Project description:mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared to vaccination in the absence of prior infection. However, the immune mechanisms by which this ‘hybrid immunity’ is generated and maintained are unknown. While genetic variation in spike glycoprotein effectively subverts neutralizing antibodies, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive or convalescent individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells as well as polyfunctional Th1 responses were similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multi-modal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.
Project description:In this work, we performed a fully descriptive analysis N- and O- linked glycosylation of SARS-COV-2 S glycoprotein. We investigated that dual-functionalized Ti-IMAC material enable the simultaneous enrichment and separation of neutral and sialyl glycopeptides of a recombinant SARS-CoV-2 S glycoprotein from HEK293, which will eliminate the signal suppression of neutral glycopeptides to sialyl glycopeptides and improve the glycoform coverage of S protein. We have profiled 19 of its 22 potential N-glycosylated sites with 398 unique glycoforms in dual-functional Ti-IMIAC approach that is 1.6-fold of that in conventional HILIC method. We also identified O-linked glycosylation site that was not found in dual-functional Ti-IMIAC approach. In addition, we have also identified mannose-6-phosphate (M6P) glycosylation, which substantially expands the current knowledge of the spike protein’s glycosylation and enables the investigation of the influence of mannose-6-Phosphate on its cell entry.