Cytotoxic T Cells Targeting Spike Glycoprotein Are Associated with Hybrid Immunity to SARS-CoV-2

mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared with vaccination in the absence of prior infection. However, the immune mechanisms by which this hybrid immunity is generated and maintained are unknown. Whereas genetic variation in spike glycoprotein effectively subverts neutralizing Abs, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled human T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive (n 5 13) or -convalescent (n 5 17) 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 and polyfunctional Th₁ responses was similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multimodal 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.