A pseudovirus-based platform to measure neutralizing antibodies in Mexico using SARS-CoV-2 as proof-of-concept

Abstract

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 American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen’s entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of 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.

High-Content Imaging-Based Assay for SARS-CoV-2-Neutralizing Antibodies

“…Different groups standardized methods like ours, which were also demonstrated to produce results in agreement with the PRNT and identify neutralizing escape when the samples were tested against VoCs [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 23 , 31 ]. PRNT requires a BSL-3, rendering it non-safety compatible since it requires live viruses, is laborious, time-consuming, and is not well adapted to high-throughput studies [ 22 , 33 ].…”

“…When tested with human convalescent plasma, their assay was comparable to the gold standard plaque reduction neutralization test (PRNT) and is being used in a phase I/II clinical trial [ 10 , 14 ]. Other groups developed similar assays using the lentiviral approach, which has the advantage of being manipulated at BSL-2 laboratories [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Based on the importance of evaluating the neutralizing capacity of human serum and developing new vaccines, when considering the variation among different populations, we standardized and validated an imaging-based high-content assay, which has been shown to be safe, high-throughput compatible, and able to determine the neutralizing potency of plasma in two days, which is half of the time required by the PRNT [ 22 ].…”

Characterisation of the Antibody Response in Sinopharm (BBIBP-CorV) Recipients and COVID-19 Convalescent Sera from the Republic of Moldova

“…In the current study, we developed an IgG ELISA with recombinant SARS-CoV-2 spike RBD and two BSL-2-safe assays with no infectious pseudovirus lacking the no-structural proteins. The pseudovirus system for screening NAbs and antivirals is a widely used method [ 46 , 47 , 48 , 49 ]. Most reports validate protocols based on ELISA and FC [ 28 , 32 , 50 ].…”

Exploring immune evasion of SARS-CoV-2 variants using a pseudotyped system

“…Fortunately, PsVs effectively mitigate this constraint. PsVs can infect susceptible cells but only undergo a single replication cycle within the host, making them generally safer and easier to use for experimental purposes compared to the wild-type (WT) virus [ 12 , 13 ]. Importantly, the structural conformation of pseudoviral surface proteins closely resembles that of natural viral proteins, facilitating efficient invasion of host cells [ 14 ].…”