Validation of a severe acute respiratory syndrome coronavirus 2 microneutralization assay for evaluation of vaccine immunogenicity.

As variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, assessment of vaccine immunogenicity remains a critical factor to support continued vaccination. To this end, an in vitro microneutralization (MN50) assay was validated to quantitate SARS-CoV-2 neutralizing antibodies against prototype and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5, and XBB.1.5) in human serum. For the prototype strain, the MN50 assay met acceptance criteria for inter-/intra-assay precision, specificity, linearity, and selectivity. The assay was robust against changes to virus/serum incubation time, cell seeding density, virus content per well, cell passage number, and serum interference. Analyte in serum samples was stable up to five freeze/thaw cycles and for up to 12 months of storage at -80 ± 10 °C. Similar results were observed for the variant-adapted MN50 assays. The conversion factor to convert assay result units to WHO international standard units (IU/mL) was determined to be 0.62 for the prototype strain. This MN50 assay will be useful for vaccine immunogenicity analyses in clinical trial samples, enabling assessment of vaccine immunogenicity for ancestral and variant strains as variant-adapted vaccines are developed.

Altered IgG4 Antibody Response to Repeated mRNA versus Protein COVID Vaccines.

Repeated mRNA SARS-CoV-2 vaccination has been associated with increases in the proportion of IgG4 in spike-specific antibody responses and concurrent reductions in Fcγ-mediated effector functions that may limit control of viral infection. Here, we assessed anti-Spike total IgG, IgG1, IgG2, IgG3 and IgG4, and surrogate markers for antibody-dependent cellular phagocytosis (ADCP, FcγRIIa binding), antibody-dependent cellular cytotoxicity (ADCC, FcγRIIIa binding), and antibody-dependent complement deposition (ADCD, C1q binding) associated with repeated SARS-CoV-2 vaccination with NVX-CoV2373 (Novavax Inc., Gaithersburg, MD). The NVX-CoV2373 protein vaccine did not induce notable increases in spike-specific IgG4 or negatively impact surrogates for Fcγ effector responses. Conversely, repeated NVX-CoV2373 vaccination uniquely enhanced IgG3 responses which are known to exhibit strong affinity for FcγRIIIa and have previously been linked to potent neutralization of SARS-CoV-2. Subsequent investigations will help to understand the immunological diversity generated by different SARS-CoV-2 vaccine types and have the potential to reshape public health strategies.

Validation of a Pseudovirus Neutralization Assay for Severe Acute Respiratory Syndrome Coronavirus 2: A High-Throughput Method for the Evaluation of Vaccine Immunogenicity.

The evaluation of coronavirus disease 2019 (COVID-19) vaccine immunogenicity remains essential as the severe acute respiratory syncytial virus 2 (SARS-CoV-2) pandemic continues to evolve and as additional variants emerge. Neutralizing antibodies are a known correlate of protection for SARS-CoV-2 vaccines. A pseudovirus neutralization (PNT) assay was developed and validated at Novavax Clinical Immunology Laboratories to allow for the detection of neutralizing antibodies in vaccine clinical trial sera. The PNT assay was precise, accurate, linear, and specific in measuring SARS-CoV-2 neutralization titers in human serum for ancestral strain and the Omicron subvariants BA.5 and XBB.1.5, with an overall geometric coefficient of variation of ≤43.4%, a percent relative bias within the expected range of -60% to 150%, and a linearity value of R2 > 0.98 for all three strains. This pseudovirus assay will be useful for the analysis of vaccine clinical trial samples to assess vaccine immunogenicity. Future work will focus on modifying the assay for emerging variants, including XBB.1.16, EG.5.1, BA.2.86, and any other variants that emerge in the ongoing pandemic.