The numerous and ever evolving prevalence of SARS-CoV-2 variants post-pandemic stresses the importance of vaccination strategies to consider the breadth and longevity of the protection conferred. Immune imprinting is a novel concern regarding the efficacy of ancestral vaccinations against highly variant viruses, such as SARS-CoV-2, stressing the importance for a greater investigation into whether prior ancestral vaccinations are capable of inducing robust cross-reactive responses to current novel viral variants. Currently, imprinting studies are focused on neutralising antibody responses, however concern for T-cell imprinting has been suggested for memory T-cells. Although such studies have demonstrated a functional importance for T-cell responses in combating SARS-CoV-2 infection, the leading assay for assessing functional central memory T-cell responses, the cultured ELISpot, has minimal representation within the SARS-CoV-2 literature.
Our laboratory has prior defined a separation of ex vivo and cultured ELISpot responses, highlighting a potential disparity between effector and central memory T-cell responses, stressing the significance for a multi-armed approach of ex vivo and cultured ELISpot analysis to assess each respective repertoire when describing vaccine conferred immunity. Herein, we present the utilisation of our cultured ELISpot protocol optimised specifically to investigate the functional cross-reactive central memory T-cell interferon gamma responses to variant SARS-CoV-2 spike protein peptides. Our investigation specifically assessed SARS-CoV-2 spike protein responses comparing the ancestral vaccination target (Wuhan) with the two SARS-CoV-2 variants targeted by vaccine formulation recommendations within Australia in 2025 (XBB.1.5, and JN.1), describing the cross-reactive responses between viral variant peptides.