A CD8+ killing assay to measure T cell responses to infection with SARS-nCoV-2.

Abstract

The coordinated efforts of the innate and adaptive immune systems are essential for the effective control and clearance of novel SARS-CoV-2 (SARS-nCoV-2) infection. While the innate immune response provides an immediate but non-specific defense, the adaptive immune response offers specificity and long-lasting protection. Although anti-SARS-nCoV-2 immune responses are well-characterized, T cell responses are equally important. But, the role of cytolytic CD8+ T cell responses in vaccine-induced protection against SARS-nCoV-2 is poorly understood, partly because of a lack of robust assays which accurately measure virus-specific CD8+ T cell (CTL) cytotoxicity. The aim of this study was to develop an assay capable of measuring SARS-nCoV-2 specific CTL responses to both COVID-19 vaccines and natural infection. This study evaluated two fluorescence-based killing assays. The pseudovirus-based killing assay aimed to optimize a spike pseudotyped reporter virus to infect target cells for a SARS-nCoV-2-specific CD8+ T cell killing assay. The second assay measured cytotoxic CD8+ T cell responses by labelling antigen-presenting target cells with different concentrations of carboxyfluorescein succinimidyl ester (CFSE), whereby fluorescence loss represented killing by autologous effector cells. For the pseudovirus approach, multiple transfection methods and target cell infection conditions were tested towards achieving a robust fluorescence readout in target cells. However, the assay did not produce a sufficient fluorescence readout to measure CD8+-mediated elimination of target cells. For the CFSE-based assay, different concentrations of CFSE were tested and optimized for antigen-specific and non-specific target cells respectively, followed by optimizing co-culture with autologous CD8+ T cells, both for ex vivo killing measurements or following cultured expansion of T cells. The assay was validated using peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH). The assay was then used to compare SAR-nCoV-2 specific CTL killing responses in PLWH and without HIV (PLWoH) vaccinated with Oxford/AstraZeneca, Coronavac or Ad26COV2.S. SARS-nCoV-2-specific CD8+ killing activity was generally low in COVID-19 vaccine recipients. Marginally higher responses were observed in PLWH receiving vector-based vaccines Oxford/AstraZeneca and Ad26COV2.S, than PLWoH. Overall, a CFSE-based CD8+ killing assay that can measure COVID-19 vaccine responses was successfully developed. Additionally, this assay can be adapted to measure CTL responses to other viruses and natural viral infections.