ANALYSIS OF EPITOPE-SPECIFIC T CELLS IN SARS-CoV-2 INFECTION AND VACCINATION

Background: T cells play a critical role in the adaptive immune response to SARS-CoV-2 in both infection and vaccination. Identifying T cell epitopes and understanding how T cells recognize these epitopes can help inform future vaccine design and provide insight into T cell recognition of newly emerging variants. Here, we identified SARS-CoV-2 specific T cell epitopes, analyzed epitope-specific T cell repertoires, and characterized the potency and cross-reactivity of T cell clones across different common human coronaviruses (HCoVs). Method(s): SARS-CoV-2-specific T cell epitopes were determined by IFNgamma ELISpot using PBMC from convalescent individuals with mild/moderate disease (n=25 for Spike (S), Nucleocapsid (N) and Membrane (M)), and in vaccinated individuals (n=27 for S). Epitope-specific T cells were isolated based on activation markers following a 6-hour peptide stimulation, and scRNAseq was performed for TCR repertoire analysis. T cell lines were generated by expressing recombinant TCRs in Jurkat cells and activation was measured by CD69 upregulation. Result(s): We identified multiple immunodominant T cell epitopes across S, N and M proteins in convalescent individuals. In vaccinated individuals, we detected many of the same dominant S-specific epitopes at similar frequencies as compared to convalescent individuals. T cell responses to peptide S205 (amino acids 817-831) were observed in 56% and 59% of individuals following infection and vaccination, respectively, while 20% and 19% of individuals responded to S302 (a.a. 1205-1219) following infection and vaccination, respectively. For S205, a CD4+ T cell response, we confirmed 8 unique TCRs and determined the minimal epitope to be a 9mer (IEDLLFNKV). While TCR genes TRAV8-6*01 and TRBV30*01 were commonly utilized across the TCRs, we did identify TCRs with unique immunogenetic properties with different potencies of cross-reactivity to other HCoVs. For S302, a CD8+ T cell response, we identified two unique TCRs with different immunogenetic properties that recognized the same 9mer (YIKWPWYIW) and cross-reacted with different HCoV peptides (Figure 1). Conclusion(s): These data identify immunodominant T cell epitopes following SARS-CoV-2 infection and vaccination and provide a detailed analysis of epitope-specific TCR repertoires. The prospect of developing a vaccine that broadly protects against multiple human coronaviruses is bolstered by the identification of conserved immunodominant SARS-CoV-2 T cell epitopes that cross react with multiple other HCoVs.

Redirection of T cells via chimeric antigen receptor (CAR) to control the invasive fungal infections

Invasive fungal infections (IFI) are associated with high rates of morbidity and mortality, and immunocompromised hosts are often affected. Candida albicans is among the main cause of IFIs in the last decades, and Paracoccidioides brasiliensis is found in most of the IFIs identified in the South America. Rhizopus oryzae causes mucormycosis that increased in the COVID-19 pandemic. Host immune response against IFIs depend of the effector activity of T cells, which is compromised in immunodeficient patients. However, chimeric antigen receptor (CAR) technology can redirect T cells to target any antigen inducing the cell activation, which can be applied in immunocompromised patient as done in cell therapy against cancer. We developed a CAR (M-CAR) specific to a carbohydrate on the fungal cell wall, and Jurkat cells expressing M-CAR after lentiviral transduction using a multiplicity of infection (MOI) of 1, 3, 5 or 10 had its recognition capacity evaluated against C. albicans, P. brasiliensis, and R. oryzae. CAR expression increased in a MOI dependent-manner, and M-CAR Jurkat cells produced high levels of IL-2 in the presence of hyphae form of C. albicans,P. brasiliensis yeast, and R. oryzae spores. These findings evidenced the capacity of M-CAR to recognize these fungi inducing T cell activation. This work opened new perspectives to evaluate the fungicidal activity of human T and NK cells expressing M-CAR in response to species of fungi studied. Keywords: Chimeric Antigen Receptor (CAR), T cells, invasive fungal infections.

Identification of SARS-CoV-2 neutralizing antibodies using engineered bacteriophage bioconjugates

Novel severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection, raised in late 2019 and unabated for more than two years, still makes challenges for modern society. New variants of SARS-CoV-2 tend to arise often and to displace previous ones, hence the development of a high-throughput and universal platform allowing the selection of antibodies with the highest neutralization and binding activity is of interest. Using our previously developed public platform for high-throughput B cell specificity screening, based on a combination of phage display and flow cytometry techniques, we constructed phage library with surface-exposed RBD (the major target for the development of neutralizing antibodies) peptides. We then constructed lentiviral scFv libraries in Jurkat cells. For these libraries, we used B cells from three donor groups: (i) SARS-CoV-2 recovered patients;(ii) Sputnik V vaccinated donors and (iii) healthy unvaccinated donors. We compared repertoires of RBD-specific antibodies in recovered and vaccinated donors. In addition, we compared the antibody repertoires selected with full-length RBD molecule and phage RBD peptides library. Selected clones with anchored scFvs were screened for neutralizing activity. The best clones from each sample were obtained as full-length immunoglobulins G and characterized for their neutralization activity. Our data indicate that the investigated technique can be successfully used for a comprehensive search for virus-neutralizing antibodies.