ABSTRACT
Background: To understand T-cell responses to SARS-CoV-2, it is essential to define the contribution of infection versus immunization to virus-specific hybrid immunity. Here, we characterized the breadth and magnitude of T-cell responses to the entire SARS-CoV2 proteome over a 2-year follow-up period in infected and vaccinated (CoV2+Vac+) and vaccinated and infected (Vac+CoV2+) individuals. Method(s): We selected samples from 38 (19 CoV2+ and 19 CoV2-, time1, T1) ProHEpiC-19 cohort participants, a prospective, longitudinal study starting in March 2020 involving 7,776 healthcare workers in Spain. Longitudinal samples were available from 10 of them after a 3-dose mRNA vaccination, including 5 CoV2+Vac+ and 5 Vac+CoV2+, at 824.5 and 250.5 days from symptoms onset (DfSO, time 2, T2). We measured the breadth and magnitude of IFN-y T-cell responses by ELISpot assay in cryopreserved PBMCs, using a 15-mer overlapping peptide (OLP) library of 2,790 SARS-CoV-2 peptides in 100 pools. Result(s): We identified immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the SARS-CoV2 proteome. We observed an increased breadth of T-cell responses (responding pools over the entire region) to S1 (44 - 30%) and S2 (31 - 40%) in CoV2+Vac+ and Vac+CoV2+, respectively. In addition, CoV2+Vac+ had an exclusive and sustained response to M. We found significantly stronger responses in CoV2+Vac+ (P=0.0313). Particularly the total magnitude was greater in CoV2+Vac+ vs. Vac+CoV2+ in S1 (4476.88 vs. 1498.53), Env (457.34 vs. 250.50), and M (455.13 vs. 0.00) but not in S2 and nsp3. The total number of peptides for deconvolution was higher in CoV2+Vac+ (32 peptides) than in Vac+CoV2+ (3 peptides) during the follow-up. Seventy-five percent of the responses targeted S, and 25% M, ORF1a, and Env. Conclusion(s): These results profile immunodominant T-cell responses in S1, S2, nsp3, Env, NC, and M proteins across the entire SARS-CoV2 proteome. The data delineate differences in the number of T-cell responses primed hybrid immunity by infection previous to vaccination (CoV2+Vac+), being broader and of higher magnitude and underlining an exclusive T-cell response to the M region. Overall, these findings identify differences in long-term T-cell hybrid immunity primed by infection or vaccination, which may have implications in protection from re-infection and vaccine design.