Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence has prompted booster immunizations. However, repeated antigen exposure effects on SARS-CoV-2 memory T cells are poorly understood. Here, we utilize MHC-multimers with scRNAseq to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two, or three antigen exposures, including vaccination, primary, and breakthrough infection. Exposure order determined the distribution between spike- and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7-CD45RA+ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.

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SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoV) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes generation of SARS-CoV-2 neutralizing antibodies in mice. Together, these data suggest pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

SARS-CoV-2 mRNA vaccines, including Pfizer/Biontech BNT162b2, were shown to be effective for COVID-19 prevention, eliciting both robust antibody responses in naive individuals and boosting pre-existing antibody levels in SARS-CoV-2-recovered individuals. However, the magnitude, repertoire, and phenotype of epitope-specific T cell responses to this vaccine, and the effect of vaccination on pre-existing T cell memory in SARS-CoV-2 convalescent patients, are still poorly understood. Thus, in this study we compared epitope-specific T cells elicited after natural SARS-CoV-2 infection, and vaccination of both naive and recovered individuals. We collected peripheral blood mononuclear cells before and after BNT162b2 vaccination and used pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses to several immunodominant epitopes, including a spike-derived epitope cross-reactive to common cold coronaviruses. Comparing responses after infection or vaccination, we found that T cells responding to spike-derived epitopes show similar magnitudes of response, memory phenotypes, TCR repertoire diversity, and αβTCR sequence motifs, demonstrating the potency of this vaccination platform. Importantly, in COVID-19-recovered individuals receiving the vaccine, pre-existing spike-specific memory cells showed both clonal expansion and a phenotypic shift towards more differentiated CCR7-CD45RA+ effector cells. In-depth analysis of T cell receptor repertoires demonstrates that both vaccination and infection elicit largely identical repertoires as measured by dominant TCR motifs and receptor breadth, indicating that BNT162b2 vaccination largely recapitulates T cell generation by infection for all critical parameters. Thus, BNT162b2 vaccination elicits potent spike-specific T cell responses in naive individuals and also triggers the recall T cell response in previously infected individuals, further boosting spike-specific responses but altering their differentiation state. Overall, our study demonstrates the potential of mRNA vaccines to induce, maintain, and shape T cell memory through vaccination and revaccination.

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