A High-Throughput Broad Neutralizing Antibody Assay for Detecting SARS-CoV-2 Variant Immunity in Population

ABSTRACT Detecting neutralizing antibodies (NAbs) to SARS-CoV-2 variants that are evolved is crucial to know the escape of host immunity to the newly arising variants. To address this need, we developed a high-throughput broad neutralizing antibody (bNAb) assay using flow cytometry with magnetic-fluorescent microspheres for detecting NAbs against diverse SARS-CoV-2 variants. The assay is rapid, reliable, 35-fold more sensitive than Luminex technology. Our results highly correlated with IgG serological assay (R = 0.90) , the FDA-approved cPass sVNT assay (R = 0.92), pseudovirus-based neutralizing assay (R = 0.96, 0.66, 0.65) and live virus based neutralization assay (R = 0.79, 0.64). When applied to 56 healthy individuals receiving third-dose vaccines (18 CoronaVac; 38 ZF2001) and 35 HIV patients with breakthrough infection of COVID-19 (16 with CD4+ < 350 cells/µL; 19 with CD4+ > 500 cells/µL), results showed that the Omicron BA.1-BA.5 variants exhibited significant resistance to inactivated vaccines in healthy individuals. In HIV patients, the breakthrough infection of Omicron BA.5.2 or BF.7 variants can induce broad neutralizing activity to non-Omicron and Omicron variants together with vaccination. Notably, the levels of NAbs against most of SARS-CoV-2 variants are much lower in the decreased immunity of HIV patients (CD4+ < 350 cells/µL) compared to the recovered immunity (CD4+ > 500 cells/µL), indicating that maintenance of the immune system is crucial for NAb production. Altogether, our high-throughput proteomics platform represents a powerful tool for the detection of bNAbs in the population and may inform the development of more effective COVID-19 vaccines and vaccination strategies in the future.