Discovery of novel HLA-B*57:01 restricted T-cell antigens through genome-wide screening of Epstein-Barr virus

Anuradha Sooda

A relationship between latent viral infections and drug hypersensitivity reactions (HSR) has been observed in several clinical situations. Abacavir hypersensitivity has been shown by several groups to be an HLA-B*57:01 restricted CD8+ T cell dependent process. Although compelling evidence suggests that carriage of HLA-B*57:01 is necessary, it is not sufficient for abacavir hypersensitivity. Based on previous structural and functional studies, it was hypothesised that heterologous immune responses of the pre-existing anti-viral T cell responses mediate abacavir hypersensitivity. Human herpesviruses (HHV) have been posited to be associated with the development of HSR because they have co-evolved with the human major histocompatibility complex and they persist in most people as lifelong latent, asymptomatic infections. In addition, they are reactivated in immunocompromised individuals and have been implicated in drug-viral interactions such as associated with Epstein-Barr virus (EBV). EBV is the most common HHV infecting more than 95% of the world’s population. The EBV genome is approximately 172 kb in length and encodes 89 proteins. However, the extent to which these proteins are surveyed by host-derived T cells has not been fully explored. The objective of this body of work was to generate ORFeome-wide map of memory CD8+ T cell responses for the identification of HLA-B*57:01 restricted antigens which could be involved in the cross-reactivity of abacavir drug hypersensitivity. Mapping revealed 14 novel HLA-B*57:01 restricted EBV antigens and confirmed the previously identified epitopes EBNA3B (VSFIEFVGW), EBNA2 (LASAMRML), and LMP1 (IALYLQQNW). A novel immunodominant epitope was detected in EBNA3C (QSRGDENRGW). To identify cross-reactive anti-viral T cell receptors, single cells specific for the immunodominant epitopes of EBNA3B (VSF) and EBNA3C (QSR) were sorted based on tetramer staining. Antigen-specific T cells were sequenced for the T cell receptors cloned into Jurkat cells. A modified heterologous T cell receptor reporter assay was developed to study the T cell responses against HLA-B*57:01 restricted EBV immunodominant epitopes. Dose-dependent T cell responses were detected against each EBV epitope, however, the TCRs were not cross-reactive to abacavir plus self-peptide presented in the context of HLA-B*57:01. The presence of abacavir was shown to modulate the EBV-specific TCR responses to the two EBV epitopes, which suggested the non-covalent binding of abacavir to HLA-B*57:01 may have down modulated the TCR responses. Although unable to demonstrate cross-reactivity of the EBV-specific TCRs with the abacavir plus self-peptide, this work has provided a road map for future experiments to elucidate the role of heterologous immunity in the immunopathogenesis of drug hypersensitivity and other aberrant acquired immune responses in humans.

Discovery of novel HLA-B*57:01 restricted T-cell antigens through genome-wide screening of Epstein-Barr virus

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