The ongoing COVID-19 pandemic is fueled by the emergence of SARS-CoV-2 variants with increased transmissibility and immune evasion capabilities. The B.1.351 variant, first identified in South Africa, harbors mutations that confer resistance to neutralization by some antibodies and convalescent sera, raising concerns about the effectiveness of current vaccines and therapeutic interventions. Therefore, it is crucial to identify and characterize potent neutralizing antibodies that can overcome these mutations and provide broader protection against emerging variants. This research focuses on identifying and characterizing such antibodies from a reservoir of SARS-CoV-2 RBD-specific monoclonal antibodies (mAbs). The study aims to evaluate the neutralizing potency of these mAbs against both the original SARS-CoV-2 strain and the B.1.351 variant, particularly focusing on their ability to bind and neutralize the virus despite the presence of immune escape mutations. The ultimate goal is to identify promising candidates for the development of effective therapeutic agents against COVID-19.

The literature extensively documents the emergence of SARS-CoV-2 variants with mutations in the Spike (S) protein, particularly within the receptor-binding domain (RBD). Studies show that these mutations can significantly impact the virus's ability to evade neutralization by antibodies generated through natural infection or vaccination. Several papers highlight the reduced effectiveness of certain neutralizing antibodies and convalescent sera against variants like B.1.351 and P.1. Conversely, research also identifies potent neutralizing antibodies that maintain activity against these variants, underscoring the need to investigate and develop antibody-based therapies with broad neutralization capabilities. The existing literature provides a foundation for understanding the challenge of immune escape and the necessity for developing novel strategies to combat emerging SARS-CoV-2 variants. This includes the identification and characterization of broadly neutralizing antibodies that can overcome the limitations of current therapies.

The study employed a rationally designed platform to identify neutralizing antibodies from a reservoir of SARS-CoV-2 RBD-specific mAbs obtained from COVID-19 convalescent individuals. Neutralizing potency was initially assessed using qRT-PCR to quantify inhibitory concentrations (IC50s) against the SARS-CoV-2 virus. Plaque-reduction neutralization testing (PRNT) was used to determine the neutralizing potency of the top 10 mAbs against authentic SARS-CoV-2 and B.1.351 viruses. Competitive ELISA and surface plasmon resonance (SPR) assays were performed to characterize the epitopes recognized by the mAbs and their ability to inhibit the interaction between the RBD and ACE2 receptor. Peptide ELISA was used to identify linear binding regions within the denatured RBD. Cryo-electron microscopy (cryo-EM) was employed to determine the three-dimensional structures of the antibody-antigen complexes to understand the molecular mechanisms of neutralization at a high resolution. In vivo prophylactic efficacy was evaluated in hACE2 transgenic mice challenged with authentic SARS-CoV-2 and B.1.351 viruses. The mice were treated with the mAbs before the viral challenge, and weight loss and viral loads were monitored. The detailed experimental procedures are provided in the supplementary information.

Three potent neutralizing mAbs, 58G6, 510A5, and 1369, demonstrated remarkable efficacy against both authentic SARS-CoV-2 and the B.1.351 variant. 58G6 and 1369 target overlapping steric regions (S470-495) on the RBD, including the E484K mutation, while 58G6 also binds to another region (S450-458). Epitope mapping revealed at least two independent epitopes on the RBD. 58G6 was found to recognize a linear region (S450-457) within the denatured RBD, 58G6 and 510A5 showed prophylactic efficacy in hACE2 transgenic mice. Both antibodies protected mice from weight loss and significantly reduced viral loads after challenge with both SARS-CoV-2 and the B.1.351 variant. Cryo-EM analysis revealed detailed molecular interactions between the antibodies and the RBD, providing insights into the mechanisms of neutralization. Specifically, the IC50 values against SARS-CoV-2 ranged from 1.285 to 9.174 ng/mL, and the neutralizing efficacy was not compromised by the B.1.351 mutations for 58G6 and 510A5. The SPR assay confirmed comparable binding affinity of 58G6 to the B.1.351 S1 subunit, while 510A5 and 1369 showed higher affinity to the SARS-CoV-2 S1 subunit. The cryo-EM structures revealed that the antibodies bind to the RBD in the 'up' conformation, potentially blocking ACE2 binding.

The identification of potent neutralizing antibodies like 58G6 and 510A5 that maintain efficacy against the B.1.351 variant addresses a critical need in the fight against COVID-19. The fact that these antibodies neutralize the variant despite mutations in key epitopes suggests that they target conserved or less mutable regions of the RBD. The in vivo efficacy data provides strong evidence for the potential of these mAbs as therapeutic agents. The structural analysis provides valuable insights into the mechanisms of neutralization, which could inform the design of future antibody-based therapies. The study also highlights the potential of convalescent plasma as a source of potent neutralizing antibodies. The findings are significant because they provide promising leads for the development of broadly effective therapeutic interventions against SARS-CoV-2 and its emerging variants. Future studies should focus on further optimization of these antibodies, including engineering for enhanced stability and manufacturability, as well as clinical evaluation of safety and efficacy.

This study successfully identified and characterized two potent neutralizing antibodies, 58G6 and 510A5, which effectively neutralize authentic SARS-CoV-2 and the B.1.351 variant, offering promising therapeutic options against COVID-19. The in vivo efficacy demonstrated in hACE2 transgenic mice further supports their clinical potential. Future research should focus on preclinical and clinical development to evaluate their safety and efficacy in human patients.

The study's sample size was limited to 74 patients, which may not fully represent the diversity of antibody responses in a larger population. While the study showed prophylactic efficacy in hACE2 transgenic mice, the results may not fully translate to humans. Further studies are needed to confirm the results in more comprehensive animal models and in human clinical trials. The correlation between specific mAbs and the blood samples from which they were isolated is difficult to establish due to the antibody isolation methods.