Inhibitor screening using microarray identifies the high capacity of neutralizing antibodies to Spike variants in SARS-CoV-2 infection and vaccination

Abstract

Rationale: Mutations of SARS-CoV-2, which is responsible for coronavirus disease 2019 (COVID-19), could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. Methods: The SARS-CoV-2 spike variant protein microarrays were prepared by printing 72 S variants onto a chemically-modified glass slides. The neutralization potential of purified anti-S antibodies and serum from convalescent COVID-19 patients and vaccinees to S variants were assessed with the mSAIS assay. Results: We identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n = 104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and five prevalent S variants (D614G, B.1.1.7, B.1.351, P.1, B.1.617.2), thus demonstrating that high antibody diversity is associated with high NAb titers. Conclusions: Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.

Keywords: SARS-CoV-2; Spike; microarray; mutation; neutralizing antibody.

Figure 1

Development of the multiplexed spike-ACE2 inhibitor screening (mSAIS) assay. (A) A schematic illustration of the microarray-based mSAIS assay and potential biomedical applications; (B, C) Intra- and inter-array reproducibility of the mSAIS assay in the absence and presence of neutralizing antibodies, respectively. The r correlation of fluorescent signals within and between different arrays were calculated.

Figure 2

SARS-CoV-2 Spike-ACE2 interaction mapping across different S variants using the Spike variant protein microarray. (A) Detection of ACE2 binding at different concentrations to Spike variants immobilized on the array. Cy5-labeled ACE2 was added to the array at two-fold serial dilutions from 0 to 20 µg/mL, and the binding of ACE2 to the array was measured via the Cy5 tag. The EC50 was calculated using GraphPad Prism software 8.3 based on the fluorescent signal intensity. (B) Distribution of S-ACE2 binding affinities (EC50) across different SARS-CoV-2 S variants. (C) Fold changes of binding affinity between the wild-type and mutated S proteins. The x-axis represents the S variants and the y-axis represents the log2 transformed EC50 ratio between the wild-type and mutated S proteins. The red frame indicates the mutations that located at the RBD/ACE2 binding interface. (D) shows the binding curves of S variants different concentrations of Cy5 labeled ACE2. The EC50s are indicated. (E) Structural analysis of escape mutations within the RBD (gray color) that interact with ACE2 (blue color). RBD mutations (PDB ID: 6M0J) are labeled in red.

Figure 3

Heterogeneous responses of purified anti-RBD antibodies to SARS-CoV-2 mutations. (A-C) Distribution of neutralization activity (IC50) of anti-RBD antibodies #73, #21, and #53 to the S variants measured by the mSAIS assay, respectively. The x-axis represents the S variants and the y-axis represents the neutralization activity (IC50) of the antibody to each spike variant. (D, E) Fold changes of the neutralization activity (IC50) of antibodies #21 and #53 between the wild-type and mutant S proteins, respectively. The x-axis represents the S variants and the y-axis represents the log2 transformed IC50 ratio between the wild-type and mutated S proteins. (F, G) Structural analysis of mutations located at the RBD-ACE2 interaction interface that are resistant and sensitive to antibodies #21 and #53, respectively. The resistant and sensitive mutations are defined as ≥ 2-fold decrease or increase in neutralization activity (IC50) compared with the wild-type S protein. RBD is colored in gray, ACE2 is colored in blue, and RBD mutations are labeled in red (PDB ID: 6M0J). The red frame indicates the mutations at the RBD/ACE2 binding interface.

Figure 4

Heterogeneous responses of serological NAbs to SARS-CoV-2 S mutations in convalescent COVID-19 patients. (A) Hierarchical cluster analysis of serum NAbs to S variants from convalescent COVID-19 patients (n = 25) detected with the mSAIS assay. The rainbow color from white to red correspond to the EC50 from low to high, respectively. (B) Neutralization (EC50) of convalescent serum against S variants in the high NAb-titer group (n = 6) was compared to the low NAb-titer group (n = 15). (C) Comparison of escape mutations between convalescent COVID-19 patients with high and low NAb titers with EC50 thresholds set at 10, 30 and 50, respectively. (D) Neutralizing antibody titers (EC50) of convalescent sera against SARS-CoV-2 S-RBD variants using the mSAIS assay. The red dotted line indicates the median EC50 of serum to wild-type RBD. (E) Comparison of NAb titers between the wild-type and D614G S proteins. (F) Comparison of NAb titers between the wild-type S protein and S variants with known escape mutations. (G) Comparison of NAb titers between the wild-type S protein and S variants with newly identified escape mutations. Escape mutations have a significantly lower EC50 than wild-type mutations as determined using an unpaired t test with a p-value ≤ 0.05. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5

Heterogeneous responses of serological NAbs to the SARS-CoV-2 S mutations in vaccinated individuals. (A) Hierarchical cluster analysis of serum NAbs to S variants in vaccinees (n = 30) detected with the mSAIS assay. The rainbow color from white to red corresponds to the EC50 from low to high, respectively. (B) Neutralization (EC50) of vaccinee serum against S variants in the high NAb-titer group (n = 13) compared to the low NAb-titer group (n = 17). Data are median with all points. (C) Comparison of escape mutations between vaccinees with high and low NAb titers with EC50 thresholds set at 10, 20 and 30, respectively. (D) Neutralizing antibody titers (EC50) of vaccinee sera against SARS-CoV-2 S-RBD variants using the mSAIS assay. The red dotted line indicates the median EC50 of serum to wild type RBD. (E) Comparison of NAb titers between the wild-type and D614G S proteins. (F) Comparison of NAb titers between the wild-type S protein and S variants with known escape mutations. (G) Comparison of NAb titers between the wild-type S protein and S variants with newly-identified escape mutations. (H) Detection of NAb titers of vaccinee serum (n = 30, Table S3) to prevalent SARS-CoV-2 variants using the pseudovirus neutralization assay. (I) Hierarchical cluster analysis of the NAbs titers of vaccinee serum against prevalent SARS-CoV-2 variants. The rainbow color from white to red correspond the NAb titers from low to high, respectively. Escape mutations have a significantly lower EC50 than wild-type mutations as determined using an unpaired t test with a p-value ≤ 0.05. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 6

The NAb titer is associated with the diversity of anti-S antibodies in the serum of vaccinees. (A) A comparison of levels of antibodies that target the S1, RBD and S2 ECD between the high (n = 13) and low (n = 17) NAb titer groups. The data were measured by the SARS-CoV-2 proteome microarray and are plotted as the mean with the error bars representing the standard deviation (SD). (B) Identification of vaccine-induced IgG antibodies to epitopes on the S protein. (C) The total number of nonredundant immunogenic peptides on the S protein in the high (n = 13) and low (n = 17) NAb titer groups. A p-value (p) ≤ 0.05 calculated with the Mann-Whitney test was considered significant. *, P < 0.05; **, P < 0.01. NTD, N-terminal domain; RBD, receptor binding domain; S1/S2, S1/S2 protease cleavage site; S1, S1 Subunit; S2 ECD, spike S2 extracellular domain. The NAb titers were measured by the mSAIS assay and grouped in Figure 5.

Figure 7

Validation of anti-S antibody diversity in 104 vaccinees. (A) A comparison of levels of antibodies that target the S proteins and its domains measured by the SARS-CoV-2 proteome microarray between group 1 (n = 16), group 2 (n = 36), and group 3 (n = 52) with different NAb titers. The data plotted were the mean with the error bars representing the standard deviation (SD). (B) Landscape of vaccine-induced IgG antibody epitopes on the S protein. (C) The total number of nonredundant immunogenic peptides on the S protein in the three groups. A p-value (p) ≤ 0.05 by Mann-Whitney test was significant. *, P < 0.05, **; P < 0.01; ***, P < 0.001; ****, P < 0.0001. NTD, N-terminal domain; RBD, receptor binding domain; CTD, C-terminal domain; S1/S2, S1/S2 protease cleavage site; S1, S1 Subunit; S2 ECD, spike S2 extracellular domain. The NAb titers (ND50) was measured using pseudovirus neutralization assay.