The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has become a worldwide pandemic, and there is a pressing need for the rapid development of novel therapeutic strategies. SARS-CoV-2 viral entry is mediated by interaction between the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and host cellular receptor, human angiotensin converting enzyme 2 (ACE2). The lack of a high throughput screening (HTS) platform for candidate drug screening means that no targeted COVID-19 treatments have been developed to date. To overcome this limitation, we developed a novel, rapid, simple, and HTS binding assay platform to screen potential inhibitors of the RBD-ACE2 complex. Three "neutralizing" mouse monoclonal antibodies capable of blocking the RBD-ACE2 interaction were identified using our binding assay and pseudovirus neutralization assay followed by further validation with the Focus Reduction Neutralization Test (FRNT), which analyzes the neutralization capacity of samples in the presence of live SARS-CoV-2. Furthermore, the consistency of our binding assay and FRNT results (R² = 0.68) was demonstrated by patients' serum, of which were COVID-19 positive (n = 34) and COVID-19 negative (n = 76). Several small molecules selected for their potential to inhibit the Spike-ACE2 complex in silico were also confirmed with the binding assay. In addition, we have evaluated vaccine efficacy using binding assay platform and validated through pseudovirus neutralization assay. The correlation between binding assay & psuedovirus assay of the post vaccinated serum showed well correlated (R² = 0.09) Moreover, our binding assay platform successfully validated different Spike RBD mutants. These results indicate that our binding assay can be used as a platform for in vitro screening of small molecules and monoclonal antibodies, and high-throughput assessment of antibody levels after vaccination. When conducting drug screening, computer virtual screening lacks actual basis, construction of pseudoviruses is relatively complicated, and even FRNT requires a P3 laboratory. There are few methods to determine the competitiveness of the target drug and SRBD or ACE2. Our binding assay can fill this gap and accelerate the process and efficiency of COVID-19 drug screening.

Keywords: ACE2; Binding assay; COVID-19; FRNT; Inhibitor screening; Neutralization antibody; RBD; SARS-CoV-2; Spike-mutant; Vaccine.

Fig. 1

The dose responsiveness and specificity of S1RBD-ACE2 and ACE2-S1RBD binding assay. (A) Schematic diagram of the S1RBD-ACE2 binding assay. (B) Detection of ACE2- S1RBD binding interaction where ACE2 protein was coated on a microplate and S1RBD concentration was varied. (C) Schematic diagram of the ACE2-S1RBD binding assay. (D) Detection of S1RBD-ACE2 binding interaction where S1RBD protein was coated on a microplate and ACE2 concentration was varied. (E) Selectivity of binding assay for non-SARS viruses in patient sera. Data points represent individual patients seropositive for the virus indicated. Binding inhibition is the percentage of signal (OD450) in the presence of seropositive sera relative to no sera. In B and D, the average of three independent experiments is shown. Error bars indicate standard error of the mean (SEM).

Fig. 2

Validation of pseudovirus system. At 24 h post infection, pseudotype viral entry into A549 was determined by measuring relative luciferase units (RLU) in cell lysates and calculating the ratio of RLU relative to a control group with no serum. (A) Pseudotypes rVSV/spike (S) virus and rVSV/G (G) virus (no S control) were treated with normal or COVID-19-positive patient sera at different dilutions (100–400 fold). (B) Western blot analysis of cell lysates infected with pseudotype rVSV/spike (S) virus and rVSV/G (G) virus. M = protein marker. Blot was probed with mouse anti-SARS-CoV-2 S1RBD monoclonal antibody (clone 1F10-D4-B1; 1:1000 dilution). Three independent experiments were performed for and pseudotype at each dilution; error bars indicate standard deviation (SD). (C) The pseudovirus assay was validated using 13 serum samples: five from normal healthy donors (COVID-19 (−ve), and eight from patients with COVID-19 (+ve). Different titers (100, 200, 400-fold) of S pseudovirus were tested. Data were normalized with no serum value.

Fig. 3

Neutralization antibodies and drugs screening using S1RBD-ACE2 binding assay and pseudovirus harboring the SARS-CoV-2 S protein. (A) Comparison of the consistency of the results of three different neutralizing antibodies and one antibody against Nucleocapsid (N) protein as negative control using binding assay and pseudovirus assay. All antibodies were raised in mice and clone numbers are indicated for each. (B) Binding assay and pseudovirus in the presence of five small molecules (0.4 μg/ml) with known ability to interfere with the S1RBD-ACE2 interaction as well as two that target unrelated proteins (see also Table 1). (C) Titration of neutralizing antibodies in binding assay. (D) Titration of neutralizing antibodies in pseudovirus assay. Inhibition was calculated as percentage of signal (OD₄₅₀ or RLU for binding assay and pseudovirus assay, respectively) in the presence of inhibitor (antibody or small molecule) relative to no inhibitor. The average of 3 independent experiments was performed with error bars indicating SEM.

Fig. 4

Evaluation of S1RBD-ACE2 binding assay and pseudovirus assay using COVID-19 patient sera. (A) Study subjects used for validation of binding assay. (B) ROC curve generated for S1RBD-ACE2 binding assay of each patient serum sample. (C) ROC curve generated for pseudovirus inhibition test in determining SARS-CoV-2 neutralization activity. (D) Correlation between binding assay and pseudovirus assay using COVID-19 (+ve) and COVID-19 (−ve) serum samples. (E) Correlation between binding assay and pseudovirus assay using COVID-19 (+ve) serum samples alone. Inhibition was calculated as the fraction of signal (OD₄₅₀ or RLU for binding assay and pseudovirus assay, respectively) in the presence of serum relative to no serum.

Fig. 5

Box and whisker plot FRNT50 in COVID-19 negative (−ve) and COVID-19 positive (+ve) serum samples. Whiskers represent min to max values and all data points are shown.

Fig. 6

Evaluation of vaccine efficacy using S1RBD-ACE2 binding assay and pseudovirus assay. (A) ACE2 binding inhibition (%) and (B) pseudovirus neutralization (%) in subjects pre- and post-vaccination. (C) Correlation study between binding assay and pseudovirus assay in post vaccinated sample. Whiskers represent min to max values and all data points are shown.