Cryo-EM Reveals How FMDV O/Cathay Variants Evade Neutralizing Antibodies

Foot-and-mouth disease virus (FMDV) remains one of the most economically consequential pathogens of livestock, and the porcinophilic O/Cathay topotype has become a persistent concern in East and Southeast Asia. A new study published in PLOS Pathogens has now dissected, at near-atomic resolution, how recently circulating O/Cathay variants have accumulated mutations that allow them to slip past the neutralizing antibodies induced by infection and vaccination. By combining serological analysis of emerging field isolates with a panel of porcine-derived neutralizing monoclonal antibodies and cryo-electron microscopy, the researchers identified two antigenic regions whose structural remodeling underlies the antigenic drift that has progressively compromised vaccine performance.

The O/Cathay topotype is unusual among FMDV lineages. First recognized in the early 1990s, it has been strongly associated with pigs, a host in which the virus typically causes severe clinical disease, and it has repeatedly given rise to variants that spread across borders. Over decades of circulation, these viruses have continuously fixed amino acid substitutions on their outer capsid proteins, the very surfaces that antibodies recognize. The consequence is antigenic drift: sera collected from animals immunized with classical vaccine strains neutralize recent isolates less and less efficiently. The new work set out to determine precisely which capsid changes drive this escape and how they alter the molecular interface between virus and antibody.

The team first examined neutralization escape at the population level. When recent O/Cathay variants recovered in China were tested against porcine immune sera, they showed markedly reduced sensitivity compared with older strains, confirming that the drift observed in sequence data translates into genuine functional evasion of herd immunity. To trace this effect to specific viral determinants, the investigators generated a panel of seven porcine-derived neutralizing monoclonal antibodies, abbreviated pnAbs, from immune animals. Because these antibodies arise from the natural host species, they provide a biologically faithful readout of the antibody specificities that a vaccine must elicit to remain protective.

Mapping experiments with this antibody panel revealed two antigenic regions harboring the crucial determinants of escape. The first centers on VP1, the capsid protein that carries the conserved arginine-glycine-aspartic acid, or RGD, motif used by the virus to engage its cellular receptor. Immediately downstream of this motif, at the +2 position, several variants have replaced glutamine with histidine or arginine. Although this residue sits just outside the receptor-binding motif itself, the charge change it introduces modulates recognition by multiple pnAbs, including pO18-34, pO18-40 and pO18-43, and simultaneously reduces sensitivity to vaccine-induced sera. In effect, a single substitution adjacent to the receptor-binding site reshapes a surface that antibodies rely on for grip without abolishing receptor engagement.

The second antigenic region proved more surprising. Cryo-electron microscopy structures of the antibody pO18-29 in complex with two very different viruses, the ancient O/HN/CHA/93 strain isolated in 1993 and the recent variant O/18074, revealed a novel VP3-centered antigenic structure. VP3 sits closer to the threefold axis of the viral capsid and had not previously been appreciated as a dominant target for porcine neutralizing antibodies in this topotype. The structures showed that the antibody grips a surface assembled around VP3, and that two positions on this protein, residue 73 and residue 134, have been altered in circulating variants: valine at position 73 is replaced by lysine, and lysine at position 134 is replaced by threonine.

The structural comparison explained how these substitutions translate into antibody escape. In the older strain, the side chains at positions 73 and 134 participate in a network of hydrophobic interactions, hydrogen bonds and a salt bridge that anchors the antibody’s combining site onto the capsid. Replacing valine with the bulkier, positively charged lysine at position 73 disrupts the hydrophobic packing, while the lysine-to-threonine change at position 134 removes a charged contact and its associated hydrogen-bonding capacity. Together these mutations dismantle the chemical scaffolding that holds the antibody-virus complex together, weakening binding enough that neutralization fails even though the overall capsid architecture remains intact.

Perhaps the most consequential finding is that the two positions do not act independently. Analyses across the antibody panel showed that residues 73 and 134 synergistically regulate recognition by four of the seven pnAbs, pO18-2, pO18-18, pO18-28 and pO18-29. Neither substitution alone fully accounts for the escape phenotype; rather, the combination of changes produces a cooperative remodeling of the antigenic surface that no single mutation could achieve. This kind of epistatic interaction has important implications for vaccine design, because it means that predicting escape from single-residue comparisons between vaccine and field strains may substantially underestimate the antigenic distance that has accumulated.

The study also clarifies why serum neutralization titers have declined against recent O/Cathay isolates. The +2 residue change on VP1 affects a set of antibodies distinct from those targeting the VP3-centered site, meaning the variants have layered escape mechanisms across at least two antigenic regions. An animal vaccinated with a classical strain produces a polyclonal mixture of antibodies; if a substantial fraction of that mixture depends on contacts disrupted by either the VP1 or VP3 changes, the overall neutralizing capacity of the serum drops sharply, even if some antibody specificities remain effective. This layered drift helps explain the pattern of diminishing vaccine efficacy documented in the field.

From a surveillance standpoint, the identification of these determinants provides concrete molecular markers to monitor. Sequencing the VP1 +2 position and VP3 residues 73 and 134 in circulating isolates offers a rapid way to gauge how far a given field strain has drifted from existing vaccine components, and it gives vaccine manufacturers defined targets for antigen matching. Because the VP3-centered epitope is recognized by antibodies from multiple independent porcine donors in the panel, it appears to represent an immunodominant specificity rather than an idiosyncratic one, strengthening the case that changes at these positions are broadly relevant to population-level immunity.

The authors frame their findings as a molecular framework for the rational design of next-generation, broad-spectrum FMDV vaccines against emerging O/Cathay variants. By knowing exactly which capsid surfaces porcine neutralizing antibodies engage, and which substitutions dismantle those interactions, vaccine antigens can be engineered to either preserve the critical contacts or to present drifted versions that elicit antibodies capable of neutralizing the circulating variants. As O/Cathay topotype viruses continue to evolve under immune pressure, structural immunology of this kind offers a way to anticipate, rather than merely react to, the antigenic moves of one of the world’s most important animal pathogens.

Subject of Research: Structural basis of antigenic drift and antibody escape in foot-and-mouth disease virus O/Cathay topotype variants

Article Title: Structural basis of antigenic drift in FMDV O/Cathay topotype variants revealed by porcine-derived single B-cell neutralizing antibodies

Article References: Li, F., Wu, S., Huang, S., Lv, L., Cao, Y., Bao, H., Sun, P., Bai, X., Yang, J., Zha, J., Zhao, Q., Zhang, H., Li, D., He, J., Liu, Z., Li, P., Lu, Z., Lei, D., & Li, K. (2026). Structural basis of antigenic drift in FMDV O/Cathay topotype variants revealed by porcine-derived single B-cell neutralizing antibodies. PLOS Pathogens, 22(10), e1014650. https://doi.org/10.1371/journal.ppat.1014650

Image Credits: AI Generated

DOI: 10.1371/journal.ppat.1014650

Keywords: foot-and-mouth disease virus, FMDV, O/Cathay topotype, antigenic drift, neutralizing antibodies, cryo-electron microscopy, VP1, VP3, vaccine escape, porcine monoclonal antibodies, RGD motif, vaccine design