U.S. patent application number 17/459313 was filed with the patent office on 2021-12-23 for adenoviral vectors encoding hepatitis b viral antigens fused to herpes virus glycoprotein d and methods of using the same.
The applicant listed for this patent is VIRION THERAPEUTICS, LLC, THE WISTAR INSTITUTE. Invention is credited to Hildegund CJ ERTL, Colin Stephen MAGOWAN.
Application Number | 20210393770 17/459313 |
Document ID | / |
Family ID | 1000005880534 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210393770 |
Kind Code |
A1 |
ERTL; Hildegund CJ ; et
al. |
December 23, 2021 |
ADENOVIRAL VECTORS ENCODING HEPATITIS B VIRAL ANTIGENS FUSED TO
HERPES VIRUS GLYCOPROTEIN D AND METHODS OF USING THE SAME
Abstract
Provided herein are non-naturally occurring variants of the
hepatitis B virus (HBV) Core protein, the HBV polymerase N-terminal
domain, and the HBV polymerase C-terminal domain, as well as
immunogenic fragments thereof. Fusion proteins comprising the HBV
variants fused to a herpes simplex virus (HSV) glycoprotein (gD)
sequence, as well as methods of using the fusion proteins, are also
provided.
Inventors: |
ERTL; Hildegund CJ;
(Villanova, PA) ; MAGOWAN; Colin Stephen; (Bishop,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VIRION THERAPEUTICS, LLC
THE WISTAR INSTITUTE |
Newark
Philadelphia |
DE
PA |
US
US |
|
|
Family ID: |
1000005880534 |
Appl. No.: |
17/459313 |
Filed: |
August 27, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2021/012630 |
Jan 8, 2021 |
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17459313 |
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62958809 |
Jan 9, 2020 |
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62958827 |
Jan 9, 2020 |
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62967104 |
Jan 29, 2020 |
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62967242 |
Jan 29, 2020 |
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63064506 |
Aug 12, 2020 |
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63064571 |
Aug 12, 2020 |
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63112202 |
Nov 11, 2020 |
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63112219 |
Nov 11, 2020 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/12 20180101;
C12N 15/86 20130101; C12N 7/00 20130101; A61K 2039/53 20130101;
A61K 39/292 20130101 |
International
Class: |
A61K 39/29 20060101
A61K039/29; C12N 15/86 20060101 C12N015/86; A61P 31/12 20060101
A61P031/12; C12N 7/00 20060101 C12N007/00 |
Claims
1. A nucleic acid molecule comprising: a nucleotide sequence
encoding an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof,
a nucleotide sequence encoding an HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 179 or an
immunogenic fragment thereof, and a nucleotide sequence encoding an
HBV Core protein comprising the amino acid sequence of SEQ ID NO:
180 or an immunogenic fragment thereof.
2. The nucleic acid molecule of claim 1, encoding a fusion protein
comprising the amino acid sequence of SEQ ID NO: 174.
3. The nucleic acid molecule of claim 1, further comprising a
nucleotide sequence encoding: an N-terminal HSV gD sequence or a
variant thereof, a C-terminal HSV gD sequence or a variant thereof,
or both.
4. The nucleic acid molecule of claim 3, comprising: a nucleotide
sequence encoding an N-terminal HSV gD sequence or a variant
thereof; a nucleotide sequence encoding an HBV fusion protein
comprising an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 178 or an immunogenic fragment thereof;
an HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 179 or an immunogenic fragment thereof; an
HBV Core protein comprising the amino acid sequence of SEQ ID NO:
180 or an immunogenic fragment thereof; and a nucleotide sequence
encoding a C-terminal HSV gD sequence or a variant thereof.
5. The nucleic acid molecule of claim 4, wherein the nucleotide
sequence encodes an HBV fusion protein comprising the amino acid
sequence of SEQ ID NO: 174.
6. The nucleic acid molecule of claim 5, wherein the nucleotide
sequence encodes an N-terminal HSV gD sequence comprising the amino
acid sequence of SEQ ID NO: 12.
7. The nucleic acid molecule of claim 5, wherein the nucleotide
sequence encodes an N-terminal HSV gD sequence comprising amino
acid residues 26-269 of SEQ ID NO: 12.
8. The nucleic acid molecule of claim 5, wherein the nucleotide
sequence encodes a C-terminal HSV gD sequence comprising the
transmembrane domain of the HSV gD.
9. The nucleic acid molecule of claim 8, wherein the nucleotide
sequence encodes a C-terminal HSV gD sequence comprising the amino
acid sequence of SEQ ID NO: 13.
10. The nucleic acid molecule of claim 4, comprising: a nucleotide
sequence encoding an N-terminal HSV gD sequence comprising amino
acid residues 26-269 of SEQ ID NO: 12, a nucleotide sequence
encoding an HBV fusion protein comprising: an HBV polymerase
N-terminal domain comprising the amino acid sequence of SEQ ID NO:
178 or an immunogenic fragment thereof, an HBV polymerase
C-terminal domain comprising the amino acid sequence of SEQ ID NO:
179 or an immunogenic fragment thereof, and an HBV Core protein
comprising the amino acid sequence of SEQ ID NO: 180 or an
immunogenic fragment thereof; and a nucleotide sequence encoding a
C-terminal HSV gD sequence comprising the amino acid sequence of
SEQ ID NO: 13.
11. The nucleic acid molecule of claim 10, wherein the nucleotide
sequence encodes an N-terminal HSV gD sequence comprising the amino
acid sequence of SEQ ID NO: 12.
12. The nucleic acid molecule of claim 10, wherein the nucleotide
sequence encodes an HBV fusion protein comprising the amino acid
sequence of SEQ ID NO: 174.
13. The nucleic acid molecule of claim 10, wherein the nucleotide
sequence encodes a fusion protein comprising the amino acid
sequence of SEQ ID NO: 185.
14. The nucleic acid molecule of claim 12, wherein the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 176.
15. The nucleic acid molecule of claim 13, wherein the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 184.
16. A virus comprising the nucleic acid molecule of claim 13.
17. The virus of claim 16, wherein the virus is an adenovirus.
18. The virus of claim 17, wherein the adenovirus is an AdC6 or
AdC7.
19. A vaccine comprising the virus of claim 16.
20. A method of inducing an immune response to HBV in a subject,
the method comprising providing to the subject an effective amount
of the nucleic acid molecule of claim 13 to thereby induce an
immune response to HBV.
21. The method of claim 20, wherein the nucleic acid molecule is in
an AdC6 virus.
22. The method of claim 20, wherein the nucleic acid molecule is in
an AdC7 virus.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US2021/012630, filed on Jan. 8, 2021, which
claims priority to U.S. Provisional Application No. 62/958,809,
filed Jan. 9, 2020, U.S. Provisional Application No. 62/958,827,
filed Jan. 9, 2020, U.S. Provisional Application No. 62/967,242,
filed Jan. 29, 2020, U.S. Provisional Application No. 62/967,104,
filed Jan. 29, 2020, U.S. Provisional Application No. 63/064,506,
filed Aug. 12, 2020, U.S. Provisional Application No. 63/064,571,
filed Aug. 12, 2020, U.S. Provisional Application No. 63/112,202,
filed Nov. 11, 2020, and U.S. Provisional Application No.
63/112,219, filed Nov. 11, 2020, the disclosure of each of which is
hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. The ASCII copy, created
on Jan. 7, 2021, is named 111876_000036_SL.txt and is 151,446 bytes
in size.
FIELD OF THE INVENTION
[0003] Disclosed herein are non-naturally occurring variants of the
hepatitis B virus (HBV) Core protein, the HBV polymerase N-terminal
domain, and the HBV polymerase C-terminal domain, as well as
immunogenic fragments thereof and fusion proteins comprising the
same.
BACKGROUND OF THE INVENTION
[0004] The World Health Organization estimates that, in 2015, 257
million people were living with chronic hepatitis B infection
(defined as hepatitis B surface antigen positive) and that
hepatitis B resulted in an estimated 887,000 deaths, mostly from
cirrhosis and hepatocellular carcinoma (i.e., primary liver
cancer). Assuming that women of reproductive age constitute 25.3%
of the world's population (United Nations data), adults chronically
infected may include 65 million women of childbearing age who can
potentially transmit HBV to their babies (WHO Global Hepatitis
Report 2017. Available at:
apps_who_int/iris/bitstream/handle/10665/255016/9789241565455-eng.pdf;jse-
ssionid=D78616700ED7322D4109CA4541FB94EA?sequence=1). The overall
incidence rate in 2016 was 1.0 case per 100,000 population (Centers
for Disease Control and Prevention. Viral Hepatitis
Surveillance--United States, 2017. Atlanta: US Department of Health
and Human Services, Centers for Disease Control and Prevention;
2019. Available at:
www_cdc_gov/hepatitis/statistics/2017surveillance/index.htm.). In
2017 alone, a total of 3,407 cases of acute hepatitis B were
reported to the Centers for Disease Control and Prevention
(CDC).
[0005] Despite the availability of a prophylactic HBV vaccine, the
burden of chronic HBV infection continues to be a significant unmet
worldwide medical problem, due to suboptimal treatment options and
sustained rates of new infections in most parts of the developing
world.
SUMMARY OF THE INVENTION
[0006] Provided herein is a hepatitis B virus (HBV) Core protein
comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof.
[0007] Also provided is an HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof.
[0008] An HBV polymerase C-terminal domain comprising the amino
acid sequence of SEQ ID NO: 10 or an immunogenic fragment thereof
is also disclosed.
[0009] Fusion proteins comprising: an N-terminal herpes simplex
virus (HSV) glycoprotein (gD) sequence or a variant thereof; the
disclosed HBV Core protein, HBV polymerase N-terminal domain, HBV
polymerase C-terminal domain, or immunogenic fragments thereof; and
a C-terminal HSV gD sequence or a variant thereof are also
provided.
[0010] Also provided herein are fusion proteins comprising: an
N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or
a variant thereof; combinations of the disclosed HBV Core protein,
HBV polymerase N-terminal domain, HBV polymerase C-terminal domain,
and/or immunogenic fragments thereof; and a C-terminal HSV gD
sequence or a variant thereof.
[0011] Nucleic acid molecules encoding the disclosed proteins or
fusion proteins, vectors comprising the nucleic acid molecules, and
vaccines comprising the disclosed vectors are disclosed herein.
[0012] Also provided herein are methods of inducing an immune
response to HBV in a subject, the method comprising providing to
the subject an effective amount of any of the disclosed fusion
proteins, nucleic acid molecules, vectors, or vaccines to thereby
induce an immune response to HBV.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The summary, as well as the following detailed description,
is further understood when read in conjunction with the appended
drawings. For the purpose of illustrating the disclosed proteins,
vaccines, and methods, there are shown in the drawings exemplary
embodiments of the proteins, vaccines, and methods; however, the
proteins, vaccines, and methods are not limited to the specific
embodiments disclosed. In the drawings:
[0014] FIG. 1 illustrates the frequency of epitope-optimized Core
amino acids. Amino acid residues are indicated on the X-axis;
percent sequence similarity across all genomes analyzed is
indicated on the Y-axis.
[0015] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F
illustrate vaccine insert-specific T cell frequencies in C57Bl/6
mice following intramuscular (i.m.) injection with the indicated
doses of: replication-defective adenovirus vector of chimpanzee
serotype 6 (AdC6) containing the epitope-optimized Core sequence
genetically fused into gD (SEQ ID NO: 15) (AdC6-gDCore) (FIG. 2A
and FIG. 2D); AdC6 containing the epitope-optimized polymerase
C-terminal domain sequence genetically fused into gD (SEQ ID NO:
19) (AdC6-gDPolC) (FIG. 2B and FIG. 2E); and AdC6 containing the
epitope-optimized polymerase N-terminal domain sequence genetically
fused into gD (SEQ ID NO: 17) (AdC6-gDPolN) (FIG. 2C and FIG. 2F).
Mice were bled 14 days after the injection and T cell frequencies
to the various HBV inserts were analyzed by intracellular cytokine
staining (ICS) for interferon (IFN)-.gamma. upon stimulation of
cells with overlapping peptides representing the HBV sequences.
Control cells were cultured without peptides. Graphs show results
for individual mice with medians indicated by the lines. FIG. 2A-2C
show insert-specific CD8+ T cell frequency; FIG. 2D-2F show
insert-specific CD4+ T cell frequency.
[0016] FIG. 3A, FIG. 3B, and FIG. 3C illustrate T cell frequencies
in different mouse strains (A: C57Bl/6 mice; B: BALB/c mice; C:
HLA-A2 transgenic (tg) mice) to pools of peptides representing the
indicated HBV sequence. Results were obtained with splenocytes
harvested 4 weeks after immunization and tested by ICS for
IFN-.gamma.. Peptides were arranged in matrices so that recognition
of 2 pools identified one peptide. The graphs show responses to the
different pools; responses to a pool containing all peptides are
shown to the right. Background frequencies obtained without the
peptides were subtracted. Pools that were deemed to elicit a
response and peptides identified in response to different pools are
listed at the bottom of each figure. CD8+ T cell and CD4+ T cell
responses are shown for BALB/c mice; CD8+ T cell responses are
shown for HLA-A2 tg mice, which carry a human MEW class I molecule
but mouse MHC class II molecules. T cells were gated on activated
CD44+ cells. Each consecutively numbered "peptide" consists of 15
amino acids beginning on the 1.sup.st, 6.sup.th, 11.sup.th, etc.
amino acid of the Core, PolN, or PolC sequence. Thus, for example,
peptide 1 of Core corresponds to amino acids 1-15 of SEQ ID NO: 6
(i.e. the epitope-optimized Core amino acid sequence), peptide 2 of
Core corresponds to amino acids 6-20 of SEQ ID NO: 6, peptide 3 of
Core corresponds to amino acids 11-25 of SEQ ID NO: 6, etc.
Similarly, peptide 1 of PolN corresponds to amino acids 1-15 of SEQ
ID NO: 8 (i.e. the epitope-optimized PolN amino acid sequence),
peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO: 8,
peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO: 8,
etc. Likewise, peptide 1 of PolC corresponds to amino acids 1-15 of
SEQ ID NO: 10 (i.e. the epitope-optimized PolC amino acid
sequence), peptide 2 of PolC corresponds to amino acids 6-20 of SEQ
ID NO: 10, peptide 3 of PolC corresponds to amino acids 11-25 of
SEQ ID NO: 10, etc.
[0017] FIG. 4A, FIG. 4B and FIG. 4C show the IFN-.gamma. response
upon boosting with AdC6-gDCore (A), AdC6-gDPolC (B), and
AdC6-gDPolN (C) in C57Bl/6 mice immunized with various doses of the
indicated vectors. The left graphs show responses tested from blood
2 weeks after priming with AdC6 vector. Mice were boosted 8 weeks
later with the same doses of AdC7 vectors expressing the same
inserts. The right graphs show responses at 2 weeks after the boost
in blood.
[0018] FIG. 5A, FIG. 5B, and FIG. 5C illustrate T cell frequencies
in different mouse strains (A: C57Bl/6 mice; B: BALB/c mice; C:
HLA-A2 tg mice) to pools of peptides representing the indicated HBV
sequence. Mice were primed with AdC6 vectors expressing either of
the 3 inserts (i.e., Core, PolC, or PolN) and were boosted 8 weeks
later with AdC7 vectors expressing the same inserts. Results were
obtained with splenocytes harvested 4 weeks after the immunization
and tested by ICS for IFN-.gamma.. Peptides were arranged in
matrices so that recognition of 2 pools identified one peptide. The
graphs show responses to the different pools; responses to a pool
containing all peptides are shown to the right. Background
frequencies obtained without the peptides were subtracted. Pools
that were deemed to elicit a response and peptides identified in
response to different pools are listed at the bottom of each
figure. CD8+ T cell and CD4+ T cell responses are shown for BALB/c
mice; CD8+ T cell responses are shown for HLA-A2 tg mice which
carry a human MEW class I molecule but mouse MHC class II
molecules. T cells were gated on activated CD44+ cells. Each
consecutively numbered "peptide" consists of 15 amino acids
beginning on the 1.sup.st, 6.sup.th, 11.sup.th, etc. amino acid of
the Core, PolN, or PolC sequence. Thus, for example, peptide 1 of
Core corresponds to amino acids 1-15 of SEQ ID NO: 6 (i.e. the
epitope-optimized Core amino acid sequence), peptide 2 of Core
corresponds to amino acids 6-20 of SEQ ID NO: 6, peptide 3 of Core
corresponds to amino acids 11-25 of SEQ ID NO: 6, etc. Similarly,
peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO: 8
(i.e. the epitope-optimized PolN amino acid sequence), peptide 2 of
PolN corresponds to amino acids 6-20 of SEQ ID NO: 8, peptide 3 of
PolN corresponds to amino acids 11-25 of SEQ ID NO: 8, etc.
Likewise, peptide 1 of PolC corresponds to amino acids 1-15 of SEQ
ID NO: 10 (i.e. the epitope-optimized PolC amino acid sequence),
peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO: 10,
peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO:
10, etc.
[0019] FIG. 6 illustrates the effect of vaccination on HBV genome
copy numbers in serum upon AAV-1.3HBV challenge. A group of 3 mice
were challenged with 1.times.10.sup.10, 1.times.10.sup.11 or
1.5.times.10.sup.11 virus genomes (vg) of an adeno-associated virus
8 (AAV8)-1.3HBV vector and 8 weeks later were vaccinated with
AdC6-gDPolN. Viral titers were tested 8 weeks after vaccination and
compared to pre-vaccination titers. Viral changes from baseline for
each treatment group are shown.
[0020] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E illustrate
exemplary HBV epitope shifting experiments. FIG. 7A--mice were
immunized with the AdC6-gDPolN vaccine. Four weeks later
splenocytes were tested by intracellular cytokine staining for
IFN-.gamma. responses to peptide pools representing the PolN
sequence. T cells after stimulation were stained for T cell
markers. FIG. 7B--results obtained with the same assay using
splenocytes from mice that were challenged with 1.times.10.sup.10
vg of AAV8-1.3-HBV. Mice were vaccinated 4 weeks later and T cell
responses were tested from spleens 10 weeks later. FIG. 7C--results
obtained with the same assay using splenocytes from mice that had
been challenged with 1.5.times.10.sup.11vg of AAV8-1.3-HBV. Mice
were vaccinated 4 weeks later and T cell responses were tested from
spleen 10 weeks later. FIGS. 7A, 7B, and 7C show the frequencies of
IFN-.gamma. producing CD44+CD8+ T cells over all CD44+CD8+ T cells.
Background responses obtained by splenocytes incubated without
peptide pools were subtracted. FIG. 7D--peptide pools. FIG.
7E--individual peptide sequences. FIG. 7E discloses SEQ ID NOs:
55-68 and 189-233, respectively, in order of appearance.
[0021] FIG. 8A, FIG. 8B, and FIG. 8C show data from the same
experiment described above in FIG. 7. Based on the responses to the
peptide pools, it was determined which individual peptides (both
pools and peptides shown in FIG. 7) were positive. The graphs show
responses to all of the peptides. Each peptide was present in two
pools and therefore two values for frequencies were obtained for
each peptide; only the lower data points are shown in this
figure.
[0022] FIG. 9A, FIG. 9B, and FIG. 9C illustrate the results from
exemplary immunogenicity experiments performed on C57Bl/6 mice (n=5
per group) injected with various doses of exemplary AdC6-gDCore,
AdC6-gDPolN, or AdC6-gDPolC vectors and boosted with AdC7 vectors
containing the same insert (i.e. AdC7-gDCore, AdC7-gDPolN, or
AdC7-gDPolC vectors) two months after the first injection. FIG. 9A
illustrates antigen immunogenicity, FIG. 9B illustrates the
duration of response, and FIG. 9C illustrates the prime-boost
response.
[0023] FIG. 10 illustrates the CD8+ T cell peptide recognition of
PolN epitopes in BALB/c, C57Bl/6, and HLA-A2 transgenic mice after
vaccination with a prime of AdC6-gDPolN and a boost of AdC7-gDPolN.
CD8+ T cell peptide recognition was calculated as the fraction of
positive peptides recognized two weeks after either the prime or
the boost by the total number of overlapping 8 peptides from PolN
(59 peptides total).
[0024] FIG. 11A and FIG. 11B illustrate vaccine-induced
HBV-specific CD8+ T cell response in the liver of C57Bl/6 mice
injected with the indicated vectors. * p-value between 0.01-0.05;
*** p-value between 0.0001-0.001; via 1-way ANOVA.
[0025] FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG.
12F illustrate hematoxylin & eosin staining of liver samples
from C57Bl/6 mice injected with the indicated vectors. 20.times.
magnification. Arrows indicate areas of lymphocytic
infiltrates.
[0026] FIG. 13A and FIG. 13B illustrate vaccine-induced markers of
CD8+ T cell activation/exhaustion in the liver of C57Bl/6 mice
injected with the indicated vectors. ** p-value between 0.001-0.01;
*** p-value between 0.0001-0.001; via 1-way ANOVA.
[0027] FIG. 14A and FIG. 14B illustrate HBV viral dynamics in
C57Bl/6 mice injected with an exemplary AdC6-gDPolN vector. The
median HBV DNA VL/ml at week 4-7.3 log.sub.10 cps/mL are provided.
n=7; one mouse excluded for missing data.
[0028] FIG. 15A and FIG. 15B illustrate the impact of AAV-induced
HBV on CD8+ T cell responses in C57Bl/6 mice first injected with
10.sup.10 or 10.sup.11 vg of AAV-1.3HBV and then four weeks later
boosted with 10.sup.10 vp of an exemplary AdC6-gDPolN vector. In
FIG. 15B, each slice represents an individual epitope with size
showing the proportion of the total; only responses >0.1% were
included. Pullouts represent epitopes only recognized in
AAV8-1.3HBV infected mice.
[0029] FIG. 16 illustrates the frequencies of IFN-.gamma.-producing
CD8+ T cells for individual C57Bl/6 mice that were injected i.v.
with the 10.sup.10 vg of the AAV8-1.3HBV vectors, vaccinated 4
weeks later with 5.times.10.sup.9 vp of the AdC6-gDPolN vector, and
boosted 2 months later with the same dose of the AdC7-gDPolN
vaccine. Control mice only received the vaccine. Naive mice served
as additional controls.
[0030] FIG. 17A and FIG. 17B illustrate: A) % of CD8.sup.+ T cells
within the lymphatic infiltrates of livers of individual mice; and
B) the frequencies of PolN-tetramer.sup.+CD8.sup.+ T cells within
the same infiltrates. C57Bl/6 mice were injected i.v. with the
10.sup.10 or 10.sup.11vg of the AAV8-1.3HBV vector, were vaccinated
4 weeks later with 5.times.10.sup.9 vp of the AdC6-gDPolN vector,
and were boosted 2 months later with the same dose of the
AdC7-gDPolN vaccine. Control mice only received the vaccine. Naive
mice served as additional controls.
[0031] FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG.
18F illustrate the phenotypes of the infiltrating
tetramer.sup.+CD8.sup.+ T cells in comparison to naive (i.e.,
tetramer.sup.- CD44.sup.- CD8.sup.+) T cells analyzed with the mean
fluorescent intensity (MFI) of the indicated markers. Lines with
stars above indicate significant differences by multiple t-test.
(*) p.ltoreq.0.05-0.01, (**) p.ltoreq.0.01-0.001, (***)
p.ltoreq.0.001-0.0001, (****) p.ltoreq.0.0001.
[0032] FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, FIG. 19E, and FIG.
19F illustrate the percentage of Tee or naive CD8.sup.+ T cells
positive for the indicated markers. Lines with stars above indicate
significant differences by multiple t-test. (*) p.ltoreq.0.05-0.01,
(**) p.ltoreq.0.01-0.001, (***) p.ltoreq.0.001-0.0001, (****)
p.ltoreq.0.0001.
[0033] FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E, and FIG.
20F illustrate the CD8.sup.+ T cell response to individual peptides
spanning the PolN sequence. Total pool--response to mixtures of all
PolN peptides; Naive--response of naive mice to mixtures of all
PolN peptides. FIG. 20A and FIG. 20D show CD8.sup.+ T cell
responses of mice that received just the AdC6-gDPolN vaccine. FIG.
20B and FIG. 20E show CD8.sup.+ T cell responses of mice that were
injected with 10.sup.10 vg of AAV8-1.3HBV 4 weeks prior to
vaccination with AdC6-gDPolN. FIG. 20C and FIG. 20F show CD8.sup.+
T cell responses of mice that were injected with 10.sup.11vg of
AAV8-1.3HBV 4 weeks prior to vaccination with AdC6-gDPolN. FIGS.
20A, 20B and 20C can be used to calculate the breadth of the immune
response by individual epitopes using the peptide pools shown in
FIG. 7D and the individual peptide sequences recognized using FIG.
7E.
[0034] FIG. 21A and FIG. 21B illustrate the PolN-specific CD8+ T
cells in the spleen or liver of mice. FIG. 21A left panel shows
CD8.sup.+ T cell responses in spleen of AAV8-1.3HBV injected mice
that did or did not receive the AdC6-gDPolN vaccine at
5.times.10.sup.10 vp subsequently. FIG. 21A middle panel shows
CD8.sup.+ T cell frequencies in livers of mice that were treated
with different doses of AAV8-1.3HBV and then received vaccines in a
prime boost regimen. FIG. 21A right panel shows the levels of Tox-1
expression in PolN-specific CD8.sup.+ T cells or naive CD8.sup.+ T
cells from the same experiment. FIG. 21B illustrates %
IFN-.gamma..sup.+CD8.sup.+ T cells.
[0035] FIG. 22A and FIG. 22B illustrate A) CD8+ T cell frequencies
in the blood of mice injected with the indicated AdC6 vectors; and
B) frequencies of tetramer+CD8+ T cells.
[0036] FIG. 23 illustrates CD8+ T cell frequencies in the blood of
mice injected with the indicated AdC7 vectors.
[0037] FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D, FIG. 24E, and FIG.
24F illustrate CD8.sup.+ (FIG. 24A-FIG. 24C) and CD4+(FIG. 24D-FIG.
24F) T cell frequencies to the gDHBV2 and gDHBV3 inserts in blood
of mice injected with the indicated AdC7 vectors ("after prime")
and then boosted with the corresponding AdC6 vectors ("after
boost"). Graphs show frequencies of T cells producing IFN-.gamma.,
frequencies of T cells producing TNF-.alpha., and the sum of
frequencies of T cells producing either cytokine.
[0038] FIG. 25A and FIG. 25B illustrate the HBV DNA viral titer in
C57Bl/6 mice that were challenged with 1.times.10.sup.9 vg of
AAV8-1.3HBV and were vaccinated 4 weeks later with
1.times.10.sup.10 vp of AdC6-gDPolN ("gDPolN"), AdC6-gDHBV2
("gDHBV2"), AdC6-gDHBV3 ("gDHBV3"), or AdC6-HBV2 without gD
("HBV2"); AAV-infected, non-vaccinated animals ("naive"), and
non-AAV-infected, non-vaccinated animals (data not shown) served as
controls. FIG. 25A illustrates the viral titer for each group at
weeks 4 and 8 after AAV challenge; FIG. 25B illustrates the results
of the individual mice at weeks 4 and 8 after AAV challenge.
[0039] FIG. 26A, FIG. 26B, FIG. 26C, and FIG. 26D illustrate the
percent of parental IFN-.gamma. and/or TNF-.alpha. producing
CD8.sup.+ T cells (FIG. 26A), CD44+CD8+ T cells (FIG. 26B), CD4+ T
cells (FIG. 26C) or CD44+CD4+ T cells (FIG. 26D) two and eight
weeks after prime and two and four weeks after the boost (as the
mean) using the indicated construct.
[0040] FIG. 27A, FIG. 27B, and FIG. 27C illustrate CD8.sup.+ T
cells at multiple time points: four weeks after prime (FIG. 27A);
two weeks after the boost (FIG. 27B); and four weeks after the
boost (FIG. 27C) with the indicated constructs (PolN=gDPolN;
HBV2=gDHBV2; HBV3=gDHBV3). The graph shows the overall frequencies
of CD8.sup.+ T cells producing IFN-.gamma..sup.+ as assessed by
ICS.
[0041] FIG. 28A, FIG. 28B, and FIG. 28C illustrate
cytokine-producing CD4+ T cells at multiple time points: four weeks
after prime (FIG. 28A); two weeks after the boost (FIG. 28B); and
four weeks after the boost (FIG. 28C) with the indicated constructs
(PolN=gDPolN; HBV2=gDHBV2; HBV3=gDHBV3) as assessed by ICS. The
dashed line indicates the cut-off for positive responses, based on
the results from the naive mice.
[0042] FIG. 29A and FIG. 29B illustrate the results of tetramer
staining gated on either CD8+ T cells (FIG. 29A) or CD44+CD8+ T
cells (FIG. 29B) at four weeks after the prime with the indicated
construct (PolN=gDPolN; HBV2=gDHBV2).
[0043] FIG. 30A, FIG. 30B, FIG. 30C, FIG. 30D, FIG. 30E, and FIG.
30F illustrates the phenotypes of the tetramer+ CD8+ T cells shown
as the mean fluorescent intensity of a dye linked to the indicated
antibody: FIG. 30A--anti-PD1 antibody conjugated to BV605; FIG.
30B--anti-LAG3 antibody conjugated to BV650; FIG. 30C--anti-TIM3
antibody conjugated to Pe-Cy7-A; FIG. 30D--anti-CTLA4 antibody
conjugated to PE-A; FIG. 30E--anti-EOMES antibody conjugated to
AF488; and FIG. 30F--anti-T-bet antibody conjugated to BV786.
[0044] FIG. 31 illustrates the CD8.sup.+ T cell responses after a
prime vaccination of 5.times.10.sup.10 vp AdC7-gDHBV2 followed two
months later by vaccination with 5.times.10.sup.10 vp AdC6-gDHBV2.
Numbers on the X axis correspond to the SEQ ID NO as provided
herein.
[0045] FIG. 32 illustrates the CD8+ T cell responses after a prime
vaccination with 5.times.10.sup.9 vp AdC7-gDHBV2 followed two
months later by vaccination with 5.times.10.sup.9 vp AdC6-gDHBV2.
Numbers on the X axis correspond to the SEQ ID NO as provided
herein.
[0046] FIG. 33 shows the immunogenicity after a prime vaccination
with 5.times.10.sup.10 vp AdC7-gDHBV3 followed two months later by
vaccination with 5.times.10.sup.10 vp AdC6-gDHBV3. Numbers on the X
axis correspond to the SEQ ID NO as provided herein.
[0047] FIG. 34 illustrates the immunogenicity of the AdC6-gDHBV2
and AdC7-gDHBV2 vaccines corresponding to the SEQ ID NO (X axis) as
provided herein. Core, PolC, and PolN regions in both HBV2
constructs were immunogenic.
[0048] FIG. 35 illustrates the immunogenicity of the AdC6-gDHBV3
and AdC7-gDHBV3 vaccines corresponding to the SEQ ID NO (X axis) as
provided herein. Core, PolC, and PolN regions in both HBV3
constructs were immunogenic.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0049] The disclosed proteins, vaccines, and methods may be
understood more readily by reference to the following detailed
description taken in connection with the accompanying figures,
which form a part of this disclosure. It is to be understood that
the disclosed proteins, vaccines, and methods are not limited to
the specific proteins, vaccines, and methods described and/or shown
herein, and that the terminology used herein is for the purpose of
describing particular embodiments by way of example only and is not
intended to be limiting of the claimed proteins, vaccines, and
methods.
[0050] Unless specifically stated otherwise, any description as to
a possible mechanism or mode of action or reason for improvement is
meant to be illustrative only, and the disclosed proteins,
vaccines, and methods are not to be constrained by the correctness
or incorrectness of any such suggested mechanism or mode of action
or reason for improvement.
[0051] Throughout this text, the descriptions refer to proteins and
methods of using said proteins. Where the disclosure describes or
claims a feature or embodiment associated with a proteins, such a
feature or embodiment is equally applicable to the methods of using
said proteins. Likewise, where the disclosure describes or claims a
feature or embodiment associated with a method of using the
proteins, such a feature or embodiment is equally applicable to the
proteins.
[0052] Where a range of numerical values is recited or established
herein, the range includes the endpoints thereof and all the
individual integers and fractions within the range, and also
includes each of the narrower ranges therein formed by all the
various possible combinations of those endpoints and internal
integers and fractions to form subgroups of the larger group of
values within the stated range to the same extent as if each of
those narrower ranges was explicitly recited. Where a range of
numerical values is stated herein as being greater than a stated
value, the range is nevertheless finite and is bounded on its upper
end by a value that is operable within the context of the invention
as described herein. Where a range of numerical values is stated
herein as being less than a stated value, the range is nevertheless
bounded on its lower end by a non-zero value. It is not intended
that the scope of the invention be limited to the specific values
recited when defining a range. All ranges are inclusive and
combinable.
[0053] When values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. Reference to a particular numerical value
includes at least that particular value, unless the context clearly
dictates otherwise.
[0054] It is to be appreciated that certain features of the
disclosed proteins, vaccines, and methods which are, for clarity,
described herein in the context of separate embodiments, may also
be provided in combination in a single embodiment. Conversely,
various features of the disclosed proteins, vaccines, and methods
that are, for brevity, described in the context of a single
embodiment, may also be provided separately or in any
subcombination.
[0055] As used herein, the singular forms "a," "an," and "the"
include the plural.
[0056] Various terms relating to aspects of the description are
used throughout the specification and claims. Such terms are to be
given their ordinary meaning in the art unless otherwise indicated.
Other specifically defined terms are to be construed in a manner
consistent with the definitions provided herein.
[0057] As used herein, "immunogenic fragment thereof" refers to a
portion of the disclosed HBV Core (Core), HBV polymerase N-terminal
domain (PolN), or HBV polymerase C-terminal domain (PolC) that can
produce an immune response in a subject.
[0058] As used herein, "providing to the subject" and similar terms
indicate a procedure by which the fusion proteins, nucleic acid
molecules, vectors, or vaccines are delivered to a subject such
that target cells, tissues, or segments of the body of the subject
are contacted with the fusion proteins, nucleic acid molecules,
vectors, or vaccines. "Providing to the subject" includes
parenteral and non-parenteral routes of administration.
[0059] The term "biosimilar" (of an approved reference
product/biological drug, i.e., reference listed drug) refers to a
biological product that is highly similar to the reference product
notwithstanding minor differences in clinically inactive components
with no clinically meaningful differences between the biosimilar
and the reference product in terms of safety, purity and potency,
based upon data derived from (a) analytical studies that
demonstrate that the biological product is highly similar to the
reference product notwithstanding minor differences in clinically
inactive components; (b) animal studies (including the assessment
of toxicity); and/or (c) a clinical study or studies (including the
assessment of immunogenicity and pharmacokinetics or
pharmacodynamics) that are sufficient to demonstrate safety,
purity, and potency in one or more appropriate conditions of use
for which the reference product is licensed and intended to be used
and for which licensure is sought for the biosimilar. The
biosimilar may be an interchangeable product that may be
substituted for the reference product at the pharmacy without the
intervention of the prescribing healthcare professional. To meet
the additional standard of "interchangeability," the biosimilar is
to be expected to produce the same clinical result as the reference
product in any given patient and, if the biosimilar is administered
more than once to an individual, the risk in terms of safety or
diminished efficacy of alternating or switching between the use of
the biosimilar and the reference product is not greater than the
risk of using the reference product without such alternation or
switch. The biosimilar utilizes the same mechanisms of action for
the proposed conditions of use to the extent the mechanisms are
known for the reference product. The condition or conditions of use
prescribed, recommended, or suggested in the labeling proposed for
the biosimilar have been previously approved for the reference
product. The route of administration, the dosage form, and/or the
strength of the biosimilar are the same as those of the reference
product and the biosimilar is manufactured, processed, packed or
held in a facility that meets standards designed to assure that the
biosimilar continues to be safe, pure and potent. The biosimilar
may include minor modifications in the amino acid sequence when
compared to the reference product, such as N- or C-terminal
truncations that are not expected to change the biosimilar
performance. Biosimilars of the disclosed proteins and fusion
proteins are included within the scope of this disclosure.
[0060] The term "subject" as used herein is intended to mean any
animal, in particular, mammals. Although induction of an immune
response in mice is exemplified herein, any type of mammal can be
treated using the disclosed methods. Thus, the methods are
applicable to human and nonhuman animals, although preferably used
with mice and humans, and most preferably with humans.
[0061] The term "comprising" is intended to include examples
encompassed by the terms "consisting essentially of" and
"consisting of"; similarly, the term "consisting essentially of" is
intended to include examples encompassed by the term "consisting
of."
[0062] The following abbreviations are used herein: hepatitis B
virus (HBV); adenovirus (Ad); herpes simplex virus (HSV);
glycoprotein (gD); and virus genomes (vg).
[0063] Provided herein is a non-naturally occurring variant of the
hepatitis B virus (HBV) Core protein. The disclosed HBV Core
protein can comprise the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof. Exemplary immunogenic fragments of
SEQ ID NO: 6 include SEQ ID NOs: 20-54 provided in Table 3, below.
In some embodiments, the immunogenic fragment of the HBV Core
protein comprises the amino acid sequence of SEQ ID NO: 180. In
some embodiments, the immunogenic fragment of the HBV Core protein
comprises the amino acid sequence of SEQ ID NO: 183.
[0064] Nucleic acid molecules encoding the HBV Core protein or an
immunogenic fragment thereof are also provided. The nucleic acid
molecule can encode the HBV Core protein comprising the amino acid
sequence of SEQ ID NO: 6. In some embodiments, the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 7. The
nucleic acid molecules can encode the Core fragments provided in
Table 3. In some embodiments, the nucleic acid molecule encodes the
amino acid sequence of SEQ ID NO: 180. In some embodiments, the
nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:
183.
[0065] Vectors comprising the nucleic acid molecules encoding the
HBV Core protein or an immunogenic fragment thereof are also
provided. Suitable vectors include viral vectors, such as
lentiviral vectors, retroviral vectors, adenoviral vectors,
adeno-associated viral vectors, alphavirus replicons, herpes virus
vectors, pox virus vectors, and rhabdovirus vectors. In some
embodiments, the viral vector is an adenoviral vector. The
adenoviral vector can be a chimpanzee-derived adenoviral vector. In
some aspects, the vector is an AdC68 vector as described in Farina
S F, Gao G P, Xiang Z Q, Rux J J, Burnett R M, Alvira M R, Marsh J,
Ertl H C, Wilson J M. "Replication-defective vector based on a
chimpanzee adenovirus." J Virol. 2001 December; 75(23):11603-13. In
some aspects, the vector is an AdC7 vector as described in
Reyes-Sandoval A, Fitzgerald J C, Grant R, Roy S, Xiang Z Q, Li Y,
Gao G P, Wilson J M, Ertl H C. "Human immunodeficiency virus type
1-specific immune responses in primates upon sequential
immunization with adenoviral vaccine carriers of human and simian
serotypes" J Virol. 2004 July; 78(14):7392-9. In some aspects, the
vector is an AdC6 vector as described in Pinto A R, Fitzgerald J C,
Giles-Davis W, Gao G P, Wilson J M, Ertl H C. "Induction of CD8+ T
cells to an HIV-1 antigen through a prime boost regimen with
heterologous E1-deleted adenoviral vaccine carriers" J Immunol.
2003 Dec. 15; 171(12):6774-9.
[0066] In some embodiments, the vector comprises the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 7. In
some embodiments, the vector is an AdC6 vector comprising the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 7. In some embodiments, the vector is an AdC7 vector comprising
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 7.
[0067] In some embodiments, the vector comprises the nucleic acid
molecule that encodes the amino acid sequence of SEQ ID NO: 180. In
some aspects, the vector is an AdC6 vector. In some aspects, the
vector is an AdC7 vector. In some embodiments, the vector comprises
the nucleic acid molecule that encodes the amino acid sequence of
SEQ ID NO: 183. In some aspects, the vector is an AdC6 vector. In
some aspects, the vector is an AdC7 vector.
[0068] Vaccines comprising the vectors comprising the nucleic acid
molecules encoding the HBV Core protein or an immunogenic fragment
thereof are also disclosed. In some embodiments, the vaccine
comprises a vector comprising the nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the
vaccine comprises an AdC6 vector comprising the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 7. In
some embodiments, the vaccine comprises an AdC7 vector comprising
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 7. In some embodiments, the vaccine comprises an AdC6 vector
comprising the nucleic acid molecule that encodes the amino acid
sequence of SEQ ID NO: 180. In some embodiments, the vaccine
comprises an AdC7 vector comprising the nucleic acid molecule that
encodes the amino acid sequence of SEQ ID NO: 180. In some
embodiments, the vaccine comprises an AdC6 vector that comprises
the nucleic acid molecule that encodes the amino acid sequence of
SEQ ID NO: 183. In some embodiments, the vaccine comprises an AdC7
vector that comprises the nucleic acid molecule that encodes the
amino acid sequence of SEQ ID NO: 183.
[0069] The vaccine can further comprise a pharmaceutically
acceptable carrier or pharmaceutical acceptable excipient. As used
herein, "pharmaceutically acceptable carrier" or "pharmaceutical
acceptable excipient" includes any material which, when combined
with the disclosed fusion proteins, nucleic acids, or vectors,
allows the fusion proteins, nucleic acids, or vectors to retain
biological activity and is non-reactive with the subject's immune
system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline or normal
(0.9%) saline. Compositions comprising such carriers are formulated
by well-known conventional methods (see, for example, Remington's
Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990; and Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
[0070] Also disclosed herein are non-naturally occurring variants
of the HBV polymerase N-terminal domain (PolN) and the HBV
polymerase C-terminal domain (PolC). The disclosed HBV polymerase
N-terminal domain can comprise the amino acid sequence of SEQ ID
NO: 8 or an immunogenic fragment thereof. Exemplary immunogenic
fragments of SEQ ID NO: 8 include SEQ ID NOs: 55-113 provided in
Table 4, below. In some embodiments, the immunogenic fragment of
the HBV PolN comprises the amino acid sequence of SEQ ID NO: 178.
In some embodiments, the immunogenic fragment of the HBV PolN
comprises the amino acid sequence of SEQ ID NO: 181. The disclosed
HBV polymerase C-terminal domain can comprise the amino acid
sequence of SEQ ID NO: 10 or an immunogenic fragment thereof.
Exemplary immunogenic fragments of SEQ ID NO: 10 include SEQ ID
NOs: 114-172 provided in Table 5, below. In some embodiments, the
immunogenic fragment of the HBV PolC comprises the amino acid
sequence of SEQ ID NO: 179. In some embodiments, the immunogenic
fragment of the HBV PolC comprises the amino acid sequence of SEQ
ID NO: 182.
[0071] Nucleic acid molecules encoding the HBV polymerase
N-terminal domain or an immunogenic fragment thereof, or the HBV
polymerase C-terminal domain or an immunogenic fragment thereof,
are also provided. The nucleic acid molecule can encode the HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8. In some embodiments, the nucleic acid molecule
encoding the HBV polymerase N-terminal domain comprises the
nucleotide sequence of SEQ ID NO: 9. The nucleic acid molecules can
encode the HBV polymerase N-terminal domain fragments provided in
Table 4. The nucleic acid molecule can encode the HBV polymerase
C-terminal domain comprising the amino acid sequence of SEQ ID NO:
10. In some embodiments, the nucleic acid molecule encoding the HBV
polymerase C-terminal domain comprises the nucleotide sequence of
SEQ ID NO: 11. The nucleic acid molecules can encode the HBV
polymerase C-terminal domain fragments provided in Table 5. In some
embodiments, the nucleic acid molecule encodes the amino acid
sequence of SEQ ID NO: 178. In some embodiments, the nucleic acid
molecule encodes the amino acid sequence of SEQ ID NO: 181. In some
embodiments, the nucleic acid molecule encodes the amino acid
sequence of SEQ ID NO: 179. In some embodiments, the nucleic acid
molecule encodes the amino acid sequence of SEQ ID NO: 182.
[0072] Vectors comprising the nucleic acid molecules encoding the
HBV polymerase N-terminal domain or an immunogenic fragment thereof
or C-terminal domain or an immunogenic fragment thereof are also
provided. Suitable vectors include those described above. In some
embodiments, the vector comprises the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 9. In some
embodiments, the vector comprises the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 11. In some
aspects, the vector is an adenoviral vector. Suitable adenoviral
vectors include, for example, an AdC6 vector or AdC7 vector. In
some embodiments, the vector is an AdC6 vector comprising the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 9. In some embodiments, the vector is an AdC7 vector comprising
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 9. In some embodiments, the vector is an AdC6 vector
comprising the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 11. In some embodiments, the vector is an
AdC7 vector comprising the nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO: 11. In some embodiments, the
vector comprises the nucleic acid molecule that encodes the amino
acid sequence of SEQ ID NO: 178. In some aspects, the vector is an
AdC6 vector. In some aspects, the vector is an AdC7 vector. In some
embodiments, the vector comprises the nucleic acid molecule that
encodes the amino acid sequence of SEQ ID NO: 181. In some aspects,
the vector is an AdC6 vector. In some aspects, the vector is an
AdC7 vector. In some embodiments, the vector comprises the nucleic
acid molecule that encodes the amino acid sequence of SEQ ID NO:
179. In some aspects, the vector is an AdC6 vector. In some
aspects, the vector is an AdC7 vector. In some embodiments, the
vector comprises the nucleic acid molecule that encodes the amino
acid sequence of SEQ ID NO: 182. In some aspects, the vector is an
AdC6 vector. In some aspects, the vector is an AdC7 vector.
[0073] Vaccines comprising the vectors comprising the nucleic acid
molecules encoding the HBV polymerase N-terminal domain or an
immunogenic fragment thereof or HBV polymerase C-terminal domain or
an immunogenic fragment thereof are also disclosed. In some
embodiments, the vaccine comprises a vector comprising the nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO: 9.
The vaccine can comprise an AdC6 vector comprising the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 9. The
vaccine can comprise an AdC7 vector comprising the nucleic acid
molecule comprising the nucleotide sequence of SEQ ID NO: 9. In
some embodiments, the vaccine comprises a vector comprising the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 11. The vaccine can comprise an AdC6 vector comprising the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 11. The vaccine can comprise an AdC7 vector comprising the
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO: 11. The vaccine can further comprise a pharmaceutically
acceptable carrier or pharmaceutical acceptable excipient as
disclosed above. In some embodiments, the vaccine comprises a
vector comprising the nucleic acid molecule that encodes the amino
acid sequence of SEQ ID NO: 178. In some aspects, the vector is an
AdC6 vector. In some aspects, the vector is an AdC7 vector. In some
embodiments, the vaccine comprises a vector comprising the nucleic
acid molecule that encodes the amino acid sequence of SEQ ID NO:
181. In some aspects, the vector is an AdC6 vector. In some
aspects, the vector is an AdC7 vector. In some embodiments, the
vaccine comprises a vector comprising the nucleic acid molecule
that encodes the amino acid sequence of SEQ ID NO: 179. In some
aspects, the vector is an AdC6 vector. In some aspects, the vector
is an AdC7 vector. In some embodiments, the vaccine comprises a
vector comprising the nucleic acid molecule that encodes the amino
acid sequence of SEQ ID NO: 182. In some aspects, the vector is an
AdC6 vector. In some aspects, the vector is an AdC7 vector.
[0074] Fusion proteins comprising combinations of the disclosed HBV
Core protein or immunogenic fragments thereof, the HBV polymerase
N-terminal domain or immunogenic fragments thereof, and/or the HBV
polymerase C-terminal domain or immunogenic fragments thereof are
also provided herein. For example, the fusion protein can comprise:
[0075] (1) an HBV Core protein comprising the amino acid sequence
of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof; [0076] (2) one or
more immunogenic fragments of the HBV Core protein comprising the
amino acid sequence of SEQ ID NO: 6 and one or more immunogenic
fragments of the HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8. For example, one or more of
SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ
ID NO: 6) and one or more of SEQ ID NOs: 55-113 provided in Table 4
(immunogenic fragments of SEQ ID NO: 8); [0077] (3) an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof and an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; [0078] (4) one or more immunogenic
fragments of the HBV Core protein comprising the amino acid
sequence of SEQ ID NO: 6 and one or more immunogenic fragments of
the HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs:
20-54 provided in Table 3 (immunogenic fragments of SEQ ID NO: 6)
and one or more of SEQ ID NOs: 114-172 provided in Table 5
(immunogenic fragments of SEQ ID NO: 10); [0079] (5) an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof and an HBV
polymerase C-terminal domain comprising the amino acid sequence of
SEQ ID NO: 10 or an immunogenic fragment thereof; [0080] (6) one or
more immunogenic fragments of the HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 and one or more
immunogenic fragments of the HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 10. For example,
one or more of SEQ ID NOs: 55-113 provided in Table 4 (immunogenic
fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172
provided in Table 5 (immunogenic fragments of SEQ ID NO: 10);
[0081] (7) an HBV Core protein comprising the amino acid sequence
of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV
polymerase C-terminal domain comprising the amino acid sequence of
SEQ ID NO: 10 or an immunogenic fragment thereof; [0082] (8) one or
more immunogenic fragments of the HBV Core protein comprising the
amino acid sequence of SEQ ID NO: 6, one or more immunogenic
fragments of the HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8, and one or more immunogenic
fragments of the HBV polymerase C-terminal domain comprising the
amino acid sequence of SEQ ID NO: 10. For example, one or more of
SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ
ID NO: 6), one or more of SEQ ID NOs: 55-113 provided in Table 4
(immunogenic fragments of SEQ ID NO: 8), and one or more of SEQ ID
NOs: 114-172 provided in Table 5 (immunogenic fragments of SEQ ID
NO: 10); [0083] (9) An HBV polymerase N-terminal domain comprising
the amino acid sequence of SEQ ID NO: 178 or an immunogenic
fragment thereof, an HBV polymerase C-terminal domain comprising
the amino acid sequence of SEQ ID NO: 179 or an immunogenic
fragment thereof, and an HBV Core protein comprising the amino acid
sequence of SEQ ID NO: 180 or an immunogenic fragment thereof; or
[0084] (10) An HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment
thereof, an HBV polymerase C-terminal domain comprising the amino
acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof,
and an HBV Core protein comprising the amino acid sequence of SEQ
ID NO: 183 or an immunogenic fragment thereof.
[0085] The fusion protein can comprise an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 178 or an
immunogenic fragment thereof, an HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 179 or an
immunogenic fragment thereof, and an HBV Core protein comprising
the amino acid sequence of SEQ ID NO: 180 or an immunogenic
fragment thereof. In some embodiments, the fusion protein comprises
the amino acid sequence of SEQ ID NO: 174.
[0086] The fusion protein can comprise an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 181 or an
immunogenic fragment thereof, an HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 182 or an
immunogenic fragment thereof, and an HBV Core protein comprising
the amino acid sequence of SEQ ID NO: 183 or an immunogenic
fragment thereof. In some embodiments, the fusion protein comprises
the amino acid sequence of SEQ ID NO: 175.
[0087] Also provided herein are fusion proteins comprising a herpes
simplex virus (HSV) glycoprotein (gD) sequence and the disclosed
HBV Core protein, the HBV polymerase N-terminal domain, the HBV
polymerase C-terminal domain, or various combinations thereof.
[0088] The HSV gD is a receptor-binding glycoprotein of HSV. The gD
ectodomain is organized in two structurally and functionally
differentiated regions: the amino-terminus, which includes the
signal sequence and receptor-binding sites; and the
carboxy-terminus, which includes the pro-fusion domain and the
transmembrane domain. gD interacts with the herpesvirus entry
mediator (HVEM) receptor and the nectin receptors. Interaction of
gD with the receptors results in the down-regulation of the HVEM
receptors binding to BTLA or CD160, which are immunoinhibitory
molecules that are expressed on T cells. In some embodiments, the
disclosed fusion proteins comprising gD and the disclosed HBV Core
protein, the HBV polymerase N-terminal domain, the HBV polymerase
C-terminal domain (referred to as "gDCore," "gDPolN" or "gDPolC,"
respectively), or combinations thereof are expected to enhance a
subject's immune response against HBV to a greater extent compared
to the HBV Core and/or polymerase antigens alone (i.e. without
gD).
[0089] Suitable HSV gD proteins for use in the disclosed fusion
proteins include wild-type or mutant gD that retains the ability
to: 1) augment stimulation of a CD8+ T cell response to an antigen;
and/or 2) disrupt an HVEM-BTLA pathway activity.
[0090] The fusion proteins can comprise the HBV Core protein or an
immunogenic fragment thereof, HBV polymerase N-terminal domain or
an immunogenic fragment thereof, HBV polymerase C-terminal domain
or an immunogenic fragment thereof disclosed herein, or any
combination thereof, an N-terminal HSV gD protein sequence, and a
C-terminal HSV gD protein sequence. The HBV Core protein, HBV
polymerase N-terminal domain, and HBV polymerase C-terminal domain
can be those provided in Table 9 or the immunogenic fragments
provided in Tables 3-5. The HBV Core protein, HBV polymerase
N-terminal domain, HBV polymerase C-terminal domain, or immunogenic
fragments thereof can be inserted between the N-terminal HSV gD
protein sequence and the C-terminal HSV gD protein sequence. In
some aspects, the N-terminal HSV gD protein sequence comprises the
amino acid sequence of SEQ ID NO: 12 and the C-terminal HSV gD
protein sequence comprises the amino acid sequence of SEQ ID NO:
13. In some embodiments, the N-terminal HSV gD protein sequence
comprises amino acid residues 26-269 of SEQ ID NO: 12.
[0091] The fusion protein can comprise: [0092] an N-terminal HSV gD
sequence or a variant thereof; [0093] an HBV Core protein
comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof; and [0094] a C-terminal HSV gD
sequence or a variant thereof.
[0095] The immunogenic fragment of the HBV Core protein can
comprise any one of SEQ ID NOs: 20-54, 180, or 183.
[0096] The fusion protein can comprise: [0097] an N-terminal HSV gD
sequence or a variant thereof; [0098] an HBV Core protein
comprising the amino acid sequence of SEQ ID NO: 180 or SEQ ID NO:
183; and [0099] a C-terminal HSV gD sequence or a variant
thereof.
[0100] The fusion protein can comprise: [0101] an N-terminal HSV gD
sequence or a variant thereof; [0102] an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof; and [0103] a C-terminal HSV gD
protein sequence or a variant thereof.
[0104] The immunogenic fragment of the HBV polymerase N-terminal
domain can comprise any one of SEQ ID NOs: 55-113, 178, or 181.
[0105] The fusion protein can comprise: [0106] an N-terminal HSV gD
sequence or a variant thereof; [0107] an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 178 or SEQ
ID NO: 181; and [0108] a C-terminal HSV gD protein sequence or a
variant thereof.
[0109] The fusion protein can comprise: [0110] an N-terminal HSV gD
sequence or a variant thereof; [0111] an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; and [0112] a C-terminal HSV gD
protein sequence or a variant thereof.
[0113] The immunogenic fragment of the HBV polymerase C-terminal
domain can comprise any one of SEQ ID NOs: 114-172, 179, or
182.
[0114] The fusion protein can comprise: [0115] an N-terminal HSV gD
sequence or a variant thereof; [0116] an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 179 or SEQ
ID NO: 182; and [0117] a C-terminal HSV gD protein sequence or a
variant thereof.
[0118] The fusion protein can comprise: [0119] an N-terminal HSV gD
sequence or a variant thereof; [0120] an HBV sequence comprising:
[0121] (1) an HBV Core protein comprising the amino acid sequence
of SEQ ID NO: 6 or an immunogenic fragment thereof and an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof; [0122] (2) one or
more immunogenic fragments of the HBV Core protein comprising the
amino acid sequence of SEQ ID NO: 6 and one or more immunogenic
fragments of the HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8. For example, one or more of
SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ
ID NO: 6) and one or more of SEQ ID NOs: 55-113 provided in Table 4
(immunogenic fragments of SEQ ID NO: 8); [0123] (3) an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof and an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; [0124] (4) one or more immunogenic
fragments of the HBV Core protein comprising the amino acid
sequence of SEQ ID NO: 6 and one or more immunogenic fragments of
the HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 10. For example, one or more of SEQ ID NOs:
20-54 provided in Table 3 (immunogenic fragments of SEQ ID NO: 6)
and one or more of SEQ ID NOs: 114-172 provided in Table 5
(immunogenic fragments of SEQ ID NO: 10); [0125] (5) an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof and an HBV
polymerase C-terminal domain comprising the amino acid sequence of
SEQ ID NO: 10 or an immunogenic fragment thereof; [0126] (6) one or
more immunogenic fragments of the HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 and one or more
immunogenic fragments of the HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 10. For example,
one or more of SEQ ID NOs: 55-113 provided in Table 4 (immunogenic
fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172
provided in Table 5 (immunogenic fragments of SEQ ID NO: 10);
[0127] (7) an HBV Core protein comprising the amino acid sequence
of SEQ ID NO: 6 or an immunogenic fragment thereof, an HBV
polymerase N-terminal domain comprising the amino acid sequence of
SEQ ID NO: 8 or an immunogenic fragment thereof, and an HBV
polymerase C-terminal domain comprising the amino acid sequence of
SEQ ID NO: 10 or an immunogenic fragment thereof; or [0128] (8) one
or more immunogenic fragments of the HBV Core protein comprising
the amino acid sequence of SEQ ID NO: 6, one or more immunogenic
fragments of the HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8, and one or more immunogenic
fragments of the HBV polymerase C-terminal domain comprising the
amino acid sequence of SEQ ID NO: 10. For example, one or more of
SEQ ID NOs: 20-54 provided in Table 3 (immunogenic fragments of SEQ
ID NO: 6), one or more of SEQ ID NOs: 55-113 provided in Table 4
(immunogenic fragments of SEQ ID NO: 8), and one or more of SEQ ID
NOs: 114-172 provided in Table 5 (immunogenic fragments of SEQ ID
NO: 10); [0129] (9) an HBV polymerase N-terminal domain comprising
the amino acid sequence of SEQ ID NO: 178 or an immunogenic
fragment thereof, an HBV polymerase C-terminal domain comprising
the amino acid sequence of SEQ ID NO: 179 or an immunogenic
fragment thereof, and an HBV Core protein comprising the amino acid
sequence of SEQ ID NO: 180 or an immunogenic fragment thereof; or
[0130] (10) an HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 181 or an immunogenic fragment
thereof, an HBV polymerase C-terminal domain comprising the amino
acid sequence of SEQ ID NO: 182 or an immunogenic fragment thereof,
and an HBV Core protein comprising the amino acid sequence of SEQ
ID NO: 183 or an immunogenic fragment thereof. and [0131] a
C-terminal HSV gD protein sequence or a variant thereof.
[0132] In some embodiments, the N-terminal HSV gD sequence can
comprise at least amino acids 1-269 of HSV gD. The N-terminal HSV
gD sequence, for example, can comprise the amino acid sequence of
SEQ ID NO: 12. In some embodiments, the N-terminal HSV gD sequence
comprises amino acid residues 26-269 of SEQ ID NO: 12.
[0133] In some embodiments, the C-terminal HSV gD sequence
comprises the transmembrane domain of the HSV gD. The C-terminal
HSV gD sequence, for example, can comprise the amino acid sequence
of SEQ ID NO: 13.
[0134] The fusion protein can comprise the amino acid sequence of
SEQ ID NO: 14 (corresponding to gDCore) or an immunogenic fragment
thereof. The fusion protein can comprise the amino acid sequence of
SEQ ID NO: 16 (corresponding to gDPolN) or an immunogenic fragment
thereof. The fusion protein can comprise the amino acid sequence of
SEQ ID NO: 18 (corresponding to gDPolC) or an immunogenic fragment
thereof. In some embodiments, the amino acid sequence of any one of
SEQ ID NOs: 14, 16, or 18, or the immunogenic fragment thereof,
does not contain the N-terminal 25 amino acid signal peptide.
[0135] The fusion protein can comprise the amino acid sequence of
SEQ ID NO: 185 (gDHBV2). The fusion protein can comprise the amino
acid sequence of SEQ ID NO: 187 (gDHBV3).
[0136] Nucleic acid molecules encoding any of the disclosed fusion
proteins are also provided. In some embodiments, the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 15
(corresponding to gDCore). In some embodiments, the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 17
(corresponding to gDPolN). In some embodiments, the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 19
(corresponding to gDPolC).
[0137] The nucleic acid molecule can comprise the nucleotide
sequence of SEQ ID NO: 184 (gDHBV2). The nucleic acid molecule can
comprise the nucleotide sequence of SEQ ID NO: 186 (gDHBV3).
[0138] Vectors comprising the nucleic acid molecules encoding the
fusion proteins are also disclosed. Suitable vectors include those
described above including, for example, an adenoviral vector. In
some embodiments, the adenoviral vector is an AdC6 vector. In some
embodiments, the adenoviral vector is an AdC7 vector. The vector
can comprise the nucleotide sequence of SEQ ID NO: 184 (gDHBV2). In
some aspects, the vector is an AdC6 vector that comprises the
nucleotide sequence of SEQ ID NO: 184 (gDHBV2). In some aspects,
the vector is an AdC7 vector that comprises the nucleotide sequence
of SEQ ID NO: 184 (gDHBV2). The vector can comprise the nucleotide
sequence of SEQ ID NO: 186 (gDHBV3). In some aspects, the vector is
an AdC6 vector that comprises the nucleotide sequence of SEQ ID NO:
186 (gDHBV3). In some aspects, the vector is an AdC7 vector that
comprises the nucleotide sequence of SEQ ID NO: 186 (gDHBV3).
[0139] Vaccines comprising any of the disclosed vectors are also
provided. The vaccine can further comprise a pharmaceutically
acceptable carrier or pharmaceutical acceptable excipient as
disclosed above. The vaccine can comprise a vector comprising the
nucleotide sequence of SEQ ID NO: 184 (gDHBV2). In some aspects,
the vaccine comprises an AdC6 vector that comprises the nucleotide
sequence of SEQ ID NO: 184 (gDHBV2). In some aspects, the vaccine
comprises an AdC7 vector that comprises the nucleotide sequence of
SEQ ID NO: 184 (gDHBV2). The vaccine can comprise a vector that
comprises the nucleotide sequence of SEQ ID NO: 186 (gDHBV3). In
some aspects, the vaccine comprises an AdC6 vector that comprises
the nucleotide sequence of SEQ ID NO: 186 (gDHBV3). In some
aspects, the vaccine comprises an AdC7 vector that comprises the
nucleotide sequence of SEQ ID NO: 186 (gDHBV3).
[0140] Provided herein are methods of inducing an immune response
to HBV in a subject, the methods comprising providing to the
subject an effective amount of any of the disclosed fusion
proteins, any of the disclosed nucleic acid molecules, any of the
disclosed vectors, or any of the disclosed vaccines to thereby
induce an immune response to HBV. In some embodiments, the methods
comprise providing to the subject an effective amount of any of the
disclosed fusion proteins to thereby induce an immune response to
HBV. In some embodiments, the methods comprise providing to the
subject an effective amount of any of the disclosed nucleic acid
molecules to thereby induce an immune response to HBV. In some
embodiments, the methods comprise providing to the subject an
effective amount of any of the disclosed vectors to thereby induce
an immune response to HBV. In some embodiments, the methods
comprise providing to the subject an effective amount of any of the
disclosed vaccines to thereby induce an immune response to HBV.
[0141] The methods can comprise providing to the subject an
effective amount of a vaccine comprising an AdC6 vector, wherein
the AdC6 vector comprises a fusion protein comprising the amino
acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an
immunogenic fragment thereof. In some embodiments, the methods
further comprise providing to the subject, subsequent to providing
the vaccine comprising the AdC6 vector, a vaccine comprising an
AdC7 vector comprising a fusion protein comprising the amino acid
sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic
fragment thereof. Such prime-boost methods can comprise: [0142]
Providing to the subject a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 14, or an immunogenic fragment thereof, and subsequently
providing to the subject a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 14, or an immunogenic fragment thereof. In some
embodiments, the amino acid sequence of SEQ ID NO: 14, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide; [0143] Providing to the subject a
vaccine comprising an AdC6 vector comprising a fusion protein
comprising the amino acid sequence of SEQ ID NO: 16, or an
immunogenic fragment thereof, and subsequently providing to the
subject a vaccine comprising an AdC7 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 16, or an
immunogenic fragment thereof. In some embodiments, the amino acid
sequence of SEQ ID NO: 16, or an immunogenic fragment thereof, does
not contain the N-terminal 25 amino acid signal peptide; or [0144]
Providing to the subject a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 18, or an immunogenic fragment thereof, and subsequently
providing to the subject a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 18, or an immunogenic fragment thereof. In some
embodiments, the amino acid sequence of SEQ ID NO: 18, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide.
[0145] The methods can comprise providing to the subject an
effective amount of a vaccine comprising an AdC7 vector, wherein
the AdC7 vector comprises a fusion protein comprising the amino
acid sequence of any one of SEQ ID NOs: 14, 16, or 18, or an
immunogenic fragment thereof. In some embodiments, the methods
further comprise providing to the subject, subsequent to providing
the vaccine comprising the AdC7 vector, a vaccine comprising an
AdC6 vector comprising a fusion protein comprising the amino acid
sequence of any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic
fragment thereof. Such prime-boost methods can comprise: [0146]
Providing to the subject a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 14, or an immunogenic fragment thereof, and subsequently
providing to the subject a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 14, or an immunogenic fragment thereof. In some
embodiments, the amino acid sequence of SEQ ID NO: 14, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide; [0147] Providing to the subject a
vaccine comprising an AdC7 vector comprising a fusion protein
comprising the amino acid sequence of SEQ ID NO: 16, or an
immunogenic fragment thereof, and subsequently providing to the
subject a vaccine comprising an AdC6 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 16, or an
immunogenic fragment thereof. In some embodiments, the amino acid
sequence of SEQ ID NO: 16, or an immunogenic fragment thereof, does
not contain the N-terminal 25 amino acid signal peptide; or [0148]
Providing to the subject a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 18, or an immunogenic fragment thereof, and subsequently
providing to the subject a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 18, or an immunogenic fragment thereof. In some
embodiments, the amino acid sequence of SEQ ID NO: 18, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide.
[0149] The methods can comprise providing to the subject an
effective amount of a vaccine comprising an AdC6 vector, wherein
the AdC6 vector comprises a fusion protein comprising the amino
acid sequence of SEQ ID NO: 185 or 187, or an immunogenic fragment
thereof. In some embodiments, the methods further comprise
providing to the subject, subsequent to providing the vaccine
comprising the AdC6 vector, a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 185 or 187, or an immunogenic fragment thereof. Such
prime-boost methods can comprise: [0150] Providing to the subject a
vaccine comprising an AdC6 vector comprising a fusion protein
comprising the amino acid sequence of SEQ ID NO: 185, or an
immunogenic fragment thereof, and subsequently providing to the
subject a vaccine comprising an AdC7 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 185, or an
immunogenic fragment thereof. In some embodiments, the amino acid
sequence of SEQ ID NO: 185, or an immunogenic fragment thereof,
does not contain the N-terminal 25 amino acid signal peptide; or
[0151] Providing to the subject a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 187, or an immunogenic fragment thereof, and
subsequently providing to the subject a vaccine comprising an AdC7
vector comprising a fusion protein comprising the amino acid
sequence of SEQ ID NO: 187, or an immunogenic fragment thereof. In
some embodiments, the amino acid sequence of SEQ ID NO: 187, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide.
[0152] The methods can comprise providing to the subject an
effective amount of a vaccine comprising an AdC7 vector, wherein
the AdC7 vector comprises a fusion protein comprising the amino
acid sequence of SEQ ID NO: 185 or 187, or an immunogenic fragment
thereof. In some embodiments, the methods further comprise
providing to the subject, subsequent to providing the vaccine
comprising the AdC7 vector, a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 185 or 187, or an immunogenic fragment thereof. Such
prime-boost methods can comprise: [0153] Providing to the subject a
vaccine comprising an AdC7 vector comprising a fusion protein
comprising the amino acid sequence of SEQ ID NO: 185, or an
immunogenic fragment thereof, and subsequently providing to the
subject a vaccine comprising an AdC6 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 185, or an
immunogenic fragment thereof. In some embodiments, the amino acid
sequence of SEQ ID NO: 185, or an immunogenic fragment thereof,
does not contain the N-terminal 25 amino acid signal peptide; or
[0154] Providing to the subject a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 187, or an immunogenic fragment thereof, and
subsequently providing to the subject a vaccine comprising an AdC6
vector comprising a fusion protein comprising the amino acid
sequence of SEQ ID NO: 187, or an immunogenic fragment thereof. In
some embodiments, the amino acid sequence of SEQ ID NO: 187, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide.
[0155] The immune response induced by the disclosed methods
include, but is not limited to, T cell responses, B cell responses,
or both (i.e. cellular and/or humoral immune responses). The immune
response can be a primary immune response or a secondary immune
response. The disclosed methods can induce a subject's immune
response against HBV to a greater extent compared to the HBV Core
or polymerase antigens alone (i.e. without gD).
[0156] The disclosed methods can be used for both therapeutic
treatment and prophylactic or preventative measures and can reduce
the severity and/or frequency of symptoms, eliminate symptoms
and/or the underlying cause of the symptoms, reduce the frequency
or likelihood of symptoms and/or their underlying cause, and
improve or remediate damage caused, directly or indirectly, by HBV.
Treatment also includes prolonging survival as compared to the
expected survival of a subject not receiving treatment. Subjects to
be treated include those that have HBV as well as those prone to
have HBV or those in which HBV is to be prevented.
[0157] The amount of the disclosed fusion proteins, nucleic acid
molecules, vectors, or vaccines needed to thereby induce an immune
response to HBV (e.g. a "effective amount") may vary according to
factors such as the disease state, age, sex, and weight of the
subject, and the ability of the fusion proteins, nucleic acid
molecules, vectors, or vaccines to cause a desired response in the
subject. Exemplary indicators of an effective amount include, for
example, improved well-being of the subject and reduction,
elimination, or prevention of HBV symptoms.
[0158] Also provided is the use of any of the disclosed fusion
proteins, nucleic acid molecules, vectors, or vaccines in the
manufacture of a medicament for inducing an immune response to HBV
in a subject.
[0159] The disclosed fusion proteins, nucleic acid molecules,
vectors, or vaccines for use in inducing an immune response to HBV
in a subject is also provided.
Examples
[0160] The following examples are provided to further describe some
of the embodiments disclosed herein. The examples are intended to
illustrate, not to limit, the disclosed embodiments.
Generation of an Epitope-Optimized Core Sequence
[0161] Hepatitis B virus (HBV) can be grouped into several
genotypes, based on phylogenic clustering. To assist in the
development of an antigen insert for a multi-genotype HBV vaccine
for patients with chronic infections, a preliminary bioinformatics
evaluation of the genes encoding the HBV Core and HBV polymerase
across genotypes A, B, C and D was conducted.
[0162] The Core amino acid sequences from the four major HBV clades
were downloaded as aligned ClustalW sequences from Hepatitis B
Virus database (HBVdb) (release version 45.0; last updated on Aug.
2, 2018). The amino acid sequences represented thousands of HBV
genomes inputted from users across Europe, as summarized in the
following table.
TABLE-US-00001 TABLE 1 Number of unique Core genomes analyzed
Genotype HBV Gene Unique Genomes Analyzed HBV genotype A Core 1,482
HBV genotype B 2,800 HBV genotype C 2,768 HBV genotype D 1,579
[0163] "Consensus" Core sequences were first identified for each
genotype using the Shannon Entropy tool hosted by the Los Alamos
National Laboratory
(www.hiv.lanl.gov/content/sequence/ENTROPY/entropy), which
calculated the variation and frequency at each amino acid position.
These calculations were repeated for each genotype, generating four
"consensus" Core sequences, one for each genotype analyzed (SEQ ID
NOs: 1-4):
TABLE-US-00002 Genotype A Consensus (SEQ ID NO: 1)
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSP
HHTALRQAILCWGELMTLATWVGNNLeDPASRDLVVNYVNTNMGLKIRQL
LWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVR
RRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC- Genotype B Consensus (SEQ ID
NO: 2) MDIDpYKEFGASvELLSFLPSDFFPSiRDLLDTAsALYREALESPEHCSP
HHTALRQAIlCWGELMNLATWVGSNLeDPASRELVVsYVNVNMGLKiRQL
LWFHISCLTFGRETVLEYLVSFGVWIRTPpAYRPpNAPILSTLPETTVVR
RRGRSPRRRTPSPRRRRSQSPRRRRSQSREsQC- Genotype C Consensus (SEQ ID NO:
3) MDIDpYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSP
HHTALRQAILCWGELMNLATWVGSNLEDPASRELVVsYVNVNMGLKiRQl
LWFHISCLTFGRETVLEYLVSFGVWIRTPpAYRPPNAPILSTLPETTVVR
RRGRSPRRRTPSPRRRRSQSPRRRSQSRESQC - Genotype D Consensus (SEQ ID NO:
4) MDIDPYKEFGAtVELLSFLPsDFFPSVRDLLDTASALYReALESPEHCSP
HHTALRQAILCWGeLMtLATWVGgNLEDPaSRDLVVSYVNTNmGLKFRQL
LWFHISCLTFGReTViEYLVSFGVWIRTPpAYRPPNAPILSTLPETTVvR
RRGRSPRRRTPSPRRRTSQSPRRRRSQSRESQC- (Bold, underlined residues
represent amino acids having less than 90% frequency).
[0164] The above "consensus" Core sequences were combined to
generate an epitope-optimized Core sequence. Conserved amino acids
were identified at each amino acid residue of the Core protein from
each genotype (A, B, C and D) and the frequency and variation
within a given sample of genotype genomes was determined. To select
amino acids at sites of variation, each variation was tested using
epitope prediction algorithms across multiple HLA types and the
most immunogenic sequence was selected. Specifically: [0165] (1)
Each residue across the four genotypes that was identical were
maintained. The genome weighted frequency was also calculated to
inform the variability with spacer added, where applicable, to
align the sequences for diversity. [0166] (2) Residues that were
not identical across the four genotypes were identified and the
amino acid diversity was recorded (see Table 2). The initial Core
sequence (SEQ ID NO: 5) is provided below, with the residues that
were not identical across the four genotypes labeled as
X.sub.1-X.sub.11 and the residues having less than 90% frequency in
bold, underlined font:
TABLE-US-00003 [0166] MDIDPYKEFGA VELLSFLPSDFFPS
DLLDTASALYREALESPEHCS PHHTALRQAILCWGELM LATWVG NLeDPASR LVV YVN
NMGLK RQLLWFHISCLTFGRETV EYLVSFGVWIRTPPAYRPPNAPI LSTLPETTVVRRR
GRSPRRRTPSPRRRRSQSPRRRRSQSRESQC
TABLE-US-00004 TABLE 2 Residues that were not identical across the
four genotypes Residue # X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5
X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10 X.sub.11 Genotype A T V T
N D N T I L D R Consensus A-Consensus 95.4% 98.2% 96.0% 94.0% 99.5%
98.9% 98.3% 98.8% 98.2% 95.8% 99.5% Frequency Genotype B S i N S E
s V i L -- -- Consensus B-Consensus 97.1% 89.7% 93.5% 90.1% 91.5%
75.2% 93.6% 80.1% 99.4% -- -- Frequency Genotype C S I N S E s V i
L -- -- Consensus C-Consensus 99.4% 90.3% 97.3% 96.3% 97.0% 83.7%
97.8% 78.7% 98.8% -- -- Frequency Genotype D t V t g D S T F i --
-- Consensus D-Consensus 75.7% 97.0% 87.5% 58.7% 98.4% 93.7% 96.5%
99.1% 77.8% -- -- Frequency
[0167] (3) To determine the final amino acid at these positions,
epitope prediction algorithms were used to select the appropriate
amino acid. For amino acids that showed variability between the
genotypes, amino acids that were present in 3 of the genotypes were
selected or an MHC class I epitope prediction software was used to
select the most immunogenic amino acids. This approach maximized
the potential immunogenicity across the greatest number of HLA
types. The epitope-optimized Core sequence across all genotypes and
within genotypes is shown below (SEQ ID NO: 6):
TABLE-US-00005 [0167]
DIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPH
HTALRQAILCWGELMTLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLL
WFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRR
RDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC
[0168] The average variation at each site across all genomes,
weighted by the number of clade-specific genomes analyzed, was
calculated and showed areas and residues of higher and greater
conservation. FIG. 1.
Generation of Epitope-Optimized Polymerase Sequences
[0169] An epitope-optimized polymerase sequence was generated from
the four major HBV clades as discussed above for the Core sequence.
Because the polymerase is long, two fragments--an N-terminal
fragment (from which a highly variable segment between the
genotypes was removed) and a C-terminal fragment--were generated.
Both fragments are approximately 300 amino acids in length. The
epitope-optimized polymerase amino acid sequences are shown below
and in Table 9:
TABLE-US-00006 Epitope-optimized HBV polymerase N-terminal amino
acid sequence (SEQ ID NO: 8):
PLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIP
WTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQEDIVDRCKQFVGPLTV
NEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTL
WKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLT
HLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVV
DFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLH PAAMP
Epitope-optimized HBV polymerase C-terminal amino acid sequence
(SEQ ID NO: 10): HLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLYVSLLLLYK
TFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSWGSLPQDHIIQKIKECF
RKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTF
SPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTF
VAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAAN
WILRGTSFVYVPSALNPADDPSRGRLGLSRPLLRLPFRPTTGRTSLYAVS PSV
Generation of AdC6 and AdC7 Vectors Expressing the
Epitope-Optimized Core and Polymerase Sequences
[0170] The genes encoding the epitope-optimized Core or polymerase
amino acid sequences were cloned into transfer vectors that
contained the herpes simplex virus (HSV) glycoprotein D (gD)
sequence under the control of the CMV promoter. The genes were then
cloned into the E1-deleted, E3 ORF 3, 4, 5, 6, and 7-deleted
replication deficient adenoviral vector (as described in
PCT/US2017/043315) to generate the following vectors: [0171] AdC6
containing the epitope-optimized Core sequence fused to gD
(AdC6-gDCore); [0172] AdC6 containing the epitope-optimized
polymerase N-terminal sequence fused to gD (AdC6-gDPolN); [0173]
AdC6 containing the epitope-optimized polymerase C-terminal
sequence fused to gD (AdC6-gDPolC); [0174] AdC7 containing the
epitope-optimized Core sequence fused to gD (AdC7-gDCore); [0175]
AdC7 containing the epitope-optimized polymerase N-terminal
sequence fused to gD (AdC7-gDPolN); and [0176] AdC7 containing the
epitope-optimized polymerase C-terminal sequence fused to gD
(AdC7-gDPolC).
[0177] Correct clones were identified by restriction enzyme digest
and the cloning sites were sequenced. Vectors were rescued and
expanded in HEK 293 cells, purified by cesium chloride (CsCl)
gradient centrifugation, and the vector concentration (vp) was
determined by spectrophotometry. Vectors were titrated for
infectious units upon their expansion in serial dilutions in HEK
293 cells, followed by isolation and reverse transcription of RNA
and a nested hexon-specific PCR reaction. Genetic integrity of the
vectors was determined by restriction enzyme digest followed by gel
electrophoresis of purified viral DNA. Protein expression was
determined by Western blotting using gD-specific antibodies.
Genetic stability was determined by serial passages (12-15) of the
vectors in HEK 293 cells followed by restriction enzyme digest of
purified viral DNA and gel electrophoresis.
Testing of Immunogenicity of Vaccines in Mice
[0178] C57Bl/6, BALB/c, and HLA-A2 tg mice (n=5 per group) were
injected with various concentrations of each of the above vectors.
Naive mice served as controls. Mice were bled at different times
after the injection and frequencies of insert-specific CD8+ and
CD4+ T cells were determined by intracellular cytokine staining
(ICS) for IFN-.gamma.. Two months after the first injection,
AdC6-immune mice were boosted with the heterologous vector (AdC7)
expressing the same insert. Frequencies of HBV-specific T cells
were tested again. Results after priming are shown in FIGS. 2A-2F
and FIG. 3A (C57Bl/6 mice), FIG. 3B (BALB/c mice) and FIG. 3C
(HLA-A2 mice). Results after the boost are shown in FIGS. 4A-4C and
5A-5B.
[0179] C57Bl/6 mice showed a very robust CD8+ T cell response to
the epitope-optimized polymerase N-terminal sequence and lower
responses to the epitope-optimized polymerase C-terminal sequence
and the epitope-optimized Core sequence, while CD4+ responses were
better against the epitope-optimized Core sequence and the
epitope-optimized polymerase C-terminal sequence (FIG. 2A-FIG. 2F).
Epitope mapping in C57Bl/6 mice showed higher and broader responses
to PolN than PolC (FIG. 3A). Within PolN a total of 14 peptides
were recognized by CD8+ T cells while within PolC only two adjacent
peptides, which most likely reflect one epitope, were recognized.
CD4+ T cells failed to respond to PolN or PolC. This pattern was
largely mirrored in BALB/c mice, where CD8+ T cell responses were
highest against PolN with recognition of 12 peptides followed by
PolC with recognition of 4 peptides (FIG. 3B). Responses to Core
were low but surprisingly broad with recognition of 10 peptides
(FIG. 3B). BALB/c CD4+ T cells responded best to Core with
recognition of 15 peptides with lower recognition of PolC (4
peptides) or PolN (2 peptides). CD8+ T cell responses were also
tested in HLA-A2 tg mice where PolN again triggered the highest
response involving 12 peptides (FIG. 3C). The response to PolC was
lower but broader (16 peptides) while only one peptide of Core was
detected (FIG. 3C). The sequences of the peptides tested in the
priming experiments are provided in Table 3 (Core peptides), Table
4 (PolN peptides), and Table 5 (PolC peptides). The peptide
composition of the peptide pools from the priming experiments are
provided in Tables 6-8. Overall these data show that the inserts
elicited detectable T cell responses that in most cases were
directed against multiple epitopes within each sequence.
TABLE-US-00007 TABLE 3 Epitope-Optimized Core Peptides SEQ ID
Peptide Amino Acid Sequence NO: Prime 1 DIDPYKEFGATVELL 20 CD8
(B/c) 2 KEFGATVELLSFLPS 21 CD8 (B/c) 3 TVELLSFLPSDFFPS 22 CD8 (B/c)
4 SFLPSDFFPSIRDLL 23 CD8 (B/c) 5 DFFPSIRDLLDTASA 24 CD8 (B/c) 6
IRDLLDTASALYREA 25 7 DTASALYREALESPE 26 CD4 (B/c) 8 LYREALESPEHCSPH
27 CD4 (B1/6); CD4 (B/c) 9 LESPEHCSPHHTALR 28 CD4 (B/c) 10
HCSPHHTALRQAILC 29 CD4 (B/c) 11 HTALRQAILCWGELM 30 CD4 (B/c) 12
QAILCWGELMTLATW 31 13 WGELMTLATWVGSNL 32 CD8 (B/c); CD4 (B/c); CD8
(HLA) 14 TLATWVGSNLEDPAS 33 CD8 (B/c); CD4 (B/c) 15 VGSNLEDPASRELVV
34 CD8 (B/c); CD4 (B/c) 16 EDPASRELVVSYVNV 35 CD8 (B/c); CD4 (B/c)
17 RELVVSYVNVNMGLK 36 CD8 (B/c); CD4 (B/c) 18 SYVNVNMGLKIRQLL 37 19
NMGLKIRQLLWFHIS 38 CD4 (B/c) 20 IRQLLWFHISCLTFG 39 CD4 (B/c) 21
WFHISCLTFGRETVI 40 CD4 (B/c) 22 CLTFGRETVIEYLVS 41 CD4 (B/c) 23
RETVIEYLVSFGVWI 42 CD4 (B/c) 24 EYLVSFGVWIRTPPA 43 25
FGVWIRTPPAYRPPN 44 26 RTPPAYRPPNAPILS 45 CD8 (B1/6) 27
YRPPNAPILSTLPET 46 28 APILSTLPETTVVRR 47 CD4 (B1/6) 29
TLPETTVVRRRDRGR 48 CD8 (B1/6) 30 TVVRRRDRGRSPRRR 49 31
RDRGRSPRRRTPSPR 50 32 SPRRRTPSPRRRRSQ 51 33 TPSPRRRRSQSPRRR 52 34
RRRSQSPRRRRRSQSR 53 35 SPRRRRRSQSRESQC 54 B/c = BALB/c; B1/6 =
C57B1/6; HLA = HLA-A2
TABLE-US-00008 TABLE 4 Epitope-Optimized PolN Peptides SEQ ID
Peptide Amino Acid Sequence NO: Prime 1 PLSYQHFRKLLLLDE 55 2
HFRKLLLLDEEAGPL 56 3 LLLDEEAGPLEEELP 57 4 EAGPLEEELPRLADE 58 5
EEELPRLADEGLNRR 59 6 RLADEGLNRRVAEDL 60 7 GLNRRVAEDLNLGNL 61 8
VAEDLNLGNLNVSIP 62 9 NLGNLNVSIPWTHKV 63 10 NVSIPWTHKVGNFTG 64 CD4
(B/c) 11 WTHKVGNFTGLYSST 65 12 GNFTGLYSSTVPVFN 66 13
LYSSTVPVFNPEWQT 67 14 VPVFNPEWQTPSFPK 68 CD4 (B/c) 15
PEWQTPSFPKIHKLQE 69 16 PSFPKIHKLQEDIVDR 70 17 IHKLQEDIVDRCKQFV 71
18 EDIVDRCKQFVGPLTV 72 19 RCKQFVGPLTVNEKRR 73 20 VGPLTVNEKRRLKLIM
74 21 VNEKRRLKLIMPARFY 75 22 RLKLIMPARFYPNVTK 76 23
MPARFYPNVTKYLPLD 77 24 YPNVTKYLPLDKGIKP 78 25 KYLPLDKGIKPYYPEH 79
26 DKGIKPYYPEHAVNHY 80 27 PYYPEHAVNHYFQTRH 81 28 HAVNHYFQTRHYLHTL
82 29 YFQTRHYLHTLWKAGI 83 30 HYLHTLWKAGILYKRE 84 31
LWKAGILYKRETTRSA 85 32 ILYKRETTRSASFCGS 86 33 ETTRSASFCGSPYSWE 87
34 ASFCGSPYSWEQELQH 88 CD8 (B1/6); CD8 (B/c); CD8 (HLA) 35
SPYSWEQELQHGSCWW 89 CD8 (B1/6); CD8 (B/c); CD8 (HLA) 36
EQELQHGSCWWLQFRN 90 37 HGSCWWLQFRNSKPCS 91 CD8 (B1/6); CD8 (B/c);
CD8 (HLA) 38 WLQFRNSKPCSEYCLT 92 CD8 (B1/6); CD8 (B/c); CD8 (HLA)
39 NSKPCSEYCLTHLVNL 93 CD8 (B1/6) 40 SEYCLTHLVNLLEDWG 94 CD8
(B1/6); CD8 (B/c); CD8 (HLA) 41 THLVNLLEDWGPCDEH 95 42
LLEDWGPCDEHGEHHI 96 43 GPCDEHGEHHIRIPRT 97 44 HGEHHIRIPRTPARVT 98
45 IRIPRTPARVTGGVFL 99 46 TPARVTGGVFLVDKNP 100 47 TGGVFLVDKNPHNTAE
101 48 LVDKNPHNTAESRLVV 102 49 PHNTAESRLVVDFSQF 103 50
ESRLVVDFSQFSRGIT 104 CD8 (B1/6); CD8 (B/c)' CD8 (HLA) 51
VDFSQFSRGITRVSWP 105 CD8 (B1/6); CD8 (B/c); CD8 (HLA) 52
FSRGITRVSWPKFAVP 106 53 TRVSWPKFAVPNLQSL 107 CD8 (B1/6); CD8 (B/c);
CD8 (HLA) 54 PKFAVPNLQSLTNLLS 108 CD8 (B1/6); CD8 (B/c); CD8 (HLA)
55 PNLQSLTNLLSSNLSW 109 CD8 (B1/6) 56 LTNLLSSNLSWLSLDV 110 CD8
(B1/6); CD8 (B/c); CD8 (HLA) 57 SSNLSWLSLDVSAAFY 111 58
WLSLDVSAAFYHIPLH 112 CD8 (B1/6); CD8 (B/c); CD8 (HLA) 59
VSAAFYHIPLHPAAMP 113 CD8 (B1/6); CD8 (B/c); CD8 (HLA) B/c = BALB/c;
B1/6 = C57B1/6; HLA = HLA-A2
TABLE-US-00009 TABLE 5 Epitope-Optimized PolC Peptides SEQ Prime
Amino Acid ID Peptide Sequence NO: 1 HLLVGSSGLSRYVAR 114 2
SSGLSRYVARLSSNSR 115 3 RYVARLSSNSRIINHQ 116 4 LSSNSRIINHQHGTMQ 117
5 RIINHQHGTMQNLHDS 118 6 QHGTMQNLHDSCSRNL 119 7 QNLHDSCSRNLYVSLL
120 8 SCSRNLYVSLLLLYKT 121 9 LYVSLLLLYKTFGRKL 122 10
LLLYKTFGRKLHLYSH 123 CD8 (HLA) 11 TFGRKLHLYSHPIILK 124 12
LHLYSHPIILKTKRWG 125 CD8 (B/c); CD8 (HLA) 13 HPIILKTKRWGYSLNF 126
CD8 (HLA) 14 KTKRWGYSLNFMGYVI 127 15 GYSLNFMGYVIGSWGS 128 CD8 (HLA)
16 FMGYVIGSWGSLPQDH 129 17 IGSWGSLPQDHIIQKI 130 18 SLPQDHIIQKIKECFR
131 19 HIIQKIKECFRKLPVN 132 20 IKECFRKLPVNRPIDW 133 21
RKLPVNRPIDWKVCQR 134 22 NRPIDWKVCQRIVGLL 135 23 WKVCQRIVGLLGFAAP
136 24 RIVGLLGFAAPFTQCG 137 25 LGFAAPFTQCGYPALM 138 26
PFTQCGYPALMPLYAC 139 CD8 (HLA) 27 GYPALMPLYACIQSKQ 140 28
MPLYACIQSKQAFTFS 141 CD8 (B/c); CD8 (HLA) 29 CIQSKQAFTFSPTYKA 142
CD8 (HLA) 30 QAFTFSPTYKAFLSKQ 143 31 SPTYKAFLSKQYLNLY 144 CD8
(B1/6); CD8 (HLA) 32 AFLSKQYLNLYPVARQ 145 CD8 (B1/6) 33
QYLNLYPVARQRPGLC 146 34 YPVARQRPGLCQVFAD 147 CD8 (HLA) 35
QRPGLCQVFADATPTG 148 CD4 (B/c) 36 CQVFADATPTGWGLAM 149 CD8 (B/c);
CD8 (HLA) 37 DATPTGWGLAMGHQRM 150 CD8 (HLA) 38 GWGLAMGHQRMRGTFV 151
39 MGHQRMRGTFVAPLPI 152 CD4 (B/c); CD8 (HLA) 40 MRGTFVAPLPIHTAEL
153 41 VAPLPIHTAELLAACF 154 42 IHTAELLAACFARSRS 155 CD8 (HLA) 43
LLAACFARSRSGAKIL 156 44 FARSRSGAKILGTDNS 157 CD8 (B/c); CD8 (HLA)
45 SGAKILGTDNSVVLSR 158 CD8 (HLA) 46 LGTDNSVVLSRKYTSF 159 47
SVVLSRKYTSFPWLLG 160 48 RKYTSFPWLLGCAANW 161 CD8 (HLA) 49
FPWLLGCAANWILRGT 162 50 GCAANWILRGTSFVYV 163 51 WILRGTSFVYVPSALN
164 CD4 (B/c) 52 TSFVYVPSALNPADDP 165 53 VPSALNPADDPSRGRL 166 54
NPADDPSRGRLGLSRP 167 55 PSRGRLGLSRPLLRLP 168 CD4 (B/c) 56
LGLSRPLLRLPFRPTT 169 57 PLLRLPFRPTTGRTSL 170 58 PFRPTTGRTSLYAVSP
171 59 TGRTSLYAVSPSV 172 B/c = BALB/c; B1/6 = C57B1/6; HLA =
HLA-A2
TABLE-US-00010 TABLE 6 Epitope-Optimized Core Pool Core Matrix A B
C D E F G 1 2 3 4 5 6 H 7 8 9 10 11 12 I 13 14 15 16 17 18 J 19 20
21 22 23 24 K 25 26 27 28 29 30 L 31 32 33 34 35
TABLE-US-00011 TABLE 7 Epitope-Optimized PolN Pool Pol N Matrix A B
C D E F G H I 1 2 3 4 5 6 7 8 J 9 10 11 12 13 14 15 16 K 17 18 19
20 21 22 23 24 L 25 26 27 28 29 30 31 32 M 33 34 35 36 37 38 39 40
N 41 42 43 44 45 46 47 48 O 49 50 51 52 53 54 55 56 P 57 58 59
TABLE-US-00012 TABLE 8 Epitope-Optimized PolC Pool Pol C Matrix A B
C D E F G H I 1 2 3 4 5 6 7 8 J 9 10 11 12 13 14 15 16 K 17 18 19
20 21 22 23 24 L 25 26 27 28 29 30 31 32 M 33 34 35 36 37 38 39 40
N 41 42 43 44 45 46 47 48 O 49 50 51 52 53 54 55 56 P 57 58
[0180] After the boost, which was tested in C57Bl/6, BALB/c and
HLA-A2 tg mice, increases in responses were mainly seen for inserts
and at vector doses that upon priming induced suboptimal responses,
i.e., for Core tested at the 1.times.10.sup.9 vp vector dose (FIG.
4A-4C). Although booster immunization failed to increase the
response to PolN or PolC when vectors were injected at high doses,
the boost nevertheless broadened the T cell responses (FIG.
5A-5C)
Immunogenicity Summary
[0181] The above results illustrate that: [0182] The vaccines are
immunogenic: PolN>PolC>Core for CD8+ T cells;
Core>PolC>PolN for CD4+ T cell responses; [0183] Immune
responses can be boosted by a heterologous vaccine carrier; [0184]
Immune responses are broad; and [0185] The breadth of the T cell
responses increases after the boost.
Effect of Vaccination on HBV Titers Low Dose AAV-1.3HBV
Challenge
[0186] A group of 3 mice were challenged with 1.times.10.sup.10,
1.times.10.sup.11 or 1.5.times.10.sup.11 vg of AAV-1.3HBV and were
vaccinated with AdC6-gDPolN 8 weeks later. Viral titers were tested
8 weeks after vaccination and compared to pre-vaccination titers.
FIG. 6 shows viral changes from baseline for each treatment
group.
Epitope Shifting
[0187] CD8+ T cells to HBV antigens become exhausted during chronic
HBV infections. Progression towards exhaustion is more rapid and
pronounced for CD8+ T cells to dominant, as compared to
subdominant, epitopes. The underlying reason is that exhaustion is
driven by overwhelming antigen-driven stimulation through the T
cell receptor; dominant epitopes are presented at higher levels on
MHC class I antigens expressed by antigen presenting cells than
subdominant epitopes with lower avidity to their restricting
elements. Typical vaccine approaches primarily induce immune
responses to dominant epitopes. Therapeutic vaccines should take
into account loss of T cells to dominant epitopes during chronic
virus infections and should be designed to favor expansion of CD8+
T cells to subdominant epitopes, which have a higher likelihood of
resisting disease-driven exhaustion, translating to superior
disease control.
[0188] The epitope profile in naive mice immunized with an
adenovirus vector comprising a nucleic acid sequence encoding the
HBV polymerase N-terminal domain (PolN) fused to the herpes simplex
virus glycoprotein D ("AdC6-gDPolN", wherein the amino acid
sequence of gDPolN is SEQ ID NO: 16) was determined. Responses in
mice that had not been pre-treated with the AAV8-1.3HBV vector were
compared to those obtained in mice infected with an AAV8 vector
expressing the 1.3HBV genome prior to vaccination with the
AdC6-gDPolN. The AAV8-1.3HBV vector induced high titers of HBV in
serum, which could drive CD8+ T cell exhaustion.
[0189] In the first series of experiments a peptide pool matrix was
used to identify epitopes in mice vaccinated with the AdC6-gDPolN
vector, but not challenged with an AAV-1.3HBV vector. A number of
regions in these naive mice were identified that elicited potent
responses (e.g. greater than 1% IFN-.gamma. production CD8+CD44+ T
cells. FIG. 7A and FIG. 8A. In the second experiment, mice were
challenged with 1.times.10.sup.10 virus genomes (vg) of the
AAV-1.3HBV vector, were vaccinated 4 weeks later with an AdC6
vector expressing the same HBV polymerase sequence (gDPolN) as in
the initial experiment in non-challenged mice, and ten weeks
thereafter the HBV PolN-specific CD8+ T cell epitope profile was
determined using peptide pool matrices on splenocytes from the mice
that had been challenged prior to vaccination. FIG. 7B and FIG. 8B.
The experiment was repeated using more stringent conditions by
challenging mice with a 1.5.times.10.sup.11 vg dose of the
AAV8-1.3HBV vector. Mice were again vaccinated 4 weeks later and
were tested approximately 10 weeks after vaccination for CD8+ T
cell responses to the peptide pool matrices. FIG. 7C and FIG. 8C.
In both experiments, compared to the results obtained from
unvaccinated mice, a shift was observed in the epitope profile in
AAV8-1.3HBV infected mice, which at the time of vaccination had
high viral loads between 10.sup.7-10.sup.9 vg per ml of serum. The
effect was more pronounced in mice that had been challenged with a
high dose of the AAV8-1.3HBV vector. In both experiments a
reduction in responses were observed. Furthermore, especially in
mice challenged with the high dose of AAV8-1.3HBV, the results
showed a loss of CD8+ T cells to many of the epitopes that showed
immunodominance in uninfected vaccinated mice (e.g. within region
represented by peptides 50 to 59, FIG. 8), a better preservation of
epitopes that were subdominant (such as those within the region
represented by peptides 2 to 8) as well as new epitopes, such as in
the region presented by peptides 10 to 29. These data confirm a
shift from recognition of dominant to recognition of subdominant
epitopes.
[0190] Based on these data, a new HBV polymerase N-terminal domain
insert (HBV PolN v2) was generated (SEQ ID NO: 173):
TABLE-US-00013 HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPK
IHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKP
YYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQH
GSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT
[0191] This insert induced CD8+ T cell responses mainly to
subdominant epitopes to which responses remain intact in mice with
high HBV viral loads.
Immunogenicity and Efficacy of gDCore, gDPolN, and gDPolC
Vaccines
[0192] The immunogenicity and efficacy the AdC6-gDCore,
AdC6-gDPolN, AdC6-gDPolC, AdC7-gDCore, AdC7-gDPolN, and AdC7-gDPolC
vaccines in an AAV8-HBV mouse model were analyzed.
Methods--Immunogenicity
[0193] C57Bl/6 mice (n=5 per group) were injected with various
doses of: AdC6-gDCore (gDCore nucleic acid sequence corresponding
to SEQ ID NO: 15); AdC6-gDPolN (gDPolN nucleic acid sequence
corresponding to SEQ ID NO: 17); or AdC6-gDPolC (gDPolC nucleic
acid sequence corresponding to SEQ ID NO: 19). Two months after the
first injection, AdC6 vector-immunized mice were boosted with AdC7
vectors containing the same insert (e.g. AdC7-gDCore, AdC7-gDPolN,
or AdC7-gDPolC). Mice were bled at 14 days and 56 days after the
injection and T cell frequencies to the various HBV inserts were
analyzed by intracellular cytokine staining (ICS) for interferon
(IFN)-.gamma. upon stimulation of cells with overlapping peptides
representing the HBV sequences. Control cells were cultured without
peptides. Frequencies and phenotype of CD8+ T cells to one
immunodominant epitope within PolN were tested for by staining with
an MHC I tetramer. The breadth and specificity of CD8+ T cell
responses to individual peptides within a target sequence was
performed via epitope mapping of splenocytes (CD8+ T cells tested
by ICS for IFN-.gamma.).
[0194] To assess CD8+ T cells in the liver, C57Bl/6 mice (n=8 per
group) received intravenous administration of 1.times.10.sup.10
viral genomes (vg) of AAV8-1.3HBV, 1.times.10.sup.11 vg of
AAV8-1.3HBV, or nothing via their tail vein, and 4 weeks later
received a single IM injection of 5.times.10.sup.9 viral particles
(vp) of AdC6-gDPolN. Eight weeks after the IM injection, mice were
sacrificed, livers were removed, and lymphocytes were isolated and
stained with T cell markers and a tetramer recognizing the T cell
receptor to an immunodominant epitope present in the PolN
sequence.
[0195] In a separate experiment, three groups of C57Bl/6 mice (n=4
per group) received a single IM injection of 5.times.10.sup.9 vp of
AdC6-gDPolN at four weeks (-) or received either intravenous
administration of 1.times.10.sup.11 viral genomes (vg) of
AAV8-1.3HBV via their tail vein with or without a single IM
injection of 5.times.10.sup.9 vp of AdC6-gDPolN four weeks later.
Approximately 2 months after administration of AAV8-1.3HBV, mice
were sacrificed, livers were removed and liver slices were prepared
from each of the three groups, stained with hematoxylin and eosin
and evaluated for lymphocytic infiltrates. From the same
experiment, cells were stained with a specific tetramer and
fluorochrome labeled antibodies to T-bet (clone 4B10, BV785 stain)
or antibodies to PD-1 (clone 29F.1A12, BF605 stain), TIM-3 (clone
RMT3-23, Pe/Cy7 stain), CTLA-4 (clone UC10-4B9, PE stain), or LAG-3
(clone C9B7W, BV650 stain). Cells were analyzed by flow cytometry
and gated on CD44+CD8 tetramer positive cells, which were then
gated on the markers. Percent marker positive cells were identified
from histograms in comparison to naive T cells.
Methods--Efficacy
[0196] AAV8-1.3HBV Vector Studies--To assess the impact of
AdC6-gDPolN on chronic HBV virus exposure, C57Bl/6 mice (n=8 per
group) were challenged intravenously via their tail vein with
1.times.10.sup.10 vg of AAV8-1.3HBV and four weeks later immunized
with a single IM injection of 5.times.10.sup.9 vp of AdC6-gDPolN.
HBV DNA viral titers were evaluated by qPCR; pre- and
post-vaccination changes from baseline (log.sub.10 copies/mL) were
reported. Viral genome copy numbers were assessed at four, six,
eight, ten, and twelve weeks after AAV8 challenge. Viral dynamics
were assessed by PCR over time and the change in log 10 in HBV
copies per mL were assessed. The number of mice showing a one, two
or three log reductions at different points after treatment was
assessed.
[0197] Impact of chronic HBV virus exposure on CD8+ T cell antigen
recognition over time--The effect of AAV8-1.3HBV on vaccine-induced
hepatic CD8+ T cells was assessed. The epitope profile in
splenocytes of naive mice immunized with a single IM injection of
5.times.10.sup.9 vp of AdC6-gDPolN was determined 4 weeks after
vaccination. Mice challenged with 1.times.10.sup.10 and
1.5.times.10.sup.11 vg of AAV8-1.3HBV and subsequently vaccinated
with 5.times.10.sup.9 vp of AdC6-gDPolN 4 weeks later had CD8+ T
cell epitope profiles in splenocytes performed 10 weeks after
vaccination (14 weeks after AAV injection). Epitope profiles
between AAV-naive and AAV-treated vaccinated animals were compared.
PolN-specific CD8+ T cells from liver were analyzed for
differentiation markers.
Results
[0198] Immunogenicity--Vaccination induced robust and sustained
CD8+ T cell responses to PolN (median frequencies over all
circulating CD8+ T cells: 6.0%) and lower responses to PolC and
core (median frequencies: 1.0% & 0.4%, respectively; FIG. 9A
and FIG. 9B). Boosting at 8 weeks increased responses to all
regions with significant changes being observed for core (p=0.007)
(FIG. 9C). FIGS. 9A-9C show % CD8+ T cells over all CD8+ T cells
for individual mice with medians indicated by the lines.
Vaccination induced broad epitope recognition by CD8+ T cells that
was further enhanced after boosting (27% to 34%; FIG. 10).
[0199] At week 12 following AdC6-gDPolN vaccination,
AAV8-1.3HBV-infected vaccinated mice showed a preferential increase
in hepatic CD8+ infiltrates (FIGS. 11A-11B and FIG. 12A-12F), a
decreased presence of vaccine-induced HBV-specific CD8+ T cells
(FIG. 11A and FIG. 11B) and slightly reduced levels of T-bet
(suggestive of loss of effector functions) (FIG. 13A-13B). FIG. 11A
shows the % CD8+ T cells over all recovered lymphocytes from
individual livers. FIG. 11B shows percent tetramer positive
CD8.sup.+ cells, which were identified from histograms in
comparison to naive T cells. No clear pattern of cellular markers
suggestive of T cell differentiation to an exhaustion phenotype was
observed, however, between vaccinated AAV1.3HBV-infected and
-uninfected mice (FIG. 13A-13B).
[0200] Efficacy--Following a single IM injection of the AdC6-gDPolN
vector, AAV8-1.3HBV-infected mice had multi-log HBV DNA declines in
serum that persisted throughout the 8-week post vaccination period
(FIG. 14). Post vaccination, median declines in serum HBV DNA viral
load levels at four and eight weeks were 0.86 and 2.69 log.sub.10
cps/mL, respectively (FIG. 14A). At week 8, all animals had a >1
log.sub.10 cps/mL, 6/7 (86%) had >2 log.sub.10 cps/mL, and 2/7
(29%) had >3 log.sub.10 cps/mL declines from baseline (FIG.
14B).
[0201] Following a single AdC6-gDPolN vector injection, distinct
CD8+ T cell recognition patterns to PolN peptides in splenocytes
were observed when AAV-HBV-infected and naive mice were compared.
FIG. 15A and FIG. 15B illustrate the results from experiments in
which mice were first injected with AAV-1.3HBV and then four weeks
later boosted with 10.sup.10 vp of the AdC6-gDPolN vector,
splenocytes were harvested 8 weeks after the immunization and
tested by ICS for IFN-.gamma. upon a short in vitro stimulation
with individual peptides spanning the sequence of PolN. Background
frequencies obtained without peptide were subtracted. FIG. 15A
shows the peptide recognition profile of mice that received the
AdC6-gDPolN vaccine only followed by those that were first injected
with the indicated doses of the AAV8-1.3HBV vector. The pie graphs
in FIG. 15B show the corresponding responses to peptides that
reached the threshold of 0.1% of all CD44.sup.+CD8.sup.+ cells
(data correspond to those in FIG. 15A). Each slice/color represents
the frequency of the response to an individual peptide with size
showing the proportion of the total; only responses greater than
0.1% were included. Pullouts indicate epitopes only recognized in
AAV8-1.3HBV infected mice. It was found that pre-treatment with AAV
reduced both the number of epitopes recognized after a single IM
prime and the magnitude of the immune response as the sum total of
IFN-.gamma. producing CD8+ T cells over the pool of CD8.sup.+ T
cells. AAV pre-treatment shifted the T cell recognition to new
epitopes, which represent roughly a third of the detectable
CD8.sup.+ T cell response. The percentage of functional
HBV-specific CD8+ T cell responses were highest in naive mice
(4.4%, FIG. 15B) but decreased in the presence of low and high dose
AAV8-1.3HBV (2.0% & 0.6%; respectively, FIG. 15B).
AAV8-1.3HBV-uninfected animals showed strong CD8+ T cell responses
to a number of epitopes, which were decreased and shifted in
AAV-HBV-infected animals to include T cell recognition of new
epitopes.
Discussion
[0202] An HBV therapeutic vaccine that targets early CD8+ T cell
activation using gD as a genetically encoded checkpoint inhibitor
was generated and was shown to: [0203] Induce potent and durable
CD8+ T cell responses to key HBV antigens (FIG. 9); [0204]
Stimulate very broad CD8+ T cell responses (FIG. 10) that included
sub-dominant epitope recognition (FIG. 15); and [0205] Achieve
sustained multi-log HBV DNA viral load reductions in an AAV mouse
model (FIG. 14) with preferential trafficking of functional CD8+ T
cells to the liver (FIGS. 11 and 12).
[0206] In the disclosed AAV studies, AAV-induced HBV infection
caused loss of CD8+ T cell recognition to dominant epitopes of PolN
following vaccination with AdC6-gDPolN (FIG. 15). Without intending
to be bound by theory, it is believed that it is the breadth of the
CD8+ T cells induced by gD and their ability to recognize
subdominant epitopes that led to a sustained immune response and
multi-log suppression of HBV.
Immunogenicity of AdC6/7-gDPolN in Blood and Liver Following
Vaccination in AAV-Induced HBV-Infected Animals
[0207] The following studies were performed to evaluate CD8.sup.+ T
cell responses to the AdC6-gDPolN vaccine in blood, spleens, and
livers of animals in the presence of pre-existing AAV-induced HBV
infection.
Experiment #1--CD8+ T Cell Responses in AAV8-1.3HBV Infected Mice:
Response Kinetics in Blood
[0208] Purpose--To assess the effect of sustained titers of HBV
antigen on CD8.sup.+ T cell responses to the gDPolN antigen as
expressed within the AdC6 vector.
[0209] Methods--C57Bl/6 mice were injected i.v. with the 10.sup.10
of the AAV8-1.3HBV vector. Four weeks later they were vaccinated
with 5.times.10.sup.9vp of the AdC6-gDPolN vector. Control mice
received only the AdC6-gDPolN vector. Naive mice served as
additional controls. Mice were boosted 2 months later with the same
dose of the AdC7-gDPolN vaccine. Blood was collected at various
times after the prime and the boost and PBMCs were tested for
IFN-.gamma.-producing CD8.sup.+ T cells.
[0210] Results--As shown in FIG. 16, mice mounted a vigorous
PolN-specific CD8.sup.+ T cell response 2 weeks after vaccination,
which gradually declined by week 8 and then increased again after
the boost. The CD8.sup.+ T cell response was more stable after the
boost than after the prime. At most time points tested responses
were lower in mice that had been injected with the AAV8-1.3HBV
vector than in the controls that had not been injected with an AAV
vector.
Experiment #2--CD8+ T Cell Responses in AAV8-1.3HBV Infected Mice:
Responses in Liver
[0211] Purpose--To assess CD8.sup.+ T cell responses including
markers indicative of T cell exhaustion in livers of
AAV8-1.3HBV-infected, vaccinated mice.
[0212] Methods--C57Bl/6 mice were injected i.v. with the 10.sup.10
or 10.sup.11vg of the AAV8-1.3HBV vectors. Four weeks later they
were vaccinated with 5.times.10.sup.9vp of the AdC6-gDPolN vector.
Control mice received only the AdC6-gDPolN vector. Naive mice
served as additional controls. Mice were boosted 2 months later
with the same dose of the AdC7-gDPolN vaccine.
[0213] To obtain hepatic lymphocytes, livers were cut into small
fragments and treated with 2 mg/ml Collagenase P, 1 mg/ml DNase I
(all from Roche, Basel Switzerland) and 2% FBS (Tissue Culture
Biologicals, Tulare, Calif.) in L15 under agitation for 1 hour.
Liver fragments were homogenized, filtrated through 70 .mu.m
strainers and lymphocytes were purified by Percoll-gradient
centrifugation and washed with DMEM supplemented with 10% FBS.
Lymphocytes were stained with a violet live/dead dye (Thermo Fisher
Scientific), anti-CD8-APC (clone 53-6.7, BioLegend),
anti-CD44-Alexa Flour 700 (clone IM7, BioLegend), anti-EOMES-Alexa
Fluor 488 (clone Dan11mag, eBioscience), anti-PD1-BV605 (clone
29F.1A12, BioLegend), anti-LAG3-BV650 (clone C9B7W, BioLegend),
anti-T-bet-BV786 (clone 4B10, BioLegend), anti-CTLA-4-PE-A (clone
UC10-4B9, BioLegend), anti-TIM-3-Pe-Cy7-A (clone RMT3-23,
BioLegend), and an APC-labeled MHC class I tetramer (NIH tetramer
Facility, Emory University, Atlanta Ga.) corresponding to amino
acids 396-404 FAVPNLQSL (SEQ ID NO: 188) (peptide 55) of the HBV
polymerase at +4.degree. C. for 30 min in the dark. Cells were
washed and were analyzed by a BD FACS Celesta (BD Biosciences, San
Jose, Calif.) and DiVa software. Post-acquisition analyses were
performed with FlowJo (TreeStar, Ashland, Oreg.).
[0214] Results--The frequencies of CD8.sup.+ T cells within the
lymphocytic liver infiltrates were analyzed. Frequencies of
CD8.sup.+ T cells within the lymphocytic liver infiltrates were
increased in vaccinated mice as compared to naive mice, and further
increases were seen in mice that prior to vaccination had been
injected with the AAV8-1.3HBV vector (FIG. 17A). Frequencies of
PolN-specific CD8.sup.+ T cells identified by staining with a
tetramer specific for an epitope present in the PolN insert were
reduced in AAV-1.3HBV-injected mice (FIG. 17B).
[0215] The phenotypes of the infiltrating tetramer.sup.+CD8.sup.+ T
cells in comparison to naive (i.e.,
tetramer.sup.-CD44.sup.-CD8.sup.+ T cells) were assessed by
determining the mean fluorescent intensity of a dye linked to a
given antibody (FIG. 18A-FIG. 18F) and by assessing the percentages
(FIG. 19A-FIG. 19F) of CD8.sup.+ T cells that were positive for the
indicated markers.
[0216] T-bet which controls a number of CD8.sup.+ T cell functions,
was reduced on hepatic CD8.sup.+ T cells from mice that had been
injected with AAV8-1.3HBV prior to vaccination in comparison the
vaccine only group. Exhaustion markers were not increased in
AAV8-1.3HBV-pre-treated groups suggesting that the observed loss of
PolN-specific CD8.sup.+ T cells in presence of HBV was unlikely to
be caused by classical CD8.sup.+ T cell exhaustion (FIG. 18A-FIG.
18F and FIG. 19A-FIG. 19F).
Experiment #3--Breadth of the PolN-Specific CD8+ T Cell Response in
AAV8-1.3HBV Infected Mice
[0217] Purpose--To assess if the presence of HBV affects the
breadth of the CD8.sup.+ T cell response to PolN expressed within
gD by the AdC vaccines.
[0218] Methods--Mice were injected i.v. with the 10.sup.10 or
10.sup.11vg of the AAV8-1.3HBV vectors and were boosted 2 months
later with the corresponding AdC7 vectors. Control mice received
only the AdC6-gDPolN vector. Mice were euthanized 10 weeks later
and the pooled splenocytes were tested against pools of peptides in
the non-AAV infected animal study. Results are provided in FIG.
20A-FIG. 20C.
[0219] In a second experiment, mice were injected i.v. with the
10.sup.10 or 10.sup.11vg of the AAV8-1.3HBV vectors. Four weeks
later they were vaccinated with 5.times.10.sup.10vp of the
AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN
vector. Naive mice served as additional controls. Splenocytes were
analyzed 6 weeks later for IFN-.gamma.-producing CD8.sup.+ T cells
in response to individual peptides spanning the PolN sequence.
Results are provided in FIG. 20D-FIG. 20F.
[0220] Results--The presence of HBV, especially high titers of HBV
such as after injection with the 10.sup.11 vg dose of AAV8-HBV1.3,
not only reduced overall CD8.sup.+ T cell responses to the PolN
sequence as presented by the AdC6-gDPolN vaccine but also caused a
shift in the epitope recognition profile.
Experiment #4--Functions of Hepatic PolN-Specific CD8+ T Cells in
AAV8-1.3HBV Infected Mice
[0221] Purpose--To evaluate if liver-infiltrating PolN-specific
CD8.sup.+ T cells remain functional in AAV8-1.3HBV infected
mice.
[0222] Methods--In the first experiment, C57BL/6 mice were injected
i.v. with 3.times.10.sup.11 vg of AAV8-1.3HBV. One group was
vaccinated 8 weeks later with 5.times.10.sup.10 vp of AdC6-gDPolN
vector. The other group was left unvaccinated. Mice were euthanized
4.5 months later and splenocytes were tested for frequencies of
CD8.sup.+ T cells producing IFN-.gamma. in response to the PolN
peptide pool.
[0223] In the second experiment, mice were injected with graded
concentrations of AAV8-1.3HBV (1.times.10.sup.10,
4.times.10.sup.10, or 1.times.10.sup.11). All mice were vaccinated
4 weeks later with 5.times.10.sup.10 vp of the AdC6-gDPolN vector.
The mice were boosted 2 months later with the same dose of the
AdC7-gDPolN vector. Mice were euthanized 2 months later and
lymphocytes were isolated from livers and tested for CD8.sup.+ T
cells producing IFN-.gamma. in response to the PolN peptide pool.
Cells were also stained with an antibody to Tox, a transcription
factor that increases in exhausted T cells.
[0224] Results--As shown in FIG. 21, vaccine-induced CD8.sup.+ T
cells remained functional in mice that had been injected with the
AAV8-1.3HBV vector.
Experiment #5--Effect of Vaccination of AAV8-1.3HBV Infected Mice
on Liver Histology
[0225] Purpose--To assess if AdC6/7-gDPolN vaccination of
AAV.8-1.3HBV-vaccinated mice causes sustained liver damage.
[0226] Methods--Mice were injected with 10.sup.10 vg of the
AAV8-1.3HPV given i.v. One month later they were vaccinated with
5.times.10.sup.9 vp of the AdC6-gDPolN vector. The mice were
boosted 2 months later with the same dose of the AdC7-gDPolN vector
given at the same dose. The mice were euthanized .about.2 months
later. Liver sections were collected and fixed in 10% formaldehyde.
Sections (.about.3 .mu.m in thickness) were prepared and stained
with Hematoxylin Eosin (H&E). They were reviewed under a light
microscope at 20.times. magnification.
[0227] Results--One out of 33 sections from mice that had received
both the AAV vector and the vaccine showed a small lymphocytic
infiltrate that was at the margin of the liver section.
[0228] As shown in FIG. 21B, following a single gDPolN vaccination
in HLA-A2-tg mice, frequencies of IFN-.gamma. producing hepatic
CD8.sup.+ T cells were reduced in mice receiving AAV as compared to
those that had only been vaccinated.
Conclusions
[0229] CD8.sup.+ T cell responses to PolN were reduced in
AAV8-1.3HBV infected mice. Nevertheless, they remained detectable.
[0230] Exhaustion markers were not increased in AAV8-1.3HBV
pre-treated animals suggesting that the observed loss of
PolN-specific CD8.sup.+ T cells in the presence of HBV was unlikely
to be caused by classical CD8.sup.+ T cell exhaustion. [0231]
AAV-induced HBV-infection caused a shift in the epitope recognition
profile of CD8.sup.+ T cell responses to PolN. [0232]
Vaccine-induced CD8.sup.+ T cells remained functional in mice that
had been previously infected with the AAV8-1.3HBV vector. [0233]
The vaccine used in a prime boost regimen did not cause overt liver
damage in HBV positive mice.
Generation of HBV PolN-PolC-Core Constructs
[0234] Two multi-antigen inserts (second generation PolN-PolC-Core
and third generation PolN-PolC-Core) were generated. The sequences
of these inserts are shown below:
TABLE-US-00014 2.sup.nd generation HBV vaccine insert ("HBV2")(Pol
N (italics)-Pol C (underlined)-Core) (SEQ ID NO: 174)
YLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGS
PYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHI
RIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVP
NLQSLTNLLSSNLSWLSLDVQAFTFSPTYKAFLSKQYLNLYPVARQRPGL
CQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKI
LGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNL
EDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWI
RTPPAYRPPNAPILSTLPETTVVRRRDRGR 3.sup.rd generation HBV vaccine
insert ("HBV3")(Pol N (italics)-Pol C (underlined)-Core) (SEQ ID
NO: 175) HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPEWQTPSFPK
IHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKP
YYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQH
GSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT
QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQR
MRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLL
GCAANWILRGTSFVYVPSALNPADDVGSNLEDPASRELVVSYVNVNMGLK
IRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPET TVVRRRDRGR
[0235] The second generation HBV ("HBV2") insert includes
immuno-dominant PolN epitopes identified from mice that had not
been infected with the AAV8-1.3HBV vector prior to vaccination.
Many of these epitopes were found to be lost in a mouse model of
chronic HBV infection brought about by pre-administering an
AAV8-1.3HBV vector (as defined in "Epitope Shifting" above). The
third generation HBV ("HBV3") insert selects for contiguous regions
of PolN that were preferentially recognized by mice with high loads
of HBV (see above). Regions of Core and PolC were selected for both
constructs using the following general formula: regions with the
highest immune responses on either prime (FIG. 3) or boost (FIG. 5)
regions in C57Bl/6, BALBc and HLA-A2 tg mice, and with the aim of
selecting a large contiguous region instead of selecting unique
epitopes and inserting spacer sequences between them.
Genetic Integrity and Stability of the 2.sup.nd and 3.sup.rd
Generation HBV Inserts (HBV2 and HBV3)
[0236] Western Blot--Purified recombinant viral vector preparations
(AdC6-gDHBV2, AdC6-gDHBV3, AdC7-gDHBV2, and AdC7-gDHBV3) were
evaluated for their ability to elicit transgene-product expression
in vitro. To that end, Western Blot assays were performed to assess
the expression of gD protein in cell lysates following cell culture
infection with the vector of interest. Adherent HEK293 cell
monolayers were infected with known quantities of the purified
vector and harvested at 48 hours post-infection, resuspended in
lysis and extraction buffer containing protease inhibitors, and
lysed by sonication. The total protein extracts were denatured by
the use of dithiothreitol as a redox agent and submitted to
electrophoresis in a 12% Bis-Tris polyacrylamide gel (PAGE).
Subsequent to protein separation by SDS-PAGE, the samples were
transferred onto an activated polyvinylidene difluoride membrane by
wet electrophoretic transfer. The membrane was immunostained for
the detection of gD protein using the primary antibody to gD
diluted to 1:1000 in saline (clone PA1-30233, Invitrogen, Carlsbad,
Calif.) for 1 h at room temperature. Membranes were washed with
1.times.TBS-T prior to incubating with HRP-conjugated goat
anti-rabbit secondary IgG (ab6721, Abcam, Cambridge UK) for 1 h at
room temperature. This was followed by the addition of a
luminol-based chemiluminescent substrate. The stained membrane was
exposed to an autoradiography film and signal emission was
evaluated after processing by an automated film developer.
Following documentation of the gD protein expression in infected
HEK293 cell lysates, the membrane was stripped and re-probed for
the presence of .beta.-actin in the total protein extract samples.
This staining step was employed to evaluate the consistency of the
PAGE sample loading step and thus better support the
semi-quantitative analysis of the in vitro stimulation of gD
protein expression by the recombinant viral vector.
[0237] Stability--To ensure the genetic integrity of the viral
construct, the genetic stability of each recombinant viral vector
lot was assessed through sequential viral passages in adherent
HEK293 cell cultures. The recombinant virus pool resulting from
each transfection was cultured under standard growing conditions
for a total of 12 passages. In the last passage, the virus pool was
expanded and the crude harvest purified by cesium chloride
gradient. Following vector purification, viral DNA was isolated
using the QIAGEN DNeasy Blood & Tissue Kit and evaluated by
restriction enzyme digest with Ase I and Bgl II, two restriction
enzymes that cleave the DNA template in distinct construct-specific
pre-defined banding patterns. After digestion, samples were
submitted to electrophoresis in 1% agarose gel containing ethidium
bromide to allow for the visualization of the digested bands,
followed by documentation of results using a digital gel imaging
system. Viral preparations that exhibited banding patterns
identical to those of an early passage virus were considered to
have maintained the original molecular clone structure and thus
deemed stable at the end of 12 viral passages.
[0238] Results--The banding patterns of viral vector DNAs remained
stable after 12 passages compared to that after 5 passages
indicating the vector genomes were stable (data not shown).
Immunogenicity of the 2.sup.nd and 3.sup.rd Generation HBV Inserts
(HBV2 and HBV3) as Expressed by AdC6 or AdC7 Vectors
[0239] Purpose--To assess CD8.sup.+ T cell responses to the HBV2
and HBV3 inserts expressed by AdC6 vectors or AdC7 vectors.
[0240] Methods--Groups of C57Bl/6 mice were injected with
5.times.10.sup.9 or 5.times.10.sup.10 vp of AdC6-gDHBV2 or
AdC6-gDHBV3 vector. Mice injected with the same doses of the
AdC6-gDPolN vector served as positive controls; naive mice served
as negative controls. Mice were bled 14 days later and PBMCs were
tested for frequencies of CD8.sup.+ T cells producing IFN-.gamma.
in response to peptide pools corresponding to the HBV inserts. Four
weeks later (6 weeks after vaccination) mice were bled again and
tested with the PolN-specific tetramer. AdC6-gDHBV3 immunized mice
were excluded as this insert lacks the epitope that corresponds to
the tetramer.
[0241] Groups of C57Bl/6 mice were injected with 5.times.10.sup.9
or 5.times.10.sup.10 vp of AdC7-gDHBV2 or 5.times.10.sup.10 vp of
AdC7-gDHBV3 vector. Naive mice served as negative controls. Mice
were bled 14 days later and PBMCs were tested for frequencies of
CD8.sup.+ T cells producing IFN-.gamma. in response to peptide
pools corresponding to the HBV inserts.
Immunogenicity of AdC7Prime/AdC6 Boost
[0242] Mice were bled .about.4 weeks later and PBMCs were retested
by ICS for CD8.sup.+ T cells producing IFN-.gamma. and/or
TNF-.alpha. in response to the peptides for the inserts. Mice were
boosted two months after the prime with the same dose of the
heterologous vector expressing the same insert. PBMCs were tested
by ICS 2 weeks later and pre- and post-boost CD8.sup.+ and
CD4.sup.+ T cell responses were compared. The AdC7-gDHBV2 vector
induced robust frequencies of CD8.sup.+ T cells producing
IFN-.gamma. and/or TNF-.alpha. after the prime. Frequencies
increased after the AdC6-gDHBV2 boost and this was especially
pronounced after the low vector doses and for CD8.sup.+ T cells
producing IFN-.gamma.. The AdC7-gDHBV3 vector was poorly
immunogenic but CD8.sup.+ T cell responses became positive after
the AdC6-gDHBV3 boost. In the same token CD4+ T cell responses were
marginal after the prime but increased after the boost. There was
no marked difference in CD4 responses to the HBV2 or HBV3
insert.
Conclusions
[0243] Both the AdC6-gDHBV2 and AdC7-gDHBV2 vectors were highly
immunogenic (FIG. 22A, FIG. 22B, and FIG. 23) and responses
increased after a boost with a heterologous AdC vector expressing
the same insert (FIG. 24). [0244] The AdC7-gDHBV2 and AdC7-gDHBV3
vectors displayed borderline immunogenicity consistent with their
design as they lack the epitope that corresponds to the tetramer
being used (FIG. 22A and FIG. 23). [0245] Boosting AdC7-gDHBV2 with
AdC6-gDHBV2 enhances CD8.sup.+ T cell responses.
Comparison of HBV DNA Viral Titers in AdC6-gDPolN, AdC6-gDHBV2,
AdC6-gDHBV3, or AdC6-HBV2 AAV-Infected Mice
Methods
[0246] Five groups of C57Bl/6 mice were challenged with
1.times.10.sup.9 vg of AAV8-1.3HBV and were vaccinated 4 weeks
later with 1.times.10.sup.10 vp of either AdC6-gDPolN (n=10),
AdC6-gDHBV2 (n=10), AdC6-gDHBV3 (n=10), or AdC6-HBV2 without gD
(n=10); AAV-infected, non-vaccinated animals ("naive") (n=10) and
non-AAV-infected, non-vaccinated animals (n=2-5) served as
controls. Viral titers were tested 4 weeks after AAV injection
(before vaccination) and compared to levels 4 weeks after
vaccination (week 8 after AAV injection).
Results
[0247] At week 8, the median HBV viral titers increased by 0.98
log.sub.10 cps/mL in naive mice, remained unchanged in AdC6-HBV2
vaccinated mice, and declined by -0.04, -1.09 and -2.13 log.sub.10
cps/mL in AdC6-gDHBV3, AdC6-gDPolN and AdC6-gDHBV2 vaccinated
animals, respectively (FIG. 25A). The results for individual mice
are shown in FIG. 25B--all AdC6-gDPolN and AdC6-gDHBV2 vaccinated
animals had greater than 1 and 2 log.sub.10 copies/mL declines,
respectively; in contrast, none of the naive, AdC6-HBV2, or
AdC6-gDHBV3 vaccinated animals had a 1 log.sub.10 copies/mL or
greater decline at Week 8.
Immunogenicity Studies for gDHBV2 and gDHBV3
[0248] The induction of CD8.sup.+ T cell responses and their
breadth to segments of HBV core and polymerase contained in either
gDHBV2 or gDHBV3 following a single prime injection or prime
followed by a boost vaccination with a heterologous vector
containing the same insert were evaluated.
Experiment 1
[0249] Purpose: Assess IFN-.gamma..sup.+CD8.sup.+ T cell responses
following prime and boost vaccinations with gD-HBV2 and gD-HBV3
expressed by heterologous chimpanzee adenoviral vectors (AdC6 and
AdC7) in C57Bl/6 mice.
[0250] Methods: Four groups of five C57Bl/6 mice were immunized via
intramuscular injection as follows: (a) 5.times.10.sup.10 vp
AdC7-gDHBV2 followed two months later by 5.times.10.sup.10 vp
AdC6-gDHBV2; (b) 5.times.10.sup.9 vp AdC7-gDHBV2 followed two
months later by 5.times.10.sup.9 vp AdC6-gDHBV2; (c)
5.times.10.sup.10 vp AdC7-gDHBV3 followed two months later by
5.times.10.sup.10 vp AdC6-gDHBV3; or (d) no vaccine. Blood was
assessed by ICS for IFN-.gamma..sup.+CD8.sup.+ T cell responses 2
and 6 weeks after the prime, prior to the boost, and then 2 and 4
weeks after the boost.
[0251] Results: At all time points tested each vaccine construct
was found to induce IFN-.gamma..sup.+CD8.sup.+ T cells. FIG. 26
shows the percent of parental IFN-.gamma. and/or TNF-.alpha.
producing CD8.sup.+ T cells (FIG. 26A), CD44+CD8+ T cells (FIG.
26B), CD4+ T cells (FIG. 26C) or CD44+CD4+ T cells (FIG. 26D).
Immune responses as assessed by ICS from PBMCs of individual mice
are shown two and eight weeks after the prime, as well as two and
four weeks after the boost as the mean.
Experiment 2
[0252] Purpose: Compare IFN-.gamma..sup.+CD8.sup.+ T cell responses
following different doses of prime and boost vaccinations with
gD-HBV2 and gD-HBV3 to that with gD-PolN using heterologous
chimpanzee adenoviral vectors (AdC6 and AdC7) in C57Bl/6 mice.
[0253] Methods: Groups of C57Bl/6 mice (n=5 mice/group) were
immunized as follows:
[0254] gDPolN Groups [0255] (a) 5.times.10.sup.9 vp AdC6-gDPolN
followed three months later by 5.times.10.sup.9 vp AdC7-gDPolN; and
[0256] (b) 5.times.10.sup.10 vp AdC6-gDPolN followed three months
later by 5.times.10.sup.10 vp AdC7-gDPolN gDHBV2 Groups [0257] (c)
5.times.10.sup.9 vp AdC6-gDHBV2 followed three months later by
5.times.10.sup.9 vp AdC7-gDHBV2 and; [0258] (d) 5.times.10.sup.10
vp AdC6-gDHBV2 followed three months later by 5.times.10.sup.10 vp
AdC7-gDHBV2 gDHBV3 Groups [0259] (e) 5.times.10.sup.9 vp
AdC6-gDHBV3 followed three months later by 5.times.10.sup.9 vp
AdC7-gDHBV3 and; [0260] (f) 5.times.10.sup.10 vp AdC6-gDHBV3
followed three months later by 5.times.10.sup.10 vp AdC7-gDHBV3
[0261] No Treatment Served as Controls
[0262] For all treatment groups, immunogenicity CD8.sup.+ T cell
responses was from blood assessed by ICS for IFN-.gamma..sup.+ at
two and six weeks after the prime, prior to the boost, and then two
and six weeks after the boost. Immunogenicity was also assessed by
tetramer staining using an APC-labeled MHC class I tetramer (NIH
tetramer Facility, Emory University, Atlanta Ga.) corresponding to
amino acids 396-404 FAVPNLQSL (peptide 55) of the HBV polymerase at
week four after the prime. HBV3 does not contain the FAVPNLQSL
peptide.
[0263] Results: At all time points, each vaccine tested was found
to induce IFN-.gamma..sup.+CD8.sup.+ T cells. Results obtained with
the gDHBV2 vaccine were similar to those obtained with the gDPolN
vaccine; the gDHBV3 vaccine was less immunogenic. Upon tetramer
staining, frequencies of the specific CD8.sup.+ T cells were
comparable between the two vaccines; a number of activation markers
tended to be more highly expressed on tetramer+ CD8+ T cells from
the gDHBV2-immunized groups. FIG. 27 shows CD8.sup.+ T cells at
multiple time points: four weeks after prime (FIG. 27A); two weeks
after the boost (FIG. 27B); and four weeks after the boost (FIG.
27C). The graph shows the overall frequencies of CD8.sup.+ T cells
producing IFN-.gamma..sup.+ as assessed by ICS.
[0264] FIG. 28 shows cytokine-producing CD4+ T cells at multiple
time points: four weeks after prime (FIG. 28A); two weeks after the
boost (FIG. 28B); and four weeks after the boost (FIG. 28C) as
assessed by ICS. The dashed line indicates the cut-off for positive
responses, based on the results from the naive mice.
[0265] FIG. 29 shows the results of tetramer staining gated on
either CD8+ T cells (FIG. 29A) or CD44+CD8+ T cells (FIG. 29B) at
four weeks after the prime.
[0266] FIG. 30 shows the phenotypes of the tetramer+ CD8+ T cells
shown as the mean fluorescent intensity of a dye linked to the
indicated antibody: FIG. 30A anti-PD1 antibody conjugated to BV605;
FIG. 30B anti-LAG3 antibody conjugated to BV650; FIG. 30C
anti-TB/13 antibody conjugated to Pe-Cy7-A; FIG. 30D anti-CTLA4
antibody conjugated to PE-A; FIG. 30E anti-EOMES antibody
conjugated to AF488; and FIG. 30F anti-T-bet antibody conjugated to
BV786.
Experiment 3
[0267] The breadth of responses were assessed from pooled
splenocytes of vaccinated C57BL/6 mice which were tested by ICS
against the individual peptides present in the HBV vaccine
inserts.
[0268] Methods: Four groups of five C57Bl/6 mice were immunized via
intramuscular injection as follows: (a) 5.times.10.sup.10 vp
AdC7-gDHBV2 followed two months later by 5.times.10.sup.10 vp
AdC6-gDHBV2; (b) 5.times.10.sup.9 vp AdC7-gDHBV2 followed two
months later by 5.times.10.sup.9 vp AdC6-gDHBV2; (c)
5.times.10.sup.10 vp AdC7-gDHBV3 followed two months later by
5.times.10.sup.10 vp AdC6-gDHBV3; or (3) no vaccine. Animals were
sacrificed eight weeks after the boost and pooled splenocytes were
assessed by ICS for IFN-.gamma..sup.+CD8.sup.+ T cell responses to
individual HBV2 or HBV3 peptides (cut-off for positive responses
set at 0.1%).
[0269] Results: Independent of the dose, the prime boost regimen
with the gDHBV2 vaccines induced responses to several epitopes
within core and polymerase. FIG. 31 shows the CD8.sup.+ T cell
responses after a prime vaccination of 5.times.10.sup.10 vp
AdC7-gDHBV2 followed two months later by vaccination with
5.times.10.sup.10 vp AdC6-gDHBV2. Numbers on the X axis correspond
to the SEQ ID NO as provided herein. FIG. 32 shows the CD8+ T cell
responses after a prime vaccination with 5.times.10.sup.9 vp
AdC7-gDHBV2 followed two months later by vaccination with
5.times.10.sup.9 vp AdC6-gDHBV2. Numbers on the X axis correspond
to the SEQ ID NO as provided herein. FIG. 33 shows the
immunogenicity after a prime vaccination with 5.times.10.sup.10 vp
AdC7-gDHBV3 followed two months later by vaccination with
5.times.10.sup.10 vp AdC6-gDHBV3. Numbers on the X axis correspond
to the SEQ ID NO as provided herein.
Experiment 4
[0270] The breadth of responses were assessed from pooled
splenocytes of vaccinated BALB/c mice which were tested by ICS
against the individual peptides present in the HBV vaccine
inserts.
[0271] Methods: Five groups of five BALB/c mice were immunized via
intramuscular injection as follows: (a) 5.times.10.sup.10 vp
AdC6-gDHBV2; (b) 5.times.10.sup.10 vp AdC6-gDHBV3; (c)
5.times.10.sup.10 vp AdC7-gDHBV2; (d) 5.times.10.sup.10 vp
AdC7-gDHBV3; or (e) no vaccine. 12 weeks post vaccination animals
were sacrificed, spleens were collected and pooled splenocytes were
assessed by ICS for IFN-.gamma..sup.+CD8.sup.+ T cell responses to
individual HBV2 or HBV3 peptides (cut-off for positive responses
set at 0.1%).
[0272] Results: At week 12, each vaccine construct was found to be
immunogenic across multiple regions of the Core and Polymerase
genes delivered by the vaccine. FIG. 34 shows the immunogenicity of
the AdC6-gDHBV2 and AdC7-gDHBV2 vaccines corresponding to the SEQ
ID NO (X axis) as provided herein. Core, PolC, and PolN regions in
both HBV2 constructs were immunogenic. FIG. 35 shows the
immunogenicity of the AdC6-gDHBV3 and AdC7-gDHBV3 vaccines
corresponding to the SEQ ID NO (X axis) as provided herein. Core,
PolC, and PolN regions in both HBV3 constructs were
immunogenic.
Experiment 5
[0273] Methods: Five groups of C57Bl/6 mice were challenged with
1.times.10.sup.9 vg of AAV8-1.3HBV and were vaccinated 4 weeks
later ("prime vaccination") with 1.times.10.sup.10 vp of either
AdC6-gDPolN (n=10), AdC6-gDHBV2 (n=10), AdC6-gDHBV3 (n=10), or
AdC6-HBV2 without gD (n=10); AAV-infected, non-vaccinated animals
(n=10) and non-AAV-infected, non-vaccinated animals (n=2-5) serve
as controls. Mice will be bled at various times after the injection
and frequencies of insert-specific CD8+ and CD4+ T cells will be
determined by intracellular cytokine staining (ICS) for
IFN-.gamma.. PCR will be performed at 2 weeks, 6 weeks, and 8 weeks
after the prime vaccination, and a T cell assay will be performed
at 4 weeks after the prime vaccination.
[0274] At 8 weeks following the prime vaccination, mice will be
boosted with AdC7 vectors containing the same antigenic insert used
in the prime vaccination ("boost vaccination") and blood and serum
will be tested for CD8+/CD4+ T cell as previously described at
different time points after vaccination. PCR will be performed at 2
weeks, 6 weeks, and 10 weeks after the boost vaccination, and a T
cell assay will be performed at 4 weeks and 12 weeks after the
boost vaccination.
[0275] Those skilled in the art will appreciate that numerous
changes and modifications can be made to the preferred embodiments
of the invention and that such changes and modifications can be
made without departing from the spirit of the invention. It is,
therefore, intended that the appended claims cover all such
equivalent variations as fall within the true spirit and scope of
the invention.
[0276] The disclosures of each patent, patent application, and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
TABLE-US-00015 TABLE 9 Sequences Sequence Genotype A
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASAL Consensus
YREALESPEHCSPHHTALRQAILCWGELMTLATWVGN (SEQ ID NO: 1)
NLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFG
RETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTV
VRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC Genotype B
MDIDpYKEFGASvELLSFLPSDFFPSiRDLLDTAsAL Consensus
YREALESPEHCSPHHTALRQAIlCWGELMNLATWVGS (SEQ ID NO: 2)
NLeDPASRELVVsYVNVNMGLKiRQLLWFHISCLTFG
RETVLEYLVSFGVWIRTPpAYRPpNAPILSTLPETTV
VRRRGRSPRRRTPSPRRRRSQSPRRRRSQSREsQC Genotype C
MDIDpYKEFGASVELLSFLPSDFFPSIRDLLDTASAL Consensus
YREALESPEHCSPHHTALRQAILCWGELMNLATWVGS (SEQ ID NO: 3)
NLEDPASRELVVsYVNVNMGLKiRQlLWFHISCLTFG
RETVLEYLVSFGVWIRTPpAYRPPNAPILSTLPETTV
VRRRGRSPRRRTPSPRRRRSQSPRRRSQSRESQC Genotype D
MDIDPYKEFGAtVELLSFLPsDFFPSVRDLLDTASALYReAL Consensus
ESPEHCSPHHTALRQAILCWGeLMtLATWVGgNLEDPaSRDL (SEQ ID NO: 4)
VVSYVNTNmGLKFRQLLWFHISCLTFGReTViEYLVSFGVWI
RTPpAYRPPNAPILSTLPETTVvRRRGRSPRRRTPSPRRRRS QSPRRRRSQSRESQC Initial
Core MDIDPYKEFGA VELLSFLPSDFFPS DLLDTASALYREAL sequence
ESPEHCSPHHTALRQAILCWGELM LATWVG NLeDPASR (SEQ ID NO: 5) LVV YVN
NMGLK RQLLWFHISCLIFGRETV EYLVSF GVWIRTPPAYRPPNAPILSTLPETTVVRRR
GRSPRRRT PSPRRRRSQSPRRRRSQSRESQC Epitope-
DIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALE optimized Core
SPEHCSPHHTALRQAILCWGELMTLATWVGSNLEDPASRELV amino acid
VSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIR sequence
TPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRR (SEQ ID NO: 6)
SQSPRRRRSQSRESQC Epitope-
GACATCGACCCCTACAAGGAGTTCGGCGCCACCGTGGAGCTG optimized Core
CTGAGCTTCCTGCCCAGCGACTTCTTCCCCAGCATCAGGGAC nucleotide
CTGCTGGACACCGCCAGCGCCCTGTACAGGGAGGCCCTGGAG sequence
AGCCCCGAGCACTGCAGCCCCCACCACACCGCCCTGAGGCAG (SEQ ID NO: 7)
GCCATCCTGTGCTGGGGCGAGCTGATGACCCTGGCCACCTGG
GTGGGCAGCAACCTGGAGGACCCCGCCAGCAGGGAGCTGGTG
GTGAGCTACGTGAACGTGAACATGGGCCTGAAGATCAGGCAG
CTGCTGTGGTTCCACATCAGCTGCCTGACCTTCGGCAGGGAG
ACCGTGATCGAGTACCTGGTGAGCTTCGGCGTGTGGATCAGG
ACCCCCCCCGCCTACAGGCCCCCCAACGCCCCCATCCTGAGC
ACCCTGCCCGAGACCACCGTGGTGAGGAGGAGGGACAGGGGC
AGGAGCCCCAGGAGGAGGACCCCCAGCCCCAGGAGGAGGAGG
AGCCAGAGCCCCAGGAGGAGGAGGAGCCAGAGCAGGGAGAGC CAGTGC Epitope-
PLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNL optimized
GNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQ polymerase N-
EDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDK terminal amino
GIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFC acid sequence
GSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWG (SEQ ID NO: 8)
PCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDF
SQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAF YHIPLHPAAMP Epitope-
CCCCTGAGCTACCAGCACTTCAGGAAGCTGCTGCTGCTGGAC optimized
GAGGAGGCCGGCCCCCTGGAGGAGGAGCTGCCCAGGCTGGCC polymerase N-
GACGAGGGCCTGAACAGGAGGGTGGCCGAGGACCTGAACCTG terminus
GGCAACCTGAACGTGAGCATCCCCTGGACCCACAAGGTGGGC nucleotide
AACTTCACCGGCCTGTACAGCAGCACCGTGCCCGTGTTCAAC sequence
CCCGAGTGGCAGACCCCCAGCTTCCCCAAGATCCACCTGCAG (SEQ ID NO: 9)
GAGGACATCGTGGACAGGTGCAAGCAGTTCGTGGGCCCCCTG
ACCGTGAACGAGAAGAGGAGGCTGAAGCTGATCATGCCCGCC
AGGTTCTACCCCAACGTGACCAAGTACCTGCCCCTGGACAAG
GGCATCAAGCCCTACTACCCCGAGCACGCCGTGAACCACTAC
TTCCAGACCAGGCACTACCTGCACACCCTGTGGAAGGCCGGC
ATCCTGTACAAGAGGGAGACCACCAGGAGCGCCAGCTTCTGC
GGCAGCCCCTACAGCTGGGAGCAGGAGCTGCAGCACGGCAGC
TGCTGGTGGCTGCAGTTCAGGAACAGCAAGCCCTGCAGCGAG
TACTGCCTGACCCACCTGGTGAACCTGCTGGAGGACTGGGGC
CCCTGCGACGAGCACGGCGAGCACCACATCAGGATCCCCAGG
ACCCCCGCCAGGGTGACCGGCGGCGTGTTCCTGGTGGACAAG
AACCCCCACAACACCGCCGAGAGCAGGCTGGTGGTGGACTTC
AGCCAGTTCAGCAGGGGCATCACCAGGGTGAGCTGGCCCAAG
TTCGCCGTGCCCAACCTGCAGAGCCTGACCAACCTGCTGAGC
AGCAACCTGAGCTGGCTGAGCCTGGACGTGAGCGCCGCCTTC TACCACATCCCCCTG
CACCCCGCCGCCATGCCC Epitope-
HLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLY optimized
VSLLLLYKTFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSW polymerase C-
GSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAP terminal amino
FTQCGYPALMPLYACIQSKQAFTFSPTYKAFLSKQYLNLYPV acid sequence
ARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAE (SEQ ID NO: 10)
LLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWI
LRGTSFVYVPSALNPADDPSRGRLGLSRPLLRLPFRPTTGRT SLYAVSPSV Epitope-
CACCTGCTGGTGGGCAGCAGCGGCCTGAGCAGGTACGTGGCC optimized
AGGCTGAGCAGCAACAGCAGGATCATCAACCACCAGCACGGC polymerase C-
ACCATGCAGAACCTGCACGACAGCTGCAGCAGGAACCTGTAC terminal
GTGAGCCTGCTGCTGCTGTACAAGACCTTCGGCAGGAAGCTG nucleotide
CACCTGTACAGCCACCCCATCATCCTGAAGACCAAGAGGTGG sequence
GGCTACAGCCTGAACTTCATGGGCTACGTGATCGGCAGCTGG (SEQ ID NO: 11)
GGCAGCCTGCCCCAGGACCACATCATCCAGAAGATCAAGGAG
TGCTTCAGGAAGCTGCCCGTGAACAGGCCCATCGACTGGAAG
GTGTGCCAGAGGATCGTGGGCCTGCTGGGCTTCGCCGCCCCC
TTCACCCAGTGCGGCTACCCCGCCCTGATGCCCCTGTACGCC
TGCATCCAGAGCAAGCAGGCCTTCACCTTCAGCCCCACCTAC
AAGGCCTTCCTGAGCAAGCAGTACCTGAACCTGTACCCCGTG
GCCAGGCAGAGGCCCGGCCTGTGCCAGGTGTTCGCCGACGCC
ACCCCCACCGGCTGGGGCCTGGCCATGGGCCACCAGAGGATG
AGGGGCACCTTCGTGGCCCCCCTGCCCATCCACACCGCCGAG
CTGCTGGCCGCCTGCTTCGCCAGGAGCAGGAGCGGCGCCAAG
ATCCTGGGCACCGACAACAGCGTGGTGCTGAGCAGGAAGTAC
ACCAGCTTCCCCTGGCTGCTGGGCTGCGCCGCCAACTGGATC
CTGAGGGGCACCAGCTTCGTGTACGTGCCCAGCGCCCTGAAC
CCCGCCGACGACCCCAGCAGGGGCAGGCTGGGCCTGAGCAGG
CCCCTGCTGAGGCTGCCCTTCAGGCCCACCACCGGCAGGACC
AGCCTGTACGCCGTGAGCCCCAGCGTG N- terminal HSV
MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF gD sequence
RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO: 12)
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR Signal peptide
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA in italics
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN QRTVAVYSLKIAGWHGP
C-terminal HSV GPKAPYTSTLLPPELSETPNATQPELAPEDPEDSALLEDPVG gD
sequence TVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIAGAVGGSL (SEQ ID NO: 13)
LAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQPSSHQPLF Y gDCore amino
MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequence
RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY Core underlined
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 14)
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptide
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italics
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN
QRTVAVYSLKIAGWHGPDIDPYKEFGATVELLSFLPSDFFPS
IRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTL
ATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTF
GRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRR
DRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQCGPKAPYTST
LLPPELSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPN
WHIPSIQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGI
VYWMHRRTRKAPKRIRLPHIREDDQPSSHQPLFY gDCore nucleic
atggggggggctgccgccaggttgggggccgtgattttgttt acid sequence
gtcgtcatagtgggcctccatggggtccgcggcaaatatgcc Core underlined
ttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 15)
cgcggcaaagaccttccggtcctggaccagctgaccgaccct
ccgggggtccggcgcgtgtaccacatccaggcgggcctaccg
gacccgttccagccccccagcctcccgatcacggtttactac
gccgtgttggagcgcgcctgccgcagcgtgctcctaaacgca
ccgtcggaggccccccagattgtccgcggggcctccgaagac
gtccggaaacaaccctacaacctgaccatcgcttggtttcgg
atgggaggcaactgtgctatccccatcacggtcatggagtac
accgaatgctcctacaacaagtctctgggggcctgtcccatc
cgaacgcagccccgctggaactactatgacagcttcagcgcc
gtcagcgaggataacctggggttcctgatgcacgcccccgcg
tttgagaccgccggcacgtacctgcggctcgtgaagataaac
gactggacggagattacacagtttatcctggagcaccgagcc
aagggctcctgtaagtacgccctcccgctgcgcatccccccg
tcagcctgcctctccccccaggcctaccagcagggggtgacg
gtggacagcatcgggatgctgccccgcttcatccccgagaac
cagcgcaccgtcgccgtatacagcttgaagatcgccgggtgg
cacgggcccgacatcgacccctacaaggagttcggcgccacc
gtggagctgctgagcttcctgcccagcgacttcttccccagc
atcagggacctgctggacaccgccagcgccctgtacagggag
gccctggagagccccgagcactgcagcccccaccacaccgcc
ctgaggcaggccatcctgtgctggggcgagctgatgaccctg
gccacctgggtgggcagcaacctggaggaccccgccagcagg
gagctggtggtgagctacgtgaacgtgaacatgggcctgaag
atcaggcagctgctgtggttccacatcagctgcctgaccttc
ggcagggagaccgtgatcgagtacctggtgagcttcggcgtg
tggatcaggaccccccccgcctacaggccccccaacgccccc
atcctgagcaccctgcccgagaccaccgtggtgaggaggagg
gacaggggcaggagccccaggaggaggacccccagccccagg
aggaggaggagccagagccccaggaggaggaggagccagagc
agggagagccagtgcgggcccaaggccccatacacgagcacc
ctgctgcccccggagctgtccgagacccccaacgccacgcag
ccagaactcgccccggaagaccccgaggattcggccctcttg
gaggaccccgtggggacggtggcgccgcaaatcccaccaaac
tggcacatcccgtcgatccaggacgccgcgacgccttaccat
cccccggccaccccgaacaacatgggcctgatcgccggcgcg
gtgggcggcagtctcctggcagccctggtcatttgcggaatt
gtgtactggatgcaccgccgcactcggaaagccccaaagcgc
atacgcctcccccacatccgggaagacgaccagccgtcctcg caccagcccttgttttactag
gDPolN amino MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid
sequence RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY PolN underlined
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 16)
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptide
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italics
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN
QRTVAVYSLKIAGWHGPPLSYQHFRKLLLLDEEAGPLEEELP
RLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVP
VFNPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLI
MPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLW
KAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKP
CSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFL
VDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTN
LLSSNLSWLSLDVSAAFYHIPLHPAAMPGPKAPYTSTLLPPE
LSETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPS
IQDAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMH
RRTRKAPKRIRLPHIREDDQPSSHQPLFY* gDPolN nucleic
atggggggggctgccgccaggttgggggccgtgattttgttt acid sequence
gtcgtcatagtgggcctccatggggtccgcggcaaatatgcc PolN underlined
ttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 17)
cgcggcaaagaccttccggtcctggaccagctgaccgaccct
ccgggggtccggcgcgtgtaccacatccaggcgggcctaccg
gacccgttccagccccccagcctcccgatcacggtttactac
gccgtgttggagcgcgcctgccgcagcgtgctcctaaacgca
ccgtcggaggccccccagattgtccgcggggcctccgaagac
gtccggaaacaaccctacaacctgaccatcgcttggtttcgg
atgggaggcaactgtgctatccccatcacggtcatggagtac
accgaatgctcctacaacaagtctctgggggcctgtcccatc
cgaacgcagccccgctggaactactatgacagcttcagcgcc
gtcagcgaggataacctggggttcctgatgcacgcccccgcg
tttgagaccgccggcacgtacctgcggctcgtgaagataaac
gactggacggagattacacagtttatcctggagcaccgagcc
aagggctcctgtaagtacgccctcccgctgcgcatccccccg
tcagcctgcctctccccccaggcctaccagcagggggtgacg
gtggacagcatcgggatgctgccccgcttcatccccgagaac
cagcgcaccgtcgccgtatacagcttgaagatcgccgggtgg
cacgggccccccctgagctaccagcacttcaggaagctgctg
ctgctggacgaggaggccggccccctggaggaggagctgccc
aggctggccgacgagggcctgaacaggagggtggccgaggac
ctgaacctgggcaacctgaacgtgagcatcccctggacccac
aaggtgggcaacttcaccggcctgtacagcagcaccgtgccc
gtgttcaaccccgagtggcagacccccagcttccccaagatc
cacctgcaggaggacatcgtggacaggtgcaagcagttcgtg
ggtcccctgaccgtgaacgagaagaggaggctgaagctgatc
atgcccgccaggttctaccccaacgtgaccaagtacctgccc
ctggacaagggcatcaagccctactaccccgagcacgccgtg
aaccactacttccagaccaggcactacctgcacaccctgtgg
aaggccggcatcctgtacaagagggagaccaccaggagcgcc
agcttctgcggcagcccctacagctgggagcaggagctgcag
cacggcagctgctggtggctgcagttcaggaacagcaagccc
tgcagcgagtactgcctgacccacctggtgaacctgctggag
gactggggtccctgcgacgagcacggcgagcaccacatcagg
atccccaggacccccgccagggtgaccggcggcgtgttcctg
gtggacaagaacccccacaacaccgccgagagcaggctggtg
gtggacttcagccagttcagcaggggcatcaccagggtgagc
tggcccaagttcgccgtgcccaacctgcagagcctgaccaac
ctgctgagcagcaacctgagctggctgagcctggacgtgagc
gccgccttctaccacatccccctgcaccccgccgccatgccc
gggcccaaggccccatacacgagcaccctgctgcccccggag
ctgtccgagacccccaacgccacgcagccagaactcgccccg
gaagaccccgaggattcggccctcttggaggaccccgtgggg
acggtggcgccgcaaatcccaccaaactggcacatcccgtcg
atccaggacgccgcgacgccttaccatcccccggccaccccg
aacaacatgggcctgatcgccggcgcggtgggcggcagtctc
ctggcagccctggtcatttgcggaattgtgtactggatgcac
cgccgcactcggaaagccccaaagcgcatacgcctcccccac
atccgggaagacgaccagccgtcctcgcaccagcccttgttt tactag gDPolC amino
MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequence
RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY PolC underlined
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR (SEQ ID NO: 18)
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA Signal peptide
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA in italics
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN
QRTVAVYSLKIAGWHGPHLLVGSSGLSRYVARLSSNSRIINH
QHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILKT
KRWGYSLNPMGYVIGSWGSLPQDHIIQKIKECFRKLPVNRPI
DWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFS
PTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGH
QRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLS
RKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLG
LSRPLLRLPFRPTTGRTSLYAVSPSVGPKAPYTSTLLPPELS
ETPNATQPELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQ
DAATPYHPPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRR
TRKAPKRIRLPHIREDDQPSSHQPLFY gDPolC nucleic
atggggggggctgccgccaggttgggggccgtgattttgttt acid sequence
gtcgtcatagtgggcctccatggggtccgcggcaaatatgcc PolC underlined
ttggcggatgcctctctcaagatggccgaccccaatcgcttt (SEQ ID NO: 19)
cgcggcaaagaccttccggtcctggaccagctgaccgaccct
ccgggggtccggcgcgtgtaccacatccaggcgggcctaccg
gacccgttccagccccccagcctcccgatcacggtttactac
gccgtgttggagcgcgcctgccgcagcgtgctcctaaacgca
ccgtcggaggccccccagattgtccgcggggcctccgaagac
gtccggaaacaaccctacaacctgaccatcgcttggtttcgg
atgggaggcaactgtgctatccccatcacggtcatggagtac
accgaatgctcctacaacaagtctctgggggcctgtcccatc
cgaacgcagccccgctggaactactatgacagcttcagcgcc
gtcagcgaggataacctggggttcctgatgcacgcccccgcg
tttgagaccgccggcacgtacctgcggctcgtgaagataaac
gactggacggagattacacagtttatcctggagcaccgagcc
aagggctcctgtaagtacgccctcccgctgcgcatccccccg
tcagcctgcctctccccccaggcctaccagcagggggtgacg
gtggacagcatcgggatgctgccccgcttcatccccgagaac
cagcgcaccgtcgccgtatacagcttgaagatcgccgggtgg
cacgggccccacctgctggtgggcagcagcggcctgagcagg
tacgtggccaggctgagcagcaacagcaggatcatcaaccac
cagcacggcaccatgcagaacctgcacgacagctgcagcagg
aacctgtacgtgagcctgctgctgctgtacaagaccttcggc
aggaagctgcacctgtacagccaccccatcatcctgaagacc
aagaggtggggctacagcctgaacttcatgggctacgtgatc
ggcagctggggcagcctgccccaggaccacatcatccagaag
atcaaggagtgcttcaggaagctgcccgtgaacaggcccatc
gactggaaggtgtgccagaggatcgtgggcctgctgggcttc
gccgcccccttcacccagtgcggctaccccgccctgatgccc
ctgtacgcctgcatccagagcaagcaggccttcaccttcagc
cccacctacaaggccttcctgagcaagcagtacctgaacctg
taccccgtggccaggcagaggcccggcctgtgccaggtgttc
gccgacgccacccccaccggctggggcctggccatgggccac
cagaggatgaggggcaccttcgtggcccccctgcccatccac
accgccgagctgctggccgcctgcttcgccaggagcaggagc
ggcgccaagatcctgggcaccgacaacagcgtggtgctgagc
aggaagtacaccagcttcccctggctgctgggctgcgccgcc
aactggatcctgaggggcaccagcttcgtgtacgtgcccagc
gccctgaaccccgccgacgaccccagcaggggcaggctgggc
ctgagcaggcccctgctgaggctgcccttcaggcccaccacc
ggcaggaccagcctgtacgccgtgagccccagcgtggggccc
aaggccccatacacgagcaccctgctgcccccggagctgtcc
gagacccccaacgccacgcagccagaactcgccccggaagac
cccgaggattcggccctcttggaggaccccgtggggacggtg
gcgccgcaaatcccaccaaactggcacatcccgtcgatccag
gacgccgcgacgccttaccatcccccggccaccccgaacaac
atgggcctgatcgccggcgcggtgggcggcagtctcctggca
gccctggtcatttgcggaattgtgtactggatgcaccgccgc
actcggaaagccccaaagcgcatacgcctcccccacatccgg
gaagacgaccagccgtcctcgcaccagcccttgttttactag HBV PolN v2
HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPE amino acid
WQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF sequence
YPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL (SEQ ID NO:
YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYC 173)
LTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT HBV2 amino acid
YLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETT sequence
RSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVN (SEQ ID NO:
LLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNRENTAES 174)
RLVVDFSQFSRGITRVSWPKFAVPNIQSLTNILSSNISWLSL (Pol N
DVQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPT (italics)-Pol C
GWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILG (underlined)-
TDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPAD Core)
DVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGR
ETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDR GR HBV3 amino acid
HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNIGNIPE sequence
WQTPSFPKIHIQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF (SEQ ID NO:
YPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL 175)
YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYC (Pol N
LTHLVNILEDWGPCDEHGEHHIRIPRTPARVTQAFTFSPTYK (italics)-Pol C
AFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMR (underlined)-
GTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYT Core)
SFPWLLGCAANWILRGTSFVYVPSALNPADDVGSNLEDPASR
ELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGV
WIRTPPAYRPPNAPILSTLPETTVVRRRDRGR HBV2 nucleic
tatctgccgctggataaaggcattaaaccgtattatccggaacatgcggtgaaccattatttt
acid sequence
cagacccgccattatctgcataccctgtggaaagcgggcattctgtataaacgcgaaacc (SEQ
ID NO: acccgcagcgcgagcttttgcggcagcccgtatagctgggaacaggaactgcagcatg
176) gcagctgctggtggctgcagtttcgcaacagcaaaccgtgcagcgaatattgcctgaccc
atctggtgaacctgctggaagattggggaccgtgcgatgaacatggcgaacatcatattc
gcattccgcgcaccccggcgcgcgtgaccggcggcgtgtttctggtggataaaaacccg
cataacaccgcggaaagccgcctggtggtggattttagccagtttagccgcggcattacc
cgcgtgagctggccgaaatttgcggtgccgaacctgcagagcctgaccaacctgctgag
cagcaacctgagctggctgagcctggatgtgcaggcgtttacctttagcccgacctataaa
gcgtttctgagcaaacagtatctgaacctgtatccggtggcgcgccagcgcccgggcctg
tgccaggtgtttgcggatgcgaccccgaccggctggggcctggcgatgggccatcagc
gcatgcgcggcacctttgtggcgccgctgccgattcataccgcggaactgctggcggcg
tgctttgcgcgcagccgcagcggcgcgaaaattctgggcaccgataacagcgtggtgct
gagccgcaaatataccagctttccgtggctgctgggctgcgcggcgaactggattctgcg
cggcaccagctttgtgtatgtgccgagcgcgctgaacccggcggatgatgtgggcagca
acctggaagatccggcgagccgcgaactggtggtgagctatgtgaacgtgaacatggg
cctgaaaattcgccagctgctgtggtttcatattagctgcctgacctttggccgcgaaaccg
tgattgaatatctggtgagctttggcgtgtggattcgcaccccgccggcgtatcgcccgcc
gaacgcgccgattctgagcaccctgccggaaaccaccgtggtgcgccgccgcgatcgg ggccgc
HBV3 nucleic
cattttcgcaaactgctgctgctggatgaagaagcgggaccgctggaagaagaactgcc acid
sequence gcgcctggcggatgaaggcctgaaccgccgcgtggcggaagatctgaacctgggcaa
(SEQ ID NO:
cctgccggaatggcagaccccgagctttccgaaaattcatctgcaggaagatattgtggat 177)
cgctgcaaacagtttgtgggaccgctgaccgtgaacgaaaaacgccgcctgaaactgatt
atgccggcgcgcttttatccgaacgtgaccaaatatctgccgctggataaaggcattaaac
cgtattatccggaacatgcggtgaaccattattttcagacccgccattatctgcataccctgt
ggaaagcgggcattctgtataaacgcgaaaccacccgcagcgcgagcttttgcggcagc
ccgtatagctgggaacaggaactgcagcatggcagctgctggtggctgcagtttcgcaa
cagcaaaccgtgcagcgaatattgcctgacccatctggtgaacctgctggaagattgggg
accgtgcgatgaacatggcgaacatcatattcgcattccgcgcaccccggcgcgcgtga
cccaggcgtttacctttagcccgacctataaagcgtttctgagcaaacagtatctgaacctg
tatccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcgaccccga
ccggctggggcctggcgatgggccatcagcgcatgcgcggcacctttgtggcgccgct
gccgattcataccgcggaactgctggcggcgtgctttgcgcgcagccgcagcggcgcg
aaaattctgggcaccgataacagcgtggtgctgagccgcaaatataccagctttccgtgg
ctgctgggctgcgcggcgaactggattctgcgcggcaccagctttgtgtatgtgccgagc
gcgctgaacccggcggatgatgtgggcagcaacctggaagatccggcgagccgcgaa
ctggtggtgagctatgtgaacgtgaacatgggcctgaaaattcgccagctgctgtggtttc
atattagctgcctgacctttggccgcgaaaccgtgattgaatatctggtgagctttggcgtgt
ggattcgcaccccgccggcgtatcgcccgccgaacgcgccgattctgagcaccctgcc
ggaaaccaccgtggtgcgccgccgagatcgaggccgc HBV2 PolN amino
YLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETT acid sequence
RSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVN (SEQ ID NO:
LLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAES 178)
RLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSL DV HBV2 PolC amino
QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGW acid sequence
GLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTD (SEQ ID NO:
NSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD 179) HBV2 Core amino
VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRE acid sequence
TVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRG (SEQ ID NO: R 180) HBV3
PolN amino HFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLPE acid sequence
WQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARF (SEQ ID NO:
YPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGIL 181)
YKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYC
LTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT HBV3 PolC amino
QAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGW acid sequence
GLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTD (SEQ ID NO:
NSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD 182) HBV3 Core amino
VGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRE acid sequence
TVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRG (SEQ ID NO: R 183)
gD-HBV2 nucleic atggggggggctgccgccaggttgggggccgtgattttgttt acid
sequence gtcgtcatagtgggcctccatggggtccgcggcaaatatgcc (SEQ ID NO:
ttggcggatgcctctctcaagatggccgaccccaatcgcttt 184)
cgcggcaaagaccttccggtcctggaccagctgaccgaccct
ccgggggtccggcgcgtgtaccacatccaggcgggcctaccg
gacccgttccagccccccagcctcccgatcacggtttactac
gccgtgttggagcgcgcctgccgcagcgtgctcctaaacgca
ccgtcggaggccccccagattgtccgcggggcctccgaagac
gtccggaaacaaccctacaacctgaccatcgcttggtttcgg
atgggaggcaactgtgctatccccatcacggtcatggagtac
accgaatgctcctacaacaagtctctgggggcctgtcccatc
cgaacgcagccccgctggaactactatgacagcttcagcgcc
gtcagcgaggataacctggggttcctgatgcacgcccccgcg
tttgagaccgccggcacgtacctgcggctcgtgaagataaac
gactggacggagattacacagtttatcctggagcaccgagcc
aagggctcctgtaagtacgccctcccgctgcgcatccccccg
tcagcctgcctctccccccaggcctaccagcagggggtgacg
gtggacagcatcgggatgctgccccgcttcatccccgagaac
cagcgcaccgtcgccgtatacagcttgaagatcgccgggtgg
cacgggccctatctgccgctggataaaggcattaaaccgtat
tatccggaacatgcggtgaaccattattttcagacccgccat
tatctgcataccctgtggaaagcgggcattctgtataaacgc
gaaaccacccgcagcgcgagcttttgcggcagcccgtatagc
tgggaacaggaactgcagcatggcagctgctggtggctgcag
tttcgcaacagcaaaccgtgcagcgaatattgcctgacccat
ctggtgaacctgctggaagattggggaccgtgcgatgaacat
ggcgaacatcatattcgcattccgcgcaccccggcgcgcgtg
accggcggcgtgtttctggtggataaaaacccgcataacacc
gcggaaagccgcctggtggtggattttagccagtttagccgc
ggcattacccgcgtgagctggccgaaatttgcggtgccgaac
ctgcagagcctgaccaacctgctgagcagcaacctgagctgg
ctgagcctggatgtgcaggcgtttacctttagcccgacctat
aaagcgtttctgagcaaacagtatctgaacctgtatccggtg
gcgcgccagcgcccgggcctgtgccaggtgtttgcggatgcg
accccgaccggctggggcctggcgatgggccatcagcgcatg
cgcggcacctttgtggcgccgctgccgattcataccgcggaa
ctgctggcggcgtgctttgcgcgcagccgcagcggcgcgaaa
attctgggcaccgataacagcgtggtgctgagccgcaaatat
accagctttccgtggctgctgggctgcgcggcgaactggatt
ctgcgcggcaccagctttgtgtatgtgccgagcgcgctgaac
ccggcggatgatgtgggcagcaacctggaagatccggcgagc
cgcgaactggtggtgagctatgtgaacgtgaacatgggcctg
aaaattcgccagctgctgtggtttcatattagctgcctgacc
tttggccgcgaaaccgtgattgaatatctggtgagctttggc
gtgtggattcgcaccccgccggcgtatcgcccgccgaacgcg
ccgattctgagcaccctgccggaaaccaccgtggtgcgccgc
cgcgatcggggccgcgggcccaaggccccatacacgagcacc
ctgctgcccccggagctgtccgagacccccaacgccacgcag
ccagaactcgccccggaagaccccgaggattcggccctcttg
gaggaccccgtggggacggtggcgccgcaaatcccaccaaac
tggcacatcccgtcgatccaggacgccgcgacgccttaccat
cccccggccaccccgaacaacatgggcctgatcgccggcgcg
gtgggcggcagtctcctggcagccctggtcatttgcggaatt
gtgtactggatgcaccgccgcactcggaaagccccaaagcgc
atacgcctcccccacatccgggaagacgaccagccgtcctcg caccagcccttgttttactag
gD-HBV2 amino MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid
sequence RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO:
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR 185)
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN
QRTVAVYSLKIAGWHGPYLPLDKGIKPYYPEHAVNHYFQTRH
YLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQ
FRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARV
TGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPN
LQSLTNLLSSNLSWLSLDVQAFTFSPTYKAFLSKQYLNLYPV
ARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAE
LLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWI
LRGTSFVYVPSALNPADDVGSNLEDPASRELVVSYVNVNMGL
KIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNA
PILSTLPETTVVRRRDRGRGPKAPYTSTLLPPELSETPNATQ
PELAPEDPEDSALLEDPVGTVAPQIPPNWHIPSIQDAATPYH
PPATPNNMGLIAGAVGGSLLAALVICGIVYWMHRRTRKAPKR IRLPHIREDDQPSSHQPLFY*
gD-HBV3 nucleic atggggggggctgccgccaggttgggggccgtgattttgttt acid
sequence gtcgtcatagtgggcctccatggggtccgcggcaaatatgcc (SEQ ID NO:
ttggcggatgcctctctcaagatggccgaccccaatcgcttt 186)
cgcggcaaagaccttccggtcctggaccagctgaccgaccct
ccgggggtccggcgcgtgtaccacatccaggcgggcctaccg
gacccgttccagccccccagcctcccgatcacggtttactac
gccgtgttggagcgcgcctgccgcagcgtgctcctaaacgca
ccgtcggaggccccccagattgtccgcggggcctccgaagac
gtccggaaacaaccctacaacctgaccatcgcttggtttcgg
atgggaggcaactgtgctatccccatcacggtcatggagtac
accgaatgctcctacaacaagtctctgggggcctgtcccatc
cgaacgcagccccgctggaactactatgacagcttcagcgcc
gtcagcgaggataacctggggttcctgatgcacgcccccgcg
tttgagaccgccggcacgtacctgcggctcgtgaagataaac
gactggacggagattacacagtttatcctggagcaccgagcc
aagggctcctgtaagtacgccctcccgctgcgcatccccccg
tcagcctgcctctccccccaggcctaccagcagggggtgacg
gtggacagcatcgggatgctgccccgcttcatccccgagaac
cagcgcaccgtcgccgtatacagcttgaagatcgccgggtgg
cacgggccccattttcgcaaactgctgctgctggatgaagaa
gcgggaccgctggaagaagaactgccgcgcctggcggatgaa
ggcctgaaccgccgcgtggcggaagatctgaacctgggcaac
ctgccggaatggcagaccccgagctttccgaaaattcatctg
caggaagatattgtggatcgctgcaaacagtttgtgggaccg
ctgaccgtgaacgaaaaacgccgcctgaaactgattatgccg
gcgcgcttttatccgaacgtgaccaaatatctgccgctggat
aaaggcattaaaccgtattatccggaacatgcggtgaaccat
tattttcagacccgccattatctgcataccctgtggaaagcg
ggcattctgtataaacgcgaaaccacccgcagcgcgagcttt
tgcggcagcccgtatagctgggaacaggaactgcagcatggc
agctgctggtggctgcagtttcgcaacagcaaaccgtgcagc
gaatattgcctgacccatctggtgaacctgctggaagattgg
ggaccgtgcgatgaacatggcgaacatcatattcgcattccg
cgcaccccggcgcgcgtgacccaggcgtttacctttagcccg
acctataaagcgtttctgagcaaacagtatctgaacctgtat
ccggtggcgcgccagcgcccgggcctgtgccaggtgtttgcg
gatgcgaccccgaccggctggggcctggcgatgggccatcag
cgcatgcgcggcacctttgtggcgccgctgccgattcatacc
gcggaactgctggcggcgtgctttgcgcgcagccgcagcggc
gcgaaaattctgggcaccgataacagcgtggtgctgagccgc
aaatataccagctttccgtggctgctgggctgcgcggcgaac
tggattctgcgcggcaccagctttgtgtatgtgccgagcgcg
ctgaacccggcggatgatgtgggcagcaacctggaagatccg
gcgagccgcgaactggtggtgagctatgtgaacgtgaacatg
ggcctgaaaattcgccagctgctgtggtttcatattagctgc
ctgacctttggccgcgaaaccgtgattgaatatctggtgagc
tttggcgtgtggattcgcaccccgccggcgtatcgcccgccg
aacgcgccgattctgagcaccctgccggaaaccaccgtggtg
cgccgccgagatcgaggccgcgggcccaaggccccatacacg
agcaccctgctgcccccggagctgtccgagacccccaacgcc
acgcagccagaactcgccccggaagaccccgaggattcggcc
ctcttggaggaccccgtggggacggtggcgccgcaaatccca
ccaaactggcacatcccgtcgatccaggacgccgcgacgcct
taccatcccccggccaccccgaacaacatgggcctgatcgcc
ggcgcggtgggcggcagtctcctggcagccctggtcatttgc
ggaattgtgtactggatgcaccgccgcactcggaaagcccca
aagcgcatacgcctcccccacatccgggaagacgaccagccg
tcctcgcaccagcccttgttttactag gD-HBV3 amino
MGGAAARLGAVILFVVIVGLHGVRGKYALADASLKMADPNRF acid sequence
RGKDLPVLDQLTDPPGVRRVYHIQAGLPDPFQPPSLPITVYY (SEQ ID NO:
AVLERACRSVLLNAPSEAPQIVRGASEDVRKQPYNLTIAWFR 187)
MGGNCAIPITVMEYTECSYNKSLGACPIRTQPRWNYYDSFSA
VSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQFILEHRA
KGSCKYALPLRIPPSACLSPQAYQQGVTVDSIGMLPRFIPEN
QRTVAVYSLKIAGWHGPHFRKLLLLDEEAGPLEEELPRLADE
GLNRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGP
LTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNH
YFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHG
SCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIP
RTPARVTQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFA
DATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSG
AKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSA
LNPADDVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISc
LTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVV
RRRDRGRGPKAPYTSTLLPPELSETPNATQPELAPEDPEDSA
LLEDPVGTVAPQIPPNWHIPSIQDAATPYHPPATPNNMGLIA
GAVGGSLLAALVICGIVYWMHRRTRKAPKRIRLPHIREDDQP SSHQPLFY*
Embodiments
[0277] The following list of embodiments is intended to complement,
rather than displace or supersede, the previous descriptions.
[0278] Embodiment 1. A hepatitis B virus (HBV) Core protein
comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof. [0279] Embodiment 2. The HBV Core
protein of embodiment 1, wherein the immunogenic fragment comprises
any one of SEQ ID NOs: 20-54. [0280] Embodiment 3. A hepatitis B
virus (HBV) Core protein comprising the amino acid sequence of SEQ
ID NO: 180 or an immunogenic fragment thereof, or the amino acid
sequence of SEQ ID NO: 183 or an immunogenic fragment thereof.
[0281] Embodiment 4. A nucleic acid molecule encoding the HBV Core
protein of any one of embodiments 1-3. [0282] Embodiment 5. The
nucleic acid molecule of embodiment 4, wherein the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 7. [0283]
Embodiment 6. A vector comprising the nucleic acid molecule of
embodiment 4 or 5. [0284] Embodiment 7. The vector of embodiment 6,
wherein the vector is an adenoviral vector. [0285] Embodiment 8.
The vector of embodiment 7, wherein the adenoviral vector is an
AdC6 vector or AdC7 vector. [0286] Embodiment 9. A vaccine
comprising the vector of any one of embodiments 6-8. [0287]
Embodiment 10. A HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment
thereof. [0288] Embodiment 11. The HBV polymerase N-terminal domain
of embodiment 10, wherein the immunogenic fragment comprises any
one of SEQ ID NOs: 55-113. [0289] Embodiment 12. A HBV polymerase
N-terminal domain comprising the amino acid sequence of SEQ ID NO:
178 or an immunogenic fragment thereof, or the amino acid sequence
of SEQ ID NO: 181 or an immunogenic fragment thereof. [0290]
Embodiment 13. A HBV polymerase C-terminal domain comprising the
amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment
thereof. [0291] Embodiment 14. The HBV polymerase C-terminal domain
of embodiment 13, wherein the immunogenic fragment comprises any
one of SEQ ID NOs: 114-172. [0292] Embodiment 15. A HBV polymerase
C-terminal domain comprising the amino acid sequence of SEQ ID NO:
179 or an immunogenic fragment thereof, or the amino acid sequence
of SEQ ID NO: 182 or an immunogenic fragment thereof. [0293]
Embodiment 16. A nucleic acid molecule encoding the HBV polymerase
of any one of embodiments 10-15. [0294] Embodiment 17. The nucleic
acid molecule of embodiment 16, wherein the nucleic acid molecule
comprises the nucleotide sequence of SEQ ID NO: 9. [0295]
Embodiment 18. The nucleic acid molecule of embodiment 16, wherein
the nucleic acid molecule comprises the nucleotide sequence of SEQ
ID NO: 11. [0296] Embodiment 19. A vector comprising the nucleic
acid molecule of any one of embodiments 16-18. [0297] Embodiment
20. The vector of embodiment 19, wherein the vector is an
adenoviral vector. [0298] Embodiment 21. The vector of embodiment
20, wherein the adenoviral vector is an AdC6 vector or AdC7 vector.
[0299] Embodiment 22. A vaccine comprising the vector of any one of
embodiments 19-21. [0300] Embodiment 23. A fusion protein
comprising: [0301] one or more of an HBV Core protein comprising
the amino acid sequence of SEQ ID NO: 6 or an immunogenic fragment
thereof, an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof,
and an HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 10 or an immunogenic fragment thereof.
[0302] Embodiment 24. The fusion protein of embodiment 23,
comprising: [0303] (1) an HBV Core protein comprising the amino
acid sequence of SEQ ID NO: 6 or an immunogenic fragment thereof
and an HBV polymerase N-terminal domain comprising the amino acid
sequence of SEQ ID NO: 8 or an immunogenic fragment thereof; [0304]
(2) one or more of SEQ ID NOs: 20-54 (immunogenic fragments of SEQ
ID NO: 6) and one or more of SEQ ID NOs: 55-113 (immunogenic
fragments of SEQ ID NO: 8); [0305] (3) an HBV Core protein
comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof and an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; [0306] (4) one or more of SEQ ID NOs:
20-54 (immunogenic fragments of SEQ ID NO: 6) and one or more of
SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO: 10);
[0307] (5) an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 8 or an immunogenic fragment thereof
and an HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 10 or an immunogenic fragment thereof;
[0308] (6) one or more of SEQ ID NOs: 55-113 (immunogenic fragments
of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172
(immunogenic fragments of SEQ ID NO: 10); [0309] (7) an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof, an HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof, and an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; or [0310] (8) one or more of SEQ ID
NOs: 20-54 (immunogenic fragments of SEQ ID NO: 6), one or more of
SEQ ID NOs: 55-113 (immunogenic fragments of SEQ ID NO: 8), and one
or more of SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO:
10). [0311] Embodiment 25. A fusion protein comprising: [0312] an
HBV polymerase N-terminal domain comprising the amino acid sequence
of SEQ ID NO: 178 or an immunogenic fragment thereof, an HBV
polymerase C-terminal domain comprising the amino acid sequence of
SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 180 or an
immunogenic fragment thereof. [0313] Embodiment 26. The fusion
protein of embodiment 25, comprising the amino acid sequence of SEQ
ID NO: 174. [0314] Embodiment 27. A fusion protein comprising:
[0315] an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof,
an HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 182 or an immunogenic fragment thereof, and
an HBV Core protein comprising the amino acid sequence of SEQ ID
NO: 183 or an immunogenic fragment thereof. [0316] Embodiment 28.
The fusion protein of embodiment 27, comprising the amino acid
sequence of SEQ ID NO: 175. [0317] Embodiment 29. A fusion protein
comprising: [0318] an N-terminal herpes simplex virus (HSV)
glycoprotein (gD) sequence or a variant thereof [0319] an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof; and [0320] a C-terminal HSV gD
sequence or a variant thereof. [0321] Embodiment 30. The fusion
protein of embodiment 29, wherein the immunogenic fragment
comprises any one of SEQ ID NOs: 20-54. [0322] Embodiment 31. A
fusion protein comprising: [0323] an N-terminal HSV gD sequence or
a variant thereof [0324] an HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof; and [0325] a C-terminal HSV gD
protein sequence or a variant thereof. [0326] Embodiment 32. The
fusion protein of embodiment 31, wherein the immunogenic fragment
comprises any one of SEQ ID NOs: 55-113. [0327] Embodiment 33. A
fusion protein comprising: [0328] an N-terminal HSV gD sequence or
a variant thereof [0329] an HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; and [0330] a C-terminal HSV gD
protein sequence or a variant thereof. [0331] Embodiment 34. The
fusion protein of embodiment 33, wherein the immunogenic fragment
comprises any one of SEQ ID NOs: 114-172. [0332] Embodiment 35. A
fusion protein comprising: [0333] an N-terminal HSV gD sequence or
a variant thereof [0334] an HBV sequence comprising: [0335] (1) an
HBV Core protein comprising the amino acid sequence of SEQ ID NO: 6
or an immunogenic fragment thereof and an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof; [0336] (2) one or more of SEQ ID NOs:
20-54 (immunogenic fragments of SEQ ID NO: 6) and one or more of
SEQ ID NOs: 55-113 (immunogenic fragments of SEQ ID NO: 8); [0337]
(3) an HBV Core protein comprising the amino acid sequence of SEQ
ID NO: 6 or an immunogenic fragment thereof and an HBV polymerase
C-terminal domain comprising the amino acid sequence of SEQ ID NO:
10 or an immunogenic fragment thereof; [0338] (4) one or more of
SEQ ID NOs: 20-54 (immunogenic fragments of SEQ ID NO: 6) and one
or more of SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO:
10); [0339] (5) an HBV polymerase N-terminal domain comprising the
amino acid sequence of SEQ ID NO: 8 or an immunogenic fragment
thereof and an HBV polymerase C-terminal domain comprising the
amino acid sequence of SEQ ID NO: 10 or an immunogenic fragment
thereof; [0340] (6) one or more of SEQ ID NOs: 55-113 (immunogenic
fragments of SEQ ID NO: 8) and one or more of SEQ ID NOs: 114-172
(immunogenic fragments of SEQ ID NO: 10); [0341] (7) an HBV Core
protein comprising the amino acid sequence of SEQ ID NO: 6 or an
immunogenic fragment thereof, an HBV polymerase N-terminal domain
comprising the amino acid sequence of SEQ ID NO: 8 or an
immunogenic fragment thereof, and an HBV polymerase C-terminal
domain comprising the amino acid sequence of SEQ ID NO: 10 or an
immunogenic fragment thereof; or [0342] (8) one or more of SEQ ID
NOs: 20-54 (immunogenic fragments of SEQ ID NO: 6), one or more of
SEQ ID NOs: 55-113 (immunogenic fragments of SEQ ID NO: 8), and one
or more of SEQ ID NOs: 114-172 (immunogenic fragments of SEQ ID NO:
10) and [0343] a C-terminal HSV gD protein sequence or a variant
thereof. [0344] Embodiment 36. A fusion protein comprising: [0345]
an N-terminal HSV gD sequence or a variant thereof; [0346] an HBV
Core protein comprising the amino acid sequence of SEQ ID NO: 180
or an immunogenic fragment thereof, or the amino acid sequence of
SEQ ID NO: 183 or an immunogenic fragment thereof; and [0347] a
C-terminal HSV gD sequence or a variant thereof. [0348] Embodiment
37. A fusion protein comprising: [0349] an N-terminal HSV gD
sequence or a variant thereof [0350] an HBV polymerase N-terminal
domain comprising the amino acid sequence of SEQ ID NO: 178 or an
immunogenic fragment thereof, or the amino acid sequence of SEQ ID
NO: 181 or an immunogenic fragment thereof; and [0351] a C-terminal
HSV gD protein sequence or a variant thereof. [0352] Embodiment 38.
A fusion protein comprising: [0353] an N-terminal HSV gD sequence
or a variant thereof [0354] an HBV polymerase C-terminal domain
comprising the amino acid sequence of SEQ ID NO: 179 or an
immunogenic fragment thereof, or the amino acid sequence of SEQ ID
NO: 182 or an immunogenic fragment thereof; and [0355] a C-terminal
HSV gD protein sequence or a variant thereof. [0356] Embodiment 39.
A fusion protein comprising: [0357] an N-terminal HSV gD sequence
or a variant thereof [0358] an HBV sequence comprising: [0359] (1)
an HBV polymerase N-terminal domain comprising the amino acid
sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, an
HBV polymerase C-terminal domain comprising the amino acid sequence
of SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV
Core protein comprising the amino acid sequence of SEQ ID NO: 180
or an immunogenic fragment thereof; or [0360] (2) an HBV polymerase
N-terminal domain comprising the amino acid sequence of SEQ ID NO:
181 or an immunogenic fragment thereof, an HBV polymerase
C-terminal domain comprising the amino acid sequence of SEQ ID NO:
182 or an immunogenic fragment thereof, and an HBV Core protein
comprising the amino acid sequence of SEQ ID NO: 183 or an
immunogenic fragment thereof; and [0361] a C-terminal HSV gD
protein sequence or a variant thereof. [0362] Embodiment 40. The
fusion protein of embodiment 39, wherein the HBV sequence comprises
an HBV polymerase N-terminal domain comprising the amino acid
sequence of SEQ ID NO: 178 or an immunogenic fragment thereof, an
HBV polymerase C-terminal domain comprising the amino acid sequence
of SEQ ID NO: 179 or an immunogenic fragment thereof, and an HBV
Core protein comprising the amino acid sequence of SEQ ID NO: 180
or an immunogenic fragment thereof. [0363] Embodiment 41. The
fusion protein of embodiment 40, wherein the HBV sequence comprises
the amino acid sequence of SEQ ID NO: 174. [0364] Embodiment 42.
The fusion protein of embodiment 39, wherein the HBV sequence
comprises an HBV polymerase N-terminal domain comprising the amino
acid sequence of SEQ ID NO: 181 or an immunogenic fragment thereof,
an HBV polymerase C-terminal domain comprising the amino acid
sequence of SEQ ID NO: 182 or an immunogenic fragment thereof, and
an HBV Core protein comprising the amino acid sequence of SEQ ID
NO: 183 or an immunogenic fragment thereof [0365] Embodiment 43.
The fusion protein of embodiment 42, wherein the HBV sequence
comprises the amino acid sequence of SEQ ID NO: 175. [0366]
Embodiment 44. The fusion protein of any one of embodiments 29-43,
wherein the N-terminal HSV gD sequence comprises the amino acid
sequence of SEQ ID NO: 12. [0367] Embodiment 45. The fusion protein
of any one of embodiments 29-43, wherein the N-terminal HSV gD
sequence comprises amino acid residues 26-269 of SEQ ID NO: 12.
[0368] Embodiment 46. The fusion protein of any one of embodiments
29-45, wherein the C-terminal HSV gD sequence comprises the
transmembrane domain of the HSV gD. [0369] Embodiment 47. The
fusion protein of any one of embodiments 29-46, wherein the
C-terminal HSV gD sequence comprises the amino acid sequence of SEQ
ID NO: 13. [0370] Embodiment 48. The fusion protein of any one of
embodiments 29-47, wherein the fusion protein comprises the amino
acid sequence of any one of SEQ ID NO: 14 or an immunogenic
fragment thereof, SEQ ID NO: 16 or an immunogenic fragment thereof,
or SEQ ID NO: 18 or an immunogenic fragment thereof. [0371]
Embodiment 49. The fusion protein of any one of embodiments 39-47,
wherein the fusion protein comprises the amino acid sequence of SEQ
ID NO: 185. [0372] Embodiment 50. The fusion protein of any one of
embodiments 39-47, wherein the fusion protein comprises the amino
acid sequence of SEQ ID NO: 187. [0373] Embodiment 51. A nucleic
acid molecule encoding the fusion protein of any one of embodiments
23-50. [0374] Embodiment 52. The nucleic acid molecule of
embodiment 51, wherein the nucleic acid molecule comprises the
nucleotide sequence of any one of SEQ ID NOs: 15, 17, or 19. [0375]
Embodiment 53. The nucleic acid molecule of embodiment 51, wherein
the nucleic acid molecule comprises the nucleotide sequence of SEQ
ID NO: 176.
[0376] Embodiment 54. The nucleic acid molecule of embodiment 51,
wherein the nucleic acid molecule comprises the nucleotide sequence
of SEQ ID NO: 177. [0377] Embodiment 55. The nucleic acid molecule
of embodiment 51, wherein the nucleic acid molecule comprises the
nucleotide sequence of SEQ ID NO: 184. [0378] Embodiment 56. The
nucleic acid molecule of embodiment 51, wherein the nucleic acid
molecule comprises the nucleotide sequence of SEQ ID NO: 186.
[0379] Embodiment 57. A vector comprising the nucleic acid molecule
of any one of embodiments 51-56. [0380] Embodiment 58. The vector
of embodiment 57, wherein the vector is an adenoviral vector.
[0381] Embodiment 59. The vector of embodiment 58, wherein the
adenoviral vector is an AdC6 vector or AdC7 vector. [0382]
Embodiment 60. A vaccine comprising the vector of any one of
embodiments 57-59. [0383] Embodiment 61. A method of inducing an
immune response to HBV in a subject, the method comprising
providing to the subject an effective amount of the fusion protein
of any one of embodiments 23-50, the nucleic acid molecule of any
one of embodiments 51-56, the vector of any one of embodiments
57-59, or the vaccine of embodiment 60 to thereby induce an immune
response to HBV. [0384] Embodiment 62. The method of embodiment 61,
wherein the vaccine comprises an AdC6 vector comprising a fusion
protein comprising the amino acid sequence of any one of SEQ ID
NOs: 14, 16, or 18, or an immunogenic fragment thereof. [0385]
Embodiment 63. The method of embodiment 62, further comprising
providing to the subject, subsequent to providing the vaccine
comprising the AdC6 vector, a vaccine comprising an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
any one of SEQ ID NOs: 14, 16, or 18, or an immunogenic fragment
thereof. [0386] Embodiment 64. The method of embodiment 61, wherein
the vaccine comprises an AdC7 vector comprising a fusion protein
comprising the amino acid sequence of any one of SEQ ID NOs: 14,
16, or 18, or an immunogenic fragment thereof. [0387] Embodiment
65. The method of embodiment 64, further comprising providing to
the subject, subsequent to providing the vaccine comprising the
AdC7 vector, a vaccine comprising an AdC6 vector comprising a
fusion protein comprising the amino acid sequence of any one of SEQ
ID NOs: 14, 16, or 18, or an immunogenic fragment thereof. [0388]
Embodiment 66. The method of embodiment 61, wherein the vaccine
comprises an AdC6 vector comprising a fusion protein comprising the
amino acid sequence of SEQ ID NO: 185. [0389] Embodiment 67. The
method of embodiment 66, further comprising providing to the
subject, subsequent to providing the vaccine comprising the AdC6
vector, a vaccine comprising an AdC7 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 185.
[0390] Embodiment 68. The method of embodiment 61, wherein the
vaccine comprises an AdC7 vector comprising a fusion protein
comprising the amino acid sequence of SEQ ID NO: 185. [0391]
Embodiment 69. The method of embodiment 68, further comprising
providing to the subject, subsequent to providing the vaccine
comprising the AdC7 vector, a vaccine comprising an AdC6 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 185. [0392] Embodiment 70. The method of embodiment 61,
wherein the vaccine comprises an AdC6 vector comprising a fusion
protein comprising the amino acid sequence of SEQ ID NO: 187.
[0393] Embodiment 71. The method of embodiment 70, further
comprising providing to the subject, subsequent to providing the
vaccine comprising the AdC6 vector, a vaccine comprising an AdC7
vector comprising a fusion protein comprising the amino acid
sequence of SEQ ID NO: 187. [0394] Embodiment 72. The method of
embodiment 61, wherein the vaccine comprises an AdC7 vector
comprising a fusion protein comprising the amino acid sequence of
SEQ ID NO: 187. [0395] Embodiment 73. The method of embodiment 72,
further comprising providing to the subject, subsequent to
providing the vaccine comprising the AdC7 vector, a vaccine
comprising an AdC6 vector comprising a fusion protein comprising
the amino acid sequence of SEQ ID NO: 187. [0396] Embodiment 74.
The method of any one of embodiments 61-73, wherein the amino acid
sequence of any one of SEQ ID NOs: 14, 16, 18, 185, or 187, or an
immunogenic fragment thereof, does not contain the N-terminal 25
amino acid signal peptide.
Sequence CWU 1
1
2331185PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Met 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 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 Asp Arg Gly Arg
Ser Pro Arg Arg145 150 155 160Arg Thr Pro Ser Pro Arg Arg Arg Arg
Ser Gln Ser Pro Arg Arg Arg 165 170 175Arg Ser Gln Ser Arg Glu Ser
Gln Cys 180 1852183PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 2Met 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 1803182PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 3Met 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 Ser Gln 165 170 175Ser Arg Glu Ser
Gln Cys 1804183PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 4Met 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 Gly 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 1805184PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
polypeptide"VARIANT(12)..(12)/replace="S"VARIANT(27)..(27)/replace="I"VAR-
IANT(66)..(66)/replace="N"VARIANT(73)..(73)/replace="S" or
"G"VARIANT(82)..(82)/replace="E"VARIANT(86)..(86)/replace="S"VARIANT(90).-
.(90)/replace="V"VARIANT(96)..(96)/replace="F"VARIANT(115)..(115)/replace=-
"I"VARIANT(152)..(153)/replace=" "SITE(1)..(184)/note="Variant
residues given in the sequence have no preference with respect to
those in the annotations for variant positions" 5Met 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 Asp Leu Leu Asp Thr 20 25 30Ala Ser
Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser 35 40 45Pro
His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu 50 55
60Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala Ser65
70 75 80Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys
Ile 85 90 95Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly
Arg Glu 100 105 110Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile Arg Thr Pro 115 120 125Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile
Leu Ser Thr Leu Pro Glu 130 135 140Thr Thr Val Val Arg Arg Arg Asp
Arg Gly Arg Ser Pro Arg Arg Arg145 150 155 160Thr Pro Ser Pro Arg
Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg 165 170 175Ser Gln Ser
Arg Glu Ser Gln Cys 1806184PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 6Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu
Leu Leu Ser1 5 10 15Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp
Leu Leu Asp Thr 20 25 30Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser
Pro Glu His Cys Ser 35 40 45Pro His His Thr Ala Leu Arg Gln Ala Ile
Leu Cys Trp Gly Glu Leu 50 55 60Met Thr Leu Ala Thr Trp Val Gly Ser
Asn Leu Glu Asp Pro Ala Ser65 70 75 80Arg Glu Leu Val Val Ser Tyr
Val Asn Val Asn Met Gly Leu Lys Ile 85 90 95Arg Gln Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu 100 105 110Thr Val Ile Glu
Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120 125Pro Ala
Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu 130 135
140Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg
Arg145 150 155 160Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro
Arg Arg Arg Arg 165 170 175Ser Gln Ser Arg Glu Ser Gln Cys
1807552DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 7gacatcgacc cctacaagga
gttcggcgcc accgtggagc tgctgagctt cctgcccagc 60gacttcttcc ccagcatcag
ggacctgctg gacaccgcca gcgccctgta cagggaggcc 120ctggagagcc
ccgagcactg cagcccccac cacaccgccc tgaggcaggc catcctgtgc
180tggggcgagc tgatgaccct ggccacctgg gtgggcagca acctggagga
ccccgccagc 240agggagctgg tggtgagcta cgtgaacgtg aacatgggcc
tgaagatcag gcagctgctg 300tggttccaca tcagctgcct gaccttcggc
agggagaccg tgatcgagta cctggtgagc 360ttcggcgtgt ggatcaggac
cccccccgcc tacaggcccc ccaacgcccc catcctgagc 420accctgcccg
agaccaccgt ggtgaggagg agggacaggg gcaggagccc caggaggagg
480acccccagcc ccaggaggag gaggagccag agccccagga ggaggaggag
ccagagcagg 540gagagccagt gc 5528305PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 8Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu
Asp Glu Glu1 5 10 15Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala
Asp Glu Gly Leu 20 25 30Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly
Asn Leu Asn Val Ser 35 40 45Ile Pro Trp Thr His Lys Val Gly Asn Phe
Thr Gly Leu Tyr Ser Ser 50 55 60Thr Val Pro Val Phe Asn Pro Glu Trp
Gln Thr Pro Ser Phe Pro Lys65 70 75 80Ile His Leu Gln Glu Asp Ile
Val Asp Arg Cys Lys Gln Phe Val Gly 85 90 95Pro Leu Thr Val Asn Glu
Lys Arg Arg Leu Lys Leu Ile Met Pro Ala 100 105 110Arg Phe Tyr Pro
Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile 115 120 125Lys Pro
Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg 130 135
140His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg
Glu145 150 155 160Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr
Ser Trp Glu Gln 165 170 175Glu Leu Gln His Gly Ser Cys Trp Trp Leu
Gln Phe Arg Asn Ser Lys 180 185 190Pro Cys Ser Glu Tyr Cys Leu Thr
His Leu Val Asn Leu Leu Glu Asp 195 200 205Trp Gly Pro Cys Asp Glu
His Gly Glu His His Ile Arg Ile Pro Arg 210 215 220Thr Pro Ala Arg
Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro225 230 235 240His
Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser 245 250
255Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu
260 265 270Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu
Ser Leu 275 280 285Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu His
Pro Ala Ala Met 290 295 300Pro3059915DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 9cccctgagct accagcactt caggaagctg ctgctgctgg
acgaggaggc cggccccctg 60gaggaggagc tgcccaggct ggccgacgag ggcctgaaca
ggagggtggc cgaggacctg 120aacctgggca acctgaacgt gagcatcccc
tggacccaca aggtgggcaa cttcaccggc 180ctgtacagca gcaccgtgcc
cgtgttcaac cccgagtggc agacccccag cttccccaag 240atccacctgc
aggaggacat cgtggacagg tgcaagcagt tcgtgggccc cctgaccgtg
300aacgagaaga ggaggctgaa gctgatcatg cccgccaggt tctaccccaa
cgtgaccaag 360tacctgcccc tggacaaggg catcaagccc tactaccccg
agcacgccgt gaaccactac 420ttccagacca ggcactacct gcacaccctg
tggaaggccg gcatcctgta caagagggag 480accaccagga gcgccagctt
ctgcggcagc ccctacagct gggagcagga gctgcagcac 540ggcagctgct
ggtggctgca gttcaggaac agcaagccct gcagcgagta ctgcctgacc
600cacctggtga acctgctgga ggactggggc ccctgcgacg agcacggcga
gcaccacatc 660aggatcccca ggacccccgc cagggtgacc ggcggcgtgt
tcctggtgga caagaacccc 720cacaacaccg ccgagagcag gctggtggtg
gacttcagcc agttcagcag gggcatcacc 780agggtgagct ggcccaagtt
cgccgtgccc aacctgcaga gcctgaccaa cctgctgagc 840agcaacctga
gctggctgag cctggacgtg agcgccgcct tctaccacat ccccctgcac
900cccgccgcca tgccc 91510303PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 10His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val
Ala Arg Leu1 5 10 15Ser Ser Asn Ser Arg Ile Ile Asn His Gln His Gly
Thr Met Gln Asn 20 25 30Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val
Ser Leu Leu Leu Leu 35 40 45Tyr Lys Thr Phe Gly Arg Lys Leu His Leu
Tyr Ser His Pro Ile Ile 50 55 60Leu Lys Thr Lys Arg Trp Gly Tyr Ser
Leu Asn Phe Met Gly Tyr Val65 70 75 80Ile Gly Ser Trp Gly Ser Leu
Pro Gln Asp His Ile Ile Gln Lys Ile 85 90 95Lys Glu Cys Phe Arg Lys
Leu Pro Val Asn Arg Pro Ile Asp Trp Lys 100 105 110Val Cys Gln Arg
Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr 115 120 125Gln Cys
Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser 130 135
140Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser
Lys145 150 155 160Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg
Pro Gly Leu Cys 165 170 175Gln Val Phe Ala Asp Ala Thr Pro Thr Gly
Trp Gly Leu Ala Met Gly 180 185 190His Gln Arg Met Arg Gly Thr Phe
Val Ala Pro Leu Pro Ile His Thr 195 200 205Ala Glu Leu Leu Ala Ala
Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys 210 215 220Ile Leu Gly Thr
Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser225 230 235 240Phe
Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr 245 250
255Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser
260 265 270Arg Gly Arg Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro
Phe Arg 275 280 285Pro Thr Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser
Pro Ser Val 290 295 30011909DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 11cacctgctgg tgggcagcag cggcctgagc aggtacgtgg
ccaggctgag cagcaacagc 60aggatcatca accaccagca cggcaccatg cagaacctgc
acgacagctg cagcaggaac 120ctgtacgtga gcctgctgct gctgtacaag
accttcggca ggaagctgca cctgtacagc 180caccccatca tcctgaagac
caagaggtgg ggctacagcc tgaacttcat gggctacgtg 240atcggcagct
ggggcagcct gccccaggac cacatcatcc agaagatcaa ggagtgcttc
300aggaagctgc ccgtgaacag gcccatcgac tggaaggtgt gccagaggat
cgtgggcctg 360ctgggcttcg ccgccccctt cacccagtgc ggctaccccg
ccctgatgcc cctgtacgcc 420tgcatccaga gcaagcaggc cttcaccttc
agccccacct acaaggcctt cctgagcaag 480cagtacctga acctgtaccc
cgtggccagg cagaggcccg gcctgtgcca ggtgttcgcc 540gacgccaccc
ccaccggctg gggcctggcc atgggccacc agaggatgag gggcaccttc
600gtggcccccc tgcccatcca caccgccgag ctgctggccg cctgcttcgc
caggagcagg 660agcggcgcca agatcctggg caccgacaac agcgtggtgc
tgagcaggaa gtacaccagc 720ttcccctggc tgctgggctg cgccgccaac
tggatcctga ggggcaccag cttcgtgtac 780gtgcccagcg ccctgaaccc
cgccgacgac cccagcaggg gcaggctggg cctgagcagg 840cccctgctga
ggctgccctt caggcccacc accggcagga ccagcctgta cgccgtgagc 900cccagcgtg
90912269PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 12Met Gly Gly Ala Ala
Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu
His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30Ser Leu Lys
Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45Val Leu
Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60Ile
Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile65 70 75
80Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu
85 90 95Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu
Asp 100
105 110Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met
Gly 115 120 125Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr
Glu Cys Ser 130 135 140Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg
Thr Gln Pro Arg Trp145 150 155 160Asn Tyr Tyr Asp Ser Phe Ser Ala
Val Ser Glu Asp Asn Leu Gly Phe 165 170 175Leu Met His Ala Pro Ala
Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190Val Lys Ile Asn
Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205Arg Ala
Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215
220Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val
Asp225 230 235 240Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn
Gln Arg Thr Val 245 250 255Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp
His Gly Pro 260 26513127PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 13Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro
Glu Leu Ser1 5 10 15Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro
Glu Asp Pro Glu 20 25 30Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr
Val Ala Pro Gln Ile 35 40 45Pro Pro Asn Trp His Ile Pro Ser Ile Gln
Asp Ala Ala Thr Pro Tyr 50 55 60His Pro Pro Ala Thr Pro Asn Asn Met
Gly Leu Ile Ala Gly Ala Val65 70 75 80Gly Gly Ser Leu Leu Ala Ala
Leu Val Ile Cys Gly Ile Val Tyr Trp 85 90 95Met His Arg Arg Thr Arg
Lys Ala Pro Lys Arg Ile Arg Leu Pro His 100 105 110Ile Arg Glu Asp
Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 115 120
12514580PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 14Met Gly Gly Ala Ala
Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu
His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30Ser Leu Lys
Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45Val Leu
Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60Ile
Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile65 70 75
80Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu
85 90 95Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu
Asp 100 105 110Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe
Arg Met Gly 115 120 125Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu
Tyr Thr Glu Cys Ser 130 135 140Tyr Asn Lys Ser Leu Gly Ala Cys Pro
Ile Arg Thr Gln Pro Arg Trp145 150 155 160Asn Tyr Tyr Asp Ser Phe
Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175Leu Met His Ala
Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190Val Lys
Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200
205Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro
210 215 220Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr
Val Asp225 230 235 240Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu
Asn Gln Arg Thr Val 245 250 255Ala Val Tyr Ser Leu Lys Ile Ala Gly
Trp His Gly Pro Asp Ile Asp 260 265 270Pro Tyr Lys Glu Phe Gly Ala
Thr Val Glu Leu Leu Ser Phe Leu Pro 275 280 285Ser Asp Phe Phe Pro
Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala 290 295 300Leu Tyr Arg
Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His305 310 315
320Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu
325 330 335Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg
Glu Leu 340 345 350Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys
Ile Arg Gln Leu 355 360 365Leu Trp Phe His Ile Ser Cys Leu Thr Phe
Gly Arg Glu Thr Val Ile 370 375 380Glu Tyr Leu Val Ser Phe Gly Val
Trp Ile Arg Thr Pro Pro Ala Tyr385 390 395 400Arg Pro Pro Asn Ala
Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val 405 410 415Val Arg Arg
Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser 420 425 430Pro
Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser 435 440
445Arg Glu Ser Gln Cys Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu
450 455 460Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu
Leu Ala465 470 475 480Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu
Asp Pro Val Gly Thr 485 490 495Val Ala Pro Gln Ile Pro Pro Asn Trp
His Ile Pro Ser Ile Gln Asp 500 505 510Ala Ala Thr Pro Tyr His Pro
Pro Ala Thr Pro Asn Asn Met Gly Leu 515 520 525Ile Ala Gly Ala Val
Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys 530 535 540Gly Ile Val
Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg545 550 555
560Ile Arg Leu Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln
565 570 575Pro Leu Phe Tyr 580151743DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 15atgggggggg ctgccgccag gttgggggcc gtgattttgt
ttgtcgtcat agtgggcctc 60catggggtcc gcggcaaata tgccttggcg gatgcctctc
tcaagatggc cgaccccaat 120cgctttcgcg gcaaagacct tccggtcctg
gaccagctga ccgaccctcc gggggtccgg 180cgcgtgtacc acatccaggc
gggcctaccg gacccgttcc agccccccag cctcccgatc 240acggtttact
acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg
300gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc
ctacaacctg 360accatcgctt ggtttcggat gggaggcaac tgtgctatcc
ccatcacggt catggagtac 420accgaatgct cctacaacaa gtctctgggg
gcctgtccca tccgaacgca gccccgctgg 480aactactatg acagcttcag
cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540cccgcgtttg
agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag
600attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc
cctcccgctg 660cgcatccccc cgtcagcctg cctctccccc caggcctacc
agcagggggt gacggtggac 720agcatcggga tgctgccccg cttcatcccc
gagaaccagc gcaccgtcgc cgtatacagc 780ttgaagatcg ccgggtggca
cgggcccgac atcgacccct acaaggagtt cggcgccacc 840gtggagctgc
tgagcttcct gcccagcgac ttcttcccca gcatcaggga cctgctggac
900accgccagcg ccctgtacag ggaggccctg gagagccccg agcactgcag
cccccaccac 960accgccctga ggcaggccat cctgtgctgg ggcgagctga
tgaccctggc cacctgggtg 1020ggcagcaacc tggaggaccc cgccagcagg
gagctggtgg tgagctacgt gaacgtgaac 1080atgggcctga agatcaggca
gctgctgtgg ttccacatca gctgcctgac cttcggcagg 1140gagaccgtga
tcgagtacct ggtgagcttc ggcgtgtgga tcaggacccc ccccgcctac
1200aggcccccca acgcccccat cctgagcacc ctgcccgaga ccaccgtggt
gaggaggagg 1260gacaggggca ggagccccag gaggaggacc cccagcccca
ggaggaggag gagccagagc 1320cccaggagga ggaggagcca gagcagggag
agccagtgcg ggcccaaggc cccatacacg 1380agcaccctgc tgcccccgga
gctgtccgag acccccaacg ccacgcagcc agaactcgcc 1440ccggaagacc
ccgaggattc ggccctcttg gaggaccccg tggggacggt ggcgccgcaa
1500atcccaccaa actggcacat cccgtcgatc caggacgccg cgacgcctta
ccatcccccg 1560gccaccccga acaacatggg cctgatcgcc ggcgcggtgg
gcggcagtct cctggcagcc 1620ctggtcattt gcggaattgt gtactggatg
caccgccgca ctcggaaagc cccaaagcgc 1680atacgcctcc cccacatccg
ggaagacgac cagccgtcct cgcaccagcc cttgttttac 1740tag
174316701PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 16Met Gly Gly Ala Ala
Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu
His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30Ser Leu Lys
Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45Val Leu
Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60Ile
Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile65 70 75
80Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu
85 90 95Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu
Asp 100 105 110Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe
Arg Met Gly 115 120 125Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu
Tyr Thr Glu Cys Ser 130 135 140Tyr Asn Lys Ser Leu Gly Ala Cys Pro
Ile Arg Thr Gln Pro Arg Trp145 150 155 160Asn Tyr Tyr Asp Ser Phe
Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175Leu Met His Ala
Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190Val Lys
Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200
205Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro
210 215 220Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr
Val Asp225 230 235 240Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu
Asn Gln Arg Thr Val 245 250 255Ala Val Tyr Ser Leu Lys Ile Ala Gly
Trp His Gly Pro Pro Leu Ser 260 265 270Tyr Gln His Phe Arg Lys Leu
Leu Leu Leu Asp Glu Glu Ala Gly Pro 275 280 285Leu Glu Glu Glu Leu
Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg 290 295 300Val Ala Glu
Asp Leu Asn Leu Gly Asn Leu Asn Val Ser Ile Pro Trp305 310 315
320Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr Val Pro
325 330 335Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys Ile
His Leu 340 345 350Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val
Gly Pro Leu Thr 355 360 365Val Asn Glu Lys Arg Arg Leu Lys Leu Ile
Met Pro Ala Arg Phe Tyr 370 375 380Pro Asn Val Thr Lys Tyr Leu Pro
Leu Asp Lys Gly Ile Lys Pro Tyr385 390 395 400Tyr Pro Glu His Ala
Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu 405 410 415His Thr Leu
Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg 420 425 430Ser
Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln 435 440
445His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser
450 455 460Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp
Gly Pro465 470 475 480Cys Asp Glu His Gly Glu His His Ile Arg Ile
Pro Arg Thr Pro Ala 485 490 495Arg Val Thr Gly Gly Val Phe Leu Val
Asp Lys Asn Pro His Asn Thr 500 505 510Ala Glu Ser Arg Leu Val Val
Asp Phe Ser Gln Phe Ser Arg Gly Ile 515 520 525Thr Arg Val Ser Trp
Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 530 535 540Thr Asn Leu
Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser545 550 555
560Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Gly Pro
565 570 575Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser
Glu Thr 580 585 590Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp
Pro Glu Asp Ser 595 600 605Ala Leu Leu Glu Asp Pro Val Gly Thr Val
Ala Pro Gln Ile Pro Pro 610 615 620Asn Trp His Ile Pro Ser Ile Gln
Asp Ala Ala Thr Pro Tyr His Pro625 630 635 640Pro Ala Thr Pro Asn
Asn Met Gly Leu Ile Ala Gly Ala Val Gly Gly 645 650 655Ser Leu Leu
Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Met His 660 665 670Arg
Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg 675 680
685Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 690 695
700172106DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 17atgggggggg
ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc
gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat
120cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc
gggggtccgg 180cgcgtgtacc acatccaggc gggcctaccg gacccgttcc
agccccccag cctcccgatc 240acggtttact acgccgtgtt ggagcgcgcc
tgccgcagcg tgctcctaaa cgcaccgtcg 300gaggcccccc agattgtccg
cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360accatcgctt
ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac
420accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca
gccccgctgg 480aactactatg acagcttcag cgccgtcagc gaggataacc
tggggttcct gatgcacgcc 540cccgcgtttg agaccgccgg cacgtacctg
cggctcgtga agataaacga ctggacggag 600attacacagt ttatcctgga
gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660cgcatccccc
cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac
720agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc
cgtatacagc 780ttgaagatcg ccgggtggca cgggcccccc ctgagctacc
agcacttcag gaagctgctg 840ctgctggacg aggaggccgg ccccctggag
gaggagctgc ccaggctggc cgacgagggc 900ctgaacagga gggtggccga
ggacctgaac ctgggcaacc tgaacgtgag catcccctgg 960acccacaagg
tgggcaactt caccggcctg tacagcagca ccgtgcccgt gttcaacccc
1020gagtggcaga cccccagctt ccccaagatc cacctgcagg aggacatcgt
ggacaggtgc 1080aagcagttcg tgggtcccct gaccgtgaac gagaagagga
ggctgaagct gatcatgccc 1140gccaggttct accccaacgt gaccaagtac
ctgcccctgg acaagggcat caagccctac 1200taccccgagc acgccgtgaa
ccactacttc cagaccaggc actacctgca caccctgtgg 1260aaggccggca
tcctgtacaa gagggagacc accaggagcg ccagcttctg cggcagcccc
1320tacagctggg agcaggagct gcagcacggc agctgctggt ggctgcagtt
caggaacagc 1380aagccctgca gcgagtactg cctgacccac ctggtgaacc
tgctggagga ctggggtccc 1440tgcgacgagc acggcgagca ccacatcagg
atccccagga cccccgccag ggtgaccggc 1500ggcgtgttcc tggtggacaa
gaacccccac aacaccgccg agagcaggct ggtggtggac 1560ttcagccagt
tcagcagggg catcaccagg gtgagctggc ccaagttcgc cgtgcccaac
1620ctgcagagcc tgaccaacct gctgagcagc aacctgagct ggctgagcct
ggacgtgagc 1680gccgccttct accacatccc cctgcacccc gccgccatgc
ccgggcccaa ggccccatac 1740acgagcaccc tgctgccccc ggagctgtcc
gagaccccca acgccacgca gccagaactc 1800gccccggaag accccgagga
ttcggccctc ttggaggacc ccgtggggac ggtggcgccg 1860caaatcccac
caaactggca catcccgtcg atccaggacg ccgcgacgcc ttaccatccc
1920ccggccaccc cgaacaacat gggcctgatc gccggcgcgg tgggcggcag
tctcctggca 1980gccctggtca tttgcggaat tgtgtactgg atgcaccgcc
gcactcggaa agccccaaag 2040cgcatacgcc tcccccacat ccgggaagac
gaccagccgt cctcgcacca gcccttgttt 2100tactag 210618699PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 18Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu
Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala
Leu Ala Asp Ala 20 25 30Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg
Gly Lys Asp Leu Pro 35 40 45Val Leu Asp Gln Leu Thr Asp Pro Pro Gly
Val Arg Arg Val Tyr His 50 55 60Ile Gln Ala Gly Leu Pro Asp Pro Phe
Gln Pro Pro Ser Leu Pro Ile65 70 75 80Thr Val Tyr Tyr Ala Val Leu
Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95Asn Ala Pro Ser Glu Ala
Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110Val Arg Lys Gln
Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125Gly Asn
Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135
140Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg
Trp145 150 155 160Asn Tyr
Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170
175Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu
180 185 190Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu
Glu His 195 200 205Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu
Arg Ile Pro Pro 210 215 220Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln
Gln Gly Val Thr Val Asp225 230 235 240Ser Ile Gly Met Leu Pro Arg
Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255Ala Val Tyr Ser Leu
Lys Ile Ala Gly Trp His Gly Pro His Leu Leu 260 265 270Val Gly Ser
Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn 275 280 285Ser
Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn Leu His Asp 290 295
300Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys
Thr305 310 315 320Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile
Ile Leu Lys Thr 325 330 335Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met
Gly Tyr Val Ile Gly Ser 340 345 350Trp Gly Ser Leu Pro Gln Asp His
Ile Ile Gln Lys Ile Lys Glu Cys 355 360 365Phe Arg Lys Leu Pro Val
Asn Arg Pro Ile Asp Trp Lys Val Cys Gln 370 375 380Arg Ile Val Gly
Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly385 390 395 400Tyr
Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala 405 410
415Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Leu
420 425 430Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln
Val Phe 435 440 445Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met
Gly His Gln Arg 450 455 460Met Arg Gly Thr Phe Val Ala Pro Leu Pro
Ile His Thr Ala Glu Leu465 470 475 480Leu Ala Ala Cys Phe Ala Arg
Ser Arg Ser Gly Ala Lys Ile Leu Gly 485 490 495Thr Asp Asn Ser Val
Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp 500 505 510Leu Leu Gly
Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val 515 520 525Tyr
Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg 530 535
540Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr
Thr545 550 555 560Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val
Gly Pro Lys Ala 565 570 575Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu
Leu Ser Glu Thr Pro Asn 580 585 590Ala Thr Gln Pro Glu Leu Ala Pro
Glu Asp Pro Glu Asp Ser Ala Leu 595 600 605Leu Glu Asp Pro Val Gly
Thr Val Ala Pro Gln Ile Pro Pro Asn Trp 610 615 620His Ile Pro Ser
Ile Gln Asp Ala Ala Thr Pro Tyr His Pro Pro Ala625 630 635 640Thr
Pro Asn Asn Met Gly Leu Ile Ala Gly Ala Val Gly Gly Ser Leu 645 650
655Leu Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg
660 665 670Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg
Glu Asp 675 680 685Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 690
695192100DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 19atgggggggg
ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc
gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat
120cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc
gggggtccgg 180cgcgtgtacc acatccaggc gggcctaccg gacccgttcc
agccccccag cctcccgatc 240acggtttact acgccgtgtt ggagcgcgcc
tgccgcagcg tgctcctaaa cgcaccgtcg 300gaggcccccc agattgtccg
cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360accatcgctt
ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac
420accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca
gccccgctgg 480aactactatg acagcttcag cgccgtcagc gaggataacc
tggggttcct gatgcacgcc 540cccgcgtttg agaccgccgg cacgtacctg
cggctcgtga agataaacga ctggacggag 600attacacagt ttatcctgga
gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660cgcatccccc
cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac
720agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc
cgtatacagc 780ttgaagatcg ccgggtggca cgggccccac ctgctggtgg
gcagcagcgg cctgagcagg 840tacgtggcca ggctgagcag caacagcagg
atcatcaacc accagcacgg caccatgcag 900aacctgcacg acagctgcag
caggaacctg tacgtgagcc tgctgctgct gtacaagacc 960ttcggcagga
agctgcacct gtacagccac cccatcatcc tgaagaccaa gaggtggggc
1020tacagcctga acttcatggg ctacgtgatc ggcagctggg gcagcctgcc
ccaggaccac 1080atcatccaga agatcaagga gtgcttcagg aagctgcccg
tgaacaggcc catcgactgg 1140aaggtgtgcc agaggatcgt gggcctgctg
ggcttcgccg cccccttcac ccagtgcggc 1200taccccgccc tgatgcccct
gtacgcctgc atccagagca agcaggcctt caccttcagc 1260cccacctaca
aggccttcct gagcaagcag tacctgaacc tgtaccccgt ggccaggcag
1320aggcccggcc tgtgccaggt gttcgccgac gccaccccca ccggctgggg
cctggccatg 1380ggccaccaga ggatgagggg caccttcgtg gcccccctgc
ccatccacac cgccgagctg 1440ctggccgcct gcttcgccag gagcaggagc
ggcgccaaga tcctgggcac cgacaacagc 1500gtggtgctga gcaggaagta
caccagcttc ccctggctgc tgggctgcgc cgccaactgg 1560atcctgaggg
gcaccagctt cgtgtacgtg cccagcgccc tgaaccccgc cgacgacccc
1620agcaggggca ggctgggcct gagcaggccc ctgctgaggc tgcccttcag
gcccaccacc 1680ggcaggacca gcctgtacgc cgtgagcccc agcgtggggc
ccaaggcccc atacacgagc 1740accctgctgc ccccggagct gtccgagacc
cccaacgcca cgcagccaga actcgccccg 1800gaagaccccg aggattcggc
cctcttggag gaccccgtgg ggacggtggc gccgcaaatc 1860ccaccaaact
ggcacatccc gtcgatccag gacgccgcga cgccttacca tcccccggcc
1920accccgaaca acatgggcct gatcgccggc gcggtgggcg gcagtctcct
ggcagccctg 1980gtcatttgcg gaattgtgta ctggatgcac cgccgcactc
ggaaagcccc aaagcgcata 2040cgcctccccc acatccggga agacgaccag
ccgtcctcgc accagccctt gttttactag 21002015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu
Leu1 5 10 152115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 21Lys Glu Phe Gly Ala Thr
Val Glu Leu Leu Ser Phe Leu Pro Ser1 5 10 152215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro
Ser1 5 10 152315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 23Ser Phe Leu Pro Ser Asp
Phe Phe Pro Ser Ile Arg Asp Leu Leu1 5 10 152415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser
Ala1 5 10 152515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 25Ile Arg Asp Leu Leu Asp
Thr Ala Ser Ala Leu Tyr Arg Glu Ala1 5 10 152615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro
Glu1 5 10 152715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Leu Tyr Arg Glu Ala Leu
Glu Ser Pro Glu His Cys Ser Pro His1 5 10 152815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu
Arg1 5 10 152915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 29His Cys Ser Pro His His
Thr Ala Leu Arg Gln Ala Ile Leu Cys1 5 10 153015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu
Met1 5 10 153115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 31Gln Ala Ile Leu Cys Trp
Gly Glu Leu Met Thr Leu Ala Thr Trp1 5 10 153215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 32Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Ser Asn
Leu1 5 10 153315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 33Thr Leu Ala Thr Trp Val
Gly Ser Asn Leu Glu Asp Pro Ala Ser1 5 10 153415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val
Val1 5 10 153515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 35Glu Asp Pro Ala Ser Arg
Glu Leu Val Val Ser Tyr Val Asn Val1 5 10 153615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 36Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu
Lys1 5 10 153715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 37Ser Tyr Val Asn Val Asn
Met Gly Leu Lys Ile Arg Gln Leu Leu1 5 10 153815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 38Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile
Ser1 5 10 153915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 39Ile Arg Gln Leu Leu Trp
Phe His Ile Ser Cys Leu Thr Phe Gly1 5 10 154015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 40Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val
Ile1 5 10 154115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 41Cys Leu Thr Phe Gly Arg
Glu Thr Val Ile Glu Tyr Leu Val Ser1 5 10 154215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Arg Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp
Ile1 5 10 154315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 43Glu Tyr Leu Val Ser Phe
Gly Val Trp Ile Arg Thr Pro Pro Ala1 5 10 154415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 44Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro
Asn1 5 10 154515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 45Arg Thr Pro Pro Ala Tyr
Arg Pro Pro Asn Ala Pro Ile Leu Ser1 5 10 154615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 46Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu
Thr1 5 10 154715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 47Ala Pro Ile Leu Ser Thr
Leu Pro Glu Thr Thr Val Val Arg Arg1 5 10 154815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 48Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly
Arg1 5 10 154915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 49Thr Val Val Arg Arg Arg
Asp Arg Gly Arg Ser Pro Arg Arg Arg1 5 10 155015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro
Arg1 5 10 155115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 51Ser Pro Arg Arg Arg Thr
Pro Ser Pro Arg Arg Arg Arg Ser Gln1 5 10 155215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 52Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg
Arg1 5 10 155316PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 53Arg Arg Arg Ser Gln Ser
Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg1 5 10 155415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Ser Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln
Cys1 5 10 155515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 55Pro Leu Ser Tyr Gln His
Phe Arg Lys Leu Leu Leu Leu Asp Glu1 5 10 155615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro
Leu1 5 10 155715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 57Leu Leu Leu Asp Glu Glu
Ala Gly Pro Leu Glu Glu Glu Leu Pro1 5 10 155815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp
Glu1 5 10 155915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 59Glu Glu Glu Leu Pro Arg
Leu Ala Asp Glu Gly Leu Asn Arg Arg1 5 10 156015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 60Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu Asp
Leu1 5 10 156115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 61Gly Leu Asn Arg Arg Val
Ala Glu Asp Leu Asn Leu Gly Asn Leu1 5 10 156215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 62Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val Ser Ile
Pro1 5 10 156315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 63Asn Leu Gly Asn Leu Asn
Val Ser Ile Pro Trp Thr His Lys Val1 5 10 156415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 64Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr
Gly1 5 10 156515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 65Trp Thr His Lys Val Gly
Asn Phe Thr Gly Leu Tyr Ser Ser Thr1 5 10 156615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 66Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr Val Pro Val Phe
Asn1 5 10 156715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 67Leu Tyr Ser Ser Thr Val
Pro Val Phe Asn Pro Glu Trp Gln Thr1 5 10 156815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro
Lys1 5 10 156916PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 69Pro Glu Trp Gln Thr Pro
Ser Phe Pro Lys Ile His Lys Leu Gln Glu1 5 10 157016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 70Pro Ser Phe Pro Lys Ile His Lys Leu Gln Glu Asp Ile Val
Asp Arg1 5 10 157116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 71Ile His Lys Leu Gln Glu
Asp Ile Val Asp Arg Cys Lys Gln Phe Val1 5 10 157216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 72Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro Leu
Thr Val1 5 10 157316PRTArtificial Sequencesource/note="Description
of
Artificial Sequence Synthetic peptide" 73Arg Cys Lys Gln Phe Val
Gly Pro Leu Thr Val Asn Glu Lys Arg Arg1 5 10 157416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 74Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu
Ile Met1 5 10 157516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 75Val Asn Glu Lys Arg Arg
Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr1 5 10 157616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 76Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val
Thr Lys1 5 10 157716PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 77Met Pro Ala Arg Phe Tyr
Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp1 5 10 157816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 78Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile
Lys Pro1 5 10 157916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 79Lys Tyr Leu Pro Leu Asp
Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His1 5 10 158016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 80Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn
His Tyr1 5 10 158116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 81Pro Tyr Tyr Pro Glu His
Ala Val Asn His Tyr Phe Gln Thr Arg His1 5 10 158216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 82His Ala Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His
Thr Leu1 5 10 158316PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 83Tyr Phe Gln Thr Arg His
Tyr Leu His Thr Leu Trp Lys Ala Gly Ile1 5 10 158416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 84His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys
Arg Glu1 5 10 158516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 85Leu Trp Lys Ala Gly Ile
Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala1 5 10 158616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 86Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys
Gly Ser1 5 10 158716PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 87Glu Thr Thr Arg Ser Ala
Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu1 5 10 158816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 88Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu
Gln His1 5 10 158916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 89Ser Pro Tyr Ser Trp Glu
Gln Glu Leu Gln His Gly Ser Cys Trp Trp1 5 10 159016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 90Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe
Arg Asn1 5 10 159116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 91His Gly Ser Cys Trp Trp
Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser1 5 10 159216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 92Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys
Leu Thr1 5 10 159316PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 93Asn Ser Lys Pro Cys Ser
Glu Tyr Cys Leu Thr His Leu Val Asn Leu1 5 10 159416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 94Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp
Trp Gly1 5 10 159516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 95Thr His Leu Val Asn Leu
Leu Glu Asp Trp Gly Pro Cys Asp Glu His1 5 10 159616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 96Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His
His Ile1 5 10 159716PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 97Gly Pro Cys Asp Glu His
Gly Glu His His Ile Arg Ile Pro Arg Thr1 5 10 159816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 98His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg
Val Thr1 5 10 159916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 99Ile Arg Ile Pro Arg Thr
Pro Ala Arg Val Thr Gly Gly Val Phe Leu1 5 10 1510016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 100Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys
Asn Pro1 5 10 1510116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 101Thr Gly Gly Val Phe
Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu1 5 10
1510216PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 102Leu Val Asp Lys Asn Pro His Asn Thr
Ala Glu Ser Arg Leu Val Val1 5 10 1510316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 103Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser
Gln Phe1 5 10 1510416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 104Glu Ser Arg Leu Val
Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr1 5 10
1510516PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 105Val Asp Phe Ser Gln Phe Ser Arg Gly
Ile Thr Arg Val Ser Trp Pro1 5 10 1510616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 106Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala
Val Pro1 5 10 1510716PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 107Thr Arg Val Ser Trp
Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu1 5 10
1510816PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 108Pro Lys Phe Ala Val Pro Asn Leu Gln
Ser Leu Thr Asn Leu Leu Ser1 5 10 1510916PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 109Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu
Ser Trp1 5 10 1511016PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 110Leu Thr Asn Leu Leu
Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val1 5 10
1511116PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 111Ser Ser Asn Leu Ser Trp Leu Ser Leu
Asp Val Ser Ala Ala Phe Tyr1 5 10 1511216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 112Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro
Leu His1 5 10 1511316PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 113Val Ser Ala Ala Phe
Tyr His Ile Pro Leu His Pro Ala Ala Met Pro1 5 10
1511415PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 114His Leu Leu Val Gly Ser Ser Gly Leu
Ser Arg Tyr Val Ala Arg1 5 10 1511516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 115Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn
Ser Arg1 5 10 1511616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 116Arg Tyr Val Ala Arg
Leu Ser Ser Asn Ser Arg Ile Ile Asn His Gln1 5 10
1511716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 117Leu Ser Ser Asn Ser Arg Ile Ile Asn
His Gln His Gly Thr Met Gln1 5 10 1511816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 118Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn Leu His
Asp Ser1 5 10 1511916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 119Gln His Gly Thr Met
Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu1 5 10
1512016PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 120Gln Asn Leu His Asp Ser Cys Ser Arg
Asn Leu Tyr Val Ser Leu Leu1 5 10 1512116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 121Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr
Lys Thr1 5 10 1512216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 122Leu Tyr Val Ser Leu
Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu1 5 10
1512316PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 123Leu Leu Leu Tyr Lys Thr Phe Gly Arg
Lys Leu His Leu Tyr Ser His1 5 10 1512416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 124Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile
Leu Lys1 5 10 1512516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 125Leu His Leu Tyr Ser
His Pro Ile Ile Leu Lys Thr Lys Arg Trp Gly1 5 10
1512616PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 126His Pro Ile Ile Leu Lys Thr Lys Arg
Trp Gly Tyr Ser Leu Asn Phe1 5 10 1512716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 127Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr
Val Ile1 5 10 1512816PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 128Gly Tyr Ser Leu Asn
Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Ser1 5 10
1512916PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 129Phe Met Gly Tyr Val Ile Gly Ser Trp
Gly Ser Leu Pro Gln Asp His1 5 10 1513016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 130Ile Gly Ser Trp Gly Ser Leu Pro Gln Asp His Ile Ile Gln
Lys Ile1 5 10 1513116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 131Ser Leu Pro Gln Asp
His Ile Ile Gln Lys Ile Lys Glu Cys Phe Arg1 5 10
1513216PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 132His Ile Ile Gln Lys Ile Lys Glu Cys
Phe Arg Lys Leu Pro Val Asn1 5 10 1513316PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 133Ile Lys Glu Cys Phe Arg Lys Leu Pro Val Asn Arg Pro Ile
Asp Trp1 5 10 1513416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 134Arg Lys Leu Pro Val
Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg1 5 10
1513516PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 135Asn Arg Pro Ile Asp Trp Lys Val Cys
Gln Arg Ile Val Gly Leu Leu1 5 10 1513616PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 136Trp Lys Val Cys Gln Arg Ile Val Gly Leu Leu Gly Phe Ala
Ala Pro1 5 10 1513716PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 137Arg Ile Val Gly Leu
Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly1 5 10
1513816PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 138Leu Gly Phe Ala Ala Pro Phe Thr Gln
Cys Gly Tyr Pro Ala Leu Met1 5 10 1513916PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 139Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr
Ala Cys1 5 10 1514016PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 140Gly Tyr Pro Ala Leu
Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln1 5 10
1514116PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 141Met Pro Leu Tyr Ala Cys Ile Gln Ser
Lys Gln Ala Phe Thr Phe Ser1 5 10 1514216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 142Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr
Lys Ala1 5 10 1514316PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 143Gln Ala Phe Thr Phe
Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln1 5 10
1514416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 144Ser Pro Thr Tyr Lys Ala Phe Leu Ser
Lys Gln Tyr Leu Asn Leu Tyr1 5 10 1514516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 145Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala
Arg Gln1 5 10 1514616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 146Gln Tyr Leu Asn Leu
Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys1 5 10
1514716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 147Tyr Pro Val Ala Arg Gln Arg Pro Gly
Leu Cys Gln Val Phe Ala Asp1 5 10 1514816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 148Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro
Thr Gly1 5 10 1514916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 149Cys Gln Val Phe Ala
Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met1 5 10
1515016PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 150Asp Ala Thr Pro Thr Gly Trp Gly Leu
Ala Met Gly His Gln Arg Met1 5 10 1515116PRTArtificial
Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 151Gly Trp Gly Leu Ala Met Gly His Gln
Arg Met Arg Gly Thr Phe Val1 5 10 1515216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 152Met Gly His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu
Pro Ile1 5 10 1515316PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 153Met Arg Gly Thr Phe
Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu1 5 10
1515416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 154Val Ala Pro Leu Pro Ile His Thr Ala
Glu Leu Leu Ala Ala Cys Phe1 5 10 1515516PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 155Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser
Arg Ser1 5 10 1515616PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 156Leu Leu Ala Ala Cys
Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu1 5 10
1515716PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 157Phe Ala Arg Ser Arg Ser Gly Ala Lys
Ile Leu Gly Thr Asp Asn Ser1 5 10 1515816PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 158Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val Leu
Ser Arg1 5 10 1515916PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 159Leu Gly Thr Asp Asn
Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe1 5 10
1516016PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 160Ser Val Val Leu Ser Arg Lys Tyr Thr
Ser Phe Pro Trp Leu Leu Gly1 5 10 1516116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 161Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala
Asn Trp1 5 10 1516216PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 162Phe Pro Trp Leu Leu
Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr1 5 10
1516316PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 163Gly Cys Ala Ala Asn Trp Ile Leu Arg
Gly Thr Ser Phe Val Tyr Val1 5 10 1516416PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 164Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala
Leu Asn1 5 10 1516516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 165Thr Ser Phe Val Tyr
Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro1 5 10
1516616PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 166Val Pro Ser Ala Leu Asn Pro Ala Asp
Asp Pro Ser Arg Gly Arg Leu1 5 10 1516716PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 167Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ser
Arg Pro1 5 10 1516816PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 168Pro Ser Arg Gly Arg
Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro1 5 10
1516916PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 169Leu Gly Leu Ser Arg Pro Leu Leu Arg
Leu Pro Phe Arg Pro Thr Thr1 5 10 1517016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 170Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr
Ser Leu1 5 10 1517116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 171Pro Phe Arg Pro Thr
Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro1 5 10
1517213PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 172Thr Gly Arg Thr Ser Leu Tyr Ala Val
Ser Pro Ser Val1 5 10173200PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 173His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly
Pro Leu Glu1 5 10 15Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn
Arg Arg Val Ala 20 25 30Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp
Gln Thr Pro Ser Phe 35 40 45Pro Lys Ile His Leu Gln Glu Asp Ile Val
Asp Arg Cys Lys Gln Phe 50 55 60Val Gly Pro Leu Thr Val Asn Glu Lys
Arg Arg Leu Lys Leu Ile Met65 70 75 80Pro Ala Arg Phe Tyr Pro Asn
Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95Gly Ile Lys Pro Tyr Tyr
Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110Thr Arg His Tyr
Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125Arg Glu
Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135
140Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg
Asn145 150 155 160Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu
Val Asn Leu Leu 165 170 175Glu Asp Trp Gly Pro Cys Asp Glu His Gly
Glu His His Ile Arg Ile 180 185 190Pro Arg Thr Pro Ala Arg Val Thr
195 200174380PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 174Tyr Leu Pro Leu Asp
Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala1 5 10 15Val Asn His Tyr
Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys 20 25 30Ala Gly Ile
Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys 35 40 45Gly Ser
Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp 50 55 60Trp
Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr65 70 75
80His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly
85 90 95Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly
Gly 100 105 110Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu
Ser Arg Leu 115 120 125Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile
Thr Arg Val Ser Trp 130 135 140Pro Lys Phe Ala Val Pro Asn Leu Gln
Ser Leu Thr Asn Leu Leu Ser145 150 155 160Ser Asn Leu Ser Trp Leu
Ser Leu Asp Val Gln Ala Phe Thr Phe Ser 165 170 175Pro Thr Tyr Lys
Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro 180 185 190Val Ala
Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr 195 200
205Pro Thr Gly Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr
210 215 220Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala
Ala Cys225 230 235 240Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu
Gly Thr Asp Asn Ser 245 250 255Val Val Leu Ser Arg Lys Tyr Thr Ser
Phe Pro Trp Leu Leu Gly Cys 260 265 270Ala Ala Asn Trp Ile Leu Arg
Gly Thr Ser Phe Val Tyr Val Pro Ser 275 280 285Ala Leu Asn Pro Ala
Asp Asp Val Gly Ser Asn Leu Glu Asp Pro Ala 290 295 300Ser Arg Glu
Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys305 310 315
320Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg
325 330 335Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile
Arg Thr 340 345 350Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu
Ser Thr Leu Pro 355 360 365Glu Thr Thr Val Val Arg Arg Arg Asp Arg
Gly Arg 370 375 380175410PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 175His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly
Pro Leu Glu1 5 10 15Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn
Arg Arg Val Ala 20 25 30Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp
Gln Thr Pro Ser Phe 35 40 45Pro Lys Ile His Leu Gln Glu Asp Ile Val
Asp Arg Cys Lys Gln Phe 50 55 60Val Gly Pro Leu Thr Val Asn Glu Lys
Arg Arg Leu Lys Leu Ile Met65 70 75 80Pro Ala Arg Phe Tyr Pro Asn
Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95Gly Ile Lys Pro Tyr Tyr
Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110Thr Arg His Tyr
Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125Arg Glu
Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135
140Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg
Asn145 150 155 160Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu
Val Asn Leu Leu 165 170 175Glu Asp Trp Gly Pro Cys Asp Glu His Gly
Glu His His Ile Arg Ile 180 185 190Pro Arg Thr Pro Ala Arg Val Thr
Gln Ala Phe Thr Phe Ser Pro Thr 195 200 205Tyr Lys Ala Phe Leu Ser
Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala 210 215 220Arg Gln Arg Pro
Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr225 230 235 240Gly
Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val 245 250
255Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala
260 265 270Arg Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser
Val Val 275 280 285Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu
Gly Cys Ala Ala 290 295 300Asn Trp Ile Leu Arg Gly Thr Ser Phe Val
Tyr Val Pro Ser Ala Leu305 310 315 320Asn Pro Ala Asp Asp Val Gly
Ser Asn Leu Glu Asp Pro Ala Ser Arg 325 330 335Glu Leu Val Val Ser
Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg 340 345 350Gln Leu Leu
Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr 355 360 365Val
Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro 370 375
380Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu
Thr385 390 395 400Thr Val Val Arg Arg Arg Asp Arg Gly Arg 405
4101761140DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 176tatctgccgc
tggataaagg cattaaaccg tattatccgg aacatgcggt gaaccattat 60tttcagaccc
gccattatct gcataccctg tggaaagcgg gcattctgta taaacgcgaa
120accacccgca gcgcgagctt ttgcggcagc ccgtatagct gggaacagga
actgcagcat 180ggcagctgct ggtggctgca gtttcgcaac agcaaaccgt
gcagcgaata ttgcctgacc 240catctggtga acctgctgga agattgggga
ccgtgcgatg aacatggcga acatcatatt 300cgcattccgc gcaccccggc
gcgcgtgacc ggcggcgtgt ttctggtgga taaaaacccg 360cataacaccg
cggaaagccg cctggtggtg gattttagcc agtttagccg cggcattacc
420cgcgtgagct ggccgaaatt tgcggtgccg aacctgcaga gcctgaccaa
cctgctgagc 480agcaacctga gctggctgag cctggatgtg caggcgttta
cctttagccc gacctataaa 540gcgtttctga gcaaacagta tctgaacctg
tatccggtgg cgcgccagcg cccgggcctg 600tgccaggtgt ttgcggatgc
gaccccgacc ggctggggcc tggcgatggg ccatcagcgc 660atgcgcggca
cctttgtggc gccgctgccg attcataccg cggaactgct ggcggcgtgc
720tttgcgcgca gccgcagcgg cgcgaaaatt ctgggcaccg ataacagcgt
ggtgctgagc 780cgcaaatata ccagctttcc gtggctgctg ggctgcgcgg
cgaactggat tctgcgcggc 840accagctttg tgtatgtgcc gagcgcgctg
aacccggcgg atgatgtggg cagcaacctg 900gaagatccgg cgagccgcga
actggtggtg agctatgtga acgtgaacat gggcctgaaa 960attcgccagc
tgctgtggtt tcatattagc tgcctgacct ttggccgcga aaccgtgatt
1020gaatatctgg tgagctttgg cgtgtggatt cgcaccccgc cggcgtatcg
cccgccgaac 1080gcgccgattc tgagcaccct gccggaaacc accgtggtgc
gccgccgcga tcggggccgc 11401771230DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 177cattttcgca aactgctgct gctggatgaa gaagcgggac
cgctggaaga agaactgccg 60cgcctggcgg atgaaggcct gaaccgccgc gtggcggaag
atctgaacct gggcaacctg 120ccggaatggc agaccccgag ctttccgaaa
attcatctgc aggaagatat tgtggatcgc 180tgcaaacagt ttgtgggacc
gctgaccgtg aacgaaaaac gccgcctgaa actgattatg 240ccggcgcgct
tttatccgaa cgtgaccaaa tatctgccgc tggataaagg cattaaaccg
300tattatccgg aacatgcggt gaaccattat tttcagaccc gccattatct
gcataccctg 360tggaaagcgg gcattctgta taaacgcgaa accacccgca
gcgcgagctt ttgcggcagc 420ccgtatagct gggaacagga actgcagcat
ggcagctgct ggtggctgca gtttcgcaac 480agcaaaccgt gcagcgaata
ttgcctgacc catctggtga acctgctgga agattgggga 540ccgtgcgatg
aacatggcga acatcatatt cgcattccgc gcaccccggc gcgcgtgacc
600caggcgttta cctttagccc gacctataaa gcgtttctga gcaaacagta
tctgaacctg 660tatccggtgg cgcgccagcg cccgggcctg tgccaggtgt
ttgcggatgc gaccccgacc 720ggctggggcc tggcgatggg ccatcagcgc
atgcgcggca cctttgtggc gccgctgccg 780attcataccg cggaactgct
ggcggcgtgc tttgcgcgca gccgcagcgg cgcgaaaatt 840ctgggcaccg
ataacagcgt ggtgctgagc cgcaaatata ccagctttcc gtggctgctg
900ggctgcgcgg cgaactggat tctgcgcggc accagctttg tgtatgtgcc
gagcgcgctg 960aacccggcgg atgatgtggg cagcaacctg gaagatccgg
cgagccgcga actggtggtg 1020agctatgtga acgtgaacat gggcctgaaa
attcgccagc tgctgtggtt tcatattagc 1080tgcctgacct ttggccgcga
aaccgtgatt gaatatctgg tgagctttgg cgtgtggatt 1140cgcaccccgc
cggcgtatcg cccgccgaac gcgccgattc tgagcaccct gccggaaacc
1200accgtggtgc gccgccgaga tcgaggccgc 1230178170PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 178Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro
Glu His Ala1 5 10 15Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His
Thr Leu Trp Lys 20 25 30Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg
Ser Ala Ser Phe Cys 35 40 45Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu
Gln His Gly Ser Cys Trp 50 55 60Trp Leu Gln Phe Arg Asn Ser Lys Pro
Cys Ser Glu Tyr Cys Leu Thr65 70 75 80His Leu Val Asn Leu Leu Glu
Asp Trp Gly Pro Cys Asp Glu His Gly 85 90 95Glu His His Ile Arg Ile
Pro Arg Thr Pro Ala Arg Val Thr Gly Gly 100 105 110Val Phe Leu Val
Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu 115 120 125Val Val
Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp 130 135
140Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu
Ser145 150 155 160Ser Asn Leu Ser Trp Leu Ser Leu Asp Val 165
170179125PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 179Gln Ala Phe Thr Phe
Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln1 5 10 15Tyr Leu Asn Leu
Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln 20 25 30Val Phe Ala
Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His 35 40 45Gln Arg
Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala 50 55 60Glu
Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile65 70 75
80Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe
85 90 95Pro Trp
Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser 100 105
110Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp 115 120
12518085PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 180Val Gly Ser Asn Leu
Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser1 5 10 15Tyr Val Asn Val
Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe 20 25 30His Ile Ser
Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr Leu 35 40 45Val Ser
Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro 50 55 60Asn
Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg65 70 75
80Arg Asp Arg Gly Arg 85181200PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 181His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly
Pro Leu Glu1 5 10 15Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn
Arg Arg Val Ala 20 25 30Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp
Gln Thr Pro Ser Phe 35 40 45Pro Lys Ile His Leu Gln Glu Asp Ile Val
Asp Arg Cys Lys Gln Phe 50 55 60Val Gly Pro Leu Thr Val Asn Glu Lys
Arg Arg Leu Lys Leu Ile Met65 70 75 80Pro Ala Arg Phe Tyr Pro Asn
Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95Gly Ile Lys Pro Tyr Tyr
Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110Thr Arg His Tyr
Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125Arg Glu
Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135
140Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg
Asn145 150 155 160Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu
Val Asn Leu Leu 165 170 175Glu Asp Trp Gly Pro Cys Asp Glu His Gly
Glu His His Ile Arg Ile 180 185 190Pro Arg Thr Pro Ala Arg Val Thr
195 200182125PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 182Gln Ala Phe Thr Phe
Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln1 5 10 15Tyr Leu Asn Leu
Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln 20 25 30Val Phe Ala
Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His 35 40 45Gln Arg
Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala 50 55 60Glu
Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile65 70 75
80Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe
85 90 95Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr
Ser 100 105 110Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp
115 120 12518385PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 183Val Gly Ser Asn Leu
Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser1 5 10 15Tyr Val Asn Val
Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe 20 25 30His Ile Ser
Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr Leu 35 40 45Val Ser
Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro 50 55 60Asn
Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg65 70 75
80Arg Asp Arg Gly Arg 851842331DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 184atgggggggg ctgccgccag gttgggggcc gtgattttgt
ttgtcgtcat agtgggcctc 60catggggtcc gcggcaaata tgccttggcg gatgcctctc
tcaagatggc cgaccccaat 120cgctttcgcg gcaaagacct tccggtcctg
gaccagctga ccgaccctcc gggggtccgg 180cgcgtgtacc acatccaggc
gggcctaccg gacccgttcc agccccccag cctcccgatc 240acggtttact
acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg
300gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc
ctacaacctg 360accatcgctt ggtttcggat gggaggcaac tgtgctatcc
ccatcacggt catggagtac 420accgaatgct cctacaacaa gtctctgggg
gcctgtccca tccgaacgca gccccgctgg 480aactactatg acagcttcag
cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540cccgcgtttg
agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag
600attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc
cctcccgctg 660cgcatccccc cgtcagcctg cctctccccc caggcctacc
agcagggggt gacggtggac 720agcatcggga tgctgccccg cttcatcccc
gagaaccagc gcaccgtcgc cgtatacagc 780ttgaagatcg ccgggtggca
cgggccctat ctgccgctgg ataaaggcat taaaccgtat 840tatccggaac
atgcggtgaa ccattatttt cagacccgcc attatctgca taccctgtgg
900aaagcgggca ttctgtataa acgcgaaacc acccgcagcg cgagcttttg
cggcagcccg 960tatagctggg aacaggaact gcagcatggc agctgctggt
ggctgcagtt tcgcaacagc 1020aaaccgtgca gcgaatattg cctgacccat
ctggtgaacc tgctggaaga ttggggaccg 1080tgcgatgaac atggcgaaca
tcatattcgc attccgcgca ccccggcgcg cgtgaccggc 1140ggcgtgtttc
tggtggataa aaacccgcat aacaccgcgg aaagccgcct ggtggtggat
1200tttagccagt ttagccgcgg cattacccgc gtgagctggc cgaaatttgc
ggtgccgaac 1260ctgcagagcc tgaccaacct gctgagcagc aacctgagct
ggctgagcct ggatgtgcag 1320gcgtttacct ttagcccgac ctataaagcg
tttctgagca aacagtatct gaacctgtat 1380ccggtggcgc gccagcgccc
gggcctgtgc caggtgtttg cggatgcgac cccgaccggc 1440tggggcctgg
cgatgggcca tcagcgcatg cgcggcacct ttgtggcgcc gctgccgatt
1500cataccgcgg aactgctggc ggcgtgcttt gcgcgcagcc gcagcggcgc
gaaaattctg 1560ggcaccgata acagcgtggt gctgagccgc aaatatacca
gctttccgtg gctgctgggc 1620tgcgcggcga actggattct gcgcggcacc
agctttgtgt atgtgccgag cgcgctgaac 1680ccggcggatg atgtgggcag
caacctggaa gatccggcga gccgcgaact ggtggtgagc 1740tatgtgaacg
tgaacatggg cctgaaaatt cgccagctgc tgtggtttca tattagctgc
1800ctgacctttg gccgcgaaac cgtgattgaa tatctggtga gctttggcgt
gtggattcgc 1860accccgccgg cgtatcgccc gccgaacgcg ccgattctga
gcaccctgcc ggaaaccacc 1920gtggtgcgcc gccgcgatcg gggccgcggg
cccaaggccc catacacgag caccctgctg 1980cccccggagc tgtccgagac
ccccaacgcc acgcagccag aactcgcccc ggaagacccc 2040gaggattcgg
ccctcttgga ggaccccgtg gggacggtgg cgccgcaaat cccaccaaac
2100tggcacatcc cgtcgatcca ggacgccgcg acgccttacc atcccccggc
caccccgaac 2160aacatgggcc tgatcgccgg cgcggtgggc ggcagtctcc
tggcagccct ggtcatttgc 2220ggaattgtgt actggatgca ccgccgcact
cggaaagccc caaagcgcat acgcctcccc 2280cacatccggg aagacgacca
gccgtcctcg caccagccct tgttttacta g 2331185776PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 185Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu
Phe Val Val1 5 10 15Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala
Leu Ala Asp Ala 20 25 30Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg
Gly Lys Asp Leu Pro 35 40 45Val Leu Asp Gln Leu Thr Asp Pro Pro Gly
Val Arg Arg Val Tyr His 50 55 60Ile Gln Ala Gly Leu Pro Asp Pro Phe
Gln Pro Pro Ser Leu Pro Ile65 70 75 80Thr Val Tyr Tyr Ala Val Leu
Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95Asn Ala Pro Ser Glu Ala
Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110Val Arg Lys Gln
Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125Gly Asn
Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135
140Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg
Trp145 150 155 160Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp
Asn Leu Gly Phe 165 170 175Leu Met His Ala Pro Ala Phe Glu Thr Ala
Gly Thr Tyr Leu Arg Leu 180 185 190Val Lys Ile Asn Asp Trp Thr Glu
Ile Thr Gln Phe Ile Leu Glu His 195 200 205Arg Ala Lys Gly Ser Cys
Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220Ser Ala Cys Leu
Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp225 230 235 240Ser
Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250
255Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Tyr Leu Pro
260 265 270Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val
Asn His 275 280 285Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp
Lys Ala Gly Ile 290 295 300Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala
Ser Phe Cys Gly Ser Pro305 310 315 320Tyr Ser Trp Glu Gln Glu Leu
Gln His Gly Ser Cys Trp Trp Leu Gln 325 330 335Phe Arg Asn Ser Lys
Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val 340 345 350Asn Leu Leu
Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His 355 360 365Ile
Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu 370 375
380Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Val Val
Asp385 390 395 400Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser
Trp Pro Lys Phe 405 410 415Ala Val Pro Asn Leu Gln Ser Leu Thr Asn
Leu Leu Ser Ser Asn Leu 420 425 430Ser Trp Leu Ser Leu Asp Val Gln
Ala Phe Thr Phe Ser Pro Thr Tyr 435 440 445Lys Ala Phe Leu Ser Lys
Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg 450 455 460Gln Arg Pro Gly
Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly465 470 475 480Trp
Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val Ala 485 490
495Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg
500 505 510Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val
Val Leu 515 520 525Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly
Cys Ala Ala Asn 530 535 540Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr
Val Pro Ser Ala Leu Asn545 550 555 560Pro Ala Asp Asp Val Gly Ser
Asn Leu Glu Asp Pro Ala Ser Arg Glu 565 570 575Leu Val Val Ser Tyr
Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln 580 585 590Leu Leu Trp
Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 595 600 605Ile
Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 610 615
620Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr
Thr625 630 635 640Val Val Arg Arg Arg Asp Arg Gly Arg Gly Pro Lys
Ala Pro Tyr Thr 645 650 655Ser Thr Leu Leu Pro Pro Glu Leu Ser Glu
Thr Pro Asn Ala Thr Gln 660 665 670Pro Glu Leu Ala Pro Glu Asp Pro
Glu Asp Ser Ala Leu Leu Glu Asp 675 680 685Pro Val Gly Thr Val Ala
Pro Gln Ile Pro Pro Asn Trp His Ile Pro 690 695 700Ser Ile Gln Asp
Ala Ala Thr Pro Tyr His Pro Pro Ala Thr Pro Asn705 710 715 720Asn
Met Gly Leu Ile Ala Gly Ala Val Gly Gly Ser Leu Leu Ala Ala 725 730
735Leu Val Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg Thr Arg Lys
740 745 750Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp Asp
Gln Pro 755 760 765Ser Ser His Gln Pro Leu Phe Tyr 770
7751862421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 186atgggggggg
ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60catggggtcc
gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat
120cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc
gggggtccgg 180cgcgtgtacc acatccaggc gggcctaccg gacccgttcc
agccccccag cctcccgatc 240acggtttact acgccgtgtt ggagcgcgcc
tgccgcagcg tgctcctaaa cgcaccgtcg 300gaggcccccc agattgtccg
cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360accatcgctt
ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac
420accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca
gccccgctgg 480aactactatg acagcttcag cgccgtcagc gaggataacc
tggggttcct gatgcacgcc 540cccgcgtttg agaccgccgg cacgtacctg
cggctcgtga agataaacga ctggacggag 600attacacagt ttatcctgga
gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660cgcatccccc
cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac
720agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc
cgtatacagc 780ttgaagatcg ccgggtggca cgggccccat tttcgcaaac
tgctgctgct ggatgaagaa 840gcgggaccgc tggaagaaga actgccgcgc
ctggcggatg aaggcctgaa ccgccgcgtg 900gcggaagatc tgaacctggg
caacctgccg gaatggcaga ccccgagctt tccgaaaatt 960catctgcagg
aagatattgt ggatcgctgc aaacagtttg tgggaccgct gaccgtgaac
1020gaaaaacgcc gcctgaaact gattatgccg gcgcgctttt atccgaacgt
gaccaaatat 1080ctgccgctgg ataaaggcat taaaccgtat tatccggaac
atgcggtgaa ccattatttt 1140cagacccgcc attatctgca taccctgtgg
aaagcgggca ttctgtataa acgcgaaacc 1200acccgcagcg cgagcttttg
cggcagcccg tatagctggg aacaggaact gcagcatggc 1260agctgctggt
ggctgcagtt tcgcaacagc aaaccgtgca gcgaatattg cctgacccat
1320ctggtgaacc tgctggaaga ttggggaccg tgcgatgaac atggcgaaca
tcatattcgc 1380attccgcgca ccccggcgcg cgtgacccag gcgtttacct
ttagcccgac ctataaagcg 1440tttctgagca aacagtatct gaacctgtat
ccggtggcgc gccagcgccc gggcctgtgc 1500caggtgtttg cggatgcgac
cccgaccggc tggggcctgg cgatgggcca tcagcgcatg 1560cgcggcacct
ttgtggcgcc gctgccgatt cataccgcgg aactgctggc ggcgtgcttt
1620gcgcgcagcc gcagcggcgc gaaaattctg ggcaccgata acagcgtggt
gctgagccgc 1680aaatatacca gctttccgtg gctgctgggc tgcgcggcga
actggattct gcgcggcacc 1740agctttgtgt atgtgccgag cgcgctgaac
ccggcggatg atgtgggcag caacctggaa 1800gatccggcga gccgcgaact
ggtggtgagc tatgtgaacg tgaacatggg cctgaaaatt 1860cgccagctgc
tgtggtttca tattagctgc ctgacctttg gccgcgaaac cgtgattgaa
1920tatctggtga gctttggcgt gtggattcgc accccgccgg cgtatcgccc
gccgaacgcg 1980ccgattctga gcaccctgcc ggaaaccacc gtggtgcgcc
gccgagatcg aggccgcggg 2040cccaaggccc catacacgag caccctgctg
cccccggagc tgtccgagac ccccaacgcc 2100acgcagccag aactcgcccc
ggaagacccc gaggattcgg ccctcttgga ggaccccgtg 2160gggacggtgg
cgccgcaaat cccaccaaac tggcacatcc cgtcgatcca ggacgccgcg
2220acgccttacc atcccccggc caccccgaac aacatgggcc tgatcgccgg
cgcggtgggc 2280ggcagtctcc tggcagccct ggtcatttgc ggaattgtgt
actggatgca ccgccgcact 2340cggaaagccc caaagcgcat acgcctcccc
cacatccggg aagacgacca gccgtcctcg 2400caccagccct tgttttacta g
2421187806PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 187Met Gly Gly Ala Ala
Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val1 5 10 15Ile Val Gly Leu
His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30Ser Leu Lys
Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45Val Leu
Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60Ile
Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile65 70 75
80Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu
85 90 95Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu
Asp 100 105 110Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe
Arg Met Gly 115 120 125Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu
Tyr Thr Glu Cys Ser 130 135 140Tyr Asn Lys Ser Leu Gly Ala Cys Pro
Ile Arg Thr Gln Pro Arg Trp145 150 155 160Asn Tyr Tyr Asp Ser Phe
Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175Leu Met His Ala
Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190Val Lys
Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200
205Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro
210 215 220Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr
Val Asp225 230 235 240Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu
Asn Gln Arg Thr Val 245 250 255Ala Val Tyr Ser Leu
Lys Ile Ala Gly Trp His Gly Pro His Phe Arg 260 265 270Lys Leu Leu
Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu Glu Glu Leu 275 280 285Pro
Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu 290 295
300Asn Leu Gly Asn Leu Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys
Ile305 310 315 320His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln
Phe Val Gly Pro 325 330 335Leu Thr Val Asn Glu Lys Arg Arg Leu Lys
Leu Ile Met Pro Ala Arg 340 345 350Phe Tyr Pro Asn Val Thr Lys Tyr
Leu Pro Leu Asp Lys Gly Ile Lys 355 360 365Pro Tyr Tyr Pro Glu His
Ala Val Asn His Tyr Phe Gln Thr Arg His 370 375 380Tyr Leu His Thr
Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr385 390 395 400Thr
Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu 405 410
415Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro
420 425 430Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu
Asp Trp 435 440 445Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg
Ile Pro Arg Thr 450 455 460Pro Ala Arg Val Thr Gln Ala Phe Thr Phe
Ser Pro Thr Tyr Lys Ala465 470 475 480Phe Leu Ser Lys Gln Tyr Leu
Asn Leu Tyr Pro Val Ala Arg Gln Arg 485 490 495Pro Gly Leu Cys Gln
Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly 500 505 510Leu Ala Met
Gly His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu 515 520 525Pro
Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg 530 535
540Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val Leu Ser
Arg545 550 555 560Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala
Ala Asn Trp Ile 565 570 575Leu Arg Gly Thr Ser Phe Val Tyr Val Pro
Ser Ala Leu Asn Pro Ala 580 585 590Asp Asp Val Gly Ser Asn Leu Glu
Asp Pro Ala Ser Arg Glu Leu Val 595 600 605Val Ser Tyr Val Asn Val
Asn Met Gly Leu Lys Ile Arg Gln Leu Leu 610 615 620Trp Phe His Ile
Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu625 630 635 640Tyr
Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg 645 650
655Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val
660 665 670Arg Arg Arg Asp Arg Gly Arg Gly Pro Lys Ala Pro Tyr Thr
Ser Thr 675 680 685Leu Leu Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala
Thr Gln Pro Glu 690 695 700Leu Ala Pro Glu Asp Pro Glu Asp Ser Ala
Leu Leu Glu Asp Pro Val705 710 715 720Gly Thr Val Ala Pro Gln Ile
Pro Pro Asn Trp His Ile Pro Ser Ile 725 730 735Gln Asp Ala Ala Thr
Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met 740 745 750Gly Leu Ile
Ala Gly Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val 755 760 765Ile
Cys Gly Ile Val Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro 770 775
780Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp Asp Gln Pro Ser
Ser785 790 795 800His Gln Pro Leu Phe Tyr 8051889PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 188Phe Ala Val Pro Asn Leu Gln Ser Leu1
518915PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 189Pro Glu Trp Gln Thr Pro Ser Phe Pro
Lys Ile His Leu Gln Glu1 5 10 1519015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 190Pro Ser Phe Pro Lys Ile His Leu Gln Glu Asp Ile Val Asp
Arg1 5 10 1519115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 191Ile His Leu Gln Glu Asp
Ile Val Asp Arg Cys Lys Gln Phe Val1 5 10 1519215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 192Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro Leu Thr
Val1 5 10 1519315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 193Cys Lys Gln Phe Val Gly
Pro Leu Thr Val Asn Glu Lys Arg Arg1 5 10 1519415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 194Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile
Met1 5 10 1519515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 195Asn Glu Lys Arg Arg Leu
Lys Leu Ile Met Pro Ala Arg Phe Tyr1 5 10 1519615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 196Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val Thr
Lys1 5 10 1519715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 197Pro Ala Arg Phe Tyr Pro
Asn Val Thr Lys Tyr Leu Pro Leu Asp1 5 10 1519815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 198Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys
Pro1 5 10 1519915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 199Tyr Leu Pro Leu Asp Lys
Gly Ile Lys Pro Tyr Tyr Pro Glu His1 5 10 1520015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 200Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His
Tyr1 5 10 1520115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 201Tyr Tyr Pro Glu His Ala
Val Asn His Tyr Phe Gln Thr Arg His1 5 10 1520215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 202Ala Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr
Leu1 5 10 1520315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 203Phe Gln Thr Arg His Tyr
Leu His Thr Leu Trp Lys Ala Gly Ile1 5 10 1520415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 204Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg
Glu1 5 10 1520515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 205Trp Lys Ala Gly Ile Leu
Tyr Lys Arg Glu Thr Thr Arg Ser Ala1 5 10 1520615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 206Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly
Ser1 5 10 1520715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 207Thr Thr Arg Ser Ala Ser
Phe Cys Gly Ser Pro Tyr Ser Trp Glu1 5 10 1520815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 208Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln
His1 5 10 1520915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 209Pro Tyr Ser Trp Glu Gln
Glu Leu Gln His Gly Ser Cys Trp Trp1 5 10 1521015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 210Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg
Asn1 5 10 1521115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 211Gly Ser Cys Trp Trp Leu
Gln Phe Arg Asn Ser Lys Pro Cys Ser1 5 10 1521215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 212Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu
Thr1 5 10 1521315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 213Ser Lys Pro Cys Ser Glu
Tyr Cys Leu Thr His Leu Val Asn Leu1 5 10 1521415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 214Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp
Gly1 5 10 1521515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 215His Leu Val Asn Leu Leu
Glu Asp Trp Gly Pro Cys Asp Glu His1 5 10 1521615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 216Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His
Ile1 5 10 1521715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 217Pro Cys Asp Glu His Gly
Glu His His Ile Arg Ile Pro Arg Thr1 5 10 1521815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 218Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val
Thr1 5 10 1521915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 219Arg Ile Pro Arg Thr Pro
Ala Arg Val Thr Gly Gly Val Phe Leu1 5 10 1522015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 220Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn
Pro1 5 10 1522115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 221Gly Gly Val Phe Leu Val
Asp Lys Asn Pro His Asn Thr Ala Glu1 5 10 1522215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 222Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Val
Val1 5 10 1522315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 223His Asn Thr Ala Glu Ser
Arg Leu Val Val Asp Phe Ser Gln Phe1 5 10 1522415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 224Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile
Thr1 5 10 1522515PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 225Asp Phe Ser Gln Phe Ser
Arg Gly Ile Thr Arg Val Ser Trp Pro1 5 10 1522615PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 226Ser Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Val
Pro1 5 10 1522715PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 227Arg Val Ser Trp Pro Lys
Phe Ala Val Pro Asn Leu Gln Ser Leu1 5 10 1522815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 228Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu
Ser1 5 10 1522915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 229Asn Leu Gln Ser Leu Thr
Asn Leu Leu Ser Ser Asn Leu Ser Trp1 5 10 1523015PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 230Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp
Val1 5 10 1523115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 231Ser Asn Leu Ser Trp Leu
Ser Leu Asp Val Ser Ala Ala Phe Tyr1 5 10 1523215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 232Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu
His1 5 10 1523315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 233Ser Ala Ala Phe Tyr His
Ile Pro Leu His Pro Ala Ala Met Pro1 5 10 15
* * * * *