Development and Validation of a Standardized Pseudotyped Virus based Neutralization Assay for Assessment of Anti-Nipah Virus Neutralizing Activity in Candidate Nipah Vaccines

Background: An effective vaccine against Nipah virus (NiV) is crucial due to its high fa-tality rate and recurrent outbreaks in South and Southeast Asia. Vaccine development is challenged by the lack of validated accessible neutralization assays, as virus culture re-quires BSL-4 facilities, restricting implementation in resource-limited settings. To address this, we standardized and validated a pseudotyped virus neutralization assay (PNA) for assessing NiV-neutralizing antibodies in BSL-2 laboratories. Methods: The NiV-PNA was validated following international regulatory standards, us-ing a replication-defective recombinant Vesicular stomatitis virus (rVSV) backbone de-pendent pseudotyped virus. Assessments included sensitivity, specificity, dilutional line-arity, relative accuracy, precision, and robustness. The assay was calibrated using the WHO International Standard for anti-NiV antibodies and characterized reference sera to ensure reliable performance. Findings: The developed NiV-PNA showed 100% sensitivity and specificity, with a posi-tive correlation to a calibrated reference assay (R² = 0.8461). Dilutional linearity (R2 = 0.9940) and accuracy (98.18%) were confirmed across the analytical titer range of 11-1728 IU/mL. The assay also exhibited high precision, with intra-assay and intermediate preci-sion geometric coefficients of variation of 6.66% and 15.63%, respectively. Robustness testing demonstrated minimal variation across different pseudotyped virus lots, incuba-tion times, and cell counts. Conclusion: The validated NiV-PNA is a reproducible and scalable assay platform for quantifying NiV neutralizing antibodies, offering a safer alternative to virus culture. Its validation and integration into the CEPI Centralized Laboratory Network will enhance global capacity for vaccine evaluation and outbreak preparedness.