Identification of targets of CD4 and CD8 T cell reactivity conserved across Sarbecoviruses and recognized across different animal species

PROJECT SUMMARY The overarching goal of Project 2 is to discover and design vaccine antigen candidates that confer broad cross- protective T cell immunity to multiple coronavirus (CoV) species with pandemic potential. Cross-reactivity at the T cell level has been unequivocally demonstrated between SARS-CoV-2, other Sarbecoviruses, and other more distantly related CoV in our previous studies. By incorporating strategies that explicitly elicit cellular immunity into vaccines generated by Project 3, cross-protection against future pandemics emerging from CoV reservoirs may be possible. Specific Aim 1 will define targets of CD4+ and CD8+ T cell reactivity that are broadly conserved across different Sarbecoviruses, Merbecoviruses, and other betacoronaviruses of concern by (i) identifying conserved regions across the entire CoV proteome, including all mature non-structural proteins (nsp) and open reading frames (ORFs) for each of these taxonomic groups, (ii) performing a meta-analysis of immunogenicity data to catalog known T cell epitopes for these antigens to define which antigen subregions are recognized following natural infection by different CoV species, and (iii) integrating these data to select a set of candidate conserved T cell epitope regions from each of the three taxonomic groups for experimental evaluation. This bioinformatic analysis will assign specific quantitative scores, including sequence conservation scores, human immunogenicity scores, and cross-reactivity for human T cells scores to various proteins subregions. Specific Aim 2 will test human T cells directed against epitopes from Aim 1 for their capacity to recognize homologous sequences from different Sarbecoviruses, Merbecoviruses, and other betacoronaviruses of concern by (i) synthesizing the conserved epitope regions defined in Aim 1 from prototype viruses and homologous peptides from each of the different taxonomic groups evaluated in Aim 1, (ii) eliciting T cell lines from COVID-19 convalescent human donors and human subjects with NL63 and OC43-reactive memory T cells using SARS-CoV-2-, NL63-, and OC43-derived epitopes, (iii) testing these epitope-specific T cell lines for cross- reactivity with corresponding homologous peptides from each of the three taxonomic groups using an approach developed in our laboratory to evaluate CoV cross-reactivity, and (iv) performing bioinformatic analyses to predict immunogenicity in our rodent models. Based on this experimental work, we will prioritize potential candidates, and deliver lists of candidates to the overall Program lead in Core A for incorporation into two vaccine platforms in Project 3 and testing for immunogenicity and cross-protection in Core B. This project will produce the first comprehensive evaluation of T cell immunogenicity of the entire CoV proteome across a broad cross- section of CoVs with pandemic potential using a tight integration of bioinformatics prediction and experimental validation to create antigens for vaccine design that elicit broad cross-protective cellular immunity to complement the humoral immunity elicited through Project 1.