Use of HLA-A*11:01 restricted hepatitis B virus (HBV) peptide for identifying HBV-specific CD8+ T cells.

Abstract

This invention relates to peptides and their ability to identify and bind to T cells specific to HBV-infected hepatocytes. In a first aspect, a peptide comprising an amino acid sequence selected from the group consisting of: STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR, wherein the peptide is derived from hepatitis B virus nucleus 169 and is capable of binding HLA-A*1101, and when binding to HLA-A*1101, is capable of identifying T cells specific to hepatitis B virus. In a second aspect, a T cell expressing a T cell receptor (TCR) molecule, wherein the TCR molecule comprises an amino acid sequence selected from the group consisting of: CASGDSNSPLHF, CASGGQIVYEQYF, CSARGGRGGDYTF, and CASSQDWTEAFF, the T cell receptor being capable of binding to the peptide according to the first aspect of the invention.

Description

[0001] This invention relates to peptides and their ability to identify and bind to T cells that are specific to HBV-infected hepatocytes.

[0002] The listing or discussion of previously published documents in this specification is not necessarily an admission that such documents are part of the prior art or general common sense.

[0003] Any document mentioned in this article is hereby incorporated in its entirety by reference.

[0004] Chronic HBV infection (CHB) remains a major health concern and a leading cause of hepatocellular carcinoma (HCC) worldwide. Despite the availability of effective vaccines, there is no cure for CHB, and many patients are diagnosed only in later stages of the disease with limited therapeutic efficacy. The estimated mortality rate from viral hepatitis has increased by more than 50% in the past decade. CHB progresses to a series of stages defined by a few clinical parameters with limited relevant immunological evidence. Acquired immunity is not fully understood as young patients progress from the immune-tolerant stage (IT, or HBeAg+ chronic infection, high viremia, but limited liver inflammation) to the progressively immune-active stage (IA, or HBeAg+ chronic hepatitis, high viremia and high liver inflammation), and for some, spontaneously becoming HBeAg-inactive carriers (InA, or HBeAg-chronic infection, low to undetectable viral load and limited liver inflammation). Some argue that these definitions, which may affect early treatment opportunities, should be revised.

[0005] Despite their low frequency in most CHB patients, virus-specific T cell responses have garnered significant attention from HBV immunologists. Pioneering experiments in CHB patients and animal models, such as HBV-challenged chimpanzees, have confirmed the indispensable role of virus-specific CD8+ T cells in viral control. Historically, mapping of potential virus-specific CD8+ T cells against HBV has primarily focused on the HLA-A*02:01-restricted epitope. For example, multiple studies targeting single epitopes of A*02:01-restricted HBVcore18-27-specific CD8+ T cells have provided numerous hints for immunotherapy. However, in Asia, where HBV infection is highly prevalent, the dominant allele in common East Asian populations is A*11:01, whose immunogenicity against chronic HBV is poorly defined. Therefore, there is an unmet need to investigate the existence of A*11:01-restricted HBV-specific CD8+ T cells in CHB. Furthermore, regardless of MHC restriction, information about the unmanipulated phenotype of HBV-specific T cells is lacking due to their extremely low frequency.

[0006] In murine chronic lymphocytic choriomeningitis virus (LCMV) infection, prolonged and elevated viral antigen exposure, coupled with the upregulation of multiple inhibitory receptors on virus-specific T cells, has led to the definition of a T cell exhaustion state. The similarity to exhausted T cells (TEX) observed in human chronic viral infections provides an explanation for the dysfunction of the immune response but also points to valuable targets for enhancing host immunity. There is evidence that such TEX cells arise from altered developmental pathways of memory T cells. In addition to several defects described in detail, the hallmark of T cell exhaustion is the gradual loss of functional capacity, which is associated with the cumulative expression of inhibitory receptors during sustained antigen stimulation. This impaired T cell subset has been described in HIV and HCV infections; however, results from other studies do not necessarily conform to this model. Therefore, by simultaneously measuring a wide range of inhibitory receptors and memory-related markers, we aimed to evaluate the degree to which the characteristics of HBV-specific T cells match this “graded T cell exhaustion” model in human CHB.

[0007] Here, to overcome the challenges associated with identifying and deeply profiling unmanipulated HBV-specific T cells, mass cytometry combined with a highly multiplexed peptide-MHC (pMHC) tetramer strategy was used to simultaneously screen and query 562 A*11:01-restricted T cell candidate epitopes. Using self-validated automated tetramer deconvolution and unsupervised high-dimensional analysis, virus-specific CD8+ T cells targeting HBVpol387 and HBVcore169 were found to exhibit complex phenotypic characteristics and T cell receptor sequence usage that co-evolve with HBV infection status. Based on high-dimensional trajectory analysis, the characteristics of HBV-specific T cells from the blood were also found to indicate the degree of viral control in patients from two separately analyzed cohorts.

[0008] The inventors have identified the amino acid sequences and compositions of peptides and variants derived from HLA-A1101-restricted HBVcore169 (also known as HBVcore141). Such epitopes can be used to induce cellular responses and trigger multifunctional antiviral T-cell activity against HBV. Specifically, this invention discloses isolated peptides comprising seven oligopeptides, including STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR. These isolated peptides can be used as antigens in epitope-based therapeutics / vaccines or immunotherapies to prevent and / or treat hepatitis B virus (HBV) infection in patients.

[0009] Furthermore, the inventors have identified amino acid and nucleotide sequences containing four HLA-A*11:01-restricted epitope-reactive T-cell receptors (TCRs) specific to HBVcore169: CASGDSNSPLHF, CASGGQIVYEQYF, CSARGGRGGDYTF, and CASSQDWTEAFF. For HLA-A11-1 positive patients, such TCR sequences can be constructed using adoptive transfer immunotherapy and encoded onto T cells to target HBV-infected hepatocytes.

[0010] The general structure of T cell receptors (TCRs), their domain structures, and the organization of the genes encoding them are well known, for example, see Chapter 11 of *Immunology*, 2nd edition (1994), Janis Kuby, WH Freeman and Co., New York, USA, and Garcia et al. (1999) Ann. Rev. *Immunol*. 17, 369-397. A common class of natural TCRs is the αβ class, in which a TCR consists of a single α chain and a single β chain, forming a heterodimer associated with the T cell membrane. Each α and β chain consists of regions from the N-terminus to the C-terminus, namely a leader sequence, a variable region, a constant region, a linker sequence, a transmembrane region, and a cytoplasmic tail region. The variable region of the α chain is called the Vα region, and the variable region of the β chain is called the Vβ region. Similarly, the constant region of the α chain is called the CI region, and the constant region of the β chain is called the Cβ region. The function of αβ TCRs is to recognize and bind peptides presented in HLA molecules within cells in vivo. Generally speaking, TCRs cannot recognize and bind to peptides unless they are presented by specific HLA molecules, and they cannot recognize HLA molecules unless they present specific peptides. T cells with occult-specific TCRs will target cells that present specific peptides in specific HLA molecules on the cell (i.e., peptide-HLA complexes), and this is based on the main principle of T cell immunity.

[0011] The peptide-HLA complex is recognized by the V region, a combination of the α and β chains of the TCR. Specifically, the complementarity-determining region (CDR) of the V region mediates the recognition of the peptide-HLA complex. The V region of the α and β chains of the native TCR consists of FR1, CDR1, FR2, CDR2, FR3, and CDR3, from the N-terminus to the C-terminus, where FR stands for "backbone region" and CDR stands for "complementarity-determining region." The FRs and CDRs of the α and β chains are different. Notably, the CDR3 of the β chain is encoded by the V(D)J segment, giving it higher sequence diversity than that of the α chain. It is also well known that the β chain CDR3 is a key determinant for TCR recognition of peptide-MHC complexes.

[0012] In a first aspect of the invention, a peptide comprising an amino acid sequence selected from the group consisting of: STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR is provided, wherein the peptide is derived from hepatitis B virus nucleus 169 and is capable of binding HLA-A*1101, and when binding HLA-A*1101, is capable of identifying T cells specific to hepatitis B virus.

[0013] Whether a peptide binds to HLA-DRB1*04 can be determined using any method known in the art.

[0014] The recognition of harmful pathogens or pathogenic mutations within the body's own tissues occurs through two mechanisms within the human immune system. Antibody molecules expressed by B cells bind in a highly specific manner to biomolecules normally expressed on the surface of invading microorganisms or cells different from the body's own, and these molecules are labeled in a way that triggers an appropriate immune response. In addition to the antibody response, pathogens and pathogenic diseases are detected due to the unique proteins expressed by the pathogen or mutation. These proteins are broken down into small peptide fragments in human cells through a natural and continuous protein degradation system. These peptide fragments bind to specific molecules expressed on all cell surfaces (called MHC class I and MHC class II molecules). In humans, these molecules are called HLA molecules and are numbered based on the large number of alleles present in the population.

[0015] In humans, peptide fragments derived from pathogens bind to specific HLA molecules and are transported to the cell surface. These peptides are captured in a specific conformation that allows for detection via T cell receptors (“TCRs”) expressed on the surface of T cells. Through natural selection and developmental processes related to the immune system’s ability to detect danger signals associated with the presence of foreign organisms, the human body produces T cells with TCRs that can recognize and distinguish peptide fragments derived from harmful pathogens from peptides derived from harmless microorganisms or from the body’s own healthy tissues.

[0016] MHC class I molecules present peptides, primarily derived from proteins already present in the cell, to CD8+ T cells, also known as cytotoxic T cells or CTLs. Peptides binding to MHC class I molecules are typically 8-10 amino acids long. MHC class II molecules present peptides derived from proteins already endocytosed from the extracellular environment or from other organisms. MHC class II molecules present peptides to CD4+ T cells, also known as T helper cells, although CD4+ T cells can also have direct cytotoxic functions. Peptides binding to MHC class II molecules are relatively unrestricted in length, but class II peptides typically fall within the 13-17 amino acid range, for example, 14, 15, or 16 amino acids.

[0017] A particular advantage of the peptide of the present invention is that it binds to the HLA-A*1101 molecule presented on an HLA allele that appears to be prevalent in Asian patient populations (statistics that are not sufficiently studied).

[0018] The term "peptide" includes not only molecules in which amino acid residues are linked by peptide (-CO-NH-) bonds, but also molecules in which peptide bonds are reversed. Such reverse-peptide mimics can be prepared using methods known in the art, such as those described by Mézière et al. (1997) J. Immunol. 159, 3230-3237. This method involves preparing pseudopeptides containing changes in scaffold orientation rather than side chain orientation. Mézière et al. (1997) showed that these pseudopeptides are useful, at least for MHC class II and T helper cell responses. Reverse-peptides containing NH-CO bonds instead of CO-NH peptide bonds are more resistant to proteolysis. We also include any protein or polypeptide that, due to its origin or derived source, is not associated with the naturally associated components that accompany it in its native state; and is substantially free of other proteins from the same source. Protein purification techniques known in the art can be used to render the protein substantially free of naturally associated components or to purify it substantially by separation. The peptides / proteins obtained in this invention may be "substantially purified," which means that the proteins are substantially free of contaminants, for example, at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminants.

[0019] Similarly, peptide bonds can be omitted, provided that a suitable linker portion is used that retains the spacing between the Cα atoms of the amino acid residues; it is particularly preferred if the linker portion has substantially the same charge distribution and substantially the same peptide bond planarity.

[0020] It should be understood that peptides can be conveniently blocked at their N-terminus or C-terminus to help reduce their sensitivity to exonuclease hydrolysis. Similarly, it should be understood that the peptides of the present invention may be in salt form or may be esters or amides containing additional -OH or -COOH groups or -NH2 groups. The peptides of the present invention are defined in the claims and will include any variations.

[0021] By "variants" of a given amino acid sequence, we mean that one, two, or three side chains of the amino acid residues are altered (e.g., by replacing them with another naturally occurring amino acid residue or some other side chain) so that the peptide can still bind to HLA molecules in substantially the same way as a peptide composed of the given amino acid sequence. For example, a peptide can be modified to at least maintain (if not improve) its ability to interact with and bind to HLA-A*1101, and to at least maintain (if not improve) its ability to generate activated CD8*T cells that recognize hepatitis E virus. Typically, amino acid substitutes are conserved in nature and come from classes such as Gly, Ala; Ile, Leu, Val; Ser, Thr; Tyr, Phe, Trp; Glu, Asp; Gln, Asn, His, Met, Cys, Ser.

[0022] Peptides of at least 15 amino acids are preferred. Therefore, the present invention also includes peptides of 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, containing an amino acid sequence selected from the group consisting of: STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR. As described above, the peptides of the present invention are capable of binding to HLA-A*1101.

[0023] Those amino acid residues that are not essential for interaction with T cell receptors can be modified by replacing them with another amino acid, the incorporation of which substantially does not affect T cell responsiveness and does not eliminate binding to the relevant HLA allele.

[0024] The peptides of the present invention (and the peptides used in the present invention) are less than 5,000, typically about 4,000, 3,000, or 2,000. In terms of the number of amino acid residues, the peptides of the present invention may have fewer than 30, 20, 19, 18, 17, 16, 15, 14, 13, or 12.

[0025] It should be understood from the following that in some applications, the peptides of the present invention may be used directly (i.e., they are not produced by expression of polynucleotides in the patient's cells or in the cells given to the patient); in such applications, it is preferred that the peptides have fewer than 30 or 25 or 24 or 23 or 22 or 21 or 20 or 19 or 18 or 17 or 16 or 15 or 14 or 13 or 12 residues.

[0026] The peptides of this invention are capable of binding to HLA-A*1101. Selective binding of the peptide to HLA-A*1101 is particularly preferred.

[0027] More preferably, the peptide of the present invention is a peptide that can be used to generate specific CD8+ T cells that mediate the specific killing of hepatitis B virus.

[0028] The peptide (HBVcore169) of this invention is particularly useful in immunotherapies against hepatitis B virus. Specifically, the combination of the peptide with specific HLA molecules can be used to select and define appropriate T cells, and track them once introduced into a patient. Particularly preferred is that, in all immunotherapies of this invention, the patient to be treated is a patient carrying class I HLA-A*1101 (i.e., having a class I HLA-A*1101 positive genotype) and possessing antigen-presenting cells expressing HLA-A*1101.

[0029] The peptides of the present invention (and the peptides used in the present invention) are peptides that bind to HLA-A*1101, and when these bind, the HLA-A*1101-peptide complex (when present on the surface of suitable antigen-presenting cells) can elicit a T cell-mediated immune response that mediates or contributes to the immune system's attack on hepatitis B virus. Specifically, the production of cytokines by CD8+ T cells can mediate the attack on hepatitis B virus.

[0030] Peptides (at least those containing peptide bonds between amino acid residues) can be synthesized using any method well known in the art, such as the Fmoc-polyamide mode for solid-phase peptide synthesis disclosed by Lu et al. (1981) J. Org. Chem. 46, 3433 and references therein. Reagents used for peptide synthesis are generally available from commercial suppliers of chemical and biological reagents. Purification can be achieved by any or a combination of techniques such as size exclusion chromatography, ion exchange chromatography, and (primarily) reversed-phase high-performance liquid chromatography. Analysis of peptides can be performed using thin-layer chromatography, reversed-phase high-performance liquid chromatography, amino acid analysis after acid hydrolysis, and analysis by rapid atomic bombardment (FAB) mass spectrometry.

[0031] In another aspect of the invention, a method for selecting hepatitis B virus antigen-specific T cells is provided, the method comprising contacting a population of T cells with a peptide according to a first aspect of the invention. Preferably, the method comprises contacting the population of T cells with the peptide or polypeptide of the invention presented in an HLA-A*1101 molecule to which the peptide is bound.

[0032] In one implementation, the T cell population is derived from individuals who have been exposed to the hepatitis B virus.

[0033] It should be understood that the peptides of the present invention can be used to generate T-cell amplification specific to hepatitis B virus in HLA-A*1101-positive patients, and there are several ways in which the present invention can be used.

[0034] The understanding that the peptide of the present invention binds to the HLA-A*1101 molecule and is recognized only by T cells in this case means that the generation of peptide-MHC multimers that directly bind to T cells can occur. This reagent can be used to directly select hepatitis B virus-specific cells from large-scale cultures for infusion of highly purified cells into patients.

[0035] This invention also includes the use of MHC multimers, along with other (to be defined) specific multimers, for the purpose of fully characterizing the product prior to infusion to ensure its purity and safety. Following infusion of hepatitis B virus T cells into a patient, the peptide / HLA-A*1101 multimer can be used directly in vitro to monitor the remodeling of hepatitis B virus-specific immunity in the patient.

[0036] Another application of this technology will be to replace the innate antigen-presenting capacity of the patient's own cells to present the peptides of the present invention in order to amplify antigen-presenting cells. Artificial antigen-presenting cells can be used, which consist of cell lines lacking all MHC but pulsed with HLA-A*1101 peptides, or artificial antigen-presenting cells that can be engineered to provide the co-stimulation required for T cell amplification along with the HLA-A*1101 peptide combination, in order to stimulate and amplify hepatitis B virus-specific T cells.

[0037] Appropriate methods for selecting hepatitis B virus-specific T cells include using ELISPOT analysis to confirm responsive T cells. Blood is obtained from HLA-A*1101 donors or patients. Peripheral blood mononuclear cells (PBMCs) are isolated by centrifugation in Biocoll separation medium (Biochrom, Berlin, Germany) and used directly after preparation or frozen for later use. Cells are cultured in RPMI 1640 containing L-glutamine (Invitrogen, Karlsruhe, Germany), supplemented with 10% heat-inactivated mixed human serum and 100 U / ml penicillin-streptomycin (Invitrogen, Karlsruhe, Germany). Hepatitis B virus-specific T cell lines are generated by incubating 1 × 10⁷ intact PBMCs per well for 7 days in 6-well plates containing FHT peptide antigen. Lymphocyte cultures were supplemented with 5 U / ml IL-2 (Proleukin, Chiron, Ratingen, Germany) every other day, and culture medium was added as needed. T cell clones were generated by repeatedly stimulating PBMCs with 1 μA / ml FHT peptide weekly for 4 weeks. Subsequently, T cell clones were generated by limiting dilution in 96-well plates and expanded using a rapid expansion protocol as described by Beck et al. This small-scale culture system can be scaled up and is suitable for a “closed system” to generate clinical-grade T cells suitable for reinfusion into patients.

[0038] Suitable methods for selecting hepatitis B virus-specific T cells include the use of a fluorescence activated cell sorting system (FACS). After exposure of donor or patient PBMCs to the peptides of this invention, responsive T cells are labeled based on activation markers or behavioral characteristics. Labeling is achieved using antibodies specific to activation markers or secreted cytokines, and such antibodies are conjugated to fluorescent dyes. Cells can then be isolated and selected using a flow cytometer equipped for FACS analysis.

[0039] Alternatively, the labeling of responsive T cells is based on the binding of an MHC multimer (HLA-A*1101) conjugated with a fluorescent marker to specific TCRs on the surface of hepatitis B virus-specific T cells.

[0040] Suitable methods for selecting T cells include the cytokine secretion assay system manufactured by Miltenyi Biotec.

[0041] Methods for preparing and using peptide MHC multimers are described, for example, in Altman et al. (1996) Science 274, 94-96; Kuabel et al. (2002) Nature Medicine 8, 631-637; and Neudorfer et al. (2007) J. Immunol. Methods 320, 119-131.

[0042] Fluorescently labeled MHC polymer / peptide complexes can be used to assess the purity of T cell populations.

[0043] Suitable methods for selecting T cells also include an MHC multimer system, available from Proimmune and Stage Pharmaceutical, which functions by artificially constructing HLA molecules that bind to the peptides of the present invention in the present context. These soluble, individual HLA molecules can be constructed in a multimer configuration such that a single multimer contains 4-5 HLA molecules, each loaded with the peptides of the present invention. These multimers can also be attached to magnetic beads as described above. The multimers are released into a blood sample, and the HLA:peptide construct binds to T cell receptors that recognize the peptides, thus labeling T cells that will recognize hepatitis B virus and initiate an immune response against it. The cell sample is then passed through a magnetic column, and the labeled cells are retained and then released.

[0044] As should be understood from the foregoing, the present invention may include a complex comprising an HLA-A*1101 molecule bound to a peptide according to the first aspect of the invention. Conveniently, this complex is a soluble complex and does not bind to cells.

[0045] Preferably, the peptide in the complex is any of the peptides of the present invention, but may be any other peptide of the present invention that will form the complex and can be used to elicit an anti-hepatitis B virus T cell response. As is common practice, the complex can be used to isolate hepatitis B virus-specific T cells. The complex can also be used to identify hepatitis B virus-specific T cells in a sample.

[0046] Typically, peptides are presented on dendritic cells or monocytes, which then present the antigen to T cells. The T cells then secrete cytokines, which in turn activate monocytes and neutrophils to enhance the killing of hepatitis B virus.

[0047] In one aspect of the invention, a T cell expressing a T cell receptor (TCR) molecule is provided, wherein the TCR molecule comprises an amino acid sequence selected from the group consisting of: CASGDSNSPLHF, CASGGQIVYEQYF, CSARGGRGGDYTF, and CASSQDWTEAFF.

[0048] The invention also includes T cells transfected with a polynucleotide or expression vector expressing the aforementioned TCR or a functionally equivalent molecule, preferably CD8+ T cells. The T cells can be obtained from the patient, or, in the case of an allogeneic HSCT patient, from a closely matched donor in terms of HLA type.

[0049] More specifically, the T cells of the present invention are used to induce antiviral T cell activity against the hepatitis B virus nuclear epitope 169, wherein the polynucleotide or expression vector of the present invention has been introduced into the T cells, preferably patient-derived T cells, such that the T cells express the encoded TCR molecule.

[0050] In addition to TCR molecules, functionally equivalent molecules of TCRs are also included in this invention. These include any molecule that is functionally equivalent to a TCR and performs the same function as a TCR. Specifically, such molecules include the genetically engineered three-domain single-stranded TCRs described by Chung et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658 and prepared by the methods mentioned above.

[0051] Typically, TCR or its functional equivalent recognizes human class I HLA molecules expressed on the surface of antigen-presenting cells and loaded with a peptide according to the first aspect of the present invention.

[0052] In various embodiments of the invention, hepatitis B virus-specific T cells can be isolated for further use. While sufficient numbers of specific T cells can be directly isolated for therapeutic purposes using certain techniques, it may be necessary to expand or clone the T cells to produce a adequate number. For adoptive immunotherapy, techniques that allow for the direct isolation of sufficient numbers of cells are generally preferred, as this can be achieved within a day (while cell expansion may take several weeks).

[0053] The appropriate procedure for identifying pathogen-specific donor clones is described in Perruccio et al. (2005) Blood 106, 4397-4406.

[0054] Hepatitis B virus-specific T cells targeting the peptides of this invention can be used for treatment.

[0055] It should be understood that those skilled in the art can readily design and synthesize TCRs according to the invention using any naming system, provided that the frame region (i.e., the region not replaced by the CDR) is compatible with the CDR, as is well known in the art.

[0056] “TCR molecule”, we include any molecule containing a given CDR and also containing an FR appropriately located within the molecule, such that the TCR forms a recognition site (binding site) for HLA-A*11:01 that presents any of the following peptides: STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR.

[0057] The TCR molecule is particularly preferred if it contains the precise amino acid sequence CASGDSNSPLHF, CASGGQIVYEQYF, CSARGGRGGDYTF, and CASSQDWTEAFF. When variants of this precise sequence are present, they preferably differ by one, two, or three (preferably one or two) amino acids. Typically, in these variants, the substituted amino acid is replaced by a conserved amino acid. Conserved amino acids include the following groupings: G, A; S, A, T; F, Y, W; D, E; N, Q; and I, L, V.

[0058] In various implementations, the amino acids encode the TCR β chain portion, particularly CDR1, CDR1, and CD3 of the β chain portion.

[0059] In another aspect of the invention, at least one polynucleotide encoding a TCR molecule as defined above is provided. In one embodiment, the polynucleotide comprises the sequences SEQ ID NO 1 to 16 listed in the table below.

[0060]

[0061]

[0062] The polynucleotide can be DNA or RNA, and may or may not contain introns. Typically, the polynucleotide does not contain introns in the region encoding the target polypeptide. It should be understood that, due to the degeneracy of the genetic code, different polynucleotides can encode the same polypeptide.

[0063] It should be understood that polynucleotides are typically used in HLA-A*1101-specific environments.

[0064] The present invention also provides expression vectors containing polynucleotides encoding the peptides and TCR molecules of the present invention. Such expression vectors allow expression of the target polypeptide when present in suitable host cells. Preferably, the expression vector is an expression vector capable of expressing the polypeptide in mammalian cells. More preferably, the expression vector is an expression vector capable of expressing the polypeptide in T cells such as human CTLs. Typically, the expression vector contains a promoter that is active in a particular cell type and can be controllable (e.g., inducible).

[0065] It should be understood that expression vectors are typically used within an HLA-A*1101-specific environment. The vector is preferably a retroviral vector capable of transfecting mammalian host cells such as human T cells. Typically, the vector is a lentiviral vector.

[0066] Other suitable expression vectors include virus-based vectors, such as retroviral, adenoviral, vaccinia virus vectors, lentiviral vectors, or replication-defective MV vectors. Suitable general cloning vectors include plasmids, bacteriophages (including λ and filamentous bacteriophages), phage particles, and granules.

[0067] Methods for manipulating, altering, and cloning nucleic acid molecules are well known in the field, and such techniques, including PCR methods, are described, for example, by Sambrook J and Russell, DW, Molecular Cloning, A Laboratory Manual, 3rd Edition, 2001, Cold Spring Harbor Laboratory Press.

[0068] Another aspect of the invention provides a host cell comprising a polynucleotide encoding the peptide and TCR molecule of the invention, or a vector of the invention. The host cell may contain a polynucleotide or vector encoding only the α-chain portion or only the β-chain portion. However, if the host cell is to produce the TCR molecule of the invention, it contains one or more polynucleotides or vectors encoding both the α-chain and β-chain portions.

[0069] In various implementation schemes, the host cell is a T cell derived from the individual.

[0070] The host cell can be any cell type, such as bacterial cells (e.g., *Escherichia coli*, *Bacillus subtilis*, and *Salmonella typhimurium*), yeast cells (e.g., *Saccharomyces cerevisiae* and *Schizosaccharomyces pombe*), insect cells, plant cells, or mammalian cells (e.g., CHO cells, COS cells, and other mammalian cells such as antigen-presenting cells), and methods for introducing polynucleotides into such cells are well known in the art. Typically, bacterial cells (such as *E. coli* cells) are used for the general propagation and manipulation of the polynucleotides and vectors of the present invention. Other host cells can be used to express the TCR molecules of the present invention, and specifically, the cell can be a mammalian cell, such as a human cell. As described below regarding treatment methods using the TCR molecules of the present invention, it is particularly desirable for the host cell to be a T cell, such as (and preferably) a T cell derived from a patient to be treated (typically a patient with a malignancy expressing WT1).

[0071] Typically, retroviral vectors (or vectors as appropriate) encoding the TCR molecule of this invention are used based on their ability to infect mature human CD4+ or CD8+ T lymphocytes and mediate gene expression: the retroviral vector system Kat is a preferred option (see Finer et al. (1994) Blood 83, 43). Following the protocol described by Roberts et al. (1994) Blood 84, 2878-2889, which is incorporated herein by reference, purified CD8+ T lymphocytes isolated from peripheral blood of cancer patients were infected with a high-titer facultative retrovirus. The use of an anti-CD3 antibody to trigger proliferating T cells facilitates retroviral integration and stable expression of the single-chain TCR. The combination of anti-CD3 and anti-CD8 antibodies can be more effective than anti-CD3 antibodies alone. Other suitable systems for introducing genes into CTLs are described by Moritz et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4318-4322, which is incorporated herein by reference. CTL transfection has also been described by Eshhar et al. (1993) Proc. Natl. Acad. Sci. USA 90, 720-724 and Hwu et al. (1993) J. Exp. Med. 178, 361-366. Commercially available nuclear factor systems, supplied by AMAXA GmbH, Germany, can be used for T cell transfection. Retroviral transduction of human CD8+ T cells is described in Stanislawski (2001) Nat. Immunol. 2, 962. Cloning methods and gene manipulation are well known in the art and are described in detail in standard manuals such as Sambrook and Russell (2001) Molecular Cloning, a laboratory manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, USA.

[0072] In another aspect of the invention, T cells for inducing antiviral T cell activity against hepatitis B virus according to an earlier aspect of the invention are provided, as well as the use of said T cells in the preparation of a medicament for inducing antiviral T cell activity against hepatitis B virus.

[0073] In one embodiment, the hepatitis B virus expression comprises an epitope containing an amino acid sequence selected from the group consisting of: STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR.

[0074] The phrase "inducing antiviral T cell activity against hepatitis B virus" should be understood as meaning that the antigen-binding site of the present invention induces or enhances any one or more activities of T cells, including but not limited to stimulating NK cell proliferation, cytotoxicity or maturation; stimulating B cell and T cell proliferation or differentiation; stimulating antibody production and affinity maturation in B cells; stimulating CD8+ T cell cytotoxicity; stimulating interferon-γ production in T cells and NK cells; inhibiting dendritic cell (DC) activation and maturation; inhibiting the release of inflammatory mediators from mast cells; enhancing macrophage phagocytosis; inhibiting TReg cell generation or survival; and stimulating bone marrow progenitor cell proliferation.

[0075] The peptides, TCR molecules, polypeptides, polynucleotides, expression vectors, and T cells of the present invention can be packaged and presented for use as medicines. Specifically, they are used to treat or combat hepatitis B virus infection. "Hepatitis B virus infection" includes treating patients with hepatitis B virus infection. We also include administering the peptides, TCR molecules, polypeptides, polynucleotides, expression vectors, and T cells (alone or in combination with suitable HLA-matched antigen-presenting cells, such as dendritic cells, B cells, monocytes, or synthetic APCs, or present within or on suitable HLA-matched antigen-presenting cells, such as dendritic cells, B cells, monocytes, or synthetic APCs) to patients not only with hepatitis B virus infection but also at risk of hepatitis B virus infection.

[0076] Patients at risk of hepatitis B virus infection include those with compromised or depleted immune systems, such as those who have undergone allogeneic HSCT, organ transplant recipients, autoimmune patients receiving immunosuppressive drugs, patients with inherited immune disorders, AIDS patients, or patients undergoing cancer chemotherapy or leukemia. Therefore, it should be understood that “fighting” includes preventing (or aiding in the prevention of) hepatitis B virus infection and providing prophylactic treatment to patients.

[0077] In another aspect of the invention, a method for treating hepatitis B virus infection in an individual is provided, the method comprising administering to the individual an effective amount of a peptide according to a first aspect of the invention, or a polynucleotide according to various aspects of the invention described above, or T cells according to one aspect of the invention described above.

[0078] The term "effective dose" or "therapeutic effective dose" means a dose sufficient to treat, suppress, or alleviate one or more symptoms of the disease state being treated, or otherwise provide the desired pharmacological and / or physiological effects, particularly enhancing the T-cell response to a selected antigen. The precise dose will vary depending on a variety of factors, such as subject dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment administered.

[0079] In another aspect of the invention, a pharmaceutical composition is provided comprising a peptide according to a first aspect of the invention, or a polynucleotide according to one aspect of the invention, or a T cell according to one aspect of the invention, and a pharmaceutically acceptable carrier.

[0080] Pharmaceutically acceptable carriers are generally defined as materials suitable for administration to a subject, wherein the carrier is biologically harmless or otherwise does not cause undesirable effects. Such carriers are typically inert components of the drug. Generally, the carrier is administered to the subject along with the active ingredient without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing the carrier. Suitable pharmaceutical carriers are described in Martin, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, Pa., (1990), which is incorporated herein by reference in its entirety.

[0081] In a more specific form of this disclosure, pharmaceutical compositions are provided comprising therapeutically effective amounts of peptides, polynucleotides, and T cells, as well as pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions include diluents with varying buffer contents (e.g., phosphates, Tris-HCl, acetates), pH, and ionic strength, and additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and fillers (e.g., lactose, mannitol). Materials may be incorporated into granular formulations of polymeric compounds (e.g., but not limited to polylactic acid or polyglycolic acid) or into liposomes. Hyaluronic acid may also be used. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance of the disclosed compositions. The compositions may be formulated, for example, in liquid form or as a dry powder, such as a lyophilized form.

[0082] It should be understood that the pharmaceutical compositions provided according to this disclosure can be administered in any manner known in the art. Preferably, the pharmaceutical compositions for administration are administered by injection, oral administration, or via the pulmonary or nasal route. In various embodiments, the antisense polynucleotide is delivered via intravenous, intra-arterial, intraperitoneal, intramuscular, or subcutaneous administration. In various embodiments, the composition is suitable for parenteral administration to a patient, either naked or in combination with a delivery agent. The carrier may be selected from the group consisting of: nanoparticles, such as polymer nanoparticles; liposomes, such as pH-sensitive liposomes, antibody-conjugated liposomes; viral vectors, cationic lipids, polymers, UsnRNA (such as U7 snRNA), and cell-penetrating peptides.

[0083] The compositions or molecules of the present invention may be administered orally, rectally, via mucosa, intestine, muscle, subcutaneously, intramedullary, intrathecal, directly intraventricular, intravenously, intravitreal, intraperitoneally, intranasally, or intraocularly.

[0084] The compositions or molecules of the present invention encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other compound that, when administered to animals (including humans), can (directly or indirectly) provide a biologically active metabolite or residues thereof. Therefore, for example, this disclosure also relates to prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0085] The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of the compounds of the present invention: that is, a salt that retains the desired biological activity of the parent compound without conferring undesirable toxicological effects upon it.

[0086] Preferred examples of pharmaceutically acceptable salts for polynucleotides include, but are not limited to: (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, and polyamines (such as spermine and spermidine); (b) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid; (c) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. The pharmaceutical compositions of this disclosure can be administered in various ways, depending on whether local or systemic treatment is required and depending on the area to be treated. Administration can be local (including ocular and mucosal, including rectal delivery), pulmonary (e.g., by inhalation of powders or aerosols (including via nebulizers, intratracheal, intranasal, epidermal, and transdermal)), oral, or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, such as intrathecal or intraventricular administration. Polynucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly suitable for oral administration.

[0087] The pharmaceutical formulations of this disclosure, which can be conveniently presented in unit dosage forms, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. Generally, formulations are prepared by homogenizing the active ingredient with a liquid carrier, or a subdivided solid carrier, or both, and then, if necessary, shaping the product.

[0088] This disclosure also contemplates combination therapy with other therapeutic agents. Examples of therapeutic agents that can be delivered concurrently with the compositions of this disclosure include, but are not limited to, glucocorticoids (e.g., but not limited to prednisone and deflazacort), angiotensin-converting enzyme inhibitors, beta-adrenergic receptor blockers, antifibrotic agents, and combinations thereof.

[0089] In some embodiments, the present invention can be used in gene therapy, for example, by using a vector (e.g., an expression vector) containing the polynucleotide of the present invention to direct the expression of the polynucleotide in a suitable host cell. Such vectors can be used, for example, to amplify the polynucleotide in a host cell to produce a usable amount, and for expressing proteins using recombinant technologies. In some embodiments, the vector is an expression vector in which the polynucleotide of the present invention is operatively linked to a polynucleotide containing an expression control sequence.

[0090] The peptides of the present invention, alone or in combination with antigens from other pathogens, can be used to activate immune cells within blood or tissue samples or their cell derivatives obtained from a patient or donor without significant further selection or purification of cell types (“unselected cell formulations”), with the aim of infusing unselected cell formulations into a patient for the treatment or prevention of hepatitis B virus infection, whether in a targeted manner or as one of several pathogens that can cause infection in the patient.

[0091] The method for combating or treating hepatitis B virus infection, as well as the pharmaceutical composition and medicine of the present invention, can be combined with other anti-hepatitis B virus treatments.

[0092] In another aspect of the invention, a vaccine against hepatitis B virus infection is provided, the vaccine comprising a peptide according to the first aspect of the invention or a polynucleotide or T cell according to various aspects of the invention described above, and is packaged and presented for use as a medicine.

[0093] In another aspect of the invention, a method for combating hepatitis B virus infection in a patient carrying HLA A*1101 is provided, the method comprising: (a) obtaining T cells from the patient; (b) introducing a polynucleotide encoding a TCR molecule according to one aspect of the invention into the T cells; and (c) introducing the T cells generated in step (b) into the patient.

[0094] Transfected T cells can help fight hepatitis B virus. Preferably, the patient to be treated carries class I HLAA*1101.

[0095] In various implementations, polynucleotides are transfected or introduced into T cells via electroporation. Other suitable systems for introducing genes into T cells are described in Moritz et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4318-4322. T cell transfection is also described in Eshhar et al. (1993) Proc. Natl. Acad. Sci. USA 90, 720-724 and Hwu et al. (1993) J. Exp. Med. 178, 361-366.

[0096] Methods for introducing nucleic acids into T cells (these methods are well known in the art and are routine practice) include transformation, transfection, electroporation, nuclear injection, or fusion with vectors such as liposomes, micelles, ghost cells, and protoplasts. Host T cells can be isolated and / or purified. T cells can also be cells transformed in vivo to induce transient or permanent expression of peptides in vivo. T cells can also be isolated cells transformed in vitro and introduced post-transformation, such as those that produce peptides in vivo for therapeutic purposes.

[0097] The polynucleotides of the present invention (particularly SEQ ID Nos. 1 to 16) can be introduced into T cells using transfection methods well known in the art. These methods include sonication, electroporation, electroporation, osmotic shock, calcium phosphate precipitation and DEAE-dextran transfection, lipid-mediated delivery, passive delivery, etc. The language “transfect T cells” is intended to include any means by which nucleic acid molecules can be introduced into T cells. The term “transfection” encompasses a variety of techniques that can be used to introduce nucleic acids into mammalian cells, including electroporation, calcium phosphate precipitation, DEAE-dextran treatment, lipid transfection, microinjection, and viral infection. Suitable methods for transfecting mammalian cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory press (1989)) and other laboratory textbooks.

[0098] Viral vectors can also be used to introduce polynucleotides into T cells. Such viral vectors include, for example, recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-1. Retroviral and adeno-associated virus vectors are generally understood as recombinant gene delivery systems selected for in vivo exogenous gene transfer (particularly to humans). Alternatively, they can be used to introduce exogenous genes into T cells. These vectors provide efficient delivery of genes into T cells, and the transferred nucleic acids are stably integrated into the host cell's chromosomal DNA.

[0099] Another viral gene delivery system that can be used in this invention utilizes an adenovirus-derived vector. The genome of the adenovirus can be manipulated to encode and express the target gene product, but it is inactivated due to its ability to replicate during the normal lysis viral life cycle. Another viral vector system that can be used to deliver nucleic acid molecules containing the target gene is adeno-associated virus.

[0100] Polynucleotides can be carried and delivered into T cells by cellular delivery mediators. Such mediators include, for example, cationic liposomes (Lipofectamines). TM Or derived (e.g., antibody-conjugated) polylysine conjugates, bacitracin S, artificial viral envelopes. These mediators can deliver nucleic acids incorporated into plasmids, vectors, or viral DNA. In one specific embodiment, efficient introduction of nucleic acid molecules into primary T lymphocytes is achieved by transfecting primary T lymphocytes with adeno-associated virus plasmid DNA complexed into cationic liposomes, as described by Philip, R. et al. (1994) Mol. Cell. Biol. 14, 2411.

[0101] In another embodiment of the invention, the polynucleotide may be delivered in the form of a soluble molecular complex. This complex contains a nucleic acid releasably bound to a carrier comprising a nucleic acid conjugate and a cell-specific conjugate, the nucleic acid binding to surface molecules of specific T cells and having a size suitable for subsequent internalization into the cell.

[0102] In another embodiment of the invention, polynucleotides are introduced into T cells by particle bombardment.

[0103] In various embodiments, polynucleotides can be passively delivered (i.e., delivered without additional transfection reagents) to T cells, particularly during T cell expansion. As used herein, expansion includes the generation of daughter cells from transfected neural stem cells in a container and under conditions well known in the art. Expansion can be performed in the presence of suitable culture media and cell growth factors. Polynucleotides can be passively delivered to T cells in cultures (e.g., culture plates, dishes, multi-well plates, etc., but not limited to) under serum-depleted conditions (including 0% serum conditions). Such conditions include cells cultured in standard serum-depleted media tested in the art, which are commercially available from many companies including Invitrogen and HyClone. In one example, cells are first seeded in serum medium, then the serum medium is replaced with serum-depleted medium containing the triple oligonucleotide complex of this disclosure for 24 hours, and then the serum-depleted medium is replaced with serum medium.

[0104] In various embodiments, the transfection reagent may be selected from the group consisting of polymers, lipids, lipid-polymers and / or combinations thereof and / or derivatives thereof containing a cell-targeting portion or an intracellular-targeting portion and / or a membrane destabilizing component and one or more delivery enhancers.

[0105] In another aspect of the invention, the peptide according to the first aspect of the invention can be used to produce monoclonal or polyclonal antibodies on a patient-specific or mass production basis, wherein the antibody is used to prevent or treat hepatitis B virus infection or to induce a primary or secondary humoral or cellular immune response against hepatitis B virus in a patient. The antibody will comprise conventional derivatives of antibodies specific to the aforementioned peptide. Preferably, the antibody recognizes the peptide of the invention when presented by a class I HLA-A*1101 molecule.

[0106] In order to fully understand the invention and readily put it into practice, preferred embodiments of the invention will now be described by way of non-limiting examples, with reference to the accompanying drawings.

[0107] Figure 1 Comprehensive epitope mapping of HBV using a highly multiplexed pMHC tetramer strategy. (A) Experimental workflow. Deep sequencing based on viral and epitope predictions generated a 562-plex pMHC tetramer library. This library included 484 putative A*11:01-restricted HBV peptides and 78 known control peptides derived from other common viruses or autoantigens. The entire library was encoded using 1001 unique combinations of quadruple SAv-metal encoding. A self-validated tetramer deconvolution algorithm automatically identified signals on patient T cells using statistical measurements. Validated antigen-specific CD8+ T cells targeting the four viral epitopes are shown. (B) Mean frequency of HBV-specific CD8+ T cells from all patients tested for four different viral proteins. Graphs show only detectable epitopes. The numbers at the bottom indicate the number of epitopes detected / screened for each viral protein. (C) Epitope nomenclature and annotations used in this report are shown. * Indicates peptide clusters containing more than one peptide (Table S1). The peptide sequences in bold are previously unpublished sequences based on the Immunological Epitope Database (IEDB). (D) Color-coded frequencies of four antigen-specific CD8+ T cells in each patient group. (E) The heatmap shows the expression of four HBV-specific CD8+ T cell markers. Boxes highlight the distinguishing markers for each patient group.

[0108] Figure 2Multivariate memory atlases of HBVpol387 and HBVcore169-specific CD8+ T cells associated with HBV clinical staging. (A) Unsupervised Phenograph clustering of all detected antigen-specific CD8+ T cell subsets in the patient group. n=20, 4 patients per group. Nineteen cell clusters objectively identified by Phenograph are color-coded as shown, and the expression levels of detected cellular proteins are illustrated. (B) Visualization of Phenograph clustering of nine major cell clusters of HBVpol387-specific CD8+ T cells. The proportion of cell clusters within HBVpol387-specific CD8+ T cells in individuals from each patient group is shown. (C) The same analytical strategy for HBVcore169-specific CD8+ T cells is shown. (D) A bar chart indicates differences in the expression of T cell memory-related markers (CD27, CD28, CD45RO, CD127, and CXCR3), inhibitory receptors (PD-1 and TIGIT), and CD57 on HBVcore169-specific CD8+ T cells. Error bars are medians and ranges, and values ​​from individuals are applied. (E) A representative contour plot shows the expression levels of markers on HBVcore169-specific CD8+ T cells between patient groups. Patients are color-coded as shown in the figure. (F) Logistic regression (top) of eight phenotypic markers that show significant differences between patient groups is stacked relative to pseudotime estimated using Scorpius. Trends of these eight cellular markers are visualized using logistic regression (black solid line, bottom). Points are individuals color-coded by clinical stage, and the expression levels of these markers on HBVcore169-specific CD8+ T cells are shown.

[0109] Figure 3 Unsupervised analysis reveals a complex model of inhibitory receptors (exhaustion markers) in CHB. (A) In a 2D plot, One-SENSE objectively visualizes the association between three distinct T cell classes (differentiation + TNFR, inhibition, and transport) and cellular protein expression levels. Dots are color-coded selected virus-specific CD8+ T cells. Boxes annotate epitopes enriched in a given region. (B) Mean fraction of the number of inhibitory receptors co-expressed on four HBV-specific CD8+ T cells in the patient group. The graph is from representative experiments of all nine inhibitory receptors. (C) Mean co-expression of inhibitory receptors on four HBV-specific CD8+ T cells in the patient group. The graph consists of three experiments simultaneously measuring eight inhibitory receptors (without TIGIT). Each dot represents an individual.

[0110] Figure 4The nonlinear correlation between pluripotency and inhibitory receptors in PBMCs of One-SENSE (A) patients was measured for functional capacity by stimulating them with corresponding viral peptides in vitro for 10 days. A classification (functional, inhibitory, and differentiation + TNFR) analysis of One-SENSE revealed diverse pluripotent virus-specific CD8+ T cell subsets and their corresponding inhibitory receptor co-expression. Dots represent different virus-specific CD8+ T cells annotated. Five distinct major functional subsets were labeled based on a heatmap and color-coded as shown in the figure. (B) The expression levels of T cell function, inhibitory receptors, and TNFR co-stimulatory receptors were compared among these five functional subsets. (C) A bar chart shows the proportion of each functional subset in HBVpol387 and HBVcore169-specific CD8+ T cells in the patient group. n = 5 for each group except IT = 4.

[0111] Figure 5 Unsupervised quantification of HBV-specific TCRs is associated with disease staging in an epitope-dependent manner. (A) TCRdist measurements of epitope-specific TCRs were clustered by unsupervised Phenograph analysis and then predicted by t-SNE. Each point represents a TCR clone. Twenty-eight TCR sequence clusters on the t-SNE map are labeled. (B) Sequence motifs of representative TCR sequence clusters are shown (labeled with dashed boxes of varying sizes). The mean linkage dendrogram for each TCR in a given cluster is presented and color-coded according to generation probability. TCR markers show the frequency of the V and J regions with the CDR3β sequence in the middle. The bottom bar is the source region as shown in the figure. Light gray represents the V region. Red represents N-insertion. Black represents the diversity of D. Dark gray represents the J region. (C) The percentage of receptors in the total epitope-specific TCRs of the twenty-eight TCR sequence clusters is shown. (D) The proportions of TCR clusters 27 (C27) and 15 (C15) in four different epitope-specific TCRs. (E) Measurement of TCRdiv diversity for each epitope-specific TCR in the patient group. (F) Stacked bar charts show the top eleven TCR clones in an individual patient. The frequency and sequence of common TCR clones are presented. (G) 3D PCA prediction depicts the clinical stage of patients using epitope-specific TCR repertoires, tetramer responses, and cellular characteristics from the same individual. (H) Correlation between the frequency of HBVcore169-specific CD8+ T cells and TCRdiv diversity measures.

[0112] Figure 6Phenotypic dynamics and machine learning-aided modeling of HBVcore169-specific CD8+ T cells. (A) The longitudinal cohort includes a total of 14 patients (n=8 for HBeAg- and n=6 for HBeAg+). The mean frequency of all detectable HBVcore169-specific CD8+ T cells at different time points is shown. Each point represents a patient with detectable HBVcore169-specific CD8+ T cells. (B) Dynamics of HBVcore169-specific CD8+ T cells in two representative patients. (C) Phenotypic dynamics of HBVcore169-specific CD8+ T cells are shown using One-SENSE. Numbers are frequencies and boxes are annotated as shown in the figure. (D) Fractions of memory (blue boxes) and terminal effector (red boxes) cells in each patient at longitudinal time points. (E) Graphs show changes in the expression of selective cellular markers on HBVcore169-specific CD8+ T cells at longitudinal time points in patients (early and late thick stacked bar graphs in D). Two time points (early and late) were selected to roughly match the time points between patients based on drug intervention. "Early" was the pre-treatment time point except for one patient (HBeAg+04, whose earliest time point was 3 months post-treatment), while "late" was approximately 30 months post-treatment. Figures show patients with detectable HBVcore169-specific CD8+ T cells at both the early and late time points. Statistical analyses were used to compare cellular marker expression between the two time points (early and late, solid lines) or between patient groups (HBeAg+ and HBeAg-, dashed lines). (F) Logistic model (grey dashed line) of cellular marker expression (dependent variable) against SVM-predicted pseudotime (independent variable). Points represent the expression levels of cellular markers on HBVcore169-specific CD8+ T cells at different longitudinal time points in different patients. (G) Statistical analysis of SVM-predicted pseudotime during progression at longitudinal time points in patients. Nonparametric paired t-tests were used.

[0113] Figure 7 Comprehensive epitope mapping strategy and experimental workflow.

[0114] (A) HBV was deep sequenced using next-generation sequencing (NGS). HLA-A*11:01 restriction epitopes were predicted based on shared sequences using NetMHC (v3.4). (B) 562 peptides were clustered based on sequence homology and further assigned unique combinations of four SAv-metal encodings. (C) These unique combinations of four SAv-metal mixtures with two different encoding conformations were prepared using an automated liquid handling robot. Patient PBMCs were stained using a pMHC tetramer library encoding four SAv-metals (for both encoding conformations). (D) Tetramer-positive cells were identified using an automated tetramer deconvolution algorithm, and tetramer signals between the two encoding conformations were calculated for their correspondences.

[0115] Figure 8 Quality and detection of antigen-specific CD8+ T cells obtained using highly multiplexed pMHC tetramer staining and mass flow cytometry.

[0116] (A) Staining quality of quadruple SAv-metal-encoded pMHC tetramers obtained using fourteen different SAv-metal channels from a representative CHB donor. PBMCs from the same vials from each donor were stained in parallel with the same 562-plex pMHC tetramer instead of two different SAv-metal-encoded conformations, as shown in the figure. (B) Quantities of selected HBV-specific CD8+ T cells detected at various clinical stages of HBV infection using a highly multiplexed pMHC tetramer strategy. The graph shows the frequency of antigen-specific CD8+ T cells for fifteen predicted HBV epitope clusters and six representative known control viral epitopes (shaded boxes). Bold epitope sequences indicate previously unpublished sequences. * indicates that the epitope cluster contains more than one peptide (related to Supplementary Table 1). The dashed line on the y-axis represents 0.002.

[0117] Figure 9 The overall magnitude of antigen-specific CD8+ T cell responses in each clinical stage during HBV infection.

[0118] (A) Top panel: Total number of different epitopes derived from the four hepatitis B virus proteins (enveloping, polymerase, nucleus, and x) detected in each individual patient at each clinical stage. Bottom panel: Sum of the frequency (%) of each antigen-specific CD8+ T cells detected against the four different hepatitis B virus proteins in each independent patient at each clinical stage. = No significant difference.

[0119] Figure 10 Validation of a highly multi-combination pMHC tetramer strategy in HLA-A*11:01 and non-HLA-A*11:01 donors.

[0120] (A) Cells from each donor were stained with selected 120-plex pMHC tetramers encoded by three different SAV-metals using two different encoding configurations (Supplementary Table 1). Experiments were performed independently, and cells were stained and gated for live CD3+, dump-(CD4+CD19+CD16+), and CD8+. Bar graphs indicate the frequency of each epitope. (B) Representative scatter plots show pMHC tetramer-positive cells and the signal of SAV metals encoded by them through different combinations of the nine metal tags SAV.

[0121] Figure 11 : Validation and reproducibility of antigen-specific CD8+ T cells using flow cytometry and serological measurements from healthy donors.

[0122] (A) Correlation of frequencies detected between FACS (single fluorescent dye encoded) and CyTOF (combined metal encoded) experiments. Each point represents a single patient. (B) Representative FACS dot plot of selected HBV epitopes. Numbers represent the frequency of total CD8+ T cells. (C) Left, HBsAb (anti-HBsAg antibody) titer in healthy donors (HD). Right, frequency of HBVpol387-specific CD8+ T cells in HD under different HBV serum (HBsAb, HBcAb, and HBeAb) states. Serum antibody levels against different HBV viral antigens were measured by ELISA. Red circles indicate the only individual who tested positive for both HBcAb and HBeAb.

[0123] Figure 12 In vitro expansion of antigen-specific CD8+ T cells after peptide stimulation.

[0124] (A) PBMCs from different patient groups were amplified with the corresponding viral peptides for 10 days. The frequency of antigen-specific CD8+ T cells was determined in the same manner as in in vitro pMHC tetramer staining experiments. The numbers above each graph indicate significant p-values.

[0125] Figure 13 Expression levels of nine different inhibitory receptors on antigen-specific CD8+ T cells.

[0126] (A) Expression levels of inhibitory receptors on CD8+ T cells specific to the selected antigen. The numbers on the x-axis indicate the peptide cluster number (epitope) for each specific antigen (see Table _S1). Colored dots represent four selective HBV epitopes (090_HBV-P-282, 106_HBV-P-387, 178_HBV-C-169, and 283_HBV-C-195v2). Color legends indicate different HBV clinical stages. Shaded areas represent control viral epitopes. Statistical significance is shown only for selected epitopes. p-values ​​less than 0.0001 (short notation) or other values ​​(long notation) are indicated.

[0127] Figure 14 Epitope frequency of HBeAg-seroconversion factor in a longitudinal patient cohort.

[0128] (A) Deep sequencing analysis of HBV viral DNA revealed different dynamics in viral mutations on selective epitopes identified by a highly multiplexed pMHC tetramer strategy. The longitudinal patient cohort included treatment-naïve CHB patients who spontaneously underwent HBeAg-seroconversion (S, bottom) or non-seroconversion (C, top) within similar timeframes. The proportion of HBVpol387 (LVVDFSQFSR) in the viral population sequenced at each time point (top right) was capped at 1. This epitope remained constant across all patients, and no changes were observed. Epitope IDs and sequences are listed in Table S1. Detailed epitope mutation data are available in Table S4.

[0129] Figure 15 Cellular characteristics of HBV-specific CD8+ T cell subset clusters identified by Phenograph and enrichment strategies.

[0130] (A) Subpopulation enrichment strategy for eight major Phenograph subsets of HBV-specific CD8+ T cells. A gating strategy was defined in representative experiments, and then qualified markers for each subset cluster were applied to three different experimental batches to identify the proportion of each subset cluster within HBVpol387 and HBVcore169-specific CD8+ T cells. Shaded areas are as follows: Figure 2 Target clusters are color-coded as shown in Figure A. Black dots represent cells within the target cluster. Gray dots represent other antigen-specific CD8+ T cells. Numbers indicate frequency.

[0131] Figure 16 Unsupervised Phenograph clustering analysis identifies multifactorial T cell heterogeneity of HBV-specific CD8+ T cells.

[0132] (A) A representative diagram of cell clusters shows that HBVpol387 (LVVDFSQFSR)-specific CD8+ T cells were enriched in different regions in the patient group. (Compared to...) Figure 2 A and B are related. Black dots are HBVpol387-specific CD8+ T cells from the donors shown. Gray dots are combinations of all antigen-specific CD8+ T cells from twenty individuals (including all patient groups). Numbers indicate the proportion of salient clusters within HBVpol387-specific CD8+ T cells. (B) Proportion of three cells (C7, C11, and C14) in HBVpol387-specific CD8+ T cells. Proportion of cell cluster C2 in HBVcore169-specific CD8+ T cells. (C) Unsupervised Phenograph clustering showing phenotypic differences between HBVpol282 and HBVcore195. Stacked bar plots show nineteen cell clusters ( Figure 2 A) Distribution in individuals within the patient group. (D) pMHC tetramer intensity was quantified by averaging the median number of metals encoding the four different SAV-metals on tetramer-positive cells. The graph shows the normalized z-scores of tetramer intensity on selected virus-specific CD8+ T cells (left) and HBVcore169-specific CD8+ T cells (right) in each patient group. (E) Cell cluster proportion and cell marker expression of HBVcore169-specific CD8+ T cells derived from an IT patient (IT07) whose frequency (0.00193%) was just below the applied cutoff value. (F) Patients derived from... Figure 1 Hierarchical clustering of cellular marker expression of EVEBNA3B-specific CD8+ T cells in individuals with the same D.

[0133] Figure 17 Co-expression of inhibitory receptors on virus-specific CD8+ T cells.

[0134] (A) Average number of inhibitory receptors co-expressed on CD8+ T cells of different antigens in an individual. The graph is a combination of four independent experiments and does not measure the cellular marker TIGIT. Each point represents one individual. (B) Average number of inhibitory receptors co-expressed on CD8+ T cells of different antigens in an individual. The graph is from an experiment that measured all nine inhibitory receptors.

[0135] Figure 18 : Heterogeneous pluripotent subset of virus-specific CD8+ T cells.

[0136] (A) Detailed One-SENSE functional clusters of different antigen-specific CD8+ T cells in the patient group. Each patient group has n = 4–5. Functional subsets are as follows: Figure 4A is marked as shown. (B) Correlation between co-expression of symbiotic granzymes A and K and 2B4 and TIGIT on virus-specific CD8+ T cells. The proportion of nonfunctional subsets (black) is associated with persistent expression of HVEM on virus-specific CD8+ T cells. Dots represent different virus-specific CD8+ T cells from individual patients. (C) Representative contour plot of HBVenv304-specific CD8+ T cells shows heterogeneous pluripotency among patients.

[0137] Figure 19 Diverse characteristics of epitope-specific TCRβ repertoires obtained using TCRdist.

[0138] (A) presents nine TCR motif clusters identified by Phenograph and illustrates representative TCR motifs using an average linkage dendrogram based on the TCRdist algorithm. Figure 5 (B) Shows a measure of TCRdiv diversity in the total CD8+ T cell TCRβ repertoire across patient groups. (C) Length (aa, amino acid) of CDR3β in total CD8+ T cells and epitope-specific CD8+ T cells in patient groups. Error bars are for mean and SEM. Statistical analysis was performed using Gaussian fitting and the null hypothesis “one curve fits all groups”.

[0139] Figure 20 Dynamics of cellular responses and viral mutations in HBVcore169-specific CD8+ T cells in a longitudinal patient cohort.

[0140] (A) shows the liver inflammation scores of two patients (HBeAg+03 and HBeAg-01) from a longitudinal patient cohort. ALT, alanine aminotransferase. AST, aspartate aminotransferase. AFP, alpha-fetoprotein. ALP, alkaline phosphatase. (Compared to...) Figure 6 A. (B) Specific tetrameric responses of seven different peptides in cluster 178 were resolved using PBMCs before and after HBeAg+03 and HBeAg-01 treatment (see Table S1). Cells were uniformly divided using flow cytometry and independently stained with the corresponding (as shown above) pMHC tetramers. Numbers indicate the frequency of CD8+ T cells. Viruses from paired serum samples were sequenced to determine variants (in frequency) of the epitope (left). WT, wild type. Identical sequences were color-coded as shown in the figure.

[0141] Figure 21 : Staining quality of cell markers, including nine inhibitory receptors, obtained using mass flow cytometry.

[0142] (A) The dot plot shows the expression levels of antigen-specific (left) markers detected on CD8+ T cells in patient PBMCs relative to the whole (right) PBMCs. The top plot shows in vitro staining. The bottom plot shows cells from in vitro peptide stimulation. For better visualization of the dot plot, all detected antigen-specific CD8+ T cells were pooled from 18 patients (left plot, n = 4–5 per patient group, including IT, IA, InA, and R). HBVcore169-specific CD8+ T cells expressing PD-1 from CHB patients are also shown. The graphs are from two independent experiments (in vitro and in vitro).

[0143] The invention will be described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto, but is limited only by the claims.

[0144] Any reference numerals in the claims should not be construed as limiting the scope. The described drawings are illustrative only and not restrictive. For illustrative purposes, some elements may be enlarged and not drawn to scale in the drawings.

[0145] As used herein, unless the context otherwise requires, the term "comprise" and its variations, such as "comprising," "comprises," and "comprised," are not intended to exclude additional additives, components, integers, or steps. As used herein, unless the context otherwise requires, "comprise" and "include" or its variations, such as "including," are used interchangeably.

[0146] When referring to singular nouns, the use of indefinite or definite articles such as "a," "an," and "the" includes the plural form of the noun unless explicitly stated otherwise. Furthermore, the terms first, second, third, etc., in the specification and claims are used to distinguish similar elements and not necessarily for sequential or chronological order. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or shown herein.

[0147] The following terms or definitions are provided only to aid in understanding the invention. Unless explicitly defined herein, all terms used herein have the same meaning to those skilled in the art. Practitioners shall draw particular attention to the definitions and terms in the works of Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0148] The definitions provided herein should not be construed as having a scope smaller than that understood by one of ordinary skill in the art. Example

[0149] Materials and methods

[0150] 1. Separation of patient samples and PBMCs

[0151] Patients with HBV infection were recruited with full informed consent from the Division of Gastroenterology and Hepatology at the National University Health System, Singapore (Table S3). The study was approved by the relevant local ethics review committee, and appropriate patient recruitment and sampling were completed at the hospital. Up to 60 ml of blood was collected for further peripheral blood mononuclear cell (PBMC) isolation using FiColl separation (Ficoll-PaquePLUS, GE Healthcare). All patients had clinical, serological, and pathological evidence of chronic hepatitis B infection, with detectable HBsAg and HBV DNA, and no positive results for HIV-1, HIV-2, or HCV. Three groups of CHB patients were enrolled in the study: immune-tolerant patients (IT, HBV DNA > 2000 IU / ml, ALT < 40 IU / ml, HBeAg+), immune-active patients (IA, HBV DNA > 2000 IU / ml, ALT > 40 IU / ml, HBeAg+), inactive carriers (InA, HBV DNA < 200 IU / ml or undetectable, ALT < 40 IU / ml, HBeAg-), and a group of acutely regressed patients (R, undetectable HBV DNA, HBsAg- and anti-HBc antibody+). Each CHB patient had at least three consecutive time points with consistent pathological and serological evidence indicating the mentioned clinical stage. At the time of blood collection, all patients were receiving treatment without any antiviral drugs or clinical intervention. Patients receiving entecavir (ETV) were longitudinally followed and enrolled. Serum and pathological scores (serum HBV DNA, HBeAg, and HBsAg) and liver function tests were determined by a hospital's clinical laboratory or ELISA. Blood was recruited from anonymous healthy donors under the institutional review committee of the Singapore Immunology Network (SIgN). Healthy cord blood samples were purchased from the Singapore Cord Blood Bank under the institutional review committee, and HIV-1 and HIV-2, HTLV-I and HTLV-II, HCV, CMV, HBsAg, and anti-HBc antibodies were undetectable. HLA-A*11:01 was confirmed by genotyping services from BGI Genomics.

[0152] Supplementary Table 3: List of patient samples and clinical and serological information.

[0153]

[0154] Table S3

[0155] 2. 562-plex combination (quadruple / triple SAV-metal encoding) pMHC tetramer

[0156] Fourteen different SAv-metals were prepared by labeling streptavidin with fourteen different metal isotopes. Similar to previous work reported by EWNewell et al., combined tetramer staining and flow cytometry analysis facilitated T cell epitope mapping and characterization. Each SAv-metal was diluted to 20 μg / ml in EDTA-free W-buffer on the same day as pMHC tetramerization. Two different conformations encoding the tetrad SAv-metals encoding the 562-plex pMHC tetramer were generated using an R-based script of the 14-choose-4 scheme (1001 combinations). The script was then uploaded to a TECAN Freedom EVO200 automated liquid dispensing robot to prepare designed combinations of tetrad SAv-metal mixtures (each mixture containing four different SAv-metals) in 2 ml 96-well deep-well plates. To form pMHC tetramers, each HLA-A*1101 monomer (562 different pMHC monomers) was randomly assigned to four different SAv-metals. To achieve a 1:4 streptavidin to pMHC ratio, the four-plex SAv-metal mixture was added to the corresponding pMHC monomer in four stepwise additions, each incubated for 10 minutes at room temperature. Finally, 10 μM D-biotin was added to the reaction, and the mixture was incubated for another 10 minutes at room temperature to saturate unbound streptavidin. Using an Amicon 50 kDa Millipore concentrator, the 562-plex pMHC tetramers were combined and concentrated in 10% FBS CyFACS buffer to 5 μg / ml for each pMHC tetramer. The total protein volume in each concentrator was limited to 300 μg. Multiple centrifugations at 700 x g for 5 minutes were performed to achieve the desired concentration and volume. The 562-plex tetramer was then filtered through a 0.1 μm Millipore tube at 2000 x g for 25 minutes. The mixture of tetramers was kept on ice and then centrifuged in a 1.5 ml Eppendorf tube at 14,000 x g for 1 minute to remove any remaining aggregates before staining.

[0157] For each independent 562-plex combined pMHC tetramer staining experiment, the combined streptavidin protein encoding was re-scrambled.

[0158] For the selected experiments, a 9-choose-3 (84 combinations) or 8-choose-3 (56 combinations) protocol was used to cover 120-plex (40 peptide clusters) or 50-plex (17 peptide clusters) combinations of triple-encoded pMHC tetramer staining, with the preferred option (Table S1) for further phenotypic analysis or in vitro peptide stimulation (Table S2).

[0159] Supplementary Table 1. Detected HLA-A*1101 restriction epitopes and their frequencies in various patient groups List

[0160]

[0161]

[0162]

[0163]

[0164] Table S1

[0165]

[0166]

[0167]

[0168]

[0169] Table S1 - Continued

[0170]

[0171]

[0172]

[0173]

[0174] Table S1 - Continued

[0175] Supplementary Table 2: List of antibody staining groups used for mass cytometry and high-dimensional flow cytometry data analysis

[0176]

[0177]

[0178] Table S2

[0179] 3. High levels of pMHC tetramer, antibody staining, and CD8 T cell enrichment.

[0180] Frozen PBMCs were thawed and washed with complete RPMI (10% FBS + 1% penicillin / streptomycin / L-glutamine + 1% 1M HEPES) (Gibco, Invitrogen) and incubated at 37°C for 3 hours. Upon recovery, cells were harvested and seeded into untreated 96-well plates, approximately 10 million cells per patient, and homogenized into two separate wells for two conformations of 562-plex combined pMHC tetramer staining. Cells were incubated with 50 μM dasatinib at 37°C and 5% CO2 for 30 minutes to prevent TCR(30) downregulation. Cells were washed with CyFACS buffer (PBS solution of 2 mM EDTA + 0.05% sodium azide + 4% FBS) and incubated with 200 mM cisplatin (Pt-195) on ice for 5 minutes, or with rhodium (Rh-103) at room temperature for 20 minutes (Table S2) for viability measurements. After washing once with CyFACS buffer, cells from the same donor in individual wells were stained for 1 hour at room temperature with 50 μl of a mixture containing the same 562-plex pMHC tetramer but a completely different SAV-metal-encoded conformation in the presence of 1:100 Fc blocking agent (Biolegend). Following incubation, cells were washed twice with CyFACS buffer and resuspended on ice in 50 μl of a 1:10 mixture of T cell or CD8T cell enrichment kit (STEMCELL) antibody in CyFACS buffer for 30 minutes. Cells were then washed and stained with 50 μl of primary antibody mixture (Table S2 and...). Figure 21Stain on ice for 30 minutes. Excess antibody was removed by washing cells twice with CyFACS buffer, and cells were resuspended on ice for 15 minutes with 4 μl enrichment beads (STEMCELL) + 46 μl CyFACS buffer. After staining, cells were washed with PBS and fixed overnight at 4°C with 200 μl of 2% PFA (paraformaldehyde, Electron Microscopy Sciences). The next day, PFA was removed and cells were incubated at room temperature for 10 minutes with permeation buffer (Biolegend), then resuspended in 50 μl of intracellular antibody mixture at room temperature for 30 minutes. For subsequent dual-quality-tagged cell barcoding, 2 mM bromoacetamide benzyl-EDTA (BABE; Dojindo) containing 0.5 mM PbCl2 was dissolved in HEPES buffer, and each sample was given a unique combination of metal-barcodes (BABE-Pd-102, BABE-Pd-104, BABE-Pd-106, BABE-Pd-108, BABE-Pd-110) for 30 minutes on ice. After incubation on ice with CyFACS buffer for 5 minutes, cells were labeled for 20 minutes at room temperature using iridium DNA chelators (Ir-191 / 193, Fluidigm DVS) in 2% PFA. Cells were then washed with CyFACS buffer and treated with EasySep. TM Magnet (STEMCELL) negatively selected CD8 T cells according to the manufacturer's instructions. The enriched cells were washed twice with MilliQ water and prepared for mass cytometry collection.

[0181] 4. Statistical Analysis

[0182] Unless otherwise specified, nonparametric analysis of variance (ANOVA) was used for grouped comparisons. A p-value < 0.05 obtained by nonparametric ANOVA allows for subsequent multiple comparison tests. p-values ​​were calculated using Prism software (GraphPad). All error bars are median and SEM.

[0183] 5. HBV genome amplification and library construction

[0184] Seven untreated HBeAg non-seroconversion factors and eight HBeAg seroconversion factors (including genotypes B and C) were recruited from patients with chronic HBV infection at the National University of Singapore Health System. Multiple longitudinal serum samples (5 to 15 time points per patient) were obtained from each patient throughout the HBeAg seroconversion event. All serum samples were analyzed by deep sequencing of the entire HBV viral genome. Similar to previous descriptions, full-length amplicones of the HBV genome were generated using primers (5'-GCTCTTCTTTTTCACCTCTGCCTAATCA-3' and 5'-GCTCTTCAAAAAGTTGCATGGTGCTGG-3'). PfuUltra was used. TM II Fusion HS DNA polymerase (Stratagene, La Jolla, California, USA) was used for polymerase chain reaction (PCR) according to the manufacturer's instructions. A 3.1 kb fragment was extracted from a 1% agarose gel prepared in 1×TBE buffer using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA), and the concentration of the extracted product was measured using a NanoDrop ND 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was fragmented into 100–300 bp fragments using Covaris S2 (Covaris, Woburn, MA, USA). (Cuting conditions: duty cycle: 20%; intensity: 5; cycles per burst: 200; time: 110 s). After fragmentation, the samples were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). The DNA1000 chip was used with a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to examine the size and quality of fragment products. For library construction, the KAPA Library Preparation Kit (KAPA Biosystems) was used according to the manufacturer's instructions. Library construction included end repair, A-tailing, adaptor ligation, and a final PCR step incorporating the index into the sample. Illumina TrueSeq adaptors and indexes (Illumina, San Diego, CA, USA) were used. PfuUltra was used according to the manufacturer's instructions. TMII Fusion HS DNA polymerase was used for this final PCR step. The sample was then cleaned using an Agenthe AMPure XP (Beckman Coulter) at a 1:1 bead-to-sample ratio. To check the size and concentration of the ligation products, a 2100 Bioanalyzer and a DNA 1000 chip were used. The quality and quantity of the products were determined by running quantitative PCR. Reactants were prepared using the KAPA Library Quantification Kit (KAPA Biosystems) and run on a LightCycler 480II Real-Time Thermal Cyclist (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's instructions. The samples were sequenced at the Genome Institute of Singapore on an Illumina HiSeq 2500 to obtain multiplexed 101 bp paired end reads.

[0185] 6. SNV analysis of viral epitopes

[0186] We modified the reference genome P121214 so that ambiguous positions (e.g., R / W / Y) were randomly replaced with one of the relevant bases (A / C / G / T). 101 bp paired end reads were mapped to the modified reference genome using BWA-MEM version 0.7.10-r789, and single nucleotide variants (SNVs) were invoked using LoFreq version 2.1.2. For all samples, the average coverage depth was approximately 10⁴–10⁵. SNVs were filtered by frequency (>5%), SNV quality (>1000), and coverage depth (>100) to remove false positives. SNVs within PCR primer regions were also ignored due to the high error rate. SNVs passing through the quality filter were then sorted, and only non-synonymous mutations (customized according to the script) with a frequency change >20% between early and late time points were retained. These SNVs were candidates for adaptive epitopes and matched with known epitope sequences for further tetramer experiments.

[0187] 7. HBV epitope prediction and peptide synthesis

[0188] The common HBV sequence from each patient was further identified and translated into the amino acid sequence of each open reading frame (ORF). Then, using NetMHC software (v3.4 server, http: / / www.cbs.dtu.dk / services / NetMHC / ), predicted binding factors (peptides) limited to HLA-A*1101 were generated based on the common sequences derived from HBV proteins (nuclear, polymerase, x, and envelope), including all possible binding variants of 8-, 9-, 10-, and 11-meric peptides above the binding threshold (for scores >0.4, weak binding was predicted, and for scores >0.6, strong binding was predicted). The prediction protocol generated 484 unique HLA-A*1101-restricted HBV epitopes, along with 78 known HLA-A*1101-restricted epitopes derived from other pathogens or endogenous proteins, thus yielding a total of 562 distinct pMHC tetramers (Table S1) for subsequent highly multiplexed pMHC tetramer mapping. Previously unidentified epitope sequences were reported using the Immunoepitaxy Database (IEDB). All peptides were synthesized by Mimotopes (Australia) with a purity >85%.

[0189] 8. Peptide sequence similarity and cluster allocation

[0190] To avoid misinterpreting cross-reactive T-cell epitopes in a 562-plex pMHC library composed of all viral proteins and variants, the library sequences were merged and loaded into a Biostring-based R writing environment. Similar to BLAST (Basic Local Alignment Search), a biological sequence and matching algorithm was performed to perform alignments and calculate peptide binding scores based on their sequence similarity. A total of 284 peptide clusters were assigned, and the peptides within each cluster were listed (Table S1). Peptides within the same cluster were then assigned the same quadruple SAv-metal encoding for a highly multiplicative pMHC tetramer strategy.

[0191] 9. Generation of HLA-A*11:01 monomer and 562-plex pMHC libraries

[0192] Inclusion bodies (76, 77) of HLA-A*1101 were generated and refolded with the UV-cleavable peptide H-RVFA(J)SFIK-OH, where J is the ANP (3-amino-3-(2-nitrophenyl)propionic acid) linker. The protein was purified and biotinylated, and stored at -20°C in PBS + 50% glycerol. In 96-well plates, peptide exchange was performed with 0.1 mg / ml of HLA-A*1101 monomer in 100 μl of PBS containing 25 μM of the target peptide. The reactants were exposed to 365 nm UV irradiation twice for 5 minutes using UVP CL-1000 UV crosslinking agent, and the plates were further sealed and stored overnight at 4°C to complete the exchange.

[0193] 10. Production and metal labeling of streptavidin (SAv)

[0194] Streptoacidin with free cysteine ​​residues separated by glycine linkers was used for recombinant expression. Briefly, purified streptavidin was prepared internally and stored as frozen aliquots in 10 mM TCEP in 20 mM HEPES (pH 7.2) buffer at -80°C. After conjugation using a DN3 polymer labeling kit and filtration through a 0.1 μm filter (Amicon), the metal-tagged streptavidin conjugate (SAv-metal) was transferred to a new 30 kDa concentrator (Merck) for five washes with EDTA-free W buffer. SAv-metal was adjusted to a final concentration of 200 μg / mL before the formation of the tetrameric pMHC complex.

[0195] 11. Antibody-metal conjugation

[0196] Purchase purified antibodies free of carrier proteins as listed (Table S2). As previously described, conjugate 50 μg or 100 μg of antibody with a metal-attached maleimide-conjugated DN3 MAXPAR (Fluidigm DVS) chelating polymer according to the manufacturer's instructions (Fluidigm DVS). All metal isotopes were purchased from Fluidigm DVS or TRACE Sciences International Inc., as listed (Table S2).

[0197] 12. Mass Flow Cytometry and Data Preprocessing

[0198] All experiments were performed using a CyTOF2 (Fluidigm DVS) system. Cells were washed twice with MilliQ water, filtered, and immediately acquired by mass cytometry at a rate of 300–350 cells / sec. Two percent of each of the four EQ beads (Fluidigm DVS) were mixed with the cell suspension. To normalize the CyTOF2 signal variations, the output FCS file was normalized based on the previously described added beads. The normalized FCS file was then loaded into a Unix-based R script, and all zero values ​​were randomized to values ​​between 0 and -1 using a uniform distribution.

[0199] 13. Self-validation and automatic unconvolution of antigen-specific T cells

[0200] After data preprocessing, live CD8+ T cells were gated using FlowJo v9.7.6 (Tree Star Inc.), and each sample was debarcoded using a Boolean gate based on dual-quality-labeled cell barcodes. Two SAV-metal-encoded configurations from the same donor were independently barcoded and output. For optimal automated identification of tetramer-positive cells, multiple safety parameters and thresholds were constructed and subjectively defined using R scripts. Figure 1 and Figure 7 In short, the threshold for each SAv-metal channel was manually defined by gating the tetramer-negative population across all fourteen SAv-metal channels. Based on the threshold for each SAv-metal channel (threshold X = Tx and threshold Y = Ty), the safety factor then objectively identifies the tetramer-positive population using preset geometric criteria (Y / X slope = k, X / Y slope = k, and width = w). Figure 7 In this report, all antigen-specific CD8+ T cells identified using a highly multi-combination pMHC tetramer strategy must first pass both a threshold and an autogating strictness parameter. Secondly, the four corresponding SAv-metals encoded on each pMHC tetramer must have exclusive metal intensities higher than the remainder of the ten SAv-metal channels. A deconvolution algorithm excludes any tetramer-positive cells with fewer or more than four SAv-metal encodings. Signal concordance of tetramer-positive cells identified in two different SAv-metal encoding configurations from the same donor is further calculated using statistical simulations. Figure 7 (where p < 0.05 was considered a confidence level.) Finally, antigen-specific CD8+ T cells that passed all the tests mentioned above at a frequency > 0.002 of total CD8+ T cells were selected for further high-dimensional data analysis.

[0201] 14. Visualization of high-dimensional cell counting data

[0202] Validated antigen-specific CD8+ T cells were individually output from each donor for dimensionality reduction analysis. Detailed methodologies for t-SNE and One-SENSE can be found elsewhere. In short, t-SNE and One-SENSE were performed using custom R scripts based on the “flowCore” and “Rtsne” packages downloaded from the Comprehensive R Architecture Network (CRAN). All data were transformed using the “logicleTransform” function with w=0.25, t=16409, m=4.5, and a=0 as input parameters to roughly match the scaling historically used in FlowJo. Cellular markers analyzed by t-SNE and One-SENSE are shown (Table S2). For One-SENSE, cellular markers were subjectively assigned to each T cell category (“differentiation + TNFR”, “exhaustion”, and “transport”) for classification analysis. The heatmap of the comparison represents the distribution of marker-positive cells as a percentage on each “bin” along an axis (category) constructed from cells residing in a small numerical range. The positive groups of the markers were determined manually, and 250 bins were used to combine markers of the same category for each dimension.

[0203] One-SENSE's 3D visualization is based on the analysis of one-SENSE and constructed from multiple consecutive 3D images supported by the "RGL" software package. The consecutive image sequences are then combined using Sequmago (AppleScript) to generate a 3D movie.

[0204] A 3-parameter logistic model was used, with logistic regression performed using the drc R package (v 3.0.1). The support vector machine was trained using the e1071 R package (v 1.6-8) with default parameters (e-regression with radial ε, γ = 1 / 7 and ε = 0.1), using the 7 parameters common to both datasets.

[0205] 15. Flow cytometry and cell sorting

[0206] Cells were prepared in the same manner as in mass cytometry experiments. After incubation with dasatinib, cells were washed with PBS and incubated in the dark on ice for 20 min with 50 μl of Live / Dead-Pacific Orange (Thermo Fisher). For single fluorescent dye-tagged pMHC tetramers, peptide exchange and tetramer formation were performed in the dark using the same method as for metal-tagged pMHC tetramers. PE-SAv (eBioscience), PE-Cy7-SAv (eBioscience), PE-Cy5-SAv (eBioscience), BV650-SAv (BD), and APC-SAv (Biolegend) were diluted to 20 μg / ml in PBS and added to the pMHC monomers loaded with different peptides in the same manner as described above. Cells were then washed and stained with the tetramer mixture in the dark under the same conditions as in mass cytometry experiments. After washing twice with FACS buffer, cells were stained in FACS buffer for 30 minutes in the dark on ice with primary antibodies Pacific Blue-CD14 (Biolegend), Pacific Blue-CD16 (Biolegend), Pacific Blue-CD19 (Biolegend), Alexa Fluor 700-CD3 (Biolegend), FITC-CD4 (Biolegend), and QD605-CD8 (Thermo Fisher). Cells were then washed twice, filtered, and analyzed using an LSR Tortessa (BD). Fluorescent dye-tagged tetramer-positive cells were stained using the same method and sorted in real-time using an Aria II 5 laser system (BD).

[0207] 16. TCR high-throughput sequencing and prototyping analysis

[0208] Five populations (comprising virus-specific CD8+ T cells stained with four different tetramers plus total CD8+ T cells from each donor) were sorted in real time, and genomic DNA was freshly extracted using a Qiagen blood and tissue kit according to the manufacturer's instructions. Cells from three donors in each patient group (a total of five groups and 75 samples) were used for TCR sequencing. Briefly, the TCR β chain was amplified using bias-controlled two-step multiplex PCR on the ImmunoSEQ platform (Adaptive Biotechnologies). The first PCR amplified the CDR3 region of the sorted T cells, followed by an adaptor sequence added in a second PCR, and then sequencing was performed via next-generation sequencing (NGS). Productive reads were generated by the reduction of amplification and sequencing bias. 1000, 5000, 1500, 2000, and 10000 cells were sorted in real time for HBVpol282-, HBVpol387-, HBVcore169-, and HBVcore195-specific CD8+ T cells, and total CD8+ T cells, respectively.

[0209] The TCR sequence was then output from the ImmunoSEQ analyzer, and the gene was defined using the IMGT / HighV-Quest nomenclature. CDR3β length analysis was performed using Prism with Gaussian fitting and the null hypothesis "one curve fits all". The null hypothesis was rejected, indicating statistical significance.

[0210] 17. TCRdist measurement for quantitative epitope-specific libraries

[0211] The quantifiable distance (TCRdist) of epitope-specific TCRβ was calculated as the similarity-weighted Hamming distance (53) between the contact regions of CDR1, CDR2, and CDR3 of two recently developed TCRs. The TCRdist pipeline (https: / / github.com / phbradley / tcr-dist) was downloaded and installed in Python. TCRβ sequences were loaded and executed using the additional commands "--make_fake_alpha" and "--make_fake_quals". An unsupervised visualization of t-SNE was constructed based on the kernel PCA coordinates of the TCR clones and the TCRdist distance matrix generated by the TCRdist pipeline using a custom R script. An executable of the unsupervised clustering Phenograph algorithm (Rphenograph) was installed in R (https: / / github.com / JinmiaoChenLab / Rphenograph). The diversity of epitope-specific TCRs was measured by considering sequence similarity and identity using the library diversity measure (TCRdiv) of the generalized Simpson diversity index.

[0212] 18. In vitro virus-specific CD8+ T cell expansion

[0213] PBMCs from patients were thawed and recovered, then resuspended in AIM-V medium (containing 2% human AB serum, 1% penicillin / streptomycin / L-glutamine, and 1% 1M HEPES) containing 20 IU / ml recombinant human IL-2 (R&D) (Gibco, Invitrogen). Cells were pulsed with the corresponding HBV or control peptide at 1 μM / 1 million cells in 200 μl of medium in 96-well round-bottom tissue culture plates and cultured at 37°C for 10 days. Half of the medium was replaced every three days as a supplement without any peptide. On day 10, cells were restimulated for 7 hours at 37°C in the presence of brefidobacterium A (eBioscience), monensin (eBioscience), and 0.5 μg / ml anti-CD107a, with or without peptide. After incubation, cells were collected and stained with 50-plex pMHC tetramer (Table S1) and surface antibody. As shown in Table S2, intracellular cytokine staining was performed on the second day. All staining, cell bar coding, and CD8 T cell enrichment were performed in the same manner as the in vitro staining described above.

[0214] 19. Enzyme-linked immunosorbent assay (ELISA)

[0215] Paired serum samples from patients were serially diluted in PBS, and the levels of HBeAg, HBsAg, HBeAb, HBsAb, and HBcAb were determined using quantitative sandwich ELISA kits (Abnova and MyBioSource) according to the manufacturer's instructions.

[0216] result

[0217] 1. Comprehensive HBV epitope mapping

[0218] To generate a comprehensive HBV-targeting pMHC library, viral DNA was isolated from serum samples of 15 longitudinal CHB patients and deep sequenced to identify common viral sequences and common variants. Figure 1 A and Figure 7 A, see “Materials and Methods”). Sequences were loaded onto the NetMHC platform to predict potential A*11:01 restriction binding factors. 484 unique putative HBV epitopes above the predicted “weak binding” threshold were combined with 78 known epitopes derived from other common antigens, resulting in a total of 562 peptides listed in Table S1. These peptides were then analyzed using a pairwise matching algorithm (…). Figure 7B and Table S1) analyzed sequence homology to group relatively similar peptides into the same cluster. The resulting 284 peptide clusters were randomly assigned to unique combinations encoded by tetra-stranded mycoagulant-metal (SAv-metal) proteins. Figure 1 A and Figure 7 C). This method avoids the misinterpretation of the combined pMHC tetramer strategy (29), which would result from T cells expected to cross-react with multiple minor variants of the same peptide. The combined library encoding the 562-plex pMHC tetramer was simultaneously probed on peripheral lymphocytes from each patient ( Figure 7 C)(30). To increase the confidence of the detection, patient cells were uniformly partitioned and independently queried using the same 562-plex pMHC tetramer library with two completely different SAv-metal encoding configurations. Figure 7 C- Figure 7 D and S2A). Along with >26 cellular markers (Table S2), the signal of 562-plex pMHC tetramer was determined by mass cytometry. Tetramer-positive events were identified unbiasedly using a self-validated automated tetramer deconvolution algorithm (Materials and Methods, ...). Figure 7 D). The correspondences between matched tetramers from the two coding configurations were calculated using bootstrap statistical analysis (see Materials and Methods). Figure 1 A and Figure 7 D). Finally, for those validated antigen-specific CD8 samples that pass all deconvolution criteria between the two configurations. + T cells are counted, and such methods can be validated by the correlation of control epitopes (e.g., CMV and EBV) detected between conformations. Figure 7 (and S2A). Using this targeted strategy, we were able to detect many unidentified candidate epitopes and their variants in CHB patients, as well as previously identified epitopes (Table S1).

[0219] It was hypothesized that antigen-specific T-cell responses could vary across different clinical stages and reflect CHB disease progression. Therefore, this strategy was applied to map potential T-cell epitopes in three CHB patient groups (IT, IA, and InA) and one group of patients with acute remission (R) (Table S3). There was no difference in the overall magnitude of antigen specificity detected between the patient groups. Figure 9 However, in all patients tested, T cells were detected to have more epitopes specific to polymerase (P) and nucleus (C) compared to envelope protein (S) and x (X) proteins, including the four epitopes observed most frequently. Figure 1B). These are HBV-P-282 (cluster 090, 4 peptide variants), HBV-P-387 (cluster 106, 1 unique peptide), HBV-C-169 (cluster 178, 7 peptide variants), and HBV-C-195v2 (cluster 283, 1 unique peptide). Figure 1 BC Figure 8 (and Table S1).

[0220] Several experiments were conducted to validate and evaluate the HBV relevance of these four epitopes. For three of these epitopes, antigen-specific T cells were detected in some healthy donor (HD) or cord blood (CB) samples. Figure 1 D), but not detected in patients with HLA-mismatch ( Figure 10 Further analysis of these cells, confirmed by fluorescent flow cytometry and pMHC tetramer staining, showed consistent results. Figure 11 A- Figure 11 B). These T cells can also proliferate upon stimulation by the corresponding viral peptides. Figure 12 Unlike other cells, those from IT and HD appeared unresponsive. While the results clearly confirmed the specificity of T cells stained with pMHC tetramers loaded with HBV-P-282 and HBV-C-195v2 peptides, these cells exhibited largely the same phenotype as the original cells. Figure 1 E). Due to the non-random nature of TCR recombination, T cells specific to these epitopes are likely to be primordial T cells with a relatively high precursor frequency and similar to the previously described HCV-specific CD8. + Original precursor.

[0221] The previously identified epitope HBV-P-387 was observed at a relatively high frequency in CHB and also in half of the tested HD samples, but to a lesser extent in CB samples. As further described in later sections, the heterogeneous phenotype of these cells is highly variable among patients. Figure 1 E), indicating an associated HBV-specific immune response. Given the high prevalence of HBV in Singapore, where HD is collected, we speculate that such unexpected detection of HBV-specific T cells may be due to high coverage of vaccination or subclinical infection without the development of anti-HBc antibodies (HBcAbs), as reported by healthy workers exposed to HBV. Figure 1 D and Figure 11 C). This remains to be determined. Finally, among the validated epitopes, HBV-C-169-specific T cells were detected only in individuals infected with HBV, and were elevated in patients with viral control (InA and R) compared to patients with high viral load (IT). Figure 1D), in which these cells are undetectable. These HBV-specific T cells exhibit different phenotypic characteristics depending on the HBV infection status. Figure 1 E). These results suggest that we should further evaluate HBV. pol387 and HBV core169 Specific CD8 + Characteristics of T cells.

[0222] 2. HBV-specific CD8 + High-dimensional phenotypic analysis of T cells

[0223] For HBV pol387 and HBV core169 Specific CD8 + Further analysis of T cells was conducted because a high degree of phenotypic heterogeneity was observed in patients at different stages of CHB. Although one of the peptides within HBV-P-387 (LVVDFSQFSR) and HBV-C-169 (STLPETTVVRR) has been previously reported, the phenotype of these reactive T cells has not yet been investigated. HBV pol387 and HBV core169 Specific CD8 + T cells are unique to CD8, which is specific to HBV. + T cells expressed higher levels of TIGIT compared to T cells. Figure 13 ), and HBV core169 Specific CD8 + Increased PD-1 expression on T cells Figure 13 ).

[0224] Unsupervised high-dimensional t-SNE visualization and Phenograph cell clustering were applied to characterize virus-specific CD8 in individuals from a large number of parallel-run samples. + T cell phenotype ( Figure 2 Based on the expression levels of markers typically associated with T cell exhaustion, indicating T cell activation, differentiation, transport, and inhibitory receptors, 19 cell clusters were objectively identified and annotated. Figure 2 A).

[0225] Through this analysis, BV was observed. pol387 Specific CD8 + Significant heterogeneity of T cells. In many cases, even within individual patients, several different populations specific to this single epitope can be observed. Despite this diversity of phenotypes, the epitope sequence is highly conserved across all patients and across HBV viral sequencing time points within the past decade. Figure 14 Table S4). Using cluster-specific gating strategies ( Figure 15Quantification of intracellular clusters specific to each T-cell antigen was performed in all experimental batches to test compositional differences associated with HBV infection status. Except for cluster 8 (C8), which was formed by HBV infection... pol387 Specific CD8 + All cell clusters occupied by T cells expressed 2B4, which exhibits a heterogeneous phenotype indicating different T cell memory states, while only cluster 9 (C9) showed elevated PD-1 expression. Figure 2 Regarding its relationship with infection status, it was observed that expression of CXCR3... + CD27 hi CD127 hi Significant enrichment of cells with a C13-like phenotype (~80%) in IT patients. Figure 2 A- Figure 2 B and Figure 16 A- Figure 16 C). Significant differences in C8 were also observed in IA patients, which was mediated by CD45RO. hi CCR4 + HVEM + With T CM Phenotypic co-expression was highlighted. Phenotypic similarity regions C6+C17 were preferentially occupied by InA, which was not present in IT patients. Figure 2 B and Figure 16 A- Figure 16 C). This subgroup is similar to terminal effect memory RA (TEMRA), but expresses CD127. Int This may indicate a specific and sustained T-cell response in patients with InA rather than IT. Given that these cells proliferate less in IT patients ( Figure 12 It also expressed several memory-related biomarkers, suggesting that they had undergone but not fully activated, and that such inactivation may be regulated by 2B4 in a PD-1-independent manner. Figure 2 A).

[0226] Supplementary Table 4: Frequency of viral mutations on selective epitopes in longitudinal cohorts of patients undergoing HBeA-seroconversion

[0227]

[0228] Table S4

[0229] Supplementary Table 4 (continued)

[0230]

[0231]

[0232] Table S4 continued

[0233]

[0234]

[0235] Table S4 continued

[0236] Composed of seven peptide sequence variants ( Figure 1 C and Table S1), HBV c ore 169 Specific CD8 + T cells had a significantly higher frequency in patients with viral control (InA and R). Figure 1 D) and pMHC tetramer staining intensity ( Figure 16 D). No T cells specific to this epitope were detected in IT patients or HD. Figure 1 D), the difference being that one IT patient had just below the applied cutoff frequency (0.002%) and detectable cells (0.00193%). Figure 16 E). t-SNE and Phenograph analyses showed that C8 and C11 were significantly enriched in InA and R, while C1 and C4 were tended to be more prevalent in IA and InA patients. This is consistent with hierarchical clustering ( Figure 1 E) Consistent, hierarchical clustering based on HBV c ore 169 Specific CD8 + T-cell phenotypes separate individuals into different clinical stages. Figure 1 E), this is for parallel analysis of EBV-specific CD8 + No T cells were observed. Figure 16 F). Compared with the increased expression of CD57, PD-1, and TIGIT in these cells observed in IA patients, HBV from InA and R patients... c ore 169 Specific CD8 + T cells significantly expressed CD27, CD28, CD45RO, CD127, and CXCR3. Figure 2 D- Figure 2 E), indicating that they are long-lived memory T cells associated with high viral control. However, it is noteworthy that these cells from R patients, despite the presumed viral clearance, express relatively high levels of PD-1 and TIGIT. Nevertheless, these cells derived from R patients differ from those from IA patients in the expression of other memory-related biomarkers and CD57. Importantly, PD-1 is expressed in HBV from R patients. c ore 169 Specific CD8 + T cells exhibited high levels of IL-7R (CD127), indicating that they are not T cells. EXHowever, these may be long-lived memory cells. It is highly likely that this type of PD-1 expression represents a sign of activation or adaptation rather than exhaustion, and is induced by strong TCR viral antigen conjugation. Figure 16 D). R patients may not have completely cleared the virus, and these HBV expressing PD-1... c ore 169 Specific CD8 + T cells still actively suppress the virus. In contrast, HBV from InA patients... c ore 169 Specific CD8 + T cells exhibited moderate levels of CD127 and CXCR3, as well as similar levels of CD27, CD28, and CD45RO compared to R patients. These cells in InA also showed reduced PD-1 expression compared to other groups. Figure 2 D- Figure 2 E). This can be explained by the low to undetectable viral load during the InA phase, resulting in less TCR viral antigen binding compared to IA patients with high viral loads.

[0237] Finally, Scorpius (R. Cannoodt et al., SCORPIUS improves trajectory inference and identifies novel modules in dendritic cell development.bioRxiv) (a trajectory inference method) was applied to calculate HBV levels in three clinical stages using patient-by-patient expression of eight statistically significant phenotypic biomarkers. core169 Specific CD8 + The trajectory of T cells ( Figure 2 F). Our analysis showed that decreased expression of PD-1, TIGIT, and CD57, along with increased expression of CD27, CD28, CXCR3, CD45RO, and CD127, was associated with the inferred infection status. Furthermore, patients with viral control-related status (InA and R) were well-separated and oriented towards the end of the trajectory, while IA patients were on the opposite side. Therefore, the highly heterogeneous HBV-specific CD8 expression during CHB progression was demonstrated using several high-dimensional analysis methods. + T cells, and targeting HBV pol387 and HBV core169 Such multifactorial cellular responses can characterize patients into their clinical stages.

[0238] 3. Multifactorial interactions of inhibitory receptors on virus-specific T cells

[0239] Next, the relationships between various classes of cellular markers expressed by each of the analyzed antigen-specific T cells were evaluated, with particular focus on nine different inhibitory receptors. To directly assess these relationships, one-dimensional singular expression (One-SENSE) via nonlinear random embedding was employed. One-SENSE works by reducing the dimension of each class of marker to a one-dimensional t-SNE map, which can be plotted together with markers of alternative classes mapped into an additional one-dimensional t-SNE map. In this way, cells are individually aligned based on their class expression, and the relationships between classes can then be visually visualized and described.

[0240] In this context, the designated categories (Table S2) are: "differentiation + TNFR" (differentiation markers and the tumor necrosis factor receptor superfamily), "inhibitory" (inhibitory receptors), and "transport" (chemokine receptors). These three derivation axes objectively represent cell subpopulations with co-expression of all possible proteins. Figure 3 A). Plot and compare four different epitopes (including HBV). pol282 HBV pol387 and HBV core169 Epitopes derived from EBV (EBV) EBNA3B )) Specific T cell characteristics. Generally, among the observed combinations of inhibitory receptors expressed by antigen-specific T cells in patients, a subset exhibits HVEM. int 2B4 + TIGIT + CD160 + PD-1 lo And mainly composed of HBV pol387 and EBV EBNA3B Specific CD8 + T cell subpopulation contribution. One-SENSE analysis also showed 2B4 and HVEM lo It is expressed by most antigen-specific T cells, but the expression of LAG-3, TIM-3 and CTLA-4 is limited. Figure 3 A and Figure 13 Additionally, it was found that most PD-1... + The cells do not express 2B4 and TIGIT, but instead express CD160.

[0241] By mapping cells based on the patient group, attention should be paid to HBV. pol387 and HBV core169 Specific CD8 + The phenotype of T cells (blue and red) is most significantly influenced by the infection status, and the distinctive features of these highly diverse cells are labeled. This heterogeneity can be identified by HBV. pol387 and HBVc ore 169 Specific CD8 + T cells are optimally presented. HBV from IT. pol387 They exhibit relatively homogeneous phenotypes in terms of memory-related receptors and transport receptors, but differ in the co-expression of four different inhibitory receptors. Figure 3 A) In other clinical stages, for HBV pol387 Specific CD8 + For T cells, greater diversity was observed in memory-related biomarkers compared to effector-related biomarkers, and these also exhibited complex relationships with patterns of co-expressed inhibitory receptors. Figure 3 A) HBV core169 Specific CD8 + Similar examinations of T cells were slightly limited by the limited cell count, but also showed a greater degree of heterogeneity than expected. Nevertheless, this performance was consistent with the above. Figure 2 ).

[0242] The number of inhibitory receptors co-expressed on these cells was further quantified using a Boolean strategy. Figure 3 B- Figure 3 C). Consistent with One-SENSE analysis, no HBV-specific cell subsets accumulating all inhibitory receptors were detected. Figure 3 A- Figure 3 C and Figure 11 Although HBV can be confirmed via One-Sensor. pol387 Specific CD8 + Different co-expression of inhibitory receptors on T cells was observed, but there was no difference in the number of cells accumulated on these receptors in the patient group. Conversely, compared to patients with viral control, IA patients showed higher levels of HBV... core169 Specific CD8 + T cells have a significantly higher number of inhibitory receptors ( Figure 3 C). Together with the visualization of One-SENSE, our data show a highly heterogeneous antigen-specific phenotype, rather than a simple accumulation of so-called “exhaustion markers” during CHB, and that the different co-expression of inhibitory receptors has different relationships with cell differentiation and transport features that can be associated with disease stage.

[0243] 5. HBV-specific CD8 + T cell function

[0244] To address the relationship between functional capacity and inhibitory receptors, the patient's cells were pulsed and expanded using short-term in vitro peptide stimulation, followed by assessment of functional responses using intracellular cytokine staining. Figure 12One-SENSE analysis was used to analyze the relationships between virus-specific CD8 based on the categories of "function", "inhibition", and "differentiation + TNFR". + T cells are characterized as five major heterogeneous functional subsets ( Figure 4 Tables A and S2). For each category (axis of the One-SENSE chart), all possible cell subpopulations are described using heatmap lines and descriptive markers. A biaxial plot of the most relevant markers for these five functionally distinct subpopulations is also shown. Figure 4 B), and quantified the relative composition of these subgroups ( Figure 4 C). In summary, this analysis highlights the inhibitory receptors and virus-specific CD8 receptors during antigen recall in CHB. + Nonlinear relationship between the functional capabilities of T cells.

[0245] Regardless of the patient group, there are multifunctional subgroups ( Figure 4 A, green box) and mainly composed of MIP-1β expression + GrzA + GrzK lo Perforated protein + Instead of expressing CD107a lo HBV pol387 and HBV env304 Specific CD8 + T cell contribution ( Figure 4 A- Figure 4 B and Figure 18 A). Another population of these cells (blue boxes) with other similar phenotypic characteristics were significantly less able to produce cytokines, but for GrzA... + GrzK + Perforated protein Int The absence of the degranulation marker CD107a suggests that this is a substitute form of dysfunctional T cells associated with the expression of 2B4 and TIGIT, rather than PD-1. Figure 4 A- Figure 4 B and Figure 18 B). These cells are mainly composed of HBV from CHB patients. env304 Specific CD8 + T cell composition ( Figure 4 A and Figure 18 A), which has greatly expanded in response to in vitro peptide stimulation (A), Figure 12 Unlike other inhibitory receptors, CD160 and HVEM are significantly reduced in effector cells after TCR stimulation. Persistent HVEM is primarily caused by HBV. pol282 Non-functional (black box) subsets of specific cells are expressed ( Figure 4 A- Figure 4 B and Figure 18 A- Figure 18 B). These naive and unresponsive T cells present different types of dysfunctional T cells, possibly related to their expression of BTLA and CD160 prior to antigen recall. Figure 13 It was also found that some of the HBV from R patients... env304 Specific CD8 + T cells possess CD8 specificity for EBV and IAV. + T cell-like functional characteristics, the EBV and IAV-specific CD8 + T cells are PD-1 - LAG-3 - TIM-3 lo Expressing IFN-γ + TNF-α hi MIP-1β hi GM-CSF hi (yellow box) Figure 4 A and Figure 18 A and Figure 18 C). Moreover, HBV core169 Specific CD8 + T cells exist in a unique, multifunctional subset (red box), which collectively produce a variety of non-cytolytic and cell recruitment factors (GrzA). - GrzK - IFN-γ + TNF-α lo MIP-1β + GM-CSF int CD107a), although they co-express five inhibitory receptors, including PD-1 ( Figure 4 A- Figure 4 B). Interestingly, this subgroup exhibited high levels of TNFR co-stimulatory receptors (OX40, GITR, 4-1BB, and CD27), suggesting a stronger activation and memory state. The main difference observed between patient groups in this analysis was HBV. core169 Specific CD8 + Characteristics of T cells. Patients with better viral control (R>InA>IA) showed a significantly higher frequency of HBV. core169 Specific CD8 + T cell multifunctional subsets. In contrast, for MIP1-β + Multifunctional subpopulation of HBV core169 Specific CD8 + The frequency of T cells showed an opposite trend ( Figure 4 C).

[0246] Together with the data mentioned above, we can conclude that HBV core169 Specific CD8 + T-cell immune responses are associated with viral control. This analysis also demonstrated the role of virus-specific CD8 during CHB. + There is a complex, ordered, rather than a simple linear, relationship between inhibitory receptors on T cells and their functional capacity.

[0247] 6. Clinical staging-dependent landscape of virus-specific TCR

[0248] How to select T-cell receptors during CHB is largely unknown. Therefore, at each clinical stage, the selection of T-cell receptors based on pMHC tetramer staining (…) Figure 11 B) Sorting and sequencing epitope-specific TCRs (HBV) pol282 HBV pol387 HBV core169 and HBV core195 The β chain of the epitopes. To map the TCR landscape of these epitopes, TCRdist, an algorithm that generates a distance matrix to quantify and obtain the relative motif similarity of TCRs based on their amino acid sequences (P. Dash et al., Quantifiable predictive features define epitope-specific T cell receptor repertoires). Using the TCRdist distance matrix, similar TCRs were clustered using an unsupervised Phenograph clustering algorithm (JHLevine et al., Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis), and then visualized using a t-SNE dimensionality reduction algorithm. Figure 5 A). Subsequently, the sequence motifs that form the basis of each TCR sequence cluster can be presented ( Figure 5 B and Figure 19 A), and the composition of these clusters can be quantitatively determined in terms of the antigen specificity of each sequence derived from the patient. Figure 5 C). TCR clusters 15 and 27 were found to be more prominent in HBV compared to other epitopes. pol282 Significantly increased in specific TCR ( Figure 5 C- Figure 5D), indicating that these motifs are important determinant clusters for identifying this HBV epitope and can be associated with the original phenotype. Furthermore, the relative use of each of these TCR sequence clusters differed significantly among patients grouped by HBV infection status. TCR cluster 15, dominated by TRBV5-6, was observed in all patients except IA patients, while IA patients were conversely rich in TRBV3-2. + TCR cluster 12. Interestingly, TCR cluster 27 connects only to the highly conservative CDR3 via TRBJ2-6. Figure 19 A). The cluster used in the TCR sequence is for HBV. core169 and HBV pol387 More diverse than specific T cells. Nevertheless, HBV core169 Specific TCR sequences also differ between patient groups. TRBV3-2 + TCR cluster 12 (also rich in HBV) pol282 Specific TCRs were similarly observed in IA patients with HBV. core169 Specific TCR enrichment. Additionally, HBV from R and InA. core169 The specific TCRs showed significantly higher TRBV6-6. + Cluster 5 and TRBV28 + Using Cluster 9 ( Figure 5 C).

[0249] The library diversity and density of each TCR sequence from various cell populations were calculated using the TCR diversity measure (TCRdiv) (P. Dash et al., Quantifiable predictive features define epitope-specific T cell receptor repertoires). Various patterns of this measurement were observed in epitopes and patient groups. Figure 5 E). It is worth noting that HBV pol387 TCRdiv score of specific TCR and HBV in patient group pol387 Specific CD8 + The proportion of cell clusters 13 in T cells shows a strikingly similar pattern. Figure 2 B), which can be attributed to its relative enrichment in the IT and HD groups. Importantly, both epitope-specific TCRs and ontological TCRs from HD exhibit greater overall diversity. Figure 19 B), which separates them from CHB patients at the molecular level. (Compared to total CD8) + T cell homogeneity compared to ( Figure 19C) CDR3 length was skewed between epitope and patient groups. Overall, these findings are consistent with previously described phenotypic differences observed in different patient groups. Therefore, our analysis suggests that biased TCR repertoire use during CHB is epitope- and clinical stage-dependent.

[0250] 7. HBV present in patients with controlled viral load core169 Specific TCR clones

[0251] Focus on HBV core169 Specific responses and queries of recently curated TCR databases revealed several previously unidentified common HBV strains shared among individuals in a clinical stage-dependent manner. core169 Specific TCRβ clones ( Figure 5 F). Specifically, in all three InA patients tested, the universal common clone CASGDSNSPLHF was present in the first three TCR clones. Two other specific common clones, CASGGQIVYEQYF and CSARGGRGGDYTF, were identified in each of the two InA patients. An additional specific common clone, CASSQDWTEAFF, was found at a low frequency in the two acute remission patients. These common TCR clones were not shared across the patient groups, further highlighting the difference in the quality of T-cell responses occurring between acute and chronic viral infections. The failure to detect common clones in IA patients suggests that the presence of common TCRs is essential for HBV control. By using PCA to combine HBV from the same donor... pol387 and HBV core169 The characteristics of specific TCR repertoires and cellular responses can depict a patient's clinical status. Figure 5 G). Finally, the observed HBV was found. core169 Specific CD8 + The frequency of T cells is negatively correlated with the diversity of their TCR repertoire. Figure 5 H), which indicates selective amplification of T cell clones after viral clearance.

[0252] 8. HBV core169 Specific CD8 + Phenotypic dynamics of T cells

[0253] To assess the characteristic changes in antigen-specific T cells during infection, patients (n=14, HLA-A*1101) who received entecavir (ETV) over several years were examined. + Selected HBV epitopes were studied in a longitudinal cohort of patients. ETV is a nucleotide analog that inhibits viral replication and leads to improved viral control in most patients. Although it does not inhibit HBeAg production by infected hepatocytes, it can also induce HBeAg seroconversion and the establishment of anti-HBeAg antibodies (HBeAb) in some patients, which are serological markers of further improved viral suppression. For 14 patients with detectable HBV...core169 Specific CD8 + Ten patients with T-cell T-cell abnormalities ( Figure 6 A) We compared these cells in patients who subsequently lost HBeAg and produced HBeAb during the study (HBeAg... - (n=6) and those patients who did not lose HBeAg and produced HBeAb (HBeAg + The characteristics of HBV in patients (n=4) are consistent with previous work. core169 Specific CD8 + The frequency of T cells decreased ( Figure 6 B and Figure 20 A). Further experiments were conducted to dissect longitudinal viral mutations and tetrameric responses at this given epitope. Figure 20 B) Showing these HBV-specific CD8 + T cells can recognize variants beyond those shared in the database. Based on analysis of these cells at multiple time points, detailed characteristics can be tracked over time, and significant changes in these cell phenotypes are often observed. Figure 6 C). For example, at early time points, HBeAg from patients + 03 (patients who did not lose HBeAg during the study period) HBV core169 Specific CD8 + T cells exhibited terminally differentiated effector phenotypes (CD57). + CD45RA + CCR7 - However, at a later time point, more than half of these phenotypic changes occurred. In contrast, those from HBeAg... - Patient (HBeAg) - 01. HBV in patients who have lost HBeAg and established HBeAb core169 Specific CD8 + T cells exhibited the memory T cell phenotype (CD27) at all test time points. + CD127 + CD45RO + This effect persisted for more than 6 years after treatment. Similar trends were observed in other patients studied, and detectable HBV levels were quantitatively described. core169 Specific CD8 + T cells ( Figure 6 D).

[0254] In addition to summarizing these cellular compositions based on their memory phenotypes and effector phenotypes over time ( Figure 6D), and also focused on the same biomarkers that showed the greatest variation in patients in our cross-sectional cohort ( Figure 1 E and Figure 2 D- Figure 2 E). Generally speaking, HBV core169 Specific CD8 + The fraction of memory cell subsets in T cells is associated with lower HBeAg levels over time. Figure 6 D). This is influenced by increased expression of T-cell memory-related markers (CD27, CD127, CD45RO, and CXCR3). Figure 6 E) and HBV core169 Specific CD8 + The reduced expression of CD57 on T cells supports similar findings from cross-sectional cohorts of InA and R patients. Figure 1 E and Figure 2 D). In patients maintaining high HBeAg levels, HBV infection occurred after viral suppression. core169 Specific CD8 + T cells exhibited significantly lower levels of CD57 and higher levels of CD27 and CD127, while those derived from HBeAg... - The patient's cells already possessed these cellular characteristics (CD57) prior to treatment. lo CD27 hi CD127 hi CD45RO + This will continue for several years. To further quantify these changes, trajectory analysis using Scorpius was applied to these samples. To examine the reproducibility of trajectory detection in this longitudinal cohort compared to the cross-sectional cohort (…),… Figure 2 F), using a support vector machine (SVM) to map data from longitudinal samples onto a quasi-temporal metric developed using Scorpius from the cross-sectional queue ( Figure 2 F, see Methods). In other words, using data from the cross-sectional cohort as the training set, we compute pseudotimes across patient time points in the test set (longitudinal cohort, ...). Figure 6 F). Among these seven cellular markers, HBV was significantly lower in two independent patient cohorts. core169 Specific CD8 + The trajectory of T cells is consistent (even when Scorpius is run independently without SVM). Besides validating the trajectory model on this independent cohort, this also allows the assumption that the evolution of HBV-specific T cell phenotypes will be tracked in terms of the degree of viral control under test. In fact, HBV... core169 Specific CD8 + The T-cell phenotype did indeed show the expected progression of all patients along this pseudo-timescale during treatment. Figure 6 (G). This suggests that virus-specific T-cell responses associated with viral control improve during antiviral therapy for each patient, as would be expected. Additionally, patients who lost HBeAg and established HBeAb had a more progressive phenotype (higher T-cell memory marker expression, lower PD-1 and TIGIT expression) at earlier time points compared to non-HBeAg seroconversion patients, indicating that these patients had better virus-specific T-cell responses at the start of treatment. Therefore, our data show that HBV expressing increased cellular markers associated with long-term T-cell memory development but with reduced expression of CD57 and the two inhibitory receptors PD-1 and TIGIT... core169 Specific CD8 + T cells are associated with viral control, and such machine learning-assisted models can have predictive value for prognosis in CHB.

[0255] discuss

[0256] By fully utilizing a highly multiplexed pMHC tetramer staining strategy, mass cytometry, and unsupervised high-dimensional analysis, we investigated 562 unique A*11:01-restricted candidate epitopes during HBV progression. Analysis of HBV-specific T cell responses is challenging due to the extremely low frequency of these cells. In this regard, we demonstrate the importance of studying both the specificity and phenotypic characterization of antigen-specific T cells to validate their involvement in HBV-specific immune responses. Furthermore, our data highlight the heterogeneity of virus-specific T cell responses associated with disease stage and provide a quantifiable analysis of an HBV-specific TCRβ repertoire corresponding to cellular phenotypes during chronic viral infection.

[0257] Host defense against HBV relies on an immune response primarily driven by virus-specific T cells. The number of A*11:01-restricted epitopes detected using this comprehensive approach is relatively limited compared to epitopes reported in the A*02:01 case. Some epitope-specific T cells may be detectable only in the liver but not in the periphery. Further research on HBV-specific intrahepatic lymphocytes is needed. The well-described A*02:01-restricted HBV has been confirmed. core18-27 Specific CD8 + The presence and frequency of T cells are associated with viral control. Therefore, numerous therapies have been developed based on these T cells, including blocking overexpressed PD-1 to restore T cell function, adoptive transfer of engineered virus-specific T cells, and TCR-like (TCR-L) antibodies to directly deliver interferon-α (IFN-αα) to infected hepatocytes. Here, the evidence presented shows that A*11:01-restricted HBV... core169 Specific CD8+ The specific responses and characteristics of T cells used in this study are associated with viral control. Comparative analysis showed that these cells exhibited different characteristics across different clinical stages. Furthermore, high-dimensional trajectory analysis allowed for the use of HBV... core169 Specific CD8 + T-cell characteristics were used to assign values ​​to each patient along an objective, pseudo-time metric. HBV core169 The fundamental characteristics of specific T cells along this trajectory were consistent in both independent patient cohorts, both showing a correlation with viral control. Based on this metric, it can also be inferred that these antiviral-treated patients and those with the most progressive T cells exhibiting viral control once HBV develops... core169 A specific memory T cell response qualifies for the safe discontinuation of antiviral drugs, consistent with recent reports demonstrating the predictive utility of HBV-specific T cells (63). This is significant because even state-of-the-art serological measures cannot accurately predict such outcomes. Overall, our findings should influence HBV immunotherapy design and could be used for HBV-based treatments. core169 Specific CD8 + Phenotypic responses of T cells can be used to predict patient clinical outcomes. The utility of this approach can also be expected to extend to other epitopes associated with viral control, derived from HBVcore or limited to other HLA alleles.

[0258] By comprehensively probing HBV epitopes in many HBV-infected patients, the inventors here were unable to identify T cells expressing all inhibitory receptors. EX Or clear evidence of “tissue T cell exhaustion.” Instead, unsupervised visualization using One-SENSE revealed a complex nonlinear relationship between the expression of inhibitory receptors. Furthermore, the dysfunction of HBV-specific T cells was not correlated with the linear accumulation of inhibitory receptors, suggesting that these cells are not completely functionally inert. One explanation is that these HBV-specific T cells do not conform well to the T cell patterns reported in LCMV-specific T cells. EX The definition of T cells is not limited to a few distinct subsets that are mostly absent, with the remaining dysfunctional T cells expressing various combinations of inhibitory receptors. Based on the current data, it is proposed that these T cell characteristics (at least in peripheral blood) better align with the description of functional adaptation in CHB. Nevertheless, it is worth noting that our functional assessment relied on in vitro peptide stimulation due to the rare detection of HBV-specific T cells, and this may limit its relevance to in vivo responses. Unlike during chronic LCMV infection, where T cells... EX The maintenance of the phenotype requires sustained and high antigen levels, as seen in HBV patients with InA who express high levels of CD27 and IL-7Rα (CD127).core169 Specific CD8 + T cells are not T cells. EX And it can persist for decades in the presence of limited amounts of viral antigen. HBV-specific T cells from these patients are PD-1. int TIGIT int Memory-related markers were elevated and showed functional capabilities seemingly associated with viral control. On the other hand, in IA patients with high and fluctuating viremia, HBV-specific T cells were detected to better match T cells. EX The expected characteristics included strong co-expression of PD-1 and TIGIT, and limited expression of memory-related markers such as CD127. Additionally, the inventors found high expression of CD57 in HBV-specific T cells from IA patients, a cellular marker indicating highly differentiated effector cells with low proliferative capacity, suggesting these cells are not long-lived memory cells. Overall, the cellular characteristics of HBV-specific T cells in IA stages were consistent with persistently high antigen exposure. In the later stages (InA) of patients with better viral control, HBV-specific T cells exhibited a memory phenotype with lower CD57 expression but elevated expression of CXCR3, CD45RO, CD27, and CD127. Detectable HBV was lacking in IT patients with high viremia. core169 Specific CD8 + T cell counts suggest that they may be largely absent, and this specific T cell deficiency may contribute to minimal liver inflammation at this stage. Future research involving larger volumes of blood samples from IT patients or using more sensitive methods could help address this issue. Further investigation into the expression levels of EOMES and T-bet may better define the true T cells. EX Epigenetic modifications are important for addressing this aspect in CHB. It is also important to note that this report is limited to circulating HBV-specific CD8. + Further analysis of T cells is needed to examine the exhaustion characteristics of intrahepatic lymphocytes to address this issue.

[0259] Previous studies have shown associations between different inhibitory receptors and T cell differentiation, which is relatively inconsistent with the current One-SENSE analysis, which objectively reveals a more complex relationship between co-expressed inhibitory receptors and T cell differentiation on several virus-specific T cells across multiple clinical stages of HBV infection. This is because many studies of chronic viral infection in humans often examine fewer than four inhibitory receptors on a limited number of virus-specific T cells from a single patient type within T cell subsets defined by only a few differentiation-related markers. Secondly, conventional analyses using hierarchical gating on biaxial dot plots to assess cellular protein expression levels can easily underestimate phenotypic complexity.

[0260] Despite the existence of HBV core169 Epitopes exhibit TCR sequence diversity specific to T cells, but several publicly known clones were detected in relatively high abundance across multiple patients. Consistent with CMV-specific TCR repertoires, it was noted that these previously unidentified public TCRs differed when derived from patients exhibiting viral control (R and InA) versus viremia (IA), indicating the functional importance of these T cells. Public virus-specific TCR clones can be selected during viral clearance (i.e., from IA to InA) and effector response convergence. As previously reported for CMV and EBV infections, virus-specific CD8... + T cells do not express IL-7Rα (CD127) until T cell memory has been established, and this selection is believed to be driven by binding to high-affinity TCR viral epitopes. This is consistent with HBV-positive InA and R patients carrying a common TCR and exhibiting elevated expression of T cell memory-related markers (including CD127). core169 Specific CD8 + The consistent characteristics of the T cells suggest their long-lived self-renewal capacity to maintain a pool of memory T cells after viral antigen reduction. Similarly, hepatic and peripheral common TCR clones have been associated with viral clearance in HCV-infected chimpanzees.

[0261] Despite the significant challenges associated with detecting HBV-specific T cells due to its low prevalence, this invention explores the previously unrecognized complexity of virus-specific T cells in lifelong human HBV infection. core169 Specific CD8 + The cellular response of T cells and the TCR sequences used are correlated with the status of HBV infection and can be used as indicators of the relative degree of viral control. Therefore, the results presented here have important implications for the development of new biomarkers, treatment strategies, and immunotherapies targeting HBV cure.

[0262] While preferred embodiments of the invention have been described in the foregoing description, those skilled in the art will understand that many variations or modifications can be made to the details of the design or construction without departing from the invention.

Claims

1. A peptide consisting of an amino acid sequence selected from the group consisting of: STLPETTVIRR, STPPETTVVRR, STLPETTVVGR, and STIPETTVVRR, wherein the peptide is derived from hepatitis B virus nucleus 169 and is capable of binding HLA-A*1101, and when binding HLA-A*1101, is capable of identifying T cells specific to hepatitis B virus.

2. Use of the peptide according to claim 1 in the preparation of a pharmaceutical agent for identifying hepatitis B virus antigen-specific T cells.

3. A method for selecting hepatitis B virus antigen-specific T cells, the method comprising contacting a population of T cells with the peptide according to claim 1.

4. Use of the peptide according to claim 1 in the preparation of a pharmaceutical composition for identifying hepatitis B virus antigen-specific T cells.

5. A pharmaceutical composition comprising the peptide according to claim 1 and a pharmaceutically acceptable carrier.

6. A vaccine against hepatitis B virus infection, comprising the peptide according to claim 1.

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