Validation of an MLV-based SARS-CoV-2 pseudovirus neutralization assay substantiates L455S-mediated antibody escape.

The ongoing evolution of SARS-CoV-2 has generated variants with increased transmissibility and immune evasion, primarily driven by mutations in the receptor-binding domain (RBD). Recent Omicron sublineages, including JN.1, have multiple spike substitutions, including L455S, which is associated with antibody evasion and persists in descendant and currently circulating lineages. While live virus neutralization assays (VNTs) remain the gold standard for evaluating immune responses, their biosafety requirements and technical complexity limit large scale application, prompting an increased reliance on pseudovirus-based systems. In this study, we compared murine leukemia virus (MLV)-based pseudovirus neutralization assays (pVNTs) with VNTs across multiple SARS-CoV-2 variants. We also assessed the contribution of the L455S substitution to immune escape, a mutation consistently implicated as a key determinant of JN.1-associated immune evasion. NT titers obtained with the MLV system showed strong correlation with those of live virus assays, supporting its reliability for variant-specific neutralization studies. Introduction of the L455S mutation into two Omicron variants resulted in substantial reductions in neutralization titers, supporting its immune-evasive properties. Together, these findings validate the MLV-based pseudovirus system as a robust NT surrogate and demonstrate its suitability for evaluating emerging mutations linked to variant evolution, such as the L455S mutation, which played a key role in enabling JN.1 to become dominant.