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.

Proteomics Platform Reveals Broad-Spectrum Nanobodies for SARS-CoV-2 Variant Neutralization

“…To identify nanobodies that bind to different spike variants, we screened 44 nanobodies using a SARS-CoV-2 spike variant microarray printed with 72 wild-type and mutated spike variants . Three anti-spike antibodies (#21, #51, and #73) employed in our previous studies served as positive controls, while the SARS-CoV-2 nucleocapsid protein included in the microarray acted as the negative control (Supporting Information Figure S3). , With this platform, we could visualize nanobodies with varying binding capabilities to S1 and RBD mutated proteins. Representative fluorescent detection images are displayed in Figure d, where five nanobodies (C-85, C-122, C-189, C-24, and C-282) exhibited distinct binding patterns to the 72 spike variants.…”

“…Third, the experiment of our microarray-based binding and neutralizing assay can be completed within 2 h, which can significantly save time and cost for nanobody screening against multi-SARS-CoV-2 variants. Notably, the results obtained by microarray-based binding and neutralizing assays were well in accordance with those of ELISA and pseudovirus-based assays, respectively. , To minimize the occurrence of false positives and false negatives during HT screening, we included the positive controls (anti-spike antibodies #21, #51, #73) and negative control (nucleocapsid protein) in our microarray experiments (Figures d,e and ), which can be employed to judge the specificity of nanobody’s binding and neutralization. In addition, all candidates selected by microarray will be validated by a bead-based multiplexed assay, a pseudovirus-based neutralization assay, and SPR analysis (Figures and ).…”

A highly sensitive bead-based flow cytometric competitive binding assay to detect SARS-CoV-2 neutralizing antibody activity

“…To overcome the limits of the aforementioned viral infection-based nAb assays, a strategy based on competitive binding (CB) to S1 or RBD of S protein between SARS-CoV-2 nAb and human ACE2 was established recently (14)(15)(16)(17)(18)(19)(20).…”

“…To overcome the limits of the aforementioned viral infection-based nAb assays, a strategy based on competitive binding (CB) to S1 or RBD of S protein between SARS-CoV-2 nAb and human ACE2 was established recently ( 14 – 20 ). The CB-based nAb assays usually have a 1-2 hours turnaround time, need less amounts of recombinant proteins, and can be converted into high throughput detection methods by combining with a magnetic bead based chemiluminescent or multiplex immunoassay ( 14 , 16 , 18 ).…”

“…In the CB-based nAb assays, either ACE2 protein ( 14 , 19 ) or RBD, S1 subunit of S protein, or even the whole S protein ( 15 – 18 , 20 ) was immobilized to catch the corresponding soluble counter-partner protein ( 14 – 19 ). However, the detection performance of these different immobilization strategies was never comparatively analyzed.…”