4). cells upon transduction. Here we comprehensively describe the 1st development of a BSL-2 safe NAbs-measuring practical assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We KRT17 applied the optimized assay inside a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% level of AZ7371 sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the 1st report of a BSL-2 safe pseudovirus-based practical assay developed in Mexico to measure NAbs, and a cornerstone strategy necessary to measure NAbs with a functional assay in limited resources settings. Subject terms: Microbiology techniques, Virology Intro Humoral immunity provides essential protection against viruses, including memory contributing to prevent future infections. In particular, neutralizing antibodies (NAbs) specifically target epitopes on viral membrane proteins, interfering with cell receptor binding1. Measuring the neutralizing potential of serum samples against a viral pathogen informs on effective virus-specific humoral immunity. This is relevant to: AZ7371 1-monitor levels of partial protection within an asymptomatic human population, 2-evaluate the humoral immunity effectiveness of existing and novel vaccines against growing pathogens, 3-test prospective restorative monoclonal NAbs and, overall, 4-contribute to understand immunity against viral pathogens. Despite numerous viral pandemics influencing Mexico, such as the influenza caused by H1N1 and COVID-19 caused by SARS-CoV-2, no practical assay has been developed on-site to allow timely measurements of NAbs using a practical assay. For example, all currently published SARS-CoV-2 NAbs data from your Mexican population have been acquired either internationally using unique disease in BLS-3 laboratories2, or nationally after importation of a surrogate ELISA-based kit3C5. Both alternatives are lengthy, expensive, and restricted by reagents availability and ongoing collaborations, respectively. This in turn could hamper the timely evaluation of the effectiveness of the national vaccination program and the monitoring of seroprevalence, both important elements in the control of COVID-19. With this context, to measure NAbs using a practical assay, serum samples are co-incubated with SARS-CoV-2 viral particles (VP) inside a BSL-3 laboratory, after which the ability of these VP to infect target cells in vitro is definitely measured6. A serums NAb titer negatively correlates with the level of VP-mediated illness. An alternative strategy to using authentic virus is to produce non-replicative VP that communicate the SARS-CoV-2 Spike (S) or RBD on their surface and that include a reporter gene delivered to target cells upon transduction7C9. These pseudotyped VP can then be used in BSL-2, and have been widely used to measure NAbs against a range of potentially fatal viruses, including influenza (H7N9), MERS-CoV, HCV, and SARS-CoV-2 and recent variants10C13. Advantages of using pseudotyped AZ7371 VP to measure NAbs, include their possible use in lower biosafety containment level, the facility of upscaling for high-throughput measurements at a lower cost, as well as the opportunity to customize the viral glycoprotein to match emerging variants. Here, we harness the current COVID-19 pandemic like a proof-of-concept, to develop a BLS-2-safe, practical SARS-CoV-2 NAbs titering assay in Mexico. The assay is based on non-replicative SARS-CoV-2 S pseudotyped lentiviral particles integrating Nanoluc Luciferase (Nluc) into transduced cells AZ7371 genomes, and all methods of development are sequentially explained. The assay facilitated quantification of effective humoral immunity to SARS-CoV-2 in COVID-19 convalescent individuals and BNT162b2 vaccinated individuals, and was validated against a commercially available surrogate ELISA assay, generally used in currently published Mexican studies. Results Development and production of a SARS-CoV-2 pseudotyped lentivirus To develop a BSL-2-ready assay to investigate neutralizing antibodies to SARS-CoV-2 in Mexico, we 1st produced SARS-CoV-2 S-pseudotyped VP. We optimized a previously reported 3rd-generation lentiviral system (Fig.?1) by using.