A Novel dual Influenza virus and SARS-CoV-2 neutralisation assay

Influenza and SARS-CoV-2 are co-circulating during the traditional influenza season giving rise to potential co-infections; currently estimated at 2.4%. Additionally, this means that the uptake of both vaccines (including SARS-CoV-2 boosters) will be encouraged within the same timeframe. Currently, patients receive separate vaccines in different arms on the same or different days. Induction of neutralising antibodies (NAbs) is used as the measure of efficacy for SARS-CoV-2 vaccinations while, the hemagglutination inhibition (HI) assay is the gold standard for seasonal influenza vaccine assessment. However, with the advent of novel universal influenza vaccine and dual SARS-CoV-2/Influenza vaccine approaches it is possible that functional NAb assays will become an essential component of a vaccine assessment toolbox. This assay is capable of distinguishing NAb responses during both dual vaccination development, and as part of co-infection and imprinting studies. Taking advantage of existing pseudotyped viruses developed by us, we present herein a dual neutralisation assay, which utilises 2 different luciferase (Renilla and Firefly) reporter lentiviral vectors. We configured assays with various combinations of SARS-CoV-2 and influenza subtype/variant combinations and compared sensitivity of this dual approach to neutralisation levels seen in single virus pseudotype Micro-neutralisation (pMN) in a double-blinded test of monoclonal antibody cocktails, and subsequently with a pre-screened serum panel. Mono and dual neutralisation pMN identified correctly positive and negative samples and IC50 values were all within 2-fold of each other, suggesting that specificity and sensitivity are retained post-multiplex. As we have taken a pseudovirus based approach there is the potential to tailor to specific vaccination candidates or currently circulating strains of each virus. The use of this versatile assay could form part of the toolbox of analysis of dual vaccination candidates and investigating responses during co-infection including studies assessing the impact of immune imprinting in the future.