Validation of CPE - based microneutralization assay for detecting Sars-CoV-2 Victoria Wild type virus and OMICRON JN.1 variant for new vaccine immunogenicity study.

During the COVID-19 pandemic, several SARS-CoV-2 variants of concern (VOCs) emerged. As reported by the WHO, the development of COVID-19 vaccines to reduce the elevated risk associated with the spread of the virus. The Micro-Neutralization assay (MN), currently considered the gold-standard, is the most specific and sensitive serological assay capable of evaluating and detecting, functional neutralizing antibodies (nAbs) to develop new vaccines. In this study, a live virus-based MN assay is presented for the quantification of SARS-CoV-2-specific nAbs in human serum samples by classical read-out by checking the percentage of cytopathic effect (CPE) in the cell monolayer. The project main task is delineating the procedures and criteria utilized for validating the Microneutralization (MN) assay against both the Sars-CoV-2 Wild type and OMICRON JN.1variant. Validation protocol adheres to EMA's and FDA's Validation on Analytical Procedures guidelines. The virus is provided by the EVAg. The virus was propagatedin VERO E6 cells at Vismederi Research Srl, following VisMederi Research's Standard Operative Procedure. The protocol reports the assessment of the following validation parameters: Specificity and Sensitivity, Precision, Linearity, Relative Accuracy, Robustness, Range, using SARS-CoV-2 specific neutralizing antibody obtained from a vaccinated healthy donors. The validation process of the MN test for SARS-CoV-2 met all the acceptability criteria outlined in the validation protocol. Specifically, the assay was shown to be: sensitive, specific, precise, linear, accurate, repeatable, and robust. Consequently, the procedure is considered reliable and valid for its intended use. Furthermore, the current findings confirm the capability of the MN CPE-based assay to detect SARS-CoV-2 specific neutralizing antibodies in human serum samples using the SARS-CoV-2 Omicron JN.1 variant as the viral input, also suggesting cross-protection. Consistent with published literature, the neutralization titers are consistently lower with Omicron JN.1 compared to the ancestral SARS-CoV-2 strain.