RESUMEN
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.
RESUMEN
As the COVID-19 pandemic continues, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge. Immunogenicity evaluation of vaccines and identification of correlates of protection for vaccine effectiveness is critical to aid the development of vaccines against emerging variants. Anti-recombinant spike (rS) protein immunoglobulin G (IgG) quantitation in the systemic circulation (serum/plasma) is shown to correlate with vaccine efficacy. Thus, an enzyme-linked immunosorbent assay (ELISA)-based binding assay to detect SARS-CoV-2 (ancestral and variant strains) anti-rS IgG in human serum samples was developed and validated. This assay successfully met acceptance criteria for inter/intra-assay precision, specificity, selectivity, linearity, lower/upper limits of quantitation, matrix effects, and assay robustness. The analyte in serum was stable for up to 8 freeze/thaw cycles and 2 years in -80 °C storage. Similar results were observed for the Beta, Delta, and Omicron BA.1/BA.5/XBB.1.5 variant-adapted assays. Anti-rS IgG assay results correlated significantly with neutralization and receptor binding inhibition assays. In addition, usage of international reference standards allows data extrapolation to WHO international units (BAU/mL), facilitating comparison of results with other IgG assays. This anti-rS IgG assay is a robust, high-throughput method to evaluate binding IgG responses to S protein in serum, enabling rapid development of effective vaccines against emerging COVID-19 variants.
RESUMEN
Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway.
