(M1330-02-12) Multi-Strain Sars-Cov-2 Neutralization Assay Qualification

Purpose: The creation of dependable tests to detect neutralizing antibodies proved crucial in the worldwide fight against the COVID-19 pandemic. Assays using SARS-CoV-2 pseudotyped viruses for neutralization (PsVNAs) have proven to be essential in evaluating the effectiveness of COVID-19 vaccines. In this context, we present the validation of three distinct PsVNAs designed to identify neutralizing antibodies targeting three different SARS-CoV-2 strains (D614G, Omicron BA.1, and Omicron BA.4/5) in human blood serum.

Methods: The validation procedure for these PsVNAs involved an extensive evaluation of critical factors to confirm their suitability for testing clinical samples. This evaluation included the accuracy within and between assays, establishing the ranges for quantification, specificity to human serum, robustness across different assay plates, and sample stability under a range of condition.

Results: Among the primary considerations were the precision assessment, which involved testing at least 20 human serum samples with neutralizing antibody titers spanning the analytical ranges. These samples were tested over a minimum of 3 separate days by at least two different analysts. The geometric coefficient of variation (GCV%) for both inter-assay and intra-assay was < 50% for three PsVNAs. Linearity experiments were performed to assess the relative accuracy and assay quantitation ranges within defined lower and upper limit of quantitation (LLOQ and ULOQ), ensuring accurate measurement of antibody levels. Specificity testing confirmed minimal cross-reactivity and false positives, enhancing the reliability of vaccine studies. Neutralizing antibody titer values of samples with homologous interferent (Spike protein) demonstrated dose-dependent effect of interferent. And the titer values of samples with heterologous interferent were within 2-fold of the samples without interferent. Ruggedness testing demonstrated consistent results at the maximum sample throughput at 10 plates per run per analyst, further validating the assays’ reliability.  Additionally, the stability test showed no significant loss of sample integrity for up to six freeze-thaw cycles. Samples after heat-inactivation were found stable at 5 ± 3 ℃ for up to seven days. These results support the practicality of clinical sample handling for PsVNA analysis.

Conclusion: Three pseudotyped virus-based viral neutralization assays have been effectively qualified for use against three different SARS-CoV-2 strains (D614G, Omicron BA.1, and Omicron BA.4/5) in human serum samples. Following qualification, these assays were used in assessing immunogenicity of novel combination mRNA vaccines for respiratory diseases during the 2022/2023 evaluation period.

Acknowledgements: Disclaimer: this work is organized and funded by Moderna, Inc. Authors MZ, PP, CC, LZ, and PCT are employees of Moderna, Inc., and may hold stock/stock options in the company.