Initial blots are presented in Sup. BSL-3 assay with live computer virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be designed to display a selected pathogens entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of ZL0420 a BSL-2 safe NAbs-measuring functional 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 applied the optimized assay in a BSL-2 facility to measure 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. Subject terms: Microbiology techniques, Virology Introduction Humoral immunity provides crucial 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 ZL0420 neutralizing potential of serum samples against a viral pathogen informs on effective virus-specific humoral immunity. This is relevant to: 1-monitor levels of partial protection within an asymptomatic populace, 2-evaluate the humoral immunity efficacy of existing and novel vaccines against emerging pathogens, 3-test prospective therapeutic monoclonal NAbs and, overall, 4-contribute to understand immunity against viral pathogens. Despite various viral pandemics affecting Mexico, such as the influenza caused by H1N1 and COVID-19 caused by SARS-CoV-2, no functional assay has been developed on-site to allow timely measurements of NAbs using a functional assay. For example, all currently published SARS-CoV-2 NAbs data from the Mexican population have been obtained either internationally using initial computer virus 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 ZL0420 evaluation of the effectiveness of the national vaccination program and the monitoring of seroprevalence, both important aspects in the control of COVID-19. In this context, to measure NAbs using a functional assay, serum samples are co-incubated with SARS-CoV-2 viral particles (VP) in a BSL-3 laboratory, after which the ability of these VP to infect target cells in vitro is usually measured6. A serums NAb titer negatively correlates with the level of VP-mediated contamination. An alternative strategy to using authentic virus is to produce non-replicative VP that express 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 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 as a proof-of-concept, to develop a BLS-2-safe, functional 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 genomes, and all actions of development are sequentially described. The assay facilitated quantification of effective humoral MGC5276 immunity to SARS-CoV-2 in COVID-19 convalescent patients and BNT162b2 vaccinated individuals, and was validated against a commercially available surrogate ELISA assay, commonly used in currently published Mexican studies. Results Development and production of a SARS-CoV-2 pseudotyped lentivirus To ZL0420 develop a BSL-2-ready assay to investigate neutralizing antibodies to SARS-CoV-2 in ZL0420 Mexico, we first produced SARS-CoV-2 S-pseudotyped VP. We optimized a previously reported 3rd-generation lentiviral system (Fig.?1) by using the reporter gene Nluc which is more stable and provides 100??brighter luminescence compared to fLuc5,9..
