Identification of natural Zika virus peptides presented on the surface of paediatric brain tumour cells by HLA classI

Despite decades of research, survival from brain cancer has scarcely improved and is drastically lower than that of other cancers. Novel therapies, such as immunotherapy, hold great promise for treating brain tumours and are desperately needed. Zika virus (ZIKV) infects and kills aggressive cancer cells with stem-like properties (CSCs) from both paediatric and adult brain tumours. Whilst T cell recruitment into ZIKV-infected brain tumours is becoming well documented, the specific mechanisms through which they are activated are poorly understood. We address this by employing a combined LC-MS/MS global proteome and immunopeptidome approach to describe, for the first time, human leukocyte antigen (HLA) presentation of ZIKV peptides on the surface of infected brain tumour cells. We first show that HLA class I (HLA-I) antigen processing & presentation is the most highly enriched immune response pathway in the global proteome of aggressive paediatric USP7-ATRT brain tumour cells following ZIKV infection. We identify USP7-ATRT cells as a good immunopeptidome model due to their homozygous of the globally most common HLA-A allotype (A*02:01). We predict the majority of the 19 ZIKV peptides that we identify here to strongly bind and be presented by HLA-A*02:01. We show that immunopeptide presentation corresponds with cellular ZIKV protein abundance, with ten peptides arising from the most abundant viral protein; non-structural protein 3 (NS3). Specifically, we show the ZIKV NS3 helicase domain to be a rich source of peptides. Finally, we verify that the identified ZIKV peptides do not mimic predicted peptides of the human proteome. The ZIKV peptides we identify here are potential candidates for developing novel epitope-specific brain tumour immunotherapies, and our findings provide potential insight into the efficacious cytotoxic T cell response that oncolytic ZIKV virotherapy can induce against brain tumours. Author SummaryViruses can attack cancer through two mechanisms: 1) infecting and killing the cancer cell and 2) activating the immune system against the tumour. Zika virus (ZIKV) uses both mechanisms to fight brain cancer. Here, we employ a powerful proteomic technique to identify fragments of viral proteins (peptides) presented by cell surface receptors on brain cancer cells infected with ZIKV. In the human body, immune system cells such as T cells recognise and become activated in response to these viral peptides and subsequently attack the infected patient tumour. We identify 19 ZIKV peptides, three of which have been shown previously to elicit T cell responses, four identified elsewhere, and twelve are novel. Our work helps delineate a component of how ZIKV acts as an immunotherapy, the T cell-specific immune response that the virus raises to promote clearance of brain tumours. The significance of our study is that the ZIKV peptides we identify may lead to the development of a novel brain tumour immunotherapy.