O02 Discovery of Novel HLA Class I Presented Hepatitis B Peptides Using an Immunopeptidomics Approach

Background and Aims An estimated 250 million people worldwide are infected with chronic hepatitis B (HBV), the leading cause of hepatocellular carcinoma (HCC) worldwide. The display of HBV derived peptides on HLA class I molecules is crucial for immune cell mediated responses and control of HBV. Most HBV peptides that have previously been characterised are restricted to HLA alleles not representative of HBV high prevalence regions and were identified by computational methods which do not consider the antigen presentation pathway. Methods We used a mass spectrometry-based approach (immunopeptidomics) to discover novel HBV peptides presented on a diverse set of HLA allotypes. Cell lines expressing single HLA class I of choice (HLA-A*02:01, -B*08:01, -B*35:01 and Cw06:02) and individual HBV proteins, HBsAg, Pol, HBcAg and HBx were generated and expanded to 108 cells. Cells were lysed, peptides eluted using a pan HLA class I antibody (W6/32) and then analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Mass spectra data were then searched against a database of possible peptides that could be derived from the host and/or HBV proteomes, to identify HBV derived peptides presented on HLA class I. We then performed exogenous peptide loading on cells expressing single HLA class I using a selection of fluorescently- labelled HLA class I binding HBV peptides discovered using the approach above. Results A total of 55 unique HBV-derived peptides were discovered, 38 (69.09%) of which were novel peptides. Importantly, binding prediction using netMHCpan4.1 revealed that 32 peptides (58.18%) were predicted to be strong binders, whereas 23 peptides (41.82%) were predicted to be weak binders. We identified numerous novel HBV-derived antigens presented on HLA-B*08:01, -B*35:01 or Cw06:02. Twenty-three peptides were derived from HBsAg, 29 peptides were derived from Pol and three peptides were derived from HBcAg. No HBx derived peptides were identified. Subsequent testing of fluorescent-derivatives of a small panel of the HBV-derived antigens confirmed their ability to bind to the specific HLA. Conclusion An immunopeptidomics approach is a promising new technique for HBV peptide discovery, which considers all aspects of the antigen presentation pathway. Furthermore, the majority of these peptides are predicted to be strong binders to HLA class I. The large numbers of HBV peptides presented on HLA -B and -C allotypes that are both novel and predicted to be strong binders is encouraging for therapeutic vaccine design. These peptides have the potential to trigger immune responses from a broad array of immune cell types and across populations with a more diverse range of HLA class I allotypes.