The COVID-19 pandemic has caused devastating health, social, and economic consequences globally. While effective vaccines have been developed, SARS-CoV-2 continues to circulate, with antigenic drift leading to variants that exhibit increased infectivity and escape from neutralizing antibodies induced by infection or vaccination. Most potent neutralizing antibodies target the receptor-binding domain (RBD) of the spike protein, but these are often vulnerable to mutations in the receptor-binding site (RBS), frequently observed in variants like Beta, Delta, and Omicron. However, a subset of broadly neutralizing antibodies (bnAbs) target highly conserved regions on the spike protein, neutralizing circulating variants of concern (VOCs), variants of interest (VOIs), and other SARS-related sarbecoviruses. Identifying these cross-neutralizing antibodies is crucial for combating the current pandemic and preparing for future outbreaks. Although potent cross-neutralizing antibodies exist, they are rare compared to SARS-CoV-2-specific neutralizing antibodies. These cross-neutralizing antibodies often target highly conserved sites on the spike protein such as the CR3022 site and N343 proteoglycan site in the RBD, and the S2 domain. While structures of cross-neutralizing antibodies targeting the CR3022 site have been reported, the extent to which the human immune system can develop effective protection against all current and future SARS-CoV-2 variants and other sarbecoviruses remains unclear. This study aimed to identify recurring motifs in antibodies that achieve broad neutralization, furthering our understanding of the immune response and informing the design of effective vaccines and therapeutics.

Previous research has highlighted the challenge posed by SARS-CoV-2 variants that escape neutralization by many antibodies. Studies have shown that several potent neutralizing antibodies target the receptor-binding domain (RBD) of the viral spike protein, but these are often susceptible to mutations within the receptor-binding site (RBS). A number of studies have characterized broadly neutralizing antibodies (bnAbs) that target conserved epitopes on the spike protein, offering a potential strategy for developing pan-sarbecovirus countermeasures. These bnAbs have shown efficacy against multiple SARS-CoV-2 variants and related coronaviruses. Structural studies have elucidated the mechanisms by which these bnAbs achieve broad neutralization. However, identifying and characterizing these cross-neutralizing antibodies remains a significant challenge. The study builds upon previous work that revealed the structures of cross-neutralizing antibodies binding to the conserved CR3022 site, showcasing their ability to neutralize a broad range of sarbecoviruses. This study adds to this body of knowledge by uncovering a shared motif in these broadly neutralizing antibodies.

The study utilized a multi-faceted approach combining structural analysis, computational sequence analysis, and functional assays. First, the researchers determined the crystal structure of the cross-neutralizing antibody ADI-62113 in complex with the SARS-CoV-2 RBD to a resolution of 2.6 Å. This allowed for a detailed analysis of the antibody-antigen interaction interface. Structural comparison of ADI-62113 with another previously characterized cross-neutralizing antibody, COVA1-16, revealed a shared YYDRxG motif within the heavy chain complementarity-determining region 3 (CDR H3). This motif was central to the antibody's interaction with a conserved site on the RBD. A computational search was then conducted through publicly available antibody sequence databases to identify other antibodies containing this or homologous motifs within CDR H3. This search involved defining a pattern encompassing the YYDRxG motif with length constraints to ensure that the motif was positioned correctly to interact with the RBD. The search yielded 153 antibodies with a YYDRxG pattern, and after further curation, a subset of 100 antibodies isolated from COVID-19 patients and mRNA vaccinees was selected for further analysis. The researchers performed immunoglobulin gene analysis to determine the origin of the identified antibodies. Yeast surface display was used to assess the binding kinetics of ADI-62113 and other antibodies to a panel of sarbecovirus RBDs. This technique allowed the researchers to measure the apparent dissociation constant (KD) for each antibody-RBD pair. Enzyme-linked immunosorbent assays (ELISAs) were employed to measure the binding of antibodies to various SARS-CoV-2 variant RBDs. Pseudovirus neutralization assays were conducted to determine the neutralization potency of antibodies against SARS-CoV-2, its variants of concern, and SARS-CoV. This involved generating pseudoviruses expressing spike proteins from different viruses and then measuring their ability to infect ACE2-expressing cells in the presence of antibodies. Statistical analysis using methods such as non-parametric Kruskal-Wallis tests and four-parameter logistic regression were used for data analysis and comparison.

The study's key findings include: 1. ADI-62113, a potent cross-neutralizing antibody, binds with high affinity to a broad spectrum of sarbecoviruses, demonstrating its potential as a pan-sarbecovirus therapeutic. 2. Structural analysis revealed that ADI-62113 binds to a highly conserved CR3022 site on the SARS-CoV-2 RBD, with CDR H3 playing a dominant role in the interaction. This site is largely unaffected by mutations in VOCs like Omicron. 3. Comparison of ADI-62113 and COVA1-16 identified a recurring YYDRxG motif in CDR H3, contributing significantly to the broad neutralization. This motif forms a conserved structure facilitated by a β-bulge, enhancing interactions with conserved RBD residues. 4. A computational search of antibody sequence databases revealed 100 antibodies from COVID-19 patients and vaccinees containing the YYDRxG motif or homologous sequences, many of which neutralize SARS-CoV-2 variants and SARS-CoV. The IGHD3-22 gene was highly enriched in these antibodies. 5. Analysis of these antibodies' ability to neutralize SARS-CoV-2 variants demonstrated that many retain their neutralization activity against challenging variants including Omicron. 6. The study confirmed that two representative antibodies (MOD8_P2_IgG_B11-P1369 and PZF12_P2_IgG_F7-P1369), selected solely based on sequence data, exhibit broad neutralization against SARS-CoV-2 variants and SARS-CoV, though with varying potencies against Omicron and SARS-CoV. 7. Analysis of antibody sequences from various donors (infected, vaccinated, or both) revealed that YYDRxG-containing antibodies are frequently elicited but are present at low serum abundance.

This study provides compelling evidence that the YYDRxG motif in CDR H3 represents a common solution used by the human immune system to generate broadly neutralizing antibodies against sarbecoviruses. The recurring nature of this motif across multiple antibodies, its association with potent neutralization of SARS-CoV-2 variants including Omicron, and its location within a conserved epitope on the RBD all suggest its importance. The findings highlight the potential to identify potent bnAbs based solely on sequence information, significantly accelerating the discovery and development of effective therapies and vaccines. The observation that this motif is found in antibodies from both infected individuals and vaccine recipients suggests that vaccines may be designed to specifically target the generation of these antibodies, achieving broader protection. The relatively low abundance of these antibodies in serum warrants further investigation into strategies to enhance their production and improve their efficacy. The study's success in identifying broadly neutralizing antibodies solely from sequence analysis offers a valuable tool for future research, potentially streamlining the process of antibody discovery for emerging viral threats.

This study demonstrates the importance of the recurring YYDRxG motif in generating broadly neutralizing antibodies against SARS-CoV-2 variants and other sarbecoviruses. The identification of this motif allows for rapid identification of potentially potent neutralizing antibodies using solely sequence data, significantly accelerating the development of vaccines and therapeutics. Future research should focus on strategies to enhance the production of these antibodies and to further elucidate the mechanisms underlying their broad neutralization capacity. Further studies investigating the design of vaccines that specifically elicit YYDRxG antibodies are also warranted.

The study primarily focuses on the analysis of antibodies identified from sequence databases, with neutralization data not available for all identified antibodies. The limited number of antibodies tested for neutralization against all VOCs may not fully represent the neutralization breadth of all YYDRxG-containing antibodies. The study's findings might be limited by the availability and bias in publicly available antibody sequence data.