HBV-specific T cell responses

Abstract

The present disclosure relates to methods for generating an immune response for treating or preventing hepatitis B virus infection. The present disclosure also relates to methods for generating MHC-E and / or MHC-II restricted CD8+ T cells for treating or preventing hepatitis B virus infection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 858,764, filed on June 7, 2019, which is hereby incorporated by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under Grant Nos. R01 AI117802, R01 AI129703, R01 AI140888, and P51OD011092 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] References to sequence listings submitted electronically

[0006] The contents of the electronically submitted ASCII text file Sequence Listing (name 4153_011PC01_Seqlisting_ST25; size: 4,288 bytes; and creation date: June 5, 2020) submitted with this application are incorporated herein by reference in their entirety. Background Art

[0007] Chronic hepatitis B virus infection (CHB) is a major global health problem, affecting 247 million individuals worldwide and causing 887,000 deaths each year. Although effective preventive vaccines are available, 10 to 15% of individuals do not respond adequately to vaccination and are not protected against hepatitis B virus (HBV) infection (Joint Committee on Vaccination and Immunisation. Hepatitis B. In Immunisation Against Infectious Disease, 3rd ed. p. 468.). CHB can lead to progressive liver dysfunction, cirrhosis, and in some cases hepatocellular carcinoma. There are multiple treatment options for CHB, including PEGylated IFNα and reverse transcriptase inhibitors (Bhattacharya, D., and CLThio. 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.).

[0008] The development of cellular immunotherapy strategies for CHB is supported by the fact that 90 to 95% of adults with acute HBV infection mount a broad, highly functional HBV-specific T cell response and subsequently clear the 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 who progress to CHB show a narrowly focused, low-frequency, and functionally exhausted HBV-specific T cell response (Bedoletti, 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-betinduction. J. Exp. Med. 211: 2047-2059.). Therefore, many immunotherapy strategies currently under development focus on enhancing HBV-specific T cell immunity.

[0009] Immunotherapies currently under investigation aim to harness the immune system to better target HBV-infected hepatocytes 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 obstacle faced by HBV immunotherapy is T cell immune tolerance (Zong et al. 2019. Breakdown of adaptive immunotolerance induces hepatocellular carcinoma in HBsAg-tg mice. Nature Communications 10: 221; Kong et al. 2014. γδT cells drive myeloid-derived suppressor cell-mediated CD8+ Tcell 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 fail to do so, is not fully understood. However, this may be partly a result of the immune tolerance environment of the liver. Therefore, in order to successfully clear CHB by immunotherapy, T cell tolerance must be overcome. Unfortunately, no immunotherapy to date has consistently achieved this goal, and this reality has been exacerbated by the lack of physiologically relevant CHB animal models.

[0010] T cell-based immunotherapy for CHB must provide durable reversal of T cell exhaustion or sustained viral suppression. Given the difficulty in 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 a de novo response is limited by patient-specific HLA expression and the HBV peptides presented by these molecules on the surface of hepatocytes. In contrast, if a ubiquitously expressed unconventional MHC-Ib T cell restriction element that does not contribute to natural, acute HBV-specific immune responses can present HBV antigens on the surface of hepatocytes, it can be targeted to elicit a completely different set of T cell responses that are not usually present in HBV infection. Therefore, there is an urgent global need to develop a curative treatment for HBV. SUMMARY OF THE INVENTION

[0012] The present disclosure relates to methods for generating an immune response against hepatitis B virus (HBV) in a subject, the method comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to elicit a CD8+ T cell response against the HBV antigen, wherein the CMV vector does not express active UL128, UL1 30, UL146, and UL147 proteins, or orthologs 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 a method of treating chronic HBV infection in a subject, the method comprising administering to the subject a CMV vector expressing an HBV antigen in an amount effective to elicit a CD8+ T cell response against the HBV antigen, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins, or orthologs thereof. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0014] The present disclosure also relates to a CMV vector expressing an HBV antigen for generating an immune response against HBV in a subject, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their orthologs. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0015] The present disclosure also relates to a CMV vector expressing an HBV antigen for treating chronic HBV infection in a subject, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their orthologs. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0016] The present disclosure also relates to the use of a CMV vector expressing an HBV antigen in the preparation of a medicament for generating an immune response against HBV in a subject, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their orthologs. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0017] The present disclosure also relates to the use of a CMV vector expressing an HBV antigen in the preparation of a medicament for treating chronic HBV infection, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their orthologs. In one embodiment, the HBV antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

[0018] 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 is 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% identical to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the hepatitis B virus antigen is 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% identical to TALRQAILCWGELMT (SEQ ID NO: 18).

[0019] In another embodiment, at least 10% of the CD8+ T cells triggered by the CMV vector are restricted by MHC-E or its ortholog or MHC-II or its ortholog. 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 triggered by the CMV vector are restricted by MHC-E or its ortholog or MHC-II or its ortholog. In another embodiment, less than 10% of the CD8+ T cells triggered by the CMV vector are restricted by MHC Ia class or its ortholog. In another embodiment, some CD8+ T cells restricted by MHC-E recognize peptides shared with at least 90% of other objects immunized with the vector. In some embodiments, CD8+ T cells restricted by MHC-E recognize MHC-E supertopes. In some embodiments, the MHC-E superepitope is 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% identical to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the MHC-E superepitope is 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% identical to TALRQAILCWGELMT (SEQ ID NO: 18).

[0020] 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 a recombinant CMV vector comprising a nucleic acid expressing an HBV antigen to a first subject in an amount effective to generate a group of CD8+ T cells that recognize the MHC-E / peptide complex, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins, or orthologs thereof; (b) identifying a first CD8+ TCR from the group of CD8+ T cells, wherein the first CD8+ TCR recognizes the MHC-E / HBV antigen-derived peptide complex; (c) isolating one or more CD8+ T cells from a second subject; and (d) transfecting the one or more CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises the first CD8+ CDR3α and CDR3β of TCR, thereby generating CD8+ T cells that recognize MHC-E / HBV antigen peptide complexes.

[0021] 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 group of CD8+ T cells from a first subject, wherein the first subject has been administered a recombinant CMV vector comprising a nucleic acid expressing an HBV antigen in an amount effective to generate a group of CD8+ T cells that recognize MHC-E / peptide complexes, wherein the CMV vector does not express active UL128, UL130, UL146, and UL147 proteins, or orthologs thereof; (b) identifying a first CD8+ TCR from the first group of CD8+ T cells, wherein the first CD8+ TCR recognizes an MHC-E / HBV antigen-derived peptide complex; (c) isolating a second group of CD8+ T cells from a second subject; and (d) transfecting the second group of CD8+ T cells with an expression vector, wherein the expression vector comprises a nucleic acid sequence encoding a second CD8+ TCR and a promoter operably linked to the nucleic acid sequence encoding the second CD8+ TCR, wherein the second CD8+ TCR comprises the first CD8+ CDR3α and CDR3β of TCR, thereby generating CD8+ T cells that recognize MHC-E / HBV antigen peptide complexes.

[0022] In one embodiment, the recombinant CMV vector is a recombinant human CMV vector or a recombinant rhesus monkey 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 is 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% identical to PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the hepatitis B virus antigen is 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% identical to TALRQAILCWGELMT (SEQ ID NO: 18).

[0023] In one embodiment, the first CD8+ T cell recognizes a specific MHC-E superepitope. In another embodiment, the second CD8+ T cell recognizes a specific MHC-E superepitope. In some embodiments, the MHC-E superepitope is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18). In some embodiments, the MHC-E superepitope is PSVRDLLDTASALYR (SEQ ID NO: 17). In some embodiments, the MHC-E superepitope is TALRQAILCWGELMT (SEQ ID NO: 18).

[0024] 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.

[0025] In one embodiment, the first object is a human or non-human primate. In another embodiment, the object is a non-human primate and the second object is a human, and wherein the second CD8+ TCR is a chimeric non-human primate-human CD8+ TCR comprising the non-human primate CDR3α and CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises non-human primate CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR. In another embodiment, the second CD8+ TCR comprises CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR. In another embodiment, the nucleic acid sequence encoding the second CD8+ TCR is the same as 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 CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of the first CD8+ TCR.

[0026] In one embodiment, administering the CMV vector to the first subject comprises administering the CMV vector to the first subject intravenously, intramuscularly, intraperitoneally or orally. In another embodiment, the transfected CD8+ T cells are also administered to the second subject to treat or prevent HBV infection.

[0027] The present disclosure also relates to CD8+ T cells produced by the methods described herein.

[0028] The present disclosure also relates to methods for treating or preventing hepatitis B infection in a subject, the method comprising administering to a subject in need thereof a CD8+ T cell as described herein. The present disclosure also relates to CD8+ T cells for use in methods for 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 preparation of a medicament for a method for treating or preventing hepatitis B infection in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A Shown are the frequencies of HBV antigen-specific CD8+ T cell responses of four rhesus macaques (RM) vaccinated with strain 68-1 RhCMV expressing HBV core, surface and polymerase antigens (RhCMV / HBV 68-1). Figure 1B CD8+ T cell responses to individual peptides of HBV core antigen (HBcAg) are shown. Each HBcAg 15-mer is represented by a box, which is color-coded as shown to indicate MHC restriction. Figure 1C Shown are the responses of CD8+ T cells isolated from vaccinated RM against K562 cells transfected with HLA-E or Mamu-E after addition of HBcAg peptide.

[0031] Figure 2A Shown is the staining of the MHC-E specific antibody 4D12 on the MHC transfected cell line. The 4D12 staining was compared with the matched IgG isotype control. In addition, the cells were stained with the pan-MHC-I specific antibody W6 / 32. Figure 2B 4D12 staining of human and RM primary hepatocytes one day after liver perfusion and plating is shown. Mouse IgG1 isotype was used to control for non-specific antibody binding in primary hepatocytes. Figure 2C Shown from Figure 2B Quantification of the percentage of MHC-E+ primary hepatocytes in vitro. Figure 2D Shown are co-staining of surface MHC-I, MHC-E or MHC-II and intracellular HBcAg in human donor primary hepatocytes four days after infection with HBV. Figure 2E Shown from Figure 2D Quantification of the percentage of HBV+ primary hepatocytes in vitro.

[0032] Figure 3A RM1 and RM2 are shown compared to uninfected RMs from two unrelated RM donors (RM8 and RM9). After incubation with HBV or HBV-infected PH, the percentage of HBV-specific CD8+ T cells restricted by MHC-I, MHC-II and MHC-E in the splenocytes from the RM1 and RM2 and the effectors sorted by CD8β. Responding T cells were identified by staining for CD3, CD8 and IFN-γ. The MHC restriction of responding CD8+ T cells was identified by the following MHC blockers: W6 / 32 antibody (pan-MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II) or HLA-DR antibody (MHC-II). Figure 3B The percentage of HBV-specific CD8+ T cells restricted by MHC-I, MHC-II, and MHC-E in splenocytes and CD8β-sorted effectors from RM1 and RM2 incubated with HBV-uninfected or HBV-infected primary hepatocytes from human donors (HD1 and HD2) is shown. Responding CD8+ T cells were identified by CD3, Cd8, and IFN-γ. MHC restriction of responding CD8+ T cells was also identified by the following MHC blockers: W6 / 32 antibody (pan-MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II), or HLA-DR antibody (MHC-II).

[0033] Figure 4 is a bar graph showing the conservation of MHC-E-binding superepitopes in HBV core antigen among global HBV strains. 6,203 whole genome HBV sequences covering all known HBV genotypes were translated and targeted against core protein 7 ( Figure 4A ) and core protein 14 ( Figure 4B ) The amino acids were aligned. DETAILED DESCRIPTION

[0034] I. Terminology

[0035] Unless otherwise indicated, technical terms are used according to conventional usage.

[0036] 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 Sheet, including U.S. Provisional Patent Application No. 62 / 858,764, filed on June 7, 2019, are hereby incorporated 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 as being so incorporated by reference.

[0037] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and its variants such as "comprising" and "containing" will be interpreted as having an open, inclusive meaning, i.e., "including, but not limited to". "Consisting of" will mean excluding more than trace elements of other ingredients and substantial method steps disclosed herein. The term "consisting essentially of" limits the scope of the claims to the specified materials or steps, or those that do not materially affect the basic characteristics of the claimed invention. For example, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from separation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc. Similarly, a protein consists essentially of a specific amino acid sequence when it includes additional amino acids that account for up to 20% of the length of the protein and do not substantially affect the activity of the protein (e.g., do not change the activity of the protein by more than 50%). Embodiments defined by each transition term are within the scope of the present invention.

[0038] Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. For ease of access to multiple embodiments of the present disclosure, the following explanation of specific terms is provided.

[0039] Antigen: As used herein, the terms "antigen" or "immunogen" are used interchangeably and refer to a substance, usually a protein, that is capable of inducing an immune response in a subject. The term also refers to a protein that is immunologically active in the sense that upon administration to a subject (directly or by administering a nucleotide sequence or vector encoding the protein to a subject), the protein is capable of eliciting a humoral and / or cellular type immune response against the protein.

[0040] Antigen-specific T cells: CD8+ or CD4+ lymphocytes that recognize specific antigens. Generally speaking, antigen-specific T cells bind specifically to a specific antigen presented by an MHC molecule, but not to other antigens presented by the same MHC.

[0041] Administration: As used herein, the term "administering" means providing or administering a medicament, such as a composition comprising an effective amount of a CMV vector comprising 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.

[0042] Effective amount: As used herein, the term "effective amount" refers to an amount of an agent sufficient to produce a desired response (e.g., to reduce or eliminate the signs or symptoms of a disorder or disease or to induce an immune response to an antigen), such as a CMV vector containing a heterologous antigen or a transfected CD8+ T cell that recognizes a peptide complex derived from MHC-E / heterologous antigen, a peptide complex derived from MHC-II / heterologous antigen, or a peptide complex derived from MHC-I / heterologous antigen. In some examples, an "effective amount" is an amount for treating (including preventing) one or more symptoms and / or potential causes of any disorder or disease. An effective amount can be a therapeutically effective amount, including an amount that prevents the development of one or more signs or symptoms of a particular disease or disorder (e.g., one or more signs or symptoms associated with an infectious disease).

[0043] Heterologous antigen: As used herein, the term "heterologous antigen" refers to any protein or fragment thereof that is not derived from CMV. The heterologous antigen may be any antigen derived from HBV.

[0044] Immunogenic peptide: A peptide that contains an allele-specific motif or other sequence (e.g., N-terminal repeats) such that the peptide 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.

[0045] In one embodiment, sequence motifs or other methods (such as neural networks or polynomial determinations known in the art) are used to identify immunogenic peptides. Generally speaking, an algorithm is used to determine the "binding threshold" of a peptide to select those with a high probability of binding with a certain affinity and a score that will have immunogenicity. The algorithm is based on the effect of MHC binding to a specific amino acid at a specific position, the effect of antibody binding to a specific amino acid at a specific position, or the effect of binding to a specific replacement in a motif-containing peptide. In the context of immunogenic peptides, "conserved residues" are residues that appear at a significantly higher frequency than would be expected from a random distribution at a specific position of the peptide. In one embodiment, conserved residues are residues for which the MHC structure can provide a contact point with an immunogenic peptide.

[0046] Mutation: As used herein, the term "mutation" refers to any difference between a nucleic acid or polypeptide sequence and a normal, shared or "wild-type" sequence. A mutant is any protein or nucleic acid sequence that contains a mutation. In addition, cells or organisms with mutations may also be referred to as mutants. Some types of coding sequence mutations include point mutations (differences in a single nucleotide or amino acid); silent mutations (differences in nucleotides that do not result in amino acid changes); deletions (differences in which one or more nucleotides or amino acids are missing, up to and including the deletion of the entire coding sequence of a gene); frameshift mutations (the deletion of some nucleotides that are not divisible by 3 results in differences in amino acid sequence changes). 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 changes in amino acids at specific positions in an amino acid sequence relative to the wild type.

[0047] Nucleotide sequence or nucleic acid sequence: The terms "nucleotide sequence" and "nucleic acid sequence" refer to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence, including but not limited to messenger RNA (mRNA), a DNA / RNA hybrid, or a synthetic nucleic acid. A nucleic acid may be single-stranded, or partially or completely double-stranded (duplex). A duplex nucleic acid may be a homoduplex or a heteroduplex.

[0048] Operably linked: As used herein, the term "operably linked" refers to a first nucleic acid sequence being operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a manner that affects the second nucleic acid sequence. Operably linked DNA sequences may be continuous, or they may function at a distance.

[0049] Promoter: As used herein, the term "promoter" may refer to any of a number of nucleic acid control sequences that direct the transcription of a nucleic acid. Generally speaking, a eukaryotic promoter includes a necessary nucleic acid sequence near the transcription start site, such as a TATA element in the case of a polymerase II type promoter, or any other specific DNA sequence recognized by one or more transcription factors. Expression by a promoter may also be regulated by an enhancer or repressor element. Many examples of promoters are available and well known to those of ordinary skill in the art. A nucleic acid comprising a promoter operatively linked to a nucleic acid sequence encoding a specific polypeptide may be referred to as an expression vector.

[0050] Recombinant: As used herein, the term "recombinant" when referring to a nucleic acid or polypeptide refers to a nucleic acid or polypeptide having a sequence that does not occur naturally or has a sequence that is prepared by artificially combining two or more otherwise separated sequence fragments, such as a CMV vector containing a heterologous antigen. This artificial combination is usually achieved by chemical synthesis, or more commonly by artificial manipulation of isolated nucleic acid fragments, such as by genetic engineering techniques. Recombinant polypeptides can also refer to polypeptides that have been prepared using recombinant nucleic acids, including recombinant nucleic acids transferred to a host organism that is not the natural source of the polypeptide (e.g., nucleic acids encoding polypeptides that form a CMV vector containing a heterologous antigen).

[0051] Pharmaceutically acceptable carrier: As used herein, the "pharmaceutically acceptable carrier" used is conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995 describes compositions and preparations suitable for drug delivery of the compositions disclosed herein. Generally, the nature of the carrier will depend on the specific mode of administration adopted. For example, parenteral preparations generally contain injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as carriers. For solid compositions (e.g., powders, pills, tablets or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0052] 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). The coding sequence from a nucleic acid indicates the sequence of the protein encoded by the nucleic acid.

[0053] Polypeptide: The terms "protein," "peptide," "polypeptide," and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The term also encompasses amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or biologically active component.

[0054] Orthologs of a protein are typically characterized by possessing greater than 75% sequence identity, which is counted when the full length of the amino acid sequence of a particular protein is aligned using ALIGN set to default parameters. Proteins with even greater similarity to a reference sequence will show an increased percentage of identity when evaluated by this method, such as at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 98% sequence identity. In addition, sequence identity can be compared over the full length of a particular domain of a disclosed peptide.

[0055] Sequence identity / similarity: As used herein, identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as identity or similarity between the sequences. Sequence identity can be measured as a percentage of identity; the higher the percentage, the higher the sequence identity. Sequence similarity can be measured as a percentage identity or similarity (taking into account conservative amino acid substitutions); the higher the percentage, the higher the sequence similarity. Polypeptides or protein domains thereof that have a significant amount of sequence identity and that function identically or similarly to one another (e.g., proteins that perform the same function in different species or mutant forms of proteins that do not change the function of the protein or its magnitude) can be referred to as "homologs".

[0056] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv Appl Math 2, 482 (1981); Needleman & Wunsch, J Mol Biol 48, 443 (1970); Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988); Higgins & Sharp, Gene 73, 237-244 (1988); Higgins & 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). Additionally, detailed considerations of sequence alignment methods and homology calculations are presented in Altschul et al., J Mol Biol 215, 403-410 (1990).

[0057] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., (1990) supra) is available from several sources, including the National Center for Biotechnology Information, NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894, and the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0058] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, the specified output file will display these homology regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not display the aligned sequences.

[0059] Once aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in the two sequences. The percentage of sequence identity is determined by dividing the number of matches by the length of the sequence shown in the identification sequence or by the articulated length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence shown in the identification sequence) and then multiplying the resulting value by 100. For example, when aligned with a test sequence having 1154 nucleotides, a nucleic acid sequence having 1166 matches has 75.0% identity with the test sequence (1166 ÷ 1554 * 100 = 75.0). The percentage of sequence identity values ​​are rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Length values ​​will always be integers. In another example, a target sequence comprising a 20 nucleotide region that aligns with 20 consecutive nucleotides from the identified sequence below comprises a region that shares 75% sequence identity with the identified sequence (ie, 15÷20*100=75).

[0060] For comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function is used using the default BLOSUM62 matrix (gap existence penalty of 11 and each residue gap penalty of 1) set to default parameters. The typical characteristics of homologues are: using NCBI Basic Blast 2.0 to perform gapped blastp on the amino acid sequence and databases (e.g., nr database, swissprot database, and patent sequence database) with a count of at least 70% sequence identity. Queries searched with the blastn program are filtered with DUST (Hancock & Armstrong, ComputAppl Biosci 10, 67-70 (1994.)) Other programs use SEG. In addition, manual comparisons can be performed. When evaluated by this method, proteins with even greater similarity will show an increased identity percentage, such as having at least about 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the protein.

[0061] When comparing short peptides (less than about 30 amino acids), the Blast 2 sequence function is used for comparison, using the PAM30 matrix (open gap 9, extended gap 1 penalty) set to default parameters. When evaluated by this method, proteins with even greater similarity to the reference sequence will show an increased identity percentage, for example, with a protein having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity. When the sequence identity is less than the entire sequence, homologues will generally have at least 75% sequence identity in a short window of 10 to 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 through such a short window are described on the NCBI website.

[0062] 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. However, due to the degeneracy of the genetic code, nucleic acid sequences that do not show a high degree of identity can still encode identical or similar (conservative) amino acid sequences. This degeneracy can be used to make changes in the nucleic acid sequence to produce multiple nucleic acid molecules that all encode substantially the same protein. For example, such a homologous nucleic acid sequence can have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity with a nucleic acid encoding a protein.

[0063] Subject: As used herein, the term "subject" refers to a living multicellular vertebrate organism, a class that includes humans and non-human mammals.

[0064] Superepitope: As used herein, the term "superepitope" or "superepitope peptide" refers to an epitope or peptide that is recognized by T cells in more than about 90% of the population, regardless of the MHC haplotype, i.e., in the presence or absence of a given MHC-I, MHC-II or MHC-E allele.

[0065] 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 reference to a disease, pathological condition, or symptom also refer to any observable beneficial effect of the treatment. The beneficial effect can be demonstrated, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters known in the art that are specific to a particular disease. Prophylactic treatment is a treatment administered to a subject who does not show signs of the disease or only shows early signs, with the goal of reducing the risk of developing the pathology. Therapeutic treatment is a treatment administered to a subject after the signs and symptoms of the disease have already occurred.

[0066] Vaccine: An immunogenic composition that can be administered to a mammal (e.g., a human) to confer immunity (e.g., active immunity) against a disease or other pathological condition. Vaccines can be used prophylactically or therapeutically. Thus, vaccines can be used to reduce the likelihood of developing a disease (e.g., a tumor or pathological infection) or to reduce the severity of the symptoms of a disease or condition, to limit the progression of a disease or condition (e.g., a tumor or pathological infection), or to limit the recurrence of a disease or condition (e.g., a tumor). In some embodiments, the vaccine is a replication-deficient CMV that expresses HBV antigens.

[0067] Vector: A nucleic acid molecule of a specific sequence can be incorporated into a vector, which is then introduced into a host cell to produce a transformed host cell. The vector may contain a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. The vector may also contain one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression. The vector may be a viral vector, such as a CMV vector. The viral vector may be constructed from a wild-type or attenuated virus (including a replication-deficient virus).

[0068] II. Methods for treating and preventing hepatitis B infection

[0069] Disclosed herein are methods for treating or preventing hepatitis B virus infection. The methods involve 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.

[0070] The antigen derived from hepatitis B virus may 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.

[0071] In some embodiments, CMV vectors do not express active UL128, UL130, UL146 and UL147 proteins because there is a mutation in the nucleic acid sequence encoding UL128, UL130, UL146 and UL147 or their homologues or their orthologues (CMV homologous genes that infect other species). The mutation can be any mutation that causes the active protein to lack expression. Such mutations may include point mutations, frameshift mutations, deletions (truncated mutations) less than the sequence of all encoded proteins, or the deletion of the nucleic acid sequence of all encoded proteins, or any other mutation.

[0072] In other examples, the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins because there is a nucleic acid sequence in the vector that comprises an antisense or RNAi sequence (siRNA or miRNA) that inhibits the expression of UL128, UL130, UL146 and UL147 proteins. Mutations and / or antisense and / or RNAi can be used in any combination to produce a CMV vector lacking active UL128, UL130, UL146 and UL147.

[0073] In some embodiments, the CD8+ T cell response triggered by the vector is characterized by having at least 10% of CD8+ T cells against HBV epitopes presented by MHC-E. In other 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 CD8+ T cells are restricted by MHC-E. In some embodiments, HBV-specific CD8+ T cells restricted by MHC-E recognize peptides shared by at least 90% of other subjects immunized with the vector. In some embodiments, CD8+ T cells are directed against HBV super epitopes presented by MHC-E. In some embodiments, the CD8+ T cell response triggered by the vector is characterized by having at least 10% of CD8+ T cells against epitopes presented by MHC-II. In other 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 HBV-specific CD8+ T cells restricted by MHC-II recognize HBV peptides shared by at least 90% of other subjects immunized with the vector. In some embodiments, the HBV-specific CD8+ T cells are directed against HBV superepitopes presented by MHC-II.

[0074] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from a CD8+ T cell primed by a CMV vector, wherein the CD8+ T cell receptor recognizes a peptide complex derived from an MHC-E / HBV antigen. 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 superepitope of HBV.

[0075] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from a CD8+ T cell primed by a CMV vector, wherein the CD8+ T cell receptor recognizes a peptide complex derived from an MHC-II / HBV antigen. 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 superepitope of HBV.

[0076] Also disclosed herein is a method for producing CD8+ T cells that recognize MHC-E-HBV peptide complexes. The method involves administering a CMV vector to a first subject (or animal) in an amount effective to produce a CD8+ T cell group that recognizes an MHC-E / HBV peptide complex. In some embodiments, the CMV vector comprises a first nucleic acid sequence encoding at least one HBV antigen and does not express active UL128, UL130, UL146, and UL147 proteins or their orthologs. In some embodiments, the HBV antigen may be a hepatitis B virus core, envelope, surface, or polymerase antigen.

[0077] The method also includes: identifying a first CD8+ T cell receptor from the CD8+ T cell group, wherein the first CD8+ T cell receptor recognizes a peptide complex derived from an MHC-E / HBV antigen. In some embodiments, the first CD8+ T cell receptor is identified by DNA or RNA sequencing. In some embodiments, the method may also include transfecting one or more CD8+ T cells with an expression vector, wherein 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 T cell receptor, wherein the second CD8+ T cell receptor comprises CDR3α and CDR3β of the first CD8+ T cell receptor, thereby generating one or more transfected CD8+ T cells that recognize a peptide complex derived from an MHC-E / HBV antigen. One or more CD8+ T cells transfected with the expression vector may be isolated from the first subject or the second subject.

[0078] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from a CD8+ T cell primed by a CMV vector, wherein the CD8+ T cell receptor recognizes a peptide complex derived from an MHC-E / HBV antigen. In some embodiments, the CD8+ T cell receptor is identified by RNA or DNA sequencing. In some embodiments, the method further comprises recognizing an HBV-specific CD8+ T cell receptor that is a super epitope of MHC-E.

[0079] Also disclosed are transfected CD8+ T cells that recognize MHC-E-HBV peptide complexes prepared by a process comprising the following steps: (1) administering a CMV vector to a first subject in an amount effective to generate a CD8+ T cell set that recognizes the MHC-E / HBV peptide complex, wherein the recombinant CMV vector comprises at least one HBV antigen; (2) identifying a first CD8+ T cell receptor from the CD8+ T cell set, wherein the first CD8+ T cell receptor recognizes the MHC-E / HBV antigen-derived peptide complex; (3) isolating one or more CD8+ T cells from the first subject or the second subject; and (4) transfecting one or more CD8+ T cells isolated from the first or second subject with an expression vector, thereby generating transfected T cells that recognize the MHC-E-HBV peptide complex. The CMV vector comprises a first nucleic acid sequence encoding at least one HBV antigen and does not express active UL128, UL130, UL146, and UL147 proteins 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 CDR3α and CDR3β of the first CD8+ T cell receptor. The hepatitis B antigen may be a hepatitis B virus core, envelope, surface or polymerase antigen.

[0080] In some embodiments, the CD8+ T cell response triggered by the CMV vector is characterized by having at least 10% of CD8+ T cells against HBV epitopes presented by MHC-II. In other 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 CD8+ T cells are restricted by MHC-II. In some embodiments, CD8+ T cells restricted by MHC-II recognize HBV peptides shared by at least 90% of other subjects immunized with the vector. In some embodiments, CD8+ T cells are directed against HBV super epitopes presented by MHC-II.

[0081] In some embodiments, the method further comprises identifying a CD8+ T cell receptor from a CD8+ T cell primed by a CMV / HBV vector, wherein the CD8+ T cell receptor recognizes a peptide complex derived from an MHC-II / HBV antigen. 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 superepitope.

[0082] When used as an expression vector, the human or animal CMV vector is inherently non-pathogenic in a selected subject, such as a human. In some embodiments, the CMV vector has been modified to make it non-pathogenic (cannot be transmitted within or between hosts) in a selected subject.

[0083] As described herein, the HBV antigen may be any HBV protein or fragment thereof.

[0084] The recombinant CMV vector disclosed herein may be derived from a human cytomegalovirus vector, a rhesus cytomegalovirus vector or a cynomolgus monkey vector.

[0085] The recombinant CMV vector disclosed herein can be used as an immunogenic composition, an immune composition or a vaccine composition comprising a recombinant CMV virus or a vector and a pharmaceutically acceptable carrier or diluent. An immune composition comprising a recombinant CMV virus or a vector (or its expression product) triggers a local or systemic immune response. The response may but need not be protective. An immunogenic composition comprising a recombinant CMV virus or a vector (or its expression product) also triggers a local or systemic immune response that may but need not be protective. Vaccine compositions trigger local or systemic protective responses. Therefore, the terms "immune composition" and "immunogenic composition" include "vaccine compositions" (because the first two terms can be protective compositions).

[0086] The recombinant CMV vectors disclosed herein can be used in a method of inducing an immune response in a subject, comprising administering to the subject an immunogenic composition, an immune composition, or a vaccine composition comprising a recombinant CMV virus or vector and a pharmaceutically acceptable carrier or diluent.

[0087] The CMV vector disclosed herein can be used in therapeutic compositions comprising recombinant CMV viruses or vectors and pharmaceutically acceptable carriers or diluents. The CMV vector disclosed herein can be prepared by inserting DNA comprising a sequence encoding HBV antigens into the essential or non-essential region of the CMV genome. The method may also include deleting one or more regions from the CMV genome. The method may include in vivo recombination. Therefore, the method may include in the presence of a donor DNA comprising a heterologous DNA sequence flanking a DNA sequence homologous to the CMV genome, transfecting cells with CMV DNA in a cell-compatible medium, thereby introducing heterologous DNA into the genome of CMV, and then optionally reclaiming CMV modified by in vivo recombination. The method may also include cutting CMV DNA to obtain cut CMV DNA, connecting heterologous DNA to cut CMV DNA to obtain hybrid CMV-heterologous DNA, transfecting cells with hybrid CMV-heterologous DNA, and then optionally reclaiming CMV modified by the HBV DNA present. Since recombination in vivo is understood, the method also provides a plasmid comprising a donor DNA that is not naturally present in CMV and that encodes a polypeptide that is foreign to CMV, the donor DNA being within a segment of CMV DNA that is otherwise colinear with an essential or non-essential region of the CMV genome, so that DNA from an essential or non-essential region of CMV is flanked by the donor DNA. When desired, HBV DNA can be inserted into CMV to produce recombinant CMV in any orientation that results in stable integration of the DNA and its expression.

[0088] The DNA encoding the HBV antigen in the recombinant CMV vector may further include a promoter. The promoter may be from any source, such as herpes virus, including endogenous cytomegalovirus (CMV) promoters, such as human CMV (HCMV), rhesus CMV (RhCMV), mouse or other CMV promoters. The promoter may also be a non-viral promoter, such as the EF1α promoter. The promoter may be a truncated transcriptionally active promoter comprising a region transactivated by a transactivator provided by a virus, and the minimum promoter region of the full-length promoter from the truncated transcriptionally active promoter is derived. The promoter may be composed of a DNA sequence association corresponding to a minimum promoter and an upstream regulatory sequence. The minimum promoter is composed of a CAP site plus an ATA frame (a minimum sequence for basic transcription levels; unregulated transcription levels); an "upstream regulatory sequence" is composed of an upstream element and an enhancer sequence. In addition, the term "truncated" means that the full-length promoter is not completely present, i.e., some parts of the full-length promoter have been removed. In addition, the truncated promoter can be derived from a herpes virus, such as MCMV or HCMV, such as HCMV-IE or MCMV-IE. Based on base pairs, the size from the full-length promoter can be reduced by up to 40%, and even up to 90%. The promoter can also be a modified non-viral promoter. Regarding the HCMV promoter, reference is made to U.S. Patent Nos. 5,168,062 and 5,385,839. Regarding transfection of cells with plasmid DNA for expression therefrom, reference is made to Feigner et al. (1994), J Biol. Chem. 269, 2550-2561. And, regarding direct injection of plasmid DNA as a simple and effective method for vaccination against a variety of infectious diseases, reference is made to Science, 259: 1745-49, 1993. Therefore, within the scope of the present disclosure, the vector can be used by direct injection of the vector DNA.

[0089] 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 may also contain a functionally truncated polyadenylation signal; for example, a truncated but still functional SV40 polyadenylation signal. It is indeed surprising that the truncated polyadenylation signal is functional, given that nature provides a larger signal. The truncated polyadenylation signal solves the problem of insertion size limitations of recombinant viruses (e.g., CMV). The expression cassette may also contain HBV DNA associated with the virus or system into which it is inserted; and the DNA may be HBV DNA as described herein.

[0090] For HBV antigens for use in vaccines or immunogenic compositions, see also Stedman's Medical Dictionary (24th ed., 1982, e.g., for definitions of vaccines (for a list of antigens for use in vaccine formulations); such antigens or epitopes of interest from those antigens may be used. For HBV antigens, one skilled in the art can select antigens and their encoding DNA without undue experimentation based on knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as 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.

[0091] One method of determining the T epitope of HBV antigens involves epitope mapping. Overlapping peptides of heterologous antigens are produced by oligopeptide synthesis. The individual peptides are then tested for their ability to bind to antibodies elicited by the native protein or to induce T cell or B cell activation. This method is particularly useful in T cell epitope mapping because T cells recognize short linear peptides complexed with MHC molecules.

[0092] The immune response to HBV antigens is usually generated as follows: T cells recognize the protein only when it is cut into smaller peptides and presented in a complex called "major histocompatibility complex (MHC)" located on the surface of another cell. There are two types 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; they 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, Qa-lb in mice).

[0093] It is noteworthy that the DNA comprising the sequence encoding the HBV antigen itself may include a promoter for driving expression in a CMV vector, or the DNA may be limited to the coding DNA of a heterologous antigen. The construct may be placed in such a direction relative to an endogenous CMV promoter so that it is effectively connected to the promoter and is therefore expressed. In addition, multiple copies of the DNA encoding heterologous antigens may be carried out or strong or early promoters or early and late promoters, or any combination thereof, may be used to amplify or improve expression. Therefore, the DNA encoding heterologous antigens may be appropriately positioned relative to the CMV endogenous promoter, or those promoters may be translocated to be inserted into another position together with the DNA encoding the heterologous antigen. Nucleic acids encoding more than one heterologous antigen may be packaged in a CMV vector.

[0094] Also disclosed are pharmaceutical compositions and other compositions comprising the disclosed CMV vectors. Such pharmaceutical compositions and other compositions can be formulated for any administration process known in the art. Such pharmaceutical compositions can be administered parenterally (intradermal, intramuscular, subcutaneous, intravenous or other). Administration can also be by mucosal routes, such as oral, nasal, genital, etc.

[0095] The disclosed pharmaceutical composition can be prepared according to standard techniques known to those skilled in the art of medicine. Taking into account such factors as the variety or species, age, sex, weight and symptoms of a particular patient and the route of administration, such compositions can be administered with dosages and techniques known to those skilled in the art of medicine. The composition can be administered alone, or it can be co-administered or sequentially administered with other CMV vectors or other immune, antigenic or vaccine or therapeutic compositions. Such other compositions may contain purified natural antigens or epitopes or antigens or epitopes expressed from recombinant CMV or another vector system; and administered in consideration of the above factors.

[0096] Some examples of compositions include liquid preparations for administration through an orifice, such as oral, nasal, anal, genital, such as vaginal, etc., such as suspensions, syrups or elixirs; and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injection administration), such as sterile suspensions or emulsions. In such compositions, the recombinant can be mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, etc.

[0097] Antigenic compositions, immune compositions or vaccine compositions may typically include an adjuvant and an amount of CMV vector or expression product to elicit a desired response. In human applications, alum (aluminum phosphate or aluminum hydroxide) is a commonly used adjuvant. Saponin and its purified components Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications are toxic, which limits their potential use in human vaccines. Chemically defined preparations such as muramyl dipeptides, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al., J Immunol. 147: 410-415 (1991), such as those described by Miller et al., J Exp. Med. 176: 1739-1744 (1992), encapsulation of proteins in proteoliposomes, and encapsulation of proteins in lipid vesicles such as Novasome lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be used.

[0098] The composition can be packaged in a single dosage form for immunization by parenteral (e.g., intramuscular, intradermal or subcutaneous) administration or by peroral administration, such as translingual (e.g., oral), intragastric, transmucosal including intraoral, intraanal, intravaginal, etc. Again, the effective dose 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 breed or species, age, sex, weight, condition and nature of the host, and LD 50 and other known screening processes that do not require undue experimentation. The dosage of the expressed product can range from a few micrograms to hundreds of micrograms, for example, 5 to 500 μg. The CMV vector can be administered in any suitable amount to achieve expression at these dosage levels. In some non-limiting examples: the CMV vector can be administered in an amount of at least 10 2 pfu; thus, the CMV vector may be administered in at least this amount; or in about 10 2 pfu to about 10 7 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 solution. "About" may mean within 1%, 5%, 10% or 20% of a defined value.

[0099] It should be understood that the proteins of the present disclosure and the nucleic acids encoding them may differ from the exact sequences shown and described herein. Thus, the present disclosure contemplates deletions, additions, truncations, and substitutions of the sequences shown, provided that the sequences function according to the methods of the present disclosure. In this regard, substitutions are generally conservative in nature, i.e., those that occur within a family of amino acids. For example, amino acids are generally divided into the following four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, and histidine; (3) non-polar - 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 reasonable to predict that a single replacement of leucine with isoleucine or valine, or vice versa; a replacement of aspartic acid with glutamic acid, or vice versa; a replacement of threonine with serine, or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on biological activity. Therefore, proteins having an amino acid sequence substantially identical to the protein but with a small number of amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the scope of the present disclosure.

[0100] The nucleotide sequences of the present disclosure can be codon optimized, for example, codons can be optimized for use in human cells. For example, any viral or bacterial sequence can be so altered. Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by changing these codons to correspond to codons commonly used in the desired subject, enhanced expression of heterologous antigens can be achieved as described in Andre et al., J Virol. 72: 1497-1503, 1998.

[0101] Nucleotide sequences encoding functional and / or antigenic equivalent variants and derivatives of the CMV vector and the glycoprotein contained therein are considered. These functional equivalent variants, derivatives and fragments show the ability to retain antigenic activity. For example, changes in the DNA sequence that do not change the encoded amino acid sequence, and lead to conservative substitutions of amino acid residues, deletions or additions of one or more amino acids, and replacement of amino acid residues by amino acid analogs are changes that do not significantly affect the properties of the encoded polypeptide. Conservative amino acid replacements are 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.

[0102] Sequence identity or homology is determined by comparing the sequences when aligned so that overlap and identity are maximized and sequence gaps are minimized. In particular, sequence identity can be determined using any of a variety of mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, as modified in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

[0103] Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers & 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 may be used. Another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

[0104] According to the present disclosure, WU-BLAST (Washington University BLAST) version 2.0 software is advantageously used.WU-BLAST version 2.0 executable programs 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 in turn based on the public domain NCBI-BLAST version 1.4 (Altschul & 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 & States, 1993; Nature Genetics 3:266-272; Karlin & Altschul, 1993; Proc. Natl. Acad. Sci. USA 90:5873-5877; all of which are incorporated herein by reference).

[0105] The various recombinant nucleotide sequences and antibodies and / or antigens of the present disclosure are prepared using standard recombinant DNA and cloning techniques. Such techniques are well known to those skilled in the art. For example, see "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al. 1989).

[0106] The nucleotide sequences of the present disclosure may be inserted into a "vector". The term "vector" is widely used and understood by those skilled in the art, and the term "vector" as used herein is used in accordance with the meaning understood by those skilled in the art. For example, those skilled in the art typically use the term "vector" to refer to a carrier that allows or facilitates the transfer of a nucleic acid molecule from one environment to another or allows or facilitates the manipulation of a nucleic acid molecule.

[0107] Any vector that allows the expression of the viruses of the present disclosure can be used in accordance with the present disclosure. In certain embodiments, the disclosed viruses can be used in vitro (e.g., using a cell-free expression system) and / or in cultured cells grown in vitro to produce encoded heterologous antigens (e.g., pathogen-specific antigens, HIV antigens, hepatitis B antigens, and antibodies), which can then be used in a variety of applications, such as the production of protein vaccines. For such applications, any vector that allows the expression of the virus in vitro and / or in cultured cells can be used.

[0108] For the disclosed heterologous antigen to be expressed, the protein coding sequence of the heterologous antigen should be "operably linked" to a regulatory sequence or nucleic acid control sequence that directs 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 the coding sequence and the nucleic acid control sequence or promoter are covalently linked in such a way that the expression or transcription and / or translation of the coding sequence is placed under the influence or control of the nucleic acid control sequence. A "nucleic acid control sequence" can be any nucleic acid element, such as, but not limited to, a promoter, an enhancer, an IRES, an intron, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto. The term "promoter" is used herein to refer to a group of transcription control modules that are clustered around the start site of RNA polymerase II and result in the expression of the encoded protein when operably linked to the protein coding sequence of the present disclosure. The expression of the transgenic of the present disclosure can be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when exposed to some specific external stimuli such as, but not limited to, antibiotics (e.g., tetracycline), hormones (e.g., ecdysone), or heavy metals. The promoter may 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 may be used for expression of the transgenic of the present disclosure. For example, suitable promoters and / or enhancers may be selected from the Eukaryotic Promoter Database (EPDB).

[0109] The vector used according to the present disclosure may contain appropriate gene regulatory regions, such as promoters or enhancers, so that the antigens of the present disclosure can be expressed.

[0110] The CMV vectors described herein may include mutations that prevent inter-host transmission, thereby preventing the virus from infecting other subjects that are immunocompromised or may face complications due to CMV infection. The CMV vectors described herein may also include mutations that result in presentation of immunodominant and non-immunodominant epitopes and atypical MHC restrictions. However, the mutations in the CMV vectors described herein do not affect the ability of the vector to reinfect the subject previously infected with CMV. Such CMV mutations are described in, for example, U.S. Patent Publications 2013-013676S, 2010-0142S23, 2014-014103S and PCT Application Publication WO 2014 / 13S209, all of which are incorporated herein by reference.

[0111] The disclosed CMV vector can be administered in vivo, for example, for the purpose of generating an immunogenic response, including a CD8+ immune response, including an immune response characterized by a high percentage of CD8+ T cells responding to be restricted by MHC-E or MHC-II (or its homologues or orthologues). For example, in some instances, it may be desirable to use the disclosed CMV vector in laboratory animals (e.g., rhesus monkeys) for preclinical testing of immunogenic compositions and vaccines using RhCMV. In other instances, it is desirable to use the disclosed CMV vector in human subjects, such as in clinical trials of immunogenic compositions using HCMV and in actual clinical applications.

[0112] For such in vivo applications, the disclosed CMV vector is administered as a component of an immunogenic composition that also includes a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition of the present disclosure can be used to stimulate an immune response to a heterologous antigen including a pathogen-specific antigen, and can be used as one or more components of a prophylactic or therapeutic vaccine for a pathogen-specific antigen for preventing, improving or treating pathogenic infection. The nucleic acid and vector of the present disclosure are particularly useful for providing a genetic vaccine, i.e., a vaccine for delivering a nucleic acid encoding an antigen of the present disclosure to an object (e.g., a human), so that the antigen is subsequently expressed in the object to elicit an immune response.

[0113] The immunization plan (or scheme) for animals (including people) is well known and can be easily determined for specific objects and immunogenic compositions. Therefore, the immunogen can be applied to the object once or more times. Preferably, there is a time interval of setting between the separate application of the immunogenic composition. Although the interval of each object is different, it is usually 10 days to several weeks, and is usually 2 weeks, 4 weeks, 6 weeks or 8 weeks. For people, the interval is usually 2 to 6 weeks. In a particularly advantageous embodiment of the present disclosure, the interval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 ​​weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58 weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks. Immunization schemes usually have 1 to 6 administrations of immunogenic compositions, but may have as little as one or twice or four times. The method of inducing an immune response may also include administering an adjuvant with an immunogen. In some cases, annual, two-year or other long interval (5 to 10 years) booster immunizations may supplement the initial immunization scheme. The present method also includes a variety of prime-boost schemes. In these methods, one or more prime-boost immunizations are followed by one or more booster immunizations. The actual immunogenic composition of each immunization may be the same or different, and the type of immunogenic composition (e.g., comprising protein or expression vector), approach and immunogenic preparation may also vary. For example, if an expression vector is used for the first immunization and booster step, it may be the same or different type (e.g., DNA or bacterial or viral expression vector). A useful first immunization-boosting scheme provides two prime-boost immunizations, four weeks apart, followed by two booster immunizations in the 4th and 8th weeks after the last prime-boost immunization. It should also be apparent to those skilled in the art that there are a variety of permutations and combinations, including using the DNA, bacterial and viral expression vectors of the present disclosure to provide prime and boost regimens. CMV vectors can be reused to simultaneously express different antigens derived from different pathogens.

[0114] Example

[0115] Example 1: RHCMV / HBV-vaccinated rhesus monkeys initiate MHC-E restricted CD8+ T cell responses against HBV antigens

[0116] RhCMV strain 68-1 vectors engineered to express antigenic targets elicited broad effector-memory CD8+ T cell responses restricted by nonclassical molecules MHC-E, monomorphic MHC class Ib molecules normally involved in NK cell signaling, or MHC-II. RhCMV vaccine vectors expressing simian immunodeficiency virus (SIV) antigens protected 50% of RMs after repeated low-dose intrarectal and intravaginal challenges with the highly pathogenic strain SIVmac239.

[0117] Here, it was determined whether the RhCMV / HBV-induced, unconventionally restricted CD8+ T cell responses recognize HBV-infected hepatocytes. Targeting these unique CD8+ T cell-restricted molecules will constitute a new paradigm for the treatment of HBV infection, and considering the extreme conservation of MHC-E in humans and macaques, it can theoretically be universally applied 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. Immuno 1. 200: 49-60).

[0118] Four rhesus macaques (RM) were vaccinated with strain 68-1 RhCMV 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 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 the C-terminal HA epitope and PCR-mediated mutagenesis with S obtained from the plasmid pCMV-S2 / S (Michel, ML, et al. 1995. DNA-mediated immunization to the hepatitis B Surface antigen in mice: aspects of the humoral response mimicking hepatitis B Viral infection in humans. Proe. Natl. Acad. Sci. USA 92: 5307-5311.) The C-terminal 228 amino acids of Ag were fused to generate fusion S / PolN (left forward primer: 59-CATCGAGCTAGCACCATGGAGAACATCACATCAGG-39 (SEQ ID NO: 1), left reverse primer: 59-GTGTTGATAGGATAGGGGAATGTATACCCAAAGAC-39 (SEQ ID NO: 2); right forward primer: 59-GTCTTTGGGTATACATTCCCCTATCCTATCAACAC-39 (SEQ ID NO: 3), right reverse primer: 59-GGAATCGTCGACTCAAGCGTAATCTGGAACATCGTATGGGTAAAGATTGACGATAAGGGAGAGGCAG-39 (SEQ ID NO: 4)).

[0119] The final PCR product was blunt-ended into pJet vector (Thermo Fisher Scientific) as a template for bacterial artificial chromosome (BAC) recombineering or blunt-ended into pORI to evaluate expression. The C-terminal 416 amino acids of the polymerase obtained from 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 HA epitope-tagged 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 the core protein was PCR amplified from 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 the core protein using KpnI and SalI to generate a fusion protein of HBV core and the C-terminus of the polymerase (core / PolC). The KpnI site added a 2-amino acid (GT) linker between the two proteins.To generate 68-1RhCMV / Core / PolC and 68-1RhCMV / S / PolN, a modified galactokinase (galK) selection system was used to replace the pp71-encoding Rh110 gene in the 68-1RhCMV BAC (Chang, WLW, et al. 2003. Cloning of the full-length rhesus cytomegalovirus genome as an infectious and self-excisable bacterial artificial chromosome for analysis of viral pathogenesis. J. Virol. 77: 5073-5083). This is 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 was recently demonstrated that Rh110 substitution can be used to elicit a robust Ag response while attenuating the 68-1RhCMV vector (Marshall, EE, et al. 2019. Enhancing safety of cytomegalovirus-based vaccine vectors by engaging host intrinsic immunity. Sci. Transl. Med. 11: eaaw260310.1126 / scitranslmed.aaw2603).

[0120] To delete Rh110, competent SW105 bacteria containing the 68-1RhCMV BAC were electroporated with a PCR product containing a galK / kanamycin cassette with 50-bp flank homology to Rh110. The 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 HBV S-PolN fusion or an HBV core-PolC fusion with the same flank homology to Rh110 was electroporated and the bacteria were plated on 2-deoxy-galactose (DOG) chloramphenicol minimal medium plates with glycerol as a carbon source for negative selection. The PCR primers used 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). NO: 11), Rh110 core / PolC reverse: 59-CAAAATATTATTACATGGTACGCAATTTATTGTCTATTTTCGTTATTTGTTTATCTAAGCGTAATCTGGAACATCGTAT-39 (SEQ ID NO: 12).

[0121] To produce 68-1RhCMV / core protein, the HBV core protein 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).

[0122] The amplified product was cloned into pORI downstream of the EF1a promoter by homologous recombination using primers with 50-bp homology to the Rh211 region (forward primer: 59-GGGAAATCACGTCATCAGGCTGGGTAGTCAACATGGGCATACGAAACTTGCCCGAATAGATGCTCTCACTTAACGGCTGACATG-39 (SEQ ID NO: 15), reverse primer: 59-CCAGAATGTGCTCTACTTTTTGGCCAGCGGGTTGGATGATTTCGCGCGTCATGGACTGCTTCACTGTAGCTTAGTACGTTAAAC-39 (SEQ ID NO: 16))

[0123] The expression cassette is inserted into the Rh211 region of 68-1RhCMV together with the Kan resistance box flanking the flip enzyme recognition target site. The PCR fragment is electroporated in the EL250 bacteria comprising RhCMV 68-1BAC, for in vivo recombination and for Kan resistance selection recombinant. The Kan resistance box is removed by temperature-induced flip enzyme recombination. The BAC gained is analyzed by the next generation sequencing on the PCR analysis of restrictive digestion, recombination site and Illumina MiSeq sequencer. The sequence analysis shows two point mutations in the S / PolN introduced during the PCR amplification, resulting in the amino acid exchange of A118T and T125M in SAg. BAC DNA was purified using alkaline lysis, phenol / chloroform extraction, and isopropanol precipitation, and virus was reconstituted by transfection of BAC DNA using rhesus fibroblasts expressing telomerized pp71 (Warming, S. et al. 2005. Simple and highlyefficient BAC recombineering using galK selection. Nucleic Acids Res. 33: e36) or primary rhesus fibroblasts using Lipofectamine 2000 (according to the manufacturer's protocol; Thermo Fisher Scientific). Expression of HBV Ag was determined by infecting telomerized RM fibroblasts with 68-1 RhCMV / surface / PolN or RhCMV / core / PolC. Cells were harvested under complete cytopathic effect and lysed in SDS sample buffer. 293T cells transfected (Lipofectamine 2000) with pORI expression plasmid containing HA-tagged HBV proteins served as positive controls. After electrophoretic separation, immunoblotting was performed with anti-HA Ab MMS-101P (Covance MMS).

[0124] Two RMs (RM1, RM2) were vaccinated with both 68-1RhCMV / surface / PolN and 68-1RhCMV / core / PolC vectors, and the other two RMs (RM3, RM4) were vaccinated with a 68-1RhCMV-based vector expressing HBV core under the EF1a promoter. CD8+ T cell responses to each Ag were monitored longitudinally by ICS using a pool of overlapping 15-mer peptides corresponding to each Ag. Longitudinal CD8+ T cell responses to these antigens were observed in the blood of vaccinated RMs ( Figure 1A ).

[0125] The MHC restriction of HBV core (HBcAg)-specific CD8+ T cell responses in these animals was characterized by intracellular cytokine staining with agents that specifically block MHC-I, MHC-II, and MHC-E presentation as previously described (Hansen et al. 2016. Broadly targeted CD8 + T cell responses restricted by majorhistocompatibility complex E. Science 351: 714-720; Hansen, et al. 2013. Cytomegalovirus vectors violate CD8+ T cell epitope recognition paradigms. Science 340: 1237874-1237874.). It was found that similar to the strain 68-1RhCMV vector expressing SIV or Mycobacterium tuberculosis antigens, the strain 68-1RhCMV / HBV vector elicited HBV-specific, MHC-E and MHC-II restricted CD8+ T cell responses targeting a broad array of HBcAg peptides ( Figure 1B ).

[0126] To further define the MHC-E restriction of RhCMV / HBV that elicits 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 cultured with Figure 1BThe blocking agent in the HBcAg was pulsed with one of three separate HBcAg 15-mer peptides identified as MHC-E restricted. Only cells expressing MHC-E can present HBcAg 15-mers to these CD8+ T cells. HBcAg-specific CD8+ T cells recognize their cognate antigen presented in the context of both HLA-E and Mamu-E ( Figure 1C These results indicate the presence of MHC-E-restricted HBV-specific CD8+ T cells in RhCMV / HBV-vaccinated RM and further support the highly functional conservation of primate MHC-E molecules.

[0127] Example 2: HBV-infected primary hepatocytes express MHC-E in vitro

[0128] Next, determine whether primary hepatocytes express MHC-E. Primary hepatocytes (PH) were isolated from three unrelated RM and three unrelated human donors (HD). To isolate RM primary hepatocytes, a single lobe of RM liver was perfused with 200 mL of pre-perfusion medium (0.5 mM EGTA (Bio-World, catalog number: 40120128-1), 10 IU / mL heparin (Fresenius Kabi, catalog number: C504730), HBSS with calcium and magnesium (Fisher Scientific, catalog number: 24-020-117)), followed by removal of the remaining EGTA by 200 mL of HBSS without calcium and magnesium (Fisher Scientific, catalog number: SH3003103). Next, 100 mL of collagenase medium (DMEM / F12 (Gibco, catalog number: 11320-082), 1 mM calcium chloride (Sigma-Aldrich, catalog number: C5670-100G), 20 mM HEPES (HyClone, catalog number: SH30237.01), 1 mg / mL collagenase IV (Sigma-Aldrich, catalog number: C9722-50MG)) warmed to 42°C was perfused into the lobe and discarded. The 150 ml collagenase medium was then recirculated through the liver lobe at a rate of 75 to 150 mL / min at 42°C for 30 minutes to 1 hour, depending on the size of the lobe. After collagenase perfusion, the liver was sliced ​​with a scalpel, rinsed with the remaining collagenase medium, and the medium was filtered through a tea strainer. PH was washed three times at room temperature in wash medium (DMEM / F12, 2% bovine growth serum (HyClone, catalog number: SH3054103), 23 mM HEPES buffer, 0.6 mg / ml glucose, 2 mM L-glutamine (HyClone, catalog number: SH3003401), 1x antibiotic / antimycotic (HyClone, catalog number: SV3007901), and 0.1 mg / mL gentamicin (LifeTechnologies, catalog number: 15750-060)) and centrifuged at 50 x g for 3 minutes between each wash. Prior to the third wash spin, PH was passed through a 70 μM filter to ensure a single cell suspension. PH was then suspended in 20 mL of 36% isotonic percoll (GE Healthcare, catalog number: 17-0891-01) in a 50 mL conical flask using PH medium as a 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. Collagen plates of hepatocytes were prepared using 0.2 mg / mL collagen R in 0.01% acetic acid (Serva, catalog number: 47254), washed with 1 mL HBSS immediately after being placed on the plate for at least 20 minutes, and then plated in 12-well plates at 2×10 per well. 5 PH was plated. The plates were placed at 37°C, 5% CO2. The next day, the wells were washed twice with HBSS and cultured in 1 ml PH medium (primary hepatocyte medium containing DMSO; PH-DMSO) supplemented with 1.8% DMSO for the remainder of the experiment.

[0129] Human donor primary hepatocytes (HD PH) were isolated from murine humanized liver and purchased from Yecuris, Inc. Humanized mice were generated using cryopreserved primary hepatocytes collected from deceased patients with the following demographic characteristics: HD1 (13 years old, female, Hispanic, HBV uninfected); HD2 (13 years old, female, Caucasian, HBV uninfected); HD3 (27 years old, male, Caucasian, HBV uninfected).

[0130] RM and HD PH MHC presentation was determined by surface expression of abundant MHC-I by the W6 / 32 clone, MHC-II by HLA-DR staining, and MHC-E by the 4D12 clone. 4D12 has been previously shown to specifically stain Mamu-E and not the classical Mamu-Ia molecules. All three human donors shared one HLA-A and one HLA-C allele (Table 1). Therefore, before proceeding, it was determined that the MHC-E-specific 4D12 clone stained only HLA-E and not HLA-A or HLA-C molecules shared between the three HDs ( Figure 2A ). The expression of MHC-E was examined by staining, and most primary hepatocytes from both species expressed MHC-E ( Figure 2B and 2C ).

[0131] Table 1. MHC genotypes of HD PH

[0132] HLA-A HLA-A HLA-B HLA-B HLA-C HLA-C HD1 02∶17 02∶01 40∶02 50∶01 03∶05 06∶02 HD2 01∶01 02∶01 15:XX* 37:XX* 03∶03 06∶02 HD3 02∶01 02∶01 57∶01 27∶05 02∶02 06∶02

[0133] XX* indicates undetermined alleles.

[0134] To determine whether HBV infection affects the expression of MHC-E on the surface of primary hepatocytes, primary hepatocytes were collected from the same HBV-infected human donor. One day after plating isolated rhesus primary hepatocytes, a replication-incompetent adenovirus serotype 5 expressing human NTCP (MOI 10) under the liver-specific TTR promoter was added to the culture for 2 days. On the second day, the cells were re-fed with 1 ml PH-DMSO medium. On the fourth day after adenoviral transduction, primary hepatocytes were washed twice in 1 ml HBSS and covered with HBV-containing medium (PH-DMSO containing 4% PEG6000, Sigma-Aldrich, catalog number: 81253-250G) with an MOI of 100 and incubated overnight. The next morning, the wells were washed three times with 1 ml HBSS and then cultured in 1 ml primary hepatocyte-DMSO for the remainder of the experiment.

[0135] One day after plating, human donor primary hepatocytes were covered and incubated overnight with HBV-containing medium (PH-DMSO containing 4% PEG6000, Sigma-Aldrich, catalog number: 81253-250G) at an MOI of 100. The next morning, the wells were washed three times with 1 ml HBSS and then cultured in 1 ml primary hepatocyte-DMSO for the remainder of the experiment.

[0136] HBV infection of human donor primary hepatocytes was determined by measuring the level of HBV envelope antigen (HBeAg) in the supernatant before cell staining. On day 4 after HBV infection, primary hepatocytes were co-stained with MHC markers (MHC-I, MHC-E, and MHC-II) and intracellular HBcAg, as this was the first time point at which intracellular HBcAg could be detected. Strong staining using the 4D12 antibody was observed on both HBV-infected and HBV-uninfected PH, indicating high levels of MHC-E expression ( Figure 2D and 2E). In contrast, HLA-DR expression was minimal or absent in primary hepatocyte samples from all three human donors, which 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.). Ex vivo manipulation of primary hepatocytes prior to assessment of surface MHC levels may induce a portion of these cells to lose MHC-E expression. However, taken together, these results show that HBV-infected primary hepatocytes express MHC-E and that MHC-E may represent a potential HBV-specific CD8+ T cell restriction element.

[0137] Example 3: MHC-E-restricted CD8+ T cells from RHCMV / HBV-vaccinated rhesus macaques recognize HBV-infected allogeneic and xenogeneic primary hepatocytes

[0138] MHC studies showed high levels of MHC-E expression on human donor and rhesus primary hepatocytes, regardless of HBV infection. Therefore, considering the high functional conservation of MHC-E in primates, it was assumed that CD8+ T cells from RhCMV / HBV-vaccinated rhesus macaques recognized allogeneic HBV-infected rhesus macaque primary hepatocytes. In support of this hypothesis, CD8+ T cells from RM1 and RM2 (bulk splenocytes and purified CD8β+ T cells) recognized HBV-infected primary hepatocytes from two unrelated rhesus macaque donors, but did not respond to HBV-uninfected primary hepatocytes ( Figure 3A ).

[0139] To more fully define the MHC restriction of CD8+ T cells recognizing HBV-infected primary hepatocyte targets, a series of recognition experiments were performed using the MHC-specific blockers shown in Figure 1 .

[0140] Before HBV infection, primary hepatocytes of one well were collected and stained as a baseline. Starting from the second day after infection, each well of HBV-infected and HBV-uninfected primary hepatocytes was collected with 0.5% trypsin-EDTA (FisherScientific, catalog number: SH30236.01) and washed twice in ice-cold FACS buffer (PBS, FisherScientific, catalog number: SH30256FS, with 10% fetal bovine serum). The cells were incubated at 4°C for 30 minutes with anti-HLA-E antibody (clone: ​​4D12, Origene, catalog number: LS-C179742), washed twice in ice-cold FACS buffer, and incubated at 4°C for 30 minutes with F(ab)2-goat anti-mouse IgG (HL)-APC (Invitrogen, catalog number: A10539). The cells were then washed twice in ice-cold PBS and incubated with pan-MHC-I-PerCP-Cy5.5 (clone: ​​W6 / 32, Biolegend Inc., catalog number: 311419), anti-HLA-DR Alexa 700 (clone: ​​L243 (BD ​​Biosciences, catalog number: 560743) and Live / Dead Fixable Yellow (Invitrogen, catalog number: L-34959) for 30 minutes at 4°C. The cells were washed in FACS buffer and fixed for one hour at room temperature using Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, catalog number: 00-5523-00). Prior to fixation, all wash spins were performed at 350×g for 3 minutes. After fixation, the cells were suspended in permeabilization buffer (eBioscience, catalog number: 00-8333-56). All wash spins after fixation were performed at 830×g for 3 minutes. The cells were then fixed using Lightning-Link R-PE kit (Innova Biosciences, catalog number: 703-0005) Primary hepatocytes were incubated with R-phycoerythrin (PE)-conjugated hepatitis B virus core antigen antibody (clone: ​​13A9, Fisher Scientific, catalog number: MA1-7606) at 4 ° C for one hour. The cells were washed three times in permeabilization buffer and then collected on Becton-Dickenson LSR-II. Analysis was performed on FlowJo X (TreeStar Inc.). In all analyses, specific MHC and HBV markers were evaluated after gating the light scattering characteristics of large complex PH.

[0141] HBV-specific CD8+ T cell responses were measured by flow cytometry ICS in mononuclear cell preparations from the spleen of RMs vaccinated with RhCMV / HBV. In brief, splenocytes or isolated CD8β+T cells were incubated with HBV-infected or HBV-uninfected primary hepatocyte targets and co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour, followed by the addition of brefeldin A (Sigma-Aldrich) for another 8 hours. Co-stimulation without primary hepatocyte target co-culture was used as a background control. The MHC restriction of the response was determined by pre-incubating the PH target for 1 hour at room temperature before co-culturing with target primary hepatocytes in the presence of pan-anti-MHC-I antibodies (25 μg / ml; clone: ​​W6-32), VL9 peptides (20uM), CLIP peptides (MHC-II-related constant chains, amino acids 89 to 100; 10 μg / ml) or anti-HLA-DR antibodies (10 μg / ml; clone: ​​L243). Stimulated cells were fixed, permeabilized and stained, and flow cytometry analysis was performed on an LSR-II instrument (BD Biosciences). Analysis was performed using FlowJo X software (Tree Star, Inc.). In all analyses, after gating the light scattering characteristics of small lymphocytes, the CD3+ group was progressively gated, and then the CD4- / CD8+ T cell subsets. The antigen-specific response frequency of the CD8+ T cell group is usually determined by the expression of intracellular IFN-γ.

[0142] HBV-infected or HBV-uninfected targets were collected on day 6 after HBV infection (MOI = 100), incubated with blocker W6 / 32 antibody (pan-MHC-I), VL9 peptide (MHC-E), CLIP (MHC-II), or HLA-DR antibody (MHC-II), and then co-cultured with splenocytes or isolated CD8β+T cells overnight. After co-culture, CD8+ T cells were intracellularly stained for IFN-γ and TNF-α to assess target recognition. CD8+ T cells that recognized HBV-infected RM PH were blocked with W6 / 32 antibody and VL9 peptide, but not with CLIP or HLA-DR, indicating that responses to HBV-infected targets were all restricted by MHC-E ( Figure 3A ).

[0143] Because MHC-E is functionally conserved in primates, it was hypothesized that CD8+ T cells from RhCMV / HBV-inoculated RMs also recognize HBV-infected HD primary hepatocytes. To test this hypothesis, similar recognition experiments were performed by incubating splenocytes and purified CD8+ T cells from the same RhCMV / HBV-inoculated rhesus macaques (RM1 and RM2) with HBV-infected human donor primary hepatocytes. As hypothesized, these CD8+ T cells recognized HBV-infected xenogeneic human donor primary hepatocytes ( Figure 3B ). As described above for the rhesus primary hepatocyte target co-culture experiments, CD8+ T cells recognizing HBV-infected primary hepatocytes from human donors were completely blocked by MHC-E binding to the VL9 peptide. In summary, these results clearly show that HBV-infected primary hepatocytes present HBV antigens in the context of MHC-E, indicating that this approach can be used to target CD8+ T cells to HBV-infected cells.

[0144] Next, the conservation of two 15-mer peptides in the HBV core antigen, core protein 7 (PSVRDLLDTASALYR; SEQ ID NO: 17) and core protein 14 (TALRQAILCWGELMT; SEQ ID NO: 18) MHC-E binding superepitopes was examined. 6,203 whole-genome HBV sequences spanning all known HBV genotypes were retrieved from the Hepatitis B Virus Database, translated, and targeted to core protein 7 ( Figure 4A ) and core protein 14 ( Figure 4B ) were used for amino acid sequence alignment. High conservation (super-epitopes) was present in two 15-mer peptides that generated MHC-E restricted CD8+ T cell responses in all animals assayed. Importantly, at two positions that were not highly conserved (core protein 7 at position 3 and core protein 14 at position 15), there was only one additional dominant amino acid at that position in global HBV strains. Thus, more than 98% of known global sequences express one of the two amino acids at these positions.

[0145] These results identify a completely new group of CD8+ T cell responses against HBV, which paves the way for the development of innovative HBV treatments. Although 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 reports of MHC-E restricted CD8+ T cell responses against HBV have been published. Therefore, it is unclear whether HBV-infected hepatocytes present HBV antigens in the context of MHC-E. These results show that MHC-E does present HBV antigens on the surface of HBV-infected cells, and CD8+ T cells from completely different primate species can recognize these MHC-E: peptide complexes.

[0146] In addition to representing a completely unique type of CD8+ T cell response against HBV, the breadth of epitopes targeted within HBcAg suggests that therapeutic vaccination with CMV / HBV vectors will elicit a broadly targeted CD8+ T cell response. Although this broad targeting has been previously shown against SIV, Mycobacterium tuberculosis, and malaria, it may be particularly effective against HBV because the vast majority of the HBV genome consists almost entirely of non-plastic overlapping reading frames. Together, these results show for the first time that MHC-E-restricted CD8+ T cells can be used to treat chronic HBV infection, either through therapeutic vaccination or adoptive immunotherapy. Sequence Listing <110> OREGON HEALTH & SCIENCE UNIVERSITY <120> HBV-specific T cell responses <130> 4153.011PC01 <150> 62 / 858,764 <151> 2019-06-07 <160> 18 <170> PatentIn Version 3.5 <210> 1 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Left reverse primer <400> 1 catcgagcta gcaccatgga gaacatcaca tcagg 35 <210> 2 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Right forward primer <400> 2 gtgttgatag gataggggaa tgtataccca aagac 35 <210> 3 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Right reverse primer <400> 3 gtctttgggt atacattccc ctatcctatc aacac 35 <210> 4 <211> 67 <212> DNA <213> Artificial sequence <220> <223> Left forward primer <400> 4 ggaatcgtcg actcaagcgt aatctggaac atcgtatggg taaagattga cgataaggga 60 gaggcag 67 <210> 5 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Forward Primer <400> 5 gtggtaccct cgaggattgg ggaccctgcg ctgaacatgg ag 42 <210> 6 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 6 tcagtcgacc taagcgtaat ctggaacatc gtatgggtac 40 <210> 7 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Forward Primer <400> 7 ctgctagcat ggacattgac ccttataaag aatttgg 37 <210> 8 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 8 ctaggtacca cattgagatt cccgagattg ag 32 <210> 9 <211> 75 <212> DNA <213> Artificial sequence <220> <223> Rh110 S / PolN forward <400> 9 gatcacgtca ttgacaccgg cctcccacca gctctcacat tctccgcatc accatggaga 60 acatcacatc aggat 75 <210> 10 <211> 79 <212> DNA <213> Artificial sequence <220> <223> Rh110 S / PolN Reverse <400> 10 caaaatatta ttacatggta cgcaatttat tgtctatttt cgttatatttgt ttattcaagc 60 gtaatctgga acatcgtat 79 <210> 11 <211> 78 <212> DNA <213> Artificial sequence <220> <223> Rh110 core / PolC forward <400> 11 gatcacgtca ttgacaccgg cctcccacca gctctcacat tctccgcatc accatggaca 60 ttgaccctta taaagaat 78 <210> 12 <211> 79 <212> DNA <213> Artificial sequence <220> <223> Rh110 core / PolC reverse <400> 12 caaaatatta ttacatggta cgcaatttat tgtctatttt cgttatatttgt ttatctaagc 60 gtaatctgga acatcgtat 79 <210> 13 <211> 55 <212> DNA <213> Artificial sequence <220> <223> Forward Primer <400> 13 ctgctagcat ggattacaag gatgacaagg acatcgaccc ttataaagaa tttgg 55 <210> 14 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 14 ctagtcgaca cattgagatt cccgagattg ag 32 <210> 15 <211> 84 <212> DNA <213> Artificial sequence <220> <223> Forward Primer <400> 15 gggaaatcac gtcatcaggc tgggtagtca acatgggcat acgaaacttg cccgaataga 60 tgctctcact taacggctga catg 84 <210> 16 <211> 84 <212> DNA <213> Artificial sequence <220> <223> Reverse primer <400> 16 ccagaatgtg ctctactttt tggccagcgg gttggatgat ttcgcgcgtc atggactgct 60 tcactgtagc ttagtacgtt aaac 84 <210> 17 <211> 15 <212> PRT <213> Artificial sequence <220> <223> antigen <400> 17 Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg 1 5 10 15 <210> 18 <211> 15 <212> PRT <213> Artificial sequence <220> <223> antigen <400> 18 Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr 1 5 10 15

Claims

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

2. Use of a CMV vector expressing an HBV antigen in the preparation of a medicament for producing an immune response against HBV in a subject, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their direct homologs, wherein the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

3. Use of a CMV vector expressing an HBV antigen in the preparation of a drug for treating chronic HBV infection, wherein the CMV vector does not express active UL128, UL130, UL146 and UL147 proteins, or their direct homologs, wherein the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17) or TALRQAILCWGELMT (SEQ ID NO: 18).

4. The use according to claim 2 or 3, wherein the hepatitis B antigen is PSVRDLLDTASALYR (SEQ ID NO: 17).

5. The use according to claim 2 or 3, wherein the hepatitis B antigen is TALRQAILCWGELMT (SEQ ID NO: 18).

6. The use of claim 2 or 3, wherein at least 10% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its ortholog.

7. The use of claim 6, wherein at least 20% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its orthologs.

8. The use of claim 6, wherein at least 30% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its orthologs.

9. The use of claim 6, wherein at least 40% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its orthologs.

10. The use of claim 6, wherein at least 50% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its ortholog.

11. The use of claim 6, wherein at least 60% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its orthologs.

12. The use of claim 6, wherein at least 75% of the CD8+ T cells primed by the CMV vector are restricted by MHC-E or its orthologs.

Images