Detection and sequencing of SARS-CoV-2 derived HLA-I bound antigenic peptides in the blood of COVID-19 patients: mapping the CD8 T cell response.

The immune response to SARS-CoV-2 will comprise both an antibody response (which it is obviously hoped will provide both immediate protection and also prolonged memory) but also crucially a killer T cell response. These cells can seek out and kill virus infected cells, whilst leaving nearby uninfected cells unharmed. Thus they remove the seat of the infection, which antibodies cannot do, and prevent the release of new virus particles. These killer T cells work by recognising small fragments of viral proteins presented to them on immune molecules called HLA class I molecules (HLA-I). Understanding which bits of the virus are being presented by HLA-I to the killer T cells will help inform upon future vaccine design, by incorporating the most targeted bits of the virus into the prospective vaccine. At present this information is not known.

We have developed a new method to identify these viral fragments using simple to access blood samples from patients, thus vastly improving on traditional methods which would require a solid lung tissue biopsy. Blood samples contain HLA-I molecules in both a soluble form and also expressed on small vesicles, released from cells in the body, including virus infected cells. Both of these are frequently raised in diseased states where inflammation is present. We will use an antibody that recognises HLA-I molecules to isolate them from patient blood (after first safely inactivating any virus in the blood samples). The small virus fragments are then released from the HLA-I molecules and analysed by a highly sensitive technique called mass spectrometry, which allows us to determine the protein sequence of the virus fragment.

Using this technique we can build a picture of which bits of the virus are being seen by these key killer T cells of the immune system. Also, because there are many thousands of different HLA-I molecules present in the human population, our system allows us to identify data which is relevant across a wide spectrum of the population. Our data will allow future vaccine design to incorporate the most relevant bits of the SARS-CoV-2 virus to promote optimal immune responses with, hopefully, long term protection being generated.

The role that CD8 cytotoxic T cells play in the clearance of viral infections cells is well established, and depends upon the recognition of short (typically 9-11 mer) antigenic viral peptide epitopes presented on HLA class I molecules (HLA-I). The design of effective future vaccines, that can induce both classical memory B cell antibody, and CD8 T cell responses therefore requires that we identify, map and include the most antigenic epitopes being presented to CD8 T cells. In this study we will take advantage of the presence in serum and plasma of both soluble HLA-I and extracellular vesicles (EV) expressing HLA-I as a source of the cellular immunopeptidome. Based on our existing recent studies that both cancer cell line derived and patient plasma EV HLA-I contain tumour associated antigenic peptides, we hypothesise that COVID-19 induced inflammation in the lungs will release excess soluble and EV HLA-I into plasma which we can utilise as a simple to access liquid biopsy, to immunoisolate the HLA-I, extract the antigenic peptides and de novo sequence by mass spectrometry. By comparison to the known viral proteome we will rapidly identify viral epitopes across a wide range of HLA-I alleles present in the population. We aim to rapidly process up to 200 patients samples to build a comprehensive map of real viral antigenic epitopes of use for future intelligent vaccine design.