Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays

Abstract

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.

Structural basis for neutralization of hepatitis A virus informs a rational design of highly potent inhibitors

“…Possibly correlated with its unusual stability, HAV capsid proteins exhibit no notable conformational changes upon binding to any NAbs. Unlike EV71 or other picornaviruses in which several distinct patches for neutralizing epitopes have been reported [20,22,23,24], all 5 NAb Fabs encircle edges of the pentameric building blocks of the virus, between the 2-fold and 3-fold axes (Fig 2A and Fig 3A). Examination of the possibility of binding of 2 arms of an immunoglobulin-G (IgG) molecule to the HAV surface showed that any 2 adjacent Fabs binding to the capsid could indeed mimic the 2 arms of a single IgG molecule (S6 Fig).…”

Towards improvements in foot-and-mouth disease vaccine performance

“…to bind to the integrin receptor) then clearly, the Mab-resistant mutants selected will not be modified at that residue. Indeed, an analysis of the interaction between a particular Mab (D9) with the intact serotype O virus, using cryo-electron microscopy, revealed interaction of the Mab with residue D147 within VP1 (part of the RGDL motif required for integrin binding) that had not been identified previously from sequencing of neutralization resistant mutants [73]. However, substitution of the adjacent residue L148 in VP1 had been shown to result in strong resistance to neutralization by the Mab D9 [64,66], thus it is not surprising that residue D147 also interacts with the antibody.…”

Functional and in silico Characterization of Neutralizing Interactions Between Antibodies and the Foot-and-Mouth Disease Virus Immunodominant Antigenic Site

“…Abs are also valuable biotechnology reagents and key molecules for FMDV diagnosis and surveillance (5). Recent FMDV publications have reported basic and applied Ab studies (6) by harnessing a wider sampling tool like nextgeneration sequencing (NGS), although a few explored the in vivo antibody (or immune receptor) repertoire (7)(8)(9). Using this approach, a broad neutralizing antibody was successfully obtained through single B-cell isolation from peripheral blood mononuclear cells of sequentially immunized bovines (10) to improve FMDV diagnostic tests.…”

“…A second serotype C Ab-FMDV structure encompassed a Cryo-EM reconstruction of Fab-capsid interactions at 30-Å resolution (13). Recently, another group reported the sequence and molecular modeling of a serotype O mAb (6).…”