Dosing interval is a major factor determining the quality of T cells induced by SARS-CoV-2 mRNA and adenoviral vaccines

Functional T cell responses are crucial for protective immunity induced by COVID-19 vaccination, but factors influencing the quality of these responses are incompletely understood. We employ an activation induced marker (AIM) assay and single-cell transcriptomic sequencing to analyze SARS-CoV-2 spike-responsive T cells following mild SARS-CoV-2 infection or following one or two doses of mRNA-LNP or adenoviral vectored COVID-19 vaccines. Our findings reveal broad functional and clonal heterogeneity in T cells generated by vaccination or infection, including multiple distinct effector populations. T cell function was largely conserved between COVID-19 vaccine platforms but was distinct compared to SARS-CoV-2 infection. Notably, the dosing interval greatly influenced the quality of T cells after two vaccine doses, particularly after mRNA-LNP vaccination, where a longer interval led to reduced inflammatory signaling and increased secondary proliferation. These insights enhance our understanding of SARS-CoV-2 specific T cells and inform the optimization of mRNA vaccination remens. Overall design: CITE-seq: Peripheral blood mononuclear cells (PBMC) were sampled from human donors before or after one or two doses of BNT162b2 or ChAdOx1 nCoV-19 vaccination (COVID-19 vaccines), or at two timepoints post mild COVID-19 infection. Within vaccine groups, individuals were vaccinated at 3-4week (short) or 8-12week (long) intervals - leading to a total of 5 study groups with six participants in each group: ChAd-short, ChAd-long, BNT-short, BNT-long, COVID-19 infected. For samples taken at timepoints after COVID-19 vaccination or infection, PBMC were incubated for 24hours with overlapping peptide pools covering the entire SARS-CoV-2 spike protein (S1 and S2). Fluorescence-assisted cytometric sorting (FACS) was used to sort CD69+ and OX-40+ and/or 4-1BB+ CD3+ lymphocytes. 10X Genomics 5' Next-GEM single-cell multi-omic sequencing was then performed on the sorted T cells, assessing gene expression, TCR and a custom panel of 8 CITE-seq markers. For PBMC samples taken pre-vaccination, a CellTrace Violet (CTV) proliferation assay was performed, whereby cells were incubated for 7-days with overlapping peptide pools covering the entire SARS-CoV-2 spike protein. CTV low CD3+ cells were sorted using FACS and sequenced as above.