Mass spectrometric identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID-19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally if not more important than humoral responses in mediating recovery and immune protection. One major challenge in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities. Our studies find that, in contrast to current convention, "immunodominant" SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from the naturally processed peptide-major histocompatibility complex (MHC) and then validating immunogenicity by determining whether such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes derived from not only structural but also nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, there are no reported T cell receptor-engineered T cell technology that can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here several SARS-CoV-2 epitopes defined by mass spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity, and demonstrate engineered TCR-redirected killing.

Identification of Novel Candidate CD8+ T Cell Epitopes of the SARS-CoV2 with Homology to Other Seasonal Coronaviruses.

Cross-reactive T cell immunity to seasonal coronaviruses (HCoVs) may lead to immunopathology or protection during SARS-CoV2 infection. To understand the influence of cross-reactive T cell responses, we used IEDB (Immune epitope database) and NetMHCpan (ver. 4.1) to identify candidate CD8+ T cell epitopes, restricted through HLA-A and B alleles. Conservation analysis was carried out for these epitopes with HCoVs, OC43, HKU1, and NL63. 12/18 the candidate CD8+ T cell epitopes (binding score of ≥0.90), which had a high degree of homology (>75%) with the other three HCoVs were within the NSP12 and NSP13 proteins. They were predicted to be restricted through HLA-A*2402, HLA-A*201, HLA-A*206, and HLA-B alleles B*3501. Thirty-one candidate CD8+ T cell epitopes that were specific to SARS-CoV2 virus (<25% homology with other HCoVs) were predominantly identified within the structural proteins (spike, envelop, membrane, and nucleocapsid) and the NSP1, NSP2, and NSP3. They were predominantly restricted through HLA-B*3501 (6/31), HLA-B*4001 (6/31), HLA-B*4403 (7/31), and HLA-A*2402 (8/31). It would be crucial to understand T cell responses that associate with protection, and the differences in the functionality and phenotype of epitope specific T cell responses, presented through different HLA alleles common in different geographical groups, to understand disease pathogenesis.

Multiplex Screening Assay for Identifying Cytotoxic CD8+ T Cell Epitopes.

The cytotoxicity of epitope-specific CD8+ T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8+ T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8+ T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8+ T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8+ T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8+ T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8+ T cell cytotoxicity in a practical manner.

Identification B and T-Cell epitopes and functional exposed amino acids of S protein as a potential vaccine candidate against SARS-CoV-2/COVID-19.

Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus that it disease spreads in over the world. Coronaviruses are single-stranded, positive-sense RNA viruses with a genome of approximately 30 KD, the largest genome among RNA viruses. Most people infected with the COVID-19 virus will experience mild to moderate respiratory illness and recover without requiring special treatment. Older people and those with underlying medical problems like cardiovascular disease, diabetes, chronic respiratory disease, and cancer are more likely to develop serious illness. At this time, there are no specific vaccines or treatments for COVID-19. So, there is an emergency need for vaccines and antiviral strategies. The spike protein is the major surface protein that it uses to bind to a receptor of another protein that acts as a doorway into a human cell. The putative antigenic epitopes may prove effective as novel vaccines for eradication and combating of COV19 infection. A combination of available bioinformatics tools are used to synthesis of such peptides that are important for the development of a vaccine. In conclusion, amino acids 250-800 were selected as effective B cell epitopes, T cell epitopes, and functional exposed amino acids in order to a recombinant vaccine against coronavirus.