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Introduction: Understanding the immune status of an individual using neutralizing antibody testing is complicated by the continued evolution of the SARS-CoV-2 virus. Previous work showed that assays developed against the wildtype strain of SARS-CoV-2 were insufficient predictors of neutralization of omicron variants, thus we developed an omicron-specific flow cytometry-based neutralizing antibody test and performed experiments to assess how well it compared to an omicron-specific PRNT assay (gold standard) and whether it could predict neutralizing activity to more recent omicron subvariants such as XBB.1.5., Methods: Accuracy of a novel flow cytometry-based neutralizing antibody (FC-NAb) assay was determined by comparison with an omicron-specific PRNT assay. A series of samples were evaluated in both the omicron FC-NAb assay and a second test was designed to assess neutralization of XBB.1.5., Results: Good concordance between the omicron FC-NAb test and the omicron PRNT was demonstrated (AUC = 0.97, p <0.001; sensitivity = 94%, specificity = 100%, PPV = 100%, and NPV = 97%). A strong linear relationship between the omicron FC-NAb and neutralization of XBB1.5 was observed (r = 0.83, p<0.001). Additionally, the omicron FC-NAb test was a very strong predictor of positive XBB1.5 NAb activity (AUC = 0.96, p<0.001; sensitivity = 94%, specificity = 90%, positive predictive value = 90%, and negative predictive values = 94%)., Discussion: Our data suggest that despite continued evolution of the SARS-CoV-2 spike protein, the omicron FC-NAb assay described here is a good predictor of XBB1.5 neutralizing activity, as evidenced by a strong correlation and good predictive performance characteristics., Competing Interests: SV, MG, SN, HL, GC, and SS are all employees and or shareholders of Pearsanta Inc. or its parent company Aditxt Inc. Pearsanta provides clinical laboratory services, including SARS-CoV-2 neutralizing antibody tests. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Varvel, Galdzicka, Nystrom, Liu, Chen, Ragan and Shabahang.)

Introduction: Neutralizing antibodies (NAbs) have been recognized as surrogates of protection against SARS-CoV-2; however, the emergence of variants/subvariants escaping neutralization suggests that laboratory assessments of NAbs against the ancestral/wild type (WT) antigens likely overestimate the degree of protection., Methods: A novel flow cytometry-based multiplex test system was developed for the simultaneous detection of NAbs of multiple SARS-CoV-2 variants. SARS-CoV-2 antibodies (Abs) including IgG, IgM, IgA isotypes were measured in the same system. Samples from negative, convalesced, vaccinated, boosted, and breakthrough infection (BTI) populations were tested for both NAbs and Abs., Results: NAbs induced by WT showed neutralization activity that correlated strongly to all variants (R 2 > 0.85) except omicron BA.1/BA.2 (R 2 <0.50). Two doses of vaccine elicited very little protective immunity against BA.1/BA.2, though a booster dose significantly improved NAbs for all variants. NAbs/Abs increased more following BTI than after a booster, suggesting that hybrid immunity (vaccination + natural immunity) was more robust to all variants including BA.1/BA.2. BTIs occurring in the omicron era led to stronger NAb responses against BA.1/BA.2 than did older BTIs. In all comparisons, the RBD antigens demonstrated greater differences between WT and BA.1/BA.2 than the spike antigens., Discussion: Taken together, we demonstrated that both Ab and NAb against multiple SARS-CoV-2 variants/subvariants can be reliably detected on the same multiplex platform. Distinguishing NAbs to the appropriate antigenic target of prevalent variants offers the best correlate of protection and aids individual decisions about the appropriateness and cadence of vaccine boosters and other exposure mitigation strategies., Competing Interests: All authors are employees or stockholders of Aditxt, Inc., which performs the tests described., (Copyright © 2022 Liu, Varvel, Chen, McConnell, Caffrey, Galdzicka and Shabahang.)