Validation of a Pseudovirus Neutralization Assay for Severe Acute Respiratory Syndrome Coronavirus 2 Omicron JN.1 and LP.8.1 Subvariant Lineage Strains with Homologous and Heterologous Matched Sera in Clinically Relevant Samples

The pseudovirus neutralization (PNT) assay is an established high-throughput, robust, and efficient BSL-2 method for detecting neutralizing antibodies (NAbs) against SARS-CoV-2 with the correlates of protection previously established for the ancestral (Wuhan) strain. The PNT assay was validated using nonmatched ancestral sera with anti-JN.1 cross-NAbs, clinically matched JN.1 sera with anti-JN.1 NAbs, or nonmatched JN.1 sera with anti-LP.8.1, KP.2, or KP.3 cross-reacting NAbs. In line with predefined validation acceptance criteria, the PNT assay was precise, with %GCV ≤ 50 in ~90–100%/200 results (40 samples/strain). The acceptance criteria were met for linearity (slope ranged from 1.041 for ancestral sera with anti-JN.1 NAbs to 1.213 for JN.1 sera with anti-KP.2 NAbs), R2 (0.9619–0.9944 for ancestral sera with anti–JN.1 NAbs), % relative bias, and total %GCV < 50 for almost all of the 15 serum samples tested for four virus strains. Human sera collected pre–COVID-19 had no detectable titer for tested Omicron JN.1 subvariants (<LLOQ) and all influenza and RSV clinical samples tested negative (<LLOQ) for SARS-CoV-2 and highly immunogenic for seasonal influenza or RSV post-vaccination, demonstrating the PNT assay specificity. Our data suggest this assay is suitable for assessing immune responses to ancestral and current SARS-CoV-2 strains and has potential for evaluating cross-reacting NAbs against emerging Omicron JN.1 subvariants.