Robust validation and performance comparison of immunogenicity assays assessing IgG and neutralizing antibodies to SARS-CoV-2.

To enable benchmarking of immunogenicity between candidate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there is a need for standardized, validated immunogenicity assays. In this article, we report the design and criteria used to validate immunogenicity assays and the outcome of the validation of serologic and functional assays for the evaluation of functional immune response and antibody titers in human serum. A quantitative cell-based microneutralization (MNT) assay, utilizing a reference standard, for detecting anti-SARS-CoV-2 spike protein-neutralizing antibodies in human serum and Meso Scale Discovery's multiplex electrochemiluminescence (MSD ECL) assay for immunoglobulin G (IgG) antibodies to SARS-CoV-2 spike, nucleocapsid, and receptor-binding domain (RBD) proteins were assessed for precision, accuracy, dilutional linearity, selectivity, and specificity using pooled human serum from coronavirus disease 2019 (COVID-19)-confirmed recovered donors. Both assays met prespecified acceptance criteria for precision, relative accuracy, dilutional linearity, selectivity, and specificity. Both assays demonstrated high specificity for the different SARS-CoV-2 antigens or virus tested, and no significant cross-reactivity with seasonal coronaviruses. An evaluation to compare the neutralizing activity in the MNT assay to the IgG measured using the MSD ECL assay showed a strong correlation between the presence of neutralizing activity and amount of antibodies against the spike and RBD proteins in sera from both convalescent and vaccinated individuals. Finally, the MNT assay was calibrated to the WHO reference standard to enable reporting of results in international units, thus facilitating comparison of immunogenicity data generated by different assays and/or laboratories. The MSD ECL assay has previously been calibrated. In conclusion, these validated assays for the evaluation of functional immune response and antibody titers following SARS-CoV-2 vaccination could provide a relatively simple standardized approach for accurately comparing immune responses to different vaccines and/or vaccination regimens.

Optimization and validation of a microcolony multiplexed opsonophagocytic killing assay for 15 pneumococcal serotypes.

Background: To streamline and improve throughput, the agar-based multiplexed opsonophagocytic killing assay (MOPA) was optimized and validated on a microcolony platform for use in the Phase III clinical trial program for V114, an MSD 15-valent pneumococcal conjugate vaccine candidate. Results & methodology: The precision, dilutional linearity and specificity of the microcolony MOPA (mMOPA) were assessed for each serotype in validation experiments. All prespecified acceptance criteria on assay performance were satisfied. Accuracy was assessed by testing 007sp and the US FDA reference panel and comparing to consensus values. The mMOPA produced comparable results to other opsonophagocytic killing assays/MOPAs. Conclusion: The mMOPA is suitable for measuring functional antibodies in adult and pediatric samples. Benefits include throughput, reduced analyst-to-analyst variability and automation potential.