A broadly reactive ultralong bovine antibody that can determine the integrity of foot-and-mouth disease virus capsids

Figures

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  Fig. 1.. (a) Double sandwich homologous ELISA of Ab117 reactivity (the complex is not pre-made before application to the ELISA plate) with SAT2 topotypes. SAT2/ZIM/7/83 VLPs with S2093Y and SAT2/EGY/4/2012 inactivated virus. (b) Antigen titration ELISA (starting from 3.43 µg ml−1 as 1/3 dilution) utilizing M377, M311 and Ab117 as both capture and detection antibody for the SAT2/EGY/4/2012 inactivated virus without and with heat treatment (56 °C for 15 min) to disrupt the capsids.

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  Fig. 2.. Fab117 binds to the interior surface of the dissociated SAT2/ZIM/7/83 pentamer. (a) Representative 2D class averages. (b) Final 3D reconstruction. (c, d) Close-up of the Fab117-SAT2/ZIM/7/83 complex density, viewed towards (c) the interior capsid surface and (d) from above the fivefold axis looking downward at the hydrophobic binding pocket. Density coloured by molecule: VP1 – blue, VP2 – turquoise, VP3 – orange and Fab117 CDR-H3 – purple.

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  Fig. 3.. Details of Fab117 binding. For clarity, only interacting residues and bonds are shown. Residues are labelled with those from the virus given a preceding chain identifier for VP1, VP2 or VP3, respectively, such that they all have four digits. (a) AlphaFold model of Fab117 with HC in magenta and LC in cyan. (b) Cα trace of the ordered ‘knob’ domain of CDR-H3 in the SAT2 pentamer–Fab117 complex. Cαs of residues contacting the antigen (within 4.0 Å) are shown as grey spheres and disulphides as yellow sticks. The CDR-H3 is orientated as in (a). (c) Fab117 (rainbow backbone) engages the hydrophobic cleft of the SAT2 pentamer shown in surface representation and coloured by hydrophobicity. (d) The tip of the Fab117 CDR-H3 is shown in purple with W138 central and SAT2 residues within 4.0 Å of the latter represented as sticks. VP2 is coloured green, VP3 red and the antibody purple, with VP2 secondary structural elements labelled. (e, f) Zoomed perspective of the interactions between Fab117 and VP2. VP2 secondary structural elements are labelled. (g) LigPlot+v2.2.8 [38] of the interaction between the Fab117 CDR-H3 and interface residues of VP2 only (thus, for instance, the contacts of W138…

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  Fig. 4.. Ab117 cross-specificity. (a) Sequence alignment of the representative FMDV serotypes [2830,52]. Residues with red background are conserved. Residues at the Fab117–SAT2/ZIM/7/83 interface are boxed. Black triangles indicate appreciable contribution to the interface, whilst red triangles indicate a hydrogen bond interaction (contributions determined using PISA). Residues directly making contact with W138 (≤4.0 Å) are indicated using an asterisk. The secondary structure of the SAT2/ZIM/7/83 proteins is indicated above the alignment. Alignment was performed using CLUSTALW [6061], and the figure was generated using ESPript [41]. (b) Heterologous sandwich ELISA shows pan-specific binding of Ab117 to thermally disrupted virus. Integrin αvβ6 was used as a universal capture for all intact viruses, whilst heated particles were trapped directly on the plates, with Ab117 applied for detection. SAT1/ZIM/22/89 inactivated virus was tested in an independent experiment. (c) Ab117 binding to SAT3/ZIM/4/81 [62] wild-type VLPs. Serum raised in animals infected with SAT3 was used as a positive control, whilst the lack of M3 binding to disrupted capsids showed the inability of this VHH to recognize…