Note: Descriptions are shown in the official language in which they were submitted.
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Methods and Compositions for Inducing an Immune Response Against Hepatitis B
Virus
(HBV)
CROSS-REFERENCE TO RELA1ED APPLICATIONS
[0001] This application claims priority to International Patent Application
No.
PCT/IB2017/058148, filed December 19, 2017, and U.S. Provisional Patent
Application
No. 62/607,439, filed December 19, 2017, the disclosures of which are
incorporated
herein by reference in their entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application contains a sequence listing, which is submitted
electronically via
EFS-Web as an ASCII formatted sequence listing with a file name "688097-413
Sequence
Listing" and a creation date of December 14, 2018, and having a size of 49.6
KB. The
sequence listing submitted via EFS-Web is part of the specification and is
herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to biotechnology. More particularly, the
invention relates
to methods and compositions for enhancing an immune response to Hepatitis B
Virus
(HBV) in a subject in need thereof.
BACKGROUND OF THE INVENTION
[0004] Hepatitis B virus (HBV) is a small 3.2-kb hepatotropic DNA virus that
encodes
four open reading frames and seven proteins. About two billion people are
infected with
HBV, and approximately 240 million people have chronic hepatitis B infection
(chronic
HBV), characterized by persistent virus and subvirus particles in the blood
for more than 6
months (1). Persistent HBV infection leads to T-cell exhaustion in circulating
and
intrahepatic HBV-specific CD4+ and CD8+ T-cells through chronic stimulation of
HBV-
specific T-cell receptors with viral peptides and circulating antigens. As a
result, T-cell
polyfunctionality is decreased (i.e., decreased levels of IL-2, tumor necrosis
factor (TNF)-
a, IFN-y, and lack of proliferation).
[0005] A safe and effective prophylactic vaccine against HBV infection has
been
available since the 1980s and is the mainstay of hepatitis B prevention (3).
The World
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Health Organization recommends vaccination of all infants, and, in countries
where there
is low or intermediate hepatitis B endemicity, vaccination of all children and
adolescents
(<18 years of age), and of people of certain at risk population categories.
Due to
vaccination, worldwide infection rates have dropped dramatically. However,
prophylactic
vaccines do not cure established HBV infection.
[0006] Chronic HBV is currently treated with IFN-a and nucleoside or
nucleotide
analogs, but there is no ultimate cure due to the persistence in infected
hepatocytes of an
intracellular viral replication intermediate called covalently closed circular
DNA
(cccDNA), which plays a fundamental role as a template for viral RNAs, and
thus new
virions. It is thought that induced virus-specific T-cell and B-cell responses
can
effectively eliminate cccDNA-carrying hepatocytes. Current therapies targeting
the HBV
polymerase suppress viremia, but offer limited effect on cccDNA that resides
in the
nucleus and related production of circulating antigen. The most rigorous form
of a cure
may be elimination of HBV cccDNA from the organism, which has neither been
observed
as a naturally occurring outcome nor as a result of any therapeutic
intervention. However,
loss of HBV surface antigens (E1BsAg) is a clinically credible equivalent of a
cure, since
disease relapse can occur only in cases of severe immunosuppression, which can
then be
prevented by prophylactic treatment. Thus, at least from a clinical
standpoint, loss of
HBsAg is associated with the most stringent form of immune reconstitution
against HBV.
[0007] For example, immune modulation with pegylated interferon (pegIFN)-a has
proven better in comparison to nucleoside or nucleotide therapy in terms of
sustained off-
treatment response with a finite treatment course. Besides a direct antiviral
effect, IFN-a is
reported to exert epigenetic suppression of cccDNA in cell culture and
humanized mice,
which leads to reduction of virion productivity and transcripts (4). However,
this therapy
is still fraught with side-effects and overall responses are rather low, in
part because IFN-a
has only poor modulatory influences on HBV-specific T-cells. In particular,
cure rates are
low (< 10%) and toxicity is high. Likewise, direct acting HBV antivirals,
namely the
HBV polymerase inhibitors entecavir and tenofovir, are effective as
monotherapy in
inducing viral suppression with a high genetic barrier to emergence of drug
resistant
mutants and consecutive prevention of liver disease progression. However, cure
of
chronic hepatitis B, defined by ElBsAg loss or seroconversion, is rarely
achieved with
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such HBV polymerase inhibitors. Therefore, these antivirals in theory need to
be
administered indefinitely to prevent reoccurrence of liver disease, similar to
antiretroviral
therapy for human immunodeficiency virus (HIV).
[0008] Therapeutic vaccination has the potential to eliminate HBV from
chronically
infected patients (5). Many strategies have been explored, but to date
therapeutic
vaccination has not proven successful.
BRIEF SUMMARY OF THE INVENTION
[0009] Accordingly, there is an unmet medical need in the treatment of
hepatitis B virus
(HBV), particularly chronic HBV, for a finite well-tolerated treatment with a
higher cure
rate. The application satisfies this need. Provided are Modified Vaccinia
Ankara (MVA)
vectors. An MVA vector of the application comprises a non-naturally occurring
nucleic
acid molecule comprising a first polynucleotide sequence encoding an HBV
polymerase
antigen comprising an amino acid sequence that is at least 98% identical to
SEQ ID NO:
4. The HBV polymerase antigen of the MVA vectors can, for example, be capable
of
inducing an immune response in a mammal against at least two HBV genotypes.
Preferably, the HBV polymerase antigen is capable of inducing a T cell
response in a
mammal against at least HBV genotypes B, C, and D. More preferably the HBV
polymerase antigen is capable of inducing a CD8 T cell response in a human
subject
against at least HBV genotypes A, B, C, and D. In an embodiment of the
application, an
HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4. In
an
embodiment of the application, the first polynucleotide sequence is at least
90% identical
to SEQ ID NO: 3. In an embodiment of the application, the first polynucleotide
sequence
comprises the polynucleotide sequence of SEQ ID NO: 3.
[0010] In an embodiment of the application, an MVA vector can further comprise
a
polynucleotide sequence encoding a signal sequence operably linked to the HBV
polymerase antigen. The signal sequence can, for example, comprise an amino
acid
sequence of SEQ ID NO: 6 or SEQ ID NO: 11. Preferably, the signal sequence is
encoded by the polynucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 10.
[0011] In an embodiment of the application, an MVA vector further comprises a
second
polynucleotide sequence encoding a truncated HBV core antigen consisting of
the amino
acid sequence of SEQ ID NO: 2. In an embodiment of the application, the second
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polynucleotide sequence is at least 90% identical to SEQ ID NO: 1. In an
embodiment of
the application, the second polynucleotide sequence comprises the
polynucleotide
sequence of SEQ ID NO: 1.
[0012] Also provided are compositions comprising an MVA vector of the
application
and a pharmaceutically acceptable carrier.
[0013] Also provided are methods of enhancing an immune response in a human
subject in need thereof. The methods comprise (a) administering to the human
subject a
first composition comprising an immunologically effective amount of an
adenovirus
vector comprising a non-naturally occurring nucleic acid molecule comprising a
first
polynucleotide sequence encoding an HBV polymerase antigen comprising an amino
acid
sequence that is at least 98% identical to SEQ ID NO:4; and (b) administering
to the
human subject a second composition comprising an immunologically effective
amount of
an MVA vector of the application; to thereby obtain an enhanced immune
response
against the HBV antigen in the human subject. In an embodiment of the
application, the
HBV polymerase antigen does not have reverse transcriptase activity and RNase
H
activity. In an embodiment of the application, the first composition is for
priming the
immune response, and the second composition is for boosting the immune
response in the
subject in need thereof. In an embodiment of the application, step (b) is
conducted 1-12
weeks after step (a). In an embodiment of the application, step (b) is
conducted 2-12
weeks after step (a). In an embodiment of the application, step (b) is
conducted at least 1
week after step (a). In an embodiment of the application, step (b) is
conducted at least 2
weeks after step (a).
[0014] In an embodiment of the application, an HBV polymerase antigen of the
first
composition is capable of inducing an immune response in the human subject
against at
least two HBV genotypes, preferably the HBV polymerase antigen is capable of
inducing
a T cell response in the human subject against at least HBV genotypes B, C,
and D, and
more preferably the HBV polymerase antigen is capable of inducing a CD8 T cell
response in the human subject against at least HBV genotypes A, B, C, and D.
[0015] In an embodiment of the application, the HBV polymerase antigen of the
first
composition comprises the amino acid sequence of SEQ ID NO: 4. The first
polynucleotide sequence of the first composition can, for example, be at least
90%
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identical to SEQ ID NO: 19. In an embodiment of the application, the first
polynucleotide sequence of the first composition comprises the polynucleotide
sequence
of SEQ ID NO: 19.
[0016] In an embodiment of the application, the nucleic acid molecule of the
adenovirus
vector in the first composition further comprises a second polynucleotide
sequence
encoding a truncated HBV core antigen consisting of the amino acid sequence of
SEQ ID
NO: 2. The second polynucleotide sequence of the first composition can, for
example, be
at least 90% identical to SEQ ID NO: 17. In an embodiment of the application,
the
second polynucleotide sequence of the first composition comprises the
polynucleotide
sequence of SEQ ID NO:17.
[0017] In an embodiment of the application, the first and second
polynucleotide
sequences of the first composition encode a fusion protein comprising the
truncated HBV
core antigen operably linked to the HBV polymerase antigen. The fusion protein
of the
first composition can, for example, comprise the truncated HBV core antigen
operably
linked to the HBV polymerase antigen via a linker. The linker of the first
composition
can, for example, comprise the amino acid sequence of (AlaGly)õ, wherein n is
an integer
of 2 to 5. Preferably the linker is encoded by a polynucleotide sequence
comprising SEQ
ID NO:14. In an embodiment of the application, the fusion protein of the first
composition comprises the amino acid sequence of SEQ ID NO:12.
[0018] In an embodiment of the application, the enhanced immune response
comprises
an enhanced antibody response against the HBV antigen in the human subject.
The
enhanced immune response can, for example, comprise an enhanced CD8+ T cell
response against the HBV antigen in the human subject. The enhanced immune
response
can, for example, comprise an enhanced CD4+ T cell response against the HBV
antigen
in the human subject.
[0019] In an embodiment of the application, the adenovirus vector is an rAd26
or
rAd35 vector.
[0020] In an embodiment of the application, a method of enhancing an immune
response in a human subject comprises (a) administering to the human subject a
first
composition comprising an immunologically effective amount of a first plasmid
comprising a first non-naturally occurring nucleic acid molecule comprising a
first
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polynucleotide sequence encoding an HBV polymerase antigen comprising an amino
acid
sequence that is at least 98% identical to SEQ ID NO: 4 and a second plasmid
comprising
a second non-naturally occurring nucleic acid molecule comprising a second
polynucleotide sequence encoding a truncated HBV core antigen consisting of
the amino
acid sequence of SEQ ID NO: 2; and (b) administering to the human subject a
second
composition comprising an immunologically effective amount of the MVA vector
of the
application; to thereby obtain an enhanced immune response against the HBV
antigen in
the human subject. In an embodiment of the application, the HBV polymerase
antigen of
the first composition does not have reverse transcriptase activity and RNase H
activity.
In an embodiment of the application, the first composition is for priming the
immune
response and the second composition is for boosting the immune response. In an
embodiment of the application, step (b) is conducted 1-12 weeks after step
(a). In an
embodiment of the application, step (b) is conducted 2-12 weeks after step
(a). In an
embodiment of the application, step (b) is conducted at least 1 week after
step (a). In an
embodiment of the application, step (b) is conducted at least 2 weeks after
step (a).
[0021] In an embodiment of the application, the HBV polymerase antigen of the
first
composition is capable of inducing an immune response in the human subject
against at
least two HBV genotypes, preferably the HBV polymerase antigen is capable of
inducing
a T cell response in the human subject against at least HBV genotypes B, C,
and D, and
more preferably the HBV polymerase antigen is capable of inducing a CD8 T cell
response in the human subject against at least HBV genotypes A, B, C, and D.
[0022] In an embodiment of the application, the HBV polymerase antigen of the
first
composition comprises the amino acid sequence of SEQ ID NO: 4. The first
polynucleotide sequence of the first composition can, for example, be at least
90%
identical to SEQ ID NO:19. In an embodiment of the application, the first
polynucleotide
sequence of the first composition comprises the polynucleotide sequence of SEQ
ID
NO:19.
[0023] In an embodiment of the application, the HBV polymerase antigen of the
first
composition comprises the amino acid sequence of SEQ ID NO: 4. In an
embodiment of
the application, the first polynucleotide sequence of the first composition is
at least 90%
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identical to SEQ ID NO: 20. The first polynucleotide sequence of the first
composition
can, for example, comprise SEQ ID NO: 20.
[0024] In an embodiment of the application, the nucleic acid molecule of the
first
plasmid of the first composition further comprises a polynucleotide sequence
encoding a
signal sequence operably linked to the EIBV polymerase antigen of the first
composition.
The signal sequence can, for example, comprise the amino acid sequence of SEQ
ID NO:
6 or SEQ ID NO: 11, preferably the signal sequence is encoded by the
polynucleotide
sequence of SEQ ID NO: 5 or SEQ ID NO: 10.
[0025] In an embodiment of the application, the second polynucleotide sequence
of the
first composition is at least 90% identical to SEQ ID NO: 18. The second
polynucleotide
sequence of the first composition can, for example, comprise the
polynucleotide sequence
of SEQ ID NO: 18.
[0026] In an embodiment of the application, the first and second
polynucleotide
sequences of the first composition further comprise a promoter sequence,
optionally one
or more additional regulatory sequences, preferably the promoter sequence
comprises the
polynucleotide sequence of SEQ ID NO: 7, and the additional regulatory
sequence is
selected from the group consisting of an enhancer sequence of SEQ ID NO: 8 or
SEQ ID
NO: 15, and a polyadenylation signal sequence of SEQ ID NO: 16.
[0027] In an embodiment of the application, the enhanced immune response
comprises
.. an enhanced antibody response against the EIBV antigen in the human
subject. The
enhanced immune response can, for example, comprise an enhanced CD8+ T cell
response against the EIBV antigen in the human subject. The enhanced immune
response
can, for example, comprise an enhanced CD4+ T cell response against the EIBV
antigen
in the human subject.
[0028] Other aspects, features and advantages of the invention will be
apparent from the
following disclosure, including the detailed description of the invention and
its preferred
embodiments and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The foregoing summary, as well as the following detailed description of
the
.. invention, will be better understood when read in conjunction with the
appended
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drawings. It should be understood that the invention is not limited to the
precise
embodiments shown in the drawings.
[0030] In the drawings:
[0031] FIGS. 1A-1B depict the genome and viral life cycle of hepatitis B
virus; FIG.
1A is a diagram of the genome of hepatitis B virus (HBV); in the native virus,
the
polymerase protein (Pol) contains the coding sequence for the envelope
proteins in a
different open reading frame; the envelope proteins (pre-S1, pre-S2, and S)
are in the same
open reading frame; FIG. 1B shows the viral life cycle of HBV;
[0032] FIGS. 2A-2C show the schematic representations of the expression
cassettes in
adenoviral and MVA vectors according to embodiments of the application; FIG.
2A
shows the expression cassette for a truncated HBV core antigen, which contains
a CMV
promoter, an intron (a fragment derived from the human ApoAl gene - GenBank
accession X01038 base pairs 295 ¨ 523, harboring the ApoAl second intron), a
human
immunoglobulin secretion signal, followed by a coding sequence for a truncated
HBV
core antigen and a SV40 polyadenylation signal; FIG. 2B shows the expression
cassette
for a fusion protein of a truncated HBV core antigen operably linked to a HBV
polymerase antigen, which is otherwise identical to the expression cassette
for the
truncated HBV core antigen except the HBV antigen; FIG. 2C shows an expression
cassette comprising a HBV core antigen operably linked to a Prl 3.5 long
promoter and an
expression cassette comprising a HBV polymerase antigen operably linked to a
PrHyb
promoter;
[0033] FIG. 3 shows a graph of ELISPOT responses of Fl mice immunized with
different combinations of HBV adenoviral vectors and HBV MVA; HBV core or
polymerase peptide pools used to stimulate splenocytes isolated from the
various
vaccinated animal groups are indicated in black (core) and grey (pol). Poll
and po12
responses were summed; the X-axis shows the adenovector dose and the presence
or
absence of the MVA boost; the number of responsive T-cells are indicated on
the y-axis
expressed as spot forming cells (SFC) per 106 splenocytes;
[0034] FIG. 4 shows a graph of intracellular cytokine staining (ICS) responses
of Fl
mice immunized with different combinations of HBV adenoviral vectors and HBV
MVA;
HBV core and polymerase peptide pools used to stimulate splenocytes isolated
from the
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various vaccinated animal groups are indicated in black (core) and grey (pol);
Poll and
po12 responses were summed; the X-axis shows the adenovirus vector dose and
the
presence or absence of the MVA boost. The percentages of CD8(+) T cells
positive for
IFN' are shown on the y-axis;
[0035] FIG. 5 shows a graph of ICS responses of Fl mice immunized with
different
combinations of HBV adenoviral vectors and HBV MVA vectors; HBV core and
polymerase peptide pools used to stimulate splenocytes isolated from the
various
vaccinated animal groups are indicated in black (core) and grey (pol); Poll
and po12
responses were summed; the X-axis shows the adenoviral vector dose and the
presence or
absence of the MVA boost; the percentages of CD4(+) T cells positive for IFN y
are
shown on the y-axis;
[0036] FIG. 6 shows a graph of ELISPOT responses of NEIPs immunized with
different
combinations of HBV adenoviral vectors and HBV MVA vectors; HBV core or
polymerase peptide pools used to stimulate PBMCs isolated from the various
vaccinated
animal groups are indicated in squares (core), circles (poll) and triangles
(po12); the X-
axis shows the different experimental groups and timepoints. The number of
responsive T-
cells are indicated on the y-axis expressed as spot forming cells (SFC) per
106 splenocytes;
background (medium + DMSO stimulation) subtracted data is shown; and
[0037] FIGS. 7A, 7B and 7C show graphs of ICS responses of NHPs immunized with
different combinations of HBV adenoviral vectors and HBV MVA vectors; HBV core
and
polymerase peptide pools used to stimulate PBMCs isolated from the various
vaccinated
animal groups are indicated in squares (core), circles (poll) and triangles
(po12); the X-
axis shows the different experimental groups and time points; the percentages
of CD4(+)
and CD8(+) T cells positive for IFN are shown on the y-axis; background
(medium +
DMSO stimulation) subtracted data is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Various publications, articles and patents are cited or described in
the
background and throughout the specification; each of these references is
herein
incorporated by reference in its entirety. Discussion of documents, acts,
materials,
devices, articles or the like which has been included in the present
specification is for the
purpose of providing context for the invention. Such discussion is not an
admission that
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any or all of these matters form part of the prior art with respect to any
inventions
disclosed or claimed.
[0039] Unless defined otherwise, all technical and scientific terms used
herein have the
same meaning as commonly understood to one of ordinary skill in the art to
which this
invention pertains. Otherwise, certain terms used herein have the meanings as
set forth in
the specification.
[0040] It must be noted that as used herein and in the appended claims, the
singular
forms "a," "an," and "the" include plural reference unless the context clearly
dictates
otherwise.
[0041] Unless otherwise stated, any numerical values, such as a concentration
or a
concentration range described herein, are to be understood as being modified
in all
instances by the term "about." Thus, a numerical value typically includes
10% of the
recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to
1.1
mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v)
to
11% (w/v). As used herein, the use of a numerical range expressly includes all
possible
subranges, all individual numerical values within that range, including
integers within
such ranges and fractions of the values unless the context clearly indicates
otherwise.
[0042] Unless otherwise indicated, the term "at least" preceding a series of
elements is
to be understood to refer to every element in the series. Those skilled in the
art will
recognize, or be able to ascertain using no more than routine experimentation,
many
equivalents to the specific embodiments of the invention described herein.
Such
equivalents are intended to be encompassed by the invention.
[0043] Throughout this specification and the claims which follow, unless the
context
requires otherwise, the word "comprise", and variations such as "comprises"
and
"comprising", will be understood to imply the inclusion of a stated integer or
step or
group of integers or steps but not the exclusion of any other integer or step
or group of
integer or step. When used herein the term "comprising" can be substituted
with the term
"containing" or "including" or sometimes when used herein with the term
"having".
[0044] When used herein "consisting of' excludes any element, step, or
ingredient not
specified in the claim element. When used herein, "consisting essentially of'
does not
exclude materials or steps that do not materially affect the basic and novel
characteristics
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of the claim. Any of the aforementioned terms of "comprising," "containing,"
"including," and "having," whenever used herein in the context of an aspect or
embodiment of the invention can be replaced with the term "consisting of' or
"consisting
essentially of' to vary scopes of the disclosure.
[0045] As used herein, the conjunctive term "and/or" between multiple recited
elements
is understood as encompassing both individual and combined options. For
instance,
where two elements are conjoined by "and/or," a first option refers to the
applicability of
the first element without the second. A second option refers to the
applicability of the
second element without the first. A third option refers to the applicability
of the first and
second elements together. Any one of these options is understood to fall
within the
meaning, and therefore satisfy the requirement of the term "and/or" as used
herein.
Concurrent applicability of more than one of the options is also understood to
fall within
the meaning, and therefore satisfy the requirement of the term "and/or."
[0046] As used herein, "subject" means any animal, preferably a mammal, most
preferably a human, to whom will be or has been treated by a method according
to an
embodiment of the application. The term "mammal" as used herein, encompasses
any
mammal. Examples of mammals include, but are not limited to, cows, horses,
sheep,
pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (1\11-
11Ps), such as
monkeys or apes, humans, etc., more preferably a human.
[0047] The terms "adjuvant" and "immune stimulant" are used interchangeably
herein,
and are defined as one or more substances that cause stimulation of the immune
system.
In this context, an adjuvant is used to enhance an immune response to the
adenovirus
and/or MVA vectors of the application.
[0048] It should also be understood that the terms "about," "approximately,"
"generally," "substantially" and like terms, used herein when referring to a
dimension or
characteristic of a component of the preferred invention, indicate that the
described
dimension/characteristic is not a strict boundary or parameter and does not
exclude minor
variations therefrom that are functionally the same or similar, as would be
understood by
one having ordinary skill in the art. At a minimum, such references that
include a
numerical parameter would include variations that, using mathematical and
industrial
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principles accepted in the art (e.g., rounding, measurement or other
systematic errors,
manufacturing tolerances, etc.), would not vary the least significant digit.
[0049] The terms "identical" or percent "identity," in the context of two or
more
nucleic acids or polypeptide sequences (e.g., HBV antigenic polypeptides and
polynucleotides that encode them), refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that
are the same, when compared and aligned for maximum correspondence, as
measured
using one of the following sequence comparison algorithms or by visual
inspection.
[0050] For sequence comparison, typically one sequence acts as a reference
sequence,
to which test sequences are compared. When using a sequence comparison
algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are
designated, if necessary, and sequence algorithm program parameters are
designated.
The sequence comparison algorithm then calculates the percent sequence
identity for
the test sequence(s) relative to the reference sequence, based on the
designated program
parameters.
[0051] Optimal alignment of sequences for comparison can be conducted, e.g.,
by the
local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981),
by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970),
by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad.
Sci. USA
85:2444 (1988), by computerized implementations of these algorithms (GAP,
BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see
generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds.,
Current
Protocols, a joint venture between Greene Publishing Associates, Inc. and John
Wiley &
Sons, Inc., (1995 Supplement) (Ausubel)).
[0052] Examples of algorithms that are suitable for determining percent
sequence
identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which
are
described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et
al. (1997)
Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST
analyses is publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring sequence
pairs
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(HSPs) by identifying short words of length W in the query sequence, which
either
match or satisfy some positive-valued threshold score T when aligned with a
word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al, supra). These initial neighborhood word hits act as
seeds for
initiating searches to find longer HSPs containing them. The word hits are
then
extended in both directions along each sequence for as far as the cumulative
alignment
score can be increased.
[0053] Cumulative scores are calculated using, for nucleotide sequences, the
parameters M (reward score for a pair of matching residues; always > 0) and N
(penalty
score for mismatching residues; always < 0). For amino acid sequences, a
scoring
matrix is used to calculate the cumulative score. Extension of the word hits
in each
direction are halted when: the cumulative alignment score falls off by the
quantity X
from its maximum achieved value; the cumulative score goes to zero or below,
due to
the accumulation of one or more negative-scoring residue alignments; or the
end of
either sequence is reached. The BLAST algorithm parameters W, T, and X
determine
the sensitivity and speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10,
M=5, N=-
4, and a comparison of both strands. For amino acid sequences, the BLASTP
program
uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)).
[0054] In addition to calculating percent sequence identity, the BLAST
algorithm also
performs a statistical analysis of the similarity between two sequences (see,
e.g., Karlin
& Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum probability
(P(N)),
.. which provides an indication of the probability by which a match between
two
nucleotide or amino acid sequences would occur by chance. For example, a
nucleic
acid is considered similar to a reference sequence if the smallest sum
probability in a
comparison of the test nucleic acid to the reference nucleic acid is less than
about 0.1,
more preferably less than about 0.01, and most preferably less than about
0.001.
[0055] A further indication that two nucleic acid sequences or polypeptides
are
substantially identical is that the polypeptide encoded by the first nucleic
acid is
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immunologically cross reactive with the polypeptide encoded by the second
nucleic
acid, as described below. Thus, a polypeptide is typically substantially
identical to a
second polypeptide, for example, where the two peptides differ only by
conservative
substitutions. Another indication that two nucleic acid sequences are
substantially
identical is that the two molecules hybridize to each other under stringent
conditions, as
described below.
[0056] As used herein, the term "enhanced" when used with respect to an immune
response, such as a CD4+ T cell response, an antibody response, or a CD8+ T
cell
response, refers to an increase in the immune response in a subject
administered with a
prime-boost combination of MVA and adenovirus vectors according to the
application,
relative to the corresponding immune response observed from the subject
administered
with an MVA vector or an adenovirus of the application alone.
[0057] As used herein, the term "CD4+ or CD8+T cell response" refers to a T
cell
immune response that is characterized by observing a high proportion of
immunogen-
specific CD4+ T cells or CD8+ T cells within the population of total
responding T cells
following vaccination. The total immunogen-specific T-cell response can be
determined
by an IFN-gamma ELISPOT assay. The immunogen-specific CD4+ or CD8+ T cell
immune response can be determined by an ICS assay.
[0058] As used herein, the term "enhanced antibody response" refers to an
increased
antibody response in a subject administered with a prime-boost combination of
MVA and
adenovirus vectors according to the application, relative to the corresponding
immune
response observed from the subject administered with an MVA vector or an
adenovirus
of the application alone.
[0059] The term "adjuvant" is defined as one or more substances that cause
stimulation
of the immune system. In this context, an adjuvant is used to enhance an
immune
response to the plasmid, adenovirus, and/or MVA vectors of the application.
[0060] As used herein, the term "antigenic gene product or fragment thereof-
or
"antigenic protein" can include a bacterial, viral, parasitic, or fungal
protein, or a
fragment thereof. Preferably, an antigenic protein or antigenic gene product
is capable of
raising in a host a protective immune response, e.g., inducing an immune
response
against a disease or infection (e.g., a bacterial, viral, parasitic, or fungal
disease or
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infection), and/or producing an immunity in (i.e., vaccinating) a subject
against a disease
or infection, that protects the subject against the disease or infection.
[0061] In an attempt to help the reader of the application, the description
has been
separated in various paragraphs or sections, or is directed to various
embodiments of the
application. These separations should not be considered as disconnecting the
substance of
a paragraph or section or embodiments from the substance of another paragraph
or
section or embodiments. To the contrary, one skilled in the art will
understand that the
description has broad application and encompasses all the combinations of the
various
sections, paragraphs and sentences that can be contemplated. The discussion of
any
embodiment is meant only to be exemplary and is not intended to suggest that
the scope
of the disclosure, including the claims, is limited to these examples. For
example, while
embodiments of HBV vectors of the application (e.g., plasmid DNA or viral
vectors)
described herein may contain particular components, including, but not limited
to, certain
promoter sequences, enhancer or regulatory sequences, signal peptides, coding
sequence
of an HBV antigen, polyadenylation signal sequences, etc. arranged in a
particular order,
those having ordinary skill in the art will appreciate that the concepts
disclosed herein
may equally apply to other components arranged in other orders that can be
used in HBV
vectors of the application. The application contemplates use of any of the
applicable
components in any combination having any sequence that can be used in HBV
vectors of
the application, whether or not a particular combination is expressly
described.
[0062] Hepatitis B Virus (HBV)
[0063] As used herein "hepatitis B virus" or "HBV" refers to a virus of the
hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus
that
encodes four open reading frames and seven proteins. See FIG. 1A. The seven
proteins
encoded by HBV include small (S), medium (M), and large (L) surface antigen
(HBsAg)
or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase
(Pol), and
Effix protein. HBV expresses three surface antigens, or envelope proteins, L,
M, and S,
with S being the smallest and L being the largest. The extra domains in the M
and L
proteins are named Pre-52 and Pre-S1, respectively. Core protein is the
subunit of the
viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse
transcriptase,
RNaseH, and primer), which takes place in nucleocapsids localized to the
cytoplasm of
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infected hepatocytes. PreCore is the core protein with an N-terminal signal
peptide and is
proteolytically processed at its N and C termini before secretion from
infected cells, as
the so-called hepatitis B e-antigen (HBeAg). Effix protein is required for
efficient
transcription of covalently closed circular DNA (cccDNA). Effix is not a viral
structural
protein. All viral proteins of HBV have their own mRNA except for core and
polymerase, which share an mRNA. With the exception of the protein pre-Core,
none of
the HBV viral proteins are subject to post-translational proteolytic
processing.
[0064] The HBV virion contains a viral envelope, nucleocapsid, and single copy
of the
partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of
core
.. protein and is covered by a capsid membrane embedded with the S, M, and L
viral
envelope or surface antigen proteins. After entry into the cell, the virus is
uncoated and
the capsid-containing relaxed circular DNA (rcDNA) with covalently bound viral
polymerase migrates to the nucleus. During that process, phosphorylation of
the Core
protein induces structural changes, exposing a nuclear localization signal
enabling
interaction of the capsid with so-called importins. These importins mediate
binding of
the core protein to nuclear pore complexes upon which the capsid disassembles
and
polymerase/rcDNA complex is released into the nucleus. Within the nucleus the
rcDNA
becomes deproteinized (removal of polymerase) and is converted by host DNA
repair
machinery to a covalently closed circular DNA (cccDNA) genome from which
overlapping transcripts encode for EIBeAg, ElBsAg, Core protein, viral
polymerase and
Effix protein. Core protein, viral polymerase, and pre-genomic RNA (pgRNA)
associate
in the cytoplasm and self-assemble into immature pgRNA-containing capsid
particles,
which further convert into mature rcDNA-capsids and function as a common
intermediate that is either enveloped and secreted as infections virus
particles or
transported back to the nucleus to replenish and maintain a stable cccDNA
pool. See
FIG. 1B.
[0065] To date, HBV is divided into four serotypes (adr, adw, ayr, ayw) based
on
antigenic epitopes present on the envelope proteins, and into eight genotypes
(A, B, C, D,
E, F, G, and H) based on the sequence of the viral genome. The HBV genotypes
are
distributed over different geographic regions. For example, the most prevalent
genotypes
in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle
East, and
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India, whereas genotype A is widespread in Northern Europe, sub-Saharan
Africa, and
West Africa.
[0066] HBV Antigens
[0067] As used herein, the terms "HBV antigen," "antigenic polypeptide of
HBV,"
"HBV antigenic polypeptide," "HBV antigenic protein," "HBV immunogenic
polypeptide," and "HBV immunogen" all refer to a polypeptide capable of
inducing an
immune response, e.g., a humoral and/or cellular mediated response, against an
HBV in a
subject. The HBV antigen can be a polypeptide of HBV, a fragment or epitope
thereof,
or a combination of multiple HBV polypeptides, portions or derivatives
thereof. An
HBV antigen is capable of raising in a host a protective immune response,
e.g., inducing
an immune response against a viral disease or infection, and/or producing an
immunity
(i.e., vaccinates) a subject against a viral disease or infection, that
protects the subject
against the viral disease or infection. For example, an HBV antigen can
comprise a
polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as
EIBeAg,
pre-core protein, 1-113sAg (S, M, or L proteins), core protein, viral
polymerase, or Effix
protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G,
and/or H, or
combination thereof.
[0068] (1) HBV Core Antigen
[0069] As used herein, each of the terms "HBV core antigen," "ElBcAg" and
"core
antigen" refers to an HBV antigen capable of inducing an immune response,
e.g., a
humoral and/or cellular mediated response, against an HBV core protein in a
subject.
Each of the terms "core," "core polypeptide," and "core protein" refers to the
HBV viral
core protein. Full-length core antigen is typically 183 amino acids in length
and includes
an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain
(amino
acids 150 to 183). The 34-residue nucleic acid binding domain is required for
pre-
genomic RNA encapsidation. This domain also functions as a nuclear import
signal. It
comprises 17 arginine residues and is highly basic, consistent with its
function. HBV
core protein is dimeric in solution, with the dimers self-assembling into
icosahedral
capsids. Each dimer of core protein has four a-helix bundles flanked by an a-
helix
domain on either side. Truncated HBV core proteins lacking the nucleic acid
binding
domain are also capable of forming capsids.
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[0070] In an embodiment of the application, an HBV antigen is a truncated HBV
core
antigen. As used herein, a "truncated HBV core antigen," refers to an HBV
antigen that
does not contain the entire length of an HBV core protein but is capable of
inducing an
immune response against the HBV core protein in a subject. For example, an HBV
core
antigen can be modified to delete one or more amino acids of the highly
positively
charged (arginine rich) C-terminal nucleic acid binding domain of the core
antigen,
which typically contains seventeen arginine (R) residues. In some embodiments
of the
application, an HBV core antigen is a truncated HBV core protein. A truncated
HBV
core antigen of the application is preferably a C-terminally truncated HBV
core protein
which does not comprise the HBV core nuclear import signal and/or a truncated
HBV
core protein from which the C-terminal HBV core nuclear import signal has been
deleted.
In an embodiment of the application, a truncated HBV core antigen comprises a
deletion
in the C-terminal nucleic acid binding domain, such as a deletion of 1 to 34
amino acid
residues of the C-terminal nucleic acid binding domain, e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, or
34 amino acid residues, preferably a deletion of all 34 amino acid residues.
In a preferred
embodiment, a truncated HBV core antigen comprises a deletion in the C-
terminal
nucleic acid binding domain, preferably all 34 amino acid residues.
[0071] According to embodiments of the application, an HBV core antigen can be
a
consensus sequence derived from multiple HBV genotypes (e.g., genotypes A, B,
C, D,
E, F, G, and H). As used herein, "consensus sequence" means an artificial
sequence of
amino acids based on an alignment of amino acid sequences of homologous
proteins,
e.g., as determined by an alignment (e.g., using Clustal Omega) of amino acid
sequences
of homologous proteins. It can be the calculated order of most frequent amino
acid
residues, found at each position in a sequence alignment, based upon sequences
of HBV
antigens (e.g., core, pol, etc.) from at least 100 natural HBV isolates. A
consensus
sequence can be non-naturally occurring and different from the native viral
sequences.
Consensus sequences can be designed by aligning multiple HBV antigen sequences
from
different sources using a multiple sequence alignment tool, and at variable
alignment
positions, selecting the most frequent amino acid. Preferably, a consensus
sequence of an
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HBV antigen is derived from HBV genotypes B, C, and D. The term "consensus
antigen" is used to refer to an antigen having a consensus sequence.
[0072] A truncated HBV core antigen according to an embodiment of the
application
lacks the nucleic acid binding function, and is capable of inducing an immune
response in
a mammal against at least two HBV genotypes. Preferably the truncated HBV core
antigen is capable of inducing a T cell response in a mammal against at least
HBV
genotypes B, C and D. More preferably, the truncated HBV core antigen is
capable of
inducing a CD8 T cell response in a human subject against at least HBV
genotypes A, B,
C and D.
[0073] In a preferred embodiment of the application, an HBV core antigen is a
consensus antigen, preferably a consensus antigen derived from HBV genotypes
B, C,
and D, more preferably a truncated consensus antigen derived from HBV
genotypes B, C,
and D. An exemplary truncated HBV core consensus antigen according to the
application consists of an amino acid sequence that is at least 90% identical
to SEQ ID
NO: 2, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%,
97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%,
99.9%, or 100% identical to SEQ ID NO: 2. SEQ ID NO: 2 is a core consensus
antigen
derived from HBV genotypes B, C, and D. SEQ ID NO: 2 contains a 34-amino acid
C-
terminal deletion of the highly positively charged (arginine rich) nucleic
acid binding
domain of the native core antigen.
[0074] In a particular embodiment of the application, an HBV core antigen is a
truncated HBV antigen consisting of the amino acid sequence of SEQ ID NO: 2.
[0075] (2) HBV Polymerase Antigen
[0076] As used herein, the term "HBV polymerase antigen," "HBV Pol antigen" or
"HBV pol antigen" refers to an HBV antigen capable of inducing an immune
response,
e.g., a humoral and/or cellular mediated response, against an HBV polymerase
in a
subject. Each of the terms "polymerase," "polymerase polypeptide," "Pol" and
"pol"
refers to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four
domains, including, from the N terminus to the C terminus, a terminal protein
(TP)
domain, which acts as a primer for minus-strand DNA synthesis; a spacer that
is
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nonessential for the polymerase functions; a reverse transcriptase (RT) domain
for
transcription; and a RNase H domain.
[0077] In an embodiment of the application, an HBV antigen comprises an HBV
Pol
antigen, or any immunogenic fragment or combination thereof. The HBV Pol
antigen
can contain further modifications to improve immunogenicity of the antigen,
such as by
introducing mutations into the active sites of the polymerase and/or RNase
domains to
decrease or substantially eliminate certain enzymatic activities.
[0078] Preferably, an HBV Pol antigen of the application does not have reverse
transcriptase activity and RNase H activity, and can be capable of inducing an
immune
response in a mammal against at least two HBV genotypes. Preferably the HBV
Pol
antigen is capable of inducing a T cell response in a mammal against at least
HBV
genotypes B, C and D. More preferably, the HBV Pol antigen is capable of
inducing a
CD8 T cell response in a human subject against at least HBV genotypes A, B, C
and D.
[0079] Thus, in some embodiments of the application, an HBV Pol antigen is an
inactivated Pol antigen. In an embodiment of the application, an inactivated
HBV Pol
antigen comprises one or more amino acid mutations in the active site of the
polymerase
domain. In another embodiment, an inactivated HBV Pol antigen comprises one or
more
amino acid mutations in the active site of the RNaseH domain. In a preferred
embodiment, an inactivated HBV pol antigen comprises one or more amino acid
mutations in the active site of both the polymerase domain and the RNaseH
domain. For
example, the "YXDD" motif in the polymerase domain of the HBV pol antigen
required
for nucleotide/metal ion binding can be mutated, e.g., by replacing one or
more of the
aspartate residues (D) with asparagine residues (N), eliminating or reducing
metal
coordination function, thereby decreasing or substantially eliminating reverse
transcriptase function. Alternatively, or in addition to mutation of the
"YXDD" motif,
the "DEDD" motif in the RNaseH domain of the HBV pol antigen required for Mg2+
coordination can be mutated, e.g., by replacing one or more aspartate residues
(D) with
asparagine residues (N) and/or replacing the glutamate residue (E) with
glutamine (Q),
thereby decreasing or substantially eliminating RNaseH function. In a
particular
embodiment, an HBV pol antigen is modified by (1) mutating the aspartate
residues (D)
to asparagine residues (N) in the "YXDD" motif of the polymerase domain; and
(2)
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mutating the first aspartate residue (D) to an asparagine residue (N) and the
first
glutamate residue (E) to a glutamine residue (N) in the "DEDD" motif of the
RNaseH
domain, thereby decreasing or substantially eliminating both the reverse
transcriptase and
RNaseH functions of the pol antigen.
[0080] In a preferred embodiment of the application, an HBV pol antigen is a
consensus antigen, preferably a consensus antigen derived from HBV genotypes
B, C,
and D, more preferably an inactivated consensus antigen derived from HBV
genotypes B,
C, and D. An exemplary HBV pol consensus antigen according to the application
comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:
4, such as
at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%,
98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
100% identical to SEQ ID NO: 4, preferably at least 98% identical to SEQ ID
NO: 4,
such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%,
99.8%, 99.9% or 100% identical to SEQ ID NO: 4. SEQ ID NO: 4 is a pol
consensus
antigen derived from HBV genotypes B, C, and D comprising four mutations
located in
the active sites of the polymerase and RNaseH domains. In particular, the four
mutations
include mutation of the aspartic acid residues (D) to asparagine residues (N)
in the
"YXDD" motif of the polymerase domain; and mutation of the first aspartate
residue (D)
to an asparagine residue (N) and mutation of the glutamate residue (E) to a
glutamine
residue (Q) in the "DEDD" motif of the RNaseH domain.
[0081] In a particular embodiment of the application, an HBV pol antigen
comprises
the amino acid sequence of SEQ ID NO: 4. In other embodiments of the
application, an
HBV pol antigen consists of the amino acid sequence of SEQ ID NO: 4.
[0082] (3) Fusion of HBV Core Antigen and HBV Polymerase Antigen
[0083] As used herein the term "fusion protein" or "fusion" refers to a single
polypeptide chain having at least two polypeptide domains that are not
normally present
in a single, natural polypeptide.
[0084] In an embodiment of the application, an HBV antigen comprises a fusion
protein comprising a truncated HBV core antigen operably linked to a HBV pol
antigen,
or a HBV pol antigen operably linked to a truncated HBV core antigen,
preferably via a
linker.
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[0085] As used herein, the term "linker" refers to a compound or moiety that
acts as a
molecular bridge to operably link two different molecules, wherein one portion
of the
linker is operably linked to a first molecule, and wherein another portion of
the linker is
operably linked to a second molecule. For example, in a fusion protein
containing a first
polypeptide and a second heterologous polypeptide, a linker serves primarily
as a spacer
between the first and second polypeptides. In one embodiment, the linker is
made up of
amino acids linked together by peptide bonds, preferably from 1 to 20 amino
acids linked
by peptide bonds, wherein the amino acids are selected from the 20 naturally
occurring
amino acids. In one embodiment, the 1 to 20 amino acids are selected from
glycine,
alanine, proline, asparagine, glutamine, and lysine. Preferably, a linker is
made up of a
majority of amino acids that are sterically unhindered, such as glycine and
alanine.
Exemplary linkers are polyglycines, particularly (Gly)5, (Gly)8; poly(Gly-
Ala), and
polyalanines. One exemplary suitable linker as shown in the Examples below is
(AlaGly)õ, wherein n is an integer of 2 to 5.
[0086] Preferably, a fusion protein of the application is capable of inducing
an immune
response in a mammal against HBV core and HBV Pol of at least two HBV
genotypes.
Preferably the fusion protein is capable of inducing a T cell response in a
mammal
against at least HBV genotypes B, C and D. More preferably, the fusion protein
is
capable of inducing a CD8 T cell response in a human subject against at least
HBV
genotypes A, B, C and D.
[0087] In an embodiment of the application, a fusion protein comprises a
truncated
HBV core antigen having an amino acid sequence at least 90%, such as at least
90%,
91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%
identical
to SEQ ID NO: 2, a linker, and a HBV pol antigen having an amino acid sequence
at least
90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%,
97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%,
99.9%, or 100%, identical to SEQ ID NO: 4.
[0088] In a preferred embodiment of the application, a fusion protein
comprises a
truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO:
2, a
linker comprising (AlaGly)õ, wherein n is an integer of 2 to 5, and a HBV Pol
antigen
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having the amino acid sequence of SEQ ID NO: 4. More preferably, a fusion
protein
according to an embodiment of the application comprises the amino acid
sequence of
SEQ ID NO: 12.
[0089] In an embodiment of the application, a fusion protein further comprises
a signal
sequence. Preferably, the signal sequence has the amino acid sequence of SEQ
ID NO: 6
or SEQ ID NO: 11. More preferably, the fusion protein comprises the amino acid
sequence of SEQ ID NO: 13.
[0090] Pobinucleotides and Vectors
[0091] In another general aspect, the application provides a non-naturally
occurring
nucleic acid molecule encoding an HBV antigen according to an embodiment of
the
application, and a vector comprising the non-naturally occurring nucleic acid.
A non-
naturally occurring nucleic acid molecule can comprise any polynucleotide
sequence
encoding an HBV antigen of the application, which can be made using methods
known in
the art in view of the present disclosure. Preferably, a polynucleotide
encodes at least
one of an HBV core antigen and an HBV polymerase antigen of the application. A
polynucleotide can be in the form of RNA or in the form of DNA obtained by
recombinant techniques (e.g., cloning) or produced synthetically (e.g.,
chemical
synthesis). The DNA can be single-stranded or double-stranded, or can contain
portions
of both double-stranded and single-stranded sequence. The DNA can, for
example,
comprise genomic DNA, cDNA, or combinations thereof. The polynucleotide can
also
be a DNA/RNA hybrid. The polynucleotides and vectors of the application can be
used
for recombinant protein production, expression of the protein in a host cell,
or the
production of viral particles. Preferably, a polynucleotide is DNA.
[0092] In an embodiment of the application, a non-naturally occurring nucleic
acid
molecule comprises a polynucleotide encoding a truncated HBV core antigen
consisting
of an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, such
as at least
90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%
identical to
SEQ ID NO: 2, preferably at least 98%, 99% or 100% identical to SEQ ID NO: 2.
In a
particular embodiment of the application, a non-naturally occurring nucleic
acid molecule
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encodes a truncated HBV core antigen comprising the amino acid sequence of SEQ
ID
NO: 2.
[0093] Examples of polynucleotide sequences of the application encoding a
truncated
HBV core antigen comprising the amino acid sequence of SEQ ID NO: 2 include,
but are
not limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO:
1, such as
at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%,
98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
100% identical to SEQ ID NO: 1, preferably at least 98%, 99% or 100% identical
to SEQ
ID NO: 1. In particular embodiments of the application, the non-naturally
occurring
nucleic acid molecule encoding a truncated HBV core antigen comprises the
polynucleotide sequence of SEQ ID NOs: 1, 17, or 18.
[0094] In an embodiment of the application, a non-naturally occurring nucleic
acid
molecule encodes a HBV polymerase antigen comprising an amino acid sequence
that is
at least 90% identical to SEQ ID NO: 4, such as at least 90%, 91%, 92%, 93%,
94%,
95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4,
preferably 100% identical to SEQ ID NO: 4. In a particular embodiment of the
application, a non-naturally occurring nucleic acid molecule encodes a HBV
polymerase
antigen comprising the amino acid sequence of SEQ ID NO: 4.
[0095] Examples of polynucleotide sequences of the application encoding a HBV
Pol
antigen comprising the amino acid sequence of SEQ ID NO: 4 include, but are
not
limited to, a polynucleotide sequence at least 90% identical to SEQ ID NO:3,
such as at
least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%,
99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%
identical to SEQ ID NO: 3, preferably at least 98%, 99% or 100% identical to
SEQ ID
NO: 3. In particular embodiments of the application, the non-naturally
occurring nucleic
acid molecule encoding a HBV pol antigen comprises the polynucleotide sequence
of
SEQ ID NOs: 3, 19, or 20.
[0096] In another embodiment of the application, a non-naturally occurring
nucleic acid
molecule encodes a fusion protein comprising a truncated HBV core antigen
operably
linked to a HBV Pol antigen, or a HBV Pol antigen operably linked to a
truncated HBV
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core antigen. In a particular embodiment, a non-naturally occurring nucleic
acid
molecule of the application encodes a truncated HBV core antigen consisting of
an amino
acid sequence that is at least 90% identical to SEQ ID NO: 2, such as at least
90%, 91%,
92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%,
99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to
SEQ ID
NO: 2, preferably 100% identical to SEQ ID NO: 2; a linker; and a HBV
polymerase
antigen comprising an amino acid sequence that is at least 90% identical to
SEQ ID NO:
4, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%,
97.5%,
98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%
or 100% identical to SEQ ID NO: 4, preferably at least 98%, 99% or 100%
identical to
SEQ ID NO: 4. In a particular embodiment of the application, a non-naturally
occurring
nucleic acid molecule encodes a fusion protein comprising a truncated HBV core
antigen
consisting of the amino acid sequence of SEQ ID NO: 2, a linker comprising
(AlaGly)n,
wherein n is an integer of 2 to 5; and a HBV Pol antigen comprising the amino
acid
sequence of SEQ ID NO: 4. In a particular embodiment of the application, a non-
naturally occurring nucleic acid molecule encodes a fusion protein comprising
the amino
acid sequence of SEQ ID NO: 12.
[0097] Examples of polynucleotide sequences of the application encoding a
fusion
protein include, but are not limited to, a polynucleotide sequence at least
90% identical to
SEQ ID NO: 1, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%,
96.5%,
97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%, 99.9% or 100% identical to SEQ ID NO: 1, preferably at least 98%, 99%
or 100%
identical to SEQ ID NO: 1, operably linked to a linker coding sequence at
least 90%
identical to SEQ ID NO: 14, such as at least 90%, 91%, 92%, 93%, 94%, 95%,
95.5%,
96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,
99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 14, preferably at
least
98%, 99% or 100% identical to SEQ ID NO: 14, which is further operably linked
a
polynucleotide sequence at least 90% identical to SEQ ID NO: 3, such as at
least 90%,
91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%
identical to
SEQ ID NO: 3, preferably at least 98%, 99% or 100% identical to SEQ ID NO: 3.
In
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particular embodiments of the application, a non-naturally occurring nucleic
acid
molecule encoding a fusion protein comprises SEQ ID NO: 1, operably linked to
SEQ ID
NO:14, which is further operably linked to SEQ ID NO: 3.
[0098] In another general aspect, the application relates to a vector
comprising an
isolated polynucleotide encoding an HBV antigen. As used herein, a "vector" is
a nucleic
acid molecule used to carry genetic material into another cell, where it can
be replicated
and/or expressed. Any vector known to those skilled in the art in view of the
present
disclosure can be used. Examples of vectors include, but are not limited to,
plasmids,
viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and
artificial
chromosomes (e.g., YACs). Preferably, a vector is a DNA plasmid. A vector can
be a
DNA vector or an RNA vector. One of skill in the art can construct a vector of
the
application through standard recombinant techniques in view of the present
disclosure.
[0099] According to embodiments of the application, a vector can be an
expression
vector. As used herein, the term "expression vector" refers to any type of
genetic
.. construct comprising a nucleic acid coding for an RNA capable of being
transcribed.
Expression vectors include, but are not limited to, vectors for recombinant
protein
expression, such as a DNA plasmid or a viral vector, and vectors for delivery
of nucleic
acid into a subject for expression in a tissue of the subject, such as a DNA
plasmid or a
viral vector. It will be appreciated by those skilled in the art that the
design of the
expression vector can depend on such factors as the choice of the host cell to
be
transformed, the level of expression of protein desired, etc.
[00100] Vectors according to embodiments of the application can contain a
variety of
regulatory sequences. As used herein, the term "regulatory sequence" refers to
any
sequence that allows, contributes or modulates the functional regulation of
the nucleic
acid molecule, including replication, duplication, transcription, splicing,
translation,
stability and/or transport of the nucleic acid or one of its derivative (i.e.
mRNA) into the
host cell or organism. In the context of the disclosure, this term encompasses
promoters,
enhancers and other expression control elements (e.g., polyadenylation signals
and
elements that affect mRNA stability).
[00101] In some embodiments, of the application, a vector is a non-viral
vector.
Examples of non-viral vectors include, but are not limited to, DNA plasmids,
bacterial
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artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc.
Preferably, a
non-viral vector is a DNA plasmid. A "DNA plasmid," which is used
interchangeably
with "DNA plasmid vector," "plasmid DNA" or "plasmid DNA vector," refers to a
double-stranded and generally circular DNA sequence that is capable of
autonomous
replication in a suitable host cell. DNA plasmids used for expression of an
encoded
polynucleotide typically comprise an origin of replication, a multiple cloning
site, and a
selectable marker, which for example, can be an antibiotic resistance gene.
Examples of
DNA plasmids suitable for use in the application include, but are not limited
to,
commercially available expression vectors for use in well-known expression
systems
(including both prokaryotic and eukaryotic systems), such as pSE420
(Invitrogen, San
Diego, Calif.), which can be used for production and/or expression of protein
in
Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be
used for
production and/or expression in Saccharomyces cerevisiae strains of yeast;
MAXBAC
complete baculovirus expression system (Thermo Fisher Scientific), which can
be used
for production and/or expression in insect cells; pcDNATM or pcDNA3 TM (Life
Technologies, Thermo Fisher Scientific), which can be used for high level
constitutive
protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies,
Thermo Fisher Scientific), which can be used for high-level transient
expression of a
protein of interest in most mammalian cells. The backbone of any commercially
available DNA plasmid can be modified to optimize protein expression in the
host cell,
such as to reverse the orientation of certain elements (e.g., origin of
replication and/or
antibiotic resistance cassette), replace a promoter endogenous to the plasmid
(e.g., the
promoter in the antibiotic resistance cassette), and/or replace the
polynucleotide sequence
encoding transcribed proteins (e.g., the coding sequence of the antibiotic
resistance gene),
by using routine techniques and readily available starting materials. (See
e.g., Sambrook
et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor
Press
(1989)).
[00102] In a preferred embodiment of the application, a DNA plasmid is an
expression
vector suitable for protein expression in mammalian host cells. Expression
vectors
suitable for protein expression in mammalian host cells include, but are not
limited to,
pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, NTC8454, etc. Preferably, the
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expression vector is based on pVAX-1, which can be further modified to
optimize protein
expression in mammalian cells. pVAX-1 is a commonly used plasmid in DNA
vaccines,
and contains a strong human immediate early cytomegalovirus (CMV-IE) promoter
followed by the bovine growth hormone (bGH)-derived polyadenylation sequence
(pA).
pVAX-1 further contains a pUC origin of replication and kanamycin resistance
gene
driven by a small prokaryotic promoter that allows for bacterial plasmid
propagation.
[00103] In an embodiment of the application, a vector is a viral vector. In
general, viral
vectors are genetically engineered viruses carrying modified viral DNA or RNA
that has
been rendered non-infectious, but still contains viral promoters and
transgenes, thus
allowing for translation of the transgene through a viral promoter. Because
viral vectors
are frequently lacking infectious sequences, they require helper viruses or
packaging lines
for large-scale transfection. Examples of viral vectors suitable for use with
the
application include, but are not limited to adenoviral vectors, Modified
Vaccinia Ankara
(MVA) vectors, adeno-associated virus vectors, pox virus vectors, enteric
virus vectors,
Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors,
Tobacco
Mosaic Virus vectors, lentiviral vectors, etc.
[00104] According to embodiments of the application, a vector, e.g., a DNA
plasmid or
a viral vector, can comprise any regulatory elements to establish conventional
function(s)
of the vector, including but not limited to replication and expression of the
HBV
antigen(s) encoded by the polynucleotide sequence of the vector. Regulatory
elements
include, but are not limited to, a promoter, an enhancer, a polyadenylation
signal,
translation stop codon, a ribosome binding element, a transcription
terminator, selection
markers, origin of replication, etc. A vector can comprise one or more
expression
cassettes. An "expression cassette" is part of a vector that directs the
cellular machinery
to make RNA and protein. An expression cassette typically comprises three
components:
a promoter sequence, an open reading frame, and a 3'-untranslated region (UTR)
optionally comprising a polyadenylation signal. An open reading frame (ORF) is
a
reading frame that contains a coding sequence of a protein of interest (e.g.,
HBV antigen)
from a start codon to a stop codon. Regulatory elements of the expression
cassette can be
operably linked to a polynucleotide sequence encoding an HBV antigen of
interest. As
used herein, the term "operably linked" is to be taken in its broadest
reasonable context,
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and refers to a linkage of polynucleotide elements in a functional
relationship. A
polynucleotide is "operably linked" when it is placed into a functional
relationship with
another polynucleotide. For instance, a promoter is operably linked to a
coding sequence
if it affects the transcription of the coding sequence.
[00105] In some embodiments, a vector comprises a promoter sequence,
preferably
within an expression cassette, to control expression of an HBV antigen of
interest. The
term "promoter" is used in its conventional sense, and refers to a nucleotide
sequence that
initiates the transcription of an operably linked nucleotide sequence. A
promoter is
located on the same strand near the nucleotide sequence it transcribes.
Promoters can be
a constitutive, inducible, or repressible. Promoters can be naturally
occurring or
synthetic. A promoter can be derived from sources including, viral, bacterial,
fungal,
plants, insects, and animals. A promoter can be a homologous promoter (i.e.,
derived
from the same genetic source as the vector) or a heterologous promoter (i.e.,
derived from
a different vector or genetic source). For example, if the vector to be
employed is a DNA
plasmid, the promoter can be endogenous to the plasmid (homologous) or derived
from
other sources (heterologous). Preferably, the promoter is located upstream of
the
polynucleotide encoding an HBV antigen within an expression cassette.
[00106] Examples of promoters suitable for use in the application include, but
are not
limited to, a promoter from simian virus 40 (SV40), a mouse mammary tumor
virus
(MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the
bovine
immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney
virus
promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV)
promoter
such as the CMV immediate early promoter (CMV-IE), Epstein Barr virus (EBV)
promoter, or a Rous sarcoma virus (RSV) promoter. Additional promoters
suitable for
use in the application include, but are not limited to, an RSV promoter, the
retrovirus
LTR, the adenovirus major late promoter, and various poxvirus promoters
including, but
not limited to the following vaccinia virus or MVA¨derived and FPV-derived
promoters:
the 30K promoter, the 13 promoter, the PrS promoter, the PrHyb, the PrS5E
promoter, the
Pr7.5K, the Pr13.5 long promoter, the 40K promoter, the MVA-40K promoter, the
FPV
40K promoter, 30k promoter, the PrSynilm promoter, the PrLE1 promoter, and the
PR1238 promoter. Additional promoters are further described in WO 2010/060632,
WO
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2010/102822, WO 2013/189611 and WO 2014/063832, and W02017/021776, which are
incorporated fully by reference herein.
[00107] A promoter can also be a promoter from a human gene such as human
actin,
human myosin, human hemoglobin, human muscle creatine, or human
metalothionein. A
promoter can also be a tissue specific promoter, such as a muscle or skin
specific
promoter, natural or synthetic.
[00108] In a preferred embodiment of the application, a promoter is a strong
eukaryotic
promoter, preferably a cytomegalovirus immediate early (CMV-IE) promoter. A
nucleotide sequence of an exemplary CMV-IE promoter is shown in SEQ ID NO: 7.
[00109] In another preferred embodiment of the application, a promoter is a
poxviral
promoter, preferably a promoter selected from PrMVA 13.5 long and/or PrHyb.
Nucleotide sequences for an exemplary Pr13.5 long promoter and a PrHyb
promoter are
shown as SEQ ID NO:25 and 26, respectively.
[00110] In some embodiments, a vector comprises additional polynucleotide
sequences
that stabilize the expressed transcript, enhance nuclear export of the RNA
transcript,
and/or improve transcriptional-translational coupling. Examples of such
sequences
include polyadenylation signals and enhancer sequences. A polyadenylation
signal is
typically located downstream of the coding sequence for a protein of interest
(e.g., an
HBV antigen) within an expression cassette of the vector. Enhancer sequences
are
regulatory DNA sequences that, when bound by transcription factors, enhance
the
transcription of an associated gene. An enhancer sequence is preferably
located upstream
of the polynucleotide sequence encoding an HBV antigen, but downstream of a
promoter
sequence within an expression cassette of the vector.
[00111] Any polyadenylation signal known to those skilled in the art in view
of the
present disclosure can be used. For example, the polyadenylation signal can be
a 5V40
polyadenylation signal (e.g., SEQ ID NO:16), LTR polyadenylation signal,
bovine
growth hormone (bGH) polyadenylation signal, human growth hormone (hGH)
polyadenylation signal, or human fi-globin polyadenylation signal. In a
preferred
embodiment of the application, a polyadenylation signal is a bovine growth
hormone
(bGH) polyadenylation signal. A nucleotide sequence of an exemplary bGH
polyadenylation signal is shown in SEQ ID NO:9.
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[00112] Any enhancer sequence known to those skilled in the art in view of the
present
disclosure can be used. For example, an enhancer sequence can be human actin,
human
myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as
one
from CMV, HA, RSV, or EBV. Examples of particular enhancers include, but are
not
limited to, Woodchuck HBV Post-transcriptional regulatory element (WPRE),
intron/exon sequence derived from human apolipoprotein Al precursor,
untranslated R-
U5 domain of the human T-cell leukemia virus type 1 (HTLV-1) long terminal
repeat
(LTR), a splicing enhancer, a synthetic rabbit 0-globin intron, or any
combination
thereof. In a preferred embodiment of the application, an enhancer sequence is
a
composite sequence of three consecutive elements of the untranslated R-U5
domain of
HTLV-1 LTR, rabbit 0-globin intron, and a splicing enhancer, which is referred
to herein
as "a triple enhancer sequence." A nucleotide sequence of an exemplary triple
enhancer
sequence is shown in SEQ ID NO: 8. Another exemplary enhancer sequence is an
ApoAl gene fragment shown in SEQ ID NO:15.
[00113] In some embodiments, a vector comprises a polynucleotide sequence
encoding
a signal peptide sequence. Preferably, the polynucleotide sequence encoding
the signal
peptide sequence is located upstream of the polynucleotide sequence encoding
an HBV
antigen. Signal peptides typically direct localization of a protein,
facilitate secretion of
the protein from the cell in which it is produced, and/or improve antigen
expression and
cross-presentation to antigen-presenting cells. A signal peptide can be
present at the N-
terminus of an HBV antigen when expressed from the vector, but is cleaved off
by signal
peptidase, e.g., upon secretion from a cell. An expressed protein in which a
signal
peptide has been cleaved is often referred to as the "mature protein." Any
signal peptide
known in the art in view of the present disclosure can be used. For example, a
signal
peptide can be a cystatin S signal peptide; an immunoglobulin (Ig) secretion
signal, such
as the Ig heavy chain gamma signal peptide SPIgG or the Ig heavy chain epsilon
signal
peptide SPIgE.
[00114] In a preferred embodiment of the application, a signal peptide
sequence is a
cystatin S signal peptide. Exemplary nucleic acid and amino acid sequences of
a cystatin
S signal peptide are shown in SEQ ID NOs: 5 and 6, respectively. Exemplary
nucleic
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acid and amino acid sequences of an immunoglobulin secretion signal are shown
in SEQ
ID NOs: 10 and 27 and SEQ ID NO: 11, respectively.
[00115] A vector, such as a DNA plasmid, can also include a bacterial origin
of
replication and an antibiotic resistance expression cassette for selection and
maintenance
of the plasmid in bacterial cells, e.g., E. coli. Bacterial origins of
replication and
antibiotic resistance cassettes can be located in a vector in the same
orientation as the
expression cassette encoding an HBV antigen, or in the opposite (reverse)
orientation.
An origin of replication (ORI) is a sequence at which replication is
initiated, enabling a
plasmid to reproduce and survive within cells. Examples of ORIs suitable for
use in the
application include, but are not limited to ColE1, pMB1, pUC, pSC101, R6K, and
15A,
preferably pUC. An exemplary nucleotide sequence of a pUC ORI is shown in SEQ
ID
NO: 21.
[00116] Expression cassettes for selection and maintenance in bacterial cells
typically
include a promoter sequence operably linked to an antibiotic resistance gene.
Preferably,
the promoter sequence operably linked to an antibiotic resistance gene differs
from the
promoter sequence operably linked to a polynucleotide sequence encoding a
protein of
interest, e.g., HBV antigen. The antibiotic resistance gene can be codon
optimized, and
the sequence composition of the antibiotic resistance gene is normally
adjusted to
bacterial, e.g., E. coli, codon usage. Any antibiotic resistance gene known to
those
skilled in the art in view of the present disclosure can be used, including,
but not limited
to, kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr), and
tetracycline
resistance gene (Tetr), as well as genes conferring resistance to
chloramphenicol,
bleomycin, spectinomycin, carbenicillin, etc.
[00117] In another particular embodiment of the application, a vector is a
viral vector,
preferably an adenoviral vector, comprising an expression cassette including a
polynucleotide encoding at least one of an HBV antigen selected from the group
consisting of an HBV pol antigen comprising an amino acid sequence at least
98%
identical to SEQ ID NO: 4, such as at least 98%, 98.5%, 99%, 99.1%, 99.2%,
99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 4,
and a
truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO:
2; an
upstream sequence operably linked to the polynucleotide encoding the HBV
antigen
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comprising, from 5' end to 3' end, a promoter sequence, preferably a CMV-IE
promoter
sequence of SEQ ID NO:7, an enhancer sequence, preferably a triple enhancer
sequence
of SEQ ID NO: 8 or an ApoAl enhancer sequence of SEQ ID NO: 15, and a
polynucleotide sequence encoding a signal peptide sequence, preferably a
cystatin S
signal having the amino acid sequence of SEQ ID NO: 6 or an immunoglobulin
secretion
signal having the amino acid sequence of SEQ ID NO: 11; and a downstream
sequence
operably linked to the polynucleotide encoding the HBV antigen comprising a
polyadenylation signal, preferably a 5V40 polyadenylation signal of SEQ ID NO:
16 or a
bGH polyadenylation signal of SEQ ID NO: 9.
[00118] In another particular embodiment of the application, a vector is a
viral vector,
preferably a MVA vector, comprising an expression cassette including a
polynucleotide
encoding at least one of an HBV antigen selected from the group consisting of
an HBV
pol antigen comprising an amino acid sequence at least 98% identical to SEQ ID
NO: 4,
such as at least 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%,
99.8%, 99.9% or 100% identical to SEQ ID NO: 4, and a truncated HBV core
antigen
consisting of the amino acid sequence of SEQ ID NO: 2; an upstream sequence
operably
linked to the polynucleotide encoding the HBV antigen comprising, from 5' end
to 3'
end, a promoter sequence, preferably a PrMVA13.5 long promoter sequence of SEQ
ID
NO: 25 or a PrHyb promoter sequence of SEQ ID NO: 26, and a polynucleotide
sequence
encoding a signal peptide sequence, preferably a cystatin S signal having the
amino acid
sequence of SEQ ID NO: 6 or an immunoglobulin secretion signal having the
amino acid
sequence of SEQ ID NO: 11; and a downstream sequence operably linked to the
polynucleotide encoding the HBV antigen comprising a polyadenylation signal or
an
early termination signal, wherein the early termination signal has a
nucleotide sequence
of SEQ ID NO: 28, or wherein the polyadenylation signal is selected from an
5V40
polyadenylation signal having a polynucleotide sequence of SEQ ID NO: 16 or a
bGH
polyadenylation signal having a polynucleotide sequence of SEQ ID NO: 9,
preferably
the downstream sequence operably linked to the polynucleotide encoding the HBV
antigen is an early termination signal having a nucleotide sequence of SEQ ID
NO: 28.
[00119] In an embodiment of the application, a vector, such as a viral vector,
encodes
an HBV Pol antigen having the amino acid sequence of SEQ ID NO: 4. Preferably,
the
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vector comprises a coding sequence for the HBV Pol antigen that is at least
90% identical
to the polynucleotide sequence of SEQ ID NO: 3, such as 90%, 91%, 92%, 93%,
94%,
95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 3,
preferably 100% identical to SEQ ID NO: 3.
[00120] In another embodiment of the application, a vector, such as a viral
vector,
encodes a truncated HBV core antigen consisting of the amino acid sequence of
SEQ ID
NO:2. Preferably, the vector comprises a coding sequence for the truncated HBV
core
antigen that is at least 90% identical to the polynucleotide sequence of SEQ
ID NO:1,
such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%,
98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or
100% identical to SEQ ID NO: 1, preferably 100% identical to SEQ ID NO: 1.
[00121] In yet another embodiment of the application, a vector, such as a
viral vector,
encodes a fusion protein comprising an HBV Pol antigen having the amino acid
sequence
of SEQ ID NO: 4 and a truncated HBV core antigen consisting of the amino acid
sequence of SEQ ID NO: 2. Preferably, the vector comprises a coding sequence
for the
fusion, which contains a coding sequence for the truncated HBV core antigen at
least
90% identical to SEQ ID NO:1, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%,
96%,
96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO:1, preferably 98%, 99% or
100%
identical to SEQ ID NO: 1, operably linked to a coding sequence for the HBV
Pol
antigen at least 90% identical to SEQ ID NO: 3, such as 90%, 91%, 92%, 93%,
94%,
95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO:3,
preferably 98%, 99% or 100% identical to SEQ ID NO: 3. Preferably, the coding
sequence for the truncated HBV core antigen is operably linked to the coding
sequence
for the HBV Pol antigen via a coding sequence for a linker at least 90%
identical to SEQ
ID NO: 14, such as 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%,
97.5%,
98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%
or 100% identical to SEQ ID NO: 14, preferably 98%, 99% or 100% identical to
SEQ ID
NO: 14. In particular embodiments of the application, the vector comprises a
coding
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sequence for the fusion having SEQ ID NO: 1 operably linked to SEQ ID NO: 14,
which
is further operably linked to SEQ ID NO: 3.
[00122] The polynucleotides and expression vectors encoding the HBV antigens
of the
application can be made by any method known in the art in view of the present
disclosure. For example, a polynucleotide encoding an HBV antigen can be
introduced
or "cloned" into an expression vector using standard molecular biology
techniques, e.g.,
polymerase chain reaction (PCR), etc., which are well known to those skilled
in the art.
[00123] Adenoviruses
[00124] In an aspect, the application provides a recombinant adenovirus
comprising a
heterologous nucleotide sequence encoding an antigenic HBV core antigen. In
another
aspect, the application provides a adenovirus comprising a heterologous
nucleotide
sequence encoding an antigenic HBV pol antigen. In another, the application
provides a
recombinant adenovirus vector comprising a first heterologous nucleotide
sequence
encoding an antigenic HBV core antigen and a second heterologous nucleotide
sequence
encoding an antigenic HBV pol antigen. In another aspect, the application
provides a
recombinant adenovirus comprising a heterologous nucleotide sequence encoding
an
antigenic HBV core-HBV pol fusion protein.
[00125] An adenovirus according to the application belongs to the family of
the
Adenoviridae and preferably is one that belongs to the genus Mastadenovirus.
It can be a
human adenovirus, but also an adenovirus that infects other species, including
but not
limited to a bovine adenovirus (e.g. bovine adenovirus 3, BAdV3), a canine
adenovirus
(e.g. CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus
(which
includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee
adenovirus
or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus
(HAdV, or
AdHu; in the application a human adenovirus is meant if referred to as Ad
without
indication of species, e.g. the brief notation "Ad5" means the same as HAdV5,
which is
human adenovirus serotype 5), or a simian adenovirus such as chimpanzee or
gorilla
adenovirus (ChAd, AdCh, or SAdV).
[00126] Most advanced studies have been performed using human adenoviruses,
and
human adenoviruses are preferred according to certain aspects of the
application. In
certain preferred embodiments, the recombinant adenovirus according to the
application
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is based upon a human adenovirus. In preferred embodiments, the recombinant
adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49
or 50.
According to a particularly preferred embodiment of the application, an
adenovirus is a
human adenovirus of one of the serotypes 26 or 35.
[00127] An advantage of these serotypes is a low seroprevalence and/or low pre-
existing neutralizing antibody titers in the human population. Preparation of
rAd26
vectors is described, for example, in WO 2007/104792 and in Abbink et al.,
(2007) Virol
81(9): 4654-63, both of which are incorporated by reference herein in their
entirety.
Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474
and
in W02007/104792 (see, e.g., SEQ ID NO:1). Preparation of rAd35 vectors is
described,
for example, in US Patent No. 7,270,811, in W000/70071, and in Vogels et al.,
(2003) J
Virol 77(15): 8263-71, all of which are incorporated by reference herein in
their entirety.
Exemplary genome sequences of Ad35 are found in GenBank Accession AC 000019
and
in W000/70071 (see, e.g., Fig. 6).
.. [00128] Simian adenoviruses generally also have a low seroprevalence and/or
low pre-
existing neutralizing antibody titers in the human population, and a
significant amount of
work has been reported using chimpanzee adenovirus vectors (e.g. U56083716;
W02005/071093; WO 2010/086189; WO 2010085984; Farina et al, 2001, J Virol 75:
11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006,
Virology
346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also
review by
Bangari and Mittal, 2006, Vaccine 24: 849- 62; and review by Lasaro and Ertl,
2009, Mol
Ther 17: 1333-39). Hence, in other preferred embodiments, the recombinant
adenovirus
according to the application is based upon a simian adenovirus, e.g. a
chimpanzee
adenovirus. In an embodiment of the application, the recombinant adenovirus is
based
upon simian adenovirus type 1, 3, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1,
29, 30, 31.1, 32,
33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or
SA7P.
[00129] Adenoviral Vectors rAd26 and rAd35
[00130] In a preferred embodiment of the application, the adenoviral vectors
comprise
capsid proteins from two rare serotypes: Ad26 and Ad35. In the typical
embodiment, the
vector is an rAd26 or rAd35 virus.
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[00131] Thus, the vectors that can be used in the application comprise an Ad26
or
Ad35 capsid protein (e.g., a fiber, penton or hexon protein). One of skill
will recognize
that it is not necessary that an entire Ad26 or Ad35 capsid protein be used in
the vectors
of the application. Thus, chimeric capsid proteins that include at least a
part of an Ad26
or Ad35 capsid protein can be used in the vectors of the application. The
vectors of the
application may also comprise capsid proteins in which the fiber, penton, and
hexon
proteins are each derived from a different serotype, so long as at least one
capsid protein
is derived from Ad26 or Ad35. In preferred embodiments, the fiber, penton and
hexon
proteins are each derived from Ad26 or each from Ad35.
[00132] One of skill will recognize that elements derived from multiple
serotypes can
be combined in a single recombinant adenovirus vector. Thus, a chimeric
adenovirus that
combines desirable properties from different serotypes can be produced. Thus,
in some
embodiments, a chimeric adenovirus of the application could combine the
absence of pre-
existing immunity of the Ad26 and Ad35 serotypes with characteristics such as
temperature stability, assembly, anchoring, production yield, redirected or
improved
infection, stability of the DNA in the target cell, and the like.
[00133] In an embodiment of the application the recombinant adenovirus vector
useful
in the application is derived mainly or entirely from Ad35 or from Ad26 (i.e.,
the vector
is rAd35 or rAd26). In some embodiments, the adenovirus is replication
deficient, e.g.
because it contains a deletion in the El region of the genome. For the
adenoviruses of the
application, being derived from Ad26 or Ad35, it is typical to exchange the E4-
orf6
coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human
subgroup
C, such as Ad5. This allows propagation of such adenoviruses in well-known
complementing cell lines that express the El genes of Ad5, such as for example
293
cells, PER.C6 cells, and the like (see, e.g. Havenga et al, 2006, J Gen Virol
87: 2135-43;
WO 03/104467). In an embodiment of the application, the adenovirus is a human
adenovirus of serotype 35, with a deletion in the El region into which the
nucleic acid
encoding the antigen has been cloned, and with an E4 orf6 region of Ad5. In an
embodiment of the application, the adenovirus is a human adenovirus of
serotype 26,
with a deletion in the El region into which the nucleic acid encoding the
antigen has been
cloned, and with an E4 orf6 region of Ad5. For the Ad35 adenovirus, it is
typical to
37
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retain the 3' end of the El B 55K open reading frame in the adenovirus, for
instance the
166 bp directly upstream of the pIX open reading frame or a fragment
comprising this
such as a 243 bp fragment directly upstream of the pIX start codon, marked at
the 5' end
by a Bsu36I restriction site, since this increases the stability of the
adenovirus because the
promoter of the pIX gene is partly residing in this area (see, e.g. Havenga et
al, 2006,
supra; WO 2004/001032).
[00134] The preparation of recombinant adenoviral vectors is well known in the
art.
Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and
in
Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26
are
found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792.
Preparation of rAd35 vectors is described, for example, in US Patent No.
7,270,811 and
in Vogels et al., (2003) J Virol 77(15): 8263-71. An exemplary genome sequence
of
Ad35 is found in GenBank Accession AC 000019.
[00135] In an embodiment of the application, the vectors useful in the
application
include those described in W02012/082918, the disclosure of which is
incorporated
herein by reference in its entirety.
[00136] Typically, a vector useful in the application is produced using a
nucleic acid
comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid,
or
baculovirus vector). Thus, the application also provides isolated nucleic acid
molecules
that encode the adenoviral vectors of the application. The nucleic acid
molecules of the
application may be in the form of RNA or in the form of DNA obtained by
cloning or
produced synthetically. The DNA may be double-stranded or single-stranded.
[00137] The adenovirus vectors useful in the application are typically
replication
defective. In these embodiments, the virus is rendered replication-defective
by deletion
or inactivation of regions critical to replication of the virus, such as the
El region. The
regions can be substantially deleted or inactivated by, for example, inserting
the gene of
interest (usually linked to a promoter). In some embodiments, the vectors of
the
application may contain deletions in other regions, such as the E2, E3 or E4
regions or
insertions of heterologous genes linked to a promoter. For E2- and/or E4-
mutated
adenoviruses, generally E2- and/or E4-complementing cell lines are used to
generate
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recombinant adenoviruses. Mutations in the E3 region of the adenovirus need
not be
complemented by the cell line, since E3 is not required for replication.
[00138] A packaging cell line is typically used to produce sufficient amount
of
adenovirus vectors of the application. A packaging cell is a cell that
comprises those
genes that have been deleted or inactivated in a replication-defective vector,
thus
allowing the virus to replicate in the cell. Suitable cell lines include, for
example,
PER. C6, 911, 293, and El A549.
[00139] As noted above, a wide variety of Hepatitis B virus (HBV) antigens
(e.g., HBV
core and HBV polymerase antigens) can be expressed in the vectors. If
required, the
heterologous gene encoding the HBV antigen can be codon-optimized to ensure
proper
expression in the treated host (e.g., human). Codon-optimization is a
technology widely
applied in the art. Typically, the heterologous gene is cloned into the El
and/or the E3
region of the adenoviral genome.
[00140] The heterologous Hepatitis B virus gene may be under the control of
(i.e.,
operably linked to) an adenovirus-derived promoter (e.g., the Major Late
Promoter) or
may be under the control of a heterologous promoter. Examples of suitable
heterologous
promoters include the CMV promoter and the RSV promoter. Preferably, the
promoter is
located upstream of the heterologous gene of interest within an expression
cassette.
[00141] MVA vectors
[00142] MVA vectors useful for the application utilize attenuated virus
derived from
Modified Vaccinia Ankara virus. The MVA vectors express a wide variety of HBV
antigens (e.g., HBV core and HBV polymerase antigens). In an aspect, the
application
provides a recombinant MVA vector comprising a heterologous nucleotide
sequence
encoding an antigenic HBV core antigen. In another aspect, the application
provides a
recombinant MVA vector comprising a heterologous nucleotide sequence encoding
an
antigenic HBV pol antigen. In an aspect, the application provides a
recombinant MVA
vector comprising a first heterologous nucleotide sequence encoding an
antigenic HBV
core antigen and a second heterologous nucleotide sequence encoding an
antigenic HBV
pol antigen. In another aspect, the application provides a recombinant MVA
vector
comprising a heterologous nucleotide sequence encoding an antigenic HBV core-
HBV
pol fusion protein.
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[00143] Modified Vaccinia Virus Ankara ("MVA")
[00144] The man-made attenuated modified vaccinia virus Ankara ("MVA") was
generated by 516 serial passages on chicken embryo fibroblasts of the Ankara
strain of
vaccinia virus (CVA) (for review see Mayr, A., et al. Infection 3, 6-14
(1975)). As a
consequence of these long-term passages, the genome of the resulting MVA virus
had
about 31 kilobases of its genomic sequence deleted and, therefore, was
described as
highly host cell restricted for replication to avian cells (Meyer, H. et al.,
J. Gen. Virol. 72,
1031-1038 (1991)). It was shown in a variety of animal models that the
resulting MVA
was significantly avirulent compared to the fully replication competent
starting material
(Mayr, A. & Danner, K., Dev. Biol. Stand. 41: 225-34 (1978)).
[00145] An MVA virus useful in the practice of the application can include,
but is not
limited to, MVA-572 (deposited as ECACC V94012707 on January 27, 1994); MVA-
575
(deposited as ECACC V00120707 on December 7, 2000), MVA-1721 (referenced in
Suter et al., Vaccine 2009), and ACAM3000 (deposited as ATCC PTA-5095 on
March
27,2003).
[00146] More preferably the MVA used in accordance with the application
includes
MVA-BN and derivatives of MVA-BN. MVA-BN has been described in International
PCT publication WO 02/042480. "Derivatives" of MVA-BN refer to viruses
exhibiting
essentially the same replication characteristics as MVA-BN, as described
herein, but
exhibiting differences in one or more parts of their genomes.
[00147] MVA-BN, as well as derivatives thereof, is replication incompetent,
meaning a
failure to reproductively replicate in vivo and in vitro. More specifically in
vitro, MVA-
BN or derivatives thereof have been described as being capable of reproductive
replication in chicken embryo fibroblasts (CEF), but not capable of
reproductive
replication in the human keratinocyte cell line HaCat (Boukamp et al (1988),
J. Cell Biol.
106:761-771), the human bone osteosarcoma cell line 143B (ECACC Deposit No.
91112502), the human embryo kidney cell line 293 (ECACC Deposit No. 85120602),
and the human cervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL-2).
Additionally, MVA-BN or derivatives thereof have a virus amplification ratio
at least two
fold less, more preferably three-fold less than MVA-575 in Hela cells and
HaCaT cell
lines. Tests and assay for these properties of MVA-BN and derivatives thereof
are
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described in WO 02/42480 (U.S. Patent application No. 2003/0206926) and WO
03/048184 (U.S. Patent application No. 2006/0159699).
[00148] The term "not capable of reproductive replication" or "no capability
of
reproductive replication" in human cell lines in vitro as described in the
previous
paragraphs is, for example, described in WO 02/42480, which also teaches how
to obtain
MVA having the desired properties as mentioned above. The term applies to a
virus that
has a virus amplification ratio in vitro at 4 days after infection of less
than 1 using the
assays described in WO 02/42480 or in U.S. Patent No. 6,761,893.
[00149] The term "failure to reproductively replicate" refers to a virus that
has a virus
amplification ratio in human cell lines in vitro as described in the previous
paragraphs at
4 days after infection of less than 1. Assays described in WO 02/42480 or in
U.S. Patent
No. 6,761,893 are applicable for the determination of the virus amplification
ratio.
[00150] The amplification or replication of a virus in human cell lines in
vitro as
described in the previous paragraphs is normally expressed as the ratio of
virus produced
from an infected cell (output) to the amount originally used to infect the
cell in the first
place (input) referred to as the "amplification ratio". An amplification ratio
of "1" defines
an amplification status where the amount of virus produced from the infected
cells is the
same as the amount initially used to infect the cells, meaning that the
infected cells are
permissive for virus infection and reproduction. In contrast, an amplification
ratio of less
than 1, i.e., a decrease in output compared to the input level, indicates a
lack of
reproductive replication and therefore attenuation of the virus.
[00151] The advantages of MVA-based vaccine include their safety profile as
well as
availability for large scale vaccine production. Preclinical tests have
revealed that MVA-
BN demonstrates superior attenuation and efficacy compared to other MVA
strains (WO
02/42480). An additional property of MVA-BN strains is the ability to induce
substantially the same level of immunity in vaccinia virus prime/vaccinia
virus boost
regimes when compared to DNA- prime/vaccinia virus boost regimes.
[00152] The recombinant MVA-BN viruses, the most preferred embodiment herein,
are
considered to be safe because of their distinct replication deficiency in
mammalian cells
and their well-established avirulence. Furthermore, in addition to its
efficacy, the
feasibility of industrial scale manufacturing can be beneficial. Additionally,
MVA-based
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vaccines can deliver multiple heterologous antigens and allow for simultaneous
induction
of humoral and cellular immunity.
[00153] MVA vectors useful for the application can be prepared using methods
known
in the art, such as those described in WO/2002/042480 and WO/2002/24224, the
relevant
disclosures of which are incorporated herein by references.
[00154] In a preferred embodiment of the application, the MVA vector(s)
comprise a
nucleic acid that encodes one or more antigenic proteins selected from the
group
consisting of HBV core antigen, HBV pol antigen, and a HBV core-HBV pol fusion
antigen.
[00155] The HBV antigen protein may be inserted into one or more intergenic
regions
(IGR) of the MVA. In an embodiment of the application, the IGR is selected
from
IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR 148/149. In an
embodiment of the application, less than 5, 4, 3, or 2 IGRs of the recombinant
MVA
comprise heterologous nucleotide sequences encoding antigenic determinants of
a HBV
core antigen and/or a HBV pol antigen. The heterologous nucleotide sequences
may,
additionally or alternatively, be inserted into one or more of the naturally
occurring
deletion sites, in particular into the main deletion sites I, II, III, IV, V,
or VI of the MVA
genome. In an embodiment of the application, less than 5, 4, 3, or 2 of the
naturally
occurring deletion sites of the recombinant MVA comprise heterologous
nucleotide
sequences encoding antigenic determinants of a HBV core antigen and/or a HBV
pol
antigen.
[00156] The number of insertion sites of MVA comprising heterologous
nucleotide
sequences encoding antigenic determinants of a HBV protein can be 1, 2, 3, 4,
5, 6, 7, or
more. In an embodiment of the application, the heterologous nucleotide
sequences are
inserted into 4, 3, 2, or fewer insertion sites. Preferably, two insertion
sites are used. In an
embodiment of the application, three insertion sites are used. Preferably, the
recombinant
MVA comprises at least 2, 3, 4, 5, 6, or 7 genes inserted into 2 or 3
insertion sites.
[00157] The recombinant MVA viruses provided herein can be generated by
routine
methods known in the art. Methods to obtain recombinant poxviruses or to
insert
exogenous coding sequences into a poxviral genome are well known to the person
skilled
in the art. For example, methods for standard molecular biology techniques
such as
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cloning of DNA, DNA and RNA isolation, Western blot analysis, RT-PCR and PCR
amplification techniques are described in Molecular Cloning, A laboratory
Manual (2nd
Ed.) (J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)), and
techniques
for the handling and manipulation of viruses are described in Virology Methods
Manual
(B.W.J. Mahy et al. (eds.), Academic Press (1996)). Similarly, techniques and
know-how
for the handling, manipulation and genetic engineering of MVA are described in
Molecular Virology: A Practical Approach (A.J. Davison & R.M. Elliott (Eds.),
The
Practical Approach Series, IRL Press at Oxford University Press, Oxford, UK
(1993)(see,
e.g., Chapter 9: Expression of genes by Vaccinia virus vectors)) and Current
Protocols in
Molecular Biology (John Wiley & Son, Inc. (1998)(see, e.g., Chapter 16,
Section IV:
Expression of proteins in mammalian cells using vaccinia viral vector)).
[00158] For the generation of the various recombinant MVAs disclosed herein,
different methods may be applicable. The DNA sequence to be inserted into the
virus can
be placed into an E. coli plasmid construct into which DNA homologous to a
section of
DNA of the MVA has been inserted. Separately, the DNA sequence to be inserted
can be
ligated to a promoter. The promoter-gene linkage can be positioned in the
plasmid
construct so that the promoter-gene linkage is flanked on both ends by DNA
homologous
to a DNA sequence flanking a region of MVA DNA containing a non-essential
locus.
The resulting plasmid construct can be amplified by propagation within E. coli
bacteria
and isolated. The isolated plasmid containing the DNA gene sequence to be
inserted can
be transfected into a cell culture, e.g., of chicken embryo fibroblasts
(CEFs), at the same
time the culture is infected with MVA. Recombination between homologous MVA
DNA
in the plasmid and the viral genome, respectively, can generate an MVA
modified by the
presence of foreign DNA sequences.
[00159] According to a preferred embodiment, a cell of a suitable cell culture
as, e.g.,
CEF cells, can be infected with a poxvirus. The infected cell can be,
subsequently,
transfected with a first plasmid vector comprising a foreign or heterologous
gene or
genes, preferably under the transcriptional control of a poxvirus expression
control
element. As explained above, the plasmid vector also comprises sequences
capable of
directing the insertion of the exogenous sequence into a selected part of the
poxviral
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genome. Optionally, the plasmid vector also contains a cassette comprising a
marker
and/or selection gene operably linked to a poxviral promoter.
[00160] Suitable marker or selection genes are, e.g., the genes encoding the
green
fluorescent protein, 0- galactosidase, neomycin-phosphoribosyltransferase or
other
markers. The use of selection or marker cassettes simplifies the
identification and
isolation of the generated recombinant poxvirus. However, a recombinant
poxvirus can
also be identified by PCR technology. Subsequently, a further cell can be
infected with
the recombinant poxvirus obtained as described above and transfected with a
second
vector comprising a second foreign or heterologous gene or genes. In case,
this gene shall
be introduced into a different insertion site of the poxviral genome, the
second vector also
differs in the poxvirus-homologous sequences directing the integration of the
second
foreign gene or genes into the genome of the poxvirus. After homologous
recombination
has occurred, the recombinant virus comprising two or more foreign or
heterologous
genes can be isolated. For introducing additional foreign genes into the
recombinant
virus, the steps of infection and transfection can be repeated by using the
recombinant
virus isolated in previous steps for infection and by using a further vector
comprising a
further foreign gene or genes for transfection.
[00161] Alternatively, the steps of infection and transfection as described
above are
interchangeable, i.e., a suitable cell can at first be transfected by the
plasmid vector
comprising the foreign gene and, then, infected with the poxvirus. As a
further
alternative, it is also possible to introduce each foreign gene into different
viruses, co-
infect a cell with all the obtained recombinant viruses and screen for a
recombinant
including all foreign genes. A third alternative is ligation of DNA genome and
foreign
sequences in vitro and reconstitution of the recombined vaccinia virus DNA
genome
using a helper virus. A fourth alternative is homologous recombination in
E.coli or
another bacterial species between a vaccinia virus genome, such as MVA, cloned
as a
bacterial artificial chromosome (BAC) and a linear foreign sequence flanked
with DNA
sequences homologous to sequences flanking the desired site of integration in
the
vaccinia virus genome.
[00162] The heterologous HBV gene (e.g., a HBV core antigen, a HBV pol
antigen,
and/or a HBV core-HBV-pol fusion protein) may be under the control of (i.e.,
operably
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linked to) one or more poxvirus promoters. In an embodiment of the
application, the
poxvirus promoter is a Pr7.5 promoter, a hybrid early/late promoter, or a PrS
promoter, a
PrS5E promoter, a synthetic or natural early or late promoter, or a cowpox
virus ATI
promoter.
[00163] Compositions, Immunogenic Combinations, and Vaccines
[00164] The application also relates to compositions, immunogenic
combinations, more
particularly kits, and vaccines comprising one or more HBV antigens,
polynucleotides,
and/or vectors encoding one more HBV antigens according to the application.
Any of the
HBV antigens, polynucleotides (including RNA and DNA), and/or vectors of the
application described herein can be used in the compositions, immunogenic
combinations
or kits, and vaccines of the application.
[00165] In a general aspect, the application provides a composition comprising
an
isolated or non-naturally occurring nucleic acid molecule (DNA or RNA)
encoding a
truncated HBV core antigen consisting of an amino acid sequence that is at
least 90%
identical to SEQ ID NO: 2 or a HBV polymerase antigen comprising an amino acid
sequence that is at least 90% identical to SEQ ID NO: 4, a vector comprising
the isolated
or non-naturally occurring nucleic acid molecule, and/or an isolated or non-
naturally
occurring polypeptide encoded by the isolated or non-naturally occurring
nucleic acid
molecule.
[00166] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring nucleic acid molecule (DNA or RNA) encoding a
truncated HBV
core antigen consisting of an amino acid sequence that is at least 90%
identical to SEQ
ID NO: 2, preferably 100% identical to SEQ ID NO: 2.
[00167] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring nucleic acid molecule (DNA or RNA) encoding a HBV pol
antigen comprising an amino acid sequence that is at least 90% identical to
SEQ ID NO:
4, preferably 100% identical to SEQ ID NO: 4.
[00168] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring nucleic acid molecule (DNA or RNA) comprising a
polynucleotide sequence encoding a truncated HBV core antigen consisting of an
amino
acid sequence that is at least 90% identical to SEQ ID NO: 2, preferably 100%
identical
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to SEQ ID NO: 2; and an isolated or non-naturally occurring nucleic acid
molecule (DNA
or RNA) comprising a polynucleotide sequence encoding a HBV pol antigen
comprising
an amino acid sequence that is at least 90% identical to SEQ ID NO: 4,
preferably 100%
identical to SEQ ID NO: 4. The coding sequences for the truncated HBV core
antigen
.. and the HBV Pol antigen can be present in the same isolated or non-
naturally occurring
nucleic acid molecule (DNA or RNA), or in two different isolated or non-
naturally
occurring nucleic acid molecules (DNA or RNA).
[00169] In an embodiment of the application, a composition comprises a viral
vector
comprising a polynucleotide encoding a truncated HBV core antigen consisting
of an
amino acid sequence that is at least 90% identical to SEQ ID NO: 2, preferably
100%
identical to SEQ ID NO: 2.
[00170] In an embodiment of the application, a composition comprises a viral
vector
comprising a polynucleotide encoding a HBV pol antigen comprising an amino
acid
sequence that is at least 90% identical to SEQ ID NO: 4, preferably 100%
identical to
SEQ ID NO: 4.
[00171] In an embodiment of the application, a composition comprises a viral
vector
comprising a polynucleotide encoding a truncated HBV core antigen consisting
of an
amino acid sequence that is at least 90% identical to SEQ ID NO: 2, preferably
100%
identical to SEQ ID NO: 2; and a viral vector comprising a polynucleotide
encoding a
HBV pol antigen comprising an amino acid sequence that is at least 90%
identical to
SEQ ID NO: 4, preferably 100% identical to SEQ ID NO: 4. The vector comprising
the
coding sequence for the truncated HBV core antigen and the vector comprising
the
coding sequence for the HBV pol antigen can be the same vector, or two
different
vectors.
[00172] In an embodiment of the application, a composition comprises a viral
vector
comprising a polynucleotide encoding a fusion protein comprising a truncated
HBV core
antigen consisting of an amino acid sequence that is at least 90% identical to
SEQ ID
NO: 2, preferably 100% identical to SEQ ID NO: 2, operably linked to a HBV Pol
antigen comprising an amino acid sequence that is at least 90% identical to
SEQ ID NO:
4, preferably 100% identical to SEQ ID NO: 4, or vice versa. Preferably, the
fusion
protein further comprises a linker that operably links the truncated HBV core
antigen to
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the HBV Pol antigen, or vice versa. Preferably, the linker has the amino acid
sequence of
(AlaGly)n, wherein n is an integer of 2 to 5.
[00173] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring truncated HBV core antigen consisting of an amino acid
sequence that is at least 90% identical to SEQ ID NO: 2, preferably 100%
identical to
SEQ ID NO: 2.
[00174] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring HBV Pol antigen comprising an amino acid sequence that
is at
least 90% identical to SEQ ID NO: 4, preferably 100% identical to SEQ ID NO:
4.
[00175] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring truncated HBV core antigen consisting of an amino acid
sequence that is at least 90% identical to SEQ ID NO: 2, preferably 100%
identical to
SEQ ID NO: 2; and an isolated or non-naturally occurring HBV Pol antigen
comprising
an amino acid sequence that is at least 90% identical to SEQ ID NO: 4,
preferably 100%
identical to SEQ ID NO: 4.
[00176] In an embodiment of the application, a composition comprises an
isolated or
non-naturally occurring fusion protein comprising a truncated HBV core antigen
consisting of an amino acid sequence that is at least 90% identical to SEQ ID
NO: 2,
preferably 100% identical to SEQ ID NO: 2, operably linked to a HBV Pol
antigen
comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:
4,
preferably 100% identical to SEQ ID NO: 4, or vice versa. Preferably, the
fusion protein
further comprises a linker that operably links the truncated HBV core antigen
to the HBV
Pol antigen, or vice versa. Preferably, the linker has the amino acid sequence
of
(AlaGly)n, wherein n is an integer of 2 to 5.
[00177] In another general aspect, the application relates to an immunogenic
combination or a kit comprising polynucleotides expressing a truncated HBV
core
antigen and an HBV pol antigen according to embodiments of the application.
Any
polynucleotides and/or vectors encoding HBV core and pol antigens of the
application
described herein can be used in the immunogenic combinations or kits of the
application.
[00178] According to embodiments of the application, the polynucleotides in a
vaccine
combination or kit can be linked or separate, such that the HBV antigens
expressed from
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such polynucleotides are fused together or produced as separate proteins,
whether
expressed from the same or different polynucleotides. In one embodiment, the
first and
second polynucleotides are present in separate viral vectors used in
combination either in
the same or separate compositions, such that the expressed proteins are also
separate
proteins, but used in combination. In another embodiment, the HBV antigens
encoded by
the first and second polynucleotides can be expressed from the same viral
vector, such
that an HBV core-pol fusion antigen is produced. Optionally, the core and pol
antigens
can be joined or fused together by a short linker. Alternatively, the HBV
antigens
encoded by the first and second polynucleotides can be expressed independently
from a
single vector using a using a ribosomal slippage site (also known as cis-
hydrolase site)
between the core and pol antigen coding sequences. This strategy results in a
bicistronic
expression vector in which individual core and pol antigens are produced from
a single
mRNA transcript. The core and pol antigens produced from such a bicistronic
expression
vector can have additional N or C-terminal residues, depending upon the
ordering of the
coding sequences on the mRNA transcript. Examples of ribosomal slippage sites
that can
be used for this purpose include, but are not limited to, the FA2 slippage
site from foot-
and-mouth disease virus (FMDV). Another possibility is that the HBV antigens
encoded
by the first and second polynucleotides can be expressed independently from
two
separate vectors, one encoding the HBV core antigen and one encoding the HBV
pol
antigen.
[00179] In a preferred embodiment, the first and second polynucleotides are
present in
separate viral vectors. Preferably, the separate vectors are present in the
same
composition.
[00180] In a particular embodiment of the application, an immunogenic
combination or
kit comprises: a first vector, preferably a DNA plasmid or a viral vector,
comprising a
polynucleotide encoding a truncated HBV core antigen consisting of an amino
acid
sequence that is at least 90% identical to SEQ ID NO: 2, preferably 100%
identical to
SEQ ID NO: 2; and a second vector, preferably a DNA plasmid or a viral vector,
comprising a polynucleotide encoding a HBV polymerase antigen comprising an
amino
acid sequence that is at least 98% identical to SEQ ID NO: 4, preferably 100%
identical
to SEQ ID NO: 4.
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[00181] In one particular embodiment of the application, the first vector is a
first DNA
plasmid and the second vector is a second DNA plasmid. Each of the first and
second
DNA plasmids comprises an origin of replication, preferably a pUC ORI of SEQ
ID NO:
21, and an antibiotic resistance cassette, preferably comprising a codon
optimized Kanr
(Kanamycin resistance) gene having a polynucleotide sequence that is at least
90%
identical to SEQ ID NO: 22, preferably under control of a bla promoter, for
instance the
bla promoter shown in SEQ ID NO: 24. Each of the first and second DNA plasmids
independently further comprises at least one of a promoter sequence, enhancer
sequence,
and a polynucleotide sequence encoding a signal peptide sequence operably
linked to the
first polynucleotide sequence or the second polynucleotide sequence.
Preferably, each of
the first and second DNA plasmids comprises an upstream sequence operably
linked to
the first polynucleotide or the second polynucleotide, wherein the upstream
sequence
comprises, from 5' end to 3' end, a promoter sequence of SEQ ID NO: 7, an
enhancer
sequence of SEQ ID NO: 8, and a polynucleotide sequence encoding a signal
peptide
sequence having the amino acid sequence of SEQ ID NO: 6. Each of the first and
second
DNA plasmids can also comprise a polyadenylation signal located downstream of
the
coding sequence of the HBV antigen, such as the bGH polyadenylation signal of
SEQ ID
NO: 9.
[00182] In one particular embodiment of the application, the first vector is a
first viral
vector and the second vector is a second viral vector. Preferably, each of the
first and
second viral vector is an adenoviral vector, more preferably an Ad26 or Ad35
vector,
comprising an expression cassette including the polynucleotide encoding the
HBV pol
antigen or the truncated HBV core antigen of the application; an upstream
sequence
operably linked to the polynucleotide encoding the HBV antigen comprising,
from 5' end
to 3' end, a promoter sequence, preferably a CMV promoter sequence of SEQ ID
NO: 7,
an enhancer sequence, preferably an ApoAI gene fragment sequence of SEQ ID NO:
15
or a triple enhancer sequence of SEQ ID NO: 8, and a polynucleotide sequence
encoding
a signal peptide sequence, preferably a cystatin S signal having the amino
acid sequence
of SEQ ID NO: 6 or an immunoglobulin secretion signal having the amino acid
sequence
of SEQ ID NO: 11; and a downstream sequence operably linked to the
polynucleotide
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encoding the HBV antigen comprising a polyadenylation signal, preferably a
SV40
polyadenylation signal of SEQ ID NO:16.
[00183] In a particular embodiment of the application, the first vector is a
first viral
vector and the second vector is a second viral vector. Preferably, each of the
first and
second viral vector is a MVA vector comprising an expression cassette
including the
polynucleotide encoding the HBV pol antigen and/or the truncated HBV core
antigen of
the application; an upstream sequence operably linked to the polynucleotide
encoding the
HBV antigen comprising, from 5' end to 3' end, a promoter sequence, preferably
a
PrMVA13.5 long promoter sequence of SEQ ID NO: 25 or a PrHyb promoter sequence
of SEQ ID NO: 26, and a polynucleotide sequence encoding a signal peptide
sequence,
preferably a cystatin S signal having the amino acid sequence of SEQ ID NO: 6
or an
immunoglobulin secretion signal having the amino acid sequence of SEQ ID NO:
11; and
a downstream sequence operably linked to the polynucleotide encoding the HBV
antigen
comprising a polyadenylation signal or an early termination signal, wherein
the early
termination signal has a nucleotide sequence of SEQ ID NO:28, or wherein the
polyadenylation signal is selected from an 5V40 polyadenylation signal having
a
polynucleotide sequence of SEQ ID NO: 16 or a bGH polyadenylation signal
having a
polynucleotide sequence of SEQ ID NO: 9, preferably the downstream sequence
operably
linked to the polynucleotide encoding the HBV antigen is an early termination
signal
having a nucleotide sequence of SEQ ID NO: 28.
[00184] In an embodiment of the application, provided is a vaccine combination
comprising (a) a first composition comprising an immunologically effective
amount of an
adenovirus vector comprising a first polynucleotide sequence encoding an HBV
polymerase antigen comprising an amino acid sequence that is at least 98%
identical to
SEQ ID NO:4, wherein the HBV polymerase antigen does not have reverse
transcriptase
activity and RNase H activity; and (b) a second composition comprising an
immunologically effective amount of a Modified Vaccinia Ankara (MVA) vector
comprising a second polynucleotide sequence encoding an HBV polymerase antigen
comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:
4,
wherein the HBV polymerase antigen does not have reverse transcriptase
activity and
RNase H activity; wherein the first composition is administered to the human
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priming the immune response, and the second composition is administered to the
human
subject one or more times for boosting the immune response.
[00185] In an embodiment of the application, provided is a vaccine combination
comprising (a) a first composition comprising an immunologically effective
amount of a
Modified Vaccinia Ankara (MVA) vector comprising a first polynucleotide
sequence
encoding an HBV polymerase antigen comprising an amino acid sequence that is
at least
98% identical to SEQ ID NO:4, wherein the HBV polymerase antigen does not have
reverse transcriptase activity and RNase H activity; and (b) a second
composition
comprising an immunologically effective amount of an adenovirus vector
comprising a
second polynucleotide sequence encoding an HBV polymerase antigen comprising
an
amino acid sequence that is at least 98% identical to SEQ ID NO: 4, wherein
the HBV
polymerase antigen does not have reverse transcriptase activity and RNase H
activity;
wherein the first composition is administered to the human subject for priming
the
immune response, and the second composition is administered to the human
subject one
or more times for boosting the immune response.
[00186] In those embodiments of the application in which an immunogenic
combination comprises a first viral vector and a second viral vector, the
amount of each
of the first and second vectors is not particularly limited. For example, the
first viral
vector and the second viral vector can be present in a ratio of 10:1 to 1:10,
by weight,
such as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,
1:6, 1:7, 1:8, 1:9,
or 1:10, by weight. Preferably, the first and second viral vectors are present
in a ratio of
1:1, by weight.
[00187] Compositions and immunogenic combinations of the application can
comprise
additional polynucleotides or vectors encoding additional HBV antigens and/or
additional
HBV antigens or immunogenic fragments thereof. However, in particular
embodiments,
the compositions and immunogenic combinations of the application do not
comprise
certain antigens.
[00188] In a particular embodiment, a composition or immunogenic combination
or kit
of the application does not comprise a ElBsAg or a polynucleotide sequence
encoding the
ElBsAg.
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[00189] In another particular embodiment, a composition or immunogenic
combination
or kit of the application does not comprise a HBV L protein or a
polynucleotide sequence
encoding the HBV L protein.
[00190] In yet another particular embodiment of the application, a composition
or
immunogenic combination of the application does not comprise a HBV envelope
protein
or a polynucleotide sequence encoding the HBV envelope protein.
[00191] Compositions and immunogenic combinations of the application can also
comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable
carrier is
non-toxic and should not interfere with the efficacy of the active ingredient.
Pharmaceutically acceptable carriers can include one or more excipients such
as binders,
disintegrants, swelling agents, suspending agents, emulsifying agents, wetting
agents,
lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and
coatings. The
precise nature of the carrier or other material can depend on the route of
administration,
e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous,
intramucosal
(e.g., gut), intranasal or intraperitoneal routes. For liquid injectable
preparations, for
example, suspensions and solutions, suitable carriers and additives include
water, glycols,
oils, alcohols, preservatives, coloring agents and the like. For solid oral
preparations, for
example, powders, capsules, caplets, gelcaps and tablets, suitable carriers
and additives
include starches, sugars, diluents, granulating agents, lubricants, binders,
disintegrating
agents and the like. For nasal sprays/inhalant mixtures, the aqueous
solution/suspension
can comprise water, glycols, oils, emollients, stabilizers, wetting agents,
preservatives,
aromatics, flavors, and the like as suitable carriers and additives.
[00192] Compositions and immunogenic combinations of the application can be
formulated in any matter suitable for administration to a subject to
facilitate
administration and improve efficacy, including, but not limited to, oral
(enteral)
administration and parenteral injections. The parenteral injections include
intravenous
injection or infusion, subcutaneous injection, intradermal injection, and
intramuscular
injection. Compositions of the application can also be formulated for other
routes of
administration including transmucosal, ocular, rectal, long acting
implantation, sublingual
administration, under the tongue, from oral mucosa bypassing the portal
circulation,
inhalation, or intranasal.
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[00193] In a preferred embodiment of the application, compositions and
immunogenic
combinations of the application are formulated for parental injection,
preferably
subcutaneous, intradermal injection, or intramuscular injection, more
preferably
intramuscular injection.
[00194] According to embodiments of the application, compositions and
immunogenic
combinations for administration will typically comprise a buffered solution in
a
pharmaceutically acceptable carrier, e.g., an aqueous carrier such as buffered
saline and
the like, e.g., phosphate buffered saline (PBS). The compositions and
immunogenic
combinations can also contain pharmaceutically acceptable substances as
required to
approximate physiological conditions such as pH adjusting and buffering
agents. In a
typical embodiment, a composition or immunogenic combination of the
application
comprising plasmid DNA can contain phosphate buffered saline (PBS) as the
pharmaceutically acceptable carrier. The plasmid DNA can be present in a
concentration
of, e.g., 0.5 mg/mL to 5 mg/mL, such as 0.5 mg/mL 1, mg/mL, 2 mg/mL, 3 mg/mL,
4
mg/mL, or 5 mg/mL, preferably at 1 mg/mL.
[00195] Compositions and immunogenic combinations of the application can be
formulated as a vaccine (also referred to as an "immunogenic composition")
according to
methods well known in the art. Such compositions can include adjuvants to
enhance
immune responses. The optimal ratios of each component in the formulation can
be
determined by techniques well known to those skilled in the art in view of the
present
disclosure.
[00196] In an embodiment of the application, an adjuvant is included in a
composition
or immunogenic combination of the application, or co-administered with a
composition
or immunogenic combination of the application. Use of an adjuvant is optional,
and may
further enhance immune responses when the composition is used for vaccination
purposes. Adjuvants suitable for co-administration or inclusion in
compositions in
accordance with the application should preferably be ones that are potentially
safe, well
tolerated and effective in humans. An adjuvant can be a small molecule or
antibody
including, but not limited to, immune checkpoint inhibitors (e.g., anti-PD1,
anti-RIM-3,
etc.), toll-like receptor inhibitors, RIG-1 inhibitors, IL-15 superagonists
(Altor
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Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro),
FLT3L
genetic adjuvant, IL-12 genetic adjuvant, and IL-7-hyFc.
[00197] Embodiments of the application also relate to methods of making
compositions
and immunogenic combinations of the application. According to embodiments of
the
application, a method of producing a composition or immunogenic combination
comprises mixing an isolated polynucleotide encoding an HBV antigen, vector,
and/or
polypeptide of the application with one or more pharmaceutically acceptable
carriers.
One of ordinary skill in the art will be familiar with conventional techniques
used to
prepare such compositions.
[00198] Methods of Inducing/Enhancing an Immune Response
[00199] In another general aspect, the application relates to a method of
inducing an
immune response against hepatitis B virus (HBV) in a subject in need thereof,
comprising
administering to the subject an immunologically effective amount of a
composition or
immunogenic composition of the application. Any of the compositions and
immunogenic
combinations of the application described herein can be used in the methods of
the
application.
[00200] The application provides an improved method of priming and boosting an
immune response to a HBV antigenic protein or immunogenic polypeptide thereof
in a
human subject using an MVA vector in combination with an adenoviral vector.
[00201] According to a general aspect of the application, a method of
enhancing an
immune response in a human subject comprises:
a. administering to the human subject a first composition comprising an
immunologically effective amount of an adenovirus vector of the
application; and
b. administering to the human subject a second composition comprising an
immunologically effective amount of a MVA vector of the application;
to thereby obtain an enhanced immune response against the HBV antigen in
the human subject.
[00202] According to another general aspect of the application, a method of
enhancing
an immune response in a human subject comprises:
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a. administering to the human subject a first composition comprising an
immunologically effective amount of a MVA vector of the application;
and
b. administering to the human subject a second composition comprising an
immunologically effective amount of an adenovirus vector of the
application;
to thereby obtain an enhanced immune response against the I-113V antigen in
the human subject.
[00203] According to another general aspect of the application, a method of
enhancing
an immune response in a human subject comprises:
a. administering to the human subject a first composition comprising an
immunologically effective amount of a first plasmid comprising a first
non-naturally occurring nucleic acid comprising a first polynucleotide
sequence encoding an I-113V pol antigen of the application, and an
immunologically effective amount of a second plasmid comprising a
second non-naturally occurring nucleic acid comprising a second
polynucleotide sequence encoding a truncated I-113V core antigen of the
application; and
b. administering to the human subject a second composition comprising an
immunologically effective amount of a MVA vector of the application;
to thereby obtain an enhanced immune response against the I-113V antigen in
the human subject.
[00204] According to another general aspect of the application, a method of
enhancing
an immune response in a human subject comprises:
a. administering to the human subject a first composition comprising an
immunologically effective amount of a MVA vector of the application;
and
b. administering to the human subject a second composition comprising an
immunologically effective amount of a first plasmid comprising a first
non-naturally occurring nucleic acid comprising a first polynucleotide
sequence encoding an I-113V pol antigen of the application, and an
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immunologically effective amount of a second plasmid comprising a
second non-naturally occurring nucleic acid comprising a second
polynucleotide sequence encoding a truncated HBV core antigen of the
application;
to thereby obtain an enhanced immune response against the HBV antigen in
the human subject.
[00205] The first composition is administered to the human subject in need
thereof to
prime the immune response, and the second composition is administered to the
human
subject in need thereof to boost the immune response. Priming and boosting the
immune
response can, for example, enhance the immune response.
[00206] According to embodiments of the application, the enhanced immune
response
comprises an enhanced antibody response against the HBV antigenic protein in
the
human subject.
[00207] Preferably, the enhanced immune response further comprises an enhanced
CD4+ response or an enhanced CD8+ T cell response against the HBV antigenic
protein
in the human subject. The enhanced CD4+ T cell response generated by a method
according to an embodiment of the application can be, for example, an increase
or
induction of a dominant CD4+ T cell response against the HBV antigenic
protein, and/or
an increase or induction of polyfunctional CD4+ T cells specific to the HBV
antigenic
protein in the human subject. The polyfunctional CD4+ T cells express more
than one
cytokine, such as two or more of IFN-gamma, IL-2 and TNF-alpha. The enhanced
CD8+
T cell response generated by a method according to an embodiment of the
application can
be, for example, an increase or induction of polyfunctional CD8+ T cells
specific to the
HBV antigenic protein in the human subject.
[00208] More preferably, the enhanced immune response resulting from a method
according to an embodiment of the application comprises an enhanced CD4+ T
cell
response, an enhanced antibody response and an enhanced CD8+ T cell response,
against
the HBV antigenic protein in the human subject.
[00209] As used herein, the term "infection" refers to the invasion of a host
by a
disease causing agent. A disease causing agent is considered to be
"infectious" when it is
capable of invading a host, and replicating or propagating within the host.
Examples of
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infectious agents include viruses, e.g., HBV and certain species of
adenovirus, prions,
bacteria, fungi, protozoa and the like. "HBV infection" specifically refers to
invasion of a
host organism, such as cells and tissues of the host organism, by HBV.
[00210] According to embodiments of the application, "inducing an immune
response"
when used with reference to the methods described herein encompasses causing a
desired
immune response or effect in a subject in need thereof against HBV or an HBV
infection.
"Inducing an immune response" also encompasses providing a therapeutic
immunity for
treating against a pathogenic agent, i.e., HBV. As used herein, the term
"therapeutic
immunity" or "therapeutic immune response" means that the HBV-infected
vaccinated
subject is able to control an infection with the pathogenic agent, i.e., HBV,
against which
the vaccination was done. In one embodiment, "inducing an immune response"
means
producing an immunity in a subject in need thereof, e.g., to provide a
therapeutic effect
against a disease, such as HBV infection. In an embodiment of the application,
"inducing
an immune response" refers to causing or improving cellular immunity, e.g., T
cell
response, against HBV. In an embodiment of the application, "inducing an
immune
response" refers to causing or improving a humoral immune response against
HBV. In
an embodiment of the application, "inducing an immune response" refers to
causing or
improving a cellular and a humoral immune response against HBV.
[00211] Typically, the administration of compositions and immunogenic
combinations
according to embodiments of the application will have a therapeutic aim to
generate an
immune response against HBV after HBV infection or development of symptoms
characteristic of HBV infection, i.e., for therapeutic vaccination.
[00212] As used herein, "an immunogenically effective amount" or
"immunologically
effective amount" means an amount of a composition, polynucleotide, vector, or
antigen
sufficient to induce a desired immune effect or immune response in a subject
in need
thereof. In one embodiment, an immunologically effective amount means an
amount
sufficient to induce an immune response in a subject in need thereof. In
another
embodiment, an immunologically effective amount means an amount sufficient to
produce immunity in a subject in need thereof, e.g., provide a therapeutic
effect against a
disease such as HBV infection. An immunologically effective amount can vary
depending upon a variety of factors, such as the physical condition of the
subject, age,
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weight, health, etc.; the particular application, e.g., providing protective
immunity or
therapeutic immunity; and the particular disease, e.g., viral infection, for
which immunity
is desired. An immunologically effective amount can readily be determined by
one of
ordinary skill in the art in view of the present disclosure.
[00213] In particular embodiments of the application, an immunologically
effective
amount refers to the amount of a composition or immunogenic combination which
is
sufficient to achieve one, two, three, four, or more of the following effects:
(i) reduce or
ameliorate the severity of an HBV infection or a symptom associated therewith;
(ii)
reduce the duration of an HBV infection or symptom associated therewith; (iii)
prevent
the progression of an HBV infection or symptom associated therewith; (iv)
cause
regression of an HBV infection or symptom associated therewith; (v) prevent
the
development or onset of an HBV infection, or symptom associated therewith;
(vi) prevent
the recurrence of an HBV infection or symptom associated therewith; (vii)
reduce
hospitalization of a subject having an HBV infection; (viii) reduce
hospitalization length
of a subject having an HBV infection; (ix) increase the survival of a subject
with an HBV
infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce
HBV
replication in a subject; and/or (xii) enhance or improve the prophylactic or
therapeutic
effect(s) of another therapy.
[00214] In other particular embodiments, an immunologically effective amount
is an
amount sufficient to reduce ElBsAg levels consistent with evolution to
clinical
seroconversion; achieve sustained ElBsAg clearance associated with reduction
of infected
hepatocytes by a subject's immune system; induce HBV-antigen specific
activated T-cell
populations; and/or achieve persistent loss of ElBsAg within 12 months.
Examples of a
target index include lower ElBsAg below a threshold of 500 copies of ElBsAg IU
and/or
higher CD8 counts.
[00215] As general guidance, an immunologically effective amount when used
with
reference to a viral vector can range from about 1 X 107 viral particles per
dose to about 1
X 1012 viral particles per dose. An immunologically effective amount can be
about 1 X
101 , about 2 X 101 , about 3 X 101 , about 4 X 101 , about 5 X 101 , about 6
X 101 ,
about 7 X 1010, about 8 X 1010, about 9 X 1010, about 1 X 1011, about 2 X
1011, about 3 X
1011, about 4 X 1011, about 5 X 1011, or about 1 X 1012 viral particles per
dose. An
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immunologically effective amount can be from one vector or from multiple
vectors. An
immunologically effective amount can be administered in a single composition,
or in
multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions
(e.g., tablets,
capsules or injectables), wherein the administration of the multiple capsules
or injections
collectively provides a subject with an immunologically effective amount. It
is also
possible to administer an immunologically effective amount to a subject, and
subsequently administer another dose of an immunologically effective amount to
the
same subject, in a so-called prime-boost regimen. This general concept of a
prime-boost
regimen is well known to the skilled person in the vaccine field. Further
booster
.. administrations can optionally be added to the regimen, as needed.
[00216] According to embodiments of the application, an immunogenic
combination
comprising two viral vectors, e.g., a first viral vector encoding an EIBV core
antigen and
second viral vector encoding an EIBV pol antigen can be administered to a
subject by
mixing both viral vectors and delivering the mixture to a single anatomic
site.
Alternatively, two separate immunizations each delivering a single expression
vector can
be performed. In such embodiments, whether both viral vectors are administered
in a
single immunization as a mixture of in two separate immunizations, the first
viral vector
and the second viral vector can be administered in a ratio of 10:1 to 1:10, by
weight, such
as 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6,
1:7, 1:8, 1:9, or
1:10, by weight. Preferably, the first and second viral vectors are
administered in a ratio
of 1:1, by weight.
[00217] In some embodiments, a subject to be treated according to the methods
of the
application is an EIBV-infected subject, particular a subject having chronic
EIBV
infection. Acute EIBV infection is characterized by an efficient activation of
the innate
immune system complemented with a subsequent broad adaptive response (e.g.,
EIBV-
specific T-cells, neutralizing antibodies), which usually results in
successful suppression
of replication or removal of infected hepatocytes. In contrast, such responses
are
impaired or diminished due to high viral and antigen load, e.g., EIBV envelope
proteins
are produced in abundance and can be released in sub-viral particles in 1,000-
fold excess
to infectious virus.
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[00218] Chronic HBV infection is described in phases characterized by viral
load, liver
enzyme levels (necroinflammatory activity), HBeAg, or ElBsAg load or presence
of
antibodies to these antigens. cccDNA levels stay relatively constant at
approximately 10
to 50 copies per cell, even though viremia can vary considerably. The
persistence of the
cccDNA species leads to chronicity. More specifically, the phases of chronic
HBV
infection include: (i) the immune-tolerant phase characterized by high viral
load and
normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-
positive
phase in which lower or declining levels of viral replication with
significantly elevated
liver enzymes are observed; (iii) the inactive ElBsAg carrier phase, which is
a low
replicative state with low viral loads and normal liver enzyme levels in the
serum that
may follow HBeAg seroconversion; and (iv) the HBeAg-negative phase in which
viral
replication occurs periodically (reactivation) with concomitant fluctuations
in liver
enzyme levels, mutations in the pre-core and/or basal core promoter are
common, such
that HBeAg is not produced by the infected cell.
[00219] As used herein, "chronic HBV infection" refers to a subject having the
detectable presence of HBV for more than 6 months. A subject having a chronic
HBV
infection can be in any phase of chronic HBV infection. In preferred
embodiments, a
chronic HBV infection referred to herein follows the definition published by
the Centers
for Disease Control and Prevention (CDC), according to which a chronic HBV
infection
is characterized by the following laboratory criteria: (i) negative for IgM
antibodies to
hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface
antigen
(HiBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B
virus DNA,
or (ii) positive for ElBsAg or nucleic acid test for HBV DNA, or positive for
HBeAg two
times at least 6 months apart.
[00220] According to particular embodiments, an immunogenically effective
amount
refers to the amount of a composition or immunogenic combination which is
sufficient to
treat chronic HBV infection.
[00221] In some embodiments, a subject having chronic HBV infection is
undergoing
nucleoside analog (NUC) treatment, and is NUC-suppressed. As used herein, "NUC-
suppressed" refers to a subject having an undetectable viral level of HBV and
stable
alanine aminotransferase (ALT) levels for at least six months. Examples of
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nucleoside/nucleotide analog treatment include I-113V polymerase inhibitors,
such as
entacavir and tenofovir. Preferably, a subject having chronic I-113V infection
does not
have advanced hepatic fibrosis or cirrhosis. Such subject would typically have
a
METAVIR score of less than 3 and a fibroscan result of less than 9 kPa. The
METAVIR
score is a scoring system that is commonly used to assess the extent of
inflammation and
fibrosis by histopathological evaluation in a liver biopsy of patients with
hepatitis B. The
scoring system assigns two standardized numbers: one reflecting the degree of
inflammation and one reflecting the degree of fibrosis.
[00222] It is believed that elimination or reduction of chronic I-113V may
allow early
disease interception of severe liver disease, including virus-induced
cirrhosis and
hepatocellular carcinoma. Thus, the methods of the application can also be
used as
therapy to treat 1413V-induced diseases. Examples of 1413V-induced diseases
include, but
are not limited to cirrhosis, cancer (e.g., hepatocellular carcinoma), and
fibrosis,
particularly advanced fibrosis characterized by a METAVIR score of 3 or
higher. In such
embodiments, an immunogenically effective amount is an amount sufficient to
achieve
persistent loss of I-113sAg within 12 months and significant decrease in
clinical disease
(e.g., cirrhosis, hepatocellular carcinoma, etc.).
[00223] Methods according to embodiments of the application further comprise
administering to the subject in need thereof another immunogenic agent (such
as another
I-113V antigen or other antigen) or another anti-I-113V agent (such as a
nucleoside analog or
other anti-I-113V agent) in combination with a composition of the application.
[00224] Methods of Delivery
[00225] Compositions and immunogenic combinations of the application can be
administered to a subject by any method known in the art in view of the
present
disclosure, including, but not limited to, parenteral administration (e.g.,
intramuscular,
subcutaneous, intravenous, or intradermal injection), oral administration,
transdermal
administration, and nasal administration. Preferably, compositions and
immunogenic
combinations are administered parenterally (e.g., by intramuscular injection
or
intradermal injection) or transdermally.
[00226] In some embodiments of the application in which a composition or
immunogenic combination comprises one or more viral vectors, administration
can be by
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injection through the skin, e.g., intramuscular or intradermal injection,
preferably
intramuscular injection. Intramuscular injection can be combined with
electroporation,
i.e., application of an electric field to facilitate delivery of the DNA
plasmids to cells. As
used herein, the term "electroporation" refers to the use of a transmembrane
electric field
pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo
electroporation, electrical fields of appropriate magnitude and duration are
applied to
cells, inducing a transient state of enhanced cell membrane permeability, thus
enabling
the cellular uptake of molecules unable to cross cell membranes on their own.
Creation of
such pores by electroporation facilitates passage of biomolecules, such as
plasmids,
oligonucleotides, siRNAs, drugs, etc., from one side of a cellular membrane to
the other.
In vivo electroporation for the delivery of DNA vaccines has been shown to
significantly
increase plasmid uptake by host cells, while also leading to mild-to-moderate
inflammation at the injection site. As a result, transfection efficiency and
immune
response are significantly improved (e.g., up to 1,000 fold and 100 fold
respectively) with
intradermal or intramuscular electroporation, in comparison to conventional
injection.
[00227] In a typical embodiment, electroporation is combined with
intramuscular
injection. However, it is also possible to combine electroporation with other
forms of
parenteral administration, e.g., intradermal injection, subcutaneous
injection, etc.
[00228] Administration of a composition, immunogenic combination or vaccine of
the
application via electroporation can be accomplished using electroporation
devices that
can be configured to deliver to a desired tissue of a mammal a pulse of energy
effective
to cause reversible pores to form in cell membranes. The electroporation
device can
include an electroporation component and an electrode assembly or handle
assembly.
The electroporation component can include one or more of the following
components of
electroporation devices: controller, current waveform generator, impedance
tester,
waveform logger, input element, status reporting element, communication port,
memory
component, power source, and power switch. Electroporation can be accomplished
using
an in vivo electroporation device. Examples of electroporation devices and
electroporation methods that can facilitate delivery of compositions and
immunogenic
combinations of the application, particularly those comprising DNA plasmids,
include
CELLECTRAO (Inovio Pharmaceuticals, Blue Bell, PA), Elgen electroporator
(Inovio
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Pharmaceuticals, Inc.) Tri-GridTM delivery system (Ichor Medical Systems,
Inc., San
Diego, CA 92121) and those described in U.S. Patent No. 7,664,545, U.S. Patent
No.
8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent
No.
6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S.
Patent No.
6,939,862, U.S. Patent No. 7,328,064, U.S. Patent No. 6,041,252, U.S. Patent
No.
5,873,849, U.S. Patent No. 6,278,895, U.S. Patent No. 6,319,901, U.S. Patent
No.
6,912,417, U.S. Patent No. 8,187,249, U.S. Patent No. 9,364,664, U.S. Patent
No.
9,802,035, U.S. Patent No. 6,117,660, and International Patent Application
Publication
W02017172838, all of which are herein incorporated by reference in their
entireties.
Also contemplated by the application for delivery of the compositions and
immunogenic
combinations of the application are use of a pulsed electric field, for
instance as described
in, e.g., U.S. Patent No. 6,697,669, which is herein incorporated by reference
in its
entirety.
[00229] In other embodiments of the application in which a composition or
immunogenic combination comprises one or more DNA plasmids, the method of
administration is transdermal. Transdermal administration can be combined with
epidermal skin abrasion to facilitate delivery of the DNA plasmids to cells.
For example,
a dermatological patch can be used for epidermal skin abrasion. Upon removal
of the
dermatological patch, the composition or immunogenic combination can be
deposited on
the abraised skin.
[00230] Methods of delivery are not limited to the above described
embodiments, and
any means for intracellular delivery can be used. Other methods of
intracellular delivery
contemplated by the methods of the application include, but are not limited
to, liposome
encapsulation, nanoparticles, etc.
[00231] Adjuvants
[00232] In some embodiments of the application, a method of inducing an immune
response against HBV further comprises administering an adjuvant. The terms
"adjuvant" and "immune stimulant" are used interchangeably herein, and are
defined as
one or more substances that cause stimulation of the immune system. In this
context, an
adjuvant is used to enhance an immune response to HBV antigens and antigenic
HBV
polypeptides of the application.
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[00233] According to embodiments of the application, an adjuvant can be
present in an
immunogenic combination or composition of the application, or administered in
a
separate composition. An adjuvant can be, e.g., a small molecule or an
antibody.
Examples of adjuvants suitable for use in the application include, but are not
limited to,
immune checkpoint inhibitors (e.g., anti-PD1, anti-RIM-3, etc.), toll-like
receptor
inhibitors, RIG-1 inhibitors, IL-15 superagonists (Altor Bioscience), mutant
IRF3 and
IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12
genetic
adjuvant, and IL-7-hyFc.
[00234] Compositions and immunogenic combinations of the application can also
be
administered in combination with at least one other anti-HBV agent. Examples
of anti-
1-1BV agents suitable for use with the application include, but are not
limited to small
molecules, antibodies, and/or CAR-T therapies that function as capsid
inhibitors, TLR
inhibitors, cccDNA inhibitors, 1-1BV polymerase inhibitors (e.g., entecavir
and tenofovir),
and/or immune checkpoint inhibitors, etc. Such anti-HBV agents can be
administered
with the compositions and immunogenic combinations of the application
simultaneously
or sequentially.
[00235] Methods of Prime/Boost Immunization
[00236] Embodiments of the application also contemplate administering an
immunologically effective amount of a composition or immunogenic combination
to a
subject, and subsequently administering another dose of an immunologically
effective
amount of a composition or immunogenic combination to the same subject, in a
so-called
prime-boost regimen Thus, in one embodiment, a composition or immunogenic
combination of the application is a primer vaccine used for priming an immune
response.
In another embodiment, a composition or immunogenic combination of the
application is
a booster vaccine used for boosting an immune response. The priming and
boosting
vaccines according to embodiments of the application can be used in the
methods of the
application described herein. This general concept of a prime-boost regimen is
well
known to the skilled person in the vaccine field. Any of the compositions and
immunogenic combinations of the application described herein can be used as
priming
and/or boosting vaccines for priming and/or boosting an immune response
against EIBV.
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[00237] According to embodiments of the application, a composition or
immunogenic
combination of the application can be administered at least once for priming
immunization. The composition or immunogenic combination can be re-
administered for
boosting immunization. Further booster administrations of the composition or
vaccine
combination can optionally be added to the regimen, as needed. An adjuvant can
be
present in a composition of the application used for boosting immunization,
present in a
separate composition to be administered together with the composition or
immunogenic
combination of the application for the boosting immunization, or administered
on its own
as the boosting immunization. In those embodiments in which an adjuvant is
included in
the regimen, the adjuvant is preferably used for boosting immunization.
[00238] An illustrative and non-limiting example of a prime-boost regimen
includes
administering a single dose of an immunologically effective amount of a
composition or
immunogenic combination of the application to a subject to prime the immune
response;
and subsequently administering another dose of an immunologically effective
amount of
a composition or immunogenic combination of the application to boost the
immune
response, wherein the boosting immunization is first administered about one to
twelve
weeks (1 to 12), about two to twelve weeks (2 to 12), about two to ten weeks
(2 to 10),
about two to six weeks (2 to 6), preferably about four weeks after the priming
immunization is initially administered, preferably about eight weeks after the
priming
immunization is initially administered. In an embodiment of the application,
the boosting
immunization is administered at least one week after the priming immunization.
In an
embodiment of the application, the boosting immunization is administered at
least two
weeks after the priming immunization. Optionally, about 10 to 14 weeks,
preferably 12
weeks, after the priming immunization is initially administered, a further
boosting
immunization of the composition or immunogenic combination, or other adjuvant,
is
administered.
[00239] Kits
[00240] The application also provides a kit comprising an immunogenic
combination
of the application. A kit can comprise the first polynucleotide and the second
polynucleotide in separate compositions, or a kit can comprise the first
polynucleotide
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and the second polynucleotide in a single composition. A kit can further
comprise one or
more adjuvants or immune stimulants, and/or other anti-HBV agents.
[00241] The ability to induce or stimulate an anti-HBV immune response upon
administration in an animal or human organism can be evaluated either in vitro
or in vivo
using a variety of assays which are standard in the art. For a general
description of
techniques available to evaluate the onset and activation of an immune
response, see for
example Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J
Wiley &
Sons Inc, National Institute of Health). Measurement of cellular immunity can
be
performed by measurement of cytokine profiles secreted by activated effector
cells
including those derived from CD4+ and CD8+ T-cells (e.g. quantification of IL-
10 or
IFN gamma-producing cells by ELISPOT), by determination of the activation
status of
immune effector cells (e.g. T cell proliferation assays by a classical [3H]
thymidine
uptake), by assaying for antigen-specific T lymphocytes in a sensitized
subject (e.g.
peptide-specific lysis in a cytotoxicity assay, etc.).
[00242] The ability to stimulate a cellular and/or a humoral response can be
determined
by antibody binding and/or competition in binding (see for example Harlow,
1989,
Antibodies, Cold Spring Harbor Press). For example, titers of antibodies
produced in
response to administration of a composition providing an immunogen can be
measured
by enzyme-linked immunosorbent assay (ELISA). The immune responses can also be
measured by neutralizing antibody assay, where a neutralization of a virus is
defined as
the loss of infectivity through reaction/inhibition/neutralization of the
virus with specific
antibody. The immune response can further be measured by Antibody-Dependent
Cellular Phagocytosis (ADCP) Assay.
EMBODIMENTS
[00243] The i application provides also the following non-limiting
embodiments.
[00244] Embodiment 1 is a Modified Vaccinia Ankara (MVA) vector comprising a
non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence
encoding an HBV polymerase antigen comprising an amino acid sequence that is
at least
98% identical to SEQ ID NO:4.
[00245] Embodiment 2 is the MVA vector of embodiment 1, wherein the HBV
polymerase antigen does not have reverse transcriptase activity and RNase H
activity.
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[00246] Embodiment 3 is the MVA vector of embodiment 1 or 2, wherein the HBV
polymerase antigen is capable of inducing an immune response in a mammal
against at
least two HBV genotypes, preferably the HBV polymerase antigen is capable of
inducing
a T cell response in a mammal against at least HBV genotypes B, C, and D, and
more
preferably the HBV polymerase antigen is capable of inducing a CD8 T cell
response in a
human subject against at least HBV genotypes A, B, C, and D.
[00247] Embodiment 4 is the MVA vector of any one of embodiments 1-3, wherein
the
HBV polymerase antigen comprises the amino acid sequence of SEQ ID NO: 4.
[00248] Embodiment 5 is the MVA vector of any one of embodiments 1-4, further
comprising a polynucleotide sequence encoding a signal sequence operably
linked to the
HBV polymerase antigen.
[00249] Embodiment 6 is the MVA vector of embodiment 5, wherein the signal
sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11,
preferably the signal sequence is encoded by the polynucleotide sequence of
SEQ ID NO:
5 or SEQ ID NO: 10.
[00250] Embodiment 7 is the MVA vector of any one of embodiments 1-6, wherein
the
first polynucleotide sequence is at least 90% identical to SEQ ID NO: 3.
[00251] Embodiment 8 is the MVA vector of embodiment 7, wherein the first
polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 3.
[00252] Embodiment 9 is the MVA vector of any one of embodiments 1-8, further
comprising a second polynucleotide sequence encoding a truncated HBV core
antigen
consisting of the amino acid sequence of SEQ ID NO: 2.
[00253] Embodiment 10 is the MVA vector of embodiment 9, wherein the second
polynucleotide sequence is at least 90% identical to SEQ ID NO: 1.
[00254] Embodiment 11 is the MVA vector of embodiment 10, wherein the second
polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 1.
[00255] Embodiment 12 is the MVA vector of any one of embodiments 9-11,
wherein
the second polynucleotide sequence further comprises a polynucleotide sequence
encoding a signal sequence operably linked to the truncated HBV core antigen.
[00256] Embodiment 13 is the MVA vector of embodiment 12, wherein the signal
sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11,
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preferably the signal sequence is encoded by the polynucleotide sequence of
SEQ ID NO:
or SEQ ID NO: 10.
[00257] Embodiment 14 is the MVA vector of any one of embodiments 9-13,
wherein
the first and second polynucleotide sequences encode a fusion protein
comprising the
5 truncated HBV core antigen operably linked to the HBV polymerase antigen.
[00258] Embodiment 15 is the MVA vector of embodiment 14, wherein the fusion
protein comprises the truncated HBV core antigen operably linked to the HBV
polymerase antigen via a linker.
[00259] Embodiment 16 is the MVA vector of embodiment 15, wherein the linker
comprises the amino acid sequence of (AlaGly)n, and n is an integer of 2 to 5,
preferably
the linker is encoded by a polynucleotide sequence comprising SEQ ID NO: 14.
[00260] Embodiment 17 is the MVA vector of embodiment 16, wherein the fusion
protein comprises the amino acid sequence of SEQ ID NO: 12.
[00261] Embodiment 18 is the MVA vector of any one of embodiments 14-17,
wherein
the fusion protein further comprises a signal sequence, preferably the signal
sequence
comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, more
preferably the signal sequence is encoded by the polynucleotide sequence of
SEQ ID NO:
5 or SEQ ID NO: 10.
[00262] Embodiment 19 is the MVA vector of any one of embodiments 1-18 further
comprising at least one promoter sequence, optionally one or more additional
regulatory
sequences, preferably the at least one promoter sequence comprises the
polynucleotide
sequence of SEQ ID NO: 25 and/or SEQ ID NO: 26, and the additional regulatory
sequence is selected from the group consisting of an enhancer sequence of SEQ
ID NO: 8
or SEQ ID NO: 15, and a polyadenylation signal sequence of SEQ ID NO: 9 or SEQ
ID
NO: 16.
[00263] Embodiment 20 is the MVA vector of any one of embodiments 1-19,
wherein
the non-naturally occurring nucleic acid molecule does not encode a HBV
antigen
selected from the group consisting of a Hepatitis B surface antigen (E1BsAg),
a HBV
envelope (Env) antigen, and a HBV L protein antigen.
[00264] Embodiment 21 is an MVA vector comprising a first non-naturally
occurring
nucleic acid molecule comprising a first polynucleotide sequence encoding an
HBV
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polymerase antigen comprising the amino acid sequence of SEQ ID NO: 4, wherein
the
first polynucleotide sequence further encodes a signal sequence comprising the
amino
acid sequence of SEQ ID NO: 6, and wherein the first polynucleotide sequence
further
comprises a promoter sequence comprising the polynucleotide sequence of SEQ ID
NO:
26, and a second non-naturally occurring nucleic acid molecule comprising a
second
polynucleotide sequence encoding a truncated HBV core antigen consisting of
the amino
acid sequence of SEQ ID NO: 2, wherein the second polynucleotide sequence
further
encodes a signal sequence comprising the amino acid sequence of SEQ ID NO: 11,
and
wherein the second polynucleotide sequence further comprises a promoter
sequence
comprising the polynucleotide sequence of SEQ ID NO: 25.
[00265] Embodiment 22 is a composition comprising the MVA vector of any one of
embodiments 1-21 and a pharmaceutically acceptable carrier.
[00266] Embodiment 23 is a method of enhancing an immune response in a human
subject, the method comprising (a) administering to the human subject a first
composition
comprising an immunologically effective amount of an adenovirus vector
comprising a
non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence
encoding an HBV polymerase antigen comprising an amino acid sequence that is
at least
98% identical to SEQ ID NO:4; and (b) administering to the human subject a
second
composition comprising an immunologically effective amount of the MVA vector
of any
one of embodiments 1-21; to thereby obtain an enhanced immune response against
the
HBV antigen in the human subject.
[00267] Embodiment 24 is the method of embodiment 23, wherein the HBV
polymerase antigen of the first composition does not have reverse
transcriptase activity
and RNase H activity.
[00268] Embodiment 25 is the method of embodiment 23 or 24, wherein the first
composition is for priming the immune response and the second composition is
for
boosting the immune response.
[00269] Embodiment 26 is the method of any one of embodiments 23-25, wherein
the
HBV polymerase antigen of the first composition is capable of inducing an
immune
response in the human subject against at least two HBV genotypes, preferably
the HBV
polymerase antigen is capable of inducing a T cell response in the human
subject against
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at least HBV genotypes B, C, and D, and more preferably the HBV polymerase
antigen is
capable of inducing a CD8 T cell response in the human subject against at
least HBV
genotypes A, B, C, and D.
[00270] Embodiment 27 is the method of any one of embodiments 23-26, wherein
the
HBV polymerase antigen of the first composition comprises the amino acid
sequence of
SEQ ID NO: 4.
[00271] Embodiment 28 is the method of any one of embodiments 23-27, further
comprising a polynucleotide sequence encoding a signal sequence operably
linked to the
HBV polymerase antigen of the first composition.
[00272] Embodiment 29 is the method of embodiment 28, wherein the signal
sequence
comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, preferably
the
signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or
SEQ ID
NO: 10.
[00273] Embodiment 30 is the method of any one of embodiments 23-29, wherein
the
first polynucleotide sequence of the first composition is at least 90%
identical to SEQ ID
NO: 19.
[00274] Embodiment 31 is the method of embodiment 30, wherein the first
polynucleotide sequence of the first composition comprises the polynucleotide
sequence
of SEQ ID NO: 19.
[00275] Embodiment 32 is the method of any one of embodiments 23-31, wherein
the
nucleic acid molecule of the adenovirus in the first composition further
comprises a
second polynucleotide sequence encoding a truncated HBV core antigen
consisting of the
amino acid sequence of SEQ ID NO: 2.
[00276] Embodiment 33 is the method of embodiment 32, wherein the second
polynucleotide sequence of the first composition is at least 90% identical to
SEQ ID NO:
17.
[00277] Embodiment 34 is the method of embodiment 33, wherein the second
polynucleotide sequence of the first composition comprises the polynucleotide
sequence
of SEQ ID NO: 17.
[00278] Embodiment 35 is the method of any one of embodiments 32-34, wherein
the
first and second polynucleotide sequences of the first composition encode a
fusion
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protein comprising the truncated HBV core antigen operably linked to the HBV
polymerase antigen.
[00279] Embodiment 36 is the method of embodiment 35, wherein the fusion
protein of
the first composition comprises the truncated HBV core antigen operably linked
to the
HBV polymerase antigen via a linker.
[00280] Embodiment 37 is the method of embodiment 36, wherein the linker of
the
first composition comprises the amino acid sequence of (AlaGly)n, and n is an
integer of
2 to 5, preferably the linker is encoded by a polynucleotide sequence
comprising SEQ ID
NO: 14.
[00281] Embodiment 38 is the method of embodiment 37, wherein the fusion
protein of
the first composition comprises the amino acid sequence of SEQ ID NO: 12.
[00282] Embodiment 39 is the method of any one of embodiments 35-38, wherein
the
fusion protein of the first composition further comprises a signal sequence,
preferably the
signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID
NO: 11,
more preferably the signal sequence is encoded by the polynucleotide sequence
of SEQ
ID NO: 5 or SEQ ID NO: 10.
[00283] Embodiment 40 is the method of any one of embodiments 23-39, wherein
the
non-naturally occurring nucleic acid molecule of the first composition further
comprises
a promoter sequence, optionally one or more additional regulatory sequences,
preferably
the promoter sequence comprises the polynucleotide sequence of SEQ ID NO: 7,
and the
additional regulatory sequence is selected from the group consisting of an
enhancer
sequences of SEQ ID NO: 8 or SEQ ID NO: 15, and a polyadenylation signal
sequence
of SEQ ID NO: 16.
[00284] Embodiment 41 is the method of any one of embodiments 23-40, wherein
the
non-naturally occurring nucleic acid molecule of the first composition does
not encode a
HBV antigen selected from the group consisting of a Hepatitis B surface
antigen
(E1BsAg), a HBV envelope (Env) antigen, and a HBV L protein antigen.
[00285] Embodiment 42 is the method of any one of claims 23-41, wherein the
enhanced immune response comprises an enhanced antibody response against the
HBV
antigen in the human subject.
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[00286] Embodiment 43 is the method of embodiment 42, wherein the enhanced
immune response comprises an enhanced CD8+ T cell response against the 1-1BV
antigen
in the human subject.
[00287] Embodiment 44 is the method of embodiment 42 or 43, wherein the
enhanced
immune response comprises an enhanced CD4+ T cell response against the 1-1BV
antigen
in the human subject.
[00288] Embodiment 45 is the method of any one of embodiments 23-44, wherein
the
adenovirus vector is an rAd26 or rAd35 vector.
[00289] Embodiment 46 is the method of any one of embodiments 23-45, wherein
step
(b) is conducted 1-12 weeks after step (a).
[00290] Embodiment 47 is the method of any one of embodiments 23-45, wherein
step
(b) is conducted 2-12 weeks after step (a).
[00291] Embodiment 48 is the method of any one of embodiments 23-45, wherein
step
(b) is conducted at least 1 week after step (a).
[00292] Embodiment 49 is the method of any one of embodiments 23-45, wherein
step
(b) is conducted at least 2 weeks after step (a).
[00293] Embodiment 50 is a vaccine combination comprising (a) a first
composition
comprising an immunologically effective amount of an adenovirus vector
comprising a
first polynucleotide sequence encoding an I-113V polymerase antigen comprising
an amino
acid sequence that is at least 98% identical to SEQ ID NO:4; and (b) a second
composition comprising an immunologically effective amount of a Modified
Vaccinia
Ankara (MVA) vector comprising a second polynucleotide sequence encoding an I-
113V
polymerase antigen comprising an amino acid sequence that is at least 98%
identical to
SEQ ID NO: 4; wherein the first composition is administered to the human
subject for
priming the immune response, and the second composition is administered to the
human
subject one or more times for boosting the immune response.
[00294] Embodiment 51 is the vaccine combination of embodiment 50, wherein the
HBV polymerase antigen of the first and second composition does not have
reverse
transcriptase activity and RNase H activity.
[00295] Embodiment 52 is the vaccine combination of embodiment 50 or 51,
wherein
the I-113V polymerase antigen of the first and second composition is capable
of inducing
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an immune response in a mammal against at least two HBV genotypes, preferably
the
HBV polymerase antigen is capable of inducing a T cell response in a mammal
against at
least HBV genotypes B, C, and D, and more preferably the HBV polymerase
antigen is
capable of inducing a CD8 T cell response in a human subject against at least
HBV
genotypes A, B, C, and D.
[00296] Embodiment 53 is the vaccine combination of any one of embodiments 50-
52,
wherein the HBV polymerase antigen of the first and second composition
comprises the
amino acid sequence of SEQ ID NO: 4.
[00297] Embodiment 54 is the vaccine combination of any one of embodiments 50-
53,
further comprising a polynucleotide sequence encoding a signal sequence
operably linked
to the HBV polymerase antigen of the first and second composition.
[00298] Embodiment 55 is the vaccine combination of embodiment 54, wherein the
signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID
NO: 11,
preferably the signal sequence is encoded by the polynucleotide sequence of
SEQ ID NO:
5 or SEQ ID NO: 10.
[00299] Embodiment 56 is the vaccine combination of any one of embodiments 50-
55,
wherein the first and second polynucleotide sequence is at least 90% identical
to SEQ ID
NO: 3.
[00300] Embodiment 57 is the vaccine combination of embodiment 56, wherein the
first and second polynucleotide sequence comprises the polynucleotide sequence
of SEQ
ID NO: 3.
[00301] Embodiment 58 is the vaccine combination of any one of embodiments 50-
57,
wherein the adenovirus vector of the first composition further comprises a
third
polynucleotide sequence and the MVA vector of the second composition further
comprises a fourth polynucleotide sequence, wherein the third and fourth
polynucleotide
sequence encode a truncated HBV core antigen consisting of the amino acid
sequence of
SEQ ID NO: 2.
[00302] Embodiment 59 is the vaccine combination of embodiment 58, wherein the
third and fourth polynucleotide sequence is at least 90% identical to SEQ ID
NO: 1.
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[00303] Embodiment 60 is the vaccine combination of embodiment 59, wherein the
third and fourth polynucleotide sequence comprises the polynucleotide sequence
of SEQ
ID NO: 1.
[00304] Embodiment 61 is a vaccine combination comprising (a) a first
composition
comprising an immunologically effective amount of a Modified Vaccinia Ankara
(MVA)
vector comprising a first polynucleotide sequence encoding an EIBV polymerase
antigen
comprising an amino acid sequence that is at least 98% identical to SEQ ID
NO:4; and
(b) a second composition comprising an immunologically effective amount of an
adenovirus vector comprising a second polynucleotide sequence encoding an EIBV
polymerase antigen comprising an amino acid sequence that is at least 98%
identical to
SEQ ID NO:4; wherein the first composition is administered to the human
subject for
priming the immune response, and the second composition is administered to the
human
subject one or more times for boosting the immune response.
[00305] Embodiment 62 is the vaccine combination of embodiment 61, wherein the
EIBV polymerase antigen of the first and second composition does not have
reverse
transcriptase activity and RNase H activity.
[00306] Embodiment 63 is the vaccine combination of embodiment 61 or 62,
wherein
the EIBV polymerase antigen of the first and second composition is capable of
inducing
an immune response in a mammal against at least two EIBV genotypes, preferably
the
EIBV polymerase antigen is capable of inducing a T cell response in a mammal
against at
least EIBV genotypes B, C, and D, and more preferably the EIBV polymerase
antigen is
capable of inducing a CD8 T cell response in a human subject against at least
EIBV
genotypes A, B, C, and D.
[00307] Embodiment 64 is the vaccine combination of any one of embodiments 61-
63,
wherein the EIBV polymerase antigen of the first and second composition
comprises the
amino acid sequence of SEQ ID NO: 4.
[00308] Embodiment 65 is the vaccine combination of any one of embodiments 61-
64,
further comprising a polynucleotide sequence encoding a signal sequence
operably linked
to the EIBV polymerase antigen of the first and second composition.
[00309] Embodiment 66 is the vaccine combination of embodiment 65, wherein the
signal sequence comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID
NO: 11,
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preferably the signal sequence is encoded by the polynucleotide sequence of
SEQ ID NO:
or SEQ ID NO: 10.
[00310] Embodiment 67 is the vaccine combination of any one of embodiments 61-
66,
wherein the first and second polynucleotide sequence is at least 90% identical
to SEQ ID
5 NO: 3.
[00311] Embodiment 68 is the vaccine combination of embodiment 67, wherein the
first and second polynucleotide sequence comprises the polynucleotide sequence
of SEQ
ID NO: 3.
[00312] Embodiment 69 is the vaccine combination of any one of embodiments 61-
68,
wherein the MVA vector of the first composition further comprises a third
polynucleotide
sequence and the adenovirus vector of the second composition further comprises
a fourth
polynucleotide sequence, wherein the third and fourth polynucleotide sequence
encode a
truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO:
2.
[00313] Embodiment 70 is the vaccine combination of embodiment 69, wherein the
third and fourth polynucleotide sequence is at least 90% identical to SEQ ID
NO: 1.
[00314] Embodiment 71 is the vaccine combination of embodiment 70, wherein the
third and fourth polynucleotide sequence comprises the polynucleotide sequence
of SEQ
ID NO: 1.
[00315] Embodiment 72 is the vaccine combination of any one of embodiments 50-
71,
which is a kit.
[00316] Embodiment 73 is a method of enhancing an immune response in a human
subject, the method comprising (a) administering to the human subject a first
composition
comprising an immunologically effective amount of a first plasmid comprising a
first
non-naturally occurring nucleic acid molecule comprising a first
polynucleotide sequence
encoding an HBV polymerase antigen comprising an amino acid sequence that is
at least
98% identical to SEQ ID NO: 4 and a second plasmid comprising a second non-
naturally
occurring nucleic acid molecule comprising a second polynucleotide sequence
encoding a
truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO:
2; and
(b) administering to the human subject a second composition comprising an
immunologically effective amount of the MVA vector of any one of embodiments 1-
21;
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to thereby obtain an enhanced immune response against the HBV antigen in the
human
subject.
[00317] Embodiment 74 is the method of embodiment 73, wherein the HBV
polymerase antigen of the first composition does not have reverse
transcriptase activity
and RNase H activity.
[00318] Embodiment 75 is the method of embodiment 73 or 74, wherein the first
composition is for priming the immune response and the second composition is
for
boosting the immune response.
[00319] Embodiment 76 is the method of any one of embodiments 73-75, wherein
the
HBV polymerase antigen of the first composition is capable of inducing an
immune
response in the human subject against at least two HBV genotypes, preferably
the HBV
polymerase antigen is capable of inducing a T cell response in the human
subject against
at least HBV genotypes B, C, and D, and more preferably the HBV polymerase
antigen is
capable of inducing a CD8 T cell response in the human subject against at
least HBV
genotypes A, B, C, and D.
[00320] Embodiment 77 is the method of any one of embodiments 73-76, wherein
the
HBV polymerase antigen of the first composition comprises the amino acid
sequence of
SEQ ID NO: 4.
[00321] Embodiment 78 is the method of any one of embodiments 73-77, further
comprising a polynucleotide sequence encoding a signal sequence operably
linked to the
HBV polymerase antigen of the first composition.
[00322] Embodiment 79 is the method of embodiment 78, wherein the signal
sequence
comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, preferably
the
signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or
SEQ ID
NO: 10.
[00323] Embodiment 80 is the method of any one of embodiments 73-79, wherein
the
first polynucleotide sequence of the first composition is at least 90%
identical to SEQ ID
NO: 20.
[00324] Embodiment 81 is the method of embodiment 80, wherein the first
polynucleotide sequence of the first composition comprises the polynucleotide
sequence
of SEQ ID NO: 20.
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[00325] Embodiment 82 is the method of embodiments 73-81, wherein the second
polynucleotide sequence of the first composition is at least 90% identical to
SEQ ID NO:
18.
[00326] Embodiment 83 is the method of embodiment 82, wherein the second
polynucleotide sequence of the first composition comprises the polynucleotide
sequence
of SEQ ID NO: 18.
[00327] Embodiment 84 is the method of any one of embodiments 73-83, wherein
the
first and second polynucleotide sequences of the first composition further
comprise a
promoter sequence, optionally one or more additional regulatory sequences,
preferably
the promoter sequence comprises the polynucleotide sequence of SEQ ID NO: 7,
and the
additional regulatory sequence is selected from the group consisting of an
enhancer
sequence of SEQ ID NO: 8 or SEQ ID NO: 15, and a polyadenylation signal
sequence of
SEQ ID NO: 16.
[00328] Embodiment 85 is the method of any one of claims 73-84, wherein the
enhanced immune response comprises an enhanced antibody response against the
HBV
antigen in the human subject.
[00329] Embodiment 86 is the method of embodiment 85, wherein the enhanced
immune response comprises an enhanced CD8+ T cell response against the IIBV
antigen
in the human subject.
[00330] Embodiment 87 is the method of embodiment 85 or 86, wherein the
enhanced
immune response comprises an enhanced CD4+ T cell response against the IIBV
antigen
in the human subject.
[00331] Embodiment 88 is the method of any one of embodiments 73-87, wherein
step
(b) is conducted 1-12 weeks after step (a).
[00332] Embodiment 89 is the method of any one of embodiments 73-87, wherein
step
(b) is conducted 2-12 weeks after step (a).
[00333] Embodiment 90 is the method of any one of embodiments 73-87, wherein
step
(b) is conducted at least 1 week after step (a).
[00334] Embodiment 91 is the method of any one of embodiments 73-87, wherein
step
.. (b) is conducted at least 2 weeks after step (a).
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[00335] Embodiment 92 is a method of enhancing an immune response in a human
subject, the method comprising (a) administering to the human subject a first
composition
comprising an immunologically effective amount of the MVA vector of any one of
embodiments 1-21; and (b) administering to the human subject a second
composition
comprising an immunologically effective amount of a first plasmid comprising a
non-
naturally occurring nucleic acid molecule comprising a first polynucleotide
sequence
encoding an HBV polymerase antigen comprising an amino acid sequence that is
at least
98% identical to SEQ ID NO: 4 and a second plasmid comprising a non-naturally
occurring nucleic acid molecule comprising a second polynucleotide sequence
encoding a
truncated HBV core antigen consisting of the amino acid sequence of SEQ ID NO:
2; to
thereby obtain an enhanced immune response against the HBV antigen in the
human
subject.
[00336] Embodiment 93 is the method of embodiment 92, wherein the HBV
polymerase antigen of the second composition does not have reverse
transcriptase
activity and RNase H activity.
[00337] Embodiment 94 is the method of embodiment 92 or 93, wherein the first
composition is for priming the immune response and the second composition is
for
boosting the immune response.
[00338] Embodiment 95 is the method of any one of embodiments 92-94, wherein
the
HBV polymerase antigen of the second composition is capable of inducing an
immune
response in the human subject against at least two HBV genotypes, preferably
the HBV
polymerase antigen is capable of inducing a T cell response in the human
subject against
at least HBV genotypes B, C, and D, and more preferably the HBV polymerase
antigen is
capable of inducing a CD8 T cell response in the human subject against at
least HBV
genotypes A, B, C, and D.
[00339] Embodiment 96 is the method of any one of embodiments 92-95, wherein
the
HBV polymerase antigen of the second composition comprises the amino acid
sequence
of SEQ ID NO: 4.
[00340] Embodiment 97 is the method of any one of embodiments 92-96, further
comprising a polynucleotide sequence encoding a signal sequence operably
linked to the
HBV polymerase antigen of the second composition.
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[00341] Embodiment 98 is the method of embodiment 97, wherein the signal
sequence
comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, preferably
the
signal sequence is encoded by the polynucleotide sequence of SEQ ID NO: 5 or
SEQ ID
NO: 10.
[00342] Embodiment 99 is the method of any one of embodiments 92-98, wherein
the
first polynucleotide sequence of the second composition is at least 90%
identical to SEQ
ID NO: 20.
[00343] Embodiment 100 is the method of embodiment 99, wherein the first
polynucleotide sequence of the second composition comprises the polynucleotide
sequence of SEQ ID NO: 20.
[00344] Embodiment 101 is the method of embodiments 92-100, wherein the second
polynucleotide sequence of the second composition is at least 90% identical to
SEQ ID
NO: 18.
[00345] Embodiment 102 is the method of embodiment 101, wherein the second
polynucleotide sequence of the second composition comprises the polynucleotide
sequence of SEQ ID NO: 18.
[00346] Embodiment 103 is the method of any one of embodiments 92-102, wherein
the first and second polynucleotide sequences of the second composition
further comprise
a promoter sequence, optionally one or more additional regulatory sequences,
preferably
the promoter sequence comprises the polynucleotide sequence of SEQ ID NO:7 ,
and the
additional regulatory sequence is selected from the group consisting of an
enhancer
sequence of SEQ ID NO: 8 or SEQ ID NO: 15, and a polyadenylation signal
sequence of
SEQ ID NO: 16.
[00347] Embodiment 104 is the method of any one of claims 92-103, wherein the
enhanced immune response comprises an enhanced antibody response against the
HBV
antigen in the human subject.
[00348] Embodiment 105 is the method of embodiment 104, wherein the enhanced
immune response comprises an enhanced CD8+ T cell response against the HBV
antigen
in the human subject.
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[00349] Embodiment 106 is the method of embodiment 104 or 105, wherein the
enhanced immune response comprises an enhanced CD4+ T cell response against
the
HBV antigen in the human subject.
[00350] Embodiment 107 is the method of any one of embodiments 92-106, wherein
step (b) is conducted 1-12 weeks after step (a).
[00351] Embodiment 108 is the method of any one of embodiments 92-106, wherein
step (b) is conducted 2-12 weeks after step (a).
[00352] Embodiment 109 is the method of any one of embodiments 92-106, wherein
step (b) is conducted at least 1 week after step (a).
[00353] Embodiment 110 is the method of any one of embodiments 92-106, wherein
step (b) is conducted at least 2 weeks after step (a).
[00354] Embodiment 111 is the method of any one of embodiments 92-106, wherein
step (b) is conducted at least 4 weeks after step (a).
[00355] Embodiment 112 is the method of any one of embodiments 92-106, wherein
step (b) is conducted at least 8 weeks after step (a).
[00356] Embodiment 113 is the method of any one of embodiments 92-106, wherein
step (b) is conducted at least 12 weeks after step (a).
EXAMPLES
[00357] The following examples of the application are to further illustrate
the nature of
the application. It should be understood that the following examples do not
limit the
invention and the scope of the invention is to be determined by the appended
claims.
[00358] Example 1: Generation of HBV Core and Pol Antigen Sequences
[00359] T-cell epitopes on the hepatitis core protein are considered important
for
elimination of hepatitis B infection and hepatitis B viral proteins, such as
polymerase,
may serve to improve the breadth of the response. Thus, hepatitis B core and
polymerase
proteins were selected as antigens for the design of a therapeutic hepatitis B
virus (HBV)
vaccine.
[00360] Derivation of HBV Core and Polymerase Antigen Consensus Sequences
[00361] HBV pol and core antigen consensus sequences were generated based on
HBV
genotypes B, C, and D. Different HBV sequences were obtained from different
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and aligned separately for core and polymerase proteins. Original sequence
alignments
for all subtypes (A to H) were subsequently limited to HBV genotypes, B, C,
and D.
Consensus sequences were defined for each protein alignment in each subtype
separately
and in all joint BCD sequences. In variable alignment positions, the most
frequent amino
acid was used in the consensus sequence.
[00362] Optimization of HBV Core Antigen
[00363] The 1-1113V core antigen consensus sequence was optimized by making
two
deletions contained in the native viral protein. The first deletion was a
deletion of the N-
terminal extension of the core protein constituting the pre-core "zinc finger"
portion,
because reports in the literature have indicated that the virus utilizes this
sequence to
induce tolerance to viral proteins in infected individuals. The second
deletion was a
deletion of thirty-four amino acids corresponding to the C-terminal highly
positively
charged segment, which is required for pre-genome encapsidation and productive
viral
positive-strand DNA synthesis in the viral life-cycle.
[00364] Optimization of the HBV Pol Antigen
[00365] The HBV pol antigen consensus sequence was optimized by changing four
residues to remove reverse transcriptase and RNAseH enzymatic activities. In
particular,
the aspartate residues (D) were changed to asparagine residues (N) in the
"YXDD" motif
of the reverse transcriptase domain to eliminate any coordination function,
and thus
nucleotide/metal ion binding. Additionally, the first aspartate residue (D)
was changed to
an asparagine residue (N) and the first glutamate residue (E) was changed to a
glutamine
residue (A) in the "DEDD" motif of the RNAseH domain to eliminate Mg2+
coordination. Additionally, the sequence of the HBV pol antigen was codon
optimized to
scramble the internal open reading frames for the envelope proteins, including
the S
protein and versions of the S protein with the N-terminal extensions pre-S1
and pre-S2.
As a result, open reading frames for the envelope proteins (pre-S1, pre-S2,
and S protein)
and the X protein were removed.
[00366] Selection of Signal Peptide for Efficient Protein Secretion
[00367] Three different signal peptides introduced in frame at the N-terminus
of the
HBV core antigen were evaluated: (1) Ig heavy chain gamma signal peptide SPIgG
(BAA75024.1); (2) the Ig heavy chain epsilon signal peptide SPIgE
(AAB59424.1); and
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(3) the Cystatin S precursor signal peptide SPCS (NP 0018901.1). Signal
peptide
cleavage sites were optimized in silico for core fusion using the Signal P
prediction
program. Secretion efficiency was determined by analyzing core protein levels
in the
supernatant. Western blot analysis of core antigen secretion using the three
different
signal peptides fused at the N-terminus demonstrated that the Cystatin S
signal peptide
resulted in the most efficient protein secretion.
[00368] Example 2: Generation of Adenoviral Vectors Expressing a Fusion of
Truncated HBV Core Antigen with HBV Pol Antigen
[00369] An adenovirus vector was created to express a fusion protein of a
truncated
HBV core antigen and a HBV pol antigen from a single open reading frame.
Additional
configurations for the expression of the two proteins (e.g., the truncated HBV
core
antigen and the HBV pol antigen), e.g. using two separate expression
cassettes, or using a
2A-like sequence to separate the two sequences, can also be envisaged.
[00370] Design of expression cassettes for adenoviral vectors
[00371] The expression cassettes (diagrammed in Fig. 2A and Fig. 2B) comprise
the
CMV promoter (SEQ ID NO:7), an intron (SEQ ID NO:15) (a fragment derived from
the
human ApoAI gene - GenBank accession X01038 base pairs 295 ¨ 523, harboring
the
ApoAI second intron), followed by the optimized coding sequence ¨ either core
alone or
the core and polymerase fusion protein preceded by a human immunoglobulin
secretion
signal coding sequence (SEQ ID NO:10), and followed by the 5V40
polyadenylation
signal (SEQ ID NO:16).
[00372] A secretion signal was included because of past experience showing
improvement in the manufacturability of some adenoviral vectors harboring
secreted
transgenes, without influencing the elicited T-cell response (mouse
experiments).
[00373] The last two residues of the Core protein (VV) and the first two
residues of the
Polymerase protein (MP) if fused results in a junction sequence (VVMP) that is
present
on the human dopamine receptor protein (D3 isoform), along with flanking
homologies.
[00374] The interjection of an AGAG linker between the core and the polymerase
sequences eliminates this homology and returned no further hits in a Blast of
the human
proteome.
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[00375] Example 3: Generation of MVA Vectors Expressing a HBV Core Antigen
and a HBV Pol Antigen
[00376] An MVA vector has been designed to encode each of the HBV Core and Pol
coding sequences of the application. Each of the HBV Core and Pol coding
sequences
were inserted into an MVA vector at IGR44/45, each under the control of a
separate
promoter. Additional configurations for the expression of the two proteins,
e.g. using a
single expression cassette wherein the Core and Pol antigen comprise a fusion
protein, or
alternatively utilizing a 2A-like sequence to separate the two sequences, can
also be
envisaged. Further, additional and/or alternative insertions sites in the MVA
vector can
also be envisaged, e.g., inserting each of the HBV Core and Pol coding
sequences into
the same or different insertion sites.
[00377] Design of expression cassettes for MVA vectors
[00378] The expression cassettes (diagrammed in Fig. 2C) are comprised of the
Pr13.51ong promoter (SEQ ID NO: 25) adjacent to and directing expression of
the HBV
Core antigen and the PrHyb promoter (SEQ ID NO: 26) adjacent to and directing
expression of the HBV Pol antigen. The HBV core coding sequence comprises SEQ
ID
NO: 1 and the HBV Pol coding sequence comprises SEQ ID NO: 3. Each of SEQ ID
NOs: 1 and 3 were modified by eliminating negative cis-acting sites and by
adjusting the
GC content. Furthermore, each of SEQ ID NOs: 1 and 3 were codon optimized for
human codon usage without affecting the amino acid sequence. Each of SEQ ID
NOs: 1
and 3 comprises an additional early termination signals (TTTTTNT (SEQ ID NO:
28))
arranged adjacent to the stop codon.
[00379] Example 4: Immunogenicity of combinations of HBV adenoviral vectors
and HBV MVA vectors in mice
[00380] Materials and Methods
[00381] Vector design: Two adenoviral vectors expressing either Core alone (an
HBV
core antigen having the amino acid sequence of SEQ ID NO: 2), or Polymerase
(an HBV
pol antigen having the amino acid sequence of SEQ ID NO: 4) in addition to
Core as a
fusion protein expressed from a single open reading frame were used. For this,
sequences
were designed in silico to provide a consensus for the B, C and D genotypes of
the
hepatitis B virus. The expression cassettes comprise the CMV promoter, an
ApoAI
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intron, a human immunoglobulin secretion signal, followed by the coding
sequence ¨
either Core alone or the Core and Polymerase fusion protein and a SV40
polyadenylation
signal.
[00382] The recombinant MVA vector is comprised of poxvirus promoter Pr13.5
(SEQ
ID NO:25) linked to the core coding sequence (nucleotide sequence of SEQ ID
NO:1,
and polypeptide sequence of SEQ ID NO:2) and PrHyb (SEQ ID NO: 26) linked to a
nucleotide sequence encoding for polymerase (nucleotide sequence of SEQ ID NO:
3,
and polypeptide sequence of SEQ ID NO: 4), both followed by a transcription
termination sequence of TTTTTNT (SEQ ID NO: 28). The core coding sequence
comprises an N-terminal immunoglobulin secretion tag (SEQ ID NO: 11), and the
polymerase coding sequence comprises an N-terminal cystatin S signal sequence
(SEQ
ID NO:6). See, e.g., FIG. 2C.
[00383] In vivo immunogenicity study in mice: To evaluate the in vivo
immunogenicity
of the combination of HBV adenoviral vectors and HBV MVA, Fl mice (C57BL/6 x
Balb/C) were prime-boost immunized intramuscularly with different vector
combinations. These immunogenicity studies focused on determining the cellular
immune responses elicited by the HBV antigens Core and Polymerase.
[00384] Antigen-specific responses were analyzed and quantified by IFN-y
enzyme-
linked immunospot (ELISPOT) and intracellular cytokine production (TNF-alpha,
IL-2
and IFN-y) was detected by flow cytometry. In these assays, isolated
splenocytes of
immunized animals were incubated with peptide pools covering the Core protein,
the Pol
protein, or the small peptide leader and junction sequence (2 g/m1 of each
peptide). In
addition a MVA specific peptide (2 g/m1) was used. The pools consist of 15-mer
peptides that overlap by 11 residues matching the genotypes ABCD consensus
sequence
of the Core and Pol adenoviral vectors. The large 94 kDa HBV Pol protein was
split in
the middle into two peptide pools. In ELISPOT, IFN-y release by a single
antigen-
specific T-cell was visualized by appropriate antibodies and subsequent
chromogenic
detection as a colored spot on the microplate referred to as spot-forming cell
(SFC). In
ICS, the percentage of cytokine-releasing cells in a particular population
(CD3-positive,
CD4-positive or CD8-positive cells) was determined.
[00385] Results
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[00386] Evaluation of immunogenicity of I-113V adenoviral vectors and EIBV MVA
combinations in mice: The purpose of the study was to evaluate the immune
response
induced by the combination of I-113V adenoviral vectors and I-113V MVA after
IM delivery
into Fl mice. The administration to Fl mice was performed as summarized in
Table 1.
Animals received one EIBV adenoviral vector immunization followed by a I-113V
MVA
immunization 8 weeks later. Splenocytes were collected one week after the last
immunization.
[00387] Table 1: Mouse Immunization Experimental Design
Group N Prime R Dose Boost Route Dose Endpt
Day 0 (1) Day 56 TCID50 Day
1 4 Core Pol fusion + Core IM 10 - 63
2 4 Core Pol fusion + Core IM 109 - 63
3 4 Core Pol fusion + Core IM 101 - 63
4 4 Core Pol fusion + Core IM 108 MVA IM 8.9 x 107
63
5 4 Core Pol fusion + Core IM 109 MVA IM 8.9 x 107
63
6 4 Core Pol fusion + Core IM 101 MVA IM 8.9 x 107
63
7 4 Core Pol fusion IM 108 - 63
8 4 Core Pol fusion IM 109 - 63
9 4 Core Pol fusion IM 101 - 63
4 Core Pol fusion IM 108 MVA IM 8.9 x 107 63
11 4 Core Pol fusion IM 109 MVA IM 8.9 x 107
63
12 4 Core Pol fusion IM 101 MVA IM 8.9 x 107 63
13 4 Empty Vector IM 101 EV IM 63
IM: intramuscular; vp: viral particles; TCID50: 50% tissue culture infective
dose; MVA:
10 Modified Vaccinia Ankara
[00388] I-113V adenoviral vectors alone and in combination with I-113V MVA
vector,
gave rise to Pol specificT cell responses in mice. Low-level core responses
were induced
by Core pol fusion + core adenoviral vectors and these responses where
amplified by
boosting with I-113V MVA vector. The combination of Core pol fusion
adenovector and
I-113V MVA vector also induced core responses (FIG. 3).
[00389] Pol responses are primarily mediated by CD8(+) T cells, whereas Core
responses primarily involve CD4(+) T cells (FIGS. 4 and 5). The combination of
Core
pol fusion + core adenovectors and I-113V MVA also induced CD8(+) T cell
driven core
responses (FIG. 4).
[00390] Conclusion: The combination of I-113V adenoviral vectors and EIBV MVA
vectors gives rise to T cell responses against core and pol in Fl mice.
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[00391] Example 5: Immunogenicity of Combinations of HBV Adenoviral Vectors
and HBV MVA Vectors in Non-Human Primates (NHPs)
[00392] In vivo immunogenicity study in NEIPs: To evaluate the in vivo
immunogenicity of the combination of HBV adenoviral vectors and HBV MVA
vectors,
Mauritian cynomolgus monkeys were prime-boost-boost immunized intramuscularly
with different vector combinations. These immunogenicity studies focused on
determining the cellular immune responses elicited by the HBV core and
polymerase
antigens.
[00393] Antigen-specific responses were analyzed and quantified by IFN-y
enzyme-
linked immunospot (ELISPOT) and intracellular cytokine production (TNF-alpha,
IL-2
and IFN-y) was detected by flow cytometry. In these assays, PBMCs of immunized
animals were incubated with peptide pools covering the Core protein or the Pol
protein
(2[1g/m1 of each peptide). The pools consist of 15-mer peptides that overlap
by 11
residues matching the genotypes ABCD consensus sequence of the Core and Pol
adenoviral vectors. The large 94 kDa HBV Pol protein was split in the middle
into two
peptide pools. In ELISPOT, IFN-y release by a single antigen-specific T-cell
was
visualized by appropriate antibodies and subsequent chromogenic detection as a
colored
spot on the microplate referred to as spot-forming cell (SFC). In
intracellular cytokine
staining (ICS), the percentage of cytokine-releasing cells in a particular
population (CD3-
positive, CD4-positive or CD8-positive cells) was determined.
[00394] Results
[00395] Evaluation of immunogenicity of HBV adenoviral vectors and HBV MVA
vectors combinations in NEIPs: The purpose of the study was to evaluate the
immune
response induced by the combination of HBV adenoviral vectors and HBV MVA
vectors
after IM delivery into Mauritian cynomolgus monkeys. The administration to
NEIPs was
performed as summarized in Table 2. Animals received one HBV adenoviral vector
immunization followed by a HBV MVA vector immunization 8 weeks later and again
followed by a HBV adenoviral vector immunization 8 weeks later. PBMCs were
collected two weeks after each immunization.
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[00396] Table 2: NHP Immunization Experimental Design
Group N Prime R Dose Boost R Dose Boost Dose R
Day 0 per Day 56 TCID50 Day 112 (vp)
vector
(vp)
1 8 Core Pol ilVI 5*1010 MVA IM 5*108 Core Pol
1*10" ilVI
fusion fusion
2 8 Core Pol ilVI 5*1010 MVA IM 5*108 Core Pol
1*10" ilVI
fusion + Core fusion
IM: intramuscular; vp: viral particles; TCID50: 50% tissue culture infective
dose; MVA:
Modified Vaccinia Ankara
[00397] Core pol fusion adenoviral vector alone and Core pol fusion + core
adenoviral
vectors in combination with HBV MVA vector, gave rise to robust Pol and Core
specific
T cell responses in NHPs. Further boosting with a Core pol fusion adenoviral
vector did
not further increase the response (FIG. 6).
[00398] Core and Pol responses in NE1Ps are mediated by both CD4(+) and CD8(+)
T
cells. The combination of Core pol fusion adenovectors + core adenovectors
(used as a
prime) and HBV MVA (used as a boost) induced the highest CD4(+) Core specific
and
CD8(+) Pol specific T cell IFN-y responses (FIG. 7).
[00399] These results demonstrate that the combination of HBV adenoviral
vectors and
HBV MVA vectors gave rise to robust T cell responses against the core and pol
antigens
in NHPs.
[00400] It will be appreciated by those skilled in the art that changes could
be made to
the embodiments described above without departing from the broad inventive
concept
thereof. It is understood, therefore, that this invention is not limited to
the particular
embodiments disclosed, but it is intended to cover modifications within the
spirit and
scope of the present invention as defined by the present description.
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