Characterization of a Broadly Neutralizing Monoclonal Antibody against SARS-CoV-2 Variants

Abstract

The constant mutation of SARS-CoV-2 has led to the emergence of new variants, which call for urgent effective therapeutic interventions. The trimeric spike (S) protein of SARS-CoV-2 is highly immunogenic with the receptor-binding domain (RBD) that binds first to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and is therefore the target of many neutralizing antibodies. In this study, we characterized a broadly neutralizing monoclonal antibody (mAb) 9G8, which shows potent neutralization against the authentic SARS-CoV-2 wild-type (WT), Alpha (B.1.1.7), and Delta (1.617.2) viruses. Furthermore, mAb 9G8 also displayed a prominent neutralizing efficacy in the SARS-CoV-2 surrogate virus neutralization test (sVNT) against the Epsilon (B.1.429/7), Kappa (B.1.617.1), Gamma (P.1), Beta (B.1.351), and Delta Plus (1.617.2.1) RBD variants in addition to the variants mentioned above. Based on our in vitro escape mutant studies, we proved that the mutations V483F and Y489H within the RBD were involved in ACE2 binding and caused the neutralizing evasion of the virus from mAb 9G8. The development of such a cross-reactive neutralizing antibody against majority of the SARS-CoV-2 variants provides an important insight into pursuing future therapeutic agents for the prevention and treatment of COVID-19.

Keywords: SARS-CoV-2 variants; cross-neutralizing antibody; escape mutant; neutralizing epitope; spike RBD.

Figure 1

Neutralization of mAbs to authentic SARS-CoV-2 WT (hCoV-19/Singapore/2/2020), Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants in Vero E6 cells. (A) The neutralizing potency of mAb 9G8 by virus microneutralization against 100 TCID₅₀ of SARS-CoV-2 WT and variants. (B) The neutralizing potency of mAb 10B12 by virus microneutralization against 100 TCID₅₀ of SARS-CoV-2 WT and variants. The half-maximal inhibitory concentration (IC₅₀) values were determined using GraphPad Prism software (v. 9). The data presented are the mean of two biological replicates ± SD of the mean.

Figure 2

Analysis of mAb 9G8 blocking the SARS-CoV-2 spike RBD variant from binding to the human ACE2 receptor by surrogate virus neutralization test (sVNT). The percent inhibition of mAb 9G8 was measured against RBD of SARS-CoV-2 WT or SARS-CoV-2 variants such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), Delta Plus/AY.1 (B.1.617.2.1), and Epsilon (B.1.429/7). The mAb 6C8 specific for nucleocapsid protein was used as a negative control. The results were expressed as the arithmetic mean of percent-blocking values of triplicates and ±SD of the mean.

Figure 3

Antigenicity-mAb 9G8-related sites. Structural overview of SARS-CoV-2 S protein at open state based on PDB: 7DDN indicating the footprints of a broadly neutralizing antibody 9G8 on the RBD of S trimers. The S trimers are colored grey, blue, and green. The mAb binding amino acid residues Val 483 and Tyr 489 are indicated in orange and pink, respectively. (A) Upright view; (B) close-up view of SARS-CoV-2 S protein at open state; (C) top-down view.