubated with FITC-conjugated goat IgG recognizing mouse IgG,
washed and analyzed by flow cytometry. Sera from mice immunized
with core particle alone did not specifically react with influenza
A virus infected cells in comparison to mock-infected cells (See,
FIG. 34, panel A). In contrast, sera from WHcAg-M2e immunized mice
recognized influenza A virus infected cells, as judged by the shift
to increased fluorescence intensity displayed in the virus-infected
cell population (See, FIG. 34, panel B). Thus, the hybrid particle
generates a physiologically relevant antibody response recognizing
influenza A virus infected cells.
[0291] Certain monoclonal antibodies targeting the M2 extracellular
domain restrict virus replication in vitro by inhibiting virus
particle budding (Hughey et al., Virol, 212:411-412, 1995). The
ability of WHcAg-M2e immunized mouse sera to inhibit influenza A
virus budding was assayed as described (Zebedee and Lamb, Proc Natl
Acad Sci USA, 86:1061-1065, 1989). Influenza A virus strain rWSN (a
M2 monoclonal antibody resistant strain), produced equivalent
numbers of infectious particles irrespective of the presence of
anti-WHcAg-M2e sera. In contrast, rWSN M-Udorn reassortant virus
possessing an RNA segment 7 from a/Udorn/72, but all other segments
derived from rWSN (a M2 monoclonal antibody sensitive strain)
consistently produced less infectious virus particles at all time
points tested when WHcAg-M2e anti sera was present (See, FIG. 35).
The reduction in virus titer indicated that sera from WHcAg-M2e
immunized mice has the ability to inhibit the production of
infectious influenza A virus particles in vitro.
Immunization/challenge studies are also done to assess the ability
of the WHcAg-M2e particles to provide protection against influenza
A infection.
[0292] Previously, the HBcAg has been used as a platform for the
M2e epitope by positioning the M2e region at the NH.sub.2-terminus
of HBcAg. The first study reported relatively low serum anti-M2e
titers (4.times.10.sup.4) after three doses of hybrid HBcAg
particles in a strong adjuvant system (Neirynck et al., Nat Med,
5:1157-1163, 1999). Nonetheless, this level of anti-M2e was
sufficient to significantly protect mice against a lethal
challenge. Another group using the same hybrid HBcAg vaccine
candidate in pigs raised less serum anti-M2e antibody
(3.times.10.sup.3), and failed to achieve protection. Similarly, a
murine study in mice using hybrid HBcAg particles without adjuvant
achieved very low anti-M2e serum titers (1:80) and challenged mice
were not protected. However, mice receiving a chemical conjugate
were protected which correlated with higher anti-M2e serum titers
(1:5, 120). Thus, it is contemplated that the quantity of
protective antibody produced is important and that a threshold
serum level is necessary and should be maintained for
antibody-mediated protection. As described herein, the M2e sequence
was inserted within the loop of WHcAg, and this prototype M2e-WHcAg
particle was found to elicit 100-fold more anti-M2e serum antibody
(3.times.10.sup.6), than the HBcAg-M2e particles shown in Table
7-1, even after fewer doses in IFA.
TABLE-US-00011 TABLE 7-1 Hybrid Core Particles Containing the
Influenza Virus M2e Sequence Antibody Titer Dose (1/dilution)
Comment Particle (adjuvant) Anti-M2e Anti-Core (reference) M2e-HBc
3 (Ribi) 4 .times. 10.sup.4 2.7 .times. 10.sup.6 protection
(Neirynck, supra, 1999) M2e-HBc 3 (adjuvant) 3 .times. 10.sup.3 --
no protection (Heinen, supra, 2002) M2e-HBc 2 (no adjuvant) 80 --
no protection (Jegerlehner, supra, 2002) HyW-IM2(-)78 2 (IFA) 3
.times. 10.sup.6 3 .times. 10.sup.6 in vitro neutralization
(present invention)
[0293] 4. Anti-Toxin Vaccine Design
[0294] Another suitable application of the WHcAg platform
technology is as a toxin subunit vaccine. One advantage of using
the WHcAg platform is that a neutralizing epitope of the toxin is
inserted into the particles. This is contemplated to be more
immunogenic than the whole toxin or toxoid, while circumventing the
expense and hazard of dealing with the whole toxin.
[0295] As a prototype, two peptidic B cell epitopes from
Staphylococcal enterotoxin B (SEB) were selected.
Anti-SEB.sub.152-161 antibodies recognize native SEB, as well as
other SE's and inhibit transcytosis of SEB, SEA, SEE and TSST-1
(Arad et al., Nat Med, 6:414-421, 2000). To prevent SE-mediated
disease, a hybrid core particle vaccine has been constructed by
inserting the SEB.sub.152-161 sequence in WHcAg at position 75, in
combination with the HyW2 C-terminus. Similarly, the
SEB.sub.140-151 peptide and antibody have also been shown to be
biologically active and highly conserved (Visvanathan et al.,
Infect Immunol, 69:875-884, 2001). Therefore, the SEB.sub.140-151
sequence is used as well, to produce WHcAg-SEB.sub.140-151 insert
particles.
[0296] B. Therapeutic Autoantibodies
[0297] The ability of the WHcAg platform to raise very high levels
of anti-insert antibody is contemplated to be useful for a number
of applications beyond the infectious disease setting. One such
application is for the production of therapeutic autoantibodies.
Several mAb-based therapies have shown encouraging results in small
animal studies and in clinical trials. For instance, mAb therapy
targeting c-erbB2 (HER 2/neu) has been used to treat breast cancer
(Pegram and Slamon, Semin Oncol, 27:13-19, 2000); antibody to
.beta.-amyloid has been used to treat an Alzheimer's-like disease
in mice (Schenk et al., Nature, 400:173-177, 1999), anti-IgE mAb
has been tested to treat allergy (Cheng, Nat Biotechnol,
18:157-162, 2000), and in human clinical trials an anti-TNF.alpha.
mAb therapy reduced the symptoms of rheumatoid arthritis and
Crohn's disease (Maini and Taylor, Annu Rev Med, 51:207-229, 2000).
However, active immunization has a number of advantages over
passive mAb therapy: i) patient convenience and cost (several
immunizations as opposed to numerous infusions, each requiring
several hours in the clinic); ii) costs for large scale mAb
production are extremely high; iii) active immunization produces
more consistent levels of antibody over time; and iv) mAb therapy
is likely to induce an inactivating antibody response. In fact,
others have begun using Papillomavirus-like-particles chemically
conjugated to self antigens to elicit therapeutic autoantibodies
(Chackerian et al., J Clin Invest, 108:415-423, 2001; and
Chackerian et al., Proc Natl. Acad Sci USA, 96:2373-2378, 1999).
Thus, several model epitopes in the context of WHcAg are used to
induce production of autoantibodies possessing therapeutic
functions.
[0298] 1. Anti-CETP Autoantibody
[0299] There is a strong inverse relationship between the plasma
concentration of cholesterol in HDLs and the development of
coronary heart disease. One therapeutic approach that has been
suggested for increasing HDL concentrations is the inhibition of
cholesteryl ester transfer protein (CETP) activity (Tall, J Lipid
Res, 34:1255-1274, 1993). The CETP functions in the plasma to lower
HDL by moving cholesteryl esters from HDLs to VLDLs and LDLs
(Barter et al., Biochem J, 208:1-7, 1982). Transient inhibition of
CETP activity in rabbits and hamsters by mAb, small molecules, or
antisense oligonucleotides (Whitlock et al., J Clin Invest,
84:129-137, 1989; Kothari et al., Atherosclerosis, 128:59-66, 1997;
and Sugano and Makino, J Biol Chem, 271:19080-19083, 1996) causes
an increase in plasma HDL. In addition, sustained inhibition of
CETP expression by antisense oligonucleotides increased plasma HDL
and reduced atherosclerotic lesions in rabbits (Sugano et al., J
Biol Chem, 273:5033-5036, 1998). In contrast, transgenic mice and
rats expressing human CETP have decreased plasma HDL (Agellon et
al., J Biol Chem, 266:10796-10801, 1991; and Herrera et al., Nat
Med, 5:1383-1389, 1999). Similarly, human populations with reduced
or absent CETP activity due to genetic mutations have markedly
elevated plasma HDL (Koizumi et al., Atherosclerosis, 58:175-186,
1985). Recently a vaccine approach was used to generate antibodies
against CETP in vivo in rabbits using a dominant linear B cell
epitope consisting of residues 461-476 of human CETP. The immunized
rabbits had reduced CETP activity, a substantial increase in HDL,
and a significant reduction in aortic atherosclerotic lesions
(Rittershaus et al., Arterioscler Thromb Vase Biol, 20:2106-2112,
2000). For this reason, the CETP.sub.461-476 sequence was inserted
within the WHcAg platform at position 74 in combination with the
HyW C-terminal platform (HyW2-CETP.sub.74). Interestingly, attempts
to insert the CETP sequence at positions 75 or 78 were unsuccessful
illustrating the versatility of the WHcAg combinatorial technology.
As shown in FIG. 36, significant levels of anti-CETP.sub.461-476
antibodies were raised in mice immunized with HyW2-CETP74, and
secondary anti-CETP.sub.461-476 antibodies also neutralize CETP
activity in vitro. Because mice do not express CETP, this does not
represent an autoantibody response. However, immunizing Tg mice
expressing human CETP (and having lowered HDL levels) with
HyW-CETP.sub.74 particles is contemplated to induce anti-CETP
antibodies capable of both neutralizing CETP activity and raising
plasma HDL levels in vivo. The ability of immunization with the
CETP.sub.461-476 peptide linked to a tetanus toxoid peptide to
raise HDL levels in vaccine recipients is currently being assessed
in human clinical trials (Rittershaus et al., supra, 2000). A
particulate WHcAg-CEPT.sub.461-476 vaccine is contemplated to be a
superior immunogen in humans as compared to a totally peptidic
vaccine.
[0300] 2. Anti-Cytokine Autoantibody
[0301] In many autoimmune diseases such as multiple sclerosis and
diabetes, Th cells play a predominant role. The Th cells function
both to help B cells produce antibody (Th.sub.2 cells) and to
mediate inflammatory responses (Th.sub.1 cells). Th.sub.1 cells
mediate their inflammatory function through the production of
cytokines, which have direct anti-pathogen effects, as well as
effects on other immune cell types. Tumor necrosis factor-alpha
(TNF.alpha.) is an inflammatory cytokine that is often produced
during autoimmune reactions and is responsible for many of the
lesions or symptoms. Therefore, B cell epitopes from TNF.alpha. are
inserted into the WHcAg platform and the effect of high levels of
anti-cytokine (e.g., anti-TNF.alpha.) antibodies is assessed in
animal models of ongoing autoimmune disease. Analogues of
TNF.alpha. and soluble receptors for TNF.alpha., as well as
anti-TNF.alpha. mAbs are currently tested with promising results in
clinical trials. However, these drugs are very expensive and
require continual dosing, whereas anti-TNF.alpha. antibodies
produced by the patient following vaccination are inexpensive and
work via the same TNF.alpha.-blocking mechanism. The murine
TNF.alpha..sub.3-22 epitope is used as a model antigen because this
region is homologous to human TNF.alpha., has been reported to
interact with the TNF.alpha. receptor and in a mouse model
vaccination with this TNF.alpha. peptide chemically conjugated to
Papillomavirus-like-particles produced anti-TNF.alpha.
autoantibodies and inhibited development of type II
collagen-induced arthritis (Chackerian et al., J Clin Invest,
108:415-423, 2001). WHcAg-TNF.alpha..sub.3-22 hybrid particles are
produced and their immunogenicity is examined using methods
disclosed herein. Moreover, the effects of anti-TNF.alpha.
autoantibody production on the induction and/or maintenance of type
II collagen-induced arthritis in a mouse model is assessed.
[0302] Although the delivery of self B cell epitopes on a
particulate structure can circumvent T cell self-tolerance and
elicit autoantibodies, this is a more complicated system than
simply eliciting antibodies to foreign epitopes. Various properties
of this system are examined using HBcAg- and HBeAg-Tg mice and T
cell receptor (TCR) double-Tg mice. For instance, autoantibodies
were elicited (anti-HBe/anti-HBc) in TCR.times.HBe/HBc-double Tg
mice by the single injection of a peptide that activates the
HBe/HBcAg-specific transgenic CD4.sup.+ T cells (See, FIG. 37).
Note that anti-HBe autoantibody production is transient lasting
approximately three months. In HBeAg-Tg mice expressing a higher
serum level of HBeAg, autoantibody production is even more
transient (e.g., 2-3 weeks). In contrast, anti-HBc autoantibody is
persistent for the life of the animal. Several characteristics of
HBeAg and HBcAg most likely explain these differences. The HBeAg is
a secreted antigen, whereas the HBcAg is a cytosolic protein
expressed in hepatocytes and relatively small amounts of HBcAg leak
out of the liver. Therefore, the presence of the autoantigen in the
serum is contemplated to clear the autoantibody via immune complex
formation and secondly via the constant exposure of HBeAg-specific
T cells to soluble HBeAg which has been shown to be
immunosuppressive through Fas-mediated apoptosis (Milich et al., J
Immunol, 160:2013-2021, 1998). However, in the WHcAg platform
system immune tolerance is predominantly relevant at the B cell
level, because the insert is a self-B cell epitope, whereas the Th
cells are specific for the WHcAg carrier. The reversibility of
autoantibody production is dependent upon whether the antigen
within the inoculum is driving antibody production mediated by
WHcAg-specific Th cells or whether at some point the endogenous
self protein itself drives autoantibody production. This is
contemplated to occur if the expanded autoreactive B cells become
efficient APCs to present the entire self protein to self-specific
Th cells to an extent capable of breaking Th cell tolerance.
[0303] 3. Alzheimer's Disease
[0304] Cleavage of amyloid precursor protein (APP) yields
amyloid-.beta.-peptide (A.beta.) a 40-42 amino acid fragment which
is present in abundance in the plaques or brain lesions found in
Alzheimer's patients. Transgenic mice expressing human A.beta.
serve as an animal model for Alzheimer's disease. Recently it was
reported that antibody production to A.beta. in these transgenic
mice inhibited plaque formation in the brains of the mice (Morgan
et al., Nature, 408:982-985, 2000). However, the A.beta. peptide
was not very immunogenic and required many immunizations to induce
rather low levels of antibody. The murine studies prompted several
clinical trials, which were halted due to a number of cases of
aseptic meningoencephalitis (Smith et al., Lancet, 359:1864-1865,
2002). Two recent studies have more carefully mapped the
specificity of the therapeutic anti-A.beta. antibodies and showed
that the beneficial effects in mice arise from antibodies
selectively directed against residues 4-10 of A.beta..sub.42, which
do not in themselves elicit an inflammatory response (Hock et al.,
Nat Med, 8:1270-1275, 2002; and McLaurin et al., Nat Med,
8:1263-1269, 2002). The A.beta..sub.4-10 epitope represents a B
cell epitope possessing no predicted T cell sites. In contrast, use
of the full-length A.beta..sub.42 is contemplated to have elicited
an inflammatory T cell response. Furthermore, it was observed that
the induction of IgG.sub.2b antibodies to A.beta..sub.4-10 had an
optimal therapeutic effect. These recent studies have revived
interest in an Alzheimer's disease vaccine. The WHcAg platform is
well suited for this purpose because it has the potential to raise
very high titer antibodies to the small A.beta..sub.4-10 epitope,
without activating A.beta.-specific T cells. The WHcAg platform
elicits a spectrum of IgG isotypes, (predominantly IgG.sub.2b
isotype), although the platform and/or formulation are manipulable
to focus antibody production to a particular IgG isotype. The
A.beta..sub.4-10 sequence and/or tandem repeats are inserted into
the WHcAg platform at various positions inside and outside the
loop, in combination with different C-termini. The platform(s)
which is most immunogenic or otherwise advantageous (e.g., IgG
isotype induction profile), is assessed in vaccination experiments
in the appropriate transgenic mouse model of Alzheimer's
disease.
[0305] C. Allergic Disorders
[0306] Simplistically, allergy occurs when exposure to an allergen
(e.g., pollen) elicits an antibody of the IgE class, as opposed to
an antibody of the IgG class. IgE antibody binds to a particular
cell type (Mast cell) and to the allergen, yielding an allergen-IgE
complex on the surface of the Mast cell, which activates the Mast
cell to release effector molecules such as histamine thereby
mediating the symptoms of an allergic response. One allergy
treatment termed desensitization involves injecting many doses of
the allergen over long periods to bias the antibody response
towards IgG rather than IgE production. The WHcAg technology is
contemplated to be useful in this application because WHcAg elicits
strong IgG responses, but not IgE responses. B cell epitopes
derived from known allergens are inserted into the WHcAg platform
and used to immunize/desensitize allergic patients. One or two
injections are contemplated to be necessary, in contrast to the
numerous injections to used in typical desensitization therapy. A
number of linear peptidic B cell epitopes have been mapped for
common allergens including: peanut allergen (Ara h 3; Rabjohn et
al., Int Arch Allergy Immunol, 128:15-23, 2002); latex allergen
hevein (Her b 5; Beezhold et al., J Allergy Clin Immunol,
107:1069-1076, 2001); brown shrimp allergen (Pen a 1; Reese et al.,
J Chromatogr B Biomed Sci Appl, 756:157-163, 2001); and the major
grass pollen allergen (Phl p 1; Suphio et al., FEBS Lett,
502:46-52, 2001). Often allergen-derived peptides lack IgE binding
capacity, yet anti-peptide IgG antibodies react with the native
allergen and inhibiting IgE from binding to the native allergen
(Focke et al., FASEB J, 15:2042-2044, 2001). This is contemplated
to occur via anti-peptide antibodies sterically hindering IgE
binding, as well as by allergen clearance by anti-peptide IgG
antibodies before IgE synthesis can occur. Hybrid WHcAg particles
containing selected allergen-specific B cell epitopes are produced
and selected for high titer IgG anti-allergen production, which is
examined for the capacity to inhibit patient IgE binding to mast or
basophil cells.
VII. Enhanced Particle Assembly Via Addition or Insertion of Acidic
Amino Acids
[0307] A. Hepadna Virus Core Antigens
[0308] During development of the present invention, the presence of
a number of highly basic amino acids (especially K, R, H) in a
candidate insert epitope was found to correlate negatively with the
assembly of hybrid WHcAg particles and hybrid HBcAg particles
(PCT/US01/25625; and Karpenko, et. al., Amino Acids, 18:329-337,
2000). As shown in Table 17 and 18, the isoelectric point (pI) of
epitope sequences inserted into the loop region, effects assembly
of hybrid WHcAg, hybrid GSHcAg and hybrid HbcAg. Previously, three
parameters of the epitope insert that prevented self-assembly of
hybrid HBcAg particles were identified: i) high epitope
hydrophobicity; ii) large epitope volume; and iii) a high
.beta.-strand index.
[0309] The pI of the wild type WHcAg loop (76-82) region is
approximately 6.14 and that of the wild type HBcAg loop
approximately 4.12. Because the wild type WHcAg and HBcAg 76-82
loop regions are acidic, the inventors predicted that epitope
inserts more positively charged than the wild type sequence may
have adverse effects on dimer formation (e.g., particle subunit)
and secondly, particle assembly (e.g., core). Theoretically, excess
positive charge in the loop may result in a repulsive force between
the monomers and negatively effect dimerization or the efficient
assembly of dimer subunits. However, an understanding of the
mechanism(s) is not necessary in order to make and use the present
invention.
[0310] Thus, several epitopes with pI's of seven or greater, which
did not permit assembly of hybrid WHcAg, hybrid GSHcAg or HBcAg
particles, were either modified to contain the acidic amino acid
glutamic acid or were bracketed by glutamic acid residues (e.g.,
linker sequences). The effect of addition of glutamic acid
substitutions or linker sequences to the insert sequence was then
tested for the ability to rescue hybrid particle assembly. As shown
in Table 18, in all cases addition of glutamic acid linker
sequences on hybrid WHcAg, hybrid GSHcAg and hybrid HbcAg rescued
particle assembly on the WHcAg, the HBcAg and the GSHcAg platforms.
Substitution of a non-acidic amino acid within the heterologous
insert (at a nonessential position) with a glutamic acid residue
also rescued hybrid particle assembly. Surprisingly even placement
of negatively charged amino acids at a distance from the positively
charged residues in the insert sequence resulted in hybrid particle
assembly.
[0311] Additionally, the effect of addition of other amino acid
residues on hybrid particle assembly was examined. A single basic
(e.g., pI=8.74) epitope sequence was selected and multiple amino
acid linkers were tested. Interestingly, peptidic linkers that
significantly lowered the insert pI (i.e., glutamic acid and
aspartic acid) permitted WHcAg hybrid particle assembly. Thus,
assembly of particles containing inserts with pIs of greater than
seven are rescued by the addition of acidic acid substitutions
and/or acidic amino acid linker sequences. Therefore, when possible
a negatively-charged epitope should be selected. However, when this
is not possible a positively-charged epitope is modified to include
acidic amino acid substitutions and/or linker sequences, in order
to obtain hybrid particles which assemble efficiently.
[0312] B. Other Self-Assembling Virus-Like-Particles (VLP)
[0313] Additional self-assembling virus-like-particles (VLP) are
finding use as vaccine carrier platforms. A major, universal
problem of VLP assembly has been the destabilizing effects of
adding or inserting foreign peptidic sequences (PCT/US01/25625,
Jegerlehner et al., Vaccine, 20:3104, 2002; Chackerian et al., J.
Clin. Invest., 108:415-423, 2001; and Casal et al., Methods
19:174-186, 1999). Hybrid VLP stability has represented such a
serious problem that users of the HBcAg platform technology
(Jegerlehner et al., supra, 2002) and the Papillomavirus platform
technology (Chackerian et. al., supra, 2001) have opted to
chemically conjugate foreign epitopes to wild type VLPs, instead of
attempting to produce hybrid particles via recombinant means. In
particular, Chackerian found that the ability of L1-self-peptide
chimeras to assemble into VLPs was highly unpredictable. Similarly,
Jegerlehner found that the size and nature of epitopes that can be
inserted into the immunodominant region of VLPs was restricted and
that VLPs containing inserts longer than 20 amino acids often
failed to assemble.
[0314] Similar to the hepadnavirus core proteins, preferred
insertion sites on many VLPs are within the immunodominant exposed
loop structures which are accessible for antibody recognition and
which may be less likely to compromise the structural integrity of
the particle, as opposed to insertions into .alpha.-helical or
.beta.-sheet regions (Sadeyen et al., Virology 309:32-40, 2003).
Thus, the inventors contemplate that the insertion of
positively-charged epitopes into the exposed loop region of other
types of hybrid cores, is also expected to negatively effect
assembly of these cores. Consequently, the inventors propose using
acidic amino acids to rescue assembly of other viral core particles
containing positively-charged epitopes. In fact, rational insertion
or substitution of acidic amino acid residues into either the
heterologous antigen of interest or the vaccine platform, is
contemplated to be useful for the following exemplary list of
viruses from which VLPs are obtained: human papillomavirus type 16
(Varsani et al., J. Virol., 77:8386-8393); human papillomavirus
type 11 (Rose et al., J Virol., 67:1936-1944, 1993); hamster
polyomavirus (Gedvilaite et al., Virol., 20:21-35, 2000); hepatitis
B virus (Pumpens et al., Intervirol., 45:24-32, 2002); yeast Ty1
and Ty3 (Roth, Yeast, 16:785-795, 2000); human immunodeficiency
virus (Wagner et al., Intervirol., 39:93-103, 1996); hepatitis C
virus (Baumert et al., Gastroenterology, 117:1397-1407, 1999);
bovine rotavirus strain C486 (Sahara et al., J. Virol.,
65:6994-6997, 1991); norwalk virus (Ball et al., Gastroenterology,
117:40-48, 1999); human parvovirus B19 (Brown et al., J. Virol.,
65:2702-2706, 1991); herpes simplex virus (Thomsen et al., J.
Virol., 68:2442-2457, 1994); poliovirus (Urakawa et al., J. Gen.
Virol., 70:1453-1463, 1989); RNA bacteriophage (Brown et al.,
Intervirol., 45:371-380, 2002); bluetongue virus (French et al., J.
Virol., 64:5695-5700, 1990); duck hepatitis B virus (Paoletti et
al., Vaccine, 20:370-376, 2001); simian immunodeficiency virus
(Yamshchikov et al., Virol., 214:50-58, 1995); rabbit hemorrhagic
disease virus (Plana-Duran et al., Arch. Virol., 141:1423-1436,
1996); hepatitis E virus (Nikura et al., Virol., 293:273-280,
2002); simian-human immunodeficiency virus (Yao, Res. Initiat.
Treat Action, 8:20-21, 2003) and bovine leukemia virus (Kakker et
al., Virol., 265:308-318, 1999).
VIII. Exemplary Hepadnavirus Core Antigen Modifications May be
Applied to Any Hepadnavirus
[0315] Exemplary modifications to hepadnavirus core antigens are
illustrated herein using orthohepadnavirus core antigens (e.g.,
from woodchuck, ground squirrel, and arctic ground squirrel)
(Tables 1, 3-1, 3-2), avihepadnavirus core antigens (e.g., from
duck, Ross' goose, Sheldgoose, heron, stork) (Table 3-3), human
hepatitis B virus core antigens (Table 4-1), and non-human primate
hepatitis virus core antigens (e.g., from orangutan, gibbon,
chimpanzee and woolly monkey) (Tables 4-2, 4-3) (see also FIG. 20
and Tables 9, 10, 17, 18). However, the invention is not limited to
these exemplary hepadnavirus core antigens, but rather includes any
hepadnavirus core antigen. The invention's illustrative
modifications are summarized in Table 7-2 below, and may be applied
to any hepadnavirus core antigen using routine methods in
combination with guidance herein, including alignment of the
hepadnavirus core antigens as shown in FIG. 46. For example, unique
restriction cloning sites may be designed to allow insertion at
desired sites, modification of the C-terminus of the hepadnavirus
core antigen, and/or use of acidic amino acid linkers and/or
substitution with acidic amino acids.
TABLE-US-00012 TABLE 7-2 Exemplary Hepadnavirus Core Protein
Combinatorial Modifications Exemplary Insertion Exemplary Exemplary
Points inside Insertion Points Acidic Amino loop at outside loop at
Exemplary Exemplary C- Acid Amino Acid Amino Acid C-Terminal
Terminal substitution.sup.(d) Species No..sup.(a) No..sup.(a),(b)
Amino Acid Modification (SEQ ID NO:) Human 76, 77, 78, 81, 44, 71,
72, 73, V.sup.149 R, K, A, RRC, 99-101 and 82 74, 75, 83, 84, SEQ
ID NO: 2-20, 85, 92, N- 22-36, 42-56, terminal and 153, 155,
C-terminal 157, 159, 161, 163-181, 183-238 Woolly 76, 77, 78, 81,
44, 71, 72, 73, V.sup.149 R, K, A, RRC, 99-101 Monkey and 82 74,
75, 83, 84, SEQ ID NO: 2-20, 85, 92, N- 22-36, 42-56, terminal and
153, 157, C-terminal 159, 161, 163-181, 183-238 Orangutan, 76, 77,
78, 81, 44, 71, 72, 73, V.sup.149 R, K, A, RRC, 99-101 Gibbon, and
and 82 74, 75, 83, 84, SEQ ID NO: 2-20, Chimpanzee 85, 92, N-
22-36, 42-56, terminal and 153, 155, C-terminal 163-181, 183-238
Woodchuck 76, 77, 78, 81, 44, 71, 72, 73, I.sup.149 R, C, K, A,
99-101 and 82 74, 75, 83, 84, RRC, and SEQ 85, 92, N- ID NOs: 2-20,
terminal and 22-36, 42-56, C-terminal 153, 155, 157, 159, 161,
163-181, 183-238 Ground 76, 77, 78, 81, 44, 71, 72, 73, I.sup.148
R, C, K, A, 99-101 Squirrel and 82 74, 75, 83, 84, RRC, and SEQ 85,
92, N- ID NOs: 2-20, terminal and 22-36, 42-56, C-terminal 153,
155, 157, 159, 161, 163-181, 183-238 Arctic 76, 77, 78, 81, 44, 71,
72, 73, I.sup.149 R, C, K, A, 99-101 Ground and 82 74, 75, 83, 84,
RRC, and SEQ Squirrel 85, 92, N- ID NOs: 2-20, terminal and 22-36,
42-56, C-terminal 153, 155, 157, 159, 161, 163-181, 183-238 Avian
91, 92, 93, 96 40, 86, 87, 88, V.sup.195 and A.sup.195 R, C, K, A,
99-101 and 97.sup.(c) 89, 90, 98, 99, RRC, and SEQ 131, 138, N- ID
NOs: 2-20, terminal and 22-36, 42-56, C-terminal 153, 155, 157,
159, 161, 163-181, 183-238 .sup.(a)Bold denotes exemplary preferred
embodiments. .sup.(b)Insertion may also be anywhere within the
1-100 amino acids that are inserted at the C-terminal, such as
insertion within one or more of SEQ ID NO: 2-20, 22-36, 42-56, 153,
183-238. .sup.(c)Insertion may be accompanied by deletion of at
least a portion of the loop. .sup.(d)Substitution may be in one or
more of the hepadnavirus core protein and the inserted heterologous
amino acid sequence of interest.
EXPERIMENTAL
[0316] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0317] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); N (Normal); mol (moles); mmol (millimoles);
(micromoles); nmol (nanomoles); g (grams); mg (milligrams); .mu.g
(micrograms); ng (nanograms); l or L (liters); ml (milliliters);
.mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); .degree. C. (degrees Centigrade); U
(units), mU (milliunits); min. (minutes); sec. (seconds); %
(percent); kb (kilobase); by (base pair); PCR (polymerase chain
reaction); WHV (woodchuck hepadnavirus); GHV (ground squirrel
hepadnavirus); HBV (human hepatitis B virus); HBcAg (hepatitis B
core antigen); WT (wild type); OS (oligosaccharide); PS
(polysaccharide); Tg (transgenic); TCR (T cell receptor); Th
(helper T cell); MHC (major histocompatibility complex); TNF (tumor
necrosis factor); IFN (interferon); mAb (monoclonal antibody), mIg
(membrane immunoglobulin); APC (antigen presenting cell); MO
(macrophage); LN (lymph node); SN (supernatant); RS (restriction
site); ELISA (enzyme linked immunosorbent assay); MOI (multiplicity
of infection); 1.degree. (primary); and 2.degree. (secondary).
[0318] The following reagents find use in the methods and
compositions of the present invention: TCR-Tg mice: 7/16-5 mice
recognizing HBcAg.sub.129-140/A.sup.b; 11/4-12 mice recognizing
HBcAg.sub.129-140/A.sup.b (Chen et al., J. Virol. 74:7587-7599,
2000); and 8/6-10 mice recognizing HBcAg.sub.120-131/A.sup.s;
HBV-Tg mice: HBcAg-Tg mice (Milich et al., J. Immunol. 152:455-466,
1994); and HBeAg-Tg mice (Milich et al., Proc. Natl. Acad. Sci.
USA, 87:6599-6603, 1190); particle-reactive antibodies (polyclonal
anti-WHcAg particles; mAb 2221, anti-WHcAg monomers; mAb 3120,
anti-HBcAg particles; and mAb 3105, anti-HBcAg loop);
insert-reactive antibodies (mAb 2A10, anti-P. falciparum NANP; mAb
2B608, anti-P. falciparum NVDP; mAb 14C2, anti-influenza M2e; mAb
TP2, anti-CETP; and mAb 2F2, anti-P. vivax repeat); CD40L plasmid
pDC406-mCD40L (ATCC No. 68872); tetanus toxin plasmid pMEC4 (Dr.
Locht, Pasteur, Lille, France); 50mer or smaller peptides
(Invitrogen); plasmids PET11d and BL21(DE3) (Stratagene); and pLEX
and K12 K802 E. coli (Invitrogen).
Example 1
Immunization of Mice
[0319] Groups of 3-5 female mice, approximately 6-8 weeks old of
various strains (either bred at the Vaccine Research Institute of
San Diego, San Diego, Calif. or obtained from Jackson Laboratories,
Bar Harbor, Me.) were immunized intraperitoneally for antibody
assays and subcutaneously for T cell assays. Antigens were injected
in saline, or absorbed in 0.1% (w/v) AIPO4 suspension, or
emulsified in IFA or the squalene water-in-oil adjuvant Montanide
ISA 720 (Seppic, Paris) depending on the experiment. Mice were bled
pre-immunization and at various times after primary and booster
immunizations for anti-insert/PS and anti-WHcAg antibody
determinations. A larger number of mice/group (at least 10) were
used to perform mouse potency (dose) studies because at limiting
antigen doses, less than 100% of mice produce antibody, and the
limiting dose was typically defined as the dose at which 50% of the
mice produce antibody.
Example 2
Antibody Assays
[0320] Anti-WHcAg or peptide antibodies were measured in pooled or
individual, murine sera by indirect solid phase ELISA using solid
phase WT WHcAg (50 ng/well) or insert peptide (0.5 .mu.g/well) and
goat anti-mouse Ig (or IgG isotype-specific) antibodies were used
as the secondary antibody. The ELISA was developed with a
peroxidase-labelled, affinity-purified swine anti-goat Ig. The data
were expressed as antibody titer representing the highest dilution
yielding three times (3.times.) the optical density of the
pre-immunization sera. Anti-PS antibodies were measured in an
identical manner on solid phase purified PSs (10 .mu.g/ml), except
that PolySorp plates (Nunc, Rosklide, Denmark) were used to coat PS
antigens. Fifty micrograms of pneumococcal cell wall polysaccharide
(C-PS) per ml of sera were added to absorb any anti-C--PS
antibodies.
Example 3
T Cell Assays
[0321] To measure T cell proliferation, groups of 3-5 mice were
primed with either 10 .mu.g of WT core, hybrid core or PS-core
conjugates by hind footpad injection. Approximately, 7-10 days
after immunization, draining popliteal lymph node (LN) cells were
harvested, and 5.times.10.sup.5 cells in 0.1 ml of Click's medium
were cultured with 0.1 ml of medium containing WT core, hybrid core
or PS-core conjugates, various synthetic peptides, or medium alone.
Cells were cultured for 96 hr at 37.degree. C. in a humidified 5%
CO.sub.2 atmosphere, and during the final 16 hr, 1 .mu.Ci of
.sup.3H-thymidine (.sup.3H-TdR; at 6.7 Ci/mmol, New England
Nuclear, Boston, Mass.) was added to each well. The cells were then
harvested onto filter strips for determination of .sup.3H-TdR
incorporation. The data were expressed as counts per minute
corrected for background proliferation in the absence of antigen
(.DELTA.cpm). The T cell nature of the proliferation was confirmed
by analyzing nylon-wool column-enriched T cells in selected
experiments. To measure cytokine production, identical culture
procedures were used with the exception that 24-72 hr supernatants
were harvested and analyzed for the presence of cytokines (IL-2,
IL-4, IFN.gamma.) in standard ELISPOT assays.
Example 4
In Vivo Antibody Production in Response to WHcAg and GSHcAg
Immunization
[0322] As discussed above, the WHcAg and the GSHcAg are
approximately 67% conserved as compared to the HBcAg at the amino
acid level. In contrast, the WHcAg and the GSHcAg are 91%
conserved. In addition, the HBcAg and the WHcAg migrate differently
in a 1% agarose gel (See, FIG. 4). Furthermore, the WHcAg and the
HBcAg do not significantly crossreact at the antibody (B cell)
level (See, FIG. 6) and are partially crossreactive at the
CD4.sup.+ T helper cell level (See, FIGS. 7-10). Similarly, the
GSHcAg and the HBcAg are not crossreactive at the B and T cell
levels (FIGS. 6, 43; Table 19, 20). Therefore, the following
studies were conducted to determine the immunogenicity of the WHcAg
and the GSHcAg for evaluation of their potential as vaccine carrier
platforms.
[0323] Briefly, eight H-2 congenic murine strains, differing in
MHC-haplotype, were immunized with equal doses of WHcAg, GSHcAg or
HBcAg (7.0 .mu.g) emulsified in incomplete Freund's adjuvant (IFA).
As shown in FIG. 5, the WHcAg elicited higher levels of anti-core
antibodies in 5 (B10.BR, B10.D1, B10.D2, B10.M and B10.RIII) of the
8 strains and equivalent anti-core antibodies in 3 strains (B10.PL,
B10.S and B10) as compared to the HBcAg (6 weeks after
administration of a single dose). The GSHcAg elicited higher levels
of anti-core antibodies than the HBcAg in all 8 strains. This
analysis lso indicated that there are no genetic nonresponders to
the WHcAg or the GSHcAg consistent with what has been previously
reported for the HBcAg (Milich and McLachlan, Science,
234:1398-1401, 1986). However, the hierarchy of responder H-2
haplotypes differs somewhat for the WHcAg and the GSHcAg as
compared to the HBcAg due to the fact that the proteins are
partially crossreactive at the CD4.sup.+ T cell level and each
protein possesses a unique repertoire of CD4.sup.+ T cell epitopes
including several shared epitopes. FIG. 6 depicts the extremely low
level of antibody crossreactivity between the HBcAg and either the
GSHcAg or the WHcAg. In contrast, the WHcAg and the GSHcAg
demonstrate significant crossreactivity. Crossreactivity between
anti-WHc and anti-HBc antibodies ranged between 0 and 0.8%.
Similarly, a panel of monoclonal antibodies (mAb) specific for the
HBcAg was found to be totally non-crossreactive with the WHcAg when
tested for binding to solid phase HBcAg and WHcAg by ELISA. The
anti-HBcAg mAB panel included #3105, #3120 (Takashi, et al., J.
Immunol., 130:2903-2911, 1983), C1-5 (Chemicon, Temecula, Calif.),
C3-1, #440 and #442 (Boehringer Mannheim, Germany), and
H40-C47.
TABLE-US-00013 TABLE 8 In Vivo Antibody Production to WHcAg and
WHeAg in Athymic Mice Antibody Titers (1/dilution) Strain Immunogen
Bleed (day) Anti-WHc Anti-WHe Balb/c (+/+) WHcAg 10 163,840 10,240
24 40 .times. 10.sup.6 2.6 .times. 10.sup.6 WHeAg 10 20,480 20,480
24 2.6 .times. 10.sup.6 655,360 Balb/c (nu/nu) WHcAg 10 10,240 640
24 10,240 0 WHeAg 10 0 0 24 0 0
Example 5
CD4.sup.+ T Cell Responses to the WHcAg
[0324] Importantly, a carrier platform must possess sufficient Th
cell (CD4.sup.+) recognition sites to ensure that every MHC
haplotype will be able to associate with at least one T cell site
in order to avoid genetic (MHC-linked) nonresponsiveness. FIG. 7
illustrates the CD4+ T cell proliferative response to WHcAg and
HBcAg upon immunization of Balb/c mice with WHcAg. The WHcAg was
able to recall a proliferative response at a relatively low in
vitro concentration of 0.12 ng/ml. Also note the low level of
crossreactivity between the WHcAg and the HBcAg. Specifically, the
HBcAg required an in vitro concentration of 80 ng/ml to recall a
proliferative response from WHcAg-primed T cells which amounts to a
666-fold difference from the recall response observed for WHcAg.
This result and additional studies indicate that the WHcAg-primed T
cells in Balb/c mice (H-2.sup.d) recognize a site(s) on WHcAg which
is not conserved on the HBcAg. Specifically, B10.D2 mice
(H-2.sup.d) recognize the p60-80 sequence of WHcAg, which is not
conserved on the HBcAg sequence (See, FIG. 8, panel B). Similarly,
the B10.M (H-2.sup.f) strain also recognizes a T cell epitope
within p60-80 on WHcAg, and WHcAg-primed T cells from this strain
are poorly crossreactive with HBcAg (See, FIG. 8, panel C).
Alternatively, if an WHcAg-specific T cell recognizes a site on
WHcAg which is highly conserved between WHcAg and HBcAg, than the
WHcAg and HBcAg epitopes will be crossreactive for that particular
T cell. Such a circumstance occurs in the B10.S (H-2.sup.s) strain
in which the dominant T cell recognition site is within p120-131, a
region which is highly conserved on HBcAg. Therefore, in strains
bearing the H-2.sup.s haplotype the WHcAg and the HBcAg are
crossreactive at the T cell (CD4.sup.+) level as shown in FIG. 8,
panel A. Thus, the T cell crossreactivity of the WHcAg and the
HBcAg has been shown herein to be variable and dependent upon the T
cell site recognized (as dictated by MHC genotype).
[0325] Additionally, the fine specificity of T cell recognition of
the WHcAg and the HBcAg in 8 different H-2 congenic strains
representing 8 separate MHC genotypes was determined using panels
of WHcAg- and HBcAg-derived synthetic peptides as antigens (See,
FIGS. 9 and 10). In the first place, all 8 MHC genotypes responded
to the WHcAg at the T cell level (no genetic nonresponders).
Secondly, in general, most strains recognize totally distinct sets
of T cell sites on WHcAg and HBcAg, and even when similar regions
are recognized, WHcAg-primed T cells rarely crossreact with
HBcAg-derived peptides and vice versa. The two exceptions are the
H-2.sup.s and the H-2.sup.b haplotypes, which predominantly
recognize the 120-131 (H-2.sup.s) and 129-140 (H-2.sup.b) sites,
both of which are highly conserved between the WHcAg and the HBcAg.
However, even in H-2.sup.s and H-2.sup.b-bearing strains a number
of additional non-crossreactive T cell sites are recognized by
WHcAg or HBcAg-primed T cells. For example, the T cells of B10
(H-2.sup.b) mice primed with WHcAg recognize non-crossreactive T
cell sites at residues 60-80, 80-90 and 105-125 in addition to the
129-140 T cell site.
[0326] Strikingly, in comparing T cell (CD4.sup.+) recognition of
WHcAg versus HBcAg, WHcAg was found to be a more efficient T cell
immunogen than HBcAg. First, in most murine strains the WHcAg
appears to possess a greater number and more potent CD4.sup.+ T
cell epitopes. Second, comparing the amount of IFN.gamma. produced
by WHcAg-primed T cells recalled with WHcAg, with the amount of
IFN.gamma. produced by HBcAg-primed T cells recalled with HBcAg
reveals that WHcAg elicits greater IFN.gamma. production in all but
one strain (i.e., B10.D1). Third, when the WHcAg is used as the
immunogen the ratio of IFN.gamma. produced after in vitro recall
with WHcAg as opposed to in vitro recall with HBcAg is always
greater than 1 (ranges between 3.0 for the B10 and B10.S strains,
and 32 for the B10.M strain). In contrast, when the HBcAg is used
as the immunogen the ratio of IFN.gamma. production after in vitro
recall with HBcAg as compared to recall with WHcAg is 1 or less in
5 of the 8 strains tested. A T cell response, which is recalled
more efficiently by a heterologous antigen than the priming antigen
is referred to as a heteroclitic T cell response. The ability of
the WHcAg to elicit a heteroclitic T cell response from
HBcAg-primed T cells in 3 strains (i.e., B10.D2, B10.PL and
B10.RIII) indicates that the WHcAg is processed and/or presented by
antigen presenting cells (APCs) more efficiently than the
HBcAg.
Example 6
Effect of WHcAg Use as a Vaccine Platform on the Anti-HBc
Diagnostic Assay
[0327] One of the advantages to using WHcAg as a vaccine platform
is because unlike HBcAg, WHcAg is not expected to elicit anti-HBc
antibodies which could compromise the use of the commercial
anti-HBc assay as a diagnostic marker of previous or current HBV
infection. To directly examine this issue murine anti-HBc, anti-WHe
and antisera to the HBcAg-based malaria vaccine candidate V12.PF3.1
and several WHcAg-based malaria-core particles were tested in a
commercial anti-HBc assay (Sorin, Italy). This assay is an
inhibition assay and positivity is measured by % inhibition. As
shown in FIG. 11, anti-HBc demonstrated 100% inhibition and
antisera to the V12-PF3.1 malaria vaccine candidate demonstrated
50% inhibition, respectively. The anti-V12-PF3.1 antisera inhibited
50% because the malaria insert in the loop disrupts one of two
dominant endogenous B cell epitopes on HBcAg (Schodel et al., J
Virol, 66:106-114, 1992). In contrast, murine antisera to native
WHcAg showed low level inhibition (16%), as did antisera to one of
the WHcAg-based hybrid-core particles (150-3RC-M74), while antisera
to a second WHcAg-based hybrid particle (HyW-M78) exhibited no
inhibition. Therefore the use of WHcAg as a vaccine platform
circumvents the problem of interference with the commercial
anti-HBc assay.
Example 7
WHcAg Effectiveness as a Vaccine Platform in HBV Chronic
Carriers
[0328] Another problem with the use of HBcAg as a vaccine platform
is the issue of immune tolerance that exists to HBcAg in chronic
carriers of the HBV. The HBV is endemic in many parts of the world
with an estimated 300-400 million HBV chronic carriers worldwide.
The HBcAg-specific CD4.sup.+ and CD8.sup.+ T cell responses in HBV
chronic carriers are severely depressed and usually undetectable
(Ferrari et al., J Immunol, 145:3442-3449, 1990). As the WHV is not
a human pathogen and because CD4.sup.+ T cell recognition of WHcAg
and HBcAg is mostly non-crossreactive, a vigorous WHcAg-specific Th
cell repertoire is contemplated to be available in HBV chronic
carriers. To explore this issue, an HBV-Tg mouse model of HBV
chronic infection was used in the following studies. HBeAg-Tg mice
produce the secreted form of the HBcAg and HBeAg-Tg mice on a
(B10.S.times.Balb/c).sub.F1 background are tolerant to the HBcAg at
the Th cell level, mimicking the immune status of HBV chronic
carriers (Milich et al., Proc. Natl. Acad. Sci. USA, 87:6599-6603,
1990).
[0329] Wild-type (+/+) mice and HBeAg-Tg mice were immunized with
either HBcAg, a HBcAg-based malaria vaccine candidate (HBc-M,
V12.PF3.1) or a WHcAg-based malaria vaccine candidate (WHc-M,
HyW-M78). Both vaccine candidates possess the same malaria
CS-derived B cell epitope (i.e., NANPNVDP(NANP).sub.3, set forth as
SEQ ID NO:75). The HBc-M vaccine candidate also possesses a
heterologous malaria-specific Th cell epitope referred to as the
malaria universal T cell site (Calvo-Calle et al., J Immunol,
159:1362-1373, 1997). As shown in FIG. 12 panel A, HBeAg-Tg mice
are immune tolerant to the HBcAg and produce no anti-HBc antibody,
whereas, the control (+/+) mice produce anti-HBc at weeks 2, 4 and
8 post immunization. Likewise, immunization with the HBc-based
V12.PF3.1 vaccine candidate also elicits significantly less
anti-HBc antibody production and less anti-NANP antibody production
in HBeAg-Tg mice as compared to the control (+/+) mice (FIG. 12,
panels B and D). Therefore, immune tolerance to the HBcAg was shown
herein to adversely affect the ability of the HBcAg to perform as a
vaccine platform for a malaria-specific B cell epitope. The low
level of anti-core and anti-NANP antibodies that are produced may
be due to the function of the heterologous universal T cell site or
a novel Th cell epitope(s) created at the junction between the
HBcAg and the inserted sequence. The (NANP).sub.3 sequence (SEQ ID
NO:68) is not a T cell epitope in (B10.S.times.Balb/c).sub.F1 mice.
Importantly, the HBeAg-Tg mice demonstrated no diminished ability
to produce anti-WHc or anti-NANP antibodies when the WHcAg-based
malaria vaccine candidate (HyW-M78) was used (FIG. 12, panels C and
E). Thus, the negative effects of immune tolerance to the HBcAg
were circumvented by using the WHcAg as a vaccine platform for a
malaria B cell epitope.
[0330] In additional studies, the HBcAg-primed T cells of
(B10.S.times.Balb/c).sub.F1 mice were found to predominantly
recognize the p120-140 sequence. The p120-140 sequence is highly
conserved between WHcAg and HBcAg, and all three antigens recall in
vitro IFN.gamma., production from HBcAg-primed T cells in +/+ mice
(FIG. 13, panel A). However, p120-140-specific T cells are
tolerized in HBeAg-Tg mice (i.e., no recall response with 120-140),
which accounts for the poor in vitro recall responses elicited by
both the HBcAg and the WHcAg compared to +/+ mice (FIG. 13, panel
D). Similarly, the in vitro T cell responses to the HBcAg and the
WHcAg are significantly reduced in HBeAg-Tg mice as compared to +/+
mice immunized with the HBcAg-based V12.PF3.1 vaccine candidate
because 120-140-specific T cells are tolerized in HBeAg-Tg mice
(FIG. 13, panels B and E). In contrast, an advantage to using the
WHcAg as a carrier platform is the presence of T cell epitopes
within the WHcAg that are unique to the WHcAg and not present on
the HBcAg, for example, residues 60-80 (W) and 80-95 (W) (FIG. 13,
panels C and F). Therefore, while the function of p120-140-specific
T cells is lost in HBeAg-Tg mice immunized with the WHcAg-based
vaccine (HyW-M78) due to the tolerizing effects of the presence of
HBeAg, T cell recognition of the WHcAg-specific T cell epitopes
(p60-80W and p80-95W) is identical in control (+/+) and HBeAg-Tg
mice. The ability of the WHcAg to recall IFN.gamma. production in
HBeAg-Tg mice is marginally decreased as compared to the HBcAg due
to the function of the p60-80 (W) and p80-95 (W) T cell sites,
which are also sufficient to promote high levels of anti-WHc and
anti-NANP antibody production in HBeAg-Tg mice as shown in FIG. 12.
Thus, the WHcAg platform is contemplated to be significantly more
effective in an HBV chronic carrier population than a vaccine based
on the HBcAg platform.
Example 8
Versatility of the WHcAg Combinatorial Technology
[0331] Although the HBcAg has been used as a carrier platform, less
than 50% of selected foreign sequences can be successfully inserted
into HBcAg (See, Jegerlehner et al., Vaccine, 20:3104, 2002; and
International Application No. PCT/US01/25625, hereby incorporated
by reference). This high failure rate is contemplated to be due to
the destabilizing effects of inserting foreign sequences on
particle assembly. To circumvent this problem, others have chosen
to chemically link foreign epitopes to wild-type particles, as
opposed to trying to incorporate the epitopes into the particles by
recombinant methods (Jegerlehner et al., supra, 2002, and
Chackerian et al., J Clin Invest, 108:415-423, 2001). In contrast,
the current invention was developed to accommodate a greater
variety of foreign epitope insertions without destabilizing
particle assembly. Specifically, successful direct insertions of
epitopes have been reported for positions 77, 78, 81, 82 and the N-
and C-termini of HBcAg (Pumpens and Grens, Intervirology,
44:98-114, 2001). On the other hand, using the WHcAg platform, in
addition to positions 77, 78, 81, and 82 within the loop region and
the N- and C-termini, a number of other internal insertion sites
outside the loop region have been identified including positions
44, 71, 72, 73, 74, 75, 76, 83, 84, 85 and 92 (See, FIG. 3).
Importantly, during development of the present invention, three HIV
epitopes, which could not be expressed and/or assembled using the
HBcAg platform, were successfully expressed and assembled in the
context of the WHcAg platform. Specifically, the WHcAg platform
rescued the HIV4.1, HIV5.1, and HIV6.1 epitopes (See, Table 10),
for which failures using HBcAg were previously reported
(International Application No. PCT/US01/25625). In short, a
relatively large library of 17 competent insertion sites on the
WHcAg platform have been identified during development of the
present invention.
[0332] Importantly, this expansion of the number of positions
available for insertion of foreign epitopes was made possible by
the generation of a library of C-terminal modifications to the
WHcAg which variably stabilize insertions at different positions.
In fact, the C-terminal modifications of the WHcAg described herein
comprise a very useful second library of 21 C-terminal
modifications. Table 1 lists the sequences of the various modified
C-termini. The C-terminal modifications were designed to eliminate
RNA/DNA binding motifs, eliminate/substitute prolines, replace the
last five C-terminal amino acids and to eliminate or conserve
non-homologous regions between HBcAg and WHcAg. Wild type or full
length WHcAg binds significant amounts of bacterial RNA/DNA, which
is undesirable for a vaccine platform. During development of the
present invention, RNA/DNA binding to the C-terminally modified
WHcAg particles has largely been eliminated. In contrast, three
different HBcAg C-termini have been previously described: full
length; truncated at residue 149, and truncated plus an added
cysteine at position 150.
[0333] The combined libraries of insertion sites and modified
C-termini accumulated for the WHcAg have permitted the successful
insertion of 22 of 24 attempted sequences (See, Table 9).
Additionally, the sequence of the inserted epitope has been found
to play a role in determining whether a given sequence can be
inserted at a given position in the context of a given C-terminus.
The sequence of selected inserts is provided in Table 10.
Therefore, three variables must be considered in designing a
WHcAg-hybrid particle: the insert position; the C-terminal
sequence; and the epitope sequence. For this reason, a rapid
screening method has been developed to examine efficacy of
expression and assembly of hybrid-core particles at the early
bacterial lysate step. This method makes feasible a combinatorial
approach involving shuffling of the insert position, and the
C-terminal modification for each epitope of interest. As shown in
Table 11, a strong correlation between the relative lysate assembly
scores and the ability to purify hybrid core particles in high
yield has been observed.
TABLE-US-00014 TABLE 9 Summary of Insert Sites, Model Epitopes, and
C-Termini Successfully Tested on the WHcAg Platform List of List of
List of Insert Sites Epitopes.sup.1 C-Termini 44-45 M FL(188) 71-72
MV 150C 72-73 IM2 150R 73-74 IM2(-) 150-2RC 74-75 FV-1 150-3RC
75-76 FV-2 150-4RC 77-78 HV-1 150-3KC 78-79 HV-2 150-3AC 81-82 HV-3
WT-R 82-83 HV-4 WT-R1 83-84 HV 4.1 WT-R2 84-85 HV 5.1 WT-R3 85-86
HV 6.1 C-long 92-93 CETP C-long(M1) C-terminal SEB C-long(M2)
N-terminal AZ C-long(M3) HCV-6 HyW HCV-10 HyW-1 HCV-17 HyW-2 HCV-18
HyW-3 HCV-24 HyW-4 EGFR VIII HyW-5 OMP-1 OMP-2 .sup.1Abbreviations
include: M, malarial CS repeat - P. falciparum; MV, malarial CS
repeat type - P. vivax; IM2, influenza A M2e extracellular domain;
IM2(-), mutant influenza A M2e domain lacking two cysteine
residues; FV, feline immunodeficiency virus-1 gp41, HV, human
immunodeficiency virus gp120; CETP, cholesteryl ester transfer
protein; SEB, staphylococcus enterotoxin B; AZ, .beta.-amyloid;
HCV, hepatitis C virus; EGFR VIII, epidermal growth factor receptor
mutant VIII; and OMP, outer membrane protein. To date 22 out of 24
epitopes tested were accommodated by the WHcAg vaccine platform
(92% success rate). This is in contrast to the less than 50%
success rate seen when using the HBcAg platform (wild type,
I.sup.149 C-terminus and C.sup.150 C-terminus) as determined
through review of the literature and through development of the
present invention.
TABLE-US-00015 TABLE 10 Primary Amino Acid Sequences of the Various
Model Epitopes Designation.sup.1 Sequence Identifier HV-1
GEIKNCSFNISTSIRGKVQKEYA SEQ ID NO: 70 FF HV-2
LTSCNTSVITQACPKVSFEPIPIH SEQ ID NO: 71 YC HV-3
PKVSFEPIPIHYCAPAGFAILKC SEQ ID NO: 72 NN HV-4
THGIRPVVSTQLLLNGSLABEE SEQ ID NO: 73 MV DRAAGQPAGDRADGQPAG SEQ ID
NO: 74 M NANPNVDPNANPNANPNANP SEQ ID NO: 75 IM2
SLLTEVETPIRNEWGCRCNDSSD SEQ ID NO: 76 IM2(--)
SLLTEVETPIRNEWGARANDSSD SEQ ID NO: 77 SEB KKKVTAQELD SEQ ID NO: 78
CETP FGFPEHLLVDFLQSLS SEQ ID NO: 79 FV-1 FYEIIMDIEQNNVQGKQGLQKL SEQ
ID NO: 80 FV-2 MELRKNGRQCGMSEKEEE SEQ ID NO: 81 EGFR VIII
LEEKKGNYVVTDH SEQ ID NO: 82 AZ-1 DAEFRHDSGYEV SEQ ID NO: 83 AZ-2
FRHDSGY SEQ ID NO: 84 HV 4.1 RIKQIGMPGGK SEQ ID NO: 85 HV 5.1
LLELDKWASL SEQ ID NO: 86 HV 6.1 EQELLELDKWASLW SEQ ID NO: 87 HCV-6
DTGFLAAL SEQ ID NO: 88 HCV-10 YCFTPSPV SEQ ID NO: 89 HCV-17
CFRKHPEA SEQ ID NO: 90 HCV-18 EATYSRCG SEQ ID NO: 91 HCV-24
HLHQNIVD SEQ ID NO: 92 .sup.1See footnote to Table 9 for a key to
epitope abbreviations.
TABLE-US-00016 TABLE 11 Positive Correlation Between Expression and
Assembly Scores and Hybrid Particle Purification.sup.1 Assembly
Expression (anti- Accessability Particle Designation (anti-pWHc)
nWHc) (anti-insert) Purification 150-3KC-M74 3 4 4 yes 150-3AC-M74
4 3 4 yes c-long(M3)-M74 3 3 4 yes 150C-M75 4 4 5 yes 150C-M77 4 3
2 yes 150C-M78 5 4 3 yes C-long-M78 3 3 4 yes HyW-M78 3 4 4 yes
HyW-M92 2 3 3 yes HyW-M(NH2) 2 3 3 yes HyW-M(COOH) 3 4 2 yes
HyW-MV78 4 3 4 yes HyW-CE74 3 3 3 yes HyW-HV-4(74) 2 2 ND yes
150C-HV-4(78) 3 2 ND yes HyW-IM2(-)78 4 4 3 yes HyW2-K75 3 3 ND yes
average 3.24 3.24 3.4 n/a 150C-IM2(74) 3 2 3 no HyW-IM2(74) 2 2 3
no WT-R-IM2(74) 3 2 2 no HyW-K78 2 2 ND no HyW-M74- 2 2 2 no
CD40L(470) average 2.4 2.0 2.5 n/a .sup.1The scores shown are
relative and designate antibody binding normalized to wild type
(WT) WHcAg or maximal (MAX) binding for the insert-specific mAbs: 5
= WT/MAX; 4 = 5X less than WT, 3 = 25X less than WT, and 2 = 125X
less than WT. ND = not determined.
Example 9
Effect of Insert Position and C-Terminus on Particle Assembly
[0334] The position of the inserted epitope within the WHcAg has
been shown herein to affect the ability of the hybrid WHcAg core to
assemble as a particle. For example, the (M) epitope in the context
of either HyW or HyW2 C-termini permitted assembly in most
positions tested with the exception of positions 21, 91 and 96
(See, Table 12). Similarly, positions 75, 76, 77, 78, 81, 82, and
83 were permissive in the context of the 150-C C-terminus. Note
that position 74 (bold-type) was not permissive in the context of
the 150-C C-terminus, but this position is rescued in the context
of HyW/HyW2 C-termini. Similarly, position 78 is not permissive for
assembly in the context of the 188-C C-terminus, but is permissive
in combination with HyW/HyW2 and 150-C. Thus, the position of the
insert can affect assembly and non-permissive insert positions can
be rescued through combination with an alternate C-terminus. This
phenomenon was not unique to malaria inserts, as similar effects
were observed with other heterologous sequences.
TABLE-US-00017 TABLE 12 Effect of Insert Position on Hybrid
Particle Assembly.sup.1 C-terminus Epitope Satisfactory Assembly
Poor/Non-Assembly HyW/HyW2 M 44, 73, 74, 75, 78, 84, 85, 21, 91, 96
92, N, C 150C M 75, 76, 77, 78, 81, 82, 83 66, 74, 79, 80, 86 188 M
74 78 HyW/HyW2 CE 74 75, 78 HyW2 FV-1 75, 78 74 HyW2 FV-2 74, 75,
78 -- 150C FV-1 75, 78 74 150C FV-2 74, 75, 78 -- HyW/HyW2 HV-4 74,
75 -- 150C HV-4 75, 78 74 .sup.1Numbers represent the amino acid
position on WHcAg that precedes the inserted epitope. Assembly was
assessed by ELISA using core assembly-dependent anti-Hc antibody.
Bold numbers depict insert positions that can be rescued by
altering the C-terminus.
[0335] A second variable influencing hybrid particle assembly is
the C-terminus of the WHcAg protein (See, Table 13). For example,
the (M) epitope inserted at position 74 results in hybrid core
particle assembly in the context of ten different C-termini,
however, five C-termini are non-permissive for assembly with (M) at
position 74. The (M) epitope in position 78 appears less
destabilizing since most C-termini are permissive including all
five of the C-termini which were non-permissive for this epitope
inserted at position 74. Therefore, non-permissive C-termini can be
rescued by altering the insert position. Interestingly, the two
non-permissive C-termini for (M) at 78 are both permissive for (M)
at 74. Without intending to limit the invention to any mechanism,
this reciprocal relationship indicates that the mechanisms of
destabilization of the (M) insert at positions 74 and 78 are
different and can be stabilized by different C-terminal sequences.
Furthermore, the HyW and HyW2 C-termini appear to be significantly
more permissive for a variety of inserted epitopes and positions
than is the 150-C C-terminus. A summary of the combinatorial
technology is depicted in Table 14. Ten of the heterologous model
epitopes that have been used are listed together with the
combination of C-terminus and insert position which resulted in an
optimal platform. Note that for these ten heterologous epitopes,
seven different combinations of C-terminus plus insert position are
represented. In short as determined during development of the
present invention, no one universal WHcAg platform suffices for all
heterologous epitopes, and thus a combinatorial approach is
necessary for the widest possible application of the WHcAg vaccine
platform technology. During development of the present invention,
various WHcAg C-termini (seven) were used in place of the wild type
HBcAg C-terminus. Specifically, three epitopes were inserted into
modified WHcAg and into modified HBcAg at five different positions.
As shown in Table 15, in all but one instance, the model epitopes
expressed as part of a hybrid HBcAg containing a WHcAg C-terminus
were assembled as virus-like particles. Therefore, the C-terminal
modifications developed for WHcAg are also useful in the context of
the HBcAg N-terminus. Similarly, the same 3 epitopes were inserted
into modified GSHcAg at four different positions using various
WHcAg C-termini (five) in place of the wild-type GSHcAg C-terminus.
As shown in Table 15, in all but one instance, the model epitopes
expressed as part of a hybrid GSHcAg containing a WHcAg C-terminus
were assembled as virus-like particles. Therefore, the C-terminal
modifications developed for WHcAg are also useful in the context of
the GSHcAg N-terminus.
TABLE-US-00018 TABLE 13 Effect of C-terminal Modification on Hybrid
Particle Assembly.sup.1 Epitope Insert Satisfactory Assembly
Poor/Non-Assembly M 74 188, 150R, 150-3RC, 150C, C-long, C-long(M1)
150-4RC, 150-3KC, C-long(M2), WT-R 150-3AC, C-long(M3), HyW HyW1,
HyW2 M 78 150C, HyW, 150-2RC, 150R, 188 150-3RC, C-long,
C-long(M1), C-long(M2), C-long(M3), WT-R CE 74 HyW 150C HV-2 75
HyW2 150C HV-3 74 HyW2 150C HV-3 75 HyW2 150C HV-4 74 HyW 150C
CD40L C 188 150C, 150R (470) IM2(-) 78 HyW 150C .sup.1Numbers
represent the amino acid position on WHcAg that precedes the
inserted epitope. Assembly was assessed by ELISA using core
assembly-dependent anti-Hc antibody. Bold numbers depict C-termini
that can be rescued by altering the insert position.
TABLE-US-00019 TABLE 14 Optimal C-Terminus and Insert Position
Combinations for Model Epitopes.sup.1 Epitope C-Terminus Insert
Position M C-long 78 MV HyW 78 CE HyW 74 FV-1 HyW2 75 FV-2 150C 74
HV-2 HyW2 75 HV-3 HyW2 75 HV-4 150C 75 IM2 WT-R 74 IM2(-) HyW 78
.sup.1The amino acid sequences of the WHcAg C-termini and of the
model epitopes are provided in Tables 1 and 10, respectively. The
optimal platform determination was based upon either the
immunogenicity of the purified hybrid particles or upon their
assembly score.
TABLE-US-00020 TABLE 15 Comparison of the WHcAg (W), GSHcAg (G) and
HBcAg (H) Vaccine Platforms.sup.1 Epitope Position C-Terminus
Platform Assembly M 92 HyW W + H + G + M 78 HyW2 W + H + G + M 83
150C W + H + G + M 78 C-long W + H + G + M 78 C-long (M3) W + H + M
78 150-3KC W + H + M 78 WT-R W + H + G + CETP 74 HyW2 W + H - G -
CETP 75 HyW2 W - H - CETP 78 HyW2 W - H - IM2(-) 78 HyW2 W + H + G
+ IM2(-) 78 150C W - H + G + .sup.1Bold type highlights differences
observed between WHcAg, GSHcAg and HBcAg platform.
Example 10
Immunogenicity of Hybrid Particles
[0336] A number of hybrid core particles containing different
epitopes inserted at different positions with varying C-termini
have been produced and purified during the development of the
present invention (See, Table 16). The in vivo humoral immune
response to the inserted epitope, as well as the WHcAg carrier, was
assessed for these particles. FIGS. 14 and 15 provide a summary of
antibody production 8 weeks after a primary (20 .mu.g), and 6 weeks
after a secondary (10 .mu.g) immunization with the various hybrid
particles emulsified in II-A for both injections. Although a
hierarchy of immunogenicity was observed, most hybrid core
particles were quite immunogenic both in terms of anti-insert and
anti-core antibody production. The primary anti-insert IgG serum
titers ranged from 1:5000 to 15.times.10.sup.6 and the secondary
anti-insert IgG serum titers ranged from 1:25,000 to
1:75.times.10.sup.6. These very high levels of anti-insert antibody
production (particularly over such a wide variety of inserted
epitopes) are unprecedented in the hybrid VLP literature, and thus
the effectiveness of the WHcAg vaccine platform was not
predictable.
[0337] Similarly, the GSHcAg can function as a carrier for inserted
epitopes as demonstrated by immunization of mice with GS-150C-M83
hybrid particles that elicited an early (4 week) anti-insert titer
of 1:5000.
TABLE-US-00021 TABLE 16 Exemplary Purified Hybrid WHcAg Particles
Particle Yield (mg/L) 188-M74 30 150C-M74 2 150R-M74 16 150C-M77 12
150C-M78 18 150C-HV4(78) 2 HyW-HV4 (74) 6 150C-3RC-M74 15
150C-3KC-M74 30 150C-3AC-M74 25 150C-M75 26 C-long-M78 16 HyW-M78
32 HyW2-IM2(-)81 30 150C-IM2(-)82 35 HyW2-SEB75 20 C-long-M3-M74 18
HyW-M74 21 HyW-M(COOH) 31 HyW-M(NH2) 16 HyW2-M75 12 HyW-IM2(-)78 10
HyW-CE74 16 HyW-K(COOH) 28 HyW2-LK75 26 HyW-M92 21 HyW-MV78 38
HyW-MV78/MF(NH2) 35 HyW2-FV-1(75) 22 188-CD40L 2 GS-150C-M78 40
GS-150C-M83 30
Example 11
Effect of Heterologous Insert Position on Immunogenicity
[0338] The immunogenicity of hybrid core particles composed of the
same HyW-modified C-terminus and the same malaria repeat epitope
was found to vary depending on where the epitope (M) was positioned
(See, FIG. 16). Particles with insertions in (position 78) or near
(position 74) the loop were more immunogenic in terms of the
anti-insert response, than were particles with inserts fused to the
N-terminus. Moreover, insert placement at the C-terminus was poorly
immunogenic both in terms of end-point serum titer and delayed
onset of antibody production. This correlation was not true for
anti-carrier antibody production, which was greater or equal for
the N- and C-terminal locations of the (M) epitope, as compared to
the internal insertions. Therefore, the position of the epitope did
not alter the overall immunogenicity of the particle and the
positional effects are due to greater surface exposure and/or
optimal spacing of the heterologous epitopes in or near the loop
region. The high anti-carrier responses to the N- and C-terminally
fused epitopes were contemplated to be due to conservation of the
native loop structure and the endogenous WHcAg B cell epitopes on
these hybrid core particles.
Example 12
Effect of C-Terminus on Immunogenicity
[0339] The immunogenicity of particles with the malaria (M) epitope
inserted at position 74 but with varied C-termini were compared as
shown in FIG. 17. Particles with the native full length (188-M74)
or with the 150-3RC C-terminus were more immunogenic in terms of
serum titers of anti-NANP antibody as well as a quicker onset (week
2) as compared to the 150 3AC and HyW C-termini. The particle
comprised of the 150R C-terminus, which lacks a cysteine, was
weakly immunogenic. The 150R-M74-hybrid particle was the least
stable in vitro (and most likely in vivo), explaining the poorer
immunogenicity results. The in vitro stability of the various other
hybrid core particles is expected to correlate with immunogenicity
in vivo.
[0340] Additionally, a bivalent hybrid core particle was
constructed, containing the Plasmodium falciparum CS repeat epitope
at the N-terminus and the P. vivax CS repeat (type 1) epitope in
the loop position 78, HyW-MV78/M (NH.sub.2). As shown in FIG. 15,
during the primary response antibodies were produced to both
inserts, although the serum titers were rather low (1:5000) as
compared to most single inserts. However, after boosting, high
titer antibodies were produced to both CS repeat sequences. Thus,
the present invention provides bivalent hybrid core particles
containing highly immunogenic epitopes at different positions on
the same particle.
Example 13
Effect of Genetics on Immunogenicity of a WHcAg-based Malaria
Vaccine
[0341] Efforts to produce P. falciparium vaccine candidates based
on the CS repeat sequences have been plagued by low immunogenicity
and severe genetic restriction characterized by low responders in
human clinical trials, and low or nonresponder murine MHC genotypes
in mouse immunization studies. To address this issue, CS-derived
CD4.sup.+ T to cell epitopes such as CS.sub.326-345 were included
(Calvo-Calle et al., J Immunol, 159:1362-1373, 1997), although
murine strains differ in responsiveness to CS.sub.326-345.
Therefore, for pathogen-specific B cell epitopes and in particular
for malaria B cell epitopes, it is imperative that the carrier
platform provide sufficient T cell helper function in the context
of a wide variety of MHC haplotypes to eliminate genetic
nonresponsiveness.
[0342] To directly examine the issue of MHC-linked restriction of
the antibody response to a WHcAg-based vaccine, B10 H-2 congenic
murine strains expressing eight different H-2 haplotypes were
immunized with a 10 .mu.g dose of a WHcAg-malaria vaccine candidate
(HyW-M78) in IFA. Both primary (1.degree., 6 weeks) and secondary
(2.degree.) anti-WHc and anti-NANP serum antibody titers were
determined as shown in FIG. 18. First and importantly, all H-2
haplotypes responded and produced both anti-WHc and anti-NANP
antibodies after a primary immunization with HyW-M78 (no
nonresponder H-2 haplotypes were identified). Secondly, all strains
at all time points produced an equal or greater antibody response
to the insert (anti-NANP) as compared to anti-WHc, with the
exception of the secondary antibody responses of the B10.S strain.
The lack of genetic nonresponders to this experimental WHcAg-based
vaccine is consistent with the absence of nonresponders to the
WHcAg platform itself at the antibody (FIG. 5) and T cell (FIGS. 9
and 10) levels as determined during development of the present
invention.
Example 14
Complexity of T Cell Recognition of Hybrid Particles
[0343] The insertion of a foreign B cell epitope is contemplated to
at times itself either represent a novel T cell site or create a
novel T cell site at the junction between the inserted sequence and
the core sequence. Also, it is contemplated that an insertion at
times disrupts an endogenous T cell site. Unexpectedly, inserting
the NANPNVDP(NANP).sub.3 epitope (SEQ ID NO:75) into the WHcAg
platform, has been observed to cause the loss and/or gain of novel
WHcAg-specific T cell sites unrelated to interrupting an endogenous
T cell site or creating a junctional T cell site, respectively. As
shown in FIG. 19, pairs of H-2 congenic mice were immunized with
wild-type WHcAg or the HyW-M78 hybrid particle containing the
malaria CS repeat epitope. T cell fine specificity was mapped using
a panel of WHcAg-derived synthetic peptides as antigens. Note that
in B10.D2 mice, immunization with HyW-M78 caused a loss of one very
strong T cell site (amino acids 90-105) and the gain of one very
strong T cell site (amino acids 80-95) as compared to WHcAg
immunization. The dominant T cell site at amino acids 60-80 was
functional for both immunogens. The loss of the amino acid 90-105
site was not obvious because the insertion at amino acids 78-79
does not directly interrupt the amino acid 90-105 sequence.
Similarly, the new T cell site at amino acids 80-95, was downstream
of the inserted sequence.
[0344] Likewise, in the B10.PL strain, two new rather weak T cell
sites were gained (amino acids 1-15 and amino acids 50-70) and a
very strong T cell site (amino acids 15-35) was converted into an
intermediate T cell site by the insertion of a malaria B cell
epitope. In the B10.RIII strain three of the four T cell sites
recognized on native WHcAg were either lost or weakened when the
HyW-M78 hybrid particle was used as the immunogen. Lastly, the B10
strain was found to recognize the (NANP).sub.n sequence as a T cell
site, as well as a B cell epitope. This insertion of a novel T cell
site caused the loss of one T cell site (amino acids 105-125), and
converted a very strong T cell site into a weak T cell site (amino
acids 60-80). Thus, the variability of T cell recognition caused by
the insertion of foreign sequences even at a distance from
endogenous T cell sites emphasizes the necessity for a carrier
platform to possess a multiplicity of T cell recognition sites
relevant to any given MHC genotype. The WHcAg satisfies this
requirement as demonstrated by the direct mapping of numerous T
cell sites relevant for each of eight different MHC genotypes, and
by the absence of nonresponder MHC haplotypes corresponding to the
HyW-M78 candidate malaria vaccine. Additionally, the recognition of
the (NANP)n sequence as a T cell site by B10 mice after
immunization with HyW-M78, indicates that the WHcAg platform serves
as a vaccine carrier for heterologous T cell (CD4+) epitopes, as
well as B cell epitopes.
Example 15
Effects of Insert pI and Linker Addition on the Assembly of Hybrid
WHcAg, Hybrid GSHcAg and Hybrid HBcAg Particles
[0345] During development of the present invention, the effect of
insert isoelectric point (pI) on assembly of hybrid hepadna virus
core particles was assessed. The predicted pI shown below was
calculated using the MacVector software program version 6.5.3,
(Oxford Molecular Group, plc). The use of other programs, such as
Protparam Tool and Compute pI/MW (available on the ExPASy
proteomics server of the Swiss Institute of Bioinformatics), for
predicting the pI of an insert peptide sequence was found to give
slightly different pI values. However, as used herein, the
predicted pI calculated using MacVector is considered to be
equivalent to the predicted pI calculated using Protparam Tool,
Compute pI/MW and any analogous algorithms.
[0346] As shown in Table 17, positively charged inserts (e.g., pI
equal to or greater than 7.0) appear to adversely effected assembly
of hybrid WHcAg or HBcAg particles. However, using the methods and
compositions described herein, the addition of acidic substitutions
or linker residues was found to be useful for neutralizing the
apparent destabilizing effect of positively-charged inserts (high
pI) on particle assembly. As shown in Table 18, the addition of
acidic residues rescued hybrid-core particle assembly on WHcAg, the
GSHcAg and HBcAg vaccine platforms. Nonetheless, an understanding
of the mechanism(s) is not necessary in order to make and use the
present invention.
[0347] Additionally, the rescue of a model positively-charged
insert was made possible through the use of either flanking
glutamic acid residues (EE-insert-EE), or flanking aspartic acid
residues (DD-insert-DD). In contrast, neither flanking nonpolar
residues (L-insert-L and P-insert-P), nor flanking uncharged polar
residues (QQ-insert-QQ, TT-insert-TT, and YY-insert-YY) were able
to convert an assembly-incompetent, positively-charged insert into
an assembly-competent insert.
TABLE-US-00022 TABLE 17 Correlation Between Insert pI and Hybrid
Particle Assembly.sup.1 Epitope Sequence Identifier pI Particles
FMDV* RYNRNAVPNLRGDLQVLAQKVARTLF SEQ ID 12.01 - NO: 93 HIV4.1*
RIKQIGMPGGK SEQ ID 11.30 - NO: 85 P. yoelii* TAVVHQLKRKH SEQ ID
11.30 - NO: 94 HIV10.1* HLLQLTVWGIKQLQAR SEQ ID 11.14 NO: 95
IgE.sub.413-435* GETYQSRVTHPHLPRALMRSTTK SEQ ID 11.13 - NO: 96
P450-1A2* GRERRPRLSDRPQLPYLEA SEQ ID 10.92 - NO: 97 HV-1
GEIKNCSFNISTSIRGKVQKEYAFF SEQ ID 9.41 - NO: 70 HV-3
PKVSFEPIPIHYCAPAGFAILKCNN SEQ ID 8.68 - NO: 72 SEB KKKVTAQELD SEQ
ID 8.63 +/- NO: 78 HV-2 LTSCNTSVITQACPKVSFEPIPIHYC SEQ ID 7.00 -
NO: 71 AZ2 FRHDSGY SEQ ID 7.00 - NO: 84 FV-2 MELRKNGRQCGMSEKEEE SEQ
ID 4.86 + NO: 81 HV-4 THGIRPVVSTQLLLNGSLAEEE SEQ ID 4.55 + NO: 73
FV-1 FYEIIMDIEQNNVQGKQGLQKL SEQ ID 4.46 + NO: 80 MV
DRAAGQPAGDRADGQPAG SEQ ID 4.20 + NO: 74 CETP FGFPEHLLVDFLQSL SEQ ID
4.11 + NO: 79 AZ1 DAEFRHDSGYEV SEQ ID 4.08 + NO: 83 IM2(--)
SLLTEVETPIRNEWGARANDSSD SEQ ID 3.95 + NO: 77 M NANPNVDPNANPNANPNANP
SEQ ID 3.43 + NO: 75 MB DPPPPNPNDPPPPNPN SEQ ID 3.22 + NO: 98
.sup.1Asterisk denotes negative assembly on HBcAg as previously
reported (PCT/US01/25625).
TABLE-US-00023 TABLE 18 Effect of Acidic Amino Acid Addition on
Assembly of Hybrid Particles INSERT Epitope Sequence.sup.1
Identifier pI ASSEMBLY WHcAg Platform SEB KKKVTAQELD SEQ ID NO: 78
8.63 +/- SEB2E EEKKKVTAQELD SEQ ID NO: 99 4.20 + EE AZ2 FRHDSGY SEQ
ID NO: 84 7.00 - AZ2E EEFRHDSGYEE SEQ ID NO: 100 4.02 + HIV4.1
RIKQIGMPGGK SEQ ID NO: 85 11.3 - HIV4.1E EERIKQIGMPGGK SEQ ID NO:
101 4.74 + EE HBcAg Platform AZ2 FRHDSGY SEQ ID NO: 84 7.00 - AZ2E
EEFRHDSGYEE SEQ ID NO: 100 4.02 + HIV4.1* RIKQIGMPGGK SEQ ID NO: 85
11.3 - HIV4.1E EERIKQIGMPGGK SEQ ID NO: 101 4.74 + EE GSHcAg
Platform AZ2 FRHDSGY SEQ ID NO: 84 7.00 + AZ2E EEFRHDSGYEE SEQ ID
NO: 100 4.02 ++ HIV4.1 RIKQIGMPGGK SEQ ID NO: 85 11.3 - HIV4.1E
EERIKQIGMPGGK SEQ ID NO: 101 4.74 + EE .sup.1Acidic substitutions
or linker additions are shown in underlined, while asterisks denote
negative assembly on HBcAg as previously reported
(PCT/US01/25625).
TABLE-US-00024 TABLE 19 Antibody crossreactivity between the HBcAg,
the WHcAg, the GSHcAg and the ArGSHcAg. ##STR00001## The indicated
antigens (i.e., recombinant core particles) were examined for
reactivity with the listed panel of antibodies. The anti-HBc
antibodies consisted of monoclonal (Mab) and the remainder were
polyclonal antibodies. (.DELTA. loop) refers to alteration or
disruption of the core loop antigenic region by insertion of a
foreign B cell epitope sequence.
TABLE-US-00025 TABLE 20 Crossreactivity of GSHcAg-primed T cells
for WhcAg and HBcAg. IL-2 (U/ml) Strain H-2 GSHcAg WHcAg HBcAg
B10.BR (k) 2,500 450 0 B10.D1 (q) 900 130 0 B10.D2 (d) 1,200 320 0
B10.M (f) 600 290 0 B10.PL (u) 2,000 400 0 B10.RIII (r) 2,400 750 0
B10.S (s) 2,500 1,800 750 B10 (b) 850 300 110 The indicated B10,
H-2 congenic strains were immunized with GSHcAg (10 .mu.g, IFA) and
4 weeks later spleen cells were incubated with GSHcAg, WHcAg or
HBcAg (0.5 .mu.g/ml) for 2 days and IL-2 in the culture media was
measured by ELISA.
Example 16
Avoiding the Problem of Pre-Existing Anti-HBc Antibodies by Using
the WHcAg or GSHcAg Platforms
[0348] Pre-existing anti-HBc antibodies are present in all chronic
or acute HBV patients as well as persons previously exposed to HBV
even though they have recovered. Pre-existing antibodies to a
carrier protein may have negative effects on the primary response
to a carrier-hapten complex because the carrier-hapten complex may
be prematurely cleared from the circulation due to the presence of
circulating anti-carrier antibodies. This is less of a concern
after secondary boosting with the carrier-hapten complex because
although anti-carrier antibodies may have been elicited by the
primary immunization anti-hapten memory B cells are also present
and require less antigen for the booster effect to occur. A way to
avoid this problem of pre-existing anti-HBc antibodies is to use a
carrier platform, which will not be recognized by natural anti-HBc
antibodies. As shown in Table 21, HBcAg particles are recognized by
anti-HBc antibodies present in the serum of chronic HBV patients
and acute HBV patients. Similarly, hybrid-HBcAg particles are also
recognized by chronic and acute HBV patient sera (data not
shown).
[0349] We have previously reported that anti-HBc antibodies in the
sera of chronic but not acute HBV-infected patients can recognize
the WHcAg and in fact suggested that this WHcAg crossreactivity
could be exploited as a diagnostic test for chronic as opposed to
acute HBV infection (Maruyama, et al., Gastroenterol.,
106:1006-1015, 1994). Indeed, as shown in Table 21, WHcAg is
recognized by anti-HBc antibodies in chronic patient sera but not
acute patient sera. This crossreactivity could compromise the
efficacy of the WHcAg platform at least in chronic HBV patients
just as the use of the HBcAg platform may be compromised in all
HBV-exposed individuals with anti-HBc antibodies.
[0350] However, as shown in Table 21, hybrid-WHcAg particles, which
would be used as a vaccine, are not recognized by chronic HBV
patient serum anti-HBc antibodies. Regardless if the heterologous B
cell epitope is inserted in the loop region, 76-82, outside the
loop region (i.e., 74,75,92) or N- or C-terminally on WHcAg, the
anti-HBc crossreactive determinant on WHcAg is destroyed on
hybrid-WHcAg particles. Therefore, pre-existing anti-HBc antibodies
in the sera of HBV-exposed individuals will not compromise the
efficacy of the WHcAg platform.
TABLE-US-00026 TABLE 21 Problem of pre-existing anti-core
antibodies in HBV-infected or previously infected patients.
##STR00002## Human sera from chronic HBV patients (6), acute HBV
patients (6) or normal non- infected blood donors (4) were diluted
1:500 and tested in ELISA using the indicated core particles as the
solid-phase ligands (20 ng/well). The data are presented as mean
O.D..sub.492 values .+-. standard deviations.
[0351] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology,
genetics, or related fields are intended to be within the scope of
the following claims.
Sequence CWU 1
1
2561188PRTWoodchuck hepatitis virus 1Met Asp Ile Asp Pro Tyr Lys
Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp
Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Thr Ala Leu
Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro His His
Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu 50 55 60Leu Thr Lys
Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln65 70 75 80Val
Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu Lys 85 90
95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln
100 105 110His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile
Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu
Ser Thr Leu Pro 130 135 140Glu His Thr Val Ile Arg Arg Arg Gly Gly
Ala Arg Ala Ser Arg Ser145 150 155 160Pro Arg Arg Arg Thr Pro Ser
Pro Arg Arg Arg Arg Ser Gln Ser Pro 165 170 175Arg Arg Arg Arg Ser
Gln Ser Pro Ser Ala Asn Cys 180 185239PRTWoodchuck hepatitis virus
2Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser Pro Arg Arg Arg Thr1 5
10 15Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser 20 25 30Gln Ser Pro Ser Ala Asn Cys 3534PRTWoodchuck hepatitis
virus 3Arg Arg Arg Cys145PRTWoodchuck hepatitis virus 4Arg Arg Arg
Arg Cys1 554PRTWoodchuck hepatitis virus 5Lys Lys Lys
Cys164PRTWoodchuck hepatitis virus 6Ala Ala Ala Cys1723PRTWoodchuck
hepatitis virus 7Ala Ala Gly Gly Ala Arg Ala Ser Arg Ser Pro Ser
Gln Ser Pro Ser1 5 10 15Gln Ser Pro Ser Ala Asn Cys
20821PRTWoodchuck hepatitis virus 8Ala Ala Gly Gly Ala Arg Ala Ser
Arg Ser Gln Ser Pro Ser Gln Ser1 5 10 15Pro Ser Ala Asn Cys
20920PRTWoodchuck hepatitis virus 9Ala Ala Gly Gly Ala Arg Ala Ser
Arg Ser Gln Ser Ser Gln Ser Pro1 5 10 15Ser Ala Asn Cys
201019PRTWoodchuck hepatitis virus 10Ala Ala Gly Gly Ala Arg Ala
Ser Arg Ser Gln Ser Ser Gln Ser Ser1 5 10 15Ala Asn
Cys1113PRTWoodchuck hepatitis virus 11Arg Arg Gly Gly Ala Arg Ala
Ser Gln Ser Ala Asn Cys1 5 101213PRTWoodchuck hepatitis virus 12Ala
Arg Gly Gly Ala Arg Ala Ser Gln Ser Ala Asn Cys1 5
101313PRTWoodchuck hepatitis virus 13Arg Ala Gly Gly Ala Arg Ala
Ser Gln Ser Ala Asn Cys1 5 101413PRTWoodchuck hepatitis virus 14Ala
Ala Gly Gly Ala Arg Ala Ser Gln Ser Ala Asn Cys1 5
101518PRTWoodchuck hepatitis virus 15Ala Ala Gly Arg Ser Pro Ser
Gln Ser Pro Ser Gln Ser Arg Glu Ser1 5 10 15Gln Cys1618PRTWoodchuck
hepatitis virus 16Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln
Ser Pro Ser Ala1 5 10 15Asn Cys1717PRTWoodchuck hepatitis virus
17Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln Ser Ser Ala Asn1
5 10 15Cys1815PRTWoodchuck hepatitis virus 18Ala Ala Gly Arg Ser
Gln Ser Pro Ser Gln Ser Ser Ala Asn Cys1 5 10 151916PRTWoodchuck
hepatitis virus 19Ala Ala Gly Arg Ser Pro Ser Gln Ser Ser Gln Ser
Ser Ala Asn Cys1 5 10 152014PRTWoodchuck hepatitis virus 20Ala Ala
Gly Arg Ser Gln Ser Ser Gln Ser Ser Ala Asn Cys1 5 1021187PRTGround
squirrel hepatitis virus 21Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly
Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro
Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Ala Ala Leu Tyr Glu Glu
Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile
Arg Gln Ala Leu Val Cys Trp Glu Glu 50 55 60Leu Thr Arg Leu Ile Thr
Trp Met Ser Glu Asn Thr Thr Glu Glu Val65 70 75 80Arg Arg Ile Ile
Val Asp His Val Asn Asn Thr Trp Gly Leu Lys Val 85 90 95Arg Gln Thr
Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln His 100 105 110Thr
Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120
125Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu
130 135 140His Thr Val Ile Arg Arg Arg Gly Gly Ser Arg Ala Ala Arg
Ser Pro145 150 155 160Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg
Ser Gln Ser Pro Arg 165 170 175Arg Arg Arg Ser Gln Ser Pro Ala Ser
Asn Cys 180 1852239PRTGround squirrel hepatitis virus 22Arg Arg Arg
Gly Gly Ser Arg Ala Ala Arg Ser Pro Arg Arg Arg Thr1 5 10 15Pro Ser
Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 20 25 30Gln
Ser Pro Ala Ser Asn Cys 352323PRTGround squirrel hepatitis virus
23Ala Ala Gly Gly Ser Arg Ala Ala Arg Ser Pro Ser Gln Ser Pro Ser1
5 10 15Gln Ser Pro Ala Ser Asn Cys 202421PRTGround squirrel
hepatitis virus 24Ala Ala Gly Gly Ser Arg Ala Ala Arg Ser Gln Ser
Pro Ser Gln Ser1 5 10 15Pro Ala Ser Asn Cys 202520PRTGround
squirrel hepatitis virus 25Ala Ala Gly Gly Ser Arg Ala Ala Arg Ser
Gln Ser Ser Gln Ser Pro1 5 10 15Ala Ser Asn Cys 202619PRTGround
squirrel hepatitis virus 26Ala Ala Gly Gly Ser Arg Ala Ala Arg Ser
Gln Ser Ser Gln Ser Ala1 5 10 15Ser Asn Cys2714PRTGround squirrel
hepatitis virus 27Arg Arg Gly Gly Ser Arg Ala Ala Ser Gln Ala Ser
Asn Cys1 5 102814PRTGround squirrel hepatitis virus 28Ala Arg Gly
Gly Ser Arg Ala Ala Ser Gln Ala Ser Asn Cys1 5 102914PRTGround
squirrel hepatitis virus 29Arg Ala Gly Gly Ser Arg Ala Ala Ser Gln
Ala Ser Asn Cys1 5 103014PRTGround squirrel hepatitis virus 30Ala
Ala Gly Gly Ser Arg Ala Ala Ser Gln Ala Ser Asn Cys1 5
103118PRTGround squirrel hepatitis virus 31Ala Ala Gly Arg Ser Pro
Ser Gln Ser Pro Ser Gln Ser Arg Glu Ser1 5 10 15Gln
Cys3218PRTGround squirrel hepatitis virus 32Ala Ala Gly Arg Ser Pro
Ser Gln Ser Pro Ser Gln Ser Pro Ala Ser1 5 10 15Asn
Cys3317PRTGround squirrel hepatitis virus 33Ala Ala Gly Arg Ser Pro
Ser Gln Ser Pro Ser Gln Ser Ala Ser Asn1 5 10 15Cys3415PRTGround
squirrel hepatitis virus 34Ala Ala Gly Arg Ser Gln Ser Pro Ser Gln
Ser Ala Ser Asn Cys1 5 10 153516PRTGround squirrel hepatitis virus
35Ala Ala Gly Arg Ser Pro Ser Gln Ser Ser Gln Ser Ala Ser Asn Cys1
5 10 153614PRTGround squirrel hepatitis virus 36Ala Ala Gly Arg Ser
Gln Ser Ser Gln Ser Ala Ser Asn Cys1 5 1037567DNAWoodchuck
hepatitis virus 37atggacatag atccctataa agaatttggt tcatcttatc
agttgttgaa ttttcttcct 60ttggacttct ttcctgacct taatgctttg gtggacactg
ctactgcctt gtatgaagaa 120gagctaacag gtagggaaca ttgctctccg
caccatacag ctattagaca agctttagta 180tgctgggatg aattaactaa
attgatagct tggatgagct ctaacataac ttctgaacaa 240gtaagaacaa
tcattgtaaa tcatgtcaat gatacctggg gacttaaggt gagacaaagt
300ttatggtttc atttgtcatg tctcactttc ggacaacata cagttcaaga
atttttagta 360agttttggag tatggatcag gactccagct ccatatagac
ctcctaatgc acccattctc 420tcgactcttc cggaacatac agtcattagg
agaagaggag gtgcaagagc ttctaggtcc 480cccagaagac gcactccctc
tcctcgcagg agaagatctc aatcaccgcg tcgcagacgc 540tctcaatctc
catctgccaa ctgctga 56738149PRTWoodchuck hepatitis virus 38Met Asp
Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn
Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25
30Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys
35 40 45Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp
Glu 50 55 60Leu Thr Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser
Glu Gln65 70 75 80Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr
Trp Gly Leu Lys 85 90 95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys
Leu Thr Phe Gly Gln 100 105 110His Thr Val Gln Glu Phe Leu Val Ser
Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu His Thr Val
Ile14539564DNAGround squirrel hepatitis virus 39atggacatag
atccctataa agaatttggt tcttcttatc agttgttgaa ttttcttcct 60ttggactttt
ttcctgatct caatgcattg gtggacactg ctgctgctct ttatgaagaa
120gaattaacag gtagggagca ttgttctcct catcatactg ctattagaca
ggccttagtg 180tgttgggaag aattaactag attaattaca tggatgagtg
aaaatacaac agaagaagtt 240agaagaatta ttgttgatca tgtcaataat
acttggggac ttaaagtaag acagacttta 300tggtttcatt tatcatgtct
tacttttgga caacacacag ttcaagaatt tttggttagt 360tttggagtat
ggattagaac tccagctcct tatagaccac ctaatgcacc cattttatca
420actcttccgg aacatacagt cattaggaga agaggaggtt caagagctgc
taggtccccc 480cgaagacgca ctccctctcc tcgcaggaga aggtctcaat
caccgcgtcg cagacgctct 540caatctccag cttccaactg ctga
56440148PRTGround squirrel hepatitis virus 40Met Asp Ile Asp Pro
Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro
Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Ala
Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro
His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Glu Glu 50 55 60Leu
Thr Arg Leu Ile Thr Trp Met Ser Glu Asn Thr Thr Glu Glu Val65 70 75
80Arg Arg Ile Ile Val Asp His Val Asn Asn Thr Trp Gly Leu Lys Val
85 90 95Arg Gln Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln
His 100 105 110Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile
Arg Thr Pro 115 120 125Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu
Ser Thr Leu Pro Glu 130 135 140His Thr Val Ile14541183PRTHuman
hepatitis B virus 41Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr
Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val
Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu
Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95Phe Arg Gln Leu Leu
Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val
Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro
Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135
140Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg
Thr145 150 155 160Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser 165 170 175Gln Ser Arg Glu Ser Gln Cys
1804234PRTHuman hepatitis B virus 42Arg Arg Arg Gly Arg Ser Pro Arg
Arg Arg Thr Pro Ser Pro Arg Arg1 5 10 15Arg Arg Ser Gln Ser Pro Arg
Arg Arg Arg Ser Gln Ser Arg Glu Ser 20 25 30Gln Cys4318PRTHuman
hepatitis B virus 43Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln
Ser Arg Glu Ser1 5 10 15Gln Cys4416PRTHuman hepatitis B virus 44Ala
Ala Gly Arg Ser Gln Ser Pro Ser Gln Ser Arg Glu Ser Gln Cys1 5 10
154515PRTHuman hepatitis B virus 45Ala Ala Gly Arg Ser Gln Ser Ser
Gln Ser Arg Glu Ser Gln Cys1 5 10 154614PRTHuman hepatitis B virus
46Ala Ala Gly Arg Ser Gln Ser Ser Gln Ser Glu Ser Gln Cys1 5
104711PRTHuman hepatitis B virus 47Arg Arg Gly Ser Gln Ser Arg Glu
Ser Gln Cys1 5 104811PRTHuman hepatitis B virus 48Ala Arg Gly Ser
Gln Ser Arg Glu Ser Gln Cys1 5 104911PRTHuman hepatitis B virus
49Arg Ala Gly Ser Gln Ser Arg Glu Ser Gln Cys1 5 105011PRTHuman
hepatitis B virus 50Ala Ala Gly Ser Gln Ser Arg Glu Ser Gln Cys1 5
105118PRTHuman hepatitis B virus 51Ala Ala Gly Arg Ser Pro Ser Gln
Ser Pro Ser Gln Ser Pro Ser Ala1 5 10 15Asn Cys5218PRTHuman
hepatitis B virus 52Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln
Ser Arg Glu Ser1 5 10 15Gln Cys5317PRTHuman hepatitis B virus 53Ala
Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln Ser Glu Ser Gln1 5 10
15Cys5415PRTHuman hepatitis B virus 54Ala Ala Gly Arg Ser Gln Ser
Pro Ser Gln Ser Glu Ser Gln Cys1 5 10 155516PRTHuman hepatitis B
virus 55Ala Ala Gly Arg Ser Pro Ser Gln Ser Ser Gln Ser Glu Ser Gln
Cys1 5 10 155614PRTHuman hepatitis B virus 56Ala Ala Gly Arg Ser
Gln Ser Ser Gln Ser Glu Ser Gln Cys1 5 1057552DNAHuman hepatitis B
virus 57atggacatcg acccttataa agaatttgga gctactgtgg agttactctc
gtttttgcct 60tctgacttct ttccttcagt acgagatctt ctagataccg cctcagctct
gtatcgggaa 120gccttagagt ctcctgagca ttgttcacct caccatactg
cactcaggca agcaattctt 180tgctgggggg aactaatgac tctagctacc
tgggtgggtg ttaatttgga agatccagca 240tccagagacc tagtagtcag
ttatgtcaac actaatatgg gcctaaagtt caggcaactc 300ttgtggtttc
acatttcttg tctcactttt ggaagagaaa ccgttataga gtatttggtg
360tctttcggag tgtggattcg cactcctcca gcttatagac caccaaatgc
ccctatccta 420tcaacacttc cggaaactac tgttgttaga cgacgaggca
ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag gtctcaatcg
ccgcgtcgca gaagatctca atctcgggaa 540tctcaatgtt ga 55258149PRTHuman
hepatitis B virus 58Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr
Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val
Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu
Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln
Ala Ile Leu Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Thr Trp Val
Gly Val Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp Leu Val Val
Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95Phe Arg Gln Leu Leu
Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val
Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro
Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135
140Glu Thr Thr Val Val1455912PRTArtificial SequenceSynthetic 59Val
Ser Phe Gly Val Trp Ile Arg Thr Pro Ala Pro1 5 106012PRTArtificial
SequenceSynthetic 60Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro
Ala1 5 106121PRTArtificial SequenceSynthetic 61Val Cys Trp Asp Glu
Leu Thr Lys Leu Ile Ala Trp Met Ser Ser Asn1 5 10 15Ile Thr Ser Glu
Gln206221PRTArtificial SequenceSynthetic 62Leu Cys Trp Gly Glu Leu
Met Thr Leu Ala Thr Trp Val Gly Gly Asn1 5 10 15Leu Glu Asp Pro
Ile206319DNAArtificial SequenceSynthetic 63ggaaattctt ctcctcgag
196424PRTArtificial SequenceSynthetic 64Met Ser Leu Leu
Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly1 5 10 15Cys Arg Cys
Asn Asp Ser Ser Asp 206518PRTPlasmodium vivax 65Ala Asn Gly Ala Gly
Asn Gln Pro Gly Ala Asn Gly Ala Gly Asp Gln1 5 10 15Pro
Gly6618PRTPlasmodium vivax 66Ala Asn Gly Ala Asp Asn Gln Pro Gly
Ala Asn Gly Ala Asp Asp Gln1 5 10 15Pro Gly6722PRTPlasmodium vivax
67Ala Pro Gly Ala Asn Gln Glu Gly Gly Ala Ala Ala Pro Gly Ala Asn1
5 10 15Gln Glu Gly Gly Ala Ala 206812PRTArtificial
SequenceSynthetic 68Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn
Pro1 5 1069260PRTMus musculus 69Met Ile Glu Thr Tyr Ser Gln Pro Ser
Pro Arg Ser Val Ala Thr Gly1 5 10 15Leu Pro Ala Ser Met Lys Ile Phe
Met Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met Ile Gly Ser
Val Leu Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp Lys Val Glu
Glu Glu Val Asn Leu His Glu Asp Phe Val 50 55 60Phe Ile Lys Lys Leu
Lys Arg Cys Asn Lys Gly Glu Gly Ser Leu Ser65 70 75 80Leu Leu Asn
Cys Glu Glu Met Arg Arg Gln Phe Glu Asp Leu Val Lys 85 90 95Asp Ile
Thr Leu Asn Lys Glu Glu Lys Lys Glu Asn Ser Phe Glu Met 100 105
110Gln Arg Gly Asp Glu Asp Pro Gln Ile Ala Ala His Val Val Ser Glu
115 120 125Ala Asn Ser Asn Ala Ala Ser Val Leu Gln Trp Ala Lys Lys
Gly Tyr 130 135 140Tyr Thr Met Lys Ser Asn Leu Val Met Leu Glu Asn
Gly Lys Gln Leu145 150 155 160Thr Val Lys Arg Glu Gly Leu Tyr Tyr
Val Tyr Thr Gln Val Thr Phe 165 170 175Cys Ser Asn Arg Glu Pro Ser
Ser Gln Arg Pro Phe Ile Val Gly Leu 180 185 190Trp Leu Lys Pro Ser
Ser Gly Ser Glu Arg Ile Leu Leu Lys Ala Ala 195 200 205Asn Thr His
Ser Ser Ser Gln Leu Cys Glu Gln Gln Ser Val His Leu 210 215 220Gly
Gly Val Phe Glu Leu Gln Ala Gly Ala Ser Val Phe Val Asn Val225 230
235 240Thr Glu Ala Ser Gln Val Ile His Arg Val Gly Phe Ser Ser Phe
Gly 245 250 255Leu Leu Lys Leu 2607025PRTArtificial
SequenceSynthetic 70Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Ser Thr
Ser Ile Arg Gly1 5 10 15Lys Val Gln Lys Glu Tyr Ala Phe Phe 20
257126PRTArtificial SequenceSynthetic 71Leu Thr Ser Cys Asn Thr Ser
Val Ile Thr Gln Ala Cys Pro Lys Val1 5 10 15Ser Phe Glu Pro Ile Pro
Ile His Tyr Cys 20 257225PRTArtificial SequenceSynthetic 72Pro Lys
Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala1 5 10 15Gly
Phe Ala Ile Leu Lys Cys Asn Asn 20 257322PRTArtificial
SequenceSynthetic 73Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu
Leu Leu Asn Gly1 5 10 15Ser Leu Ala Glu Glu Glu 207418PRTArtificial
SequenceSynthetic 74Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala
Asp Gly Gln Pro1 5 10 15Ala Gly7520PRTArtificial SequenceSynthetic
75Asn Ala Asn Pro Asn Val Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro1
5 10 15Asn Ala Asn Pro 207623PRTArtificial SequenceSynthetic 76Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys1 5 10
15Arg Cys Asn Asp Ser Ser Asp 207723PRTArtificial SequenceSynthetic
77Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Ala1
5 10 15Arg Ala Asn Asp Ser Ser Asp 207810PRTArtificial
SequenceSynthetic 78Lys Lys Lys Val Thr Ala Gln Glu Leu Asp1 5
107916PRTArtificial SequenceSynthetic 79Phe Gly Phe Pro Glu His Leu
Leu Val Asp Phe Leu Gln Ser Leu Ser1 5 10 158022PRTArtificial
SequenceSynthetic 80Phe Tyr Glu Ile Ile Met Asp Ile Glu Gln Asn Asn
Val Gln Gly Lys1 5 10 15Gln Gly Leu Gln Lys Leu 208118PRTArtificial
SequenceSynthetic 81Met Glu Leu Arg Lys Asn Gly Arg Gln Cys Gly Met
Ser Glu Lys Glu1 5 10 15Glu Glu8213PRTArtificial SequenceSynthetic
82Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His1 5
108312PRTArtificial SequenceSynthetic 83Asp Ala Glu Phe Arg His Asp
Ser Gly Tyr Glu Val1 5 10847PRTArtificial SequenceSynthetic 84Phe
Arg His Asp Ser Gly Tyr1 58511PRTArtificial SequenceSynthetic 85Arg
Ile Lys Gln Ile Gly Met Pro Gly Gly Lys1 5 108610PRTArtificial
SequenceSynthetic 86Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu1 5
108714PRTArtificial SequenceSynthetic 87Glu Gln Glu Leu Leu Glu Leu
Asp Lys Trp Ala Ser Leu Trp1 5 10888PRTArtificial SequenceSynthetic
88Asp Thr Gly Phe Leu Ala Ala Leu1 5898PRTArtificial
SequenceSynthetic 89Tyr Cys Phe Thr Pro Ser Pro Val1
5908PRTArtificial SequenceSynthetic 90Cys Phe Arg Lys His Pro Glu
Ala1 5918PRTArtificial SequenceSynthetic 91Glu Ala Thr Tyr Ser Arg
Cys Gly1 5928PRTArtificial SequenceSynthetic 92His Leu His Gln Asn
Ile Val Asp1 59326PRTArtificial SequenceSynthetic 93Arg Tyr Asn Arg
Asn Ala Val Pro Asn Leu Arg Gly Asp Leu Gln Val1 5 10 15Leu Ala Gln
Lys Val Ala Arg Thr Leu Phe 20 259411PRTArtificial
SequenceSynthetic 94Thr Ala Val Val His Gln Leu Lys Arg Lys His1 5
109516PRTArtificial SequenceSynthetic 95His Leu Leu Gln Leu Thr Val
Trp Gly Ile Lys Gln Leu Gln Ala Arg1 5 10 159623PRTArtificial
SequenceSynthetic 96Gly Glu Thr Tyr Gln Ser Arg Val Thr His Pro His
Leu Pro Arg Ala1 5 10 15Leu Met Arg Ser Thr Thr Lys
209719PRTArtificial SequenceSynthetic 97Gly Arg Glu Arg Arg Pro Arg
Leu Ser Asp Arg Pro Gln Leu Pro Tyr1 5 10 15Leu Glu
Ala9816PRTArtificial SequenceSynthetic 98Asp Pro Pro Pro Pro Asn
Pro Asn Asp Pro Pro Pro Pro Asn Pro Asn1 5 10 159914PRTArtificial
SequenceSynthetic 99Glu Glu Lys Lys Lys Val Thr Ala Gln Glu Leu Asp
Glu Glu1 5 1010011PRTArtificial SequenceSynthetic 100Glu Glu Phe
Arg His Asp Ser Gly Tyr Glu Glu1 5 1010115PRTArtificial
SequenceSynthetic 101Glu Glu Arg Ile Lys Gln Ile Gly Met Pro Gly
Gly Lys Glu Glu1 5 10 15102187PRTArctic ground squirrel hepatitis
virus 102Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln
Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro Glu Leu Asn Ala
Leu Val Asp 20 25 30Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly
Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile Arg Gln Ala Leu
Val Cys Trp Glu Glu 50 55 60Leu Thr Arg Leu Ile Ala Trp Met Ser Ala
Asn Ile Asn Ser Glu Glu65 70 75 80Val Arg Arg Val Ile Val Ala His
Val Asn Asp Thr Trp Gly Leu Lys 85 90 95Val Arg Gln Asn Leu Trp Phe
His Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110His Thr Val Gln Glu
Phe Leu Val Ser Phe Gly Val Arg Ile Arg Thr 115 120 125Pro Ala Pro
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu
His Thr Val Ile Arg Arg Arg Gly Ser Ala Arg Val Val Arg Ser145 150
155 160Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro 165 170 175Arg Arg Arg Pro Gln Ser Pro Ala Ser Asn Cys 180
185103188PRTWoodchuck hepatitis virus 103Met Asp Ile Asp Pro Tyr
Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu
Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Thr Ala
Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro His
His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu 50 55 60Leu Thr
Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln65 70 75
80Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu Lys
85 90 95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly
Gln 100 105 110His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140Glu His Thr Val Ile Arg Arg Arg Gly
Gly Ala Arg Ala Ser Arg Ser145 150 155 160Pro Arg Arg Arg Thr Pro
Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro 165 170 175Arg Arg Arg Arg
Ser Gln Ser Pro Ser Ala Asn Cys 180 185104188PRTWoodchuck hepatitis
virus 104Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln
Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala
Leu Val Asp 20 25 30Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly
Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile Arg Gln Ala Leu
Val Cys Trp Asp Glu 50 55 60Leu Thr Lys Leu Ile Ala Trp Met Ser Ser
Asn Ile Thr Ser Glu Gln65 70 75 80Val Arg Thr Ile Ile Val Asn His
Val Asn Asp Thr Trp Gly Leu Lys 85 90 95Val Arg Gln Ser Leu Trp Phe
His Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110His Thr Val Gln Glu
Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Ala Pro
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu
His Thr Val Ile Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser145 150
155 160Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro 165 170 175Arg Arg Arg Arg Ser Gln Ser Pro Ser Ala Asn Cys 180
185105187PRTWoodchuck hepatitis virus 105Met Asp Ile Asp Pro Tyr
Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu
Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Thr Ala
Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro His
His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu 50 55 60Leu Thr
Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln65 70 75
80Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu Lys
85 90 95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly
Gln 100 105 110His Thr Val Gln Glu Phe Leu Val Ser Phe Val Val Trp
Ile Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140Glu His Thr Val Ile Arg Arg Gly Gly
Ala Arg Ala Ser Arg Ser Pro145 150 155 160Arg Arg Arg Thr Pro Ser
Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg 165 170 175Arg Arg Arg Ser
Gln Ser Pro Ser Ala Asn Cys 180 185106188PRTWoodchuck hepatitis
virus 106Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln
Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala
Leu Val Asp 20 25 30Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly
Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile Arg Gln Ala Leu
Val Cys Trp Asp Glu 50 55 60Leu Thr Lys Leu Ile Ala Trp Met Ser Ser
Asn Ile Thr Ser Glu Gln65 70 75 80Val Arg Thr Ile Ile Val Asn His
Val Asn Asp Thr Trp Gly Leu Lys 85 90 95Val Arg Gln Ser Leu Trp Phe
His Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110His Thr Val Gln Glu
Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Ala Pro
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu
His Thr Val Ile Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser145 150
155 160Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro 165 170 175Arg Arg Arg Arg Ser Gln Ser Pro Ser Ala Asn Cys 180
185107188PRTWoodchuck hepatitis virus 107Met Asp Ile Asp Pro Tyr
Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu
Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp 20 25 30Thr Ala Thr Ala
Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys 35 40 45Ser Pro His
His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu 50 55 60Leu Thr
Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln65 70 75
80Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu Lys
85 90 95Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly
Gln 100 105 110His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140Glu His Thr Val Ile Arg Arg Arg Gly
Gly Ala Arg Ala Ser Arg Ser145 150 155 160Pro Arg Arg Arg Thr Pro
Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro 165 170 175Arg Arg Arg Arg
Ser Gln Ser Pro Ser Thr Asn Cys 180 185108187PRTGround squirrel
hepatitis virus 108Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser
Tyr Gln Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu
Asn Ala Leu Val Asp 20 25 30Thr Ala Ala Ala Leu Tyr Glu Glu Glu Leu
Thr Gly Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile Arg Gln
Ala Leu Val Cys Trp Glu Glu 50 55 60Leu Thr Arg Leu Ile Thr Trp Met
Ser Glu Asn Thr Thr Glu Glu Val65 70 75 80Arg Arg Ile Ile Val Asp
His Val Asn Asn Thr Trp Gly Leu Lys Val 85 90 95Arg Gln Thr Leu Trp
Phe His Leu Ser Cys Leu Thr Phe Gly Gln His 100 105 110Thr Val Gln
Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120 125Ala
Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu 130 135
140His Thr Val Ile Arg Arg Arg Gly Gly Ser Arg Ala Ala Arg Ser
Pro145 150 155 160Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser
Gln Ser Pro Arg 165 170 175Arg Arg Arg Ser Gln Ser Pro Ala Ser Asn
Cys 180 185109183PRTHuman hepatitis B virus 109Met Asp Ile Asp Pro
Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro
Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser
Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro
His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60Leu
Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala65 70 75
80Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys
85
90 95Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Ile Phe Gly
Arg 100 105 110Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr145 150 155 160Pro Ser Pro Arg Arg Arg
Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175Gln Ser Arg Glu
Ser Gln Cys 180110185PRTHuman hepatitis B virus 110Met Asp Ile Asp
Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu
Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala
Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser
Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55
60Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala65
70 75 80Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Val Gly Leu
Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro
Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val Arg Arg Arg
Asp Arg Gly Arg Ser Pro Arg Arg145 150 155 160Arg Thr Pro Ser Pro
Arg Arg Arg Arg Ser Pro Ser Pro Arg Arg Arg 165 170 175Arg Ser Gln
Ser Arg Glu Ser Gln Cys 180 185111183PRTHuman hepatitis B virus
111Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu1
5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu
Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu
His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys
Trp Gly Glu 50 55 60Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu
Glu Asp Pro Ala65 70 75 80Ser Arg Glu Leu Val Val Ser Tyr Val Asn
Val Asn Met Gly Leu Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile
Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu
Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg
Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr
Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr145 150 155
160Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175Gln Ser Arg Glu Ser Gln Cys 180112183PRTHuman hepatitis
B virus 112Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu
Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln Ala Ile
Leu Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Thr Trp Val Gly Val
Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp Leu Val Val Ser Tyr
Val Asn Thr Asn Met Gly Leu Lys 85 90 95Phe Arg Gln Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu
Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr145 150
155 160Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser 165 170 175Gln Ser Arg Glu Ser Gln Cys 180113183PRTHuman
hepatitis B virus 113Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala
Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser
Val Arg Asp Leu Leu Asp 20 25 30Thr Ala Ala Ala Leu Tyr Arg Asp Ala
Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg
Gln Ala Ile Leu Cys Trp Gly Asp 50 55 60Leu Met Thr Leu Ala Thr Trp
Val Gly Thr Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Asp Leu Val
Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys 85 90 95Phe Arg Gln Leu
Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr
Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120
125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro
130 135 140Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg
Arg Thr145 150 155 160Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg Ser 165 170 175Gln Ser Arg Glu Ser Gln Cys
180114183PRTHuman hepatitis B virus 114Met Asp Ile Asp Pro Tyr Lys
Glu Phe Gly Ala Ser Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp
Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu
Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60Leu Met Asn
Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala65 70 75 80Ser
Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90
95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg
100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile
Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu
Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val Arg Arg Arg Gly Arg
Ser Pro Arg Arg Arg Thr145 150 155 160Pro Ser Pro Arg Arg Arg Arg
Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175Gln Ser Arg Glu Ser
Gln Cys 180115183PRTChimpanzee hepatitis B virus 115Met Asp Ile Asp
Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu
Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala
Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser
Pro Asn His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55
60Leu Met Thr Leu Ala Ser Trp Val Gly Asn Asn Leu Glu Asp Pro Ala65
70 75 80Ser Arg Glu Gln Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro
Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val Arg Arg Arg
Gly Arg Ser Pro Arg Arg Arg Thr145 150 155 160Pro Ser Pro Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175Gln Ser Pro
Ala Ser Gln Cys 180116183PRTGibbon hepatitis B virus 116Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe
Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr
Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40
45Ser Pro Asn His Thr Ala Leu Arg Gln Ala Val Leu Cys Trp Gly Glu
50 55 60Leu Met Thr Leu Ala Ser Trp Val Gly Asn Asn Leu Glu Asp Pro
Ala65 70 75 80Ser Arg Glu Leu Val Val Ser Tyr Val Asn Asn Asn Met
Gly Leu Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu
Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn
Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val Arg
Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr145 150 155 160Pro Ser Pro
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175Gln
Ser Pro Ala Ser Gln Cys 180117183PRTOrangutan hepatitis virus
117Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1
5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu
Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu
His Cys 35 40 45Ser Pro Asn His Thr Ala Leu Arg Gln Ala Val Leu Cys
Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Ser Trp Val Gly Asn Asn Leu
Glu Asp Pro Ala65 70 75 80Ser Arg Glu Leu Val Val Asn Tyr Val Asn
Asn Asn Met Gly Leu Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile
Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu
Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg
Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr
Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr145 150 155
160Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser
165 170 175Gln Ser Pro Ala Ser Gln Cys 180118182PRTWoolly monkey
hepatitis virus 118Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr
Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ala Asp Phe Phe Pro Ser Val
Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu
Glu Ser Ser Asp His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln
Thr Val Leu Cys Trp Gly Glu 50 55 60Leu Met Ser Leu Ala Ser Trp Val
Gly Thr Asn Leu Glu Asp Pro Ala65 70 75 80Ala Arg Glu Leu Val Val
Ser Tyr Val Asn Asp Asn Met Gly Leu Lys 85 90 95Val Arg Gln Leu Leu
Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val
Leu Glu Tyr Leu Val Ser Phe Trp Val Trp Ile Arg Thr 115 120 125Pro
Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135
140Glu Thr Thr Val Val Arg Arg Arg Arg Pro Ser Gly Arg Arg Thr
Pro145 150 155 160Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg
Arg Arg Ser Gln 165 170 175Ser Pro Ala Ser Ser Cys 180119262PRTDuck
hepatitis virus 119Met Asp Ile Asn Ala Ser Arg Ala Leu Ala Asn Val
Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys Ile Asp Asp Leu Val
Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser
Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile
Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu
Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr Thr Ala Pro Val Pro
Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu
Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile 100 105 110Val Ser Phe
Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His Ala His 115 120 125Leu
Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135
140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala
Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu
Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile
Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val Ala Gln Gly Gly Arg
Lys Thr Ser Ser Gly Thr Arg Lys 195 200 205Pro Arg Gly Leu Glu Pro
Arg Arg Arg Lys Val Lys Thr Thr Phe Val 210 215 220Tyr Gly Arg Arg
Arg Ser Lys Ser Arg Glu Arg Arg Ala Pro Ser Pro225 230 235 240Gln
Arg Ala Gly Ser Pro Leu Pro Arg Ser Ser Ser Ser His His Arg 245 250
255Ser Pro Ser Pro Arg Lys 260120262PRTDuck hepatitis virus 120Met
Asp Ile Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10
15Asp Asp Phe Phe Pro Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp
20 25 30Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser Ile Lys Lys His Val
Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln
Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu Ser Leu Arg Ala Val
Ile Pro Pro65 70 75 80Thr Thr Thr Pro Val Pro Pro Gly Tyr Leu Ile
Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys
His Gln Glu Glu Arg Ile 100 105 110Val Ser Phe Gln Pro Asp Tyr Pro
Ile Thr Ala Arg Ile His Ala His 115 120 125Leu Lys Ala Tyr Ala Lys
Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp
Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Gln145 150 155 160Val
Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170
175Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro
180 185 190Ile Gln Val Ala Gln Gly Gly Arg Lys Thr Thr Thr Gly Thr
Arg Lys 195 200 205Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys
Thr Thr Val Val 210 215 220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu
Arg Arg Ala Pro Thr Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu
Pro Arg Ser Ser Ser Ser His His Arg 245 250 255Ser Pro Ser Pro Arg
Lys 260121262PRTDuck hepatitis virus 121Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr
Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170
175Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro
180 185 190Ile Gln Val Ala Gln Gly Gly Arg Lys Thr Ser Ser Gly Thr
Arg Lys 195 200 205Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys
Thr Thr Val Val 210 215 220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Asp
Arg Arg Ala Pro Ser Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu
Pro Arg Ser Ser Ser Ser His His Arg 245 250 255Ser Pro Ser Pro Arg
Lys 260122262PRTDuck hepatitis virus 122Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
Ala Gln Gly Gly Arg Lys Thr Ser Ser Gly Thr Arg Lys 195 200 205Pro
Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val 210 215
220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu Arg Arg Ala Pro Ser
Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu Pro Arg Ser Ser Ser
Ser His His Arg 245 250 255Ser Pro Ser Pro Arg Lys 260123262PRTDuck
hepatitis virus 123Met Asp Ile Asn Ala Ser Arg Ala Leu Ala Asn Val
Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys Ile Asp Asp Leu Val
Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser
Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile
Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu
Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr Thr Ala Pro Val Pro
Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu
Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile 100 105 110Val Ser Phe
Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His Ala His 115 120 125Leu
Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135
140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala
Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu
Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile
Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Ala Ala Gln Gly Gly Arg
Lys Thr Ser Ser Gly Thr Arg Lys 195 200 205Pro Arg Gly Leu Glu Pro
Arg Arg Arg Lys Val Lys Thr Thr Val Val 210 215 220Tyr Gly Arg Arg
Arg Ser Lys Ser Arg Glu Arg Arg Ala Pro Ser Pro225 230 235 240Gln
Arg Ala Gly Ser Pro Leu Pro Arg Ser Ser Ser Ser His His Arg 245 250
255Ser Pro Ser Pro Arg Lys 260124262PRTDuck hepatitis virus 124Met
Asp Ile Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10
15Asp Asp Phe Phe Pro Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp
20 25 30Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser Ile Lys Lys His Val
Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln
Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu Ser Leu Arg Ala Val
Ile Pro Pro65 70 75 80Thr Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile
Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys
His Gln Glu Glu Arg Ile 100 105 110Val Ser Phe Gln Pro Asp Tyr Pro
Ile Thr Ala Arg Ile His Ala His 115 120 125Leu Lys Ala Tyr Ala Lys
Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp
Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Asn145 150 155 160Val
Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170
175Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro
180 185 190Ile Gln Ala Ala Gln Gly Gly Arg Lys Thr Ser Ser Gly Thr
Arg Lys 195 200 205Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys
Thr Thr Val Val 210 215 220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu
Arg Arg Ala Pro Ser Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu
Pro Arg Ser Ser Ser Ser His His Arg 245 250 255Ser Pro Ser Pro Arg
Lys 260125262PRTRoss' goose hepatitis virus 125Met Asp Ile Asn Ala
Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe
Pro Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu
Pro Tyr Trp Arg Asn Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala
Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln
Gly Met His Glu Ile Ala Glu Ala Leu Arg Ala Ile Ile Pro Ala65 70 75
80Thr Thr Ala Pro Val Pro Gln Gly Phe Leu Val Gln His Glu Glu Ala
85 90 95Glu Glu Ile Pro Leu Gly Glu Leu Phe Arg Tyr Gln Glu Glu Arg
Leu 100 105 110Thr Asn Phe Gln Pro Asp Tyr Pro Val Thr Ala Arg Ile
His Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser
Leu Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys
Leu Leu Trp Gly Glu Pro Asn145 150 155 160Val Thr Asn Tyr Ile Ser
Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly
Lys Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln
Val Ala Gln Gly Gly Arg Asn Lys Thr Gln Gly Val Arg Lys 195 200
205Ser Arg Gly Leu Glu Pro Arg Arg Arg Arg Val Lys Thr Thr Ile Val
210 215 220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu Arg Arg Ala Pro
Thr Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu Pro Arg Thr Ser
Arg Asp His His Arg 245 250 255Ser Pro Ser Pro Arg Glu
260126262PRTHeron hepatitis virus 126Met Asp Val Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Gln
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ala Glu Thr Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
Ser Gln Ile Ala Asp Ala Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Val Pro Val Pro Glu Gly Phe Leu Ile Thr His Ser Glu Ala 85 90
95Glu Glu Ile Pro Leu Asn Asp Leu Phe Ser Asn Gln Glu Glu Arg Ile
100 105 110Val Asn Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Thr His 115 120 125Leu Arg Val Tyr Thr Lys Leu Asn Glu Gln Ala Leu
Asp Lys Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Thr145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Lys
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
Ala Gln Gly Gly Arg Asn Gln Thr Lys Gly Thr Arg Lys 195 200 205Pro
Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val 210 215
220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro
Ser225 230 235 240Gln Arg Ala Gly Ser Pro Leu Pro Arg Asn Arg Gly
Asn Gln Thr Arg 245 250 255Ser Pro Ser Pro Arg Glu
260127564DNAArctic ground squirrel hepatitis virus 127atggacatag
atccctataa agaatttggt tcatcctacc agttgttgaa ttttcttcct 60ttggacttct
ttcctgaact caatgccttg gtggacactg ctactgctct ctatgaagaa
120gaattaacag gtagggagca ctgctctcct catcacacag ctatcagaca
agctttagtt 180tgctgggaag aattaacaag attaattgcg tggatgagtg
ctaacattaa ttcagaagaa 240gtaagaagag ttatagttgc tcatgtcaat
gacacttggg gacttaaagt taggcagaat 300ttatggtttc acttatcctg
tctgactttt gggcaacaca cagtgcagga atttttagtc 360agctttggag
taaggatcag aactccggct ccttatagac ctcctaatgc acccattctc
420tcaactcttc cggaacatac agtcattagg agaagaggaa gtgcaagagt
tgttaggtcc 480cccagaagac gcactccctc tcctcgcagg agaagatctc
aatcaccgcg tcgcaggcct 540caatctccag cttccaactg ctga
564128564DNAGround squirrel hepatitis virus 128atggacatag
atccctataa agaatttggt tcttcttatc agttgttgaa ttttcttcct 60ttggactttt
ttcctgatct caatgcattg gtggacactg ctgctgctct ttatgaagaa
120gaattaacag gtagggagca ttgttctcct catcatactg ctattagaca
ggccttagtg 180tgttgggaag aattaactag attaattaca tggatgagtg
aaaatacaac agaagaagtt 240agaagaatta ttgttgatca tgtcaataat
acttggggac ttaaagtaag acagacttta 300tggtttcatt tatcatgtct
tacttttgga caacacacag ttcaagaatt tttggttagt 360tttggagtat
ggattagaac tccagctcct tatagaccac ctaatgcacc cattttatca
420actcttccgg aacatacagt cattaggaga agaggaggtt caagagctgc
taggtccccc 480cgaagacgca ctccctctcc tcgcaggaga aggtctcaat
caccgcgtcg cagacgctct 540caatctccag cttccaactg ctga
564129567DNAWoodchuck hepatitis virus 129atggacatag atccttataa
agaatttggt tcatcttatc agttgttgaa ttttcttcct 60ttggactttt ttcctgacct
taatgctttg gtggacactg ctactgcctt gtatgaagaa 120gaactaacag
gtagggaaca ttgctctccg caccatacag ctattagaca agctttagta
180tgctgggatg aattaactaa attaatagct tggatgagct ctaacataac
ttctgaacaa 240gtaagaacaa tcattgtaaa tcatgtcaat gatacctggg
gacttaaggt gagacaaagt 300ttatggtttc atttgtcatg tctcactttc
ggacaacata cagttcaaga atttttagta 360agttttggag tatggattag
gactccagct ccatatagac ctcctaatgc acccattctc 420tcgactcttc
cggaacatac agtcattagg agaagaggag gtgcaagagc ttctaggtcc
480cccagaagac gcactccctc tcctcgcagg agaagatctc aatcaccgcg
tcgcagacgc 540tctcaatctc catctaccaa ctgctga 567130567DNAWoodchuck
hepatitis virus 130atggacatag atccctataa agaatttggt tcatcttatc
agttgttgaa ttttcttcct 60ttggacttct ttcctgacct taatgctttg gtggacactg
ctactgcctt gtatgaagaa 120gagctaacag gtagggaaca ttgctctccg
caccatacag ctattagaca agctttagta 180tgctgggatg aattaactaa
attgatagct tggatgagct ctaacataac ttctgaacaa 240gtaagaacaa
tcatagtaaa tcatgtcaat gatacctggg gacttaaggt gagacaaagt
300ttatggtttc atttgtcatg tctcactttt ggacaacata cagttcaaga
atttttagta 360agttttggag tatggatcag aactccagct ccatatagac
ctcctaatgc acccattctc 420tcgactcttc cggaacatac agtcattagg
agaagaggag gtgcaagagc ttctaggtcc 480cccagaagac gcactccctc
tcctcgcagg agaagatctc aatcaccgcg tcgcagacgc 540tctcaatctc
catctgccaa ctgctga 567131564DNAWoodchuck hepatitis virus
131atggacatag atccctataa agaatttggt tcatcttatc agttgttgaa
ttttcttcct 60ttggacttct ttcctgacct taatgctttg gtggacactg ctactgcctt
gtatgaagaa 120gagctaacag gtagggaaca ttgctctccg caccatacag
ctattagaca agctttagta 180tgctgggatg aattaactaa attgatagct
tggatgagct ctaacataac ttctgaacaa 240gtaagaacaa tcatagtaaa
tcatgtcaat gatacctggg gacttaaggt gagacaaagt 300ttatggtttc
atttgtcatg tctcactttc ggacaacata cagttcaaga atttttagta
360agttttgtag tatggatcag aactccagct ccatatagac ctcctaatgc
acccattctc 420tcgactcttc cggaacatac agtcattaga agaggaggtg
caagagcttc taggtccccc 480agaagacgca ctccctctcc tcgcaggaga
agatctcaat caccgcgtcg cagacgctct 540caatctccat ctgccaactg ctga
564132567DNAWoodchuck hepatitis virus 132atggacatag atccctataa
agaatttggt tcatcttatc agttgttgaa ttttcttcct 60ttggacttct ttcctgacct
taatgctttg gtggacactg ctactgcctt gtatgaagaa 120gagctaacag
gtagggaaca ttgctctccg caccatacag ctattagaca agctttagta
180tgctgggatg aattaactaa attgatagct tggatgagct ctaacataac
ttctgaacaa 240gtaagaacaa tcatagtaaa tcatgtcaat gatacctggg
gacttaaggt gagacaaagt 300ttatggtttc atttgtcatg tctcactttc
ggacaacata cagttcaaga atttttagta 360agttttggag tatggatcag
aactccagct ccatatagac ctcctaatgc acccattctc 420tcgactcttc
cggaacatac agtcattagg agaagaggag gtgcaagagc ttctaggtcc
480cccagaagac gcactccctc tcctcgcagg agaagatctc aatcaccgcg
tcgcagacgc 540tctcaatctc catctgccaa ctgctga 567133567DNAWoodchuck
hepatitis virus 133atggacatag atccttataa agaatttggt tcatcttatc
agttgttgaa ttttcttcct 60ttggacttct ttcctgatct taatgctttg gtggacactg
ctactgcctt gtatgaagaa 120gaactaacag gtagggaaca ttgctctccg
caccatacag ctattagaca agctttagta 180tgctgggatg aattaactaa
attgatagct tggatgagct ctaacataac ttctgaacaa 240gtaagaacaa
tcattgtaaa tcatgtcaat gatacctggg gacttaaggt gagacaaagt
300ttatggtttc atttgtcatg tctcactttc ggacaacata cagttcaaga
atttttagta 360agttttggag tatggatcag gactccagct ccatatagac
ctcctaatgc acccattctc 420tcgactcttc cggaacatac agtcattagg
agaagaggag gtgcaagagc ttctaggtcc 480cccagaagac gcactccctc
tcctcgcagg agaagatctc aatcaccgcg tcgcagacgc 540tctcaatctc
catctgccaa ctgctga 567134552DNAOrangutan hepatitis virus
134atggacattg acccttataa agaatttgga gctactgtgg agttactctc
ttttttgcct 60tcggatttct ttccgtctgt cagagatcta ctcgacaccg catcagccct
gtatcgggaa 120gccttagagt ctccagaaca ttgttcacct aaccacacag
cactcaggca agcagttctg 180tgctggggtg agttaatgac tctggcttcc
tgggtgggta ataatttgga agacccagca 240tctagggaac tggtagttaa
ttatgtcaac aataatatgg ggctaaaaat cagacaacta 300ctgtggtttc
acatttcctg tcttactttt ggaagagaaa cagttttaga atatttggtg
360tcttttggag tgtggattcg cactcctcct gcgtacagac caccaaatgc
ccctatcttg 420tcaacacttc cggaaactac tgttgttaga cgaagaggca
ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag gtctcaatca
ccgcgtcgca gaagatctca atctccagct 540tcccaatgtt ag
552135549DNAWoolly monkey hepatitis virus 135atggacattg atccttataa
agaatttggc gctactgttg agttattgtc ttttttgcct 60gctgacttct ttccttccgt
acgggatttg ctggacacag cttctgctct gtatagagaa 120gccctggagt
cttccgacca ctgttcaccg caccatactg ccttgaggca gacggtactg
180tgctggggag aattaatgtc cttagcttct tgggtgggaa ctaatttgga
ggatcctgct 240gctagagaat tagtggttag ctatgtcaat gacaacatgg
gactgaaggt gagacaactc 300ctttggttcc atatttcctg tctcactttt
ggtagggaaa ctgttttgga atatctggtt 360tctttttggg tgtggatacg
cacacctcct gcatatagac cacccaatgc ccctatctta 420tcaacacttc
cggaaactac tgttgttaga cgaaggagac cctctggaag acgcactccc
480tcgcctcgca gacgaagatc tcaatcgccg cgtcgcagaa ggtctcaatc
tccagcatct 540tcctgttag 549136552DNAGibbon hepatitis B virus
136atggacattg acccttataa agaatttgga gctactgtgg agttactctc
ttttttgcct 60tctgacttct ttccgtcggt tagagatctc cttgacaccg cctcagctct
atatcgggaa 120gccctagagt ctccagaaca ttgttcacct aatcatacag
cactcaggca agctgttttg 180tgctggggtg agttgatgac tctggcttcc
tgggtgggca ataatttgga agatccagca 240tctagggaac tagtagtcag
ttatgttaat aataacatgg gtctaaaaat caggcaacta 300ttgtggtttc
acatttcctg tcttactttt ggaagagaaa ctgtccttga gtatttagtg
360tcttttggag tgtggattcg cactcctcca gcttacagac caccaaatgc
ccctatctta 420tccactcttc cggagactac tgttgttaga cgaagaggca
ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag gtctcaatcg
ccgcgtcgca gaagatctca atctccagct 540tcccaatgtt ag
552137552DNAChimpanzee hepatitis B virus 137atggacattg acccttataa
agaatttgga gctacagtgg agttactctc ttttttgcct 60tctgatttct ttccgtcggt
ccgtgatctc ctcgacaccg cctcagctct gtaccgggaa 120gccttagagt
ctccagagca ctgttcacct aaccatacag cacttaggca agctatactg
180tgctggggtg agttaatgac tctggcctcc tgggtgggca ataatttgga
agatccagca 240tccagggaac aagtagttaa ttatgtcaat accaatatgg
gtttaaagat cagacaatta 300ttgtggtttc atatttcctg tcttactttt
ggaagagaaa ctgtccttga gtatttggtg 360tcttttggag tgtggattcg
cactcccccc gcttatagac caccaaatgc ccctatctta 420tcaacacttc
cggaaactac tgttgttaga cgacgaggca ggtcccctag aagaagaact
480ccctcgcctc gcagacgaag gtctcaatcg ccgcgtcgca gaagatctca
atctccagct 540tcccaatgtt ag 552138552DNAHuman hepatitis B virus
138atggacattg acccttataa agaatttgga gctaccgtgg agttactctc
gtttttgcct 60tctgacttct ttccttcagt acgagatctt ctagataccg cctcagctct
gtatcgggat 120gccttagagt ctcctgagca ttgttcacct caccatactg
cactcaggca agcaattctt 180tgctgggggg aactaatgac tctagctacc
tgggtgggtg ttaatttgga agatccagca 240tctagggacc tagtagtcag
ttatgtcaac actaatatgg gcctaaagtt cagacaactc 300ttgtggtttc
acatttcttg tctcattttt ggaagagaaa cagttataga gtatttggtg
360tctttcggag tgtggattcg cactcctcca gcttatagac caccaaatgc
ccctatccta 420tcaacacttc cggagactac tgttgttaga cgacgaggca
ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag gtctcaatcg
ccgcgtcgca gaagatctca atctcgggaa 540tctcaatgtt ag 552139558DNAHuman
hepatitis B virus 139atggacattg acccttataa agaatttgga gctactgtgg
agttactctc gtttttgcct 60tctgacttct ttccttccgt cagagatctc ctagacaccg
cctcagctct gtatcgagaa 120gccttagagt ctcctgagca ttgctcacct
caccatactg cactcaggca agccattctc 180tgctgggggg aattgatgac
tctagctacc tgggtgggta ataatttgga agatccagca 240tctagggatc
ttgtagtaaa ttatgttaat actaacgtgg gtttaaagat caggcaacta
300ttgtggtttc atatatcttg ccttactttt ggaagagaga ctgtacttga
atatttggtc 360tctttcggag tgtggattcg cactcctcca gcctatagac
caccaaatgc ccctatctta 420tcaacacttc cggaaactac tgttgttaga
cgacgggacc gaggcaggtc ccctagaaga 480agaactccct cgcctcgcag
acgcagatct ccatcgccgc gtcgcagaag atctcaatct 540cgggaatctc aatgttag
558140552DNAHuman hepatitis B virus 140atggacattg acccgtataa
agaatttgga gcttctgtgg agttactctc ttttttgcct 60tctgacttct ttccttctat
tcgagatctc ctcgacaccg cctcagctct atatcgggag 120gccttagagt
ctccggaaca ttgttctcct catcatacag cactcaggca agctattctg
180tgttggggtg agttgatgaa tctggccacc tgggtgggaa gtaatttgga
agacccagca 240tccagggaat tagtagtcag ctatgtcaat gttaatatgg
gcctaaaaat cagacaacta 300ctgtggtttc acatttcctg tcttactttt
ggaagagaaa ctgttcttga gtatttggtg 360tcttttggag tgtggattcg
cactcctcct gcttacagac caccaaatgc ccctatctta 420tcaacacttc
cggaaactac tgttgttaga cgacgaggca ggtcccctag aagaagaact
480ccctcgcctc gcagacgaag gtctcaatcg ccgcgtcgca gaagatctca
atctcgggaa 540tctcaatgtt ag 552141552DNAHuman hepatitis B virus
141atggacattg acccttataa agaatttgga gctactgtgg agttactctc
gtttttgcct 60tctgacttct ttccttccgt acgagatctt ctagataccg ccgcagctct
gtatcgggat 120gccttagagt ctcctgagca ttgttcacct caccatactg
cactcaggca agcaattctt 180tgctggggag acttaatgac tctagctacc
tgggtgggta ctaatttaga agatccagca 240tctagggacc tagtagtcag
ttatgtcaac actaatgtgg gcctaaagtt cagacaatta 300ttgtggtttc
acatttcttg tctcactttt ggaagagaaa cggttctaga gtatttggtg
360tcttttggag tgtggattcg cactcctcca gcttatagac caccaaatgc
ccctatccta 420tcaacgcttc cggagactac tgttgttaga cgacgaggca
ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag atctcaatcg
ccgcgtcgca gaagatctca atctcgggaa 540tctcaatgtt ag 552142552DNAHuman
hepatitis B virus 142atggacattg acccgtataa agaatttgga gcttctgtgg
agttactctc ttttttgcct 60tctgacttct ttccttctat tcgagatctc ctcgacaccg
cctctgctct gtatcgggag 120gccttagagt ctccggaaca ttgttcacct
caccatacag cactcaggca agctattctg 180tgttggggtg agttgatgaa
tttggccacc tgggtgggaa gtaatttgga agacccagca 240tccagggaat
tagtagtcag ctatgtcaat gttaatatgg gcctaaaaat cagacaacta
300ttgtggtttc atatttcctg tcttactttt ggaagagaaa ctgttcttga
gtatttggtg 360tcttttggag tgtggattcg cactcctccc gcttacagac
caccaaatgc ccctatctta 420tcaacacttc cggaaactac tgttgttaga
cgacgaggca ggtcccctag aagaagaact 480ccctcgcctc gcagacgaag
gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa 540tctcaatgtt ag
552143786DNARoss' goose hepatitis virus 143atggatatca acgcttcaag
agctttagct aatgtatatg atttgccaga tgatttcttt 60ccaaagattg atgatttagt
tagagatgct aaagatgctt tagagcctta ttggagaaat 120gattcaataa
agaaacatgt tttaattgca actcactttg tggatctcat tgaggatttc
180tggcaaacca ctcagggtat gcatgaaata gcagaggcac tgagagctat
aattcctgcc 240actactgctc cagtacctca gggatttctg gtccaacacg
aagaagctga agagatacct 300ttgggtgaac tttttaggta tcaggaagaa
agactaacta actttcaacc agattatcca 360gttaccgcca gaattcatgc
tcacctgaaa gcatatgcaa aaataaatga ggaatcttta 420gatagagcta
ggagattgct ttggtggcat tataactgtt tattgtgggg cgagcctaac
480gttaccaact atatttcgag attaagaact tggttatcca cacctgaaaa
atacagagga 540aaagatgccc caaccattga agcaatcact agaccaatcc
aagtggcgca gggaggcaga 600aataagactc agggagttag aaaatctcgt
ggactcgaac ctaggagaag aagagttaaa 660accacaattg tctatgggag
aagacgttca aagtccaggg aaaggagagc ccctacaccc 720cagcgtgcgg
gctcccctct cccgcgtact tctagggacc accacagatc tccctcgcct 780agggaa
786144789DNAHeron hepatitis virus 144atggatgtca atgcttcaag
agctttagca aatgtatatg atctgccaga tgatttcttt 60cctcagattg atgatcttgt
tagagatgct aaggatgctt tagaacctta ttggaaagcc 120gaaacaataa
agaaacatgt tttaattgct actcactttg tggatttgat tgaggacttc
180tggcagacca ctcagggtat gagccaaatt gcagacgccc tccgagcagt
aattccacct 240actaccgtac cagtaccgga gggttttctc attactcata
gtgaggcaga agagatcccc 300ttgaacgatc tcttttcaaa tcaagaggag
aggatagtca atttccaacc tgactatccc 360attacagcta gaattcatac
ccacttacgt gtttatacta aattgaatga acaagctttg 420gacaaagctc
gcagattgct ttggtggcat tacaattgcc tcctctgggg agaagccact
480gttacaaatt atatttctcg cctccgtact tggctttcta ctcccgaaaa
atatcgaggc 540aaggatgccc caaccattga agcaatcact agaccaatcc
aggtggctca aggaggcaga 600aatcaaacta agggaactag aaaacctcgt
ggactcgaac ctagaagacg aaaggttaaa 660accacagttg tctatgggag
aagacgttct aagtcccgag gcaggagatc ctctccatcc 720caacgtgcgg
gctcccctct cccacgtaat cggggaaacc agacacgatc cccctcacct 780agggaatag
789145786DNADuck hepatitis virus 145atggatatca atgcttctag
agccttagca aatgtgtatg atctgcctga tgatttcttt 60ccaaaaatag atgatcttgt
aagggatgct aaagacgctt tagaacctta ttggaaatct 120gactcaataa
agaaacatgt tttaattgca actcattttg tggatcttat tgaggatttt
180tggcagacta cgcagggtat gcatgaaatc gctgaatcac taagagcagt
aattccacct 240accactgctc ctgttcctac tgggtatctc attcagcacg
aagaggcaga agagatacca 300ttaggtgatt tatttaaaca tcaagaagaa
agaatagtca gtttccaacc tgactaccca 360attacagcaa gaattcatgc
acacctaaaa gcatatgcaa aaattaacga ggaatcattg 420gatcgggcta
ggagattgct ttggtggcat tataactgtt tactgtgggg agaagctaac
480gttactaatt atatttctcg cctccgtact tggttgtcaa ctcctgaaaa
gtacagaggt 540cgagatgccc caaccattga agcaatcact agaccaatcc
aagtggctca gggaggcaga 600aaaacatctt cgggaactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacatttg tctatgggag
aagacgttca aagtccaggg aaaggagagc cccttcaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaa
786146789DNADuck hepatitis virus 146atggatatca atgcttctag
agccttagcc aatgtgtatg atctaccaga tgatttcttt 60ccaaaaatag atgatcttgt
tagagatgct aaagacgctt tagagcctta ttggaaatca 120gattcaataa
agaaacatgt tttgattgca actcactttg tggatcttat tgaagacttc
180tggcagacta cacagggcat gcatgaaata gccgaatcct taagagctgt
tatacctccc 240actactactc ctgttccacc gggttatctt attcagcacg
aagaagctga agagatacct 300ttgggagatt tatttaaaca ccaagaagaa
aggatagtga gtttccaacc cgactatcca 360attacggcta gaattcatgc
tcatttgaaa gcttatgcaa aaattaacga ggaatcactg 420gatagggcta
ggagattgct ttggtggcat tacaattgtt tactgtgggg agaagctcaa
480gttactaact atatttctcg cttgcgtact tggttgtcaa ctcctgagaa
atatagaggt 540agagatgccc cgaccattga agcaatcact agaccaatcc
aagtggctca gggaggccga 600aaaacaacta cgggtactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacagttg tctatgggag
aagacgttca aagtcccggg aaaggagagc ccctacaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaataa
789147789DNADuck hepatitis virus 147atggatatca atgcttctag
agccttagcc aatgtgtatg atctgccaga tgatttcttc 60cctaaaattg atgatcttgt
aagggatgct aaagacgcat tagaacctta ttggaaatct 120gattcaataa
agaaacatgt tttaattgca actcactttg tggatcttat tgaagacttt
180tggcagacta ctcagggtat gcatgaaatt gctgaatcct taagagctgt
aataccacct 240acgactgctc ctgtacctac tgggtatctc attcaacacg
aggaagctga agagatacct 300ttaggtgatt tatttaaaca tcaggaagaa
agaatagtca gtttccaacc tgactatcct 360attacagcaa gaattcatgc
acacctaaaa gcttatgcta aaattaatga ggaatcgttg 420gatagggcta
ggagattgct ttggtggcat tacaactgtt tactgtgggg agaagctaac
480gttactaatt atatttctcg gctccgtact tggttgtcaa ctcctgaaaa
gtacagaggc 540cgtgatgccc caaccattga agcaatcact agaccaatcc
aggtggctca gggaggcaga 600aaaacatctt cgggaactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacagttg tctatgggag
aagacgttca aagtccaggg ataggagagc cccttcaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaataa
789148789DNADuck hepatitis virus 148atggatatca atgcttctag
agccttagcc aatgtatatg atctgccaga tgatttcttt 60ccaaaaattg atgatcttgt
aagggatgcg aaagatgctt tagaacctta ttggaaatct 120gattcaataa
agaaacatgt tttaattgca actcacttcg tggatcttat tgaagacttc
180tggcagacta cacagggtat gcatgagata gctgaatcat taagagcagt
aattccacct 240accactgctc ctgtacctac ggggtatctc attcaacacg
aagaggctga agagatacct 300ttaggtgatc ttttcaaaca tcaggaagaa
aggatagtta gtttccagcc agactatccg 360attactgcta gaattcatgc
acatctaaaa gcttatgcta aaattaatga ggaatcatta 420gatcgggcta
ggagattgct ttggtggcat tacaactgtt tactgtgggg agaagctaac
480gttaccaact atatttctcg gctccgtact tggttgtcaa ctcctgaaaa
gtaccgaggc 540cgtgatgccc caaccattga agcaatcact agaccaatcc
aagtggctca gggaggcaga 600aaaacatctt cgggaactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacagttg tctatgggag
aagacgttca aagtcccggg aaaggagagc cccttcaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaataa
789149789DNADuck hepatitis virus 149atggatatca atgcttctag
agccttagcc aatgtatatg atctgccaga tgatttcttt 60ccaaaaattg atgatcttgt
aagggatgct aaagacgctt tagaacctta ctggaaatct 120gattcaataa
agaaacatgt tttgattgca actcactttg tggatcttat tgaagacttc
180tggcagacta ctcagggtat gcatgaaatt gctgaatcct taagagcagt
aataccacct 240accactgctc ctgtacctac tggatatctc attcaacacg
aggaggctga agagataccc 300ttaggtgatt tatttaaaca tcaggaagaa
agaatagtca gttttcaacc agactatcct 360attacagcaa gaattcatgc
acacctaaaa gcttatgcaa aaattaatga ggaatctttg 420gatagggcta
ggagattgct ttggtggcat tacaactgtt tactgtgggg agaagctaac
480gttactaatt acatttctcg gctccgtact tggttgtcaa ctccggaaaa
gtaccgaggc 540cgtgatgccc caaccattga agcaatcact agaccaatcc
aagcggctca gggaggcaga 600aaaacatctt cgggaactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacagttg tctatgggag
aagacgttca aagtccaggg aaaggagagc cccttcaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaataa
789150789DNADuck hepatitis virus 150atggatatca atgcttctag
agccttagcc aatgtatatg atctgccaga tgatttcttt 60ccaaaaattg atgatcttgt
aagggatgct aaagacgctt tagaacctta ctggaaatct 120gattcaataa
agaaacatgt tttgattgca actcactttg tggatcttat tgaagacttc
180tggcagacta ctcagggtat gcatgaaatt gctgaatcct taagagcagt
aataccacct 240accactgctc ctgtacctac tggatatctc attcaacacg
aggaggctga agagataccc 300ttaggtgatt tatttaaaca tcaggaagaa
agaatagtca gttttcaacc agactatcct 360attacagcaa gaattcatgc
acacctaaaa gcttatgcaa aaattaatga ggaatctttg 420gatagggcta
ggagattgct ttggtggcat tacaactgtt tactgtgggg agaagctaac
480gttactaatt acatttctcg gctccgtact tggttgtcaa ctccggaaaa
gtaccgaggc 540cgtgatgccc caaccattga agcaatcact agaccaatcc
aagcggctca gggaggcaga 600aaaacatctt cgggaactag aaaacctcgt
ggactcgaac ctagaagaag aaaagttaaa 660accacagttg tctatgggag
aagacgttca aagtccaggg aaaggagagc cccttcaccc 720caacgtgcgg
gctcccctct cccacgtagt tcgagcagcc accatagatc tccctcgcct 780aggaaataa
789151262PRTSheldgoose hepatitis virus 151Met Asp Val Asn Ala Ser
Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro
Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro
Tyr Trp Arg Ser Glu Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr
His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly
Met His Glu Ile Ala Glu Ala Leu Arg Ala Val Ile Pro Pro65 70 75
80Thr Thr Thr Pro Val Pro Pro Gly Tyr Leu Ile Gln His Glu Glu Ala
85 90 95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg
Ile 100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile
His Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser
Leu Asp Lys Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys
Leu Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser
Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Arg Tyr Arg Gly
Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln
Val Ala Gln Gly Gly Arg Asn Lys Thr Gln Gly Ser Arg Lys 195 200
205Pro Arg Gly Leu Gln Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val
210 215 220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Asp Arg Arg Ala Pro
Ser Pro225 230 235 240Gln Arg Ala Gly Ser Pro Leu Pro Arg Pro Ser
Thr Ser His His Arg 245 250 255Ser Pro Ser Pro Arg Lys
260152262PRTStork hepatitis virus 152Met Asp Val Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Gln
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ala Glu Thr Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
Ser Gln Ile Ala Asp Ala Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Thr Pro Val Pro Asp Gly Tyr Leu Ile Ser His Asn Glu Ala 85 90
95Gln Glu Leu Pro Leu Asn Asp Leu Phe Val Asn Gln Glu Glu Arg Ile
100 105 110Val Asn Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Thr His 115 120 125Leu Arg Val Tyr Thr Lys Leu Asn Glu Gln Ala Leu
Asp Lys Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ser Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Lys
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
Ala Gln Gly Ser Arg Asn Gln Thr Lys Gly Val Arg Lys 195 200 205Pro
Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val 210 215
220Tyr Gly Arg Arg Arg Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro
Ser225 230 235 240Gln Arg Ala Gly Ser Pro Ile Pro Arg Asn Arg Glu
Asn Gln Ser Arg 245 250 255Ser Ser Ser Pro Arg Glu
26015338PRTArctic ground squirrel hepatitis virus 153Arg Arg Arg
Gly Ser Ala Arg Val Val Arg Ser Pro Arg Arg Arg Thr1 5 10 15Pro Ser
Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Pro Gln 20 25 30Ser
Pro Ala Ser Asn Cys 35154149PRTArctic ground squirrel hepatitis
virus 154Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln
Leu Leu1 5 10 15Asn Phe Leu Pro Leu Asp Phe Phe Pro Glu Leu Asn Ala
Leu Val Asp 20 25 30Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly
Arg Glu His Cys 35 40 45Ser Pro His His Thr Ala Ile Arg Gln Ala Leu
Val Cys Trp Glu Glu 50 55 60Leu Thr Arg Leu Ile Ala Trp Met Ser Ala
Asn Ile Asn Ser Glu Glu65 70 75 80Val Arg Arg Val Ile Val Ala His
Val Asn Asp Thr Trp Gly Leu Lys 85 90 95Val Arg Gln Asn Leu Trp Phe
His Leu Ser Cys Leu Thr Phe Gly Gln 100 105 110His Thr Val Gln Glu
Phe Leu Val Ser Phe Gly Val Arg Ile Arg Thr 115 120
125Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro
130 135 140Glu His Thr Val Ile14515533PRTWoolly monkey hepatitis
virus 155Arg Arg Arg Arg Pro Ser Gly Arg Arg Thr Pro Ser Pro Arg
Arg Arg1 5 10 15Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
Ala Ser Ser 20 25 30Cys156149PRTWoolly monkey hepatitis virus
156Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1
5 10 15Ser Phe Leu Pro Ala Asp Phe Phe Pro Ser Val Arg Asp Leu Leu
Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Ser Asp
His Cys 35 40 45Ser Pro His His Thr Ala Leu Arg Gln Thr Val Leu Cys
Trp Gly Glu 50 55 60Leu Met Ser Leu Ala Ser Trp Val Gly Thr Asn Leu
Glu Asp Pro Ala65 70 75 80Ala Arg Glu Leu Val Val Ser Tyr Val Asn
Asp Asn Met Gly Leu Lys 85 90 95Val Arg Gln Leu Leu Trp Phe His Ile
Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu
Val Ser Phe Trp Val Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg
Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr
Val Val14515734PRTOrangutan hepatitis virus 157Arg Arg Arg Gly Arg
Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg1 5 10 15Arg Arg Ser Gln
Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Ala Ser 20 25 30Gln
Cys158149PRTOrangutan hepatitis virus 158Met Asp Ile Asp Pro Tyr
Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu Pro Ser
Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala Ser Ala
Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser Pro Asn
His Thr Ala Leu Arg Gln Ala Val Leu Cys Trp Gly Glu 50 55 60Leu Met
Thr Leu Ala Ser Trp Val Gly Asn Asn Leu Glu Asp Pro Ala65 70 75
80Ser Arg Glu Leu Val Val Asn Tyr Val Asn Asn Asn Met Gly Leu Lys
85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val Val14515934PRTGibbon
hepatitis B virus 159Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr
Pro Ser Pro Arg Arg1 5 10 15Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg
Ser Gln Ser Pro Ala Ser 20 25 30Gln Cys160149PRTGibbon hepatitis B
virus 160Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu
Leu Leu1 5 10 15Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp
Leu Leu Asp 20 25 30Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys 35 40 45Ser Pro Asn His Thr Ala Leu Arg Gln Ala Val
Leu Cys Trp Gly Glu 50 55 60Leu Met Thr Leu Ala Ser Trp Val Gly Asn
Asn Leu Glu Asp Pro Ala65 70 75 80Ser Arg Glu Leu Val Val Ser Tyr
Val Asn Asn Asn Met Gly Leu Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110Glu Thr Val Leu Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125Pro Pro Ala
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140Glu
Thr Thr Val Val14516134PRTChimpanzee hepatitis B virus 161Arg Arg
Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg1 5 10 15Arg
Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Ala Ser 20 25
30Gln Cys162149PRTChimpanzee hepatitis B virus 162Met Asp Ile Asp
Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe Leu
Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30Thr Ala
Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45Ser
Pro Asn His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55
60Leu Met Thr Leu Ala Ser Trp Val Gly Asn Asn Leu Glu Asp Pro Ala65
70 75 80Ser Arg Glu Gln Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu
Lys 85 90 95Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg 100 105 110Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr 115 120 125Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro
Ile Leu Ser Thr Leu Pro 130 135 140Glu Thr Thr Val
Val14516367PRTDuck hepatitis virus 163Ala Gln Gly Gly Arg Lys Thr
Ser Ser Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg
Lys Val Lys Thr Thr Phe Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser
Arg Glu Arg Arg Ala Pro Ser Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu
Pro Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65164195PRTDuck hepatitis virus 164Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
19516567PRTDuck hepatitis virus 165Ala Gln Gly Gly Arg Lys Thr Thr
Thr Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser Arg
Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu Pro
Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65166195PRTDuck hepatitis virus 166Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Thr Pro Val Pro Pro Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Gln145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
19516767PRTDuck hepatitis virus 167Ala Gln Gly Gly Arg Lys Thr Ser
Ser Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser Arg
Asp Arg Arg Ala Pro Ser Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu Pro
Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65168195PRTDuck hepatitis virus 168Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
19516967PRTDuck hepatitis virus 169Ala Gln Gly Gly Arg Lys Thr Ser
Ser Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser Arg
Glu Arg Arg Ala Pro Ser Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu Pro
Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65170195PRTDuck hepatitis virus 170Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Val
19517167PRTDuck hepatitis virus 171Ala Gln Gly Gly Arg Lys Thr Ser
Ser Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser Arg
Glu Arg Arg Ala Pro Ser Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu Pro
Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65172195PRTDuck hepatitis virus 172Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Ala
19517367PRTDuck hepatitis virus 173Ala Gln Gly Gly Arg Lys Thr Ser
Ser Gly Thr Arg Lys Pro Arg Gly1 5 10 15Leu Glu Pro Arg Arg Arg Lys
Val Lys Thr Thr Val Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys Ser Arg
Glu Arg Arg Ala Pro Ser Pro Gln Arg Ala 35 40 45Gly Ser Pro Leu Pro
Arg Ser Ser Ser Ser His His Arg Ser Pro Ser 50 55 60Pro Arg
Lys65174195PRTDuck hepatitis virus 174Met Asp Ile Asn Ala Ser Arg
Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe Pro Lys
Ile Asp Asp Leu Val Arg Asp Ala Lys Asp 20 25 30Ala Leu Glu Pro Tyr
Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu 35 40 45Ile Ala Thr His
Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr 50 55 60Gln Gly Met
His Glu Ile Ala Glu Ser Leu Arg Ala Val Ile Pro Pro65 70 75 80Thr
Thr Ala Pro Val Pro Thr Gly Tyr Leu Ile Gln His Glu Glu Ala 85 90
95Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys His Gln Glu Glu Arg Ile
100 105 110Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His
Ala His 115 120 125Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu
Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp Trp His Tyr Asn Cys Leu
Leu Trp Gly Glu Ala Asn145 150 155 160Val Thr Asn Tyr Ile Ser Arg
Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170 175Lys Tyr Arg Gly Arg
Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro 180 185 190Ile Gln Ala
19517567PRTRoss' goose hepatitis virus 175Ala Gln Gly Gly Arg Asn
Lys Thr Gln Gly Val Arg Lys Ser Arg Gly1 5 10 15Leu Glu Pro Arg Arg
Arg Arg Val Lys Thr Thr Ile Val Tyr Gly Arg 20 25 30Arg Arg Ser Lys
Ser Arg Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala 35 40 45Gly Ser Pro
Leu Pro Arg Thr Ser Arg Asp His His Arg Ser Pro Ser 50 55 60Pro Arg
Glu65176195PRTRoss' goose hepatitis virus 176Met Asp Ile Asn Ala
Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1 5 10 15Asp Asp Phe Phe
Pro Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp
20 25 30Ala Leu Glu Pro Tyr Trp Arg Asn Asp Ser Ile Lys Lys His Val
Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln
Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu Ala Leu Arg Ala Ile
Ile Pro Ala65 70 75 80Thr Thr Ala Pro Val Pro Gln Gly Phe Leu Val
Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu Gly Glu Leu Phe Arg
Tyr Gln Glu Glu Arg Leu 100 105 110Thr Asn Phe Gln Pro Asp Tyr Pro
Val Thr Ala Arg Ile His Ala His 115 120 125Leu Lys Ala Tyr Ala Lys
Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg 130 135 140Arg Leu Leu Trp
Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Pro Asn145 150 155 160Val
Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu 165 170
175Lys Tyr Arg Gly Lys Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro
180 185 190Ile Gln Val 19517767PRTSheldgoose hepatitis virus 177Ala
Gln Gly Gly Arg Asn Lys Thr Gln Gly Ser Arg Lys Pro Arg Gly1 5 10
15Leu Gln Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly Arg
20 25 30Arg Arg Ser Lys Ser Arg Asp Arg Arg Ala Pro Ser Pro Gln Arg
Ala 35 40 45Gly Ser Pro Leu Pro Arg Pro Ser Thr Ser His His Arg Ser
Pro Ser 50 55 60Pro Arg Lys65178195PRTSheldgoose hepatitis virus
178Met Asp Val Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1
5 10 15Asp Asp Phe Phe Pro Lys Ile Asp Asp Leu Val Arg Asp Ala Lys
Asp 20 25 30Ala Leu Glu Pro Tyr Trp Arg Ser Glu Ser Ile Lys Lys His
Val Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp
Gln Thr Thr 50 55 60Gln Gly Met His Glu Ile Ala Glu Ala Leu Arg Ala
Val Ile Pro Pro65 70 75 80Thr Thr Thr Pro Val Pro Pro Gly Tyr Leu
Ile Gln His Glu Glu Ala 85 90 95Glu Glu Ile Pro Leu Gly Asp Leu Phe
Lys His Gln Glu Glu Arg Ile 100 105 110Val Ser Phe Gln Pro Asp Tyr
Pro Ile Thr Ala Arg Ile His Ala His 115 120 125Leu Lys Ala Tyr Ala
Lys Ile Asn Glu Glu Ser Leu Asp Lys Ala Arg 130 135 140Arg Leu Leu
Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Asn145 150 155
160Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu
165 170 175Arg Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr
Arg Pro 180 185 190Ile Gln Val 19517967PRTHeron hepatitis virus
179Ala Gln Gly Gly Arg Asn Gln Thr Lys Gly Thr Arg Lys Pro Arg Gly1
5 10 15Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly
Arg 20 25 30Arg Arg Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro Ser Gln
Arg Ala 35 40 45Gly Ser Pro Leu Pro Arg Asn Arg Gly Asn Gln Thr Arg
Ser Pro Ser 50 55 60Pro Arg Glu65180195PRTHeron hepatitis virus
180Met Asp Val Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1
5 10 15Asp Asp Phe Phe Pro Gln Ile Asp Asp Leu Val Arg Asp Ala Lys
Asp 20 25 30Ala Leu Glu Pro Tyr Trp Lys Ala Glu Thr Ile Lys Lys His
Val Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp
Gln Thr Thr 50 55 60Gln Gly Met Ser Gln Ile Ala Asp Ala Leu Arg Ala
Val Ile Pro Pro65 70 75 80Thr Thr Val Pro Val Pro Glu Gly Phe Leu
Ile Thr His Ser Glu Ala 85 90 95Glu Glu Ile Pro Leu Asn Asp Leu Phe
Ser Asn Gln Glu Glu Arg Ile 100 105 110Val Asn Phe Gln Pro Asp Tyr
Pro Ile Thr Ala Arg Ile His Thr His 115 120 125Leu Arg Val Tyr Thr
Lys Leu Asn Glu Gln Ala Leu Asp Lys Ala Arg 130 135 140Arg Leu Leu
Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Thr145 150 155
160Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu
165 170 175Lys Tyr Arg Gly Lys Asp Ala Pro Thr Ile Glu Ala Ile Thr
Arg Pro 180 185 190Ile Gln Val 19518167PRTStork hepatitis virus
181Ala Gln Gly Ser Arg Asn Gln Thr Lys Gly Val Arg Lys Pro Arg Gly1
5 10 15Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val Tyr Gly
Arg 20 25 30Arg Arg Ser Lys Ser Arg Gly Arg Arg Ser Ser Pro Ser Gln
Arg Ala 35 40 45Gly Ser Pro Ile Pro Arg Asn Arg Glu Asn Gln Ser Arg
Ser Ser Ser 50 55 60Pro Arg Glu65182195PRTStork hepatitis virus
182Met Asp Val Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro1
5 10 15Asp Asp Phe Phe Pro Gln Ile Asp Asp Leu Val Arg Asp Ala Lys
Asp 20 25 30Ala Leu Glu Pro Tyr Trp Lys Ala Glu Thr Ile Lys Lys His
Val Leu 35 40 45Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp
Gln Thr Thr 50 55 60Gln Gly Met Ser Gln Ile Ala Asp Ala Leu Arg Ala
Val Ile Pro Pro65 70 75 80Thr Thr Thr Pro Val Pro Asp Gly Tyr Leu
Ile Ser His Asn Glu Ala 85 90 95Gln Glu Leu Pro Leu Asn Asp Leu Phe
Val Asn Gln Glu Glu Arg Ile 100 105 110Val Asn Phe Gln Pro Asp Tyr
Pro Ile Thr Ala Arg Ile His Thr His 115 120 125Leu Arg Val Tyr Thr
Lys Leu Asn Glu Gln Ala Leu Asp Lys Ala Arg 130 135 140Arg Leu Leu
Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ser Asn145 150 155
160Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu
165 170 175Lys Tyr Arg Gly Lys Asp Ala Pro Thr Ile Glu Ala Ile Thr
Arg Pro 180 185 190Ile Gln Val 19518322PRTArctic ground squirrel
hepatitis virus 183Ala Ala Gly Ser Ala Arg Val Val Arg Ser Pro Ser
Gln Ser Pro Gln1 5 10 15Ser Pro Ala Ser Asn Cys 2018421PRTArctic
ground squirrel hepatitis virus 184Ala Ala Gly Ser Ala Arg Val Val
Arg Ser Ser Gln Ser Pro Gln Ser1 5 10 15Pro Ala Ser Asn Cys
2018520PRTArctic ground squirrel hepatitis virus 185Ala Ala Gly Ser
Ala Arg Val Val Arg Ser Ser Gln Ser Gln Ser Pro1 5 10 15Ala Ser Asn
Cys 2018619PRTArctic ground squirrel hepatitis virus 186Ala Ala Gly
Ser Ala Arg Val Val Arg Ser Ser Gln Ser Gln Ser Ala1 5 10 15Ser Asn
Cys18714PRTArctic ground squirrel hepatitis virus 187Arg Arg Gly
Ser Ala Arg Val Val Ser Gln Ala Ser Asn Cys1 5 1018814PRTArctic
ground squirrel hepatitis virus 188Ala Arg Gly Ser Ala Arg Val Val
Ser Gln Ala Ser Asn Cys1 5 1018914PRTArctic ground squirrel
hepatitis virus 189Arg Ala Gly Ser Ala Arg Val Val Ser Gln Ala Ser
Asn Cys1 5 1019014PRTArctic ground squirrel hepatitis virus 190Ala
Ala Gly Ser Ala Arg Val Val Ser Gln Ala Ser Asn Cys1 5
1019117PRTArctic ground squirrel hepatitis virus 191Ala Ala Gly Arg
Ser Pro Ser Gln Ser Pro Gln Ser Arg Glu Ser Gln1 5 10
15Cys19217PRTArctic ground squirrel hepatitis virus 192Ala Ala Gly
Arg Ser Pro Ser Gln Ser Pro Gln Ser Pro Ala Ser Asn1 5 10
15Cys19316PRTArctic ground squirrel hepatitis virus 193Ala Ala Gly
Arg Ser Pro Ser Gln Ser Pro Gln Ser Ala Ser Asn Cys1 5 10
1519414PRTArctic ground squirrel hepatitis virus 194Ala Ala Gly Arg
Ser Gln Ser Pro Gln Ser Ala Ser Asn Cys1 5 1019515PRTArctic ground
squirrel hepatitis virus 195Ala Ala Gly Arg Ser Pro Ser Gln Ser Gln
Ser Ala Ser Asn Cys1 5 10 1519613PRTArctic ground squirrel
hepatitis virus 196Ala Ala Gly Arg Ser Gln Ser Gln Ser Ala Ser Asn
Cys1 5 1019718PRTWoolly monkey hepatitis virus 197Ala Ala Arg Pro
Ser Pro Ser Gln Ser Pro Ser Gln Ser Pro Ala Ser1 5 10 15Ser
Cys19816PRTWoolly monkey hepatitis virus 198Ala Ala Arg Pro Ser Gln
Ser Pro Ser Gln Ser Pro Ala Ser Ser Cys1 5 10 1519915PRTWoolly
monkey hepatitis virus 199Ala Ala Arg Pro Ser Gln Ser Ser Gln Ser
Pro Ala Ser Ser Cys1 5 10 1520014PRTWoolly monkey hepatitis virus
200Ala Ala Arg Pro Ser Gln Ser Ser Gln Ser Ala Ser Ser Cys1 5
1020113PRTWoolly monkey hepatitis virus 201Arg Arg Gly Ser Gln Ser
Arg Arg Ser Gln Ser Ser Cys1 5 1020213PRTWoolly monkey hepatitis
virus 202Ala Arg Gly Ser Gln Ser Arg Arg Ser Gln Ser Ser Cys1 5
1020313PRTWoolly monkey hepatitis virus 203Arg Ala Gly Ser Gln Ser
Arg Arg Ser Gln Ser Ser Cys1 5 1020413PRTWoolly monkey hepatitis
virus 204Ala Ala Gly Ser Gln Ser Arg Arg Ser Gln Ser Ser Cys1 5
1020518PRTWoolly monkey hepatitis virus 205Ala Ala Arg Arg Arg Pro
Ser Gln Ser Pro Ser Gln Ser Pro Ala Ser1 5 10 15Ser
Cys20617PRTWoolly monkey hepatitis virus 206Ala Ala Arg Arg Arg Pro
Ser Gln Ser Pro Ser Gln Ser Ala Ser Ser1 5 10 15Cys20716PRTWoolly
monkey hepatitis virus 207Ala Ala Arg Arg Arg Pro Ser Gln Ser Pro
Ser Gln Ser Ser Ser Cys1 5 10 1520814PRTWoolly monkey hepatitis
virus 208Ala Ala Arg Arg Ser Gln Ser Pro Ser Gln Ser Ser Ser Cys1 5
1020915PRTWoolly monkey hepatitis virus 209Ala Ala Arg Arg Ser Pro
Ser Gln Ser Ser Gln Ser Ser Ser Cys1 5 10 1521013PRTWoolly monkey
hepatitis virus 210Ala Ala Arg Arg Ser Gln Ser Ser Gln Ser Ser Ser
Cys1 5 1021118PRTOrangutan hepatitis virus 211Ala Ala Gly Arg Ser
Pro Ser Gln Ser Pro Ser Gln Ser Pro Ala Ser1 5 10 15Gln
Cys21216PRTOrangutan hepatitis virus 212Ala Ala Gly Arg Ser Gln Ser
Pro Ser Gln Ser Pro Ala Ser Gln Cys1 5 10 1521315PRTOrangutan
hepatitis virus 213Ala Ala Gly Arg Ser Gln Ser Ser Gln Ser Pro Ala
Ser Gln Cys1 5 10 1521414PRTOrangutan hepatitis virus 214Ala Ala
Gly Arg Ser Gln Ser Ser Gln Ser Ala Ser Gln Cys1 5
1021511PRTOrangutan hepatitis virus 215Arg Arg Gly Ser Gln Ser Pro
Ala Ser Gln Cys1 5 1021611PRTOrangutan hepatitis virus 216Ala Arg
Gly Ser Gln Ser Pro Ala Ser Gln Cys1 5 1021711PRTOrangutan
hepatitis virus 217Arg Ala Gly Ser Gln Ser Pro Ala Ser Gln Cys1 5
1021811PRTOrangutan hepatitis virus 218Ala Ala Gly Ser Gln Ser Pro
Ala Ser Gln Cys1 5 1021918PRTOrangutan hepatitis virus 219Ala Ala
Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln Ser Pro Ala Ser1 5 10 15Gln
Cys22017PRTOrangutan hepatitis virus 220Ala Ala Gly Arg Ser Pro Ser
Gln Ser Pro Ser Gln Ser Ala Ser Gln1 5 10 15Cys22116PRTOrangutan
hepatitis virus 221Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Gln
Ser Ser Gln Cys1 5 10 1522215PRTOrangutan hepatitis virus 222Ala
Ala Gly Arg Ser Gln Ser Pro Ser Gln Ser Ala Ser Gln Cys1 5 10
1522316PRTOrangutan hepatitis virus 223Ala Ala Gly Arg Ser Pro Ser
Gln Ser Ser Gln Ser Ala Ser Gln Cys1 5 10 1522414PRTOrangutan
hepatitis virus 224Ala Ala Gly Arg Ser Gln Ser Ser Gln Ser Ala Ser
Gln Cys1 5 1022523PRTDuck hepatitis virus 225Ala Ala Gly Gly Glu
Arg Gly Val Arg Ser Pro Ser Gln Ser Pro Ser1 5 10 15Arg Ser Pro Ser
Pro Arg Lys2022621PRTDuck hepatitis virus 226Ala Ala Gly Gly Glu
Arg Gly Val Arg Ser Gln Ser Pro Ser Arg Ser1 5 10 15Pro Ser Pro Arg
Lys2022720PRTDuck hepatitis virus 227Ala Ala Gly Gly Glu Arg Gly
Val Arg Ser Gln Ser Ser Arg Ser Pro1 5 10 15Ser Pro Arg
Lys2022819PRTDuck hepatitis virus 228Ala Ala Gly Gly Glu Arg Gly
Val Arg Ser Gln Ser Ser Arg Ser Ser1 5 10 15Pro Arg Lys22915PRTDuck
hepatitis virus 229Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser Pro Ser
Pro Arg Lys1 5 10 1523015PRTDuck hepatitis virus 230Ala Arg Gly Gly
Ala Arg Ala Ser Arg Ser Pro Ser Pro Arg Lys1 5 10 1523115PRTDuck
hepatitis virus 231Arg Ala Gly Gly Ala Arg Ala Ser Arg Ser Pro Ser
Pro Arg Lys1 5 10 1523215PRTDuck hepatitis virus 232Ala Ala Gly Gly
Ala Arg Ala Ser Arg Ser Pro Ser Pro Arg Lys1 5 10 1523319PRTDuck
hepatitis virus 233Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Ser
Arg Ser Pro Ser1 5 10 15Pro Arg Lys23419PRTDuck hepatitis virus
234Ala Ala Gly Arg Ser Pro Ser Gln Ser Pro Ser Ser Arg Ser Pro Ser1
5 10 15Pro Arg Glu23518PRTDuck hepatitis virus 235Ala Ala Gly Arg
Ser Pro Ser Gln Ser Pro Ser Ser Arg Ser Ser Pro1 5 10 15Arg
Glu23616PRTDuck hepatitis virus 236Ala Ala Gly Arg Ser Gln Ser Pro
Ser Ser Arg Ser Ser Pro Arg Glu1 5 10 1523716PRTDuck hepatitis
virus 237Ala Ala Gly Arg Ser Pro Ser Gln Ser Ser Arg Ser Ser Pro
Arg Glu1 5 10 1523814PRTDuck hepatitis virus 238Ala Ala Gly Arg Ser
Gln Ser Ser Arg Ser Ser Pro Arg Glu1 5 1023920PRTTetanus toxin
239Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val Pro Tyr Ile Gly Pro1
5 10 15Ala Leu Asn Ile2024021PRTTetanus toxin 240Asn Asn Phe Thr
Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala1 5 10 15Ser His Leu
Glu Gln2024121PRTTetanus toxin 241Phe Asn Asn Phe Thr Val Ser Phe
Trp Leu Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu
Glu2024214PRTTetanus toxin 242Gln Tyr Ile Lys Ala Asn Ser Lys Phe
Ile Gly Ile Thr Glu1 5 1024316PRTTetanus toxin 243Val Ser Ile Asp
Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys1 5 10
1524416PRTTetanus toxin 244Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro
Asn Asn Glu Ile Asp Ser1 5 10 1524519PRTDiptheria toxin 245Pro Leu
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala1 5 10 15Gln
Val Ile24620PRTDiptheria toxin 246Val His His Asn Thr Glu Glu Ile
Val Ala Gln Ser Ile Ala Leu Ser1 5 10 15Ser Leu Met
Val2024720PRTDiptheria toxin 247Gln Ser Ile Ala Leu Ser Ser Leu Met
Val Ala Gln Ala Ile Pro Leu1 5 10 15Val Gly Glu
Leu2024820PRTDiptheria toxin 248Gln Gly Glu Ser Gly His Asp Ile Lys
Ile Thr Ala Glu Asn Thr Pro1 5 10 15Leu Pro Ile
Ala2024920PRTDiptheria toxin 249Val Asp Ile Gly Phe Ala Ala Tyr Asn
Phe Val Glu Ser Ile Ile Asn1 5 10 15Leu Phe Gln
Val2025020PRTDiptheria toxin 250Gly Val Leu Leu Pro Thr Ile Pro Gly
Lys Leu Asp Val Asn Lys Ser1 5 10 15Lys Thr His
Ile2025120PRTPlasmodium falciparum 251Glu Tyr Leu Asn Lys Ile Gln
Asn Ser Leu Ser Thr Glu Trp Ser Pro1 5 10 15Cys Ser Val
Thr2025220PRTPlasmodium falciparum 252Asp Ile Glu Lys Lys Ile Cys
Lys Met Glu Lys Cys Ser Ser Val Phe1 5 10
15Asn Val Val Asn2025315PRTHepatitis B virus 253Phe Phe Leu Leu Thr
Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp1 5 10 1525413PRTInfluenza
virus 254Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr1 5
1025516PRTInfluenza virus 255Tyr Ser Gly Pro Leu Lys Ala Glu Ile
Ala Gln Arg Leu Glu Asp Val1 5 10 1525618PRTMeasles virus 256Lys
Leu Leu Ser Leu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly1 5 10
15Val Glu
* * * * *