Hepatitis b virus-specific t cell responses

Abstract

To provide a method for inducing an immune response against hepatitis B virus (HBV).SOLUTION: A method comprising administering to a subject a CMV vector expressing an HBV antigen in an amount effective to elicit a CD8+ T cell response to the HBV antigen, wherein the CMV vector does not express an active UL128, UL130, UL146, and UL147 protein or orthologs thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,756, filed June 7, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers R01 AI129703, R01 AI140888, and P51OD011092 awarded by the National Cancer Institute. The government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing in an ASCII text file submitted with this application (Name: 4153_011PC01_Seqlisting_ST25; Size: 4,288 bytes; Created June 5, 2020) are hereby incorporated by reference in their entirety. [Background technology]

[0004] Chronic hepatitis B virus infection (CHB) is a major global health concern, affecting 247 million people worldwide and resulting in 887,000 deaths annually. While an effective preventive vaccine is available, 10–15% of individuals do not adequately respond to vaccination and are not protected from hepatitis B virus (HBV) infection (Joint Committee on Vaccination and Immunization. Hepatitis B. In Immunization Against Infectious Disease, 3rd ed., p. 468.). CHB can lead to progressive liver dysfunction, cirrhosis, and in some cases, hepatocellular carcinoma. There are numerous treatment options for CHB, including PEG-IFN and reverse transcriptase inhibitors (Bhattacharya, D., and CL Thio. 2010. Review of hepatitis B therapeutics. Clin. Infect. Dis. 51:1201-1208.), but these treatments are rarely curative (Zhang et al., 2016. HBsAg seroclearance or seroconversion induced by PEG-interferon alpha and lamivudine or adefovir combination therapy in chronic hepatitis B treatment: a meta-analysis and systematic review. Rev Esp Enferm Dig 108:263-270.).

[0005] The development of cellular immunotherapeutic strategies for CHB is supported by the finding that 90–95% of adults with acute HBV infection demonstrate broad and highly functional HBV-specific T cell responses and subsequent definitive infection (Maini et al., 1999. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology 117:1386–1396; Phillips et al., 2010. CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions. J. Immunol. 184:287–295; Fisicaro et al., 2009. Early kinetics of innate and adaptive immune responses during hepatitis B virus infection. Gut 58:974–982). In contrast, patients progressing to CHB show narrowly focused, low frequencies of functionally exhausted HBV-specific T cell responses (Bertoletti, A., and C. Ferrari. 2016. Adaptive immunity in HBV infection. J. Hepatology 64:S71-S83; Rehermann, B., and A. Bertoletti. 2015. Immunological aspects of antiviral therapy of chronic hepatitis B virus and hepatitis C virus infections. Hepatology 61:712-721; Kurktschiev et al., 2014. Dysfunctional CD8+ T cells in hepatitis B and C are characterized by a lack of antigen-specific T-bet induction. J. Exp. Med. 211:2047-2059.).Therefore, many immunotherapeutic strategies currently under development focus on enhancing HBV-specific T cell immunity.

[0006] Immunotherapies currently under investigation are designed to harness the immune system to better target HBV-infected liver cells and include immune stimulation with pattern recognition receptor agonists, checkpoint inhibitor blockade, therapeutic vaccines, and adoptive T cell therapy (Gill, US, and PTF Kennedy. 2017. Current therapeutic approaches for HBV-infected patients. J. Hepatology 67:412-414.). A common hurdle facing HBV immunotherapy is T cell tolerance (Zong et al., 2019. Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice. Nature Communications 10:221; Kong et al., 2014. γδ T cells drive myeloid-derived suppressor cell-mediated CD8+ T cell exhaustion in hepatitis B virus-induced immunotolerance. J. Immunol. 193:1645-1653; Milich, DR. 2016. The Concept of Immune Tolerance in Chronic Hepatitis B Virus Infection Is Alive and Well. Gastroenterology 151:801-804). The initial trigger of immune tolerance, which distinguishes patients who successfully clear acute HBV viremia from those who do not, is not fully understood. However, it is likely that, in part, it is the result of the immune-permissive environment of the liver. Therefore, to successfully eliminate CHB by immunotherapy, T cell tolerance must be overcome. Unfortunately, to date, no immunotherapy has consistently achieved this goal, a reality exacerbated by the lack of physiologically relevant animal models of CHB.

[0007] T cell-based immunotherapy for CHB must result in sustained reversal of T cell exhaustion or sustained viral suppression. Given the difficulty of reversing established HBV-specific T cell dysfunction in CHB patients, the most effective way to enhance HBV-specific T cell immunity may be to generate or confer a completely unique set of T cell responses through therapeutic vaccination or adoptive T cell therapy. Unfortunately, the generation of such de novo responses is restricted by patient-specific HLA expression and HBV peptides present on the hepatocyte surface. In contrast, nonconventional MHC-Ib T cell restriction elements, which, if ubiquitously expressed, do not contribute to natural acute HBV-specific immune responses, can present HBV antigens on the hepatocyte surface and can be targeted to elicit a completely different set of T cell responses not typically seen in HBV infection. Therefore, there is an urgent and comprehensive need to develop treatments for HBV. Summary of the Invention

[0008] The present disclosure relates to a method of generating an immune response to hepatitis B virus (HBV) in a subject, comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to elicit a CD8+ T cell response to the HBV antigen, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins or their orthologues. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0009] The present disclosure also relates to a method of treating chronic HBV infection in a subject, comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to elicit a CD8+ T cell response to the HBV antigen, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins or their orthologues. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0010] The present disclosure also relates to a CMV vector expressing an HBV antigen for use in generating an immune response to HBV in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or an orthologue thereof. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0011] The present disclosure also relates to a CMV vector for use in treating chronic HBV infection in a subject expressing an HBV antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or an orthologue thereof. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0012] The present disclosure also relates to the use of a CMV vector expressing an HBV antigen in the manufacture of a medicament for use in generating an immune response to HBV in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or an orthologue thereof. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0013] The present disclosure also relates to the use of a CMV vector expressing an HBV antigen in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their orthologues. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0014] In one embodiment, the hepatitis B virus antigen is a hepatitis B virus core, envelope, surface, X, or polymerase antigen. In some embodiments, the hepatitis B virus antigen has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the hepatitis B virus antigen has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to TALRQAILCWGELMT (SEQ ID NO: 18).

[0015] In another embodiment, at least 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or its orthologue, or MHC-II or its orthologue. In another embodiment, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or its orthologue, or MHC-II or its orthologue. In another embodiment, less than 10% of the CD8+ T cells induced by the CMV vector are restricted by MHC-class 1a or its orthologue. In another embodiment, some MHC-E-restricted CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the vector. In some embodiments, MHC-E-restricted CD8+ T cells recognize an MHC-E supertope. In some embodiments, the MHC-E supertope has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the MHC-E supertope has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to TALRQAILCWGELMT (SEQ ID NO: 18).

[0016] The present disclosure also relates to a method for generating CD8+ T cells that recognize MHC-E-HBV antigen peptide complexes, the method comprising: (a) administering to a first subject a recombinant CMV vector that includes a nucleic acid that expresses an HBV antigen and does not express active UL128, UL130, UL146, and UL147 proteins or their orthologues, in an amount effective to generate a set of CD8+ T cells that recognize MHC-E / HBV antigen-derived peptide complexes; and (b) generating a first set of CD8+ T cells that recognize MHC-E / HBV antigen-derived peptide complexes. (c) identifying a CD8+ T cell from the set of CD8+ T cells; (c) isolating one or more CD8+ T cells from the second subject; and (d) transfecting one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize MHC-E / HBV antigen peptide complexes.

[0017] The present disclosure also relates to a method for generating CD8+ T cells that recognize MHC-E-HBV antigen peptide complexes, the method comprising: (a) isolating a first set of CD8+ T cells from a first subject who has been administered a recombinant CMV vector comprising a nucleic acid that expresses an HBV antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or orthologues thereof, in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / HBV antigen peptide complex; and (b) isolating a first set of CD8+ T cells from a first subject who has been administered a recombinant CMV vector comprising a nucleic acid that expresses an HBV antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein, or orthologues thereof, in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / HBV antigen peptide complex. (c) identifying a first CD8+ TCR that recognizes the peptide complex from a first set of CD8+ T cells; (c) isolating a second set of CD8+ T cells from a second subject; and (d) transfecting the second set of CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize the MHC-E / HBV antigen-peptide complex.

[0018] In one embodiment, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector. In another embodiment, the hepatitis B virus antigen is a hepatitis B virus core, envelope, surface, or polymerase antigen. In some embodiments, the hepatitis B virus antigen has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the hepatitis B virus antigen has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to TALRQAILCWGELMT (SEQ ID NO: 18).

[0019] In one embodiment, the first CD8+ T cells recognize a specific MHC-E supertope. In another embodiment, the second CD8+ T cells recognize a specific MHC-E supertope. In some embodiments, the MHC-E supertope is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18). In some embodiments, the MHC-E supertope is PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the MHC-E supertope is TALRQAILCWGELMT (SEQ ID NO: 18).

[0020] In another embodiment, the first CD8+ TCR is identified by DNA or RNA sequencing. In another embodiment, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

[0021] In one embodiment, the first subject is a human or non-human primate. In another embodiment, the subject is a non-human primate and the second subject is human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising a non-human primate CDR3α and a CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises a non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and a CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises a CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and a CDR3β of the first CD8+ TCR. In another embodiment, the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR. In another embodiment, the second CD8+ TCR is a chimeric CD8+ TCR. In another embodiment, the second CD8+ TCR comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

[0022] In one embodiment, administering the CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the CMV vector to the first subject. In another embodiment, the transfected CD8+ T cells are further administered to the second subject to treat or prevent HBV infection.

[0023] The present disclosure also relates to CD8+ T cells generated by the methods described herein.

[0024] The present disclosure also relates to methods of treating or preventing hepatitis B infection in a subject, the methods comprising administering to a subject in need thereof the CD8+ T cells described herein. The present disclosure also relates to CD8+ T cells for use in methods of treating or preventing hepatitis B infection in a subject in need thereof. The present disclosure also relates to the use of CD8+ T cells in the manufacture of a medicament in methods of treating or preventing hepatitis B infection in a subject in need thereof. [Brief explanation of the drawings]

[0025] [Figure 1A] FIG. 1 shows the frequency of HBV antigen-specific CD8+ T cell responses in four rhesus macaques (RM) inoculated with strain 68-1RhCMV expressing HBV core, surface, and polymerase antigens (RhCMV / HBV68-1). [Figure 1B] 1 shows CD8+ T cell responses to individual peptides of the HBV core antigen (HBcAg), each HBcAg 15-mer color-coded to indicate MHC restriction and represented by a box. [Figure 1C] FIG. 1 shows the response of CD8+ T cells isolated from RMs inoculated with either HLA-E or Mamu-E transfected K562 cells upon addition of HBcAg peptide. [Figure 2A]Figure 1 shows staining of MHC-transduced cell lines with the MHC-E-specific antibody 4D12. 4D12 staining was compared to a matching IgG-isotype control. Additionally, cells were stained with the pan-MHC-I-specific antibody W6 / 32. [Figure 2B] Figure 1 shows 4D12 staining of human and RM primary hepatocytes one day after liver perfusion and plating. Mouse IgG1 isotype was used to control for nonspecific antibody binding by primary hepatocytes. [Figure 2C] FIG. 2C shows quantification of the percentage of MHC-E+ primary hepatocytes from FIG. 2B. [Figure 2D] FIG. 1 shows co-staining of surface MHC-I, MHC-E, or MHC-II with intracellular HBcAg in human donor primary hepatocytes on day 4 post-HBV infection. [Figure 2E] FIG. 2D shows quantification of the percentage of HBV+ primary hepatocytes from FIG. 2D. [Figure 3A] Figure 1 shows the percentage of HBV-specific CD8+ T cells restricted by MHC-I, MHC-II, and MHC-E in splenocytes and CD8β-classified effectors from RM1 and RM2 when incubated with HBV-naive or HBV-infected PH from two unrelated RM donors (RM8 and RM9). Responding T cells were identified by staining for CD3, CD8, and IFN-γ. MHC restriction of responding CD8+ T cells was determined using the following MHC blocking agents: W6 / 32 antibody (pan MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II), or HLA-DR antibody (MHC-II). [Figure 3B]This figure shows the percentage of HBV-specific CD8+ T cells restricted by MHC-I, MHC-II, and MHC-E from splenocytes or CD8β-sorted effectors from RM1 and RM2 incubated with HBV-naive or HBV-infected primary hepatocytes from human donors (HD1 and HD2). Responding CD8+ T cells were identified by CD3, CD8, and IFN-γ. MHC restriction of responding CD8+ T cells was also determined with the following MHC blocking agents: W6 / 32 antibody (pan MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II), or HLA-DR antibody (MHC-II). [Figure 4] Figure 4A shows the conservation of MHC-E-binding supertopes of the HBV core antigen among HBV strains worldwide. 6,203 whole-genome HBV sequences spanning all known HBV genotypes were translated and amino acid aligned to Core7 (Figure 4A) and Core14 (Figure 4B). DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Terminology Unless otherwise specified, technical terms are used according to conventional usage.

[0027] All publications, patents, patent applications, internet sites, and accession numbers / database sequences (including both polynucleotide and polypeptide sequences) cited herein or listed in the application data sheets, including U.S. Provisional Patent Application No. 62 / 858,764, filed July 7, 2019, are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number / database sequence was specifically and individually indicated to be incorporated by reference as such.

[0028] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open and inclusive sense, i.e., "including, but not limited to." "Consisting of" shall mean excluding more than trace elements of other components and substantial method steps disclosed herein. The term "consisting essentially of" limits the claim to specific materials or steps, or those that do not materially affect the essential characteristics of the claimed invention. For example, a composition consisting essentially of the elements specified herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, and the like. Similarly, a protein consists essentially of a particular amino acid sequence if it includes additional amino acids that contribute up to 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., alter the activity of the protein by 50% or less). Embodiments defined by each transition phrase are included within the scope of the invention.

[0029] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0030] Antigen: As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein, that can induce an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that when administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein), the protein is capable of eliciting a humoral and / or cellular immune response against the protein.

[0031] Antigen-specific T cells: CD8 that recognize specific antigens + Lymphocytes or CD4 + Lymphocytes. Generally, antigen-specific T cells specifically bind to a particular antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0032] Administration: As used herein, the term "administration" means providing or giving an agent, such as a composition comprising an effective amount of a CMV vector containing an exogenous antigen, to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0033] Effective amount: As used herein, the term "effective amount" refers to an amount of an agent, such as a CMV vector containing a heterologous antigen or transfected CD8+ T cells that recognize an MHC-E / xenogenous antigen-derived peptide complex, an MHC-II / xenogenous antigen-derived peptide complex, or an MHC-I / xenogenous antigen-derived peptide complex, that is sufficient to reduce or eliminate the signs or symptoms of a condition or disease, or to produce a desired response, such as inducing an immune response to an antigen. In some examples, an "effective amount" is an amount that treats (including prevents) one or more symptoms and / or underlying causes of any of the disorders or diseases. An effective amount can be a therapeutically effective amount, including an amount that prevents the onset of one or more signs or symptoms of a particular disease or condition, such as one or more signs or symptoms associated with an infectious disease.

[0034] Heterologous antigen: As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. A heterologous antigen can be any antigen derived from HBV.

[0035] Immunogenic peptide: A peptide containing allele-specific motifs or other sequences, e.g., N-terminal repeats, which will bind to MHC molecules and induce a cytotoxic T lymphocyte ("CTL") response or a B cell response (e.g., antibody production) against the antigen from which the immunogenic peptide is derived.

[0036] In one embodiment, immunogenic peptides are identified using sequence motifs or other methods, such as neural nets or polynomial determination, known in the art. Typically, an algorithm is used to determine the "binding threshold" of a peptide and select peptides with scores that result in a high probability of binding with a certain affinity and are immunogenic. The algorithm is based on either the effect of a specific amino acid at a specific position on MHC binding, the effect of a specific amino acid at a specific position on antibody binding, or the effect of a specific substituent on binding in a motif-containing peptide. In the context of immunogenic peptides, a "conserved residue" is a residue that occurs significantly more frequently than would be expected by random distribution at a specific position in the peptide. In one embodiment, a conserved residue is a residue that may provide a contact point for the MHC structure with the immunogenic peptide.

[0037] Mutation: As used herein, the term "mutation" refers to any difference in a nucleic acid or polypeptide sequence from a normal, consensus, or "wild-type" sequence. A mutant is any protein or nucleic acid sequence containing a mutation. Furthermore, a cell or organism having a mutation may also be referred to as a mutant. Some types of coding sequence mutations include point mutations (individual nucleotide or amino acid differences), silent mutations (nucleotide differences that do not result in an amino acid change), deletions (differences in which one or more nucleotides or amino acids are missing, including deletions of the entire coding sequence of a gene or less), and frameshift mutations (differences in which deletion of a number of nucleotides not divisible by three results in a change in the amino acid sequence). Mutations that result in amino acid differences may also be referred to as amino acid substitution mutations. Amino acid substitution mutations can be described by the amino acid change at a specific position in the amino acid sequence compared to the wild-type.

[0038] Nucleotide sequence or nucleic acid sequence: The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence, including, but not limited to, messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid can be single-stranded or partially or completely double-stranded (duplex). A duplex nucleic acid can be a homoduplex or a heteroduplex.

[0039] Operably linked: As used herein, the term "operably linked" means that a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned so that it affects the second nucleic acid sequence. Operably linked DNA sequences may be contiguous, or they may be operably spaced apart.

[0040] Promoter: As used herein, the term "promoter" can refer to any of several nucleic acid control sequences that direct transcription of a nucleic acid. Typically, eukaryotic promoters contain necessary nucleic acid sequences near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element or some other specific DNA sequence recognized by one or more transcription factors. Expression by a promoter can be further regulated by enhancer or repressor elements. Numerous examples of promoters are available and well known to those skilled in the art. A nucleic acid containing a promoter operably linked to a nucleic acid sequence encoding a specific polypeptide can be called an expression vector.

[0041] Recombinant: As used herein, the term "recombinant" when referring to a nucleic acid or polypeptide refers to one having a sequence that is not naturally occurring or that is made by the artificial combination of two or more otherwise separated segments of sequence, such as a CMV vector containing a heterologous antigen. This artificial combination is often achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acid, for example, by genetic engineering techniques. Recombinant polypeptide can also refer to a polypeptide made using recombinant nucleic acid, including a recombinant nucleic acid (e.g., a nucleic acid encoding a polypeptide that forms a CMV vector containing a heterologous antigen) that has been introduced into a host organism that is not the natural source of the polypeptide.

[0042] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" is conventional. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1995, 19th Edition, by E.W. Martin, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. Generally, the nature of the carrier will vary depending on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as excipients. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

[0043] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Polynucleotides are composed of four bases: adenine, cytosine, guanine, and thymine / uracil (uracil is used in RNA). A coding sequence from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

[0044] Polypeptide: The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymers can be linear or branched, can comprise modified amino acids or amino acid analogs, and can be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as labeling or conjugation with a biologically active moiety.

[0045] Protein orthologs are typically characterized by having greater than 75% sequence identity across a full-length alignment with the amino acid sequence of a particular protein using ALIGN set to default parameters. Proteins with even greater similarity to the reference sequence will exhibit increasing percentage identities, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity, as assessed by this method. Additionally, sequence identity can be compared across the entire length of a particular domain of the disclosed peptide.

[0046] Sequence identity / similarity: As used herein, identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity, with the higher the percentage, the more identical the sequences. Sequence similarity is measured in terms of percentage identity or similarity (taking into account conservative amino acid substitutions), with the higher the percentage, the more similar the sequences are. Polypeptides or protein domains thereof that have a significant amount of sequence identity and function identically or similarly to each other (e.g., proteins that perform the same function in different species or mutant forms of the protein that do not change the function or magnitude of the protein) can be referred to as "homologs."

[0047] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv Appl Math 2, 482 (1981); Needleman and Wunsch, J Mol Biol 48, 443 (1970); Pearson and Lipman, Proc Natl Acad Sci USA 85, 2444 (1988); Higgins and Sharp, Gene 73, 237-244 (1988); Higgins and Sharp, CABIOS 5, 151-153 (1989); Corpet et al., Nuc Acids Res 16, 10881-10890 (1988); Huang et al., Computer App Biosci, 8, 155-165 (1992); and Pearson et al., Meth Mol Bio 24, 307-331 (1994). Furthermore, Altschul et al., J Mol Biol 215, 403-410 (1990) present a detailed discussion of sequence alignment methods and homology calculations.

[0048] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., (1990) supra) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Maryland 20894) and on the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Further information can be found on the NCBI website.

[0049] BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, the specified output file will present those homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences.

[0050] Once the alignment is complete, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches by the length or concatenated length of the sequence shown in the identified sequence (e.g., 100 consecutive nucleotides or amino acid residues from the sequence shown in the identified sequence), and then multiplying the resulting value by 100. For example, a nucleic acid sequence with 1166 matches when aligned with a test sequence having 1154 nucleotides is 75.0% identical to the test sequence (1166 ÷ 1554 × 100 = 75.0). The percent sequence identity value is rounded to the nearest whole number. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Length values are always integers. In another example, a target sequence containing a 20-nucleotide region that matches 20 consecutive nucleotides from a sequence identified as follows contains a region that shares 75% sequence identity with the identified sequence (i.e., 15÷20×100=75).

[0051] For comparison of amino acid sequences longer than approximately 30 amino acids, the Blast2 alignment function is used with the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, per-residue gap cost of 1). Homologs are typically characterized as having at least 70% sequence identity counted over a full-length alignment with the amino acid sequence using NCBI Basic Blast 2.0, gapped blastp, and databases such as the nr database, swissprot database, and patent sequence databases. Queries searched with the blastn program were filtered using DUST (Hancock and Armstrong, Comput Appl Biosci 10, 67-70 (1994)). Other programs use SEG. Additionally, manual alignments may be performed. Proteins with even higher similarity, as assessed by this method, will exhibit increasing percentage identities, such as at least approximately 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the protein.

[0052] When aligning short peptides (fewer than approximately 30 amino acids), alignments are performed using the Blast2 sequence function, employing the PAM30 matrix set to default parameters (9 penalties for open gaps, 1 for extension gaps). Proteins with even higher similarity to the reference sequence, as assessed by this method, will exhibit increasing percentage identities, such as at least approximately 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the protein. When comparing less than the entire sequence for sequence identity, homologs will typically have at least 75% sequence identity over a short window of 10–20 amino acids and may have at least 85%, 90%, 95%, or 98% sequence identity, depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described on the NCBI website.

[0053] One indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions, as described above. Nevertheless, nucleic acid sequences that do not show a high degree of identity may encode identical or similar (conserved) amino acid sequences due to the degeneracy of the genetic code. Alterations in nucleic acid sequences can be made using this degeneracy to generate multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity with the nucleic acid encoding the protein.

[0054] Subject: As used herein, the term "subject" refers to a living multi-cellular vertebrate organism, a category that includes both human and non-human mammals.

[0055] Supertope: As used herein, the term "supertope" or "supertope peptide" refers to an epitope or peptide that is recognized by T cells in greater than about 90% of the human population, regardless of MHC haplotype, i.e., in the presence or absence of a given MHC-I, MHC-II, or MHC-E allele.

[0056] Treatment: As used herein, the term "treatment" refers to an intervention that improves the signs or symptoms of a disease or pathological condition. As used herein, the terms "treatment," "treat," and "treating" in relation to a disease, pathological condition, or symptom also refer to any observable beneficial effect of treatment. A beneficial effect can be evidenced, for example, by a delay in the onset of clinical symptoms of a disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of a disease, a delay in the progression of a disease, a reduction in the number of recurrences of a disease, an improvement in the overall health or well-being of a subject, or other parameters known in the art specific to a particular disease. A preventive treatment is a treatment administered to a subject who does not show signs of a disease or who only shows early signs, with the aim of reducing the risk of developing a pathology. A therapeutic treatment is a treatment administered to a subject after signs and symptoms of a disease have occurred.

[0057] Vaccine: An immunogenic composition that can be administered to a mammal, such as a human, to confer immunity, such as active immunity, against a disease or other pathological condition. Vaccines can be used prophylactically or therapeutically. Thus, a vaccine can be used to reduce the likelihood of developing a disease (such as a tumor or pathological infection), or to reduce the severity of symptoms of a disease or condition, to limit the progression of a disease or condition (such as a tumor or pathological infection), or to limit the recurrence of a disease or condition (such as a tumor). In a specific embodiment, the vaccine is a replication-deficient CMV that expresses an HBV antigen.

[0058] Vector: A nucleic acid molecule of a specific sequence can be incorporated into a vector, and then the vector can be introduced into a host cell, thereby producing a transformed host cell. A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also contain other genetic elements known in the art, including one or more selectable marker genes and promoter elements that direct nucleic acid expression. A vector can be a viral vector, such as a CMV vector. A viral vector can be constructed from wild-type or attenuated viruses, including replication-deficient viruses.

[0059] II. Methods for Treating and Preventing Hepatitis Infection Disclosed herein are methods for treating or preventing hepatitis B virus infection, comprising administering to a subject an effective amount of at least one recombinant CMV vector comprising at least one heterologous antigen, wherein the at least one heterologous antigen comprises an antigen derived from hepatitis B virus.

[0060] Antigens derived from Hepatitis B virus can be derived from any part of the viral pathogen. Hepatitis B antigens include, but are not limited to, core protein, envelope protein, surface protein, X protein, and polymerase protein.

[0061] In some embodiments, the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins due to a mutation in the nucleic acid sequence encoding UL128, UL130, UL146, and UL147, or their homologs, or their orthologues (homologous genes of CMV that infect other species). The mutation may be any mutation that results in the lack of expression of the active protein. Such mutations may include point mutations, frameshift mutations, deletions of less than the entire protein-encoding sequence (truncating mutations), or deletions of the entire protein-encoding nucleic acid sequence, or any other mutation.

[0062] In a further example, the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein due to the presence of a nucleic acid sequence in the vector that includes an antisense or RNAi sequence (siRNA or miRNA) that inhibits expression of the UL128, UL130, UL146, and UL147 proteins. Any combination of mutations and / or antisense and / or RNAi can be used to generate CMV vectors that lack active UL128 activity, active UL130, active UL146, and active UL147.

[0063] In some embodiments, the CD8+ T cell response elicited by the vector is characterized by at least 10% of the CD8+ T cells being directed against an HBV epitope presented by MHC-E. Further examples of CD8+ T cells include at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, or at least 95% being restricted by MHC-E. In some embodiments, HBV-specific MHC-E-restricted CD8+ T cells recognize peptides shared by at least 90% of other subjects immunized with the vector. In some embodiments, the CD8+ T cells are directed against an HBV supertope presented by MHC-E. In some embodiments, the CD8+ T cell response elicited by the vector is characterized by at least 10% of the CD8+ T cells being directed against an epitope presented by MHC-II. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, or at least 95% of the CD8+ T cells are restricted by MHC-II. In some embodiments, the MHC-II-restricted HBV-specific CD8+ T cells recognize a peptide shared by at least 90% of other subjects immunized with the HBV vector. In some embodiments, the HBV-specific CD8+ T cells are directed against an HBV supertope presented by MHC-II.

[0064] In some embodiments, the method further comprises identifying a CD8+ T cell receptor on a CD8+ T cell induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-E / HBV antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes an MHC-E supertope of HBV.

[0065] In some embodiments, the method further comprises identifying a CD8+ T cell receptor on a CD8+ T cell induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-II / HBV antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes an MHC-II supertope of HBV.

[0066] Also disclosed herein is a method for generating CD8+ T cells that recognize MHC-E-HBV peptide complexes. The method includes administering to a first subject (or animal) a set of CD8+ T cells that recognize MHC-E / HBV peptide complexes in an amount effective to generate a CMV vector. In some embodiments, the CMV vector comprises a first nucleic acid sequence encoding at least one HBV antigen and does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or their orthologues. In some embodiments, the HBV antigen can be a hepatitis B virus core, envelope, surface, or polymerase antigen.

[0067] The method further includes identifying a first CD8+ T cell receptor from the set of CD8+ T cells, where the first CD8+ T cell receptor recognizes an MHC-E / HBV antigen-derived peptide complex. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, the method may further include transfecting one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ T cell receptor comprising the CDR3α and CDR3β of the first CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize an MHC-E / HBV antigen-derived peptide complex. The one or more CD8+ T cells for transfection with the expression vector may be isolated from the first subject or the second subject.

[0068] In some embodiments, the method further comprises identifying a CD8+ T cell receptor on a CD8+ T cell induced by the CMV vector, wherein the CD8+ T cell receptor recognizes an MHC-E / HBV antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises an HBV-specific CD8+ T cell receptor that recognizes an MHC-E supertope.

[0069] Also disclosed are transfected CD8+ T cells that recognize MHC-E / HBV peptide complexes, prepared by a method comprising: (1) administering to a first subject a recombinant CMV vector containing at least one HBV antigen in an amount effective to generate a set of CD8+ T cells that recognize MHC-E / HBV peptide complexes; (2) identifying from the set of CD8+ T cells a first CD8+ T cell receptor that recognizes the MHC-E / HBV antigen-derived peptide complex; (3) isolating one or more CD8+ T cells from the first or second subject; and (4) transfecting the one or more isolated CD8+ T cells from the first or second subject with an expression vector, thereby generating transfected T cells that recognize MHC-E / HBV peptide complexes. The CMV vector comprises a first nucleic acid sequence encoding at least one HBV antigen and does not express active UL128 protein, active UL130 protein, active UL146 protein, or active UL147 protein or their orthologs. The expression vector comprises a nucleic acid sequence encoding a second CD8+ T cell receptor and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ T cell receptor, wherein the second CD8+ T cell receptor comprises the CDR3α and CDR3β of the first CD8+ T cell receptor. The hepatitis B antigen can be a hepatitis B virus core, envelope, surface, or polymerase antigen.

[0070] In some embodiments, the CD8+ T cell response elicited by the CMV vector is characterized in that at least 10% of the CD8+ T cells are directed against an HBV epitope presented by MHC-II. In further examples, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 90%, at least 95%, or at least 95% of the CD8+ T cells are restricted by MHC-II. In some embodiments, the MHC-II-restricted CD8+ T cells recognize peptides shared by at least 90% of other subjects immunized with the HBV vector. In some embodiments, the CD8+ T cells are directed against an HBV supertope presented by MHC-II.

[0071] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from the CD8+ T cells induced by the CMV / HBV vector, wherein the CD8+ T cell receptor recognizes an MHC-II / HBV antigen-derived peptide complex. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises a CD8+ T cell receptor that recognizes an MHC-II-restricted HBV supertope.

[0072] When used as a vector, a human or animal CMV vector is inherently non-pathogenic in a selected subject, such as a human, hi some embodiments, the CMV vector is modified to be non-pathogenic (incapable of spreading within or from host to host) in a selected subject.

[0073] The HBV antigen can be any HBV protein or fragment thereof, as described herein.

[0074] The recombinant CMV vectors disclosed herein can be derived from a human cytomegalovirus vector, a rhesus cytomegalovirus vector, or a cynomolgus vector.

[0075] The recombinant CMV vectors disclosed herein can be used as immunogenic, immunological, or vaccine compositions comprising a recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. Immunological compositions containing a recombinant CMV virus or vector (or its expression product) induce a local or systemic immunological response. The response may be, but need not be, protective. Immunogenic compositions containing a recombinant CMV virus or vector (or its expression product) similarly induce a local or systemic immunological response that may be, but need not be, protective. Vaccine compositions induce a local or systemic protective response. Thus, the terms "immunological composition" and "immunogenic composition" include "vaccine composition" (as both terms can be protective compositions).

[0076] The recombinant CMV vectors disclosed herein can be used in methods of inducing an immunological response in a subject, comprising administering to the subject an immunogenic, immunological, or vaccine composition comprising the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.

[0077] The recombinant CMV vectors disclosed herein can be used in therapeutic compositions containing the recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent. The CMV vectors disclosed herein can be prepared by inserting DNA containing a sequence encoding an HBV antigen into an essential or non-essential region of the CMV genome. This method can further include deleting one or more regions from the CMV genome. This method can include in vivo recombination. Thus, this method can include transfecting CMV DNA into cells in a cell-compatible medium in the presence of donor DNA containing heterologous DNA flanked by DNA sequences homologous to a portion of the CMV genome, thereby introducing the heterologous DNA into the CMV genome, and then optionally recovering the modified CMV by in vivo recombination. This method can also include cleaving CMV DNA to obtain the cleaved CMV DNA, ligating the heterologous DNA to the cleaved CMV DNA to obtain a hybrid CMV-heterologous DNA, transfecting cells with the hybrid CMV-heterologous DNA, and then optionally recovering the CMV modified by the presence of HBV DNA. Because in vivo recombination is involved, this method also results in a plasmid containing donor DNA not naturally occurring in CMV that encodes a polypeptide foreign to CMV, where the donor DNA is within a segment of CMV DNA that is otherwise colinear with an essential or nonessential region of the CMV genome, such that DNA from essential or nonessential regions of CMV flanks the donor DNA. HBV DNA can be inserted into CMV to generate recombinant CMV in any orientation that results in stable integration and expression of that DNA.

[0078] The DNA encoding the HBV antigen in the recombinant CMV vector can also contain a promoter. The promoter can be derived from any source, such as a herpesvirus, including an endogenous cytomegalovirus (CMV) promoter, such as human CMV (HCMV), rhesus CMV (RhCMV), mouse, or other CMV promoter. The promoter can also be a non-viral promoter, such as the EF1α promoter. The promoter can be a truncated transcriptionally active promoter, which contains a region transactivated by a viral transactivation protein and a minimal promoter region of the full-length promoter from which the truncated transcriptionally active promoter is derived. The promoter can be composed of an assembly of DNA sequences corresponding to a minimal promoter and upstream regulatory sequences. A minimal promoter is composed of a CAP site plus an ATA box (a minimal sequence for the basal level of transcription; an unregulated level of transcription), and the "upstream regulatory sequence" is composed of multiple upstream elements and multiple enhancer sequences. Furthermore, the term "truncated" indicates that the full-length promoter is not present in its entirety, i.e., a portion of the full-length promoter has been removed. Truncated promoters can also be derived from herpesviruses such as MCMV or HCMV, e.g., HCMV-IE or MCMV-IE. Based on base pairs, the size can be reduced by up to 40%, or even up to 90%, from the full-length promoter. The promoter can also be a modified non-viral promoter. For HCMV promoters, see U.S. Patent Nos. 5,168,062 and 5,385,839. Transfecting cells with plasmid DNA to achieve cellular expression is described in Feigner et al. (1994), J. Biol. Chem. 269, 2550-2561. For direct injection of plasmid DNA as a simple and effective method for vaccination against various infectious diseases, see Science, 259:1745-49, 1993. Therefore, it is within the scope of this disclosure that vectors can be used by direct injection of vector DNA.

[0079] Also disclosed is an expression cassette that can be inserted into a recombinant virus or plasmid containing a truncated transcriptionally active promoter. The expression cassette can further contain a functional truncated polyadenylation signal, such as a truncated but functional SV40 polyadenylation signal. Considering that larger signals are provided in nature, it is quite surprising that the truncated polyadenylation signal is functional. The truncated polyadenylation signal addresses the insert size limitation problem of recombinant viruses such as CMV. The expression cassette can also contain HBV DNA related to the virus or system into which it is inserted, and the DNA can be the HBV DNA described herein.

[0080] For HBV antigens for use in vaccines or immunological compositions, see also Stedman's Medical Dictionary (24th ed., 1982), e.g., the definition of vaccine (for a list of antigens used in vaccine formulations). Such antigens or epitopes of interest from these antigens can be used. With regard to HBV antigens, one skilled in the art should be able to select antigens and their encoding DNA without undue experimentation from knowledge of the amino acids and corresponding DNA sequences of peptides or polypeptides, as well as from the properties of specific amino acids (e.g., size, charge, etc.) and the codon dictionary. Exemplary antigens include, but are not limited to, hepatitis B virus core, envelope, surface, X, or polymerase antigens.

[0081] One method for determining T epitopes of HBV antigens involves epitope mapping. Overlapping peptides of heterologous antigens are generated by oligopeptide synthesis. Individual peptides are then tested for their ability to bind to antibodies elicited by the native protein or to induce T or B cell activation. This approach has been particularly useful for mapping T cell epitopes, as T cells recognize short, linear peptides complexed with MHC molecules.

[0082] The immune response to HBV antigens generally occurs as follows: T cells recognize proteins only when they are cleaved into smaller peptides and presented in complexes called "major histocompatibility complexes (MHC)" located on the surface of other cells. There are two classes of MHC complexes, class I and class II, and each class is composed of many different alleles. Different species and individual subjects have different types of MHC complex alleles and are said to have different MHC types. One type of MHC class I molecule is called MHC-E (HLA-E in humans, Mamu-E in RM, and Qa-lb in mice).

[0083] It is known that the DNA containing the sequence encoding the HBV antigen may itself contain a promoter to drive expression in the CMV vector, or the DNA may be limited to the DNA encoding the heterologous antigen. This construct may be oriented relative to the endogenous CMV promoter so that it is operably linked to the promoter and expressed thereby. Furthermore, multiple copies of the DNA encoding the heterologous antigen, or the use of strong or early promoters, or early and late promoters, or any combination thereof, may be used to amplify or increase expression. Thus, the DNA encoding the heterologous antigen may be appropriately positioned relative to the CMV endogenous promoter, or the promoters may be translocated so that they are inserted into a different location along with the DNA encoding the heterologous antigen. Nucleic acids encoding two or more heterologous antigens can be packaged into a CMV vector.

[0084] Pharmaceutical and other compositions containing the disclosed CMV vectors are further disclosed. The aforementioned pharmaceutical and other compositions can be formulated for use in any administration procedure known in the art. The aforementioned pharmaceutical compositions can be administered via parenteral routes (intradermal, intramuscular, subcutaneous, intravenous, etc.). Administration can also be via mucosal routes, such as oral, nasal, genital, etc.

[0085] The disclosed pharmaceutical compositions can be prepared according to standard techniques well known to those skilled in the art. The compositions can be administered in dosages and by techniques well known to those skilled in the art, taking into account factors such as the race or species, age, sex, weight, and condition of the particular patient, as well as the route of administration. The compositions can be administered alone, or co-administered or sequentially with other CMV vectors, or other immunological, antigenic, vaccine, or therapeutic compositions. The other compositions can include purified native antigens or epitopes or antigens or epitopes expressed by recombinant CMV vectors or other vector systems, and are administered taking into account the above factors.

[0086] Examples of compositions include liquid preparations such as suspensions, syrups, or elixirs for administration via orifices, for example, oral, nasal, anal, or genital (e.g., vaginal) routes, and preparations for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable liquid administration), such as sterile suspensions or emulsions. In the above-mentioned compositions, the recombinant may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, or glucose.

[0087] Antigenic, immunological, or vaccine compositions typically contain an adjuvant and a certain amount of CMV vector or expression product to elicit the desired response. For human applications, alum (aluminum phosphate or aluminum hydroxide) is a typical adjuvant. Saponin and its purified components, Quil A, Freund's complete adjuvant, and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. Chemically defined preparations, such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described in Goodman-Snitkoff et al., J. Immunol. 147:410-415 (1991), encapsulation of proteins within proteoliposomes, such as those described in Miller et al., J. Exp. Med. 176:1739-1744 (1992), and encapsulation of proteins in lipid vesicles, such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH), can also be used.

[0088] The composition may be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or mucosal administration, including orifice administration, e.g., perilingual (e.g., oral), intragastric, buccal, anal, vaginal, etc. Similarly, the effective dosage and route of administration are determined by the nature of the composition, the nature of the expression product, the expression level if recombinant CMV is used directly, and known factors such as the species or race of the host, age, sex, weight, condition, and nature, as well as LD50 and other screening procedures that are known and do not require undue experimentation. The dosage of the expression product can range from a few micrograms to hundreds of micrograms, e.g., 5 μg to 500 μg. The CMV vector can be administered in any appropriate amount to achieve expression at these dosage levels. In a non-limiting example, the CMV vector is administered in an amount of at least 10 μg. 2 pfu, i.e., the CMV vector may be administered in at least this amount, or about 10 2 pfu~about 10 7The CMV vector may be administered in an amount ranging from 1 pfu to 10 pfu. Other suitable carriers or diluents may be water or buffered saline, with or without preservatives. The CMV vector may be lyophilized for resuspension at the time of administration, or may be in a dissolved state. "About" may mean within 1%, 5%, 10%, or 20% of the defined value.

[0089] It goes without saying that the proteins of the present disclosure and the nucleic acids encoding them may differ from the exact sequences specifically shown and described herein. Accordingly, the present disclosure contemplates deletions, additions, truncations, and substitutions to the sequences shown, so long as the sequences function according to the methods of the present disclosure. In this regard, substitutions are generally conservative in nature, i.e., substitutions that occur within a single family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, and histidine; (3) nonpolar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is reasonably expected that a single substitution of leucine with isoleucine or valine, or vice versa, a single substitution of aspartate with glutamate, or vice versa, a single substitution of threonine with serine, or vice versa, or similar conservative substitution of amino acids with structurally related amino acids, will not significantly affect biological activity.Therefore, proteins that have substantially the same amino acid sequence as the described proteins but have minor amino acid substitutions that do not substantially affect the immunogenicity of the proteins are included within the scope of the present disclosure.

[0090] The nucleotide sequences of the present disclosure may be codon-optimized, for example, the codons may be optimized for use in human cells. For example, any viral or bacterial sequence may be modified in this manner. Many viruses, including HIV and other lentiviruses, use many rare codons, and by modifying these codons to correspond to codons commonly used in the desired target, enhanced expression of HBV antigens can be achieved, as described by Andre et al., J. Virol. 72:1497-1503, 1998.

[0091] Nucleotide sequences encoding functionally and / or antigenically equivalent variants and derivatives of the CMV vector and the glycoproteins contained therein are contemplated. These functionally equivalent variants, derivatives, and fragments exhibit the ability to retain antigenic activity. For example, DNA sequence changes that do not alter the encoded amino acid sequence, as well as changes resulting in conservative substitutions of amino acid residues, deletions or additions of one or a few amino acids, and substitutions of amino acid residues with amino acid analogs, do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions include glycine / alanine, valine / isoleucine / leucine, asparagine / glutamine, aspartic acid / glutamic acid, serine / threonine / methionine, lysine / arginine, and phenylalanine / tyrosine / tryptophan. In one embodiment, the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.

[0092] Sequence identity or homology is determined by comparing sequences when aligned to maximize overlap and identity while minimizing sequence gaps.In particular, sequence identity can be determined using any of several mathematical algorithms.A non-limiting example of the mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, Proc.Natl.Acad.Sci.USA 1990;87:2264-2268, modified as in Karlin and Altschul, Proc.Natl.Acad.Sci.USA 1993;90:5873-5877.

[0093] Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS 1988;4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying and aligning regions of local sequence similarity is the FASTA algorithm described in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988;85:2444-2448.

[0094] Conveniently used with the present disclosure is WU-BLAST (Washington University BLAST) version 2.0 software. A WU-BLAST version 2.0 executable program for several UNIX platforms can be downloaded from ftp: / / blast.wustl.edu / blast / executables. This program is based on WU-BLAST version 1.4, which is based on the public domain NCBI-BLAST version 1.4 in tumors. (Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266:460-480; Altschul et al., Journal of Molecular Biology 1990;215:403-410; Gish and States, 1993; Nature Genetics 3:266-272; Karlin and Altschul, 1993; Proc. Natl. Acad. Sci. USA 90:5873-5877, all of which are incorporated herein by reference).

[0095] The various recombinant nucleotide sequences and antibodies and / or antigens of the present disclosure are produced using standard recombinant DNA and cloning techniques. Such techniques are well known to those skilled in the art. See, for example, "Molecular Cloning: A Laboratory Manual," 2nd Edition (Sambrook et al., 1989).

[0096] The nucleotide sequences of the present disclosure can be inserted into a "vector." The term "vector" is widely used and understood by those skilled in the art, and "vector" is used herein in accordance with its meaning to those skilled in the art. For example, a "vector" is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of a nucleic acid molecule from one environment to another, or that allows or facilitates the manipulation of a nucleic acid molecule.

[0097] Any vector that allows the virus of the present disclosure to be expressed can be used according to the present disclosure.In certain embodiments, the disclosed virus can be used in vitro (for example, using a cell-free expression system) and / or in vitro grown cultured cells to produce the encoded heterologous antigen (for example, pathogen-specific antigen, HIV antigen, Hepatitis B antigen, and antibody), and then can be used for various applications, such as the production of proteinaceous vaccines.For the aforementioned applications, any vector that allows the virus to be expressed in vitro and / or in cultured cells can be used.

[0098] To express the disclosed heterologous antigens, the protein coding sequence of the heterologous antigen must be "operably linked" to a regulatory sequence or nucleic acid control sequence that directs the transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be "operably linked" when they are covalently linked in a manner that places the expression, transcription, and / or translation of the coding sequence under the influence or control of the nucleic acid control sequence. A "nucleic acid control sequence" can be any nucleic acid element, including, but not limited to, a promoter, enhancer, IRES, intron, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence operably linked thereto. As used herein, the term "promoter" refers to a group of transcriptional control modules centered around the initiation site of RNA polymerase II that, when operably linked to a protein coding sequence of the present disclosure, directs the expression of the encoded protein. Expression of the transgenes of the present disclosure can be under the control of a constitutive promoter or an inducible promoter that initiates transcription only upon exposure to some specific external stimulus, including, but not limited to, an antibiotic such as tetracycline, a hormone such as ecdysone, or a heavy metal. The promoter can also be specific to a particular cell type, tissue or organ.Many suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer can be used for the expression of the transgene of the present disclosure.For example, suitable promoters and / or enhancers can be selected from the Eukaryotic Promoter Database (EPDB).

[0099] The vectors used in accordance with the present disclosure may include appropriate gene regulatory regions, such as promoters or enhancers, so that the antigens of the present disclosure may be expressed.

[0100] The CMV vectors described herein may contain mutations that can prevent host-to-host transmission, thereby preventing the virus from infecting immunocompromised subjects or other subjects who may face complications as a result of CMV infection. The CMV vectors described herein may also contain mutations that result in the presentation of immunodominant and non-immunodominant epitopes and non-canonical MHC restriction. However, the mutations in the CMV vectors described herein do not affect the vector's ability to reinfect subjects previously infected with CMV. The aforementioned CMV mutations are described, for example, in U.S. Patent Application Publication Nos. 2013-013676S, 2010-0142S23, 2014-014103S, and PCT Application Publication No. WO2014 / 13S209, all of which are incorporated herein by reference.

[0101] The disclosed CMV vectors can be administered in vivo, e.g., to generate immunogenic responses, including CD8+ immune responses, including immune responses characterized by a high percentage of CD8+ T cell responses restricted by MHC-E or MHC-II (or homologs or orthologs thereof). For example, in some instances, it may be desirable to use the disclosed CMV vectors in laboratory animals, such as rhesus monkeys, to conduct preclinical testing of immunogenic compositions and vaccines using RhCMV. In other instances, it will be desirable to use the disclosed CMV vectors in human subjects, such as in clinical trials and for actual clinical use of immunogenic compositions using HCMV.

[0102] For such in vivo applications, the disclosed CMV vectors are administered as a component of an immunogenic composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the immunogenic compositions of the present disclosure are useful for stimulating an immune response against heterologous antigens, including pathogen-specific antigens, and can be used as one or more components of prophylactic or therapeutic vaccines against pathogen-specific antigens for the prevention, amelioration, or treatment of pathogenic infections. The nucleic acids and vectors of the present disclosure are particularly useful for providing genetic vaccines, i.e., vaccines for delivering nucleic acids encoding the disclosed antigens to a subject, such as a human, so that the antigen is expressed in the subject and thereby elicits an immune response.

[0103] Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for a particular subject and immunogenic composition. Accordingly, the immunogen may be administered to the subject once or multiple times. Preferably, a set time interval is allowed between individual administrations of the immunogenic composition. This interval varies from subject to subject, but typically ranges from 10 days to several weeks, often 2, 4, 6, or 8 weeks. For humans, the interval is typically 2 to 6 weeks. In particularly advantageous embodiments of the present disclosure, the intervals are longer, preferably about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70 weeks. The immunization regimen typically includes one to six administrations of the immunogenic composition, but may include as few as one, two, or four administrations. The method of inducing an immune response may also include administering an adjuvant with the immunogen. In some cases, the initial immunization protocol may be supplemented with booster immunizations annually, twice a year, or at longer intervals (5-10 years). The present method also includes various prime-boost regimens. These methods involve one or more priming immunizations followed by one or more boosting immunizations. The actual immunogenic composition can be the same or different for each immunization, and the type of immunogenic composition (e.g., including a protein or expression vector), route, and formulation of the immunogen can also vary. For example, if an expression vector is used for the priming and boosting steps, it can be of the same type or different types (e.g., DNA or bacterial or viral expression vectors). One useful prime-boost regimen involves two priming immunizations spaced four weeks apart, followed by two boosting immunizations four and eight weeks after the final priming immunization.It will also be apparent to those skilled in the art that there are several permutations and combinations encompassed using the DNA, bacterial, and viral expression vectors of the present disclosure to achieve priming and boosting regimens. CMV vectors can be used repeatedly, expressing different antigens derived from different pathogens. [Example]

[0104] Example 1: RHCMV / HBV-inoculated rhesus macaques mount MHC-E-restricted CD8+ T cell responses to HBV antigens RhCMV68-1 strain vectors engineered to express antigenic targets elicit broad effector memory CD8+ T cell responses restricted by either the nonclassical molecule MHC-E, the monomorphic MHC class Ib molecule normally involved in NK cell signaling, or MHC-II. RhCMV vaccine vectors expressing simian immunodeficiency virus (SIV) antigens protected 50% of RMs from repeated low-dose intrarectal and intravaginal challenge with the highly pathogenic strain SIVmac239.

[0105] We investigated whether RhCMV / HBV-induced non-conventional restricted CD8+ T cell responses recognize HBV-infected hepatocytes. Targeting these unique CD8+ T cell-restrictive molecules may constitute a new paradigm for the treatment of HBV infection, and given the high degree of conservation of MHC-E between humans and monkeys, it could theoretically be universally applicable to all patients (Wu et al., 2018. The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques. J. Immunol. 200:49-60).

[0106] Four rhesus macaques (RM) were inoculated with the RhCMV68-1 strain expressing HBV genotype D serotype ayw core, surface, and / or polymerase antigens. Genotype D, serotype ayw HBV core, polymerase, and S Ag gene fragments were isolated by PCR from previously described plasmids (Frank Chisari, Scripps Research Institute). The N-terminal 333 amino acids of the polymerase from the plasmid pCDNA3-POL / ENV (Kakimi, K. et al., 2002. Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections. J. Virol. 76:8609-8620) were tagged with a C-terminal HA-epitope and fused by PCR-mediated mutagenesis to the C-terminal 228 amino acids of S Ag from the plasmid pCMV-S2 / S (Michel, M.L. et al., 1995. DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B virus infection in humans. Proc. Natl. Acad. Sci. USA 92:5307-5311.) to generate the fusion S / PolN (left forward primer: The left reverse primer was 59-CATCGAGCTAGCACCATGGAGAACATCACATCAGG-39 (SEQ ID NO: 1), the right forward primer was 59-GTGTTGATAGGATAGGGGAATGTATACCCAAAGAC-39 (SEQ ID NO: 2), the right reverse primer was 59-GTCTTTGGGTATACATTCCCCTATCCTATCAACAC-39 (SEQ ID NO: 3), the right reverse primer was 59-GGAATCGTCGACTCAAGCGTAATCTGGAACATCGTATGGGTAAAGATTGACGATAAGGGAGAGGCAG-39 (SEQ ID NO: 4). The final PCR product was blunt-end cloned into pJet vector (Thermo Fisher Scientific) or pORI as a template for bacterial artificial chromosome (BAC) recombineering to assess expression. The C-terminal 416 amino acids of the polymerase obtained from the plasmid pCDNA3-POL / ENV (Kakimi, K. et al., 2002. Immunogenicity and tolerogenicity of hepatitis B virus structural and nonstructural proteins: implications for immunotherapy of persistent viral infections. J. Virol. 76:8609-8620) were tagged with an HA epitope by PCR-mediated mutagenesis and inserted into pORI (forward primer: 59-GTGGTACCCTCGAGGATTGGGGACCCTGCGCTGAACATGGAG-39 (SEQ ID NO: 5), reverse primer: 59-TCAGTCGACCTAAGCGTAATCTGGAACATCGTATGGGTAC-39 (SEQ ID NO: 6)). The gene encoding Core was PCR amplified from the plasmid pCDNA-CORE ( 22 ) and inserted into pORI (forward primer: 59-CTGCTAGCATGGACATTGACCCTTATAAAGAATTTGG-39 (SEQ ID NO: 7), reverse primer: 59-CTAGGTACCACATTGAGATTCCCGAGATTGAG-39 (SEQ ID NO: 8)).The C-terminal polymerase fragment was then inserted downstream of Core using KpnI and SalI to generate a C-terminal fusion protein of HBV core and polymerase (Core / PolC). The KpnI site adds a 2-amino acid (GT) linker between the two proteins. To generate 68-1RhCMV / Core / PolC and 68-1RhCMV / S / PolN, the Rh110 gene encoding pp71 in 68-1RhCMVBAC (Chang, W.L.W. et al., 2003. Cloning of the full-length rhesus cytomegalovirus genome as an infectious and self-excisable bacterial artificial chromosome for analysis of virus pathogenesis. J. Virol. 77:5073-5083) was replaced using the modified galactokinase (galK) selection system, a two-step method that allows DNA modification without introducing unwanted heterologous sequences (Warming, S. et al., 2005. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33:e36). It has recently been demonstrated that substitutions at Rh110 can be used to attenuate the 68-1RhCMV vector while still eliciting an Ag response in healthy individuals (Marshall, EE et al., 2019. Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity. Sci. Transl. Med. 11:eaaw2603 10.1126 / scitranslmed.aaw2603).

[0107] To eliminate Rh110, competent SW105 bacteria containing the 68-1RhCMV BAC were electroporated with a PCR product containing a galK / kanamycin cassette with 50-bp of flanking homology to Rh110. Bacteria were plated on kanamycin / chloramphenicol Luria Bertani agar at 30°C for positive selection. To replace the galK / kanamycin cassette with an HBV fusion gene, a PCR product containing an HBVS-PolN fusion or an HBVCore-PolC fusion with the same flanking homology to Rh110 was electroporated, and bacteria were plated on 2-deoxy-galacto(canine)chloramphenicol minimal medium plates with glycerol as the carbon source for negative selection. PCR primers for homologous recombination were as follows: Rh110 S / PolN forward: 59-GATCACGTCATTGACACCGGCCTCCCACCAGCTCTCACATTCTCCGCATCACCATGGAGAACATCACATCAGGAT-39 (SEQ ID NO: 9), Rh110 S / PolN reverse: 59-CAAAATATTATTACATGGTACGCAATTTATTGTCTATTTTCGTTATTTGTTTATTCAAGCGTAATCTGGAACATCGTAT-39 (SEQ ID NO: 10), and Rh110 Core / PolC forward: 59-GATCACGTCATTGACACCGGCCTCCCACCAGCTCTCACATTCTCCGCATCACCATGGACATTGACCCTTATAAAGAAT-39 (SEQ ID NO: 11). Core / PolC reverse: 59-CAAAATATTATTACATGGTACGCAATTTATTGTCTATTTTCGTTATTTGTTTATCTAAGCGTAATCTGGAACATCGTAT-39 (SEQ ID NO: 12).To generate 68-1RhCMV / Core, the HBV core gene was amplified from pCDNA-CORE and an N-terminal FLAG tag was introduced by PCR (forward primer: 59-CTGCTAGCATGGATTACAAGGATGACAAGGACATCGACCCTTATAAAGAATTTGG-39 (SEQ ID NO: 13), reverse primer: 59-CTAGTCGACACATTGAGATTCCCGAGATTGAG-39) (SEQ ID NO: 14). The amplified product was cloned into pORI downstream of the EF1a promoter. This expression cassette, along with a Kan resistance cassette flanked by flippase recognition target sites, was inserted into the Rh211 region of 68-1RhCMV by homologous recombination using primers with 50 bp of homology to the Rh211 region (forward primer: 59-GGGAAATCACGTCATCAGGCTGGGTAGTCAACATGGGCATACGAAACTTGCCCGAATAGATGCTCTCACTTAACGGCTGACATG-39 (SEQ ID NO: 15); reverse primer: 59-CCAGAATGTGCTCTACTTTTTGGCCAGCGGGTTGGATGATTTCGCGCGTCATGGACTGCTTCACTGTAGCTTAGTACGTTAAAC-39 (SEQ ID NO: 16)). For in vivo recombination and Kan resistance selection of recombinants, the PCR fragment was electroporated into EL250 bacteria containing the RhCMV 68-1 BAC. The Kan resistance cassette was removed by temperature-induced flippase recombination. The resulting BAC was analyzed by restriction digestion, PCR analysis of the recombination site, and next-generation sequencing on an Illumina MiSeq sequencer. Sequence analysis revealed that two point mutations in S / PolNthat were introduced during PCR amplification, resulting in amino acid exchanges A118T and T125M in the S Ag.BAC DNA was purified using alkaline lysis, phenol / chloroform extraction, and isopropanol precipitation. Virus was reconstituted using Lipofectamine 2000 (according to the manufacturer's protocol) in telomerized pp71-expressing rhesus fibroblasts or primary rhesus fibroblasts by transfection of BAC DNA (Warming, S. et al., 2005. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33:e36). Expression of HBV Ags was confirmed by infecting telomerized RM fibroblasts with 68-1 RhCMV / Surface / PolN or RhCMV / Core / PolC. Cells were harvested upon sufficient cytopathic effect and lysed in SD sample buffer. 293T cells transfected (Lipofectamine 2000) with a pORI expression plasmid containing HA-tagged HBV proteins served as a positive control. After electrophoretic separation, immunoblotting was performed using anti-HAABMMS-101P (Covance MMS).

[0108] Two RMs (RM1, RM2) were vaccinated with both the 68-1RhCMV / Surface / PolN and 68-1RhCMV / Core / PolC vectors, and two additional RMs (RM3, RM4) were vaccinated with a 68-1RhCMV-based vector expressing HBV Core under the EF1a promoter. CD8+ T cell responses to each of the antigens were monitored longitudinally by ICS using pools of overlapping 15-mer peptides corresponding to each antigen. Long-term CD8+ T cell responses to these antigens were observed in the blood of vaccinated RMs (Figure 1A).

[0109] The MHC restriction of HBV core (HBcAg)-specific CD8+ T cell responses in these animals was characterized by intracellular cytokine staining with reagents that specifically inhibit presentation by MHC-I, MHC-II, and MHC-E as previously described (Hansen et al., 2016). Broadly targeted CD8 + T cell responses restricted by major histocompatibility complex E. Science 351:714-720; Hansen et al., 2013. Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science 340:1237874-1237874.) Similar to RhCMV strain 68-1 expressing SIV or Mycobacterium tuberculosis antigens, we found that the RhCMV strain 68-1 / HBV vector elicited HBV-specific, MHC-E- and MHC-II-restricted CD8+ T cell responses targeting a broad array of HBcAg peptides (Figure 1B).

[0110] To further confirm that MHC-E restriction of RhCMV / HBV resulted in the observed CD8+ T cell responses, splenocytes from RhCMV / HBV-vaccinated RMs were stimulated with K562 cells (MHC-Null) transduced to express a single human (HLA) or rhesus macaque (Mamu) MHC-E allele and pulsed with one of three individual HBcAg 15-mer peptides identified as MHC-E-restricted by blocking in Figure 1B. Only cells expressing MHC-E could present the HBcAg 15-mer to these CD8+ T cells. HBcAg-specific CD8+ T cells recognized their cognate antigen presented in the context of both HLA-E and Mamu-E (Figure 1C). These results demonstrate the presence of MHC-E-restricted HBV-specific CD8+ T cells in RhCMV / HBV-vaccinated RMs and further support the presence of highly functional conserved primate MHC-E molecules.

[0111] Example 2: HBV-infected primary hepatocytes express MHC-E in vitro Next, we determined whether primary hepatocytes express MHC-E. Primary hepatocytes (PH) were isolated from three unrelated RMs and three unrelated human donors (HDs). To isolate RM primary hepatocytes, a single lobe of the RM liver was perfused with 200 mL of preperfusion medium (0.5 mM EGTA (Bio-World, cat#: 40120128-1), 10 IU / ml heparin (Fresenius Kabi, cat#: C504730), HBSS with calcium and magnesium (Fisher Scientific, cat#: 24-020-117)), followed by 200 mL of HBSS without calcium and magnesium (Fisher Scientific, cat#: SH3003103) to remove residual EGTA. Next, 100 ml of collagenase medium (DMEM / F12 (Gibco, cat#: 11320-082), 1 mM calcium chloride (Sigma-Aldrich, cat#: C5670-100G), 20 mM HEPES (HyClone, cat#: SH30237.01), 1 mg / ml collagenase IV (Sigma-Aldrich, cat#: C9722-50MG)) preheated to 42°C was perfused into the liver lobe and discarded. After this, 150 ml of collagenase medium was recirculated through the liver lobe at a rate of 75–150 ml / min at 42°C for 30 min–1 h, depending on the size of the liver lobe. After collagenase perfusion, the liver was minced with a scalpel, washed with the remaining collagenase medium, and the medium was filtered through a tea strainer. The PH were washed three times at room temperature in wash medium (DMEM / F12, 2% bovine growth serum (HyClone, cat#: SH3054103), 23 mM HEPES buffer, 0.6 mg / ml glucose, 2 mM L-glutamine (HyClone, cat#: SH3003401), 1x antibiotic / antimycotic (HyClone, cat#: SV3007901), and 0.1 mg / ml gentamicin (Life Technologies, cat#: 15750-060)) with centrifugation at 50 x g for 3 min between each wash. Before the third wash spin, the PH were passed through a 70 µM filter to ensure a single-cell suspension.The PH cells were then suspended in 20 ml of 36% isotonic Percoll (GE Healthcare, cat#: 17-0891-01) in a 50 ml Erlenmeyer flask using PH medium as the diluent (DMEM / F12, 10% bovine growth serum, 23 mM HEPES buffer, 0.6 mg / ml glucose, 2 mM L-glutamine, 1x antibiotic / antimycotic, and 0.1 mg / ml gentamicin) and centrifuged at 200 x g for 7 minutes. The purified PH pellet was then resuspended in room temperature PH medium and counted. Hepatocyte collagen-loaded plates were prepared using 0.2 mg / mL collagen R (Serva, cat#: 47254) in 0.01% acetic acid. The plates were left on the plate for at least 20 minutes before being washed with 1 ml of HBSS, followed immediately by 2 x 10 centrifugation. 5 The cells were plated in 12-well plates at pH 1 / well. The plates were placed at 37°C with 5% CO2. The next day, the wells were washed twice with HBSS and cultured in 1 ml of PH medium supplemented with 1.8% DMSO (primary hepatocyte medium containing DMSO, PH-DMSO) for the remainder of the experiment.

[0112] Human donor primary hepatocytes (HD-PH) were isolated from mouse humanized livers and purchased from Yecuris, Inc. Humanized mice were generated using cryopreserved primary hepatocytes collected from patients with the following demographics: HD1 (13 years old, female, Hispanic, HBV-naive), HD2 (13 years old, female, Caucasian, HBV-naive), and HD3 (27 years old, male, Caucasian, HBV-naive).

[0113] The PH MHC presentation of RM and HD was determined by surface expression of bulk MHC-I by the W6 / 32 clone, surface expression of MHC-II by HLA-DR staining, and surface expression of MHC-E by the 4D12 clone. 4D12 was previously demonstrated to specifically stain Mamu-E and non-classical Mamu-Ia molecules. All three human donors shared one HLA-A and one HLA-C allele (Table 1). Therefore, prior to the procedure, we confirmed that the MHC-E-specific 4D12 clone stained only for HLA-E and not for HLA-A or HLA-C molecules shared among the three HDs (Figure 2A). MHC-E expression was examined by staining, and the majority of primary hepatocytes from both species expressed MHC-E (Figures 2B and 2C). Table 1. MHC genotypes in HD PH [Table 1]

[0114] To investigate whether HBV infection affects MHC-E expression on the surface of primary hepatocytes, primary hepatocytes were harvested from the same HBV-infected human donor. One day after plating, isolated rhesus primary hepatocytes were transfected with a replication-incompetent adenovirus serotype 5 expressing human NTCP (MOI 10) under the liver-specific TTR promoter and cultured for 2 days. On day 2, the cells were refed with 1 ml of PH-DMSO medium. On day 4 after adenoviral transduction, primary hepatocytes were washed twice with 1 ml of HBSS and overlaid with HBV-containing medium (PH-DMSO containing 4% PEG 6000, Sigma-Aldrich, cat#: 81253-250G) at an MOI of 100 and incubated overnight. The following morning, wells were washed three times with 1 ml of HBSS and then cultured with 1 ml of primary hepatocyte-DMSO for the remainder of the experiment.

[0115] One day after plating, human donor primary hepatocytes were overlaid with HBV-containing medium (PH-DMSO containing 4% PEG6000, Sigma-Aldrich, cat#:81253-250G) at an MOI of 100 and incubated overnight. The next morning, wells were washed three times with 1 ml of HBSS and then cultured with 1 ml of primary hepatocyte-DMSO for the remainder of the experiment.

[0116] HBV infection of human donor primary hepatocytes was confirmed by measuring the levels of HBV envelope antigen (HBeAg) in the supernatant before staining the cells. Primary hepatocytes were co-stained with four MHC markers (MHC-I, MHC-E, and MHC-II) along with cellular HBcAg on day 4 after HBV infection, as this was the time point at which intracellular HBcAg was first detected. Strong staining with the 4D12 antibody was observed in both HBV-infected and HBV-naive hepatocytes, indicating high expression of MHC-E (Figures 2D and 2E). In contrast, HLA-DR expression was minimal or absent in all three human donor primary hepatocyte samples. This is consistent with previously published results (Senaldi et al., 1991. Class I and class II major histocompatibility complex antigens on hepatocytes: importance of the method of detection and expression in histologically normal and diseased livers. J. Clin. Pathol. 44: 107-114). Although ex vivo manipulation of primary hepatocytes prior to evaluation of surface MHC levels may have induced some of these cells to lose MHC-E expression, these results nonetheless demonstrated that HBV-infected primary hepatocytes express MHC-E and that MHC-E may be a restriction element for potential HBV-specific CD8+ T cells.

[0117] Example 3: MHC-E-restricted CD8+ T cells from RHCMV / HBV-inoculated rhesus macaques recognize HBV-infected allogeneic and xenogeneic primary hepatocytes MHC studies revealed high levels of MHC-E expression on primary hepatocytes from human donors and rhesus macaques, regardless of HBV infection. Therefore, given the high functional conservation of MHC-E in primates, we hypothesized that CD8+ T cells from RhCMV / HBV-inoculated rhesus macaques would recognize allogeneic HBV-infected primary hepatocytes. Supporting this hypothesis, CD8+ T cells (bulk spleen cells and purified CD8β+ T cells) from RM1 and RM2 recognized HBV-infected primary hepatocytes from two unrelated rhesus macaque donors but did not react to HBV-uninfected primary hepatocytes (Figure 3A).

[0118] To more comprehensively determine the MHC restriction of CD8+ T cells that recognize HBV-infected primary hepatocyte targets, we performed a series of recognition experiments using the MHC-specific blocking reagents shown in Figure 1 .

[0119] Prior to HBV infection, one well of primary hepatocytes was harvested and stained as a baseline. Starting two days postinfection, one well each of HBV-infected and HBV-naive primary hepatocytes was harvested with 0.5% trypsin-EDTA (Fisher Scientific, cat#: SH30236.01) and washed twice with ice-cold FACS buffer (PBS, Fisher Scientific, cat#: SH30256FS, containing 10% fetal bovine serum). The cells were incubated with anti-HLA-E antibody (clone: 4D12, Origene, cat#: LS-C179742) for 30 minutes at 4°C, washed twice with ice-cold FACS buffer, and then incubated with F(ab)2-goat anti-mouse IgG(HL)-APC (Invitrogen, cat#: A10539) for 30 minutes at 4°C. Next, cells were washed twice with ice-cold PBS and incubated with pan-MHC-I-PerCP-Cy5.5 (clone: W6 / 32, Biolegend Inc., cat#: 311419), anti-HLA-DR Alexa700 (clone: L243 (BD Biosciences, cat#: 560743), and Live / Dead fixable yellow (Invitrogen, cat#: L-34959) for 30 minutes at 4°C. Cells were washed with FACS buffer and fixed for 1 hour at room temperature using Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, cat#: 00-5523-00). All wash spins were performed at 350x g for 3 minutes before fixation. After fixation, cells were suspended in permeabilization buffer (eBioscience, cat#: 00-8333-56). All wash spins after fixation were performed at 830x g for 3 minutes. Lightning-Link Primary hepatocytes were incubated with R-phycoerythrin (PE)-conjugated hepatitis B virus core antigen antibody (clone: 13A9, Fisher Scientific, cat#: MA1-7606) using an R-PE kit (Innova Biosciences, cat#: 703-0005) for 1 hour at 4°C. Cells were washed three times with permeabilization buffer and then harvested using a Becton-Dickenson LSR-II.Analyses were performed in FlowJo X (TreeStar Inc.) All analyses were gated on the large and complex light scatter signature of PH, followed by evaluation of specific MHC and HBV markers.

[0120] Flow cytometry (ICS) was performed on mononuclear cell preparations from the spleens of RhCMV / HBV-vaccinated RMs. Briefly, splenocytes or isolated CD8+ T cells were incubated with HBV-infected or HBV-naive primary hepatocyte targets and the costimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by the addition of brefeldin (Sigma-Aldrich) for an additional 8 hours. Costimulation without primary hepatocyte target coculture served as a background control. MHC restriction of responses was determined by preincubating the PH targets for 1 hour at room temperature in the presence of a pan anti-MHC-I antibody (25 mg / ml, clone: W6-32), VL9 peptide (20 μM), CLIP peptide (MHC-II-related invariant chain, amino acids 89–100, 10 mg / ml), or anti-HLA-DR antibody (10 mg / ml, clone: L243) before coculture with primary hepatocyte targets. Stimulated cells were fixed, permeabilized, and stained for flow cytometry analysis using an LSR-II instrument (BD Biosciences). Analysis was performed using FlowJo X software (Tree Star, Inc.). Following gating on the light scatter signature of small lymphocytes, gating was performed on the CD3+ population, followed by gating on the CD4- / CD8+ T cell subset. The frequency of antigen-specific responses in the CD8+ T cell population was routinely determined by intracellular expression of IFN-γ.

[0121] HBV-infected or HBV-naive targets were harvested on day 6 post-HBV infection (MOI = 100) and incubated with the blocking agents W6 / 32 antibody (pan MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II), or HLA-DR antibody (MHC-II), followed by overnight coculture with splenocytes or isolated CD8β+ T cells. After coculture, CD8+ T cells were intracellularly stained with IFN-γ and TNF-α to assess target recognition. CD8+ T cell recognition of HBV-infected RM PH was blocked by W6 / 32 antibody and VL9 peptide, but not by CLIP or HLA-DR, indicating that the entire response to HBV-infected targets was MHC-E restricted (Figure 3A).

[0122] Because MHC-E is functionally conserved among primates, we hypothesized that CD8+ T cells from RhCMV / HBV-inoculated RMs would also recognize HBV-infected HD primary hepatocytes. To test this hypothesis, we performed similar recognition experiments by incubating spleen cells and purified CD8+ T cells from the same RhCMV / HBV-inoculated monkeys (RM1 and RM2) with HBV-infected human donor primary hepatocytes. As hypothesized, these CD8+ T cells recognized xenogeneic HBV-infected human donor primary hepatocytes (Figure 3B). As described above for the rhesus macaque primary hepatocyte-targeting co-culture experiments, CD8+ T cell recognition of HBV-infected human donor primary hepatocytes was completely inhibited by the MHC-E-binding VL9 peptide. These results conclusively demonstrate that HBV-infected primary hepatocytes present HBV antigens in the context of MHC-E and demonstrate that this pathway can be utilized to attract CD8+ T cells to HBV-infected cells.

[0123] Next, we examined the conservation of the MHC-E-binding supertopes of two 15-mer peptides in the HBV core antigen: core 7 (PSVRDLLDTASALYR; SEQ ID NO: 17) and core 14 (TALRQAILCWGELMT; SEQ ID NO: 18). 6,203 HBV whole genome sequences spanning all known HBV genotypes were retrieved from The Hepatitis B Virus Database, translated, and aligned with core 7 (Figure 4A) and core 14 (Figure 4B). The two 15-mer peptides that elicited MHC-E-restricted CD8+ T cell responses in all animals were highly conserved (supertopes). Importantly, the two positions that were not highly conserved (position 3 of core 7 and position 15 of core 14) contained only one additional amino acid that is globally dominant across HBV strains. Therefore, more than 98% of known global sequences express one of the two amino acids at these positions.

[0124] These results identify a novel set of CD8+ T cell responses against HBV, paving the way for the development of innovative HBV therapeutics. While MHC-E-restricted CD8+ T cell responses have been identified in natural viral infections with CMV, EBV, and HCV (Joosten et al., 2016. Characteristics of HLA-E Restricted T-Cell Responses and Their Role in Infectious Diseases. Journal of Immunology Research 2016: 1-11), no MHC-E-restricted CD8+ T cell responses against HBV have been reported. Therefore, it was unclear whether HBV-infected hepatocytes present HBV antigens in the context of MHC-E. These results demonstrate that MHC-E presents HBV antigens on the surface of HBV-infected cells and that CD8+ T cells from distinct primates recognize these MHC-E:peptide complexes.

[0125] In addition to representing a completely unique type of CD8+ T cell response against HBV, the breadth of epitopes targeted within HBcAg indicates that therapeutic vaccination with a CMV / HBV vector elicits broadly targeted CD8+ T cell responses. While such broad targeting has previously been demonstrated against SIV, Mycobacterium tuberculosis, and malaria, it appears particularly effective against HBV, given that the majority of the HBV genome consists of almost exclusively non-plastic overlapping reading frames. These results demonstrate for the first time that MHC-E-restricted CD8+ T cells can be harnessed for the treatment of chronic HBV infection through vaccine therapy or adoptive immunotherapy.

Claims

1. A method for generating an immune response against hepatitis B virus (HBV) in a subject, comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to induce a CD8+ T cell response against the HBV antigen, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

2. A method for treating chronic HBV infection in a subject, comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to induce a CD8+ T cell response against the HBV antigen, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

3. A CMV vector expressing an HBV antigen for use in generating an immune response to HBV in a subject, wherein the CMV vector does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, and an active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

4. A CMV vector expressing an HBV antigen for use in treating chronic HBV infection in a subject, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

5. Use of a CMV vector expressing an HBV antigen in the manufacture of a medicament for use in generating an immune response to HBV in a subject, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

6. Use of a CMV vector expressing an HBV antigen in the manufacture of a medicament for the treatment of chronic HBV infection, wherein the CMV vector does not express active UL128 protein, active UL130 protein, active UL146 protein, and active UL147 protein or their orthologues, and the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

7. 7. The method, CMV vector for use, or use of a CMV vector in manufacture according to any one of claims 1 to 6, wherein the hepatitis B virus antigen is a hepatitis B virus core, envelope, surface, X, or polymerase antigen.

8. 8. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 7, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

9. 9. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 8, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17).

10. 9. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 8, wherein the Hepatitis B antigen is TALRQAILCWGELMT (SEQ ID NO: 18).

11. 7. The method, CMV vector for use, or use of a CMV vector in manufacture according to any one of claims 1 to 6, wherein at least 10% of CD8+ T cells induced by the CMV vector are restricted by MHC-E or an orthologue thereof, or MHC-II or an orthologue thereof.

12. 12. The method, CMV vector for use, or use of a CMV vector in manufacturing according to claim 11, wherein at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75% of the CD8+ T cells induced by the CMV vector are restricted by MHC-E or an orthologue thereof, or MHC-II or an orthologue thereof.

13. 12. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 11, wherein MHC-E restricted CD8+ T cells recognize an MHC-E supertope.

14. 14. The method, CMV vector for use, or use of a CMV vector in production according to claim 13, wherein said MHC-E supertope is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

15. 14. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 13, wherein said MHC-E supertope is PSVRDLLDTASALYR (SEQ ID NO: 17).

16. 14. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 13, wherein said MHC-E supertope is TALRQAILCWGELMT (SEQ ID NO: 18).

17. 17. The method, CMV vector for use, or use of a CMV vector in manufacture according to any one of claims 1 to 16, wherein less than 10% of CD8+ T cells induced by the CMV vector are restricted by MHC-class 1a or an orthologue thereof.

18. 17. The method, CMV vector for use, or use of a CMV vector in manufacture according to any one of claims 1 to 16, wherein a proportion of MHC-E-restricted CD8+ T cells recognize an epitope shared by at least 90% of other subjects immunized with the vector.

19. 1. A method for generating CD8+ T cells that recognize MHC-E-HBV antigenic peptide complexes, comprising: (a) administering to a first subject a recombinant CMV vector comprising a nucleic acid expressing an HBV antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or an orthologue thereof, in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex; (b) identifying a first CD8+ TCR from the set of CD8+ T cells that recognizes an MHC-E / HBV antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from the second subject; (d) transfecting one or more CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize an MHC-E / HBV antigenic peptide complex.

20. 1. A method for generating CD8+ T cells that recognize MHC-E-HBV antigenic peptide complexes, comprising: (a) isolating a first set of CD8+ T cells from a first subject who has been administered a recombinant CMV vector comprising a nucleic acid expressing an HBV antigen, and which does not express an active UL128 protein, an active UL130 protein, an active UL146 protein, or an active UL147 protein or an orthologue thereof, in an amount effective to generate a set of CD8+ T cells that recognize an MHC-E / peptide complex; (b) identifying a first CD8+ TCR from the first set of CD8+ T cells that recognizes an MHC-E / HBV antigen-derived peptide complex; (c) isolating a second set of CD8+ T cells from a second subject; (d) transfecting a second set of CD8+ T cells with an expression vector comprising a nucleic acid sequence encoding a second CD8+ TCR comprising CDR3α and CDR3β of the first CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR to generate CD8+ T cells that recognize an MHC-E / HBV antigenic peptide complex.

21. The method according to claim 19 or claim 20, wherein the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus CMV vector.

22. The method of any one of claims 19 to 21, wherein the hepatitis B virus antigen is a hepatitis B virus core, envelope, surface, or polymerase antigen.

23. 23. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 22, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

24. 24. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 23, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17).

25. 24. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 23, wherein the Hepatitis B antigen is TALRQAILCWGELMT (SEQ ID NO: 18).

26. 26. The method of any one of claims 19-25, wherein the first set of CD8+ T cells recognize a specific Hepatitis B virus antigen that is a supertope of a Hepatitis B virus core, envelope, surface, X, or polymerase antigen peptide shared by at least 90% of other subjects immunized with the vector.

27. 27. The method of claim 26, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

28. 27. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 26, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17).

29. 27. The method, CMV vector for use, or use of a CMV vector in manufacture according to claim 26, wherein the Hepatitis B antigen is TALRQAILCWGELMT (SEQ ID NO: 18).

30. 30. The method of any one of claims 19 to 29, wherein the second set of CD8+ T cells recognizes a Hepatitis B virus antigen supertope that is shared by at least 90% of other subjects immunized with the vector.

31. 31. The method of claim 30, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

32. 32. The method of claim 31, wherein the Hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17).

33. 32. The method of claim 31, wherein the Hepatitis B antigen is TALRQAILCWGELMT (SEQ ID NO: 18).

34. 34. The method of any one of claims 19 to 33, wherein the first CD8+ TCR is identified by DNA or RNA sequencing.

35. 35. The method of any one of claims 19 to 34, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

36. 36. The method of any one of claims 19 to 35, wherein the first subject and / or the second subject is a human or a non-human primate.

37. 37. The method of any one of claims 19 to 36, wherein the first subject is a non-human primate, the second subject is a human, and the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising a non-human primate CDR3α and a CDR3β of the first CD8+ TCR.

38. 38. The method of any one of claims 19 to 37, wherein the second CD8+ TCR comprises a non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

39. 39. The method of any one of claims 19 to 38, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

40. 40. The method of any one of claims 19 to 39, wherein the nucleic acid sequence encoding the second CD8+ TCR is identical to the nucleic acid sequence encoding the first CD8+ TCR.

41. The method of any one of claims 19 to 40, wherein the second CD8+ TCR is a chimeric CD8+ TCR.

42. 42. The method of any one of claims 19 to 41, wherein the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the first CD8+ TCR.

43. 43. The method of any one of claims 19 to 42, wherein the step of administering the CMV vector to the first subject comprises intravenous, intramuscular, intraperitoneal, or oral administration of the CMV vector to the first subject.

44. 44. The method of any one of claims 19 to 43, further comprising administering transfected CD8+ T cells to said second subject to treat or prevent HBV infection.

45. A CD8+ T cell generated by the method of any one of claims 19 to 44.

46. 46. A method for treating or preventing hepatitis B infection in a subject, comprising administering the CD8+ T cells of claim 45 to a subject in need of treatment or prevention of hepatitis B infection.

47. 46. The CD8+ T cell of claim 45 for use in a method for treating or preventing hepatitis B infection in a subject in need thereof.

48. 46. Use of the CD8+ T cells of claim 45 in the manufacture of a medicament for use in a method for treating or preventing hepatitis B infection in a subject in need thereof.

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