ABSTRACT
T cell immunity plays a pivotal role in the control of SARS-CoV-2 infections. Despite major efforts in quantifying SARSCoV-2-specific T cell responses, the quality of such responses, in particular of CD8+ T cells, has been marginally investigated so far, leaving the importance of CD8+ T cells during COVID-19 and for protective immunity unclear. To assess CD8+ T cell func-tionality, the identification and characterization of SARS-CoV-2- specific T cell receptors (TCRs) is indispensable. To first identify SARS-CoV-2-specific epitopes, we evaluated immunogenicity of candidate epitopes on convalescent donor material as functional and protective responses are supposed to be found. After a short in-vitro expansion and restimulation of SARS-CoV-2-specific CD8+ T cells, we were able to detect response rates ranging from 33 - 100 %. Importantly, SARS-CoV-2-specific CD8+ T cells were detected even one year after infection. For two HLA-restricted epitopes we subsequently identified TCRs by performing single-cell RNA sequencing. By re-expressing SARS-CoV-2 TCRs via CRISPR/Cas9-mediated orthotopic replacement (OTR), we confirmed functional avidity as well as cytotoxicity towards virus-infected cells. By combining experimental data with gene signatures of recent activation, we further defined a ''reactivity signature'' and a ''functionality signature'' to differentiate TCRs with high and low functionality. To test these gene signatures as a means of predicting TCR functionality in silico, we identified TCRs against nine additional immunodominant SARS-CoV-2 epitopes restricted to five different HLA class I molecules and could confirm TCR functionality. Finally, we showed that the SARS-CoV-2 TCR repertoire is highly polyclonal. In summary, our data demonstrate by single cell TCR identification in combination with OTR engineering that CD8+ T cell responses upon mild COVID-19 infection are polyclonal, long-lasting, and highly functional. We furthermore demonstrated for several TCRs highly effective cytotoxicity towards virus-infected cells, which might open options for therapeutic use with adoptive T cell therapy in COVID-19 patients.
ABSTRACT
The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens;however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and beta-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small beta-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The beta-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the beta-annulus peptide for vaccine design.