Predictive markers of SARS-CoV-2 spike-specific cytotoxic T cell activity following Omicron breakthrough infection

At a population level, evaluation of vaccine efficacy and of immunity in general focuses on measuring humoral immunity, especially antibody-mediated neutralization. However, antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular immunity are key processes for pathogen clearance and often provide broader protection against emerging variants. More information on vaccine and infection-related determinants or markers of cell-mediated cytotoxicity is needed to inform design of standardized, high throughput tests assessing these processes. We previously showed that persons infected with SARS-CoV-2 prior to vaccination (hybrid immunity) had polyfunctional CD8⁺ T cells and robust ADCC against SARS-CoV-2 spike (S), while persons infected with Omicron after vaccination (breakthrough infection) had weak ADCC. In the current study, we investigated the impact of Omicron breakthrough infection on S-specific cellular immunity, including polyfunctional CD8⁺ T cell and cytotoxic T lymphocyte (CTL) levels of 28 previously uninfected individuals vaccinated either two or three times against SARS-CoV-2. In the group with three vaccinations, Omicron breakthrough infection significantly increased the frequency of circulating SARS-CoV-2 S-specific T cells, polyfunctional interferon-gamma and interleukin-2-producing SARS-CoV-2 S-specific CD8⁺ T cells and CTL after <i>in vitro</i> stimulation. Matrix testing with overlapping S peptides indicated recognition of one to two CTL epitopes per individual and revealed two previously unreported epitopes. Circulating S-specific cellular immune response magnitude neither correlated with, nor predicted CTL activity against SARS-CoV-2 S, but polyfunctional S-specific CD8⁺ T cell frequency seven days after stimulation and antibody reactivity against determinants indicating robust ADCC were both associated with day 10 CTL activity.