The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of infections and deaths globally. The continuous evolution of SARS-CoV-2, with the emergence of variants of concern (VOCs) like Alpha, Beta, Gamma, Delta, and Omicron, poses significant challenges. Omicron, in particular, exhibits substantial resistance to neutralization by existing antibodies. The spike (S) protein of SARS-CoV-2, responsible for viral entry into host cells, is the primary target for neutralizing antibodies (nAbs). The receptor-binding domain (RBD) within the S1 subunit of the S protein is crucial for binding to the host cell receptor, angiotensin-converting enzyme 2 (ACE2). nAbs targeting the RBD are critical for blocking viral infection. However, the evolving mutations in SARS-CoV-2 raise concerns about the efficacy of mAb therapies and vaccine-induced immunity. Many approved and clinical-stage mAbs show reduced efficacy against Omicron. This necessitates research into broadly neutralizing antibodies (bnAbs) that can overcome variant-specific immune evasion. This study investigates the longitudinal antibody responses in humans following vaccination with Ad5-nCoV, characterizing the bnAbs elicited and their structural basis for neutralization.

Previous research has highlighted the immune escape capabilities of various SARS-CoV-2 variants, particularly Omicron. Studies demonstrated reduced neutralization of Omicron by several approved mAbs and a significant decrease in neutralizing activity of vaccine-induced plasma. These findings emphasize the urgent need for bnAbs that can effectively neutralize a broad range of SARS-CoV-2 variants, including Omicron. The existing classification of RBD-targeting mAbs into groups (A-F) based on their binding modes and epitope locations provides a framework for understanding the mechanisms of neutralization and immune escape. Group A-D mAbs target the receptor-binding site (RBS) with varying binding modes and RBD conformations, while group E and F mAbs target more conserved epitopes outside the RBS. The effectiveness of different vaccine platforms in eliciting bnAbs against emerging variants also remains an area of ongoing investigation.

The study involved five healthy adults vaccinated with the Ad5-nCoV COVID-19 vaccine, with a prime dose delivered via aerosol and a boost dose intramuscularly. Blood samples (plasma and PBMCs) were collected at various time points before and after vaccination. Longitudinal analysis of antibody responses was performed, focusing on neutralization activity against different SARS-CoV-2 variants including Omicron. Next-generation sequencing (NGS) was used to characterize the B cell repertoires post-vaccination. Single B-cell sorting was employed to isolate S protein-specific human mAbs. The binding and neutralizing capacities of these mAbs were assessed against various SARS-CoV-2 variants using ELISA and pseudotyped/authentic virus neutralization assays. Surface plasmon resonance (SPR) was used to determine binding kinetics. Biolayer interferometry (BLI) was employed to study antibody competition. Cryo-electron microscopy (cryo-EM) was used to determine the three-dimensional structure of the complexes formed between selected nAbs and the S protein, clarifying the structural basis for neutralization. Finally, the prophylactic and therapeutic efficacy of selected mAbs was evaluated in a K18-hACE2 transgenic mouse model challenged with the Delta variant.

The study found that Ad5-nCoV vaccination induced robust IgG and IgA binding antibodies against SARS-CoV-2 variants, including Omicron, with neutralization activity significantly improved after the boost dose. NGS analysis revealed specific increases in certain immunoglobulin gene families and somatic hypermutation after vaccination. From single B-cell sorting, nineteen mAbs were isolated, twelve of which bound to the RBD. Antibody ZWD12 exhibited potent and broad neutralization against Wuhan-Hu-1, Alpha, Beta, Gamma, Kappa, Delta, and Omicron variants by blocking spike protein binding to ACE2 and protected mice in a challenge model. Cryo-EM structural analysis revealed the binding mode of ZWD12 to the RBD. ZWD12 and ZWC6 showed complete protection in the K18-hACE2 transgenic mouse model against lethal Delta variant challenge in prophylactic and therapeutic settings. The epitope of ZWD12 shows minimal overlap with Omicron mutation sites, explaining its broad neutralization ability compared to ZWC6, which interacts with regions containing Omicron mutations.

The findings demonstrate that Ad5-nCoV vaccination can induce bnAbs with potent neutralization activity against a range of SARS-CoV-2 variants, including Omicron. The mAb ZWD12, in particular, shows exceptional breadth and potency. The structural analysis reveals the key interactions between ZWD12 and the RBD, providing insights into the mechanism of neutralization and highlighting the importance of specific residues for antibody binding and neutralization. These findings contrast with the reduced efficacy of several other approved mAbs against Omicron, highlighting the potential of ZWD12 as a therapeutic candidate. The study also suggests that Ad5-nCoV vaccination, potentially with boosting strategies, could be effective in combating Omicron and other emerging variants.

This study identified a potent broadly neutralizing monoclonal antibody, ZWD12, elicited by Ad5-nCoV vaccination, effective against multiple SARS-CoV-2 variants, including Omicron. Structural analysis revealed the mechanism of neutralization. This mAb offers promise for therapeutic development and vaccine design. Further research should focus on optimizing vaccine strategies to induce similar bnAbs and explore the clinical potential of ZWD12.

The study's sample size was relatively small, limiting the generalizability of the findings. The mouse model used may not perfectly recapitulate human immune responses. The study primarily focused on neutralization; other aspects of immune response were not comprehensively investigated.