(855) Development & clinical validation of microneutralization assays for the robust quantification of neutralizing antibodies to human cytomegalovirus.

Introduction/Rationale: mRNA-1647 is an investigational vaccine composed of six mRNAs encoding human cytomegalovirus (hCMV) gB and pentamer antigens. To evaluate vaccine immunogenicity, we developed two hCMV microneutralization (MN) assays to measure neutralizing antibodies that can block viral entry and serve as potential correlates of protection.

Methods: Cell-based MN assays were validated for fibroblast-tropic AD169 and epithelial-tropic VR1814 strains. Infection was measured as foci-forming units via immunofluorescent detection of immediate early-1 protein. Neutralization titers were expressed as ID50, the serum dilution yielding 50% inhibition relative to virus-only controls. Validation followed a prespecified plan assessing limits of quantitation (LOQs), relative accuracy, dilution linearity, precision, specificity, and stability.

Results: Dynamic LOQs were 22–7,842 (fibroblast) and 13–206,687 (epithelial), with broader linearity for the epithelial assay. Relative accuracy met the ±2-fold criterion for both assays; the overall geometric mean fold bias (GMFB) was 1.00, and all dynamic-LOQ samples fell within ±2-fold criterion, confirming accuracy. Intra-assay precision CVs were 23.7% and 20.4%, and intermediate precision CVs were 53.7% and 28.7% for fibroblast and epithelial assays, respectively demonstrating acceptable precision. Specificity was confirmed in both assays by observing a ≥4-fold titer reduction when competed with homologous antigens, while heterologous antigen competition produced ≤2-fold reductions in ≥80% and ≥60% samples in fibroblast and epithelial assay respectively. Serum samples were demonstrated to be stable through 15 freeze–thaw cycles and up to 72 hours at ambient temperature.

Conclusion: These validated hCMV MN assays demonstrated acceptable accuracy, precision, linearity, stability, and specificity across the dynamic range, enabling reliable, strain- and cell type–specific quantification of nAb responses for clinical evaluation of mRNA-1647.