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.

Keywords: COVID-19; SARS-CoV-2; assay validation; correlate of protection; human angiotensin-converting enzyme 2 (hACE2); immune evasion; immunogenicity; neutralizing antibody titers.

Figure 1

Procedure for performing the hACE2 binding inhibition assay. Ab—antibody; hACE2—human angiotensin-converting enzyme 2; His— polyhistidine; rS—recombinant spike; SARS-CoV-2—severe acute respiratory syndrome coronavirus 2; TMB—3,3′5,5′-tetramethylbenzidine.

Figure 2

hACE2 binding inhibition assay ancestral strain linearity. Two hACE2 binding inhibition-positive samples were tested undiluted or diluted in 1:2 series in 5 assay runs by 3 analysts. Linearity was then evaluated by calculating precision and accuracy of the titer at each dilution as well as the slope of linear regression lines for each sample. For the 2 different samples, (a,b), dilutions are shown from undiluted to 1:256 dilution.

Figure 3

hACE2 binding inhibition assay ancestral strain temperature and freeze/thaw stability. Samples were stored at various conditions (RT, refrigeration, or freezing) for various lengths of time, or were subjected to multiple freeze/thaw cycles, then were used in the hACE2 binding inhibition assay to determine sample stability. The acceptable range of deviation was 80% to 120% of the reference condition values (dashed lines).

Figure 4

hACE2 binding inhibition assay ancestral strain and variants quality control: performance of standards. (a) For ancestral strain, (b) Delta variant, (c) Omicron BA.1 variant, or (d) Omicron BA.5 variant. QC samples were tested in the hACE2 binding inhibition assay to examine assay performance. For ancestral strain, HQC should have a titer between 107.3 and 223.7, MQC should be between 46.3 and 90.3, and LQC should be between 25.7 and 52.2 (inclusive). For Delta variant, HQC should have a titer between 114.5 and 334.9, MQC should be between 64.8 and 191.0, and LQC should be between 13.6 and 36.8 (inclusive). For Omicron BA.1 variant, HQC should have a titer between 445.7 and 1161.2, MQC should be between 138.6 and 298.4, and LQC should be between 28.8 and 56.7 (inclusive). For Omicron BA.5 variant, HQC should have a titer between 426.9 and 1137.4, MQC should be between 201.0 and 487.9, and LQC should be between 34.8 and 63.6 (inclusive). GMT values are shown above each bar.

Figure 5

Correlation of ancestral strain and Omicron variant (BA.1) hACE2 binding inhibition with anti-rS IgG levels and neutralizing antibody titers. (a–c) Detection of anti-S IgG antibodies was performed, as previously described [18]. Linear regression analysis was performed after excluding the hACE2 binding inhibition values at LOD (for a,b) to compare results from the IgG assay and the hACE2 binding inhibition assay for (a) ancestral strain, (b) Omicron BA.1 variant, or (c) Omicron BA.5 variant. (d,e) Detection of neutralizing antibodies was performed, as previously described [18]. Linear regression analysis was performed to compare results from the microneutralization assay and the hACE2 binding inhibition assay for (d) ancestral strain or (e) Omicron BA.1 variant. Serum sample data were from Novavax clinical trials. LOD—limit of detection; Log₁₀—logarithm with base 10; MN₅₀—50% microneutralization.