The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin America, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution is to use a BSL-2-safe assay with non-replicative viral particles engineered to display a selected pathogen’s entry protein on their surface, that deliver a reporter gene into target cells upon entry protein-mediated transduction. Here we comprehensively describe the first development of a BSL-2 safe NAb-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure SARS-CoV-2 Spike NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.

Measuring the neutralizing potential of SARS-CoV-2 antigens-exposed sera informs on effective humoral immunity. This is relevant to 1-monitor levels of protection within an asymptomatic population, 2-evaluate the efficacy of existing and novel vaccines against emerging variants, 3-test prospective therapeutic monoclonal neutralizing antibodies (NAbs) and, overall, to contribute to understand SARS-CoV-2 immunity. However, the gold-standard method to titer NAbs is a functional assay of virus-mediated infection, which requires biosafety level 3 (BSL-3) facilities. As these facilities are insufficient in Latin American countries, including Mexico, scant information has been obtained about NAb in these countries during the COVID-19 pandemic. An alternative solution to acquire NAb information locally is to use non-replicative viral particles that display the SARS-CoV-2 Spike (S) protein on their surface, and deliver a reporter gene into target cells upon transduction. Here we present the development of a NAb-measuring assay based on Nanoluc-mediated luminescence measurements from SARS-CoV-2 S-pseudotyped lentiviral particle-infected cells. We applied the optimized assay in a BSL-2 facility to measure NAbs in 15 pre-pandemic, 18 COVID-19 convalescent and 32 BNT162b2 vaccinated serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. The assay highlighted heterogeneity in neutralization curves which are relevant in discussing neutralization potency dynamics. Overall, this is the first report of a BSL-2 safe functional assay to measure SARS-CoV-2 in Mexico and a cornerstone methodology necessary to measure NAb with a functional assay in the context of limited resources settings.

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