USE OF HLA-A*11:01-RESTRICTED HEPATITIS B VIRUS (HBV) PEPTIDES FOR IDENTIFYING HBV-SPECIFIC CD8+ T CELLS
20230087348 · 2023-03-23
Assignee
Inventors
Cpc classification
A61K35/17
HUMAN NECESSITIES
A61K39/292
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61K35/17
HUMAN NECESSITIES
Abstract
The present invention relates to peptides and their ability to identify and bind to T cells specific for HBV-infected hepa-tocytes. In an first aspect of the invention, there is provided a peptide comprising an amino sequence selected from the group consisting of STLPETAVVRR, STLPETAVVR, STLPETTVVRR, STLPETTVIRR, STPPETTVVRR, STLPETTVVGR and STIPETTVVRR, wherein the peptide is derived from Hepatitis B virus core169 and is capable of binding HLA-A*1101 and when bound to HLA-A*1101 s capable of identifying T cells specific for Hepatitis B virus. In a second aspect of the invention, there is provided A T cell expressing a T cell receptor (TCR) molecule, wherein the TCR molecule comprises an amino acid sequence selected from the group comprising: CASGDSNSPLHF, CASSGGQIVYEQYF, CSARGGRGGDYTF and CASSQDWTEAFF, the T cell receptor is able to bind to a pep-tide according to the first aspect of the invention.
Claims
1. A peptide comprising an amino sequence selected from the group consisting of STLPETAVVRR (SEQ ID No. 21), STLPETAVVR (SEQ ID No. 22), STLPETTVVRR (SEQ ID No. 23), STLPETTVIRR (SEQ ID No. 24), STPPETTVVRR (SEQ ID No. 25), STLPETTVVGR (SEQ ID No. 26) and STIPETTVVRR (SEQ ID No. 27), wherein the peptide is derived from Hepatitis B virus core169 and is capable of binding HLA-A*1101 and when bound to HLA-A*1101 is capable of identifying T cells specific for Hepatitis B virus.
2. Use of a peptide according to claim 1 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 a peptide according to claim 1.
4. A T cell receptor (TCR) molecule comprising an amino acid sequence selected from the group comprising: CASGDSNSPLHF (SEQ ID No. 17), CASSGGQIVYEQYF (SEQ ID No. 18), CSARGGRGGDYTF (SEQ ID No. 19) and CASSQDWTEAFF (SEQ ID No. 20), the T cell receptor is able to bind to a peptide according to claim 1.
5. The TCR molecule according to claim 4, wherein the amino acid encodes for a TCR beta chain portion.
6. A polynucleotide encoding a peptide according to a TCR molecule in claim 4.
7. The polynucleotide encoding a T cell receptor according to claim 6 has a sequence selected from the group comprising SEQ ID NOS 1 to 16.
8. An expression vector comprising a polynucleotide according to claim 6.
9. A host cell comprising a polynucleotide according to claim 6.
10. The host cell according to claim 9, wherein the host cell is a T cell derived from a patient.
11. A T cell modified to express the TCR molecule according to claim 4.
12. (canceled)
13. A pharmaceutical composition comprising a peptide according to claim 1 and a pharmaceutically acceptable carrier.
14. (canceled)
15. A vaccine against Hepatitis B virus infection comprising a peptide according to claim 1.
16. (canceled)
17. A method of treating a Hepatitis B virus infection in an individual, the method comprising administering to the individual an effective amount of a peptide according to claim 1.
18. (canceled)
19. A method of combating a Hepatitis B virus infection in a patient which carries HLA A*1101, the method comprising: (a) obtaining T cells from the patient; (b) introducing into the T cells a polynucleotide encoding a TCR molecule according to claim 4; (c) introducing the T cells produced in step (b) into the patient.
20. The method according to claim 19, wherein the polynucleotide is transfected or introduced to the T cells by electroporation.
21. A host cell comprising an expression vector according to claim 8.
22. A pharmaceutical composition comprising a T cell according to claim 10 and a pharmaceutically acceptable carrier.
23. A vaccine against Hepatitis B virus infection comprising a T cell according to claim 11.
24. A method of treating a Hepatitis B virus infection in an individual, the method comprising administering to the individual an effective amount of a T cell according to claim 11.
Description
[0106]
[0107]
[0108]
[0109]
[0110]
[0111] Correlation between the frequency and TCRdiv diversity measures of HBVcore169-specific CD8+ T cells.
[0112]
[0113]
[0114] (A) HBV was deep-sequenced using next-generation sequecing (NGS). NetMHC (v3.4) was used to predict HLA-A*11:01-restricted epitopes based on the consensus sequences. (B) 562 peptides were clustered based on the sequence homology and were further given unique combinations of four SAv-metal codes. (C) These unqie combinations of four SAv-metal mixtures for two different coding configurations were prepared by automatic liquid handling robot. The quandruple SAv-metal coded pMHC tetramer library (for two coding configurations) was used to stain on patient's PBMC. (D) Tetramer positive cells were determined by an automatic tetramer deconvolution algorithm and the tetramer signals between the two coding configurations were calculated for their correspondence.
[0115]
[0116] (A) The staining quality of quadruple SAv-metal coded pMHC tetramers using fourteen different SAv-metal channels from one representative CHB donor. The same vial of PBMC from each donor was stained in parallel with the same 562-plex pMHC tetramers but two different SAv-metal coding configurations as shown. (B) Magnitude of selected HBV-specific CD8+ T cells detected by highly multiplexed combinatorial pMHC tetramer strategy across various clinical stages of HBV infection. Plots show the frequency of antigen-specific CD8+ T cells for fifteen predictive HBV epitope clusters and six representative known control viral epitopes (shaded box). Epitope sequences in bold face indicate previously unpublished sequences. * means this epitope cluster contains more than one peptide (related to Supplementary Table 1). Dash lines on the y-axis are 0.002.
[0117]
[0118] (A) Upper panel, the total number of different epitopes derived from four hepatitis B viral proteins (envelope, polymerase, core and x) detected in each individual patient across various clinical stages. Lower panel, the sum of frequency (%) of every detected antigen-specific CD8+ T cells for four different hepatitis B viral proteins in each individual patient across various clinical stages. n.s.=no significance.
[0119]
[0120] (A) Cells from each donor were stained with selected 120-plex (Supplementary Table 1) pMHC tetramers coded with three different SAv-metal using two different sets of coding configurations. Experiments were performed independently and cells were stained and gated on live CD3+, Dump-(CD4+CD19+CD16+) and CD8+. Bar graphs indicate the frequencies for each epitope. (B) Representative dot plots show the pMHC tetramer positive cells and their signals of coded SAv-metals by different combinations of nine metal-tag SAv.
[0121]
[0122] (A) Correlations of the detected frequencies between FACS (single fluorochrome-coded) versus CyTOF (combinatorial metal-coded) based experiments. Each dot is one individual patient. (B) Representative FACS dotplots of selected HBV epitopes. The numbers are the frequencies of total CD8+ T cells. (C) Left, the hBsAb (anti-HBsAg antibody) titers in healthy donors (HD). Right, the frequency of HBVpol387-specific CD8+ T cells in HD in different HBV serological (HBsAb, HBcAb and HBeAb) status. The levels of serum antibodies against different HBV viral antigens were measured by ELISA. Red circle indicates the only individual who was tested positive for HBcAb and HBeAb.
[0123]
[0124] (A) PBMC from different patient groups were expanded by corresponded viral peptides for 10 days. The frequency of antigen-specific CD8+ T cells were determined as the same as ex vivo pMHC tetramer staining experiment. The number above each graph indicates the significant p value.
[0125]
[0126] (A) The expression level of inhibitory receptors on selected antigen-specific CD8+ T cells. The numbers on x-axis indicate the peptide cluster number for each specificity (epitopes) (see table_S1). Colored dots are the four selective HBV epitopes (090_HBV-P-282, 106_HBV-P-387, 178_HBV-C-169 and 283_HBV-C-195v2). Color legends indicate the different HBV clinical stages. Shaded areas are control viral epitopes. Statistical significances were only shown for selected epitopes. p value less than 0.0001 (short ticks) or other values (long ticks) are indicated.
[0127]
[0128] (A) Deep sequencing analysis of HBV viral DNA showed the different dynamic of viral mutations on selective epitopes identified by highly multiplexed combinatorial pMHC tetramer strategy. A longitudinal patient cohort included treatment naive CHB patients who spontaneously underwent HBeAg-seroconversion (S, bottom) or Non-seroconversion (C, upper) across the similar time frame in chronological order. HBVpol387 (LVVDFSQFSR (SEQ ID No. 28)) proportion (upper right) in the viral population sequenced at each time point, out of a maximum of 1. This epitope remained fixed in all patients and no change was observed. Epitope ID and sequence was as listed in table S1. Detailed epitope mutation data can be found in table S4.
[0129]
[0130] (A) Subset enrichment strategy for eight major Phenograph subset clusters of HBV-specific CD8+ T cells. Gating strategy was defined in a representative experiment and defining markers for each subset clusters were then applied onto three different batches of experiments to identify the proportion of each subset cluster within HBVpol387 and HBVcore169-specific CD8+ T cells. Shaded areas are the target clusters color-coded as in
[0131]
[0132] (A) Representative plots of cellular cluster showed HBVpol387(LVVDFSQFSR (SEQ ID No. 28))-specific CD8+ T cells were enriched in distinct regions across patient groups. Related to
[0133]
[0134] (A) The average numbers of co-expressed inhibitory receptors on different antigen-specific CD8+ T cells in individuals. Plots were combined of four independent experiments without the measurement of cellular marker TIGIT. Each dot is one individual. (B) The average numbers of co-expressed inhibitory receptors on different antigen-specific CD8+ T cells in individuals. Plots were from an experiment with the measurement of all nine inhibitory receptors.
[0135]
[0136] (A) The detailed One-SENSE functional clusters of different antigen-specific CD8+ T cells across patient groups. n=4.sup.˜5 per patient group. Functional subsets were labelled as in
[0137]
[0138] (A) Nine TCR motif clusters identified by Phenograph were presented, and the representative TCR motifs were shown using average-linkage dendrogram using TCRdist algorithm. Related to
[0139]
[0140] (A) The liver inflammation scores of two patients (HBeAg+03 and HBeAg-01) from a longitudinal patient cohort were shown. ALT, alanine aminotransferase. AST, aspartate aminotransferase. AFP, alpha-fetoprotein. ALP, alkaline phosphatase. Related to
[0141]
[0142] (A) Dotplots show the expression levels of markers probed on antigen-specific (left) versus global (right) CD8+ T cells in patient's PBMC. Upper panels are ex vivo staining. Lower panels are cells from in vitro peptide stimulation. For the better visualization of dotplots, all detected antigen-specific CD8+ T cells were pooled from 18 patients (left panel, n=4.sup.˜5 per patient group, including IT, IA, InA and R). PD-1-expressing HBVcore169-specific CD8+ T cells from a CHB patient is also showed. Plots were from two independent experiments (ex vivo and in vitro).
[0143] The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims.
[0144] Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.
[0145] As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps. As used herein, except where the context requires otherwise, “comprise” and “include”, or variations of the term such as “including” can be used interchangeably.
[0146] Where an indefinite or definite article is used when referring to a singular noun e.g. “a” or “an”, “the”, this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
[0147] The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to 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, N.Y. (1999), for definitions and terms of the art.
[0148] The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.
EXAMPLE
Materials and Methods
[0149] 1. Patient Samples and PBMC Isolation
[0150] Patients (table S3) with HBV infection were recruited with fully informed written consent from Division of Gastroenterology and Hepatology at National University Health System, Singapore. The respective local ethical institutional review boards approved the study, and the recruitment and sampling of suitable patients was completed at hospital. Up to 60 ml of blood was taken, peripheral blood mononucleated cells (PBMC) were further isolated by using Ficoll-separation (Ficoll-Paque PLUS, GE Healthcare). All patients had clinical, serological and virological evidences of chronic hepatitis B infection with detection of HBsAg and HBV DNA, and no positive result for the presence of HIV-1 and -2, and HCV. Three CHB patient groups, Immune Tolerant (IT, HBV DNA>2000 lu/ml, ALT<40 lU/ml, HBeAg+), Immune Active (IA, HBV DNA>2000 lu/ml, ALT>40 lU/ml, HBeAg+), Inactive Carrier (InA, HBV DNA<200 lU/ml or undetectable, ALT<40 lU/ml, HBeAg−) and one group of acute resolved patients (R, undetectable HBV DNA, HBsAg− and anti-HBc antibody+) were enrolled in this study. Each CHB patient had at least three adjacent time points indicating consistent virological and serological evidences for the referring clinical stages. All patients were treatment free from any antiviral drug or clinical intervention at the time of blood draw. Patients received Entecavir (ETV) were followed up longitudinally and enrolled. Serological and virological scores (serum HBV DNA, HBeAg and HBsAg) and liver function test were determined by clinical laboratory at hospital or ELISA. Blood from anonymous healthy donors were recruited under Singapore immunology Network (SIgN) institutional review board. Healthy cord blood samples were purchased from Singapore Cord Blood Bank under the institutional review board, without detection of HIV-1 and -2, HTLV-i and II, HCV, CMV, HBsAg and anti-HBc antibody. HLA-A*11:01 was confirmed by typing service from BGI Genomics.
TABLE-US-00002 TABLE S3 Supplementary Table 3: List of the patient samples and the clinical and serological informations. Patient DNA ALT AST Tissue types group
Age (
) (
) (
)
PBMC
11 N 32.5 (
)
Not 4.3 (
) detected
20 N 33.3 (20-60)
Not
Active (
) detected Inactive 16 N 33.3 (31-72)
(
)
13 N N/A Not N/A N/A Negative Negative >
positive detected Healthy 13 N 34 (
) N/A N/A N/A N/A N/A
Healthy Cord 10 N N/A N/A N/A N/A N/A N/A N/A N/A Blood (CB)
# of
DNA ALT AST
patient group timepoints (
) (
) (
) PBMC
8
5-7
Positive Negative
N/A Positive
6
4-8
Positive Positive N/A Negative
# of
DNA ALT patient group timepoints (log10,
) (
) Serum
8 N 4-8 8 (
) 3.3 (
)
7 N 5-7 9 (
) 48 (
)
indicates data missing or illegible when filed
[0151] 2. 562-Plex Combinatorial (Quadruple/Triple SAv-metal coded) pMHC Tetramers
[0152] Fourteen different SAv-metals were made by labelling streptavidin with fourteen different metal isotopes. Similar to previous work reported in E. W. Newell et al., Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization, each SAv-metal was diluted into 20 μg/ml in EDTA-free W-buffer on the same day of tetramerization of pMHC. Two different configurations of quadruple SAv-metal coding for 562-plex pMHC tetramers were generated using a R-based script for a 14-choose-4 scheme (for 1001 combinations). The script was then loaded onto TECAN Freedom EVO200 automatic liquid distribution robot to prepare the designed combinations of quadruple SAv-metal mixtures (each mixture contains 4 different SAv-metals) in 2 ml 96-well deep well plates. To form pMHC tetramers, each peptide-loaded HLA-A*1101 monomer (562 different pMHC monomers) was randomly assigned for four different SAv-metals. To reach a 1:4 ratio of streptavidin to pMHC, quadruple SAv-metal mixtures were added to the corresponded pMHC monomer in a stepwise manner of four additions, each has 10 min incubation at room temperature. 10 μM D-biotin was added into the reaction at the end for another 10 min at room temperature to saturate unbound streptavidins. The 562-plex pMHC tetramers were combined and concentrated down to 5 μg/ml per pMHC tetramer in 10% FBS CyFACS buffer using Amicon 50 kDa cut-off concentrator (Millipore). The total amount of protein in each concentrator was limited to 300 μg. To reach the desired concentration and volume, multiple spins were performed at 700×g for 5 min. The 562-plex tetramers were then filtered by 0.1 μm filter tube (Millipore) at 2000×g for 25 min. The cocktail of tetramers was kept on ice, and then spin at 14,000×g for 1 min in a 1.5 ml Eppendorf tube to remove the remaining aggregates prior the staining.
[0153] The combinatorial streptavidin codings were re-scrambled for every independent 562-plex combinatorial pMHC tetramer staining experiment.
[0154] For selected experiments, a 9-choose-3 (84 combinations) or 8-choose-3 (56 combinations) scheme were used to cover 120-plex (40 peptide clusters) or 50-plex (17 peptide clusters) combinatorial triple coded pMHC tetramers staining preferentially selected (table S1) for more phenotypic analysis, or in vitro peptide stimulation (table S2).
TABLE-US-00003 TABLE S1 Supplementary Table 1. List of detected HLA-A*1101-restricted epitopes and the frequency across various patient groups Source Protein 1st Clust- & Peptide Hits (Freq > 0.002%) Average of hits (%) er Position Sequence Peptides in Cluster IT IA InA R IT IA InA R Antigen Source-Hepatitis B virus 1 HBV-S- HQLDPAFK 27 (SEQ ID No. 32) 2 HBV-S- ASTNRQSGRK 94 (SEQ ID No. 33) 3 HBV-S- STNRQSGRK* STNRQSGRK STNRQSGR 5/8 7/12 4/13 1/12 0. 0. 0. 95* (SEQ ID No. (SEQ ID No. (SEQ ID No. 003228 004132 005883 34) 34) 35) 4 HBV-S- STFHQALLDPR* STFHQALLDPR TTFHQALLDPR TTFHQTLQDPR 1/12 0. 124* (SEQ ID (SEQ ID No. (SEQ ID No. (SEQ ID No. 008353 No. 36) 36) 37) 38) 5 HBV-S- TLQDPRVRALY* TLQDPRVRALY ALLDPRVRGLY 129* (SEQ ID (SEQ ID No. (SEQ ID No. No. 39) 39) 40) 6 HBV-S- TVSAISSILSK* TVSAISSILSK TVSTISSILSK TASPISSIFSK TASPISSIFSR 1/8 2/12 1/12 0.00508 0. 0. 156* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 2619 002307 005196 41) 41) 42) 43) 44) 7 HBV-S- VSAISSILSK* VSAISSILSK ASPISSIFSK VSTISSILSK ASPISSIFSR 1/12 0. 157* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 005228 45) 45) 46) 47) 48) 8 HBV-S- STISSILSK* STISSILSK SAISSILSK SPISSIFSK SPISSIFSR 1/12 1/13 0. 0. 158* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003020 015373 49) 49) 50) 51) 52) 9 HBV-S- TISSILSK* TISSILSK AISSILSK PISSIFSK 159* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 53) 53) 54) 55) 10 HBV-S- VLQAGFFLLTK VLQAGFFLLTK VLQAGFFSLTK VLQAGFFLLTR 187* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 56) 56) 57) 58) 11 HBV-S- LQAGFFLLTK* LQAGFFLLTK LQAGFFSLTK LQAGFFLLTR 1/12 0. 188* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 002898 59) 59) 60) 61) 12 HBV-S- QAGFFLLTK* QAGFFLLTK QAGFFSLTK 189* (SEQ ID No. (SEQ ID No. (SEQ ID No. 62) 62) 63) 13 HBV-S- AGFFLLTK* AGFFLLTK AGFFSLTK 1/13 0. 190* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002499 64) 64) 65) 14 HBV-S- TSLNFLGGAPK 210 (SEQ ID No. 66) 15 HBV-S- SLNFLGGAPK 211 (SEQ ID No. 67) 16 HBV-S- TSCPPICPGYR 236 (SEQ ID No. 68) 17 HBV-S- LIFLLVLLDY 264 (SEQ ID No. 69) 18 HBV-S- GTSTTSTGPCK GTSTTSTGPCK GSSTTSTGPCK 285* (SEQ ID No. (SEQ ID No. (SEQ ID No. 70) 70) 71) 19 HBV-S- SSTTSTGPCK* SSTTSTGPCK TSTTSTGPCK 286* (SEQ ID No. (SEQ ID No. (SEQ ID No. 72) 72) 73) 20 HBV-S- STTSTGPCK 287 (SEQ ID No. 74) 21 HBV-S- TTSTGPCK 1/12 1/12 0. 0. 288 (SEQ ID No. 02665 006913 75) 22 HBV-S- TSMFPSCCCTK 1/8 2/12 1/13 0. 0. 0. 304 (SEQ ID 013587 003647 004037 No. 76) 23 HBV-S- SMFPSCCCTK SMFPSCCCTK SMYPSCCCTK 305* (SEQ ID No. (SEQ ID No. (SEQ ID No. 77) 77) 78) 24 HBV-S- IPIPSSWAFAK 323 (SEQ ID No. 79) 25 HBV-S- PIPSSWAFAK 324 (SEQ ID No. 80) 26 HBV-S- IPSSWAFAK 325 (SEQ ID No. 81) 27 HBV-S- PSSWAFAK 326 (SEQ ID No. 82) 28 HBV-S- SSWAFAKY 327 (SEQ ID No. 83) 29 HBV-S- SVIWMMWY* SVIWMMWY SVIWMMWF SVIWMMWY 366* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 84) 84) 85) 86) 30 HBV-S- WMMWFWG WMMWFWG WMMWYWG 369* PSLY* PSLY PSLY (SEQ ID No. (SEQ ID No. (SEQ ID No. 87) 87) 88) 31 HBV-S- MMWFWGPS 370 LY(SEQID No. 89) 32 HBV-P- MPLSYLHFRK* MPLSYLHFRK MPLSYQHFRK 0* (SEQ ID No. (SEQ ID No. (SEQ ID No. 90) 90) 91) 33 HBV-P- PLSYLHFRK* PLSYLHFRK PLSYQHFRK 1* (SEQ ID No. (SEQ ID No. (SEQ ID No. 92) 92) 93) 34 HBV-P- LSYLHFRK* LSYLHFRK LSYQHFRK 3/8 4/12 1/13 2/12 0. 0. 0. 0. 2* (SEQ ID No. (SEQ ID No. (SEQ ID No. 004069 005406 005959 008759 94) 94) 95) 35 HBV-P- NLNVSIPWTHK 4/8 8/12 3/13 1/12 0. 0. 0. 0. 44 (SEQ ID 004481 003864 007687 003309 No. 96) 36 HBV-P- NVSIPWTHK 46 (SEQ ID No. 97) 37 HBV-P- VSIPWTHK 47 (SEQ ID No. 98) 38 HBV-P- KVGNFTGLY 54 (SEQ ID No. 99) 39 HBV-P- YSSTVPCFNPK 62 (SEQ ID No. 100) 40 HBV-P- SSTVPCFNPK 63 (SEQ ID No. 101) 41 HBV-P- STVPCFNPK 1/12 0. 64 (SEQ ID No. 002033 102) 42 HBV-P- TVPCFNPK 1/13 0. 65 (SEQ ID No. 013511 103) 43 HBV-P- QTPSFPHIHLK 74 (SEQ ID No. 104) 44 HBV-P- TPSFPHIHLK 75 (SEQ ID No. 105) 45 HBV-P- PSFPHIHLK 1/8 1/12 0.00926 0. 76 (SEQ ID No. 8851 002264 106) 46 HBV-P- SFPHIHLK 77 (SEQ ID No. 107) 47 HBV-P- YVGPLTVNEK 94 (SEQ ID No. 108) 48 HBV-P- LTINENRRLK* LTINENRRLK LTVNETRRLK LTVNENRRLK LTVNEKRRLK 1/12 0. 98* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003304 109) 109) 110) 111) 112) 49 HBV-P- TVNETRRLK* TVNETRRLK TVNENRRLK TVNEKRRLK TINENRRLK 1/8 2/12 2/13 0. 0. 0. 99* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003505 004665 007724 113) 113) 114) 115) 116) 50 HBV-P- KLIMPARFY 107 (SEQ ID No. 117) 51 HBV-P- LVMPARFY 108 (SEQ ID No. 118) 52 HBV-P- RFYPNLTK* RFYPNLTK RFYPNVTK 113* (SEQ ID No. (SEQ ID No. (SEQ ID No. 119) 119) 120) 53 HBV-P- NVTKYLPLDK 117 (SEQ ID No. 121) 54 HBV-P- VTKYLPLDK* VTKYLPLDK LTKYLPLDK 1/8 3/13 2/12 0. 0. 0. 118* (SEQ ID No. (SEQ ID No. (SEQ ID No. 004096 019158 003800 122) 122) 123) 55 HBV-P- HTVNHYFK* HTVNHYFK HTVNHYFQTR HVVDHYFQTR HVVNHYFQTR 135* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 124) 124) 125) 126) 127) 56 HBV-P- TVNHYFQTR* TVNHYFQTR VVNHYFQTR TVNHYFKTR IVNHYFQTR TVNHYFQTRHY 5/8 4/12 5/13 0. 0. 0. 136* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003132 003651 003352 128) 128) 129) 130) 131) 132) TVNHYFTRH VVHHYFQTR VVDHYFQTR VVNHYFQTRHY (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 133) 134) 135) 136) 57 HBV-P- KTRHYLHTLW KTRHYLHTLW QTRHYLHTLW 142* (SEQ ID No. (SEQ ID No. (SEQ ID No. 137) 137) 138) 58 HBV-P- RHYLHTLWK 144 (SEQ ID No. 139) 59 HBV-P- HTLWKAGILYK* HTLWKAGILYK HTLWEAGILYK HTLWKAGILY HTLWEAGILY 1/8 4/12 1/13 2/12 0. 0. 0. 0. 148* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 005276 003741 006728 037610 140) 140) 141) 142) 143) 60 HBV-P- TLWEAGILYK* TLWEAGILYK TLWKAGILYK 149* (SEQ ID No. (SEQ ID No. (SEQ ID No. 144) 144) 145) 61 HBV-P- STRSASFY 161 (SEQ ID No. 146) 62 HBV-P- RSASFYGSPY* RSASFYGSPY RSASFCGSPY 163* (SEQ ID No. (SEQ ID No. (SEQ ID No. 147) 147) 148) 63 HBV-P- SASFYGSPY* SASFYGSPY SASFCGSPY 164* (SEQ ID No. (SEQ ID No. (SEQ ID No. 149) 149) 150) 64 HBV-P- ASFYGSPY* ASFYGSPY ASFCGSPY 165* (SEQ ID No. (SEQ ID No. (SEQ ID No. 151) 151) 152) 65 HBV-P- RLVFQTSK 182 (SEQ ID No. 153) 66 HBV-P- LVFQTSER* LVFQTSER LVFQTSTR LVFQTSKR 1/13 1/12 0. 0. 183* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003668 002847 154) 154) 155) 156) 67 HBV-P- QTSERHGDK* QTSERHGDK QTSKRHGDK 1/13 1/12 0. 0. 186* (SEQ ID No. (SEQ ID No. (SEQ ID No. 015373 002615 157) 157) 158) 68 HBV-P- TSERHGDK* TSERHGDK TSKRHGDK 187* (SEQ ID No. (SEQ ID No. (SEQ ID No. 159) 159) 160) 69 HBV-P- CSQSSGILSR 197 (SEQ ID No. 161) 70 HBV-P- SQSSGILSR 198 (SEQ ID No. 162) 71 HBV-P- QSSGILSR 1/12 0. 199 (SEQ ID No. 003660 163) 72 HBV-P- GILPRSSVGPR 1/12 3/13 0. 0. 202 (SEQ ID No. 002828 072039 164) 73 HBV-P- SVGSCIQSQLR* SVGSCIQSQLR SVGPRIQSQLR SVGPCIQSQLR 208 (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 165) 165) 166) 167) 74 HBV-P- GSCIQSQLRK* GSCIQSQLRK GSCIQSQLR 1/8 0. 210* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002406 168) 168) 169) 75 HBV-P- SCIQSQLRK 211 (SEQ ID No. 170) 76 HBV-P- RIQSQLRK* RIQSQLRK GIQSQLRK CIQSQLRK 212* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 171) 171) 172) 173) 77 HBV-P- RSQFKQSR 214 (SEQ ID No. 174) 78 HBV-P- SQLRKSRLGPK 215 (SEQ ID No. 175) 79 HBV-P- QQGSMASGK 1/13 0. 227 (SEQ ID No. 004808 176) 80 HBV-P- RSMASGKPGR* RSMASGKPG RGSMASGKPG GSMARGKSG 229* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 177) 177) 178) 179) 81 HBV-P- SIRARVHPTSR 241 (SEQ ID No. 180) 82 HBV-P- RVHSSPWR* RVHSSPWR RVHPTSRR 245* (SEQ ID No. (SEQ ID No. (SEQ ID No. 181) 181) 182) 83 HBV-P- SASSASSCLY* SASSASSCLY STSSASYCLH 267* (SEQ ID No. (SEQ ID No. (SEQ ID No. 183) 183) 184) 84 HBV-P- ASSASSCLY 268 (SEQ ID No. 185) 85 HBV-P- SSASSCLY 269 (SEQ ID No. 186) 86 HBV-P- ASYCLHQSAVR* ASYCLHQSAV SSSCLHQPAV SSSCLHQSAVK ASSCLHQSAVR ASSCLYQSAVR 271* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 187) 187) 188) 189) 190) 191) 87 HBV-P- SSCLHQPAVRK* SSCLHQPAVRK SSCLYQSAVR KSSCLHQSAVR SSCLYQSAVR SSCLHQPAVR 1/12 0. 272* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 009064 192) 192) 193) 194) 195) 196) SSCLHQSAVR SYCLHQSAVRK (SEQ ID No. (SEQ ID No. 197) 198) 88 HBV-P- CLYQSAVRK* CLYQSAVRK CLYQSAVRKK CLHQPAVRKN CLHQSAVRK 274* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 199) 199) 200) 201) 202) 89 HBV-P- YQSAVRKK 276 (SEQ ID No. 203) 90 HBV-P- KTAYSHLSTSK* KTAYSHLSTSK KTAYSLISTSK KAAYSLISTSK KAAYSLNSTSK 8/8 12/ 11/ 7/12 0. 0. 0. 0. 282* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 12 13 025309 021343 020005 014202 204) 204) 205) 206) 207) 91 HBV-P- KAYSHLSSSK* KAYSHLSSSK TAYSHLSTSK TAYLISTSK AAYSLISTSK AAYSLNSTSK 283* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 208) 208) 209) 210) 211) 212) TAYSHLSTSKR TAYSLISTSKR KAYSHLSSSKR (SEQ ID No. (SEQ ID No. (SEQ ID No. 213) 214) 215) 92 HBV-P- AYSHLSSSK* AYSHLSSSK AYSHLSTSK AYSLISTSK AYSLNSTSK 284* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 216) 216) 217) 218) 219) 93 HBV-P- YSLNSTSK* YSLNSTSK YSLISTSK YSHLSSSK YSHLSTSK 285* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 220) 220) 221) 222) 223) 94 HBV-P- SLISTSKR 286 (SEQ ID No. 224) 95 HBV-P- STSKGHSSSR* STSKGHSSSR STSKGHSSSGR 289* (SEQ ID No. (SEQ ID No. (SEQ ID No. 225) 225) 226) 96 HBV-P- TSKGHSSSR 290 (SEQ ID No. 227) 97 HBV-P- SSSRHAVELR 295 (SEQ ID No. 228) 98 HBV-P- RQFPPNTSR 304 (SEQ ID No. 229) 99 HBV-P- SSARSQSER 1/13 0. 309 (SEQ ID No. 002307 230) 100 HBV-P- SVLSCWWLQFR* SVLSCWWLQFR SVLSCWWLQ PVLSCWWLQFR 318* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 231) 231) 232) 233) 101 HBV-P- VLSCWWLQFR* VLSCWWLQFR ILSCWWLQPR 319* (SEQ ID No. (SEQ ID No. (SEQ ID No. 234) 234) 235) 102 HBV-P- LSCWWLQPR* LSCWWLQPR PSCWWLQPR 320* (SEQ ID No. (SEQ ID No. (SEQ ID No. 236) 236) 237) 103 HBV-P- WLQPRNSK 324 (SEQ ID No. 238) 104 HBV-P- RVTGGVFLVDK* RVTGGVFLVDK RITGGVPLVDK 1/12 0. 367* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002254 239) 239) 240) 105 HBV-P- VTGGVFLVDK* VTGGVFLVDK ITGGVPLVDK 1/12 0. 368* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002254 241) 241) 242) 106 HBV-P- LVVDFSQFSR 5/8 9/12 9/13 9/12 0.06119 0. 0. 0. 387 (SEQ ID No. 8525 086237 055185 066603 28) 107 HBV-P- VVDFSQFSR 388 (SEQ ID No. 243) 108 HBV-P- SQFSRGSTR* SQFSRGSTR SQFSRGNTR 392* (SEQ ID No. (SEQ ID No. (SEQ ID No. 244) 244) 245) 109 HBV-P- RGNTRVSWPK* RGNTRVSWPK RGSTHVSWPK RGSTRVSWPK 1/8 2/12 1/13 1/12 0.03816 0. 0. 0. 396* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 5858 029153 005767 010167 246) 246) 247) 248) 110 HBV-P- GSTHVSWPK* GSTHVSWPK GSTRVSWPK GSTQVSWPK 1/8 1/12 0.00763 0. 397* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 3172 002506 249) 249) 250) 251) 111 HBV-P- STQVSWPK* STQVSWPK STHVSWPK STRVSWPK NTRVSWPK 2/8 1/12 0. 0. 398* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 004475 004529 252) 252) 253) 253) 254) 112 HBV-P- AAFYHLPLH 431 (SEQ ID No. 256) 113 HBV-P- LVGSSGLPR* LVGSSGLPR LVGSSGLSR 447* (SEQ ID No. (SEQ ID No. (SEQ ID No. 257) 257) 258) 114 HBV-P- GSSGLSRYVAR* GSSGLSRYVAR GSSGLPRYVAR 449* (SEQ ID No. (SEQ ID No. (SEQ ID No. 259) 259) 260) 115 HBV-P- SSGLSRYVAR* SSGLSRYVAR SSGLPRYVAR 450* (SEQ ID No. (SEQ ID No. (SEQ ID No. 261) 261) 262) 116 HBV-P- YVARLSSTSR* YVARLSSTSR YVARLSSNSR 456* (SEQ ID No. (SEQ ID No. (SEQ ID No. 263) 263) 264) 117 HBV-P- SSTSRNINY 461 (SEQ ID No. 265) 118 HBV-P- STSRIINNQHR* STSRIINNQHR STSRNINY STSRIINDQHR 462* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 266) 266) 267) 268) 119 HBV-P- RIINNQHR 1/12 1/12 0. 0. 465 (SEQ ID No. 008372 012989 269) 120 HBV-P- TMQNLHSSCS TMQNLHSSCS TMQDLHNSCS TMQNLHNSCS TMQNLHDSCSR TMQNLHDSCSR 473* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 270) 270) 271) 272) 273) 274) AMQDLHDSC (SEQ ID No. 275) 121 HBV-P- MQNLHSSCSR 474 (SEQ ID No. 276) 122 HBV-P- SSCSRNLY 479 (SEQ ID No. 277) 123 HBV-P- NLYVSLMLLYK* NLYVSLMLLYK NLYVSLLLLYK NLYVSLMLLY NLYVSLLLLY 484* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 278) 278) 279) 280) 281) 124 HBV-P- LYVSLMLLYK* LYVSLMLLYK LYVSLLLLYK 485* (SEQ ID No. (SEQ ID No. (SEQ ID No. 282) 282) 283) 125 HBV-P- YVSLLLLYK* YVSLLLLYK YVSLMLLYK YVSLMLLY VVSLLLLY 2/8 2/12 3/13 1/12 0. 0. 0. 0. 486* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 006757 003553 010339 009762 284) 284) 285) 286) 287) 126 HBV-P- VSLMLLYK* VSLMLLYK VSLLLLYK 1/12 0. 487* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002615 288) 288) 289) 127 HBV-P- SLMLLYKTYGR* SLMLLYKTYGR SLLLLYKTFGR 488* (SEQ ID No. (SEQ ID No. (SEQ ID No. 290) 290) 291) 128 HBV-P- LMLLYKTYGRK* LMLLYKTYGRK LLLLYKTFGRK 489* (SEQ ID No. (SEQ ID No. (SEQ ID No. 292) 292) 293) 129 HBV-P- MLLYKTYGRK* MLLYKTYGRK LLLYKTFGRK MLLYKTYGR 1/13 0. 490* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002855 294) 294) 295) 130 HBV-P- LLYKTFGRK* LLYKTFGRK LLYKTYGRK LLYKTFGR 4/8 5/12 5/13 1/12 0.00301 0. 0. 0. 491* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 2655 004777 009704 025620 297) 297) 298) 299) 131 HBV-P- KTYGRKLHLY* KTYGRKLHLY KTFGRKLHLY 1/8 0.002007 494* (SEQ ID No. (SEQ ID No. (SEQ ID No. 300) 300) 301) 132 HBV-P- YSHPIILGFRK 503 (SEQ ID No. 302) 133 HBV-P- PIILGFRK 506 (SEQ ID No. 303) 134 HBV-P- FTSAICSVVRR 528 (SEQ ID No. 304) 135 HBV-P- TSAICSVVRR* TSAICSVVRR TSAICSVVR 529* (SEQ ID No. (SEQ ID No. (SEQ ID No. 305) 305) 306) 136 HBV-P- SAICSVVRR* SAICSVVRR SAICSVVR 590* (SEQ ID No. (SEQ ID No. (SEQ ID No. 307) 307) 308) 137 HBV-P- AICSVVRR 531 (SEQ ID No. 309) 138 HBV-P- SVVRRAFPH 1/8 3/12 2/13 0. 0. 0. 534 (SEQ ID No. 041982 003832 005224 310) 139 HBV-P- RAFPHCLAFSY 538 (SEQ ID No. 311) 140 HBV-P- SYMDDVVLGAK 547 (SEQ ID No. 312) 141 HBV-P- YMDDVVLGAK 548 (SEQ ID No. 313) 142 HBV-P- SVQHLESLY* SVQHLESLY SVQHLESVY 558* (SEQ ID No. (SEQ ID No. (SEQ ID No. 314) 314) 315) 143 HBV-P- SVYAAVTNFLL 564 (SEQ ID No. 316) 144 HBV-P- LSLGIHLNPNK* LSLGIHLNPNK LSLGIHLNPHK 3/12 1/13 0. 0. 574* (SEQ ID No. (SEQ ID No. (SEQ ID No. 004279 002691 317) 317) 318) 145 HBV-P- SLGIHLNPNK* SLGIHLNPNK SLGIHLNPHK 575* (SEQ ID No. (SEQ ID No. (SEQ ID No. 319) 319) 320) 146 HBV-P- GIHLNPNK* GIHLNPNK GIHLNPHK GIHLNPHKTK GIHLNPNKTK 577* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 321) 321) 322) 323) 324) 147 HBV-P- YSLNFMGY 590 (SEQ ID No. 325) 148 HBV-P- GTLPQEHIVLK* GTLPQEHIVLK GTLPQEHIVQK 1/12 0. 603* (SEQ ID No. (SEQ ID No. (SEQ ID No. 004185 326) 326) 327) 149 HBV-P- TLPQEHIVLK* TLPQEHIVLK TLPQEHIVQK 604* (SEQ ID No. (SEQ ID No. (SEQ ID No. 328) 328) 329) 150 HBV-P- HIVQKIKMCFK 609 (SEQ ID No. 330) 151 HBV-P- IVQKIKMCFRK* IVQKIKMCFRK IVQKIKMCFK IVQKIKLCFRK IVQKIKMCRKK IVLKLKQCFRK 1/8 1/12 2/12 0.00227 0. 0. 610* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 8415 009584 004296 331) 331) 332) 333) 334) 335) IVQKIKQCFRK IVQKIKMCFR IVQKIKLCFR IVLKLKQCFR IVQKIKQCFR (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 336) 337) 338) 339) 340) 152 HBV-P- VQKIKMCFK* VQKIKMCFK VQKIKMCFRK VQKIKLCRFK VQKIKMCFKK VLKLKQCFRK 611* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 341) 341) 342) 343) 344) 345) VQKIKQCFRK (SEQ ID No. 346) 153 HBV-P- KIKMCFRK* KIKMCFRK KIKMCFKK KIKLCFRK KIKQCFRK KLKQCFRK 2/8 1/12 0.00504 0. 613* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 7506 005196 347) 347) 348) 349) 350) 351) 154 HBV-P- KMCFRKLPVNR* KMCFRKLPVNR KQCFRKLPINR KMCFKKLPVNR KQCFRKLPVNR KLCFRKLPVNR 615* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 352) 352) 353) 354) 355) 356) 155 HBV-P- PVNRPIDWK 1/8 3/12 0.005276 0. 622 (SEQ ID No. 000000 357) 156 HBV-P- PLYACIQAK* PLYACIQAK PLYACIQTK 655* (SEQ ID No. (SEQ ID No. (SEQ ID No. 358) 358) 359) 157 HBV-P- KQAFTFSPTYK* KQAFTFSPTYK KQAFTFSPTY 663* (SEQ ID No. (SEQ ID No. (SEQ ID No. 360) 360) 361) 158 HBV-P- QAFTFSPTYK* QAFTFSPTYK QAFTFSPTY 2/12 2/13 1/12 0. 0. 0. 664* (SEQ ID No. (SEQ ID No. (SEQ ID No. 010664 005043 002264 362) 362) 363) 159 HBV-P- AFTFSPTYK 665 (SEQ ID No. 364) 160 HBV-P- FTFSPTYK 666 (SEQ ID No. 365) 161 HBV-P- FSPTYKAFLSK* FSPTYKAFLSK FSPTYKAFLCK 668* (SEQ ID No. (SEQ ID No. (SEQ ID No. 366) 366) 367) 162 HBV-P- SPTYKAFLCK* SPTYKAFLCK SPTYKAFLSK 669* (SEQ ID No. (SEQ ID No. (SEQ ID No. 368) 368) 369) 163 HBV-P- PTYKAFLSK* PTYKAFLSK PTYKAFLCK 1/8 2/12 2/13 1/12 0.00791 0. 0. 0. 670* (SEQ ID No. (SEQ ID No. (SEQ ID No. 4106 003032 003198 002264 370) 370) 371) 164 HBV-P- TYKAFLSK* TYKAFLSK TYKAFLCK 671* (SEQ ID No. (SEQ ID No. (SEQ ID No. 372) 372) 373) 165 HBV-P- KQYLHLYPVAR* KQYLHLYPVAR KQYLNLYPVA 678* (SEQ ID No. (SEQ ID No. (SEQ ID No. 374) 374) 375) 166 HBV-P- AACFARSR 731 (SEQ ID No. 376) 167 HBV-P- GTDNSVVLSRK* GTDNSVVLSRK GTDNSVVLSR 745* (SEQ ID No. (SEQ ID No. (SEQ ID No. 377) 377) 378) 168 HBV-P- NSVVLSRK 748 (SEQ ID No. 379) 169 HBV-P- CAANWILR 765 (SEQ ID No. 380) 170 HBV-P- SALNPADDPSR 781 (SEQ ID No. 381) 171 HBV-P- GLYRPLLR* GLYRPLLR GLYRPLLRLLY GLYRPLLRLVY 795* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 382) 382) 383) 384) 172 HBV-P- LVYRPTTGR 1/12 1/13 0. 0. 803 (SEQ ID No. 002566 002594 385) 173 HBV-P- TTGRTSLY 808 (SEQ ID No. 386) 174 HBV-P- SVPSHLPVR* SVPSHLPVR SVPFHLPDR SVPSHLPDR SVPSHPPDR 820* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 387) 387) 388) 389) 390) 175 HBV-P- FASPLHVAWK 831 (SEQ ID No. 391) 176 HBV-P- ASPLHVAWK 832 (SEQ ID No. 392) 177 HBV-P- SPLHVAWK 833 (SEQ ID No. 393) 178 HBV-C- STLPETAVVRR* STLPETAVVRR STLPETAVVR STLPETTVVRR STLPETTVIRR STPPETTVVRR 4/12 7/13 9/12 0. 0. 0. 169* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 098664 010236 026518 21) 21) 22) 23) 24) 25) STLPETTVVGR STIPETTVVRR (SEQ ID No. (SEQ ID No. 21) 21) 179 HBV-C- AVVRRRCRSPR* AVVRRRCRSP TVVRRRGRSP TVIRRRGRSPR TVVGRRGRSPR 3/8 6/12 2/13 1/12 0.00546 0. 0. 0. 175* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 0555 004748 005453 004136 394) 394) 395) 396) 397) 180 HBV-X- RVCCQLDPAR 3 (SEQ ID No. 398) 181 HBV-X- SSAGPCALR* SSAGPCALR SSTGPCALR 2/13 0. 63* (SEQ ID No. (SEQ ID No. (SEQ ID No. 015895 399) 399) 400) 182 HBV-X- STGPCALR 64 (SEQ ID No. 401) 183 HBV-X- TTVNAHWNL TTVNAHWNL TTVNALGNLP TTVNAHGNLP TTVNAPGNLPK TTVNAHQVLPK 1/8 3/12 2/13 1/12 0.00231 0. 0. 0. 80* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 5941 004083 004750 007793 402) 402) 403) 404) 405) 406) TTVNAHRNLP TTVNARQVLP (SEQ ID No. (SEQ ID No. 407) 408) 184 HBV-X- TVNAHQVLPK* TVNAHQVLPK TVNAHRNLPK TVNAHWNLPK TVNAHGNLPK TVNALGNLPK 1/12 3/13 2/12 0. 0. 0. 81* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 005656 003399 002743 409) 409) 410) 411) 412) 413) TVNAPGNLPK TVNARQVLPK (SEQ ID No. (SEQ ID No. 414) 415) 185 HBV-X- VNAHWNLPK 82 (SEQ ID No. 416) 186 HBV-X- NAHWNLPK* NAHWNLPK NALGNLPK NAHQVLPK NAHGNLPK NAHRNLPK 83* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 417) 417) 418) 419) 420) 421) 187 HBV-X- ALGNLPKVLHK 84 (SEQ ID No. 422) 188 HBV-X- RQVLPKVLHK* RQVLPKVLHK HQVLPKVLHK 85* (SEQ ID No. (SEQ ID No. (SEQ ID No. 423) 423) 424) 189 HBV-X- QVLPKVLHK* QVLPKVLHK QVLPKVLHKR 1/12 0. 86* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002715 425) 425) 426) 190 HBV-X- VLPKVLHK 87 (SEQ ID No. 427) 191 HBV-X- SVMSMTDLEAY* SVMSMTDLEAY SVMSTTDLEAY SAMSTTDLEAY SAMSATDLEAY 100* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 428) 428) 429) 430) 431) 192 HBV-X- MSMTDLEAYFK* MSMTDLEAYFK MSATDLEAYFK MSTTDLEAYFAK MSMTDLEAY 1/12 1/12 0. 102* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003997 432) 432) 433) 434) 435) 193 HBV-X- STTDLEAYFK* STTDLEAYFK SMTDLEAYFK SATDLEAYFK STTDLEAY 103* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 436) 436) 437) 438) 439) 194 HBV-X- TTDLEAYFK* TTDLEAYFK MTDLEAYFK ATDLEAYFK 1/12 3/13 1/12 0. 0. 0. 104* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 002297 003752 005196 440) 440) 441) 442) 195 HBV-X- EAYFKDCVFK 108 (SEQ ID No. 441) 196 HBV-X- AYFKDCVFK 109 (SEQ ID No. 444) 197 HBV-X- RLMIFVLGGCR 127 (SEQ ID No. 445) 198 HBV-X- KVFVLGGCRHK* KVFVLGGCRHK MIFVLGGCRHK KIYVLGGCRHK KVFVLGGGR KIYVLGGCR 1/8 1/12 2/12 0. 0. 0. 129* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 005276 007552 004401 442) 442) 442) 442) 442) 442) MIFVLGGCR (SEQ ID No. 451) 199 HBV-X- YVLGGCRHK 131 (SEQ ID No. 452) 202 HBV-C- YVNVNMGLK* YVNVNMGLK YVNTNMGLK YVNVNMGPK YVNVNTGLK YVNVNMGK 1/12 1/12 0. 0. 88* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 002576 003396 453) 453) 454) 455) 456) 457) YANVNMGIK YVNVNMRLK YVNVIMGLK (SEQ ID No. (SEQ ID No. (SEQ ID No. 458) 459) 460) 265 HBV-C- CSCPTVQASK* CSCPTVQASK CSCSTVQASK CSCPTVQTSK CTCPTVQASK CSCPTVQVSK 2/13 0. 11* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 171478 461) 461) 462) 463) 464) 465) 266 HBV-C- CSTVQASK 13 (SEQ ID No. 466) 267 HBV-C- TVQASKLCLGR 15 (SEQ ID No. 467) 268 HBV-C- TVQASKLY 15v1 (SEQ ID No. 468) 269 HBV-C- RLWGMDIDPYK 25 (SEQ ID No. 469) 270 HBV-C- GMDIDPYK* GMDIDPYK GMNIDPYK GMDIDAYK GMDIDTYK 28* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 470) 470) 471) 472) 473) 271 HBV-C- VVSYVNVNMR 113 (SEQ ID No. 474) 272 HBV-C- VSYVNVNMG VSYVNVNMG VSYVNVNMG VSYVNVNTGL VSYVNVNMGIK VSYANVNMGIK 1/12 0. 114* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 002033 475) 475) 476) 477) 478) 479) VSYVNVNMR VSYVNVIMGL (SEQ ID No. (SEQ ID No. 480) 481) 273 HBV-C- VSYVNVNMR 114v6 (SEQ ID No. 482) 274 HBV-C- SYVNVNMGLK* SYVNVNMGLK SYVNVNMGPK SYVNVNTGLK GYVNVNMGLK SYVNVNMRLK 115* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 483) 483) 484) 485) 486) 487) SYVNVIMGLK (SEQ ID No. 488) 275 HBV-C- RQLLWFHISCR 126 (SEQ ID No. 489) 276 HBV-C- HISCLTFGR 132 (SEQ ID No. 490) 277 HBV-C- LTFGRETVLE* LTFGRETVLEY RTFGRETVLEY LTFGRQTVLEY 1/13 1/12 0. 0. 136* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 003706 003309 491) 491) 492) 493) 278 HBV-C- GVWIRTPPAYR* GVWIRTPPAYR GVWIRTPPAFR GVWIRTPTAY GVWIRTPSAYR GVWIRTPLAYR 1/13 0. 151* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 010397 494) 494) 495) 496) 497) 483) GVWIRAPPAYR (SEQ ID No. 499) 279 HBV-C- STLPETTVVR* STLPETTVVR STLPETTVIR STIPETTVVR 1/13 1/12 0. 0. 169v3* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 010508 004473 500) 500) 501) 502) 280 HBV-C- TVIRRRGR 175v3 (SEQ ID No. 503) 281 HBV-C- RTQSPRRR 195 (SEQ ID No. 504) 282 HBV-C- RTQSPRRRR 1/8 6/12 5/13 2/12 0. 0. 0. 0. 195v1 (SEQ ID No. 002316 006087 008052 005029 505) 283 HBV-C- RSQSPRRRRSK 8/8 12/ 6/13 6/12 0. 0. 0. 0. 195v2 (SEQ ID No. 12 010887 026361 007769 007808 506) 284 HBV-C- SQSPRRRRSK 196 (SEQ ID No. 507) control antigens 200 CMV- ATVQGQNLK 3/8 2/12 9/13 4/12 0.00422 0. 0. 0. pp65_1 (SEQ ID No. 0616 004034 145515 014064 507) 201 HCV- STNPKPQK GPP-2 (SEQ ID No. 508) 203 Sur- DLAQCFFCFK vivin- (SEQ ID No. 53 510) 204 EBV- AVFDRKSDAK 2/8 5/12 4/13 1/12 0. 0. 0. 0. EVNA3B (SEQ ID No. 121349 211809 209514 172746 511) 205 EBV- IVTDFSVIK 1/12 0. EBNA3B- (SEQ ID No. 003396 416 512) 206 HIV- AVDLSHFLK Nef-85 (SEQ ID No. 513) 207 HPV33- NTLEQTVKK E6-86 (SEQ ID No. 514) 208 DENV1- GTSGSPIVNR* GTSGSPIVNR GTSGSPIIDK GTSGSPIVDR GTSGSPIVDK GTSGSPIADK 5/8 4/12 4/13 4/12 0. 0. 0. 0. 1-PP* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 013932 006662 016159 046505 515) 515) 516) 517) 518) 519) GTSGSPIINR GTSGSPIIDK (SEQ ID No. (SEQ ID No. 520) 521) 209 CMV- SVLGPISGHVLK 2/13 0. pp65_2 (SEQ ID No. 005887 522) 210 CMV- TVRAFSRAYH 1/12 0. 2 HRINR 004067 (SEQ ID No. 523) 211 MTB- QIMYNYPAM EsxR (SEQ ID No. 524) 212 MTB- ANTMAMMAR EsxH (SEQ ID No. 525) 213 TG-MS* KSFKDILPK* KSFKDILPK RSFKDLLKK (SEQ ID No. (SEQ ID No. (SEQ ID No. 526) 526) 527) 214 TG-DG_1 AMLTAFFLR (SEQ ID No. 528) 215 TG-SAG_1 STFWPCLLR (SEQ ID No. 529) 216 TG-SAG_2 SSAYVFSVK (SEQ ID No. 530) 217 TG-DG_2 AVVSLLRLLK (SEQ ID No. 531) 218 AV-MP1_1 ASCMGLIYNR 1/8 1/13 1/12 0.00247 0. 0. (SEQ ID No. 0337 003668 018185 532) 219 IAV-MP2 RLFFKCIYRR (SEQ ID No. 533) 220 IAV-NPv1 SVQPTFSVQR 2/8 3/12 4/13 1/12 0.01239 0. 0. 0. (SEQ ID No. 4747 005795 009134 016267 534) 221 IAV-NPv2 SVQRNLPFER 4/8 4/12 6/13 3/12 0.00453 0. 0. 0. (SEQ ID No. 5774 005311 044947 014219 535) 222 IAV-PA_1 KFLPDLYDYK (SEQ ID No. 536) 223 IAV-PCS_ KLVGINMSKK 1/13 0. 1 (SEQ ID No. 003386 537) 224 IAV-PCS_ GTFEFTSFFY 2 (SEQ ID No. 538) 225 IAV-PB2_ SFSFGGFTFK 2/12 2/13 1/12 0. 0. 0. 1 (SEQ ID No. 003073 056170 008540 539) 226 IAV-PB2_ VLRGFLILGK 2 (SEQ ID No. 540) 227 DENV2-5- SQIGAGVYK 1/8 3/12 2/13 1/12 0.00262 0. 0. 0. PP (SEQ ID No. 4733 003189 002960 002847 541) 228 DENV2-7- KTFDSEYVK 1/12 0. PP (SEQ ID No. 026576 542) 229 DENV2-8- RIYSDPLALK PP (SEQ ID No. 543) 230 DENV2-9- ATVLMGLGK 1/8 1/12 1/13 0.00791 0. 0. PP (SEQ ID No. 4106 002360 002428 544) 231 DENV2- STYGWNLVR* STYGWNLVR ATYGWNLVK STYGWNIVK 5/8 7/12 4/13 3/12 0.00298 0. 0. 0. 10-PP* (SEQ ID No. (SEQ ID No. (SEQ ID No. (SEQ ID No. 4029 004907 003946 006023 545) 545) 546) 547) 232 DENV1-2- TVMDIISRR 2/8 0.00698 PP (SEQ ID No. 0396 548) 233 DENV2- RTTWSIHAK 2/8 5/12 3/13 3/12 0.00349 0. 0. 0. 11-PP (SEQ ID No. 347 003315 003655 003184 549) 234 DENV2- RQMEGEGVFK 12-PP (SEQ ID No. 550) 235 AV-MP2- KSMREEYRK 70 (SEQ ID No. 551) 236 IAV-MP1- RMVLASTTAK 178 (SEQ ID No. 552) 237 IAV-MP1- SIIPSGPLK 13 (SEQ ID No. 553) 238 EBV- SSCSSCPLSK* SSCSSCPLSK SSCSSCPLSK 6/8 12/ 11/ 8/12 0.01116 0. 0. 0. LMP2- (SEQ ID No. (SEQ ID No. (SEQ ID No. 12 13 2901 031328 061079 024404 340* 554) 554) 555) 239 DENV2-PP AVQTKPGLFK (SEQ ID No. 556) 240 DENV3-PP GAMLFLISGK (SEQ ID No. 557) 241 DENV4-PP KSGAIKVLK 1/13 0. (SEQ ID No. 002257 558) 242 DENV5-PP KTFVDLMRR 1/12 0. (SEQ ID No. 003485 559) 243 DENV6-PP MANEMGFLEK (SEQ ID No. 560) 244 DENV7- MATYGWNLVK* MATYGWNLVK MSTYGWNIVK 1/12 1/13 0. 0. PP* (SEQ ID No. (SEQ ID No. (SEQ ID No. 002438 011825 561) 561) 562) 245 DENV9- MSYTMCSGK PP (SEQ ID No. 563) 246 DENV10- MVSRLLLNR 1/12 0. PP (SEQ ID No. 004186 564) 247 DENV11- RQLANAIFK PP (SEQ ID No. 565) 248 DENV12- RVIDPRRCLK* RVIDPRRCLK RVIDPRRCMK 1/8 0.00280 PP* (SEQ ID No. (SEQ ID No. (SEQ ID No. 2527 566) 566) 567) 249 DENV15- TSGSPIIDK 1/8 0.00527 PP (SEQ ID No. 609 568) 250 DENV16- TTKRDLGMSK 1/8 2/12 0.00355 0. PP (SEQ ID No. 1109 014267 569) 251 DENV17- VTRGAVLMHK PP (SEQ ID No. 570) 252 DENV18- YVSAIAQTEK PP (SEQ ID No. 571) 253 DENV19- SPGTSGSPIIDK PP KGK (SEQ ID No. 572) 254 EBV- ATIGTAMYK 1/8 3/12 3/13 2/13 0.00415 0. 0. 0. BRLF1 (SEQ ID No. 6485 018045 078450 011428 573) 255 CMV- GPISGHVLK pp65_3 (SEQ ID No. 574) 256 IAV-MP1_ AYQKRMGVQM 2 (SEQ ID No. 575) 257 IAV-PA_2 LYASPQLEGF (SEQ ID No. 576) 258 LMAV-GP LVTFLLLCGR (SEQ ID No. 577) 258 LCMV- LVSFLLLAGR GP_1 (SEQ ID No. 578) 259 LCMV- FTNDSIISH GP_2 (SEQ ID No. 579) 260 LCMV- TTYLGPLSCK RING-Z (SEQ ID No. 580) 261 Mus AINSEMFLR musculus- (SEQ ID No. GP 581) 262 Mus LALEVARQKR LALEVARQKR TLALEVARQK musculus- (SEQ ID No. (SEQ ID No. (SEQ ID No. Insulin-1 582) 582) 583) 263 Mus TLALEVAQQK musculus- (SEQ ID No. Insulin-2 584) 264 MTB- AMGDAGGYK 1/13 0. Ag85B (SEQ ID No. 002307 585)
TABLE-US-00004 TABLE S2 Supplementary Table 2: List of the antibody staining panels used for mass cytometry and high-dimensional cytometric data analysis Metal- PBMC ex vivo panel 1, 14(SAv)-choose-4 Isotope Antibody (or label) Clone Company t-SNE Y-89 CD45 HI30 Fluidigm (DVS) Pd-102 Cell barcode Rh-103 DNA Intercalator Fluidigm (DVS) Pd-104 Cell barcode Pd-105 Cell barcode Pd-106 Cell barcode Pd-108 Cell barcode Pd-110 Cell barcode Cd-112/114 Qdt800-CD14 TuK4 Molecular Probes In-113 Empty In-115 CD57 (Differentiation) HCD57 Biolegend ✓ La-139 Va7.2 (Differentiation) 3C10 Biolegend ✓ Ce-140 CD3 UCHT1 BioXcell Pr-141 HLA-DR (Differentiation) L243 Biolegend ✓ Nd-142 CD45RO (Differentiation) UCHL1 Biolegend ✓ Nd-143 CD38 (Differentiation) HIT2 Biolegend ✓ Nd-144 CCR7 (Differentiation) 150503 R&D Systems ✓ Nd-145 CD27 (Differentiation) LG.7F9 eBioscience ✓ Nd-146 CD8b SK1 Biolegend Sm-147 CD28 (Differentiation) CD28.2 Biolegend ✓ Nd-148 SAv-Nd-148 in-house Sm-149 CXCR3 (Differentiation) 1C6 BD Bioscience ✓ Nd-150 KLRG-1 (Differentiation) 13F12F2 eBioscience ✓ Eu-151 SAv-Eu-151 in-house Sm-152 CXCR5 (Differentiation) RF8B2 BD Bioscience ✓ Eu-153 SAv-Eu-153 in-house Sm-154 HVEM (Exhaustion) 94801 R&D Systems ✓ Gd-155 CTLA-4 (Exhaustion) BNI3 BD Bioscience ✓ Gd-156 CD39 (Differentiation) A1 Biolegend ✓ Gd-157 SAv-Gd-157 in-house Gd-158 CD45RA (Differentiation) HI100 BD Bioscience ✓ Tb-159 SAv-Tb-159 in-house Gd-160 PD-1 (Exhaustion) eBioJ105 eBioscience ✓ Dy-161 SAv-Dy-161 in-house Dy-162 CD4 SK3 Biolegend Dy-162 CD19 HIB19 Biolegend Dy-162 CD56 NCAM16.2 BD Bioscience Dy-163 SAv-Dy-163 in-house Dy-164 LAG-3 (Exhaustion) 874501 R&D Systems ✓ Ho-165 SAv-Ho-165 in-house Er-166 SAv-Er-166 in-house Er-167 TIM-3 (Exhaustion) 344823 R&D Systems ✓ Er-168 SAv-Er-168 in-house Tm-169 SAv-Tm-169 in-house Er-170 2B4 (Exhaustion) C1.7 Biolegend ✓ Yb-171 SAv-Yb-171 in-house Yb-172 BTLA (Exhaustion) MIH26 eBioscience ✓ Yb-173 SAv-Yb-173 in-house Yb-174 CD160 (Exhaustion) 688327 R&D Systems ✓ Lu-175 SAv-Yb-175 in-house Yb-176 CD127 (Differentiation) A019D5 Biolegend ✓ Ir-191/193 CD161 (Differentiation) HP-3G10 Biolegend ✓ Pt-194 Pt-195 Live/Dead Pt-198 Bi-209 One-SENSE Differentiation category Exhaustion Trafficking PBMC ex vivo panel 2, , 9(SAv)-choose-3 Metal- t- Isotope Antibody (or label) Clone Company SNE Y-89 CD45 HI30 Fluidigm (DVS) Pd-102 Cell barcode Rh-103 Live/Dead Pd-104 Cell barcode Pd-105 Cell barcode Pd-106 Cell barcode Pd-108 Cell barcode Pd-110 Cell barcode Cd-112/114 Qdt800-CD14 TuK4 Molecular Probes In-113 Empty In-115 CD57 (Differentiation + TNFR) HCD57 Biolegend ✓ La-139 Va7.2 (Differentiation + TNFR) 3C10 Biolegend ✓ Ce-140 CD3 UCHT1 BioXcell Pr-141 HLA-DR (Differentiation + TNFR) L243 Biolegend ✓ Nd-142 CD27 (Differentiation + TNFR) LG.7F9 eBioscience ✓ Nd-143 CD38 (Differentiation + TNFR) HIT2 Biolegend ✓ Nd-144 CCR4 (Trafficking) 205410 R&D Systems ✓ Nd-145 CD45RA (Differentiation + TNFR) HI100 BD Bioscience ✓ Nd-146 CCR6 (Trafficking) G034E3 Biolegend ✓ Sm-147 CD45RO (Differentiation + TNFR) UCHL1 Biolegend ✓ Nd-148 CTLA-4 (Exhaustion) BNI3 BD Bioscience ✓ Sm-149 CXCR3 (Trafficking) 1C6 BD Bioscience ✓ Nd-150 KLRG-1 (Differentiation + TNFR) 13F12F2 eBioscience ✓ Eu-151 CCR7 (Differentiation + TNFR) 150503 R&D Systems ✓ Sm-152 CXCR5 (Trafficking) RF8B2 BD Bioscience ✓ Eu-153 SAv-Eu-153 in-house Sm-154 HVEM (Exhaustion) 94801 R&D Systems ✓ Gd-155 SAv-Gd-155 in-house Gd-156 CD39 (Differentiation + TNFR) A1 Biolegend ✓ Gd-157 0X40 (Differentiation + TNFR) 443318 R&D Systems ✓ Gd-158 TIGIT (Exhaustion) MBSA43 eBioscience ✓ Tb-159 GITR (Differentiation + TNFR) 110416 R&D Systems ✓ Gd-160 PD-1 (Exhaustion) eBioJ105 eBioscience ✓ Dy-161 CCR5 (Trafficking) HEK/1/85a Abeam ✓ Dy-162 CD161 (Differentiation + TNFR) HP-3G10 Biolegend ✓ Dy-162 Dy-162 Dy-163 BTLA (Exhaustion) MIH26 Fluidigm ✓ (DVS) ✓ Dy-164 LAG-3 (Exhaustion) 874501 R&D Systems Ho-165 SAv-Ho-165 in-house Er-166 SAv-Er-166 in-house Er-167 TIM-3 (Exhaustion) 874501 R&D Systems ✓ Er-168 SAv-Er-168 in-house Tm-169 SAv-Tm-169 in-house Er-170 2B4 (Exhaustion) C1.7 Biolegend ✓ Yb-171 SAv-Yb-171 in-house Yb-172 4-1BB (Differentiation + TNFR) 4B4-1 Biolegend ✓ Yb-173 SAv-Yb-173 in-house Yb-174 CD160 (Exhaustion) 688327 R&D Systems ✓ Lu-175 SAv-Yb-175 in-house Yb-176 CD127 (Differentiation + TNFR) A019D5 Biolegend ✓ Ir-191/193 DNA Intercalator (DVS) Pt-194 CD8b SKI Biolegend Pt-195 Empty Pt-198 CD4 SK3 Biolegend CD19 HIB19 Biolegend Bi-209 CD16 3G8 Fluidigm (DVS) One-SENSE Differentiation + TNFR category Exhaustion Trafficking PBMC in vitro panel, 8(SAv)-choose-3 Metal- t- Isotope Antibody (or label) Clone Company SNE Y-89 CD45 HI30 Fluidigm (DVS) Pd-102 Cell barcode Rh-103 Live/Dead Pd-104 Cell barcode Pd-105 Cell barcode Pd-106 Cell barcode Pd-108 Cell barcode Pd-110 Cell barcode Cd-112/114 Qdt800-CD14 TuK4 Molecular Probes Qdt800-CD19 HIB19 Molecular Probes In-113 Empty In-115 CD57 (Differentiation + TNFR) HCD57 Biolegend ✓ La-139 IL-15Ra (Differentiation + TNFR) R&D Systems Ce-140 CD3 UCHT1 BioXcell Pr-141 IFN-g (Function) 4S.B3 eBioscience ✓ Nd-142 CD27 (Differentiation + TNFR) LG.7F9 eBioscience ✓ Nd-143 Granzyme B (Function) CLB-GB11 Abeam ✓ Nd-144 CD107a (Function) H4A3 BD Bioscience ✓ Nd-145 CD45RA (Differentiation + TNFR) HI100 BD Bioscience ✓ Nd-146 MIP1-b (Function) D21-1351 BD Bioscience ✓ MQ1- Sm-147 IL-2 (Function) 17H12 eBioscience ✓ Nd-148 CTLA-4 (Exhaustion) BNI3 BD Bioscience ✓ Sm-149 TNF-a (Function) Mab11 eBioscience ✓ Nd-150 KLRG-1 (Differentiation + TNFR) 13F12F2 eBioscience ✓ Eu-151 CCR7 (Differentiation + TNFR) 150503 R&D Systems ✓ Sm-152 Perforin (Function) B-D48 Abeam ✓ BVD2- Eu-153 GM-CSF (Function) 21C11 Biolegend ✓ Sm-154 HVEM (Exhaustion) 94801 R&D Systems ✓ Gd-155 SAv-Gd-155 in-house Gd-156 CD39 (Differentiation + TNFR) A1 Biolegend ✓ Gd-157 OX40 (Differentiation + TNFR) 443318 R&D Systems ✓ Gd-158 TIGIT (Exhaustion) MBSA43 eBioscience ✓ Tb-159 GITR (Differentiation + TNFR) 110416 R&D System ✓ Gd-160 PD-1 (Exhaustion) eBioJ105 eBioscience ✓ Dy-161 Granzyme K (Function) GM6C3 Biolegend ✓ Dy-162 Granzyme A (Function) CB9 Biolegend ✓ Dy-162 Dy-162 Dy-163 BTLA (Exhaustion) MIH26 Fluidigm (DVS) ✓ Dy-164 LAG-3 (Exhaustion) 874501 R&D Systems ✓ Ho-165 SAv-Ho-165 in-house Er-166 SAv-Er-166 in-house Er-167 TIM-3 (Exhaustion) 874501 R&D Systems ✓ Er-168 SAv-Er-168 in-house Tm-169 SAv-Tm-169 in-house Er-170 2B4 (Exhaustion) C1.7 Biolegend ✓ Yb-171 SAv-Yb-171 in-house Yb-172 4-1BB (Differentiation + TNFR) 4B4-1 Biolegend ✓ Yb-173 SAv-Yb-173 in-house Yb-174 CD160 (Exhaustion) 688327 R&D Systems ✓ Lu-175 SAv-Yb-175 in-house Yb-176 CD127 (Differentiation + TNFR) A019D5 Biolegend ✓ Ir-191/193 DNA Intercalator Fluidigm (DVS) Pt-194 CD8b SK1 Biolegend Pt-195 Empty Pt-198 CD4 SK3 Biolegend Bi-209 CD16 3G8 Fluidigm (DVS) One-SENSE Differentiation + TNFR category Exhaustion Function
[0155] 3. Highly Multiplexed pMHC Tetramer, Antibody Staining and CD8 T cell Enrichment
[0156] Cryopreserved PBMC were thawed and washed with complete RPMI (10% FBS+1% penicillin/streptomycin/L-glutamine+1% 1M HEPES) (Gibco, Invitrogen), and rest for 3 hours at 37° C. After the recovery, cells were harvested and seeded on a non-treated 96-well plate, and about 10 million cells per patient were used and split evenly in two separated wells for two configurations of 562-plex combinatorial pMHC tetramer staining. 50 μM dasatinib was incubated with cells for 30 min at 37° C., 5% CO2, to prevent the downregulation of TCR (30). Cells were washed with CyFACS buffer (2 mM EDTA+0.05% sodium azide+4% FBS in PBS) and incubated with 200 mM cisplatin (Pt-195) for 5 min on ice, or rhodium (Rh-103) for 20 min at room temperature (table S2) for viability measurement. After wash once with CyFACS buffer, cells from the same donor in separated wells were stained with 50 μl of cocktail containing the same 562-plex pMHC tetramers but completely different SAv-metal coding configurations for 1 hour in room temperature in the presence of 1:100 Fc block (Biolegend). Cells were washed twice with CyFACS buffer after incubation, and resuspend in 50 μl of T cells or CD8 T cells enrichment kit (STEMCELL) antibody cocktail in 1:10 in CyFACS buffer for 30 min on ice. Cells were then washed, and stained with 50 μl of primary antibody cocktail (table S2 and
[0157] 4. Statistical Analysis
[0158] Non-parametric analysis of variance (ANOVA) was used for group comparison unless Indicated elsewhere. p<0.05 by non-parametric ANOVA allowed the subsequent multiple comparison test. P values were calculated using Prism software (GraphPad). All error bars are median and SEM.
[0159] 5. Amplification of HBV Genome and Library Construction
[0160] Seven treatment-naïve HBeAg non-seroconverters and eight HBeAg seroconverters of patients chronically infected by HBV (including genotype B and C) were recruited in National University Health System, Singapore. Multiple longitudinal serum samples (5 to 15 time points per patient) from each patient were taken across the event of HBeAg seroconversion. Deep sequencing analysis was performed in all serums samples by sequencing the whole HBV viral genome. Similar to previously description, primers (5′-GCTCTTCTTTTTCACCTCTGCCTAATCA-3′ (SEQ ID No. 29) and 5′-GCTCTTCAAAAAGTTGCATGGTGCTGG-3′ (SEQ ID No. 30)) were used to generate full-length amplicons of the HBV genome. Polymerase chain reaction (PCR) was performed using the PfuUltra™ II Fusion HS DNA Polymerase (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. The 3.1 kb fragment was extracted from 1% agarose gel prepared in 1×TBE buffer, using the QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif., USA) and the concentration of the extracted product was measured using the NanoDrop N. Dak. 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA). Each sample was fragmented into 100-300 bp using the Covaris S2 (Covaris, Woburn, Mass., USA). (Shearing conditions—Duty cycle: 20%; Intensity: 5; Cycles per burst: 200; Time: 110 seconds). After fragmentation, the samples were purified using the QlAquick PCR purification kit (Qiagen, Valencia, Calif., USA). The DNA 1000 Chip was used with the 2100 bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) to check the size and quality of the fragmented products. For library construction, the KAPA Library Preparation Kit (KAPA Biosystems) was used according to the manufacturer's instructions. The library construction includes end repair, A-tailing, ligation of adapters and a final PCR step that incorporates the indexes into the samples. Illumina TrueSeq adapters and indexes were used (Illumina, San Diego, Calif., USA). PfuUltra™ II Fusion HS DNA Polymerase was used for this final PCR step according to the manufacturer's instructions. The samples were then cleaned up using the Agentcourt AMPure XP (Beckman Coulter) on a 1:1 ratio of beads to sample. To check on the size and concentration of the ligated products, the 2100 Bioanalyzer with DNA 1000 Chip was used. The quality and quantity of the products were determined by running a quantitative PCR. The reactions were prepared using the KAPA Library Quantification kit (KAPA Biosystems) and the run was done on the LightCycler 480 II real time thermal cycler (Roche Applied
[0161] Science, Indianapolis, IN, USA) according to the manufacturer's instructions. Samples were sequenced in the Genome Institute of Singapore on the Illumina HiSeq 2500 to obtain multiplexed 101 bp paired-end reads.
[0162] 6. SNV Analysis on Viral Epitope
[0163] We modified the reference genome P121214 such that ambiguous positions (e.g. R/W/Y) were replaced by one of the relevant bases (A/C/G/T) randomly. 101 bp paired-end reads were mapped to the modified reference genome with BWA-MEM version 0.7.10-r789 and Single-nucleotide variant (SNV) calling was carried out with LoFreq version 2.1.2. Coverage depth averaged around 104˜105 for all samples. 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 high error rates. SNVs passing quality filters were then sorted and only non-synonymous mutations that changed >20% in frequency between the early and late time points were kept (custom per script). These SNVs were candidates for adaptive epitopes, and were matched to known epitope sequences for further tetramer experiments.
[0164] 7. HBV Epitope Prediction and Peptide Synthesis
[0165] The consensus sequences of HBV from each patient were further determined and translated into amino acid sequence for each open reading frame (ORF). The predicted binders (peptides) restricted to HLA-A*1101 were then generated by NetMHC software (v3.4 server, http://www.cbs.dtu.dk/services/NetMHC/) based on the consensus sequences derived from HBV proteins (core, polymerase, x and envelope), including all possible binding variants for 8-, 9-, 10-, and 11-mer peptides that above the binding threshold (for score>0.4 predicting weak binding and score>0.6 predicting strong binding). The prediction scheme produced 484 unique HLA-A*1101-restricted HBV epitopes, together with 78 known HLA-A*1101-restricted epitopes derived from other pathogens or self-proteins, a total of 562 different pMHC tetramers (table S1) were therefore made from these peptides for subsequent highly multiplexed combinatorial pMHC tetramer mapping. Immune Epitope Database (IEDB) was used to report previously unidentified epitope sequences. All peptides were synthesized by Mimotopes (Australia) with purity>85%.
[0166] 8. Peptides Sequence Similarity and Cluster Assignment
[0167] To avoid the incorrect interpretation from cross-reactive T cell epitopes in the 562-plex pMHC library that comprised of all viral proteins and variants, sequences of the library were pooled and loaded onto a Biostrings-based R-written environment. Similar to BLAST (Basic Local Alignment Search Tool), the biological sequence and matching algorithm performed pairwise alignment to calculate the peptide binding score based on their sequence similarity. Total of 284 peptide clusters were assigned, and the peptides in each cluster are listed (table S1). Peptides within the same cluster were then given the same quadruple SAv-metal coding for highly multiplexed combinatorial pMHC tetramer strategy.
[0168] 9. Generation of HLA-A*11:01 Monomer and 562-Plex pMHC Library
[0169] The inclusion bodies of HLA-A*1101 were produced (76, 77), and refolded with a UV-cleavable peptide H-RVFA(J)SFIK-OH (SEQ ID No. 31), where i is ANP (3-Amino-3-(2-nitrophenyl) propionic acid) linker. The protein was purified and biotinylated, and stored in PBS+50% glycerol at −20° C. Peptide exchange was performed at 0.1 mg/ml of HLA-A*1101 monomer in 100 μl PBS with 25 μM of peptide of interest in a 96-well plate. The reaction was exposed to 365 nm UV irradiation for 5 min twice using UVP CL-1000 Ultraviolet Crosslinker, the plate was further sealed and stored at 4° C. overnight to complete the exchange.
[0170] 10. Streptavidin (SAy) Production and Metal Labeling
[0171] Streptavidin with free cysteines residues separated by glycine linkers were used for recombinant expression. Briefly, purified streptavidin was made in-house and stored in 10 mM TCEP in 20 mM HEPES (pH 7.2) buffered saline as frozen aliquots at −80° C. After conjugation using DN3 polymer labeling kits and filtering using 0.1 μm inn filters (Amicon), the metal-tag streptavidin conjugates (SAv-metal) were transferred to a new 30 kD concentrator
[0172] (Merck) to perform five washes with eDTA-free W-buffer. SAv-metal was adjusted to final concentration at 200 μg/ml prior the formation of tetrameric pMHC complex.
[0173] 11. Antibody-Metal Conjugation
[0174] Purified antibodies without carrier proteins were purchased as listed (table S2). 50 or 100 μg of antibody was conjugated with metal-attached maleimide-coupled DN3 MAXPAR (Fluidigm DVS) chelating polymer according to manufacturer's instruction (Fluidigm DVS) as previously described. All metal isotopes were purchased from Fluidigm DVS or TRACE Sciences International Inc. as listed (table S2).
[0175] 12. Mass Cytometry and Data Pre-Processing
[0176] All experiments were acquired by CyTOF2 (Fluidigm DVS) systems. Cells were wash by MilliQ water twice, filtered, and immediately acquired by mass cytometry with an acquisition rate of 300˜350 cells/sec. 2% of Four EQ beads (Fluidigm DVS) were mixed with cell suspension. To normalize signal variations of CyTOF2, the output FCS files were normalized based on the added beads that has been previously described. Normalized FCS files were further loaded onto a Unix-based R-written script and all zero values were randomized into values between 0 to −1 using uniform distribution.
[0177] 13. Self-Validated Automatic Deconvolution of Antigen-Specific T Cells
[0178] After data pre-processing, lived CD8+ T cells were gated using FlowJo v9.7.6 (Tree Star Inc.) and individual samples were de-barcoded based on the dual mass-tag cellular barcodes using Boolean gates. Two SAv-metal coding configurations from the same donor were barcoded and exported independently. For optimal automatic identification of tetramer positive cells, multiple safety parameters and thresholds were built and subjectively defined using an R-written script, respectively (
[0179] 14. High-Dimensional Cytometric Data Visualization
[0180] Validated antigen-specific CD8+ T cells were exported individually from each donor for dimensionality reduction analysis. Detailed methodology for t-SNE and One-SENSE can be found elsewhere. Briefly, t-SNE and One-SENSE were performed using custom R scripts based on “flowCore” and “Rtsne” packages downloaded from The Comprehensive R Archive Network (CRAN). All data were transformed using the “logicleTransform” function and w=0.25, t=16409, m=4.5, a=0 as input parameters to roughly match scaling historically used in FlowJo. Cellular markers analyzed by t-SNE and One-SENSE were indicated (table S2). For One-SENSE, cellular markers in each T cell category (“Differentiation+TNFR”, “Exhaustion” and “Trafficking”) were subjectively assigned for categorical analysis. Aligned heatplots represent the distribution of marker positive cells in percentage on each “bin” on the axis (category) constructed by cells residing in small ranges of values. Positive population of markers was manually defined and markers of the same category were combined for each dimension using 250 bins.
[0181] The 3D visualization of One-SENSE was built from numerous consecutive 3D images supported by “rgl” package based on the three dimensions of One-SENSE analysis. The continuous image sequences were subsequently combined by Sequimago (AppleScript) to generate 3D movies.
[0182] Logistic regression was performed using the drc R package (v 3.0.1) using a 3-parameters logistic model. Support vector machines was trained on the 7 parameters common across the two datasets using the e1071 R package (v 1.6-8) using default parameters (epsilon-regression with radial kernel, gamma of 1/7 and epsilon of 0.1).
[0183] 15. Flow Cytometry and Cell Sorting
[0184] Cells were prepared as the same fashion as mass cytometry experiments. After incubation with dasatinib, cells were washed with PBS and incubated with 50 μl of Live/Dead-Pacific Orange (Thermo Fisher) on ice for 20 min in dark. For single fluorochrome-tag pMHC tetramers, peptide-exchange and tetramer formation were done using the same method as metal-tag pMHC tetramers in dark. PE-SAv (ebioscience), PE-Cy7-SAv (ebioscience), PE-Cy5-SAy (eBioscience), BV650-SAv (BD) and APC-SAv (Biolegend) were diluted to 20 μg/ml in PBS and added into pMHC monomer loaded with different peptides in the same manner as mentioned above. Cells then washed, and stained by tetramer cocktail in the same condition as mass cytometry experiment in dark. After two washed with FACS buffer, cells were stained 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) in FACS buffer for 30 min on ice in dark. Cells were then washed twice, filtered, and analyzed by LSRFortessa (BD). Fluorochrome-tag tetramer positive cells were stained using the same method and live-sorted using Aria II 5 lasers system (BD).
[0185] 16. TCR High-Throughput Sequencing and Spectratyping
[0186] Five populations, including four different tetramer-stained virus-specific CD8+ T cells plus the total CD8+ T cells from each donor were live-sorted, and genomic DNA were freshly extracted using Qiagen Blood & Tissue kit according to manufacturer's instruction. Cells from three donors per patient group, with five groups and 75 samples in total, were used for TCR sequencing. Briefly, TCβ chains were amplified using a bias-controlled two-step multiplex PCR by ImmunoSEQ platform (Adaptive Biotechnologies). The first PCR amplified the CDR3 region of sorted T cells, followed by adding the adaptor sequences in second PCR, and subsequently sequenced by next-generation sequencing (NGS). Productive reads were generated from the reduction of amplification and sequencing bias. 1000, 5000, 1500, 2000 and 10000 cells were live sorted for HBVpol282-, HBVpol387-, HBVcore169-, HBVcore195-specific and total CD8+ T cells, respectively.
[0187] TCR sequences were then exported from ImmunoSEQ Analyzer and the genes were defined by IMGT/HighV-Quest nomenclature. CDR3β length analysis was calculated by Prism using Gaussian fit with the null hypothesis “one curve fits all”. The null hypothesis was rejected with statistical significance.
[0188] 17. TCRdist Measurements for Quantifying Epitope-Specific Repertoires
[0189] Quantifiable distances of epitope-specific TCRβ (TCRdist) were computed to be the similarity-weighted Hamming distances between the contacting regions of two TCR's CDR1, 2 and 3 as recently developed (53). TCRdist pipeline was downloaded (https://github.com/phbradley/tcr-dist) and installed in Python. TCRβ sequences were loaded and executed with additional command “—make_fake_alpha” and “—make_fake_quals”. The unsupervised visualization of t-SNE maps were constructed based on the kernel PCA coordinates and the TCRdist distance matrix of TCR clones generated by TCRdist pipeline using a custom R-written script. The implementation for unsupervised clustering Phenograph algorithm, Rphenograph, was installed in R (https://github.com/JinmiaoChenLab/Rphenograph). A repertoire diversity metric (TCRdiv) that generalizes Simpson's diversity index was used to measure the diversity of epitope-specific TCRs by accounting both the similarity and identity of the sequences.
[0190] 18. In Vitro Virus-Specific CD8+ T Cells Expansion
[0191] PBMC from patients were thawed and recovered, and then resuspend in AIM-V medium (with 2% human AB serum, 1% penicillin/streptomycin/L-glutamine, 1% 1M HEPES) (Gibco, Invitrogen) with 20 lU/ml of recombinant human IL-2 (R&D). Cells were pulsed with corresponded HBV or control peptides at 1 μM per 1 million cells in 200 μl medium in 96-well round-bottom tissue culture plate and cultured for 10 days at 37° C. Half of the medium was replaced as supplementary every three days without any peptide. On day 10, cells were restimulated with or without the corresponded peptides for 7 hours in the presence of Brefeldin A (eBioscience), monensin (eBioscience) and 0.5 μg/ml anti-CD107a at 37° C. After incubation, cells were collected and then stained with the 50-plex pMHC tetramers (table S1) and surface antibodies. Intracellular cytokine staining was performed on the second day as indicated in (table S2). All staining, cellular barcoding, and CD8 T cell enrichment were done in the same manner as ex vivo staining described above.
[0192] 19. Enzyme-Linked Immunosorbent Assay (ELISA)
[0193] Paired serum samples from patients were serially diluted in PBS, and level of HbeAg, HBsAg, HBeAb, HBsAb and HBcAb were determined using quantitative sandwich ELISA kits (Abnova and MyBioSource) according to manufacturer's instruction.
Results
[0194] 1. Comprehensive HBV Epitope Mapping
[0195] To generate a comprehensive HBV targeting pMHC library, viral DNA was isolated and deep sequenced from serum samples of 15 longitudinal CHB patients to determine viral consensus sequences and common variants (
[0196] It was hypothesized that the antigen-specific T cell responses could vary across different clinical stages and reflect CHB disease progression. Therefore, this strategy to map potential T cell epitopes across three CHB patient groups (IT, IA and InA) and one group of acutely resolved patients (R) (table S3) was applied. There was no difference in the overall magnitudes of detected antigen-specificities between patient groups (
[0197] Several experiments were performed to validate and assess the HBV relevance of these four epitopes. For three out of four of these epitopes, antigen-specific T cells were detected in some healthy donor (HD) or cord blood (CB) samples (
[0198] A previously identified epitope, HBV-P-387 was observed with relatively high frequency in CHB and also in half of the HD tested, but to a lesser extent in CB samples. As described further in subsequent sections, the heterogeneous phenotypes of these cells were highly variable between patients (
[0199] 2. High-Dimensional Phenotypic Profiling of HBV-Specific CM8.sup.+ T cells
[0200] Further analyses were directed at HBV-P-387 and HBV.sub.core169-specific CD8.sup.+ T cells because of their higher degrees of phenotypic heterogeneity observed across patients at various stages of CHB. Although both HBV-P-387 (LVVDFSQFSR (SEQ ID No. 28)) and one of the peptides within the HBV-C-169 (STLPETTVVRR (SEQ ID No. 23)) have been previously reported, the phenotypes of these reactive T cells have not been investigated. Unique to HBV.sub.pol387 and HBV.sub.core169-specific CD8.sup.+ T cells is the higher expression of TIGIT compared to other HBV-specific CD8.sup.+ T cells (
[0201] Unsupervised high-dimensional t-SNE visualization and Phenograph cellular clustering were applied to describe the phenotypes of virus-specific CD8.sup.+ T cells across individuals from one large batch of samples run in parallel (
[0202] From this analysis, remarkable heterogeneity of HBV.sub.pol387-specific CD8.sup.+ T cells was observed. In many instances, several distinct populations specific for this one epitope could be seen even within individual patients. Despite such diverse phenotypes, this epitope sequence was highly conserved across all patients and time-points subjected to HBV viral sequencing over a decade (
TABLE-US-00005 TABLE S4 Supplementary Table 4: The frequency of viral mutation on selective epitopes in longitudinal patient cohort across HBeAg-seroconversion. Time Patient 6-Jan- 29-May- 4-Jun- 29-Jul- 3-Feb- 27-Jul- 10-Aug- Patient group ID Epitope Sequence 1999 2000 2001 2003 2004 2005 2006 Non- C3 003_HBV- STNRQSGRQ 0.9623 0.9608 0.9601 0.9569 0.9623 0.9607 0.9611 seroconverters S-95* (SEQ ID No. 586) 056_HBV- VVNHYFQTR 0.9704 0.9692 0.9681 0.9666 0.9698 0.9689 0.9677 P-136* (SEQ ID No. 129) 090_HBV- KAAYSLISTSK 0.9448 0.9436 0.9390 0.9396 0.9443 0.9426 0.9442 P-282* (SEQ ID No. 206) 106_HBV- LVVDFSQFSR 0.9556 0.9558 0.9542 0.9509 0.9581 0.9558 0.9556 P-387 (SEQ ID No. 28) 125 HBV- Y[V]SLMLLYK 0.9552 0.9541 0.9537 0.9514 0.9548 0.9521 0.9560 P-486* (SEQ ID No. 285) 178 HBV- STLPETTWRR 0.9601 0.9631 0.9616 0.9592 0.9618 0.9598 0.9578 C-169* (SEQ ID No. 23) 179 HBV- TWRRRGRSPR 0.9570 0.9572 0.9578 0.9556 0.9584 0.9559 0.9563 C-175* (SEQ ID No. 395) 183 HBV- TTVNAH[G]NLPK 0.9474 0.9462 0.9443 0.9411 0.9500 0.9480 0.9455 X-80* (SEQ ID No. 404) 283_HBV- RSQSPRRRRSQ 0.9568 0.9555 0.9551 0.9517 0.9557 0.9542 0.9550 C-195v2 (SEQ ID No. 587) 1-Jun- 21-Dec- 12-Oct- 16-Sep- — — 1991 1991 1992 1996 C4 003_HBV- STNRQSGRQ 0.9851 0.9605 0.9841 0.9599 S-95* (SEQ ID No. 586) 056_HBV- VVNHYFQTR 0.9784 0.9519 0.9419 0.8340 P-136* (SEQ ID No. 129) VV[D]HYFQTR 0.0106 0.0158 0.0351 0.1301 (SEQ ID No. 135) 090_HBV- KAAYSLISTSK 0.9784 0.9415 0.9656 0.9380 P-282* (SEQ ID No. 206) 106_HBV- LVVDFSQFSR 0.9805 0.9573 0.9815 0.9518 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9739 0.9460 0.9706 0.9451 P-486* (SEQ ID No. 285) 178_HBV- STLPETTWRR 0.9832 0.9622 0.9824 0.9571 C-169* (SEQ ID No. 23) 179_HBV- TVVRRRGRSPR 0.9801 0.9601 0.9807 0.9540 C-175* (SEQ ID No. 395) 183_HBV- TTVNAH[G]NLPK 0.9751 0.9412 0.9716 0.9406 X-80* (SEQ ID No. 404) 283_HBV- RSQSPRRRRSQ 0.9768 0.9538 0.9749 0.9454 C-195v2 (SEQ ID No. 587) 12-Nov- 19-Jun- 8-Sep- 21-Jun- 2-Sep- — — 1988 1990 1992 1995 1996 C5 003_HBV- STNRQSGRQ 0.9639 0.9635 0.9655 0.9632 0.9801 S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.9614 0.9594 0.9636 0.9633 0.9808 P-136* (SEQ ID No. 129) 090_HBV- KAAYSLISTSK 0.9480 0.9462 0.9494 0.9470 0.9715 P-282* (SEQ ID No. 206) 106_HBV- LVVDFSQFSR 0.9617 0.9595 0.9625 0.9638 0.9810 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9577 0.9579 0.9614 0.9606 0.9819 P-486* (SEQ ID No. 285) 178_HBV- STLPETTVIRR 0.9612 0.9615 0.9646 0.9663 0.9818 C-169* (SEQ ID No. 24) 179_HBV- TVIRRRGRSPR 0.9566 0.9597 0.9615 0.9607 0.9786 C-175* (SEQ ID No. 396) 183_HBV- TTVNAHRNLPK 0.9481 0.9477 0.9546 0.9515 0.9735 X-80* (SEQ ID No. 407) 283_HBV- RSQSPRRRRSQ 0.9611 0.9593 0.9608 0.9616 0.9760 C-195v2 (SEQ ID No. 587) 15-May- 5-Jul- 18-Jul- 25-May- 19-Jan- 10-Feb- 2-Feb- — — 2001 2002 2003 2004 2005 2006 2007 C7 003_HBV- STNRQSGRQ 0.9629 0.9630 0.9594 0.9610 0.9643 0.9624 0.9623 S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.9593 0.9600 0.9592 0.9564 0.9595 0.9586 0.9587 P-136* (SEQ ID No. 129) 090_HBV- KAAYSLISTSK 0.9454 0.9458 0.9412 0.9436 0.9471 0.9430 0.7917 P-282* (SEQ ID No. 206) KAA[H]SLISTSK 0.1527 (SEQ ID No. 588) 106_HBV- LWDFSQFSR 0.9556 0.9569 0.9530 0.9567 0.9587 0.9564 0.9566 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9494 0.9513 0.9473 0.9490 0.9517 0.9489 0.9484 P-486* (SEQ ID No. 285) 178_HBV- STLPETTVVRR 0.9595 0.9619 0.9597 0.9610 0.9611 0.9591 0.9595 C-169* (SEQ ID No. 23) 179_HBV- TVVRRRGRSPR 0.9570 0.9579 0.9579 0.9578 0.9589 0.9566 0.9529 C-175* (SEQ ID No. 395) 183_HBV- TTVNAHRNLPK 0.9430 0.9449 0.9405 0.9398 0.9484 0.9436 0.9425 X-80* SEQ ID No. 407) 283_HBV- RSQSPRRRRSQ 0.9544 0.9553 0.9570 0.9525 0.9552 0.9521 0.9493 C-195v2 (SEQ ID No. 587) Numbers indicate the frequenies of the given variants. Grey shaded sequences; are wild type sequences. Time Patient 17-Aug- 26-Oct- 12-Jun- 14-Mar- 3-Jun- 9-Feb- Patient group ID Epitope Sequence 1998 1999 2000 2001 2003 2004 HBeAg- S1 003_HBV- STNRQSGRQ 0.9799 0.9815 0.9812 0.9852 0.9584 0.9770 seroconverters S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.9854 0.9862 0.9853 0.9874 0.9626 0.9747 P-136* (SEQ ID No. 129) W[D]HYFQTR 0.0109 (SEQ ID No. 135) 090_HBV- KAAYSLISTSK 0.9748 0.9725 0.9758 0.9709 0.9420 0.9697 P-282* (SEQ ID No. 206) 106_HBV- LVVDFSQFSR 0.9822 0.9821 0.9816 0.9806 0.9526 0.9801 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9819 0.9810 0.9818 0.9547 0.9485 0.9784 P-486* (SEQ ID No. 285) 178_HBV- STLPETTWRR 0.9825 0.9827 0.9835 0.9828 0.9590 0.9820 C-169* (SEQ ID No. 23) 179_HBV- TWRRRGRSPR 0.9793 0.9804 0.9790 0.9795 0.7738 0.2946 C-175* (SEQ ID No. 395) TVVRRR[C]RSPR 0.1824 0.6858 (SEQ ID No. 589) 183_HBV- TTVNAHRNLPK 0.0628 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.9731 0.9126 0.9751 0.9484 0.9382 0.9749 (SEQ ID No. 404) TTVNA[L][G]NLP 0.0194 K (SEQ ID No. 403) 283_HBV- RSQSPRRRRSQ 0.9772 0.9786 0.9777 0.9828 0.9565 0.9779 C-195v2 (SEQ ID No. 587) 13-Aug- 29-Jun- 11-Aug- 23-Mar- 5-Feb- 13-Dec- 24-Mar- — 1991 1992 1993 1994 1998 2000 2004 S2 STNRQSGRQ 003_HBV- 0.9624 0.9794 0.9659 0.9804 0.9658 0.9858 0.9858 S-95* (SEQ ID No. 586) 056_HBV- VVNHYFQTR 0.9680 0.9815 0.9685 0.9811 0.5004 0.9811 P-136* (SEQ ID No. 129) VV[H]HYFQTR 0.3335 0.3654 (SEQ ID No. 134) VV[D]HYFQTR 0.1079 0.5556 (SEQ ID No. 135) VV[S]HYFQTR 0.0247 (SEQ ID No. 590) [l]VNHYFQTR 0.0617 (SEQ ID No. 131) 090_HBV- KAAYSLISTSK 0.9474 0.9713 0.9477 0.9727 0.9516 0.8700 0.8100 P-282* (SEQ ID No. 206) [E][T]AYS[H][L][T] 0.1082 TSK (SEQ ID No. 591) [E]AAYS[F]ISTS[E] 0.1900 (SEQ ID No. 592) 106_HBV- LVVDFSQFSR 0.9613 0.9816 0.9625 0.9822 0.9784 0.9800 0.9999 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9573 0.9827 0.9619 0.9839 0.9801 0.8944 0.9700 P-486* (SEQ ID No. 285) Y[V]SL[L]LLYK 0.0900 (SEQ ID No. 284) 178_HBV- STLPETTWRR 0.9640 0.9822 0.9647 0.9821 0.9703 0.3558 0.7700 C-169* (SEQ ID No. 23) STLPETTVVR[C] 0.0879 (SEQ ID No. 593) STLPET[A]WRR 0.5395 0.1500 (SEQ ID No. 21) STLPETTV[I]RR 0.0800 (SEQ ID No. 24) 179_HBV- TVVRRRGRSPR 0.9553 0.9704 0.8969 0.9068 0.5168 0.0873 0.7920 C-175* (SEQ ID No. 395) TVVRRR[C]RSPR 0.0654 0.0698 0.4472 0.2718 (SEQ ID No. 589) TWR[C]RGRS[T] 0.1021 R (SEQ ID No. 594) [A]WRRR[C]RSP 0.5225 0.2000 R (SEQ ID No. 394) TV[I]RRRGR[T]P 0.0080 R (SEQ ID No. 595) 183_HBV- TTVNAHRNLPK 0.0547 0.0327 0.0249 0.0198 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.8972 0.9429 0.9310 0.9712 0.9456 0.9041 0.1600 (SEQ ID No. 404) TTVNA[R][Q][V]L 0.0764 PK (SEQ ID No. 408) 283_HBV- RSQSPRRRRSQ 0.9589 0.9790 0.9639 0.9788 0.9604 0.9809 0.9809 C-195v2 (SEQ ID No. 587) 25-Aug- 29-Jun- 30-Aug- — 1992 1993 1994 S3 003_HBV- STNRQSGRQ 0.9864 0.9428 0.9839 S-95* (SEQ ID No. 586) S[N]NRQSGRQ 0.0231 (SEQ ID No. 596) 056_HBV- WNHYFQTR 0.6202 0.8098 0.9752 P-136* (SEQ ID No. 129) W[D]HYFQTR 0.3160 (SEQ ID No. 135) VV[H]HYFQTR 0.0527 0.1735 0.0114 (SEQ ID No. 134) 090_HBV- KAAYSLISTSK 0.9040 0.9318 0.9785 P-282* (SEQ ID No. 206) KAA[N]SLISTSK 0.0744 (SEQ ID No. 597) KAAYSLISTS[T] 0.0102 (SEQ ID No. 598) KAAYSL[N]STSK 0.0147 (SEQ ID No. 207) KAAYS[R]ISTSK (SEQ ID No. 599) 106_HBV- LVVDFSQFSR 0.9833 0.9820 0.9800 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.8413 0.9841 0.9812 P-486* (SEQ ID No. 285) Y[D]SL[I]LLYK 0.1217 (SEQ ID No. 600) Y[D]SLMLLYK 0.0227 (SEQ ID No. 601) 178_HBV- STLPETTWRR 0.9837 0.9830 0.9794 C-169* (SEQ ID No. 23) 179_HBV- TVVRRRGRSPR 0.9807 0.9806 0.9773 C-175* (SEQ ID No. 395) 183_HBV- TTVNAH[G]NLPK 0.9138 0.7518 0.9617 X-80* (SEQ ID No. 404) TTVN[T]H[G]NLP 0.0664 K (SEQ ID No. 602) TTVNAH[W]NLPK 0.0242 0.0127 (SEQ ID No. 402) TTVNA[P][G]NLP 0.1872 K (SEQ ID No. 405) 283_HBV- RSQSPRRRRSQ 0.9838 0.6616 0.9712 C-195v2 (SEQ ID No. 587) R[T]QSPRRRRS 0.2879 0.0118 Q (SEQ ID No. 603) RSQSPRRRRS[K] 0.0244 (SEQ ID No. 506) 4-Jan- 16-Feb- 20-Jan- 11-Jan- — 1992 1993 1994 1995 HBeAg- S4 003_HBV- STNRQSGRQ 0.9635 0.9588 0.9654 0.9790 seroconverters S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.9632 0.9600 0.9645 0.9799 P-136* (SEQ ID No. 129) 090_HBV- KAAYSLISTSK 0.9413 0.9273 0.9454 0.9252 P-282* (SEQ ID No. 206) KAAYS[R]ISTSK 0.0301 (SEQ ID No. 599) 106_HBV- LVVDFSQFSR 0.9578 0.9587 0.9555 0.9819 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9539 0.9472 0.9530 0.9815 P-486* (SEQ ID No. 285) 178_HBV- STLPETTWRR 0.9614 0.9616 0.9596 0.9805 C-169* (SEQ ID No. 23) 179_HBV- TWRRRGRSPR 0.9585 0.9569 0.9545 0.9763 C-175* (SEQ ID No. 395) 183_HBV- TTVNAHRNLPK 0.9395 0.9425 0.9397 0.8980 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.0671 (SEQ ID No. 404) 283_HBV- RSQSPRRRRSQ 0.9607 0.9588 0.9589 0.9794 C-195v2 (SEQ ID No. 587) 14-Mar- 3-Apr- — 1991 1993 S5 003_HBV- STNRQSGRQ 0.9813 0.9594 S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.8592 0.9603 P-136* (SEQ ID No. 129) W[D]HYFQTR 0.0417 (SEQ ID No. 135) W[H]HYFQTR 0.0783 (SEQ ID No. 134) 090_HBV- KAAYSLISTSK 0.9749 0.9080 P-282* (SEQ ID No. 206) KAAYSL[N]STSK 0.0367 (SEQ ID No. 207) 106_HBV- LWDFSQFSR 0.9823 0.9586 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9822 0.9560 P-486* (SEQ ID No. 285) 178_HBV- STLPETTVVRR 0.9833 0.9611 C-169* (SEQ ID No. 23) 179_HBV- TVVRRRGRSPR 0.9463 0.9413 C-175* (SEQ ID No. 395) TVVRRRGRS[T]R 0.0316 0.0101 (SEQ ID No. 604) 183_HBV- TTVNAHRNLPK 0.0289 0.0181 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.9308 0.6834 (SEQ ID No. 404) TTVNAH[WJNLPK 0.2053 (SEQ ID No. 402) TTVNAH[E]NLPK 0.0360 (SEQ ID No. 605) 283_HBV- RSQSPRRRRSQ 0.9755 0.9578 C-195v2 (SEQ ID No. 587) 28-Oct- 9-Dec- 4-Aug- 23-Feb- 6-Jun- 10-Apr- — 1991 1991 1992 1994 1996 2001 S6 003_HBV- STNRQSGRQ 0.9678 0.9796 0.9793 0.9792 0.9799 0.9825 S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.9617 0.9836 0.9848 0.9871 0.9775 0.9770 P-136* (SEQ ID No. 129) WNHY[L]QTR 0.0111 (SEQ ID No. 606) 090_HBV- KAAYSLISTSK 0.9547 0.9627 0.9565 0.9639 0.9594 0.7289 P-282* (SEQ ID No. 206) KA[S]YSLISTSK 0.0117 0.0110 0.1834 (SEQ ID No. 607) KAA[N]SLISTSK 0.0537 (SEQ ID No. 597) 106_HBV- LVVDFSQFSR 0.9810 0.9826 0.9819 0.9819 0.9818 0.9794 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9848 0.9825 0.9838 0.9719 0.9811 0.9770 P-486* (SEQ ID No. 285) 178_HBV- STLPETTVVRR 0.9830 0.9806 0.9832 0.9827 0.9812 0.9661 C-169* (SEQ ID No. 23) STLPETTV[I]RR 0.0170 (SEQ ID No. 24) 179_HBV- TVVRRRGRSPR 0.9794 0.9784 0.9779 0.9807 0.9789 0.9627 C-175* (SEQ ID No. 395) TV[I]RRRGRSPR 0.0162 (SEQ ID No. 396) 183_HBV- TTVNAHRNLPK 0.0158 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.9807 0.9753 0.9780 0.9594 0.9716 0.0872 (SEQ ID No. 404) TTVNAH[W]NLPK 0.8652 (SEQ ID No. 402) 283_HBV- RSQSPRRRRSQ 0.9835 0.9783 0.9760 0.9816 0.9113 0.2492 C-195v2 (SEQ ID No. 587) R[T]QSPRRRRS 0.0658 0.7290 Q (SEQ ID No. 603) 19-Sep- 29-Oct- 15-Sep- 19-Oct- — 1990 1991 1992 1993 S7 003_HBV- STNRQSGRQ 0.9645 0.9831 0.9841 0.9822 S-95* (SEQ ID No. 586) 056_HBV- WNHYFQTR 0.7695 0.6757 0.9503 0.9840 P-136* (SEQ ID No. 129) W[D]HYFQTR 0.0699 0.2116 0.0345 (SEQ ID No. 135) VV[H]HYFQTR 0.1436 0.1007 (SEQ ID No. 13r) 090_HBV- KAAYSLISTSK 0.9152 0.8284 0.9782 0.9792 P-282* (SEQ ID No. 206) KAA[N]SLISTSK 0.0365 0.1220 (SEQ ID No. 597) KAAYS[I]ISTSK 0.0223 (SEQ ID No. 608) 106_HBV- LVVDFSQFSR 0.9817 0.9826 0.9819 0.9850 P-387 (SEQ ID No. 28) 125_HBV- Y[V]SLMLLYK 0.9791 0.9414 0.9605 0.9848 P-486* (SEQ ID No. 285) Y[D]SLMLLYK 0.0441 0.0233 (SEQ ID No. 609) 178_HBV- STLPETTWRR 0.9798 0.9568 0.9832 0.9846 C-169* (SEQ ID No. 23) STLPE[I]TWRR 0.0270 (SEQ ID No. 610) 179_HBV- TWRRRGRSPR 0.9277 0.9381 0.9786 0.9808 C-175* (SEQ ID No. 395) TVVRRR[C]RSPR 0.0468 0.0406 (SEQ ID No. 589) 183_HBV- TTVNAHRNLPK 0.8787 0.9438 X-80* (SEQ ID No. 407) TTVNAH[G]NLPK 0.7742 0.8143 0.0994 0.0308 (SEQ ID No. 404) TTVN[T]H[G]NLP 0.1802 0.1366 K (SEQ ID No. 602) TTVNA[L][G]NLP 0.0214 K (SEQ ID No. 403) 283_HBV- RSQSPRRRRSQ 0.9733 0.9409 0.9814 0.9799 C-195v2 (SEQ ID No. 587) RSQSPRRRRS[K] 0.0406 (SEQ ID No. 506)
[0203] Comprised of seven peptide-sequence variants (
[0204] Lastly, 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 compute the trajectory of HBV.sub.core169-specific CD8.sup.+ T cells across three clinical stages using the patient-wise expression of eight phenotypic markers that showed statistical significances (
[0205] 3. Multifactorial Interrelations of Inhibitory Receptors on Virus-Specific T Cells
[0206] Next, the relationships between various categories of cellular markers expressed by each of the antigen-specific T cells analyzed, with a special focus on nine different inhibitory receptors was evaluated. To directly assess these relationships, One-Dimensional Soli-Expression by Nonlinear Stochastic Embedding (One-SENSE) was employed. One-SENSE works by reducing dimensionality of each category of markers into a one-dimensional t-SNE map that can be plotted in conjunction with alternative categories of markers mapped into additional one-dimensional t-SNE maps. In this way, cells are separately arranged based on their categorical expression and then relationships between the categories can be intuitively visualized and described.
[0207] In this case, the categories assigned (table S2) were: “Differentiation+TNFR” (markers of differentiation and tumor necrosis factor receptor superfamily), “Inhibitory” (inhibitory receptors) and “Trafficking” (chemokine receptors). The three derived axes called out the cellular subsets objectively with all possible protein co-expressions (
[0208] By plotting the cells based on the patient groups, it was noted that the phenotypes of HBV.sub.pol387 and HBV.sub.core169-specific CD8.sup.+ T cells (blue and red) were most heavily influenced by status of infection the distinguishing features of these highly diverse cells are labeled. This heterogeneity can be best presented by HBV.sub.pol387 and HBV.sub.core169-specific CD8.sup.+ T cells. HBV.sub.pol387 from IT had a relatively homogeneous phenotype of in terms of memory-associated and trafficking receptors, but varied in four distinct co-expressions of inhibitory receptors (
[0209] The numbers of co-expressed inhibitory receptors on these cells using a Boolean strategy (
[0210] Although the different co-expressions of inhibitory receptors on HBV.sub.pol387-specific CD8.sup.+ T cells can be shown by One-SENSE, there were no differences in terms of the numbers accumulated on the cells across patient groups. Conversely, IA patients had significant higher numbers of inhibitory receptors on HBV.sub.core169-specific CD8.sup.+ T cells compared to patients with viral control (
[0211] 5. Functional Capacity of HBV-Specific CD8.sup.+ T cells
[0212] To address the relationships of functional capacity and inhibitory receptors, patient's cells were pulsed and expanded using short-term in vitro peptide stimulation and then assessed their functional responses using intracellular cytokine staining (
[0213] Regardless of patient groups, a multi-functional subset was present (
[0214] Together with the abovementioned data, it was concluded that the immune responses of HBV.sub.core169-specific CD8.sup.+ T cells were linked to viral control. The analysis also displays complex orchestrations rather than a simple linear relationship between inhibitory receptors and functional capacity on virus-specific CD8.sup.+ T cells during CHB.
[0215] 6. Clinical Stage-Dependent Landscapes of Virus-Specific TCR
[0216] How T cell receptors are selected over the course of CHB is largely unknown. Hence, the pMHC tetramer-stained cells (
[0217] Repertoire diversity and density for each TCR sequence from various cell populations using a TCR diversity metric (TCRdiv) was calculated (P. Dash et al., Quantifiable predictive features define epitope-specific T cell receptor repertoires). Various patterns of this measure were observed among the epitope and patient groups (
[0218] 7. Shared HBV.sub.core169-Specific TCR Clones in Patients with Viral Control
[0219] Focusing on the HBV.sub.core169-specific response and inquiring a recently curated TCR database, several previously unidentified public HBV.sub.core169-specific TCRβ clones that were shared between individuals in a clinical stage-dependent manner were discovered (
[0220] 8. Phenotypic Dynamic of HBV.sub.core169-specific CD8.sup.+ T Cells
[0221] To assess the characteristic changes of antigen-specific T cells over the course of infection, selected HBV epitopes in a (n=14, HLA-A*1101+ patients) longitudinally studied patient cohort who received Entecavir (ETV) over the course of several years were examined. ETV is a nucleotide analogue 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 lead to HBeAg seroconversion and established anti-HBeAg antibody (HBeAb) in some patients, and this is a serological marker of further improved viral suppression. For 10 out of 14 patients who had detectable HBV.sub.core169-specific CD8.sup.+ T cells (
[0222] In addition to summarizing the composition of these cells in terms of their memory vs. effector phenotypes over time (
Discussion
[0223] By fully leveraging a highly multiplexed combinatorial pMHC tetramer staining strategy, mass cytometry and unsupervised high-dimensional analyses, we investigated 562 unique A*11:01-restricted candidate epitopes during the progression of HBV. Analysis of HBV-specific T cell responses is difficult due to the very low frequencies of these cells. In this regard, we show the importance of investigating both the specificity and phenotypic profiles of the antigen-specific T cells to verify their involvement in the HBV-specific immune response. Beyond this, our data highlights the heterogeneity of the virus-specific T cell response that was associated with disease stages and provides quantifiable analyses of HBV-specific TCRβ repertoires that corresponded to cellular phenotypes during chronic viral infection.
[0224] Host defense against HBV weighs on immune response driven largely by virus-specific T cells. The number of A*11:01-restricted epitopes detected by this comprehensive approach were relatively limited compared to the reported epitopes in the context of A*02:01. It is possible that some epitope-specific T cells were only detectable in the liver but not in the periphery. Future investigation on HBV-specific intrahepatic lymphocytes is needed. The presence and frequencies of well-described A*02:01-restricted HBV.sub.core18-27-specific CD8.sup.+ T cells have been shown to associate with viral control. Many therapeutics have been therefore developed based on this T cells, including the blockade of overexpressed PD-1 to reinstate T cell function, adoptive transfer of engineering virus-specific T cells, and TCR-like (TCR-L) antibody to deliver interferon-a (IFN-α) directly onto infected hepatocytes. Here, evidence was presented that showed that the specific responses and characteristics of A*11:01-restricted HBV.sub.core169-specific CD8.sup.+ T cells were linked to viral control, with public TCR clones used by these T cells. Comparative analysis showed that these cells had differing profiles across clinical stages. Furthermore, high-dimensional trajectory analysis allowed the use of the profiles of HBV.sub.core169-specific CD8.sup.+ T cells to assign each patient a value along an objective pseudotime metric. The underlying features of HBV.sub.169-specific T cells along this trajectory were consistent across two independent patient cohorts, both showing correlations with viral control. Based on this metric, it is also conceivable that these antiviral-treated patients and possessing T cells with the most-progressed features of viral control could be eligible for safe discontinuation of antiviral drug once they mounted HBV.sub.core169-specific memory T cells response, in line with a recent report showing the predictive utility of HBV-specific T cells (63). This is important because even the most advanced serological measures are unable to accurately predict such outcomes. Collectively, our findings should impact HBV immunotherapy design, and could be useful to predict patient's clinical outcome based on the phenotypic response of HBV.sub.core169-specific CD8.sup.+ T cells. It is also anticipated that the utility of this approach could be extended of other epitopes associated with viral control derived from HBV core or other proteins that are restricted to other HLA alleles.
[0225] By comprehensively probing HBV epitopes on numerous HBV-infected patients, the inventors here failed to identify T.sub.EX expressing all inhibitory receptors or overt evidence for “Hierarchical T cell exhaustion”. Instead, unsupervised visualization using One-SENSE showed complex non-linear relationships between the expression of inhibitory receptors. Moreover, the dysfunctionality of HBV-specific T cells did not correlate to the linear accumulation of inhibitory receptors, indicating these cells were not completely functional inert. One interpretation is that these HBV-specific T cells do not nicely fit the definition of TEx as reported in LCMV-specific T cells, but instead a different type of subset that were mostly absent, with the remaining dysfunctional T cells expressing various combinations of inhibitory receptors. Based on present data, it is proposed that these T cell profiles, at least in the peripheral blood, could fit better with the description of functional adaption in CHB. Nonetheless, it is noteworthy that our functional assessment was relied on in vitro peptide stimulation due to the rare detection of HBV-specific T cells, and this might limit its relevance to in vivo response. Unlike during chronic LCMV infection, where maintenance of the TEx phenotype requires persistent and high antigen level, HBV.sub.core169-specific CD8.sup.+ T cells expressing high level of CD27 and IL-7Rα (CD127) found in InA patients were not TEx and are likely to be maintained for decades with a limited amount of viral antigen present. HBV-specific T cells from these patients were PD-1.sup.intTIGIT.sup.int with elevated expression of memory-associated markers and functional capacities that seem to be associated with control of the virus. On the other hand, for IA patients who have high and fluctuating viremia, HBV-specific T cells were detected that better matched the expected features of T.sub.EX. These included a strong co-expression of PD-1 and TIGIT and limited expression of memory-associated markers such as CD127. Additionally, the inventors also found substantial expression of CD57 on HBV-specific T cells from IA patients, a cellular marker that is indicative of more differentiated effector cells with low proliferative capacity, suggesting these cells were not long-lived memory cells. Overall, cellular profiles of HBV-specific T cells in IA stage are consistent with persistent high antigen-exposure. At later stages (InA) in patients with better viral control, HBV-specific T cells had a memory phenotype expressing lower CD57 but elevated CXCR3, CD45RO, CD27 and CD127. The lack of the detectable HBV.sub.core169-specific CD8.sup.+ T cells in IT patients who has high viremia suggests that they might be largely deleted, and the absence of such particular T cells could contribute to the minimum liver inflammation in this stage. Future studies involve larger volume of blood samples from IT patients or using more sensitive approaches may help to address this issue. Further investigation of the expression level of EOMES and T-bet, or the epigenetic modification that better defined the bona fide TEX is important to address this aspect in CHB. It is also important to note that this report is limited to the analysis of circulating HBV-specific CD8.sup.+ T cells, further examination of the exhaustion profile in intrahepatic lymphocytes is needed to address this question.
[0226] Previous studies have suggested the link between the different inhibitory receptors and T cell differentiation, which is in relative disagreement with the present One-SENSE analysis objectively showing more complex relationships between the 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 in human chronic viral infection often examined less than four inhibitory receptors within T cell subsets that were simply defined by few differentiation-associated markers, or on limited numbers of virus-specific T cells from one type of patient. Secondly, traditional analysis using hierarchical gating on biaxial dot plots to assess the expression level of cellular protein can easily underestimate the phenotypic complexity.
[0227] Despite the TCR sequence diversity of T cells specific for the HBV.sub.core169 epitope, several public clones at relatively high abundance were detected in multiple patients. In line with the CMV-specific TCR repertoire, it was noted that these previously unidentified public TCRs were different when derived from patients showing viral control (R and InA) vs. viremia (IA), suggesting the functional importance of these T cells. The public virus-specific TCR clones may be selected over the course of viral clearance (i.e. from IA into InA) and contraction of the effector response. As previously reported for CMV and EBV infection, virus-specific CD8.sup.+ T cells do not express IL-7Rα (CD127) until T cell memory had been established, and such selection is thought to be driven by high affinity TCR-viral epitope binding. This is consistent with the characteristics of HBV.sub.core169-specific CD8.sup.+ T cells in InA and R patients who carried public TCRs and elevated expression of T cell memory-associated markers including CD127, indicating their long-lived and self-renewing ability to maintain memory T cells pool after the reduction of viral antigen. Analogously, intrahepatic and peripheral public TCR clones have been linked to viral clearance in HCV-infected chimpanzees.
[0228] Despite the challenges highlighted associated with detecting HBV-specific T cells due to their low prevalence, the present invention explores the previously unappreciated complexity of virus-specific T cells in lifelong human HBV viral infection. The cellular responses of HBV.sub.core169-specific CD8.sup.+ T cells and TCR sequences used were associated with the status of
[0229] HBV infection and could be used as an indicator of the relative extent of viral control. Thus, the results provided here could have important implications for the development of new biomarkers, treatment strategies and immunotherapy aiming at HBV cure.
[0230] Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.