Somatic Mutations Widen the Reach of Public Antibodies Against SARS-CoV-2 Variants

As SARS-CoV-2 continues to evolve, the virus accumulates mutations in the receptor-binding domain (RBD) of its Spike protein, allowing newly emerging variants to escape many of the antibodies generated by prior infection or vaccination. Among the antibodies most frequently recruited by the human immune system against this virus are those encoded by the IGHV3-53 and IGHV3-66 immunoglobulin genes. These so-called public antibodies appear repeatedly in different individuals, whether they were infected with early variants of the virus or vaccinated with vaccines based on the prototype strain. They neutralize the original Wuhan strain of SARS-CoV-2 with remarkable potency, yet most of them fail to neutralize recently circulating variants, a limitation largely attributed to immune imprinting, which anchors antibody responses to the first viral version the immune system encountered.

Only a small number of IGHV3-53/3-66 monoclonal antibodies have been shown to cross-neutralize a wide range of variants. These broadly neutralizing antibodies, or bnAbs, carry a higher burden of somatic hypermutations, the point mutations introduced into antibody genes during affinity maturation in germinal centers, than antibodies that recognize only the prototype virus. Importantly, several of these mutations recur across independent bnAbs, suggesting a reproducible evolutionary path by which prototype-specific antibodies could acquire breadth. A new study published in Immunity, Inflammation and Disease set out to answer a critical question: are the mutations commonly observed in broadly neutralizing IGHV3-53/3-66 antibodies sufficient, on their own, to confer broad neutralizing activity on antibodies that currently lack it?

The research team, led by Takeo Kuwata, approached the problem by engineering a series of mutant antibodies based on the monoclonal antibody 9-105, which potently neutralizes pre-Omicron variants of SARS-CoV-2. To identify which mutations to test, the investigators mined sequence data from two previously described broadly neutralizing antibodies, K4-66 and K1-68, together with 36 IGHV3-53/3-66 monoclonal antibodies catalogued in the CoV-AbDab database that neutralize the XBB variant. From this analysis they pinpointed nine positions in the heavy chain that are frequently mutated in broadly neutralizing antibodies, using IMGT numbering conventions.

The most frequent bnAb-associated mutations proved to be strikingly consistent across the antibody set. Y66F was found in 65.8 percent of the 38 antibodies analyzed, F28I/L/V in 63.2 percent, T29I in 52.6 percent, S36R/A/N in 52.6 percent, V55L/I/S in 47.4 percent, S40T/N in 44.7 percent, A96V/P/G in 44.7 percent, N82D/S/T in 36.8 percent, and S58P/A in 26.3 percent. Two of these positions, F28 and V55, are already mutated in 9-105. The team therefore introduced the six remaining frequent mutations, T29I, S36R, S40T, S58P, Y66F, and A96V, into 9-105 in various combinations, generating five mutant antibodies for functional testing. The N82 position was excluded because of its low mutation frequency among bnAbs and its distance from the complementarity-determining regions that form the antigen-binding interface.

When the mutant antibodies were evaluated for neutralizing activity, the results were revealing. Against the 614G variant, which carries an early Spike mutation and closely resembles the prototype virus, all of the engineered antibodies performed comparably to the parental 9-105. Against the Omicron subvariants BA.1, BA.2, and BA.4/5, however, the mutants showed markedly enhanced activity. The pattern was even more telling when the researchers tested XBB.1.5 and XBB.1.16. Neither the parental antibody nor any of the partially mutated versions, designated 9-105M1 through M4, could neutralize these variants. Only 9-105M5, which carries all six introduced mutations, succeeded, demonstrating that breadth against XBB-lineage viruses requires the cooperative effect of multiple mutations acting together rather than any single substitution.

None of the antibodies, including 9-105M5, neutralized the JN.1 variant, although 9-105M5 again displayed greater cross-neutralizing activity than the others, indicating that even the fully engineered antibody has limits against the most recent evolutionary steps of the virus. To understand the biophysical basis of the gains in breadth, the researchers used surface plasmon resonance to measure binding of the mutant antibodies to the RBDs of both the Wuhan strain and BA.4/5. Binding to the Wuhan RBD was similar across all antibodies, including the parental molecule, whereas binding to BA.4/5 RBD varied substantially among the mutants. 9-105M5 exhibited a markedly higher resonance signal than any other antibody, requiring a reduced concentration of BA.4/5 RBD for the assay, which points to substantially enhanced binding.

Dissecting the contributions of individual mutations showed that 9-105M1 and 9-105M2, which carry changes in the CDR1 and CDR2 regions respectively, showed modest increases in binding signal, while the signals of 9-105M3 and 9-105M4 were comparable to the parental antibody. Notably, the binding affinity of 9-105M3 for the BA.4/5 RBD, measured at 9.3 nanomolar, was stronger than that of the other antibodies, which ranged from 12 to 81 nanomolar. Because 9-105M3 carries the Y66F substitution, the single most frequent bnAb-associated mutation in the analysis, the authors conclude that this substitution likely contributes to increased affinity for the BA.4/5 RBD.

To confirm that bnAb-associated mutations can enhance antibodies that already possess considerable breadth, the team introduced Y66F into a second IGHV3-53/3-66 public antibody, K4-66. This antibody already carries six bnAb-associated mutations and neutralized all tested variants up to JN.1, but it lacked the Y66F substitution seen in the majority of broadly neutralizing antibodies. The resulting mutant, K4-66F, neutralized the tested variants more effectively than the parental K4-66, with neutralization potency against the Beta, BA.1, BA.4/5, and JN.1 variants enhanced by factors of roughly 2.6 to 3.4. This result demonstrates that bnAb-associated mutations can further sharpen the potency of public antibodies that have already achieved broad neutralization, refining rather than merely initiating the maturation process.

Taken together, the study demonstrates that both the breadth and the potency of IGHV3-53/3-66 public antibodies can be enhanced by introducing mutations into the IGHV3-53/3-66 gene. The findings suggest that prototype-specific public antibodies, induced by infection with early SARS-CoV-2 variants or by vaccination with the prototype strain, retain the latent potential to mature into broadly neutralizing antibodies through the accumulation of somatic hypermutations. The results also indicate that multiple mutations at critical positions within the IGHV3-53/3-66 gene are required to achieve full breadth, since partial mutation sets improved neutralization of early Omicron variants but failed to extend activity to the XBB lineages.

The work builds on and extends prior observations in the field. Antibodies using the IGHV1-58 gene have also been reported to mature into broadly neutralizing antibodies, and previous studies have shown that introducing four mutations, G27E, T29I, S58P, and Y66F, into several IGHV3-53/3-66 public antibodies broadens their activity, while other groups have highlighted the contribution of G27E and the importance of light-chain gene usage in shaping the paratope. Because IGHV3-53/3-66 public antibodies are already present in a large proportion of the population, the authors propose that promoting their maturation could serve as a practical vaccine strategy, one that would guide existing immune memory toward protection against the diverse and continually evolving variants of SARS-CoV-2 rather than requiring the immune system to start from scratch with each new antigenic challenge.

Subject of Research: Somatic hypermutations that broaden neutralization by IGHV3-53/3-66 public antibodies against SARS-CoV-2 variants

Article Title: Somatic Hypermutations Enhance Neutralization Breadth of IGHV3‐53/3‐66 Public Antibodies Against SARS‐CoV‐2 Variants

Article References: Kuwata, T., Okazaki, K., Morioka, H., Kaku, Y., Shimizu, M., Maruyama, Y., Hamdy, A. K., The Genotype to Phenotype Japan (G2P‐Japan) Consortium, Suzuki, T., Hashiguchi, T., Sato, K., & Matsushita, S. (2026). Somatic Hypermutations Enhance Neutralization Breadth of IGHV3‐53/3‐66 Public Antibodies Against SARS‐CoV‐2 Variants. Immunity, Inflammation and Disease, 14(9), Article e70527. https://doi.org/10.1002/iid3.70527

Image Credits: AI Generated

DOI: 10.1002/iid3.70527

Keywords: SARS-CoV-2, IGHV3-53, IGHV3-66, broadly neutralizing antibodies, somatic hypermutation, immune imprinting, receptor-binding domain, Omicron variants, antibody affinity maturation, vaccine strategy, public antibodies, viral escape