Joyce S. Plested

Mucosal immunity, including antibodies like immunoglobulin A (IgA), function as the body’s first line of defense in the respiratory tract, particularly against viruses. An anti-rS protein IgA enzyme-linked immunosorbent assay (ELISA) was developed using the Omicron XBB.1.5 subvariant of SARS-CoV-2 and was validated to demonstrate the suitability of the method for testing saliva from SARS-CoV-2 vaccine clinical trials. This assay successfully met acceptance criteria for inter-/intra-assay precision, specificity, selectivity, linearity, lower/upper limits of quantitation, and assay robustness. The IgA in saliva was stable for up to 7 freeze/thaw cycles, for up to 48 h at 24 °C, up to 7 days at 4 °C, up to 3 weeks at −20 °C, and up to 1 year at −80 °C. After validation using Omicron XBB.1.5 rS protein, cross-reactivity was demonstrated with the SARS-CoV-2 variant JN.1. This validated IgA assay can be a valuable tool to assess mucosal IgA levels in SARS-CoV-2 clinical trials.

The pseudovirus neutralization (PNT) assay is an established high-throughput, robust, and efficient BSL-2 method for detecting neutralizing antibodies (NAbs) against SARS-CoV-2 with the correlates of protection previously established for the ancestral (Wuhan) strain. The PNT assay was validated using nonmatched ancestral sera with anti-JN.1 cross-NAbs, clinically matched JN.1 sera with anti-JN.1 NAbs, or nonmatched JN.1 sera with anti-LP.8.1, KP.2, or KP.3 cross-reacting NAbs. In line with predefined validation acceptance criteria, the PNT assay was precise, with %GCV ≤ 50 in ~90–100%/200 results (40 samples/strain). The acceptance criteria were met for linearity (slope ranged from 1.041 for ancestral sera with anti-JN.1 NAbs to 1.213 for JN.1 sera with anti-KP.2 NAbs), R² (0.9619–0.9944 for ancestral sera with anti–JN.1 NAbs), % relative bias, and total %GCV < 50 for almost all of the 15 serum samples tested for four virus strains. Human sera collected pre–COVID-19 had no detectable titer for tested Omicron JN.1 subvariants (<LLOQ) and all influenza and RSV clinical samples tested negative (<LLOQ) for SARS-CoV-2 and highly immunogenic for seasonal influenza or RSV post-vaccination, demonstrating the PNT assay specificity. Our data suggest this assay is suitable for assessing immune responses to ancestral and current SARS-CoV-2 strains and has potential for evaluating cross-reacting NAbs against emerging Omicron JN.1 subvariants.

Immunoglobulin (Ig) A acts as a first line of defense against respiratory pathogens. Mucosal IgA in salivary and nasal passages has a rapid response to antigens and can play a protective role against reinfection. The mainstay for analyzing SARS-CoV-2 infection and vaccine efficacy has been assessment of serum IgG levels; however, validated assays for assessment of mucosal IgA in clinical samples are necessary as new and adapted measures are generated to combat immune-evasive viral variants. A mucosal IgA assay was developed and tested through assessment of IgA levels in salivary samples from participants of the 2019nCoV-314/ NCT05973006 study. These participants had previously received ≥2 mRNA-based COVID-19 vaccinations prior to enrollment and received a single intramuscular study dose of NVX-CoV2601 (XBB.1.5) or bivalent vaccine (NVX-CoV2601 + NVX-CoV2373 [Wuhan]). Salivary samples were collected prior to vaccination on day 0 and on day 28 to assess response post vaccination. Both vaccine groups elicited a significant increase in anti–SARS-CoV-2 spike IgA against XBB.1.5. Furthermore, cross-reactivity via identification of anti-JN.1 and anti-Wuhan IgA was also observed. The detection of IgA in clinical mucosal samples through this assay will be a valuable tool in supporting vaccine development.

Currently available seasonal influenza vaccines confer variable protection due to antigenic changes resulting from the accumulation of diverse mutations. The analysis of new seasonal influenza vaccines is challenging in part due to the limitations of the traditional hemagglutination inhibition (HAI) assay with A/H3N2 strains. An improved and objective novel HAI assay was developed with recombinant virus-like particles (VLPs) and an egg-derived virus as agglutinins, the oseltamivir treatment of VLPs, human red blood cells, and using an automated image reader-based analysis of hemagglutination. HAI validation was demonstrated using four VLPs and egg-derived strains, with 46–56 serum samples tested 12 times in duplicate per strain. The validated HAI assay was precise as indicated by the percent geometric coefficient of variation for intra-, inter-, and total assay precision, as well as accurate as evidenced by percent bias measurements. The assay exhibited linearity, specificity for homologous type/subtype strains, and sensitivity with a starting dilution of 1:10. Assay robustness and sample stability were demonstrated as a percentage difference compared to reference condition. Validated HAI results were equivalent for the single and duplicate sample testing and correlated well with a qualified live wild-type influenza microneutralization assay. These findings demonstrate the suitability of this high-throughput novel modified validated HAI assay for evaluating vaccine immunogenicity and efficacy.

Currently available seasonal influenza vaccines confer variable protection due to antigenic changes resulting from accumulation of diverse mutations. Analysis of new seasonal influenza vaccines is challenging in part due to limitations of the traditional hemagglutination inhibition (HAI) assay with A/H3N2 strains. An improved and objective novel HAI assay was developed using recombinant virus-like particles (VLPs) and egg-derived virus as agglutinins, oseltamivir treatment of VLPs, human red blood cells, and using an automated image reader-based analysis of hemagglutination. HAI validation was demonstrated using four VLP and egg-derived strains, with 46–56 serum samples tested 12 times in duplicate per strain. The validated HAI assay was precise as indicated by the percent geometric coefficient of variation for intra-, inter-, and total-assay precision, and accurate as evidenced by % bias measurements. The assay exhibited linearity, specificity for homologous type/subtype strains, and sensitivity with a starting dilution of 1:10. Assay robustness and sample stability were demonstrated as a % difference compared to reference condition. Validated HAI results were equivalent for singleton and duplicate sample testing and correlated well with a qualified live wild-type influenza microneutralization assay. These findings demonstrate the suitability of this high-throughput novel modified validated HAI assay, for evaluating vaccine immunogenicity and efficacy.

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.

Neutralizing antibody responses from COVID-19 vaccines are pivotal in conferring protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Effective COVID-19 vaccines and assays measuring neutralizing antibodies against emerging variants (i.e., XBB.1.5, XBB.1.16, and XBB.2.3) are needed. The use of biosafety level (BSL)-3 laboratories for live virus assays results in higher costs and a longer turnaround time; therefore, a BSL-2–based pseudovirus neutralization assay (PNT) was developed. The pseudoviruses were produced by cotransfecting cells with plasmids encoding a lentiviral backbone-expressing luciferase reporter; non-surface proteins for lentiviral production; and ancestral or Omicron (BA.1 and BA.5) SARS-CoV-2 spike (S) proteins. The PNT was developed and optimized in dose and kinetics experiments. The representative serum samples (COVID-19–convalescent or NVX-CoV2373–vaccinated participants enrolled in the 2019nCoV-101 trial) demonstrated a wide dynamic range. The neutralization data showed robust correlation with validated anti-recombinant spike IgG levels and angiotensin-converting enzyme 2 inhibition titers (ancestral). This assay is suitable for measurement of the neutralization ability in clinical samples from individuals infected with SARS-CoV-2 or immunized with a COVID-19 vaccine. The results suggest that this PNT provides a lower cost, high-throughput, rapid turnaround alternative to BSL-3–based microneutralization assays and enables the discovery and development of effective vaccines against emerging variants.

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 ancestral and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5 and XBB.1.5) in human serum. For ancestral 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 5 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 ancestral 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.