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Identification of novel HLA class I-restricted hepatitis B virus peptides and their modulation by peptide editor TAPBPR

ABSTRACT: Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma and infections remain a substantial global health problem for which no curative therapy exists. As cytotoxic CD8+ T lymphocytes are the principal effectors of natural HBV resolution, there is considerable interest in discovering novel HBV epitopes that are presented for immune recognition. Current knowledge of HBV-derived peptide presentation to CD8+ T cells is largely dominated human leukocyte antigen (HLA)-A*02:01. Our aim was to characterise novel HBV-derived peptides presented on HLA-B and -C molecules, particularly allotypes that are prevalent in regions with high HBV infection rates. Additionally, we sought to determine if the recently discovered major histocompatibility complex class I (MHC-I) peptide editor TAPBPR modulated expression of HBV-derived epitopes. To discover new HBV-derived epitopes, immunopeptidomic analysis was performed on cells expressing single HLA class I molecules transduced to express individual HBV proteins. Peptide binding assays were used to validate HLA binding of selected peptides. To determine the effect of TAPBPR on the HBV peptidome, immunopeptidomic analysis was performed on either wild-type HeLa cells expressing HBV proteins or their TAPBPRKO counterpart. The results were validated using TAPBPR peptide-exchange assays. We identified 28 novel HBV-derived peptides presented on HLA class I. Importantly, 22 of the newly discovered HBV-derived peptides were identified on cells expressing HLA-B*08:01, -B*15:03, -B*35:01, -C*06:02 and -C*12:03. Additionally, TAPBPRKO HeLa cells presented four additional and novel HBV-derived peptides. This work presents a substantial expansion of the HLA-B and HLA-C-restricted HBV-derived peptides described in the Immune Epitope Database (IEDB). Additionally, this work represents the first indication that TAPBPR shapes not only the cellular but also the viral peptide repertoire presented on MHC-I molecules.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Jens Bauer

LAB HEAD: Juliane Sarah Walz

PROVIDER: PXD078020 | Pride | 2026-07-15

REPOSITORIES: Pride

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			Action	DRS
	211004_JB_HeLa_A2_01_W6-32_Coprocessed_3Reps.msf	Msf
	211004_JB_HeLa_A2_01_W6-32_DDA_400-650MZ_QIS_CIDOT_Rep1.raw	Raw
	211004_JB_HeLa_A2_01_W6-32_DDA_400-650MZ_QIS_CIDOT_Rep2.raw	Raw
	211004_JB_HeLa_A2_01_W6-32_DDA_400-650MZ_QIS_CIDOT_Rep3.raw	Raw
	211004_JB_HeLa_TBP_KO_W6-32_CoProcessed_5Reps.msf	Msf

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Project description:The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S194-202) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C141-151) from the capsid protein were validated by our discovery platform, but both were detected at a very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P606-616) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.

Project description:The majority of peptides presented by MHC class I results from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides for MHC class I loading non-canonically. We used an im-munopeptidomics workflow combined with a prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect proteasomal processing of immune epitopes presented by A549 cells. Treatment of A549 cells with IFNγ enhanced proteasomal cleavage probability of MHC class I ligands for both, the constitutive proteasome and the im-munoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated HLA allotype-specific proteasomal cleavage probabili-ties for MHC class I ligands, peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. Peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases. The mass spectrometry raw files used in this study were originally produced for a previous publication, where they were utilized to address the influence of cigarette smoke on the antiviral T-cell immune response. Here, we have reanalyzed the data to investigate processing of MHC class I ligands by c20S or i20S, respectively. Original publication: Chen, J.; Wang, X.; Schmalen, A.; Haines, S.; Wolff, M.; Ma, H.; Zhang, H.; Stoleriu, M.G.; Nowak, J.; Nakayama, M.; et al. Antiviral CD8(+) T cell immune responses are impaired by cigarette smoke and in COPD. Eur Respir J 2023, doi:10.1183/13993003.01374-2022.

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Identification of novel HLA class I-restricted hepatitis B virus peptides and their modulation by peptide editor TAPBPR

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