The COVID-19 pandemic necessitates urgent development of effective interventions against SARS-CoV-2 variants. Monoclonal antibodies (mAbs) and nanobodies (Nbs) are promising therapeutic options. While Nbs offer advantages like stability, solubility, and cost-effective production, their broad neutralizing activity against circulating variants remained unclear. This study aims to address this gap by evaluating the efficacy of potent neutralizing Nbs against variants of concern (VOCs), including Alpha, Beta, and Gamma, and a highly evolved RBD variant (B62). The research focuses on understanding how these Nbs achieve broad neutralization through high-resolution cryo-electron microscopy structural analysis and functional assays, ultimately guiding the development of pan-coronavirus vaccines and therapeutics. The ACE2 receptor binding site (RBS) on the spike glycoprotein is a primary target of neutralizing antibodies and a hotspot for convergent mutations in circulating variants, potentially enhancing ACE2 binding and leading to immune evasion. Therefore, understanding Nb interaction with the RBS and other epitopes is crucial.

The literature review covers existing research on monoclonal antibodies and nanobodies as therapeutic agents against SARS-CoV-2. Several studies demonstrated the successful development and neutralization efficacy of mAbs isolated from convalescent plasma and nanobodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. These studies highlight the potential of these antibody formats for COVID-19 treatment, but also raise concerns about the emergence of antibody-resistant variants. Previous studies have shown that mutations in the RBD can lead to reduced efficacy of some neutralizing antibodies, emphasizing the need for broadly neutralizing agents that can combat emerging variants. This paper builds upon existing research by focusing on the detailed structural and functional characterization of highly potent nanobodies and their interaction with various SARS-CoV-2 variants.

The study employed a multifaceted approach combining various experimental techniques and computational analyses. Initially, enzyme-linked immunosorbent assays (ELISA) were performed to assess the impact of six critical RBD mutations on the binding of seven potent neutralizing Nbs. A pseudotyped virus neutralization assay was conducted to evaluate the neutralizing activity of the Nbs against the Alpha and Beta VOCs, using a wild-type SARS-CoV-2 strain as a control. To investigate the structural basis of broad neutralization, high-resolution cryo-electron microscopy (cryo-EM) was used to determine eight Nb-bound structures, involving six Nbs bound to either the prefusion-stabilized spike protein (S) or the RBD. The cryo-EM data were processed using Relion and CryoSPARC software, followed by model building, refinement, and analysis using Coot and Phenix. Molecular dynamics (MD) simulations were performed using NAMD to investigate the dynamics of Nb-RBD interactions, and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations were used to estimate the relative binding energies. Additionally, ACE2 competition assays and limited proteolysis experiments were performed to elucidate the mechanism of neutralization. Statistical analysis was performed using two-tailed student t-tests.

The study identified three classes of potent neutralizing Nbs. Class I Nbs bind to the ACE2-binding site (RBS) on the RBD and effectively block ACE2 receptor binding, though a single point mutation (E484K/Q) significantly reduces their binding affinity. Class II Nbs target highly conserved epitopes outside the RBS, exhibiting potent neutralization activity against various VOCs, including Alpha and Beta, and even the highly evolved B62 variant. They achieve neutralization by sterically hindering ACE2 binding. Class III Nbs recognize unique epitopes, likely inaccessible to larger antibodies, and employ distinct neutralization mechanisms. Nb17, a representative Class III Nb, may lock the spike protein in an all-RBD-up conformation, leading to impaired spike function, while Nb36 destabilizes the spike protein, potentially via interaction between an RBD and the adjacent N-terminal domain (NTD). Compared to monoclonal antibodies (mAbs), Nbs displayed a significantly lower probability of epitope residues coinciding with variant mutations, thus exhibiting superior resistance to VOCs. Structural comparisons revealed that while Nbs and mAbs both use hydrophobic interactions for RBD binding, Nbs utilize electrostatic interactions more extensively, have lower buried surface areas, and more efficiently exploit surface residues, particularly CDR3 loops, for high affinity binding. Class II Nbs, specifically Nb95 and Nb105, retained strong binding to SARS-CoV RBD, highlighting their potential for developing pan-sarbecovirus Nb constructs.

The findings demonstrate the remarkable resistance of potent neutralizing Nbs to the convergent mutations observed in circulating SARS-CoV-2 variants. The three classes of Nbs identified exhibit diverse mechanisms of action, with Class I primarily targeting the RBS, Class II targeting conserved epitopes, and Class III employing spike destabilization or conformational locking. The superior resistance of Nbs to VOCs, compared to mAbs, likely stems from their smaller size and ability to access epitopes less accessible to larger antibodies. This is further supported by the observation that Nbs tend to bind conserved regions of the RBD less frequently than mAbs. The discovery of conserved epitopes targeted by Class II Nbs opens up possibilities for the design of pan-sarbecovirus and pan-coronavirus therapeutics and vaccines. The unique mechanisms utilized by Class III Nbs warrant further investigation, particularly in the context of in vivo viral infection.

This study provides a comprehensive understanding of the structural basis and mechanisms of action of potent neutralizing nanobodies against SARS-CoV-2 variants. The identification of three distinct classes of epitopes, the superior resistance of Nbs to VOCs, and the elucidation of novel neutralization mechanisms, all suggest that Nbs represent a valuable and robust therapeutic strategy to combat the evolving SARS-CoV-2 pandemic. Future research should focus on further characterizing the in vivo efficacy of the identified Nbs and on developing multivalent Nb constructs targeting multiple epitopes for broader and more potent neutralization.

While this study provides extensive structural and functional characterization of the Nbs, further in vivo studies are needed to confirm their efficacy in treating COVID-19. The study mainly focused on in vitro neutralization assays and did not fully assess the Nbs' long-term effectiveness or potential for inducing immune escape. The relatively small number of Nbs analyzed might not fully represent the entire repertoire of potent neutralizing Nbs. The evolved B62 variant, while insightful, may not perfectly represent all future emerging variants. Finally, the entropy contribution to the binding free energy was not considered in the MM-PBSA calculations.