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Journal of Virology logoLink to Journal of Virology
. 2021 Nov 23;95(24):e01308-21. doi: 10.1128/JVI.01308-21

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization

Yong He a,b,c,#, Kun Li b,#, Li Wang b, Zixian Sun d, Yimei Cao b, Pinghua Li b, Pu Sun b, Huifang Bao b, Shasha Zhou b, Sheng Wang b, Xingwen Bai b, Xuerong Liu e, Lixia Zhao f, Xiuli Fan f, Zaixin Liu b,✉, Zengjun Lu b,✉, Cheng Yang a,✉, Zhiyong Lou c,✉
Editor: Susana Lópezg
PMCID: PMC8610593  PMID: 34586859

ABSTRACT

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the βB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines.

IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3E70 forms a key component of neutralizing epitopes, and the determination of the VP3E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

KEYWORDS: foot-and-mouth disease virus, bovine antibody, cross-reaction, interpentamer epitope

INTRODUCTION

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease of cloven-hoofed animals (1). The causative agent, FMD virus (FMDV), is a small nonenveloped virus containing a positive-sense single-stranded RNA that belongs to the genus Aphthovirus of the family Picornaviridae. FMDV exhibits a high degree of genetic and antigenic diversity due to the inherently error-prone nature of RNA-dependent RNA polymerase (2–4). It exists as seven immunologically distinct serotypes (A, C, O, SAT1, SAT2, SAT3, and Asia1), with numerous and constantly evolving subtypes within each serotype (5). There is no effective cross-protection between different serotypes or even heterologous strains of the same serotype based on antigenic variation, making the prevention and control of FMD more difficult.

Vaccination is thought to play a predominant role in the prevention and control of FMDV (6). Although the protective mechanism provided by FMDV vaccines has not been understood thoroughly, the indispensable role of neutralizing antibodies in protection from viral infection after vaccination is well established (7). Understanding the mechanisms of how antibodies neutralize infections and how viruses escape neutralizing antibody recognition requires detailed knowledge of not only the virus structure but also the virus–antibody interaction details at the amino acid level.

Structures of several serotypes determined at near-atomic resolution revealed that the FMDV capsid is composed of 60 copies of four structural proteins (VP1, VP2, VP3, and VP4) (8–10). Only VP1-3 are exposed on the outer surface of the virus particle, and therefore, these three proteins determine the reactivity of virus-neutralizing antibodies. Among seven serotypes, serotype A is prevalent in most FMD endemic regions and exhibits more antigenic divergence with poor intraserotype cross-neutralization (11, 12). However, there is limited structural information regarding the determinant for poor intra-serotype cross-neutralization.

FMDV A/AF/72 and FMDV A/WH/09 are vaccine strains used to prevent outbreaks caused by FMDV serotype A in China. FMDV A/AF/72 from the A22 lineage of serotype A is a traditional vaccine strain (13). FMDV A/WH/09, belonging to the SEA-97/G1 genotype of the ASIA topotype, was isolated from Wuhan in 2009 and was subsequently proven to spread to nine other areas of the Chinese mainland (14). Here, we isolated an antibody (R55) from natural bovine hosts through the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISAs) and virus neutralization tests (VNTs) showed that R55 displays cross-reactions with both FMDV A/WH/09 and FMDV A/AF/72 but only has a neutralizing effect on FMDV A/WH/09. Meanwhile, we determined the cryo-EM structures of R55 scFv in complex with FMDV A/WH/09 and A/AF/72. The near-atomic level details revealed the nature of the binding model and the key epitope for intraserotype cross-neutralization. These results inform a structure-based rationale to design broadly cross-neutralizing vaccines.

RESULTS

Characterization of anti-FMDV bovine IgG antibody R55.

Previously, we isolated a serotype O/A cross-neutralizing antibody (R50) from a recovered natural bovine host by using the single B cell antibody isolation technique (CD21+IgM− O-FMDV+A-FMDV+ B cells) (15). In this process, we also found serotype A-specific B cells (CD21+IgM− O-FMDV−A-FMDV+ B cells). We successfully amplified the IgG BCR variable gene and developed the complete bovine IgG antibody R55. To further investigate the serotype specificity of R55, we propagated purified FMDV O/Tibet/99 (hereafter named FMDV-OTi), FMDV O/Mya/98 (hereafter named FMDV-OMy), FMDV A/WH/09 (hereafter named FMDV-AWH), and FMDV A/AF/72 (hereafter named FMDV-AAF) virions and separately examined their binding abilities to R55 by enzyme-linked immunosorbent assays (ELISA). The results showed that R55 binds to both FMDV-AWH and FMDV-AAF but does not react with FMDV-OTi and FMDV-OMy, suggesting that R55 is serotype A specific (Fig. 1A). Additionally, a microneutralization assay was used to assess the neutralization potency against these two representative strains of FMDV serotype A. The results showed that R55 could efficiently neutralize FMDV-AWH infection but did not have a neutralizing effect against FMDV-AAF (virus neutralization [VN] titer >200 μg/ml) (Fig. 1B).

FIG 1.

FIG 1

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Characterization of anti-FMDV bovine IgG antibody R55. (A) Dose-dependent binding analysis of bovine IgG antibody R55 against representative FMDV serotypes O and A by ELISA. Each plot represents the mean OD450 value from duplicate wells. Error bars represent standard error of mean (SEM). (B) The neutralizing efficacy of bovine IgG antibody R55 against FMDV A/WH/09 and FMDV A/AF/72 was evaluated using a microneutralization assay. The neutralization titer (NT) represented the lowest antibody concentration required to fully prevent CPE.

Overall architecture of the FMDV virion-R55 scFv complexes.

To better understand the mechanism underlying the neutralization difference, we determined the structures of R55 scFv in complex with FMDV-AWH and FMDV-AAF. Electron micrographs of the virion-scFv complex were collected using a 200 kV Arctica D683 (FEI) with a Falcon II direct electron detector (FEI). The acquired cryo-EM images and subsequent two-dimensional (2D) classification of the extracted particle images clearly indicated that the scFv had attached to the surface of the virions (Fig. S1). The cryo-EM reconstruction showed that three R55 scFv molecules bound to the FMDV-AWH and FMDV-AAF capsid around each icosahedral 3-fold axis (Fig. 2A, B, D, and E). A total of 60 copies of R55 scFv were bound to each mature virion. The final resolution of the cryo-EM reconstruction was estimated by the gold standard Fourier shell correlation (FSC) = 0.143 criterion to be 3.95 Å for the FMDV-AWH-R55 complex and 3.91 Å for the FMDV-AAF-R55 complex (Fig. S1). In both cases, the cryo-EM densities were of sufficient quality to allow for atomic modeling of most of the FMDV capsid proteins and the variable loops of the scFv antibody that are responsible for virus recognition (Fig. S2 and S3). To obtain information on the epitope of NAb R55, the scFv molecular interactions were analyzed using CCP4, and the footprints were defined by the atoms in the virus that were closer than 4 Å to any atom in the bound scFv molecule by using RIVEM (Fig. 2C and 2F).

FIG 2.

FIG 2

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Cryo-EM structures. (A, D) The central cross-section through cryo-EM maps of the FMDV-AWH-R55 complex (A) and FMDV-AAF-R55 complex (D) are shown with icosahedral 2-, 3- and 5-fold axes. Each image in the 480-pixel boxes corresponds to 446 Å in each dimension. Scale bars, 100 Å. (B, E) Rendered images of the FMDV-AWH-R55 complex (B) and FMDV-AAF-R55 complex (E). Depth cueing with color is used to indicate the radius (< 120 Å, blue; 130–150 Å, from cyan to yellow; > 160 Å, red). The icosahedral 5- and 3-fold axes are represented by pentagons and triangles, respectively. (C, F) Footprints of R55 on the FMDV-AWH (C) and FMDV-AAF (F) surfaces. The figure shows a two-dimensional projection of the FMDV surface produced using RIVEM. The 5- and 3-fold icosahedral symmetry axes are marked as pentagons and triangles, respectively, on one icosahedral asymmetrical unit. The spherical polar angles (θ, ϕ) define the location on the icosahedral surface. The depictions are radially depth cued from blue (radius = 130 Å) to red (radius = 155 Å). The R55 footprints are shown in purple.

FMDV-AAF-R55 and FMDV-AWH-R55 interfaces.

The FMDV-AAF-R55 complex structure shows that R55 makes contact with the βB and BC/HI-loops of VP2 and with the B-B knob of VP3 within one protomer (Fig. 3A to 3D). Residues in the VP3 B-B knob (AAF-VP3D59 and AAF-VP3Y63) interact with residues in HCDR2 (VHN56) and HCDR3 (VHY102). The AAF-VP3D59 and AAF-VP3Y63 side chains form hydrogen bond contacts with the side chains of VHN56 and VHY102 (Fig. 3B). Meanwhile, residues in VP2 βB (AAF-VP2D68), BC-loop (AAF-VP2T70, AAF-VP2D72, AAF-VP2K73 and AAF-VP2H77), and HI-loop (AAF-VP2Q196) interact with residues in FR3 (VLR69), LCDR1 (VLD35), and HCDR3 (VHH100, VHT108 and VHY112) (Fig. 3C and D). The VLR69 side chain forms a hydrogen bond with the side chain atom of AAF-VP2Q196 and a potential salt bridge with AAF-VP2D68 (Fig. 3C). The AAF-VP2D72 side chain forms hydrogen bond contacts with the side chains of VHT108 and VHY112. Meanwhile, the side chain of VLD35 also forms hydrogen bond contacts with the AAF-VP2T70 and AAF-VP2K73 side chains (Fig. 3D).

FIG 3.

FIG 3

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Structure of the FMDV-AAF-R55 complex. (A) Cartoon representation of a protomer showing the interaction interface between R55 scFv and the capsid. The heavy chain and light chain of R55 are colored purple and orange, respectively. The capsid proteins VP1 to VP4 are colored blue, green, red and yellow. (B–D) Expanded views of the interaction interface spotlighting the B-B knob (B) of VP3 and βB and the HI loop (C) and BC loop (D) of VP2. Dashed lines indicate contacts with potential hydrogen bonds and salt bridges. Amino acid side chains are colored blue (nitrogen) and red (oxygen).

As is homologous to that observed in the FMDV-AAF-R55 complex, the antibody-interacting residues on the FMDV-AWH capsid are also located in the βB (AWH-VP2D68), BC-loop (AWH-VP2T70, AWH-VP2D72, AWH-VP2K73 and AWH-VP2H77), and HI-loop (AWH-VP2Q196) of VP2 and the B-B knob of VP3 (AWH-VP3D59 and AWH-VP3Y63) (Fig. 4A to 4D). The structural alignment of the FMDV-AWH and FMDV-AAF capsid proteins showed no substantial conformational changes in the VP2 βB, BC-loop, and HI-loop and the VP3 B-B knob (Fig. 5B). Meanwhile, the sequence alignment of FMDV-AWH and FMDV-AAF shows that the common residues (VP2D68, VP2T70, VP2D72, VP2K73, VP2H77, VP3D59, and VP3Y63) that contact R55 are strictly conserved (Fig. 5A), indicating that these highly conserved interaction residues may be the determinants for cross-reaction with FMDV-AWH and FMDV-AAF.

FIG 4.

FIG 4

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Structure of the FMDV-AWH-R55 complex. (A) Cartoon representation of two adjacent protomers showing the interaction interface between R55 scFv and the capsid. The heavy chain and light chain of R55 are colored purple and orange, respectively. The capsid proteins VP1 to VP4 are colored blue, green, red, and yellow. (B–D) Black dotted box shows the expanded views of the interaction interface spotlighting the B-B knob of VP3 (B) and BC loop of VP2 (D) within protomer 2. Red dotted box shows the expanded views of the interaction interface spotlighting the BC loop of VP3 within protomer 1 and the βB and HI loops of VP2 within protomer 2. (C) Dashed lines indicate contacts with potential hydrogen bonds and salt bridges. Amino acid side chains are colored blue (nitrogen) and red (oxygen).

FIG 5.

FIG 5

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Comparison of the interaction residues between FMDV-AAF and FMDV-AWH. (A) Sequence alignment of FMDV-AWH-R55 and FMDV-AAF-R55. Residues with red backgrounds are identical. Residues at the FMDV-AWH-R55 and FMDV-AAF-R55 interfaces are indicated with black and red triangles, respectively. The same contacting residues are highlighted in blue boxes. Alignment was performed by CLUSTALW, and the figure was generated by ESPript. (B) The loops from FMDV-AWH (VP2: green, VP3: red) that are involved in interactions between FMDV-AWH and R55 (VP2 βB, VP2 BC loop, VP2 HI loop, and VP3 B-B knob) are aligned with those of FMDV-AAF (gray). (C) One-step growth kinetics study was performed on BHK-21 cells. BHK-21 monolayer cells in 24-well plate were respectively infected with wildtype (FMDV A/WH/09) and mutant virus (VP3E70G) at an MOI of 1 and the samples were harvested at 0 h, 4 h, 8 h, 12 h, 16 h, and 20 h postinfection. The average log10 titers corresponding to each time points were determined by plaque assays, as indicated on the graph. The standard deviations of the titers determined for triplicate wells are indicated on the graph. (D) Plaque phenotypes of the wild-type (FMDV A/WH/09) and its mutant VP3E70G in BHK-21 cells, and the sizes were correlated with the CPE patterns. (E) The neutralizing efficacy of bovine IgG antibody R55 against wild-type (FMDV A/WH/09) and mutant VP3E70G was evaluated using a microneutralization assay. The neutralization titer (NT) represented the lowest antibody concentration required to fully prevent CPE. ** indicates a significant difference compared with the wild-type at P less than 0.05.

In contrast, the FMDV-AWH-R55 complex structure shows that R55 also makes contact with the BC loop of VP3 from the adjacent protomer, revealing an interprotomer epitope. The side chain of AWH-VP3E70 located at the VP3 BC-loop forms hydrogen bond contacts with the side chains of VLS70 and VLN72 located in the light chain FR3 (Fig. 4C). Meanwhile, the sequence alignment of FMDV-AWH and FMDV-AAF shows that there is a Glu → Gly mutation at position 70 in VP3 (Fig. 5A). Therefore, the VP3E70 amino acid may play a critical role in the neutralization of FMDV-AWH by the R55 antibody. The AWH-VP3E70 → AAF-VP3G70 mutation may be the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF.

To further validate the importance of residue VP3E70, we mutated the FMDV-AWH amino acid by FMDV-AAF’s sequence at position 70 (AWH-VP3E70 → AAF-VP3G70), rescued virions using a reverse genetics approach and evaluated the neutralization capacity using a virus neutralization test (VNT). We successfully rescued the mutated viruses with increased virus proliferation and a greater cytopathic effect (CPE) (Fig. 5C and D). The VNT results showed that the AWH-VP3E70G mutation resulted in a significant reduction (∼18-fold) in virus neutralization titer (Fig. 5E). Therefore, residue 70 of VP3 is critical to neutralization because it forms a key component of the neutralizing epitopes.

Determinant of the neutralizing differences against FMDV-AWH and FMDV-AAF.

Previously, several studies have shown that some picornaviruses (enteroviruses) form an expanded intermediate capsid with channels close to the icosahedral 2-fold axes, allowing egress of the genome (16–21). Meanwhile, our previous study found that acidification of the SVV-ANTXR1 complex to pH = 6.0 resulted in a major reconfiguration of the pentameric capsid assemblies, which resembles a potential uncoated intermediate with remarkable perforations in both the 2- and 3-fold axes (22). However, to our knowledge, there have been no studies showing a similar intermediate state for an aphthovirus (FMDV). Aphthoviruses release the genome via disassembly into pentamers. Stuart et al. demonstrated that FMDV capsids release their genome into the host cell from an acidic compartment, which will dissociate into pentamers during this process (23). The pentameric subassemblies are highly stable, but the adjacent pentamers are held together by tenuous noncovalent interactions; therefore, the interpentamer interactions are often unstable. Stabilization of interpentamer interactions is sufficient to enhance capsid stability as well as the effectiveness of vaccine preparations (24).

Structural analysis of the FMDV-AWH-R55 complex showed that R55 recognizes the βB- and BC/HI-loops of VP2 and the B-B knob, as well as the BC-loop (AWH-VP3E70) of VP3 from another adjacent pentamer, which may enhance capsid stability and prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection (Fig. 6A and B). In contrast, R55 only binds to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither strengthens the interpentamer interactions nor neutralizes the FMDV-AAF infection (Fig. 6C).

FIG 6.

FIG 6

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R55 binds to two adjacent pentamers of FMDV-AWH. (A) Surface representation of two adjacent pentamers showing the interaction interface between R55 scFv and FMDV-AWH capsid. The heavy chain and light chain of R55 are colored purple and orange, respectively. The capsid proteins VP1 to VP3 are colored blue, green, and red. (B, C) Expanded views of the interaction interface spotlighting the binding difference of the FMDV-AWH capsid and FMDV-AAF capsid. (D) Stabilities of FMDV-AAF or FMDV-AWH particles and their immune complexes with R55 determined by thermal stability assays using the SYT09 dye to detect RNA exposure. The first derivatives are shown, and the experiments were independently conducted in triplicate. (E) Footprint of the receptor [integrin (avβ6)] and R55 on the FMDV-AWH capsid surface. The GH-loop is based on the “up” conformation that observed on FMDV binding to its integrin receptor (PDB:5NEM). Residues identified for R55 are indicated in cyan, and resides (RGD peptides) identified for integrin are indicated in magenta. The border of one protomer is indicated by a yellow dotted line.

In the fluorescence-based thermal stability assay, the melting temperature remained at approximately the same level when the FMDV-AAF virion was bound with or without the R55 scFv antibody (Fig. 6D). In contrast, the binding of R55 to the FMDV-AWH virion significantly increased the thermal stability, and the melting temperature increased ∼5.5°C compared with the unbound full particle (Fig. 6D). Meanwhile, in the acid stability assay of FMDV-AWH virions with or without the R55 scFv antibody, negative stained electron micrographs showed that FMDV-AWH virions rapidly dissociated into pentamers under acidic conditions (pH = 6.0), and very few particles remained intact after 10 s (Fig. 7A). Surprisingly, the binding of R55 to FMDV-AWH significantly increased the acid stability of the virus particles, and the particle density remained high after 30 s of incubation under acidic conditions (pH = 6.0) (Fig. 7B). In contrast to FMDV-AWH, the binding of R55 to FMDV-AAF does not significantly enhance acid stability and it rapidly induces the dissociation of the virus particles into pentamers (Fig. 7C). Overall, these findings show that R55 binds to FMDV-AWH virions crossing two adjacent pentamers by interacting with this critical amino acid (AWH-VP3E70), which neutralizes viral infection by overstabilizing the virion to prevent genome release.

FIG 7.

FIG 7

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Acid stability analysis. The FMDV-AWH virus particles (A), FMDV-AWH-R55 complex (B), or FMDV-AAF-R55 complex (C) were incubated under acidic conditions (pH = 6.0) for 10 s and 30 s at 4°C. The acid stability and capsid integrity were assessed using negative stain transmission electron microscopy. Red circle, pentamer, top view. Blue circle, pentamer, side view.

DISCUSSION

Nonenveloped viruses, including picornaviruses, can be neutralized by antibodies that target different steps of viral infection by preventing virus attachment to the cellular receptor (25–27), inducing virus uncoating to release the genome (28, 29) and overstabilizing the virus to prevent genome release (30) or destabilizing the virus and physically damaging and/or aggregating it (31, 32). Previously, there have been some reports about the neutralization mechanism against FMDV. Baxt et al. demonstrated that one neutralizing antibody can bind to the intact virion and cause viral aggregation but has no effect on the binding of the virus to cellular receptors (32). An early work also revealed that the murine MAb 4C9 neutralized FMDV by altering the conformation of the virions to induce genome release (33). In addition, in a previous study, we isolated a serotype O/A cross-neutralizing antibody (R50) and found that R50 destabilized the virus particles and induced dissociation of the virus particles (15). FMDV mediates cell entry by attachment to the integrin receptor via a conserved arginine-glycine-aspartic (RGD) motif in the exposed GH loop of the capsid protein VP1 (34). The FMDV-C-S8c1-SD6 Fab complex structure shows that the murine Fab fragment SD6 binds to the VP1 GH-loop (residues 136–147) and blocks virus attachment to the cellular receptor (35, 36).

Notably, the R55 footprint on the FMDV-AAF or FMDV-AWH capsid did not overlap with the identified receptor binding sites (Fig. 6E), indicating that R55 may not directly compete with the binding of receptors. This might explain why R55 binds to the FMDV-AAF capsid but does not have neutralizing activity. FMDV-AAF virions that are bound to antibodies are still able to bind to integrin receptors, which induce internalization through the clathrin-mediated endocytosis pathway and trafficking through endosomes. Furthermore, fluorescence-based thermal stability and acid stability assays demonstrated that the binding of R55 to FMDV-AAF does not enhance viral thermal and acid stability. Therefore, endosomal acid pH can still trigger viral capsid dissociation into pentamers to release the genome into the cytoplasm and maintain viral infection (Fig. 8). In contrast, for FMDV-AWH, because the interpentamer interactions are strengthened by the H-bonds between the capsid and antibody, this may act to overstabilize the capsid and prevent the capsid dissociation required for genome release and neutralize the viral infection (Fig. 8). The fluorescence-based thermal stability assay appears consistent with this hypothesis given that the FMDV-AWH-R55 immune complex melts at an ∼5.5°C higher temperature than virus particles in the absence of R55. Meanwhile, the acid stability assay also appears consistent with this hypothesis given that the binding of R55 to FMDV-AWH significantly increases the acid stability of virus particles.

FIG 8.

FIG 8

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Proposed neutralization mechanism of the R55 antibody against FMDV-AWH. Virus-bound R55 antibodies can still bind to integrin receptors, which induces internalization through a clathrin-mediated endocytosis pathway. The internalized vesicle is then delivered to endosomes. Endosomal acid pH triggers the dissociation of FMDV-AAF into pentamers and releases the genome into the cytoplasm. For FMDV-AWH, the interpentamer interactions are strengthened by the H-bonds between the capsid and antibody, which may enhance capsid stability and prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection.

Neutralizing antibodies (NAbs) play a major role in protecting against viral infection after vaccination. In our study, the ELISA and VNT results showed that R55 can bind to both FMDV-AWH and FMDV-AAF but has only a neutralizing effect against FMDV-AWH. The results indicate that bovine vaccinated with the FMDV-AWH strain may not develop protection against FMDV-AAF strain infection, which may result in vaccination failure. Therefore, the development of polyvalent vaccines with broader protectivity is important for the control of FMD. Meanwhile, cryo-EM structures and site-directed mutagenesis show that the VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The determination of a crucial amino acid mutation (VP3E70G) involved in intra-serotype diversity may aid in the development of broadly protective vaccines.

MATERIALS AND METHODS

Virus production and purification.

Inactivated FMDV A/AF/72 and FMDV A/WH/09 antigens were kindly provided by FMD inactivated vaccine manufacturers (The Spirit Jinyu Biological Pharmaceutical Co., Ltd., Hohhot, China, and China Agricultural Vet. Bio. Science and Technology Co., Ltd.). Viral antigens were first pelleted through a cushion of 30% (wt/vol) sucrose in PBS (pH = 7.4) by centrifugation at 35,000 g for 1.5 h. The pellet was resuspended in 500 μl of PBS (pH = 7.4) and fractionated by centrifugation at 35,000 g for 4 h at 4°C. The fractions were analyzed by negative stain electron microscopy, and the fraction containing 146S particles was transferred to a 100 kDa MWCO centrifugal filter for buffer exchange with PBS (pH = 7.4) to remove the sucrose. The final 146S particles were quantified by absorbance at 260 nm (where an optical density of 7.7 = 1 mg/ml) and immediately used for subsequent experiments.

Enzyme-Linked Immunosorbent Assay.

Indirect ELISA was used to assess the reactivity of the R55 antibody with the FMDV 146S antigen. In the indirect ELISA experiments, 200 ng/well viral antigen was coated in 96-well plates overnight at room temperature. The plates were then washed three times with PBST (PBS buffer plus 0.05% Tween 20) and blocked with 1% gelatin in PBS at 37°C for 2 h. After three washes, the R55 antibody at a concentration of 0.1125–1.8 μg/ml was added and incubated at 37°C for 1 h. The plates were washed three times with PBST, and then HRP-conjugated anti-His tag antibody (GenScript, China) at a dilution of 1:5,000 was added to the wells. The plates were incubated at 37°C for 30 min and washed three times with PBST. The color was developed by adding 50 μl of TMB substrate (Pierce, Life Technology) for 10 min at room temperature. The process was stopped by adding equal volumes of 1 M H2SO4. The optical density at 450 nm (OD450) was measured on a microplate reader (Bio-Rad).

Virus Neutralizing Test.

Bovine NAb R55 was titrated for viral neutralizing activity against two representative strains of FMDV serotype A (FMDV A/WH/09 and FMDV A/AF/72) by using a microneutralization assay as previously described (37). Briefly, antibody samples were serially diluted 2-fold in 96-well cell culture plates in a total volume of 50 μl, and 100 TCID50 of FMDV in 50 μl of culture media was added to each well. After incubation for 1 h at 37°C, ∼5 × 104 BHK-21 cells in 100 μl of media were added to each well. The plates were incubated at 37°C with 5% CO2 for 48 h, fixed in acetone-methanol (volume ratio = 1:1) and stained with a 0.2% crystal violet solution. The endpoint titer was determined as the reciprocal of the last antibody dilution to fully prevent a cytopathic effect (CPE) by 100 TCID50 FMDV in each well. The neutralizing activity was expressed as the virus neutralization (VN) titer, which was calculated as the initial antibody concentration divided by the endpoint titer.

Cryo-EM sample preparation and data collection.

FMDV-AWH or FMDV-AAF and R55 scFv were both incubated at a molar ratio of 1:240 in a volume of 50 μl for 5 min at 4°C. A 3 μl aliquot of the mixture was applied to a glow-discharged carbon-coated gold grid (GIG, Au 1.2/1.3, 200 mesh; Lantuo). The grid was blotted for 5.5 s in 100% relative humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher, USA). The cryo-EM data were collected at 200 kV with an FEI Arctica (Thermo Fisher, USA) and a direct electron detector (Falcon II, Thermo Fisher) at Tsinghua University. Micrograph images were collected as movies (19 frames, 1.2 s) and recorded at −2.4 to −1.4 μm under focus at a calibrated magnification of ×110 kX, resulting in a pixel size of 0.93 Å per pixel. The data collection and refinement statistics are summarized in Table S1.

Image processing and three-dimensional reconstruction.

Similar image processing procedures were employed for all data sets. Individual frames from each micrograph movie were aligned and averaged using MotionCor2 (38) to produce drift-corrected images. Particles were picked and selected in Relion-2.1 (39), and contrast transfer function (CTF) parameters were estimated using CTFFIND4 (40) and integrated in Relion-2.1. Subsequent steps in three-dimensional reconstruction used Relion-2.1 in accordance with the recommended gold-standard refinement procedures (39). For all reconstructions, the final resolution was assessed using the standard FSC = 0.143 criterion.

Model building and refinement.

To obtain the atomic coordinates of the complex, we fitted the previously reported FMDV-AWH protomer structure (PDB ID:7D3K) (15) and homology model of the scFv fragment into the virion-scFv complex density map using UCSF Chimera (41). The fitting was further improved with real-space refinement using Phenix (42). Manual model building was performed using Coot (43) in combination with real-space refinement with Phenix (42) to adjust for the mismatches between the model and the target protein. The density maps were kept constant during the entire fitting process, and the atomic coordinates were subjected to refinement. Validation was conducted using the MolProbity function integrated within Phenix. The refinement statistics are presented in Table S1.

Rescue of site-directed FMDV mutants by reverse genetics.

Full-length cDNAs were generated by an existing pOFS plasmid that contains the whole P1 gene of FMDV A/WH/09. Site-directed mutagenesis was used to introduce nucleic acid mutations to produce full-length cDNAs with single amino acid substitutions (44). The mutant construct was confirmed by nucleotide sequencing. The site-directed FMDV mutant viruses were rescued as previously described (45). Briefly, NotI-linearized mutant plasmids were transfected into BSR/T7 cells using Lipofectamine 2000 following the manufacturer’s instructions. The transfected cells were monitored daily for the appearance of CPE. At 72 h posttransfection, the culture supernatants were harvested and passaged on BHK-21 cells. The mutant virus titers were measured using the plaque forming unit (PFU) assay. Confluent monolayers of BHK-21 cells were infected with 10-fold serially diluted FMDV samples in 6-well plates. Tragacanth gum (0.6%) was added after 1 h of incubation. Plaques were visualized at 48 h postinfection by fixing with acetone-methanol and staining with crystal violet. The amount of plaque was observed and statistically analyzed.

ThermoFluor assay.

ThermoFluor experiments were performed using a Quant Studio RT-PCR instrument (ABI, Thermo Fisher Scientific) and were used to evaluate the FMDV 146S particle stability after incubation with the scFv antibody. Each 50 μl volume was set up in thin-walled PCR plates (ABI, Thermo Fisher Scientific) using reaction mixtures containing 1.0 μg of 146S particles plus R55 scFv antibody (∼60 antibody molecules per FMDV virion) and 5 μM SYTO9 (Invitrogen, USA). The melt temperature was set from 25°C to 95°C in 0.5°C increments with intervals of 1 s for all assays. Fluorescence was read with excitation and emission wavelengths of 490 nm and 516 nm, respectively. The release of RNA and hence the dissociation of the capsids was detected by increases in the fluorescence signal. Three independent ThermoFluor assays were performed for each analysis. Data sets exported from the PCR machine were visualized using GraphPad Prism 8.0.

Acid stability assay.

Samples of purified virus prepared as described above were adjusted to pH 6.0 by 10-fold dilution in pH 6.0 PBS (phosphate-buffered saline) (137 mM NaCl, 2.7 mM KCl, 50 mM Na2HPO4 and 10 mM KH2PO4). After incubation under acidic conditions for 10 s and 30 s at 4°C, the acid stability and capsid integrity were assessed using a negative stain transmission electron micrograph. FMDV-AWH or FMDV-AAF and R55 scFv antibodies were both incubated at a molar ratio of 1:240 for 1 min at 4°C. Then, the FMDV-AWH-R55 or FMDV-AAF-R55 complex was also adjusted to pH 6.0 and incubated under acidic conditions for 10 s and 30 s at 4°C. The acid stability was also assessed using a negative stain transmission electron micrograph.

Data availability.

The cryo-EM density maps and the structures were deposited into the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) with the following accession numbers: FMDV-AWH-R55, EMD-31555, PDB 7FEI, and FMDV-AAF-R55, EMD-31556, and PDB 7FEJ. All other data supporting the findings of this study are available from the corresponding authors upon request.

ACKNOWLEDGMENTS

We thank the Computing and Cryo-EM Platforms of Tsinghua University, Branch of the National Center for Protein Sciences (Beijing) for providing facilities. This work was supported by the National Program on Key Research Project of China (2017YFC0840300 and 2020YFA0707500) and the National Natural Science Foundation of China (no. 32072873 and no. 31902288).

We declare no conflict of interest.

Z. Lou, C.Y., Z. Lu, and Z. Liu conceived the project. Z. Lou and Z. Lu designed the experiments. Y.H., K.L., L.W., Z.S.,Y.C., P.L., H.B., S.Z., S.W., X.B., X.L., L.Z., and X.F. performed virus and antibodies purification, cryo-EM data collection and processing. Y.H., K.L., Z. Lu, and Z. Lou analyzed the data. Y.H., K.L., Z. Lu, and Z. Lou wrote the manuscript. All authors discussed the experiments and read and approved the manuscript.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Fig. S1 to S3 and Table S1<br>. Download jvi.01308-21-s0001.pdf, PDF file, 0.9 MB (874.4KB, pdf)

Contributor Information

Zaixin Liu, Email: liuzaixin@caas.cn.

Zengjun Lu, Email: luzengjun@caas.cn.

Cheng Yang, Email: cheng.yang@nankai.edu.cn.

Zhiyong Lou, Email: louzy@mail.tsinghua.edu.cn.

Susana López, Instituto de Biotecnologia/UNAM.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Fig. S1 to S3 and Table S1<br>. Download jvi.01308-21-s0001.pdf, PDF file, 0.9 MB (874.4KB, pdf)

Data Availability Statement

The cryo-EM density maps and the structures were deposited into the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) with the following accession numbers: FMDV-AWH-R55, EMD-31555, PDB 7FEI, and FMDV-AAF-R55, EMD-31556, and PDB 7FEJ. All other data supporting the findings of this study are available from the corresponding authors upon request.

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