Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail

Li, Haozhou; Liu, Pan; Dong, Hu; Dekker, Aldo; Harmsen, Michiel M.; Guo, Huichen; Wang, Xiangxi; Sun, Shiqi

doi:10.1038/s41467-024-53027-5

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Published: 10 October 2024

Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail

Nature Communications volume 15, Article number: 8774 (2024) Cite this article

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Abstract

Unlike most other picornaviruses, foot-and-mouth disease (FMD) intact virions (146S) dissociate easily into small pentameric subunits (12S). This causes a dramatically decreased immunogenicity by a mechanism that remains elusive. Here, we present the high-resolution structures of 12S (3.2 Å) and its immune complex of a single-___domain antibody (VHH) targeting the particle interior (3.2 Å), as well as two 146S-specific VHHs complexed to distinct sites on the 146S capsid surface (3.6 Å and 2.9 Å). The antigenic landscape of 146S is depicted using 13 known FMD virus-antibody complexes. Comparison of the immunogenicity of 146S and 12S in pigs, focusing on the resulting antigenic sites and incorporating structural analysis, reveals that dissociation of 146S leads to structural alteration and destruction of multiple epitopes, resulting in significant differences in antibody profiles/lineages induced by 12S and 146S. Furthermore, 146S generates higher synergistic neutralizing antibody titers compared to 12S, whereas both particles induce similar total FMD virus specific antibody titers. This study can guide the structure-based rational design of novel multivalent and broad-spectrum recombinant vaccines for protection against FMD.

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Introduction

Foot-and-mouth disease (FMD), a highly relevant infectious disease of cloven-hoofed animals, has caused devastating economic losses of global livestock industry¹. In FMD-endemic areas, the substantial financial burden incurred annually is attributed to the costs of FMD vaccination and the resulting losses of production or culling livestock². The causative agent for FMD is the FMD virus (FMDV), an aphthovirus belonging to the Picornaviridae family. It encompasses seven serotypes (O, A, C, Asia 1, SAT 1–3), each with multiple constantly evolving subtypes and no cross-protection between serotypes. Notably, serotype A, which is highly prevalent, demonstrates significant genetic and antigenic heterogeneity within its subtypes³.

The mature FMD virion (146S) consists of a positive-sense RNA genome enclosed within a pseudo T = 3 icosahedral capsid. The capsid is formed through the assembly of 60 protomers. Each protomer comprises a single copy of the viral capsid proteins VP1, VP2, and VP3, along with an interior VP4 protein. Unlike picornaviruses from other genera, when exposed to mild heating (56 °C) or acidic pH (< 6.5), intact FMDV capsid degrades easily into VP4-deficient pentameric 12S particles and an insoluble aggregate of VP4^4,5.

Inactivated virions serve as the conventional FMD vaccine antigen. The vaccine potency in guinea pigs of 12S particles is 45–400-fold less than that of the intact 146S virion⁶. T-cell responses that aid neutralizing antibody production also rely on the integrity of 146S⁷. This disparity in immunogenicity contradicts with the conservation of numerous epitopes between 12S and 146S^5,8,9. The repetitiveness and particle size of viral antigens have also been shown to be the key to eliciting neutralizing antibodies (NAbs) for many viruses, but not for FMDV^10,11. Thus, although the interaction between FMDV and the host immune system is well studied¹², the molecular basis for the less efficient generation of protective antibodies by 12S is not yet fully understood.

NAbs serve as the primary protective mechanism in animals immunized with FMD vaccines^13,14. Hence, NAbs and antigenic determinants of FMDV which generate NAbs have raised significant interest. Initial delineation of FMDV antigenic sites primarily involved generating virus escape mutants utilizing NAbs. Studies with NAb escape mutants showed 5 sites where mutations are seen¹⁵. Others identified 3 mechanisms involved in neutralization^16,17. Among these mechanisms, there are NAbs targeting the GH loop region of VP1 and thereby interfere with FMDV attachment to the cellular integrin receptor. Another mechanism of NAbs is the binding to VP1 residue 173 (VP1-173), triggering extensive viral aggregation. The third mechanism comprises 146S-specific NAbs, which bind to VP3-175 and VP3-178, they neither impede attachment nor induce aggregation and their underlying neutralizing mechanism remains unresolved. Recently, we isolated 146S-specific single-___domain antibodies (VHHs) against serotypes SAT2 and Asia1¹⁸ and serotype A¹⁹ for use in tests to monitor 146S integrity during the vaccine manufacturing process. Others isolated monoclonal antibodies (mAbs) for this purpose²⁰. Those studies showed that most antibodies are capable of binding to both 146S and 12S, while antibodies that are specific to either 146S or 12S are more rarely isolated.

In recent years, X-ray crystallography and cryogenic electron microscopy (cryo-EM) studies yielded high-resolution 3D structures of FMDV 146S particles or recombinant virus-like particles (VLPs) for various serotypes and including complexes with integrin receptor or antibodies^{21,22,23,24,25,26}. These studies shed light on the neutralizing mechanism of the FMDV NAbs, which involves simultaneous steric hindrance of integrins and a second cell attachment heparan sulfate receptor, destabilizing the viral capsid²⁵, or preventing genome release by capsid overstabilization²⁷. In contrast to 146S, 12S has only been studied in low resolution (8 Å), and the comparison of the structural differences between 146S and 12S was limited to a mechanistic understanding⁵.

In this work, to understand the mechanism underlying the reduced immunogenicity of 12S, we obtain high-resolution cryo-EM structures of FMDV 12S and its immune complex with a VHH binding the interior of the particle, both at a resolution of 3.2 Å, in addition with the resolution of two 146S-specific VHHs complexed to the capsid surface at 3.6 Å and 2.9 Å. To our knowledge, these are the first observations of high-resolution structure of 12S pentamers for any picornavirus, and an antibody targeting the interior of a picornavirus. Furthermore, we conduct comparative studies of the epitope specificity of polyclonal antibodies elicited in pigs either by 146S or 12S particles. By combining these results with available FMDV complex structures, we are able to gain insights into the molecular basis of antigenicity and immunogenicity of different FMDV particle forms.

Results

Characterizations of 146S- and 12S-specific antibodies

In previous studies, we isolated nearly 50 FMDV anti-capsid llama single-___domain antibodies^18,19. Among these, M678F and M688F¹⁹ are 146S-specific for serotype A strain A/TUR/14/98, while M3F is 12S-specific with broad spectrum recognition for all seven serotypes except for SAT2¹⁸ (Fig. 1a). Cell-based neutralization assays indicated that M678F and M688F are neutralizers that both have a 50% neutralizing concentration value (IC₅₀) of 1 µM (95% CI: 0.2–2 µM) (Fig. 1b). Interestingly, a clear synergistic neutralization was achieved by a cocktail of M678F and M688F, with IC₅₀ of 0.02 µM (95% CI: 0.01–0.04 µM), indicative of distinct neutralizing mechanisms for these two VHHs. The mixture surpassed the neutralizing potency exhibited by M8 at IC₅₀ of 2 µM (Fig. 1b), which was earlier shown to be a potent NAb²⁵. Biolayer interferometry (BLI) experiments using strictly monomeric VHHs (suffix K) verified tight interactions for these three antibodies to the antigens with binding affinities (K_D) ranging from 50 to 0.7 nM (Fig. 1e–g). To explore the simultaneous binding of M678F and M688F to 146S capsid, a competitive surface-plasmon resonance (SPR) binding assay was performed. Saturation binding of either M678F or M688F to a sensor loaded with 146S capsid could not prevent the binding of the alternate VHH (Fig. 1c, d). This indicates binding of independent antigenic sites. This underlines the potential of these two antibodies in formulating cooperative two-antibody cocktails as they bind simultaneously to distinct epitopes. As expected based on its 12S specificity, M3F was non-neutralizing, as was earlier shown^28,29.

**Fig. 1: Characterization of M678F and M688F.**

Structures of 146S FMDV in complex with a two-antibody cocktail

To define the key epitopes and atomic determinants of the interactions between FMDV and its two antibodies precisely, we set out to resolve structures of FMDV mature particles (146S) in complex with M678F or M688F or both M678F and M688F. Icosahedral reconstructions of these three complexes were achieved at resolutions of 3.6 Å, 2.9 Å and 2.9 Å (Supplementary Figs. 1, 2, 3). As expected, M678F and M688F recognize two distinct antigenic sites (Fig. 2a). The density maps show that M678F and M688F target the viral particle around its 3-fold and 5-fold vertices, respectively, and thus up to 60 copies of M678F or M688F could bind to each particle (Fig. 2a–d). In the immune complex with both M678F and M688F, there are a total of 120 antibodies (60 M678F and 60 M688F) concurrently bound to each particle and the interaction modes between M678F/M688F and the particles are essentially indistinguishable to those in 146S-M678F and 146S-M688F complexes. Footprint analysis reveals that 146S-M678F and 146S-M688F extend across two adjacent protomers with a buried area of 913.3 Å² and 585.7 Å², respectively on the particle (Fig. 2b, c). All three complementary determining regions (CDRs) of M678F are involved in the engagement with the BC and HI loops from VP3 as well as VP2 HI loop (Fig. 2e and Supplementary Table 1). The 146S-M678F interaction establishes an elaborate hydrogen-bonding network, including 7 hydrogen bonds formed by Y29, D50, S30, R96, R97, and S106 from M678F and K198 and T195 from VP2 and E68, R67, N197 and Q71 from VP3, respectively. The interaction patch in M688F comprises all three CDRs: CDR1 (residue 29), CDR2 (residues 52, 54, and 55), and CDR3 (residues 103 and 114). The epitope recognized by M688F contains 17 residues, only locating in the VP1 BC, CD, EF and HI loops (Fig. 2f). Tight binding between M688F and FMDV is chiefly due to extensive hydrophilic interactions, including 7 hydrogen bonds, the antibody components of these include the side-chains of S29, R52, S54, D55, T103 and Y114, which interact with T87, S173, N43, T45, E83 and D171, respectively (Fig. 2f and Supplementary Table 2).

**Fig. 2: Cryo-EM structures of FMDV in complex with M678F or M688F.**

Double block neutralization mechanisms for a two-antibody cocktail

To shed light on the molecular basis for the synergistic neutralization of FMDV by M678F and M688F, we first explored their effects on the stability of viral particles upon binding at neutral and acidic pH. Particle stability was evaluated by PaSTRy assays³⁰ to detect exposure to viral RNA. Unlike other NAbs that destabilize/damage viral particles by receptor mimicry, binding of M678F or M688F stabilizes the particles at pH 6.5 by about 4 °C (Fig. 3a). A moderate synergy in increasing viral stability raised by M678F and M688F together, in particular at pH 5.75 was also observed. Given the fact that FMDV uncoating occurs at pH values slightly below neutrality via dissociating into pentamers to release its genome RNA, physical over-stabilization of viral particles that interfere with normal uncoating may be a possible neutralization mechanism for M678F and M688F. To further verify this speculation, we performed a negative staining EM analysis of these two NAb-virion complexes. Visual inspection of the EM micrographs showed that as many as 95% of FMDV particles incubated with mixture of M678F and M688F were intact at pH 5.75 at room temperature (Fig. 3b). In contrast to this, nearly all unbound FMDV virions had disassembled into pentameric subunits at the same condition (Fig. 3b). Recognition modes of these two antibodies reveal that 60 copies of M678F bind to each particle (Fig. 2a), around the edges of the pentameric building blocks of the virus—specifically, between the 2-fold and 3-fold axes, firmly connecting two neighboring pentamers (Fig. 3c). This binding patch is broadly similar in ___location to those observed previously for F145 antibody bound to FMDV-OTi and NAb A9 bound to EV71³¹. In general, antibodies specific for epitopes near the 3-fold axis are capable of interfering with viral stability and uncoating (e.g., triggering/impeding the conversion from mature virions to uncoating intermediate particles) or even causing physical destabilization/damage to the virions³². However, M688F targets to the FMDV outer surface within the pentameric building blocks at a site near the 5-fold axis, stably linking five adjacent VP1 subunits and constructing the “star-shaped” protrusions surrounding the mesa to stabilize the particle (Fig. 3c). These structural evidences support the experimental observation that M678F and M688F synergistically over-stabilize viral particles, subsequently interfering with viral uncoating. FMDV uses integrins as receptors to mediate viral attachment via recognizing a small cleft at the subunit interface of the integrin head²³. Although the footprints of M678F and M688F on the FMDV surface do not overlap with the integrins receptor site, superpositions of the FMDV-M678F/FMDV-M688F and FMDV-αvβ6 complex structures revealed clear clashes between the two subunits of αvβ6 and M678F/M688F (Fig. 3d). Remarkably, the structural overlay analysis reveals that M678F competes with the αv subunit and M688F occupies the β6 subunit binding site (Fig. 3d). Thereby, simultaneous binding of M678F and M688F could synergistically block the attachment of integrin to FMDV owing to the substantially stronger binding affinities of these two antibodies for FMDV when compared to that of the integrin. In summary, M678F and M688F execute their potent neutralization against FMDV infection in a double block mechanism by blocking viral attachment to host cells and interfering with viral uncoating.

**Fig. 3: Neutralization mechanism of M678F and M688F.**

High resolution structure of dissociated 12S and altered antigenicity

Native FMDV particles were subjected to low pH treatment, before purifying dissociated 12S pentamers by sucrose density gradient ultracentrifugation (see “Methods”). We determined the first high-resolution structure of 12S pentamers for FMDV at 3.2 Å, and to our knowledge for any picornavirus (Fig. 4a and Supplementary Fig. 3d). The dissociated pentamer is largely ordered, apart from the disorder of VP1 and VP2 N-termini and the loss of VP4, breaking pentamer-pentamer interactions that should have existed in intact capsid. Although the overall structure of dissociated pentamer resembles those in the 146S and 75S particles, dissociated pentamer displays a significantly increased curvature with the top pushing the VP1 external surface more outward and the edge (VP2 and VP3) more inward (Fig. 4b and Supplementary Fig. 3d), presumably due to the lack of geometric restraints raised by the particle formation. In contrast, 146S pentamers are relatively flat, driven by the pentamer-pentamer interactions during the assembly of viral capsid. These structural changes are accompanied by a 3° counterclockwise rotation of the protomeric building block, which pivots about the central shell at the icosahedral 5-fold axis (Fig. 4c). In addition to large-scale disorder (described above), small-scale conformational alterations/shifts primarily occur at the exposed regions, including a number of reported epitopes, such as DE, FG loops of VP1, BC, EF loops of VP2 and EF, HI loops of VP3 (Fig. 4d), indicating likely differences in antigenicity between the 12S and 146S, which is confirmed by several NAbs in this study (Fig. 1). Furthermore, structural details of 12S pentamer also explain our immunogenic results in pigs where the antisera from 12S exhibited ~100-fold lower neutralizing titers against FMDV than those from 146S, albeit both could elicit high and comparable binding antibody titers (Fig. 4e). For many picornaviruses, at least two (mature virions or natural empty particles or uncoating intermediate/dissociated 12S particles) predominate types of particles with various ratios are produced during natural infection³³. Remarkably, the ratios of these particles that may directly determine immunogenicity and vaccine efficacy could be affected by a number of factors, including viral strain, cell culture conditions, purification procedures and storage conditions. Structural and immunogenic characterizations of 12S pentamers provide lessons for FMDV vaccine production: the procedures for virus production and storage need to be improved to minimize amounts of antigenically altered 12S pentamers, which substantially dilutes protective antigens.

**Fig. 4: Cryo-EM structure of FMDV dissociated pentamer.**

Structural dissections for 12S-specific antibody and NAb category

To further unveil the molecular basis for precise recognitions of 12S- and 146S-specific antibodies, we determined atomic structures of dissociated pentamer in complex with M3F, a 12S-specific non-neutralizing binder (Figs. 5a and 1a). Interestingly, M3F recognizes a conformational epitope located at the inner surface within the edges of the pentamers (Fig. 5a), structurally explaining the incapability in binding to intact viral particles. The binding position for M3F is adjacent to the 3-fold axis, roughly opposite to that observed for M678F, but comparatively more far away from the 3-fold axis. M3F mainly interacts with VP3, involving βD, βE, βF, βG strands and CD, EF, FG loops in VP3 as well as the VP2 N-terminus (Fig. 5b). The epitope for M3F includes residues E52 at the VP2 N-terminus, T104 in VP3 CD loop, H108 in VP3 βD, R120 in VP3 βE, C143 in VP3 EF loop, I144, H145, A146, E147, W148 and D149 in VP3 βF, L152 and N153 in VP3 FG loop, and F156, T157, F158, S159 and P161 in VP3 βG (Fig. 5b and Supplementary Table 3). Extensive hydrophobic interactions contribute to the tight binding between M3F and 12S pentamer (Fig. 5c).

**Fig. 5: Structural topographical classification of FMDV antibodies.**

To acquire greater insight as to the antibody binding sites on the FMDV capsid, 12 FMDV NAbs and one 12S-specific binder with available structures^24,25,27, including 3 cryo-EM structures determined in this manuscript (Supplementary Fig. 3 and Supplementary Table 4) were examined. By using cluster analysis on epitope structures, the antibodies were primarily classified into five groups (I, II, III, IV and V) (Fig. 5d). Additionally, we superimposed structures of FMDV capsid from these complex structures and calculated the clash areas between any 2 antibodies (Fig. 5e). Both strategies yielded identical results. Among these, class V antibodies, represented by M3F, are 12S pentamer specific and non-neutralizing. Antibodies from class I and II, target VP1 only around the 5-fold axis, among which class I antibodies form a compact cluster at the “plateau”, whereas class II antibodies are more outspread (Fig. 5f). Notably, class II antibodies bind to the VP1 GH loop, directly competing with αvβ6^23,25. Epitopes for class III and class IV antibodies mainly locate on VP2 and VP3 surrounding the 2- and 3-fold axes, respectively (Fig. 5g). Two further 146S-specific NAbs, M691F and M702F, were earlier shown to bind the same site as M678F and M170, and are thus also class IV NAbs²⁹. Remarkably, at least 3 types of NAbs have potential to simultaneously bind to the FMDV capsid, formulating multiple-antibody cocktails to prevent viral infection, akin to polyvalent antiserum elicited by vaccination.

Antibody composition of polyvalent antisera elicited by 12S and 146S particles

Given the fact that 12S and 146S have a different antigenicity, we analyzed antibody composition of polyvalent antisera elicited by equal amounts of 12S or 146S particles in groups of 6 pigs followed for 4 weeks. NAbs emerged one week after vaccination and reached a plateau on 21 days (Fig. 4e). Serum samples on days 28 post vaccinations were selected and representative antibodies from all five classes were used for liquid-phase blocking ELISA to evaluate their antibody levels in the sera of 146S or 12S immunized pigs. Clearly, antibody titers with similar or competitive epitopes from classes I to IV NAbs were 100-1000-fold higher in 146S immunized antisera than those in 12S vaccinated antisera. By contrary, non-neutralizing Abs using a class V antibody (M3F) were similar between 12S immunized pigs and 146S immunized pigs (Fig. 5h). Antibody composition analysis for polyvalent antisera elicited by 12S and 146S particles dissects the difference in immunogenicity, by which 12S particles are incapable of inducing high levels of various NAbs, but still induce non-neutralizing Abs.

Discussion

The reduced protective efficacy of FMDV dissociated 12S has been observed for decades. However, its molecular mechanism remained elusive due to missing detailed information about alterations at the atomic level. Herein we determined the first high-resolution structure of the dissociated pentamer. Compared to the relatively flat 146S pentamer, the dissociated pentamer possesses a significantly increased curvature, which is accompanied by tectonic shifts, large-scale disorder and conformational shifts at several positions, including the DE, FG loops of VP1, BC, EF loops of VP2 and EF, HI loops of VP3, which affect epitopes of NAbs from classes I, III and IV. Class III NAb R55 and class IV NAb M678F can also exhibit 146S specificity by binding across the interface of two adjacent pentamers at the 3-fold axis, from where 12S dissociates (Fig. 5i, j). In contrast, class I NAb M688F achieves 146S specificity by binding across two adjacent protomers at the 5-fold vertex, where the dissociation leads to increased curvature, enlarging the distance between these protomers at the 5-fold vertex on 12S (Fig. 5j). These structural alterations thus explain the reduced protective efficacy of dissociated pentamers. In addition, we report the structure of the dissociated pentamer complexed to a 12S-specific non-neutralizing VHH (M3F). To our knowledge, it is the first structural observation that an antibody targets the inside of a picornavirus capsid. Surprisingly, the conformational epitope primarily comprises four β-strands in VP3, rather than exposed loops. Even in target animals immunized with 146S, high antibody titers against non-protective epitopes located at the capsid interior were generated as demonstrated by M3F LPBE. These expand our understandings in epitope distributions and antibody response ranging from the outer surface to inner surface and antigenic alterations at atomic resolution.

A high level of capsid flexibility serves as uncoating preparation for picornaviruses. It includes a “breathing” process that involves transient exposure of the N-termini of VP1 and VP4³⁴, and a pre-dissociation cell entry intermediate^35,36,37. In enteroviruses, such intermediates open pores around the 2-fold and quasi-3-fold axes, facilitating the release of the N-termini of VP1, forming a umbilical-like structure connecting the capsid and membrane^38,39, whereas VP4 forms a homo-multimeric virally encoded channel for RNA delivery to the cytoplasm⁴⁰. Belonging to same genus as FMDV, equine rhinitis A virus also exhibits a transient intact empty particle as pre-dissociation cell entry intermediate during the uncoating⁴¹. Its RNA is also protected from RNases in the endosome⁴² and suggested to use same translocation model as enteroviruses⁴⁰. We observed larger disordered regions in the VP1 N-terminus (25 amino acids), VP2 N-terminus (40 amino acids) and C-terminus (7 amino acids) in the 12S comparing to 146S. The disordered regions may provide uncoating cues for FMDV, making the dissociated 12S a byproduct following the uncoating.

Efforts of many investigators over three decades have mapped five functionally independent antigenic sites by neutralization escape mutants^17,43,44. These sites were only globally defined by identification of critical residues at specific positions. Therefore, a large gap between the observed immune escape and comprehensive epitope distributions at atomic resolution still exists. By using FMDV/antibody complex structures determined in this study as well as previously, we constructed the antigenic landscape with comprehensive epitope distribution, immunogenic and mutational heatmaps on the capsid surface of FMDV (Fig. 5f). Some of the previously identified antigenic sites, indicated by numbers 1 to 5, are related to four classes of antigenic sites assigned in our structural study. Site 1 is mainly linear and trypsin-sensitive, encompassing the GH loop and C-terminus of VP1, which is equivalent to the class II. Sites 2–5 are all conformational and trypsin-resistant except for site 5. Our study did not support the separate classification of sites 1 and 5, which both involve the VP1 GH loop. However, sites 2, 3 and 4 correspond to class III, I and IV, respectively. The classification of sites based on structures also allowed the mapping of a non-neutralizing binder to class V that evidently was not earlier identified by escape mutants. Our classification also allowed analysis of antibody responses against individual epitopes from distinct classes in the serum, and shows divergent antibody lineages were induced by immunization with 146S and 12S.

In previous studies, the therapeutic use of individual antibody and peptide vaccines targeting an individual antigenic site of FMDV demonstrated suboptimal protective efficacy against FMD in target animals^45,46. As the unique neutralizing epitopes exclusive to 146S facilitate a cooperative humoral defense against FMDV, cocktail therapies targeting these distinctive neutralizing epitopes, along with other NAbs in other classes (Fig. 5d), could be developed for emergency use during FMDV outbreaks to prevent the stamping out of livestock. Antibodies also facilitate opsonization-enhanced phagocytosis, a pivotal process for FMDV clearance in vivo⁴⁷. A recent study further indicates that the in vitro neutralizing potency of antibodies correlates with their opsonophagocytic capability in vivo. NAbs even demonstrate opsonophagocytosis effects at low affinities, highlighting the significance of NAbs in vivo⁴⁸.

In conclusion, the antigenic landscape and immunogenic characterizations unveiled in this study will provide guidance for the structure-based rational design of novel highly potent and stable vaccines for protection against FMD.

Methods

Ethics statement

The vaccination study in pigs was approved by the Institutional Review Board of Wageningen Bioveterinary Research (protocol code 2013153). Permissions for pig immunization with FMDV were granted by the Dutch Central Authority for Scientific Procedures on Animals (Project AVD401002015265).

Preparation of 146S particles and 12S particles

FMDV serotype A vaccine strains A/TUR/14/98 and A22/IRQ/24/64 (A22/IRQ) were obtained from the virus production facilities in Lelystad. Briefly, FMDV was amplified in BHK-21 cells grown in suspension in industrial-scale bioreactors, binary-ethyleneimine inactivated, and concentrated as previously described¹⁹. FMDV 146S particles were purified by fractionation using 15–45% (w/v) sucrose density gradients (SDG) in Tris-KCl buffer (300 mM KCl; 20 mM Tris.Cl, pH 7.4) for 2 h at 10 °C and 200,000 × g. The fractions containing 146S were pooled, concentrated and buffer-changed to PBS or Tris-KCl. The purified 146S content was quantified by absorbance using the extinction coefficient E_{259 nm} = 132⁴⁹ and checked for integrity by negative-stain transmission electron microscopy (TEM). SDG-purified A/HuBWH/CHA/2009 146S was provided by the virus production facilities of Lanzhou Veterinary Research Institute. The production and purification process were as for the aforementioned strains. Dissociation of 146S into 12S particles was done by either heating for 1 h at 56 °C or acid treatment, which was done by addition of 1 volume 0.1 M NaH₂PO₄ (pH < 5) to 146S particles, incubation at room temperature for 15 min, and neutralization to pH 7.4 by addition of 1 volume 0.1 M NaOH.

Vaccination of pigs with 146S and 12S antigens

SDG-purified A22/IRQ 146S particles and 12S particles derived thereof by heating were used to compare immunogenicity in pigs. Two double-oil-emulsion vaccines containing either 146S or 12S particles were prepared as described previously⁵⁰ using 0.3% BSA and 10% sucrose as excipients to stabilize FMDV particles but lacking the destabilizing excipient thiomersal. Two groups of 6 pigs each were immunized intramuscularly with either 146S or 12S vaccine containing 5 µg 146S or 12S particles per animal. Animal sera were collected on days 0, 7, 14, 21 and 28 post vaccination.

Virus neutralization test

The NAb response was measured by virus neutralization test (VNT) using primary porcine kidney cells following standard procedures⁵¹. Briefly, two-fold serial dilutions of sera in 96-well plates (50 µl/well) were mixed with 50 µl 100 tissue culture infective doses of A22/IRQ virus required to infect 50% of the wells (TCID₅₀) and incubated at 37 °C for 1 h. Then an equal volume of porcine kidney cell suspension was added and plates were placed at 37 °C for 3 days before staining the monoloayers using Amido Black. End point titers were calculated using the Spearmann-Kärber method as the log₁₀ of the reciprocal of the final serum dilution that prevents the cytopathogenic effect in 50% of the wells.

Production of VHHs and antibodies

The VHHs M3, M8²⁸, M678 and M688¹⁹ were produced in baker’s yeast with a C-terminal his6 tag, purified from culture supernatant using immobilized-metal affinity chromatography and biotinylated as previously described⁵². The VHHs M3F, M8F, M678F and M688F (suffix F) were expressed using plasmid pRL188, resulting in additional C-terminal extension with the llama heavy-chain antibody long hinge region, that contains a cysteine for partial disulfide bond formation between two VHHs, and a his6 tag. M3K, M678K and M688K (suffix K) were produced with only a his6 tag using plasmid pUR4584, resulting in strictly monomeric VHHs. Bovine antibody R55 specific for A/HuBWH/CHA/2009 146S particles²⁷ was kindly provided by dr. Zengjun Lu.

Plaque-reduction neutralization test

FMDV neutralization by VHHs was determined by a plaque-reduction neutralization test (PRNT). VHHs M678F, M688F, M8F or a cocktail of M678F and M688F were 2-fold serially diluted in Dulbecco’s Modified Eagle Medium containing 2% fetal bovine serum, with an initial VHH concentration of 2 mg/ml, or 1 mg/ml of each VHH in the cocktail. Two control wells lacking VHHs were included. After adding an equal volume of A/TUR/14/98 virus aiming at 100 plaque-forming units per well and incubation at 37 °C for 1 h, the mixture was transferred to a 6-well culture plate with a monolayer of primary lamb kidney cells. After further incubation for 1 h, the plates were covered with cell medium containing 1% methylcellulose and further incubated till the next day. Then, the overlay was removed, and the cells were stained with Amido Black to visualize plaques. Subsequently, the relative number of plaques in the wells treated with different VHH dilutions as compared to control assays without VHH was calculated. Each assay was performed in duplicate. The VHH concentration required to inhibit 50% (IC₅₀) of the number of plaques was then calculated by four-parameter nonlinear curve fitting using GraphPad Prism 8.0, assuming the same slope for all VHHs and 100% neutralization at a high dose.

FMDV particle stability assay

A thermofluor particle stability thermal release assay (PaSTRy)³⁰ was performed with a Lightcycler 480 PCR machine in 96-well plates using 1 μg SDG-purified A/TUR/14/98 146S particles in 50 µl 300 mM KCl containing 20 mM phosphate buffer at pH of 5.75 or 6.5. VHHs were added to 146S particles at 5-fold molar excess of VHHs to binding sites (1 μg) to analyze stabilizing effects. The SYTO9 (Thermo Fisher Scientific, Rockford, IL) fluorescent probe with excitation and emission wavelengths of 486 nm and 501 nm, respectively, was added to 5 µM final concentration to detect the single-stranded RNA that is released when virus capsids dissociate. The temperature was ramped from 30 °C to 95 °C in 0.11 °C increments per s. The dissociation of capsids was detected by interpreting the fluorescence signal as the minimum of the negative first-derivative of the fluorescence curve.

Binding affinity measurements

VHH affinity was analyzed by bio-layer interferometry (BLI) using the Octet Red96 System (Sartorius, USA) at 30 °C in PBS containing 0.05% Tween-20 (PBST). High precision streptavidin (SAX)-sensors (Sartorius) were loaded with biotinylated M678F (2 µg/ml) or M8F (1 µg/ml) for 300 s and subsequently with A/TUR/14/98 146S (2 µg/ml) or 12S (1 µg/ml) for 900 s, for subsequent analysis of, respectively, 146S- or 12S-specific binding of unlabeled monomeric (suffix K) VHHs. After a baseline step in PBST for 300 s, the association of serial dilutions of unlabeled VHHs was done for 300–600 s and finally dissociation for 900–1800 s. A reference sensor without unlabeled VHH was included to correct for baseline drift. The on-rate (k_a) and off-rate (k_d) were determined by global fitting of the association and dissociation phases of a series of unlabeled VHH concentrations assuming a 1:1 stoichiometry of VHH binding using Octet Analysis Studio v12.2 software (Sartorius). The equilibrium dissociation constant (K_D), a measure for affinity, was then calculated as the ratio of k_d and k_a.

Surface plasmon resonance competitive assay

To analyze the ability of 2 VHHs to bind simultaneously to FMDV, surface plasmon resonance (SPR) experiments were done using a BIAcore 8 K machine and CM5 sensor chips (Cytiva Life Sciences) in PBST. Unlabeled M678F (399 RU) or M688F (1000 RU) were immobilized individually onto a CM5 sensor chip surface using the EDC/NHS method. SDG-purified A/TUR/14/98 146S of 4 nM was injected onto the chip at a flow rate of 20 μl/min for 120 s. An injection of either 500 nM M678F or 1000 nM M688F was done sufficiently long to ensure complete blocking of antigenic sites, followed by a dual injection of the mixture of 500 nM M678F and 1000 nM M688F. Binding signals were analyzed with BIAevaluation Version 4.1 software.

Negative stain TEM

The effect of VHH binding on acid-induced 146S dissociation was analyzed by TEM. A 5-fold molar excess of VHHs was mixed with FMDV A/TUR/14/98 146S particles in PBS followed by dilution into 20 mM phosphate buffer of pH 5.75 or pH 7.0. After 10 min incubation at room temperature, these samples were deposited onto glow-discharged copper grids for 1 min. After removing the excess sample, the grids were washed twice with PBS and immediately negatively stained for 1 min with 1% phosphotungstic acid (pH 7.0). Excess stain was blotted away and the grids were loaded into a JEOL 1400Plus Transmission electron microscope for examination.

VHHs binding to FMDV in ELISA

Double antibody sandwich ELISAs using VHHs M3F, M8F, M678F and M688F to detect FMDV particles were done as earlier described⁵². In brief, 96-well polystyrene plates were coated with unlabeled VHH and subsequently incubated with 2-fold serial dilution series of FMDV particles, starting at 2 μg/ml 146S or 12S, in PBST with 1% milk (PBSTM). Bound FMDV particles were detected by subsequent incubation with the biotinylated form of same VHH as used in the coating step, and horse radish peroxidase (HRP)-conjugated streptavidin, followed by staining with 3,3’,5,5’-tetramethylbenzidine (TMB). After stopping the staining reaction by the addition of sulfuric acid, the absorbance at 450 nm (A450) was measured. The concentration with 50% binding was assessed using 4-parameter curve fitting.

Liquid phase blocking ELISAs

Pig serum samples from 21 days post vaccination were tested in five liquid phase blocking ELISAs (LPBE) using four different VHHs (M3F, M8F, M678F, M688F) or bovine mAb R55. The LPBEs were performed as described above for the VHH-based sandwich ELISAs, using several modifications that are summarized in Supplementary Table 5. Pig sera were serially 2-fold diluted from 0.9–3.3 log10 for all LPBEs. A 50 μl of diluted serum was mixed with 50 μl of a pre-titrated amount of the antigen. After 16−20 h incubation at 4 °C, 50 μl of these mixtures was transferred to pre-coated plates. After 1 h incubation at room temperature, the plates were washed and incubated with detecting (competing) antibodies. After 1 h incubation, the plates were washed and incubated with conjugates as described above for the VHH-based sandwich ELISAs, while for R55 LPBE, rabbit anti-bovine immunoglobulin−HRP (Sigma Aldrich, St. Louis, MO, USA) was used as conjugate. Control sera of known titers as well as one negative control serum (unvaccinated animal) were included on each ELISA plate as internal standards. Four wells without sera were used as antigen controls (100 % reactivity). Antibody titers were expressed as the logarithm of the reciprocal of the serum dilution giving 50% inhibition of the mean absorbance recorded in the antigen control wells. Serological data were statistically analyzed using two way mixed-effects ANOVA.

ELISAs measuring pig IgG titer

The total pig anti-FMDV IgG titer was determined by an indirect ELISA using plates coated with 1 µg/ml A22/IRQ antigen at 100 μl/well, 4 °C overnight. The antigen consisted of a mixture of 0.5 µg/ml each of 146S and 12S, that was prepared by acidification. The coated ELISA plates were blocked with PBSTM and then incubated with 3-fold dilution series of pig sera starting with a 15-fold dilution. Bound FMDV specific IgG was detected by incubation with a HRP-conjugated polyclonal rabbit anti-pig antibody (Sigma Aldrich), followed by TMB staining and measurement of the A450 as described above. Then, for each serum sample, the absorbance and serum dilution values were fitted to a 4-parameter logistic model and the serum dilution that resulted in an absorbance value equal to twice the background value observed without serum, was interpolated in this curve, using Origin pro 2019⁵³.

Cryo-EM sample preparation and data collection

Purified M678F and M688F were incubated either individually or as a mixture with purified A/TUR/14/98 146S particles (at a concentration of 0.5 mg/ml) at 4 °C for 1 min at the ratio of ~300 Abs per FMDV 146S particle. A/TUR/14/98 12S particles were prepared by acidification and purified by SDG. In total 3 μl of the purified 12S without antibody or complexed with M3F as well as 146S complexed with M678F, M688F, or the mixture M678F-M688F were applied to a freshly glow-discharged C-flat 1.2/1.3 Au grid (C-flat, CF-2/1-2 C, Protochips). Grids were blotted for 3 s in 100% relative humidity for plunge-freezing (Vitrobot; Thermo Fisher Scientific) in liquid ethane. Cryo-EM datasets were collected at 300 kV using a Titan Krios microscope (Thermo Fisher Scientific) equipped with a K2 detector (Gatan, Pleasanton, CA). Movies (32 frames, each 0.2 s, total dose 60 e − Å−2) were recorded with a defocus of between 1.5 and 2.7 μm using SerialEM, which yields a final pixel size of 1.04 Å.

Image processing, three-dimensional reconstruction, model building and refinement

Data processing was performed using RELION3⁵⁴. Briefly, a total of 3673 micrographs (12S), 5506 micrographs (12S-M3F complex), 2734 micrographs (146S-M678F complex), 501 micrographs (146S-M688F complex) and 2094 micrographs (146S-M678F-M688F complex) first underwent several steps including Motion Correction (MOTIONCORR)⁵⁵, CTF Estimation (Gctf)⁵⁶, Auto-picking and Particle Extraction from Micrographs. After that, a total of 1,088,637 particles (12S), 2,349,733 particles (12S-M3F complex), 132,191 particles (146S-M678F complex), 53,165 (146S-M688F complex) and 60,791 particles (146S-M678F-M688F complex) were subjected to two-dimensional classification. Finally, 192,654 particles of 12S and 232,071 particles of 12S-M3F complex were used for three-dimensional reconstruction with C5 symmetry imposed, while 46,708 particles of FDMV-M678F complex, 25,824 particles of FMDV-M688F complex and 46,767 particles of FMDV-M678F-M688F complex were used for the icosahedral symmetry reconstruction. The resolution of the final reconstructions was 3.2 Å, 3.2 Å, 3.6 Å, 2.9 Å and 2.9 Å, respectively, as evaluated by Fourier shell correction (threshold = 0.143 criterion). The atomic model of FMDV (PDB code: 7DSS) was initially fitted into the maps with CHIMERA and future corrected, mutated and refined manually by real-space refinement in COOT. The atomic models of M678F, M688F and M3F were built de novo into densities with structures of VHHs as a guide, using COOT. These models were further refined by positional and B-factor refinement in real space with Phenix. Refinement statistics are summarized in Supplementary Table 4.

Antibody clustering

All structures of FMDV complexed with antibodies available in the Protein Data Bank were retrieved and the FMDV protomers were aligned. The buried surface areas between each pair of antibodies were calculated as the inter-antibody distances. Subsequently, a buried surface area matrix clustering was conducted using the pheatmap package in pisa as previously described⁵⁷. Residues on the FMDV protomer within a 4 Å distance from the respective antibodies were identified as epitope residues. The binding frequency of each residue was computed to generate an epitope heatmap. Additionally, a sequence distance matrix clustering was performed using the fitch tool in PHYLIP⁵⁸.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The cryo-EM density maps of icosahedral reconstructions for 12S, 146S-M678F, 146S-M688F, 146S-M678F + M688F and 12S-M3F have been deposited in the Electron Microscopy Data Bank under accession codes: EMD-34689, EMD-34630, EMD-34636, EMD-34637 and EMD-34690, respectively. The corresponding atomic coordinates have been submitted to the Protein Data Bank with accession numbers: 8HEE, 8HBG, 8HBI, 8HBJ and 8HEG, respectively. Source data are provided with this paper.

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Acknowledgements

We thank Wim van der Poel (Wageningen University) for carefully revising the manuscript, Qianhui Zhu (University of Chinese Academy of Sciences) for help conducting SPR assay, Cynthia Baars and Marga van Hagen-van Setten (Wageningen Bioveterinary Research) for excellent technical assistance. Work was supported by the National Key Research and Development Program of China 2021YFD1800303 to S.S. and H.G., 2018YFA0900801 to X.W., National Natural Science Foundation of China (32072847, 32072859, 32301127) to S.S. and H.G., CAS (YSBR-010) and the National Science Foundation Grants (12034006, 32325004 and T2394482) to X.W., National Science Fund for Distinguished Young Scholar (No. 32325004) and the NSFS Innovative Research Group (No. 81921005) to X.W., Natural Science Foundation of Gansu Province (22JR5RA032, 23JRRA551), Postdoctoral Science Foundation Funded Project (2023M733819, 23JRRA554), Lanzhou Talent Innovation and Entrepreneurship Project(2023-RC-3) to S.S and H.G. The research done at Wageningen Bioveterinary Research was funded by the Ministry of Agriculture, Nature and Food Quality, the Netherlands (project WOT-01-002-034) to A.D.

Author information

These authors contributed equally: Haozhou Li, Pan Liu, Hu Dong.
These authors jointly supervised this work: Huichen Guo, Xiangxi Wang, Shiqi Sun.

Authors and Affiliations

State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
Haozhou Li, Hu Dong, Huichen Guo & Shiqi Sun
CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Pan Liu & Xiangxi Wang
University of Chinese Academy of Sciences, Beijing, China
Pan Liu
Wageningen Bioveterinary Research, Wageningen University & Research, Lelystad, The Netherlands
Aldo Dekker & Michiel M. Harmsen

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Haozhou Li
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Pan Liu
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Hu Dong
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Michiel M. Harmsen
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Huichen Guo
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Contributions

S.S., X.W., H.G., and M.H. conceptualized and designed the experiments. S.S. and H.G. acquired funding and supervised the project. H.L. and M.H. produced the antibodies. H.L. and D.H. purified the viruses. P.L., D.H., and H.L. performed the structural study. M.H. and A.D. conducted the animal experiment. H.L. conducted all the ELISAs, SPR, and TEM assays. M.H. performed BLI and thermofluor PaSTRy assay. A.D. performed PRNT and VNT assays. H.L. and A.D. conducted the statistics. P.L. and H.L. performed visualization. All authors analyzed data. H.L. and X.W. wrote the manuscript with input from all authors. S.S., M.H., and H.G. reviewed and edited the manuscript.

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Correspondence to Huichen Guo, Xiangxi Wang or Shiqi Sun.

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Li, H., Liu, P., Dong, H. et al. Foot-and-mouth disease virus antigenic landscape and reduced immunogenicity elucidated in atomic detail. Nat Commun 15, 8774 (2024). https://doi.org/10.1038/s41467-024-53027-5

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Received: 21 February 2024
Accepted: 24 September 2024
Published: 10 October 2024
DOI: https://doi.org/10.1038/s41467-024-53027-5

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