Optimisation of the Cultured ELISpot / Fluorospot technique for the selective investigation of SARS-CoV-2 reactive central memory T-cells

Jack Jerome ¹ Kirsty Wilson ¹ Joshuah Fialho ¹ Georgia Goodchild ¹ Monica Devi Prakash ¹ Charlie Mcleod ^2,3,4 Peter C Richmond ^4,5,6 Vasso Apostolopoulos ¹ Katie Louise Flanagan ^1,7,8 Magdalena Plebanski ^1*

• ¹ School of Health and Biomedical Sciences, RMIT University, Melbourne, Australia
• ² Centre for Child Health Research, University of Western Australia, Crawley, Australia, Perth, Australia
• ³ School of Public Health, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia
• ⁴ Wesfarmers Centre of Vaccines and Infectious Diseases, Kids Research Institute of Australia, Nedlands, Australia, Perth, Australia
• ⁵ Division of Paediatrics, School of Medicine, University of Western Australia, Perth, Western Australia, Australia
• ⁶ Department of Immunology, Perth Children’s Hospital, Nedlands WA, Perth, Australia
• ⁷ Tasmanian Vaccine Trial Centre, Clifford Craig Foundation, Launceston General Hospital, Launceston, Tasmania, Australia, Tasmania, Australia
• ⁸ School of Health Sciences and School of Medicine, University of Tasmania, Launceston, Tasmania, Australia, Tasmania, Australia

This study presents an optimised cultured ELISpot protocol for detecting central memory T-cell interferon gamma (IFNγ) responses against SARS-CoV-2 peptides following an initial priming with either peptides, or whole spike protein. Key variations optimised include the culture length, timing of exogenous survival signals (IL-2), and endpoint analysis modality and cell density to enhance assay sensitivity without compromising specificity for central memory T-cell IFNγ recall responses to cognate antigen. We noted a culture duration of 10 days, combined with a delayed IL-2 administration on day 5 to enhance assay sensitivity while maintaining response specificity towards cognate antigen when compared to shorter culture periods or earlier exogenous survival signal provision. With regards to lower-frequency T-cell interactions, as we observed with our donor SARS-CoV-2 epitope responses, our findings suggest Fluorospot to be preferable to the chromogenic ELISpot modality, and an immediate cell washing after culture collection to better facilitate cognate antigen responses. Fluorospot enabled a higher cell density while minimising the generation of visual artefacts, meanwhile immediate cell washing was critical for improving endpoint assay sensitivity. CCR7+ cell depletion was used to demonstrate our optimised protocol to selectively demonstrate central memory T-cell responses. Lastly, we provide evidence for the capacity of our assay to delineate individual responding peptides following peptide pool priming, and to explore cross-reactivity between viral variant peptides. This work advances the methodology for investigating T-cell immunity, particularly in the context of SARS-CoV-2, and emphasises the balance between enhancing specific cognate central memory responses while limiting non-specific activation.