Structural and molecular basis for foot-and-mouth disease virus neutralization by two potent protective antibodies

Outbreaks of Foot-and-mouth disease (FMD) caused by FMD virus result in significant economic losses. Vaccination is helpful, but the benefits are diminished with antigenic diversity within serotypes, instability of the immunogen and inability to confer protection for long durations. Here we have further dissected the mechanisms underpinning the protective efficacy of two previously reported neutralizing antibodies (NAbs), M8 and M170. The atomic details of the epitopes of M8 and M170 unveiled suggest that protection is conferred by disrupting the virus-receptor interactions. Consequently, administration of these NAbs conferred prophylactic and therapeutic benefit in guinea pigs, raising the possibility of administering NAbs before or during vaccination to confer immediate protection; well before the bolstering of the immune response by the vaccine. Differences in the residues and the conformation of elements making up the epitopes explain the differences in specificities of M8 and M170. An ability to bind 146S viral particles specifically, but not 12S degraded components, highlights a likely role for M170 in the quality control of vaccines.

(One-third of the neutralizing titer of the antibody plus a 2-fold diluted another antibody), which were incubated with an equal volume of virus (200 PFU/ml) at 37 °C for 1 h. The complexes were transferred to a 6-well culture plate with the momolayer BHK21 cells. After further incubating the plate for 1 h with slightly shaking every 20 min, the survival virus attached to the cell surface. The plates were covered with the gum tragacanth (2 ml/well) supplemented with 2% FBS and further incubated for 72 h. After removing the overlay, the cells were washed with PBS and fixed with 4% paraformaldehyde. Plaques were visualized by staining with 2.5% crystal violet. According to the neutralization efficiency of the experimental group to the control sample, the neutralization titers of the two antibodies were calculated by nonlinear curve fitting. The Data is presented as the mean ± SD of triplicate measurements.

Binding affinity measurements
SPR experiments were performed by a BIAcore 8k machine with CM5 sensor chips (both GE Healthcare) in PBST buffer (PBS,

FMDV challenge in guinea pigs
Guinea pigs (weight 300-400 g) were randomly divided into six groups, including therapeutic and prophylactic groups. Briefly, groups of guinea pigs were administrated intramuscularly with M8/M170 (2.5 mg/kg) 1 day before (prophylactic) or after (therapeutic) challenge with 100 50% median infective doses (100 ID 50 ) of FMDV (0.2 ml) on the left hind footpad. Guinea pigs injected intramuscularly with PBS before or after challenge were acted as control groups. All animals were examined for clinical symptoms at 1 to 10 days post-infection (DPI). No lesions were considered as full protection, and blood samples were collected for detecting the viral RNA copies by real-time quantitative PCR (RT-qPCR). In brief, the total RNA of samples was extracted by TRIzol reagent (Invitrogen) for the synthesis of cDNA TCC TTT GCA CGC CGT GGG AC 3'), and 2 μl of the cDNA template. The pcDNA 3.1 -3D plasmids was constructed and quantified as a standard sample.

Virus quantification on the cell surface by RT-PCR
The amounts of FMDV remaining on the surface of BHK21 cells after M8/M170 treatment were estimated using qPCR as previously described. Briefly

Thermofluor Assay
Thermofluor assay was performed with a MX3005p RT-PCR instrument (Agilent), SYTO9 (invitrogen) was used as fluorescent probe to detect the single-stranded RNA from virus capsid. In brief, the 50 μl reaction system includes, 2 μg purified viruses or 2 μg of viruses plus 1.5 μg of NAbs (∼120 antibody molecules per FMDV virion), or 37 °C treated viruses or virus-antibody complexes and 5 μM SYTO9 in PBS buffer solution. System program was ramped from 25 to 99 °C with fluorescence recorded in triplicate at 1 °C intervals.

Cryo-EM and data collection
Purified M8/M170 were incubated with purified FMDV particles (at a concentration of 0.5 mg/ml) at 4 °C for 1 min at the ratio of ~300 NAbs per FMDV particle. A 3 μl aliquot of the complex of FMDV and M8/M170 were applied to a freshly glow-discharged 400-mesh holey carbon-coated copper grid (C-flat, CF-2/1-2C, Protochips). Grids were blotted for 3 s in 90% relative humidity for plunge-freezing  Table S1.