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Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1/20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto VERO E6/VERO E6 TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using four-parameter logistic regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against WHO anti-SARS-CoV-2 Immunoglobulin Standards. Using WHO Standards with low, medium or high anti-SARS-CoV-2 IgG, both Micro-NT and PRNT achieved comparable NT50 values. Micro-NT was found to be highly reproducible (inter-assay CV of 11.64%). Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s 1/5000. We could also characterise immune-escape VOC observing up to 10-fold reduction in NT50 against SARS-CoV-2 Beta variant. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials. It has higher throughput (96 well format versus 12 well) and reduced infection time (18h vs 48-96h) compared to the gold standard PRNT.

Background Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry based micro-neutralisation assay to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type (D641G) and Variants of Concern (VoC) in convalescent/vaccinated populations. Methods Micro-Neutralisation assay (Micro-NT) was performed in 96-well plates using clinical isolate 2019-nCoV/Italy-INMI1, D641G (SARS-CoV-2/human/IRL/AIIDV1446/2020) and/or VOCs Beta (SARS-CoV-2/human/IRL/AIIDV1752/2021) and Omicron (SARS-Cov-2/human/IRL/AIIDV2326/2021). Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1/20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto VERO E6/VERO-E6 TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) was determined using four-parameter logistic regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against WHO anti-SARS-CoV-2 Immunoglobulin Standards. Results Using WHO Standards with low, medium or high anti-SARS-CoV-2 IgG, both Micro-NT and PRNT achieved comparable NT50 values (Table 1). Micro-NT was found to be highly reproducible (inter-assay CV of 11.39%). Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we achieved an assay sensitivity of 90% and specificity of 81%. We demonstrated that Micro-NT has broad dynamic range differentiating NT50s < 1/20 to > 1/5000 (Figure 1). We could also characterise immune-escape VoC, observing up to 10-fold reduction in NT50 against Beta (Figure 2). Table 1:NT50s of Low, Medium and High Titre Anti-SARS-CoV-2 IgG Standards measured against Live SARS-CoV-2 using PRNT and Micro-NT Neutralising Capacity of low, medium and high-titre anti-SARS-CoV-2 IgG (WHO, International Standards) against live SARS-CoV-2 (2019-nCoV/Italy-INMI1) measured using PRNT and Micro-NT Assays on Vero E6 cells, as well as the potency of NAbs in each sample in International Units (IU/ml) as determined by the WHO. Figure 1:Dynamic Range of Micro-NT Micro-NT has a broad Dynamic Range, distinguishing low (A), medium (B) and high (C) neutralising plasma samples against live SARS-CoV-2 (2019-nCoV/Italy-INMI1) from a cohort of COVID-19 convalescent individuals (AIID cohort), as well as negative samples from COVID-19 naïve samples (D). Graphs show 3 representative samples of each NT50 range. (E) shows the population distribution of 190 Convalescent plasma samples as measured by Micro-NT on Vero E6 cells. Figure 2:Reduced Neutralisation Capacities measured against SARS-CoV-2 VoC using Micro-NT Low (A), Medium (B) and High (C) anti-SARS-CoV-2 IgG (WHO Standards) show different neutralising capacities against WT (D614G) SARS-CoV-2 and variants Beta and Omicron, measured using Micro-NT on Vero-E6-TMPRSS2 cells. Conclusion Our flow-cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, an important evaluation endpoint in clinical trials. It has higher throughput (96 well format versus 12 well) and reduced infection time (18h vs 48-96h) compared to the gold standard PRNT. Disclosures All Authors: No reported disclosures.