Customizable platform of pseudovirus-based neutralization assays for rapid responses to public health challenges

Introduction: Due to the high infectivity and pathogenicity of certain viruses, tests using infectious particles must often be conducted in Biosafety Level 3 (BSL-3) labs and involve time-consuming standardizations, which limit their execution.Alternatives, such as Pseudovirus-Based Neutralization (PBN) assays, have complemented the gold-standard Plaque Reduction Neutralization Test (PRNT) and were widely adopted during the pandemic to quantify neutralizing antibodies (nAbs) in SARS-CoV-2 vaccine studies.Most SARS-CoV-2 pseudoviruses are non-replicative, express replaceable target antigens, and carry a reporter gene.As PBN assays are conducted in BSL-2, they provide increased throughput in addition to faster and more flexible responsiveness.Objectives: This study aimed to develop customizable PBN assays, standardize them using SARS-CoV-2 as a model, and compare them with PRNT.Methodology: We adopted a commercial Lentivirus (LV)-based and an in-house Vesicular Stomatitis Virus (VSV)-based pseudoviruses, both expressing the Spike protein and carrying luciferase and/or GFP ORFs.Experimental parameters were optimized for homogeneous cell monolayers and improved measurements of virus-infected cells.Samples screened by PRNT were used to assemble a panel, define control sera, and compare PBN-generated titers to those obtained by PRNT.A total of 1,200 and 320 serum cohort tests were performed using commercial and in-house assays, respectively.Results: Commercial GFP-SARS-CoV-2 XBB 1.5 pseudovirus entered HEK293T-ACE2 cells 72 hours post-infection.PBN titers from the serum panel were qualitatively equivalent to PRNT.The commercial assay showed increasing and sustained high nAb titers for Wuhan, the only circulating strain at sample collection, while Omicron XBB 1.5 showed a slight increase.Similarly, the in-house assay confirmed titers significantly higher for Wuhan than Omicron BA.1, and it was able to distinguish different titer ranges.PRNT-Wuhan panel screening confirmed the equivalency of PBN assays to each other. Conclusion:GFP-pseudoviruses findings point to the possibility of using them in mechanistic infection studies.Results from luciferase-containing particles indicate both PBN assays are comparable and PRNTequivalent, strengthening them as trustful tools.Additionally, both assays distinguished nAb titers across different value ranges, which is a valuable feature for clinical screening.Lastly, the customizable aspect of PBN platforms allows for the modification of pseudovirus target antigens, enabling quick responses to emerging outbreaks.