Rapid and Quantitative In Vitro Evaluation of SARS-CoV-2 Neutralizing Antibodies and Nanobodies

Abstract

Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on-lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 h, does not require BSL-2 facilities, and consumes only 8 μL of a low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC₅₀ values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the β and γ variants. Finally, we demonstrated that our assay enables the rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein, utilizing a set of nanobodies with known binding epitopes.

Figure 1.

Schematic of rapid in-vitro inhibition assay (RIVIA, left panel) and pseudovirus neutralization assay (pVNA) using HEK 293 cell (right panel).

Figure 2.

Optimization of RIVIA protocol. Ten-fold serial-diluted antibodies starting at 50 μg/mL were tested with four different types of SARS-CoV-2 spike proteins. (A) RBD. (B) S1 subunit. (C) S-ECD monomer. (D) S-ECD homotrimer. Error bars are obtained by duplicate measurements. The dashed lines mark 50% inhibition. The corresponding IC₅₀s are tabulated in Table S3.

Figure 3.

(A) Testing neutralizing nanobodies using RIVIA. Ten-fold serial-diluted nanobodies were tested starting at 50 μg/mL. The dashed lines provide an estimate for half neutralization. (B) Comparison of neutralizing efficacy of the strongest antibody, R001, and the strongest nanobody Nb21. Error bars are obtained by duplicate measurements. The dashed lines mark 50% inhibition. The corresponding IC₅₀s are tabulated in Table S4.

Figure 4.

Testing S-ECD homotrimer wild-type and three variants using RIVIA. Ten-fold serial-diluted antibodies and nanobodies starting at 50 μg/mL were tested. (A) Wild-type. (B) Variant Alpha Variant (B.1.1.7). (C) Beta Variant (B.1.351). (D) Gamma Variant (P.1). Error bars are obtained by duplicate measurements. The dashed lines mark 50% inhibition. The corresponding IC₅₀s are tabulated in Table S5.

Figure 5.

Screening of immune-evasive mutations on epitopes using RIVIA. Ten-fold serial-diluted nanobodies starting at 50 μg/mL were tested. (A) Nb21. (B) Nb34. (C) Nb93. (D) Nb95. Error bars are obtained by duplicate measurements. The dashed lines mark 50% inhibition. The corresponding IC₅₀s are tabulated in Table S6.