Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8⁺ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8⁺ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8⁺ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8⁺ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

SummaryFactors that govern the complex formation of memory T cells are not completelyunderstood. A better understanding of thedevelopment of memory Tcell hetero-geneity is however required to enhance vaccination and immunotherapy ap-proaches. Here we examined the impact of pathogen- and tissue-specific cueson memory CD8+T cell heterogeneity using high-dimensional single-cell mass cy-tometry and a tailored bioinformatics pipeline. We identified distinct populationsof pathogen-specific CD8+T cells that uniquely connected to a specific pathogenor associated to multiple types of acute and persistent infections. In addition, thetissue environment shaped the memory CD8+T cell heterogeneity, albeit to alesser extent than infection. The programming of memory CD8+T cell differenti-ation during acute infection is eventually superseded by persistent infection.Thus, the plethora of distinct memory CD8+T cell subsets that arise upon infec-tion is dominantly sculpted by the pathogen-specific cues and further shaped by the tissue environment.