Severe Acute Respiratory Syndrome Coronavirus 2 Receptor (Human Angiotensin-Converting Enzyme 2) Binding Inhibition Assay: A Rapid, High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation

Abstract

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show immune evasion of vaccine-derived immunity, highlighting the need for better clinical immunogenicity biomarkers. To address this need, an enzyme-linked immunosorbent assay-based, human angiotensin-converting enzyme 2 (hACE2) binding inhibition assay was developed to measure antibodies against the ancestral strain of SARS-CoV-2 and was validated for precision, specificity, linearity, and other parameters. This assay measures the inhibition of SARS-CoV-2 spike (S) protein binding to the receptor, hACE2, by serum from vaccine clinical trials. Inter- and intra-assay precision, specificity, linearity, lower limit of quantitation, and assay robustness parameters successfully met the acceptance criteria. Heme and lipid matrix effects showed minimal interference on the assay. Samples were stable for testing in the assay even with 8 freeze/thaws and up to 24 months in −80 °C storage. The assay was also adapted for variants (Delta and Omicron BA.1/BA.5), with similar validation results. The hACE2 assay showed significant correlation with anti-recombinant S immunoglobulin G levels and neutralizing antibody titers. This assay provides a rapid, high-throughput option to evaluate vaccine immunogenicity. Along with other clinical biomarkers, it can provide valuable insights into immune evasion and correlates of protection and enable vaccine development against emerging COVID-19 variants.

A Severe Acute Respiratory Syndrome Coronavirus 2 Anti-Spike Immunoglobulin G Assay: A Robust Method for Evaluation of Vaccine Immunogenicity Using an Established Correlate of Protection

“…Natural SARS-CoV-2 infection also induces anti-rS IgG as part of the body’s immune response. Levels of anti-rS IgG in the serum from both seropositive and seronegative subjects were evaluated using the anti-rS IgG assays for ancestral, Omicron BA.1, and Omicron BA.5 strains, followed by correlation with the neutralizing antibody levels (either live virus-based microneutralization assays or pseudovirus-based neutralization assays) and the hACE 2 binding inhibition assay [ 15 ].…”

“…Anti-rS IgG levels in the serum of either seropositive or seronegative subjects correlated well with the other measures of immunogenicity, including live wild-type virus/pseudovirus-based neutralization assays and hACE 2 binding inhibition assays for the ancestral and variant strains tested to date [ 15 ]. Based on these results, the validated IgG assay described here could be useful for measuring the immunogenicity of vaccines against both ancestral and emerging variants.…”

“…Each sample was assessed in the anti-rS IgG assay described here. The same samples were also tested in wild-type virus neutralization [ 13 ], pseudovirus neutralization [ 14 ], and hACE 2 binding inhibition assays [ 15 ]. Linear regression analysis was conducted using GraphPad Prism software (San Diego, CA, USA; Version 9.3.1) to determine the correlation between anti-rS IgG results and other assay results.…”

“…Microorganisms 2023, 11, x FOR PEER REVIEW 15 of 1 live wild-type virus neutralization assay was performed as previously described [13]. For (b,e), an hACE2 binding inhibition assay was performed as previously described [15]. Linear regression anal ysis was performed to compare results from the IgG assay and the other assays.…”

A Pseudovirus-Based Neutralization Assay for SARS-CoV-2 Variants: A Rapid, Cost-Effective, BSL-2–Based High-Throughput Assay Useful for Vaccine Immunogenicity Evaluation

“…For correlation with anti-S IgG antibodies and hACE2 binding inhibition, the samples were tested using a validated anti-S IgG assay (Clinical Immunology, Novavax) and using a previously published method [ 15 ] or a validated hACE2 binding inhibition assay (Clinical Immunology, Novavax) [ 16 ], followed by comparison of the data with the PNT results using linear regression analysis.…”