Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S) Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

Abstract

Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

Development of a Potent Stabilizer for Long-Term Storage of Foot-and-Mouth Disease Vaccine Antigens

“…Five protomers, each of which consists of one set of structural proteins (VP1, VP2, VP3, and VP4), assemble to form a pentamer, and 12 copies of the pentamer and the encapsidated viral RNA form a 146S particle [ 3 ]. Intact virus particles, followed by empty capsids (75S) are known to confer the most potent protective immunity to vaccinated animals compared to pentamers (12S) that are poor at conferring immune protection [ 4 ]; however, 146S can easily dissociate into less immunogenic 12S particles by weak acid or mild heat [ 4 , 5 ]. Therefore, stabilization of 146S particles is a key technique for FMD vaccine production.…”

Chimeric O1K foot-and-mouth disease virus with SAT2 outer capsid as an FMD vaccine candidate

“…To corroborate the thermofluor data generated using the mutated SAT2/O viruses we used an ELISA-based technique which uses a single-domain llama antibody (M377F VHH) that specifically recognises intact FMDV capsid (146S) 18 .…”

“…Doel and Baccarini showed that live viruses of types A and C are more stable than types O and SAT1-3, with SAT viruses being particularly sensitive to temperature 8 . To determine the thermostability of the viruses used in this study two recently reported techniques were used, (i) thermofluor analysis (also termed PaSTRy: Particle STability using Release assaY) which monitors genome release as a consequence of capsid dissociation and (ii) ELISA using a llama single-domain antibody that recognises intact FMDV particles (146S infectious virions or 75S VLPs) 15 , 16 , 18 , 23 , 24 . In these assays purified virus samples were used to standardise experimental conditions and to ensure samples predominantly consisted of 146S intact virions.…”

“…Likewise, comparable ELISA-based methodologies have been described for the quantification of intact 146S particles in vaccine preparations 25 . The llama antibody, M377F VHH, used in this study was produced as previously reported for an analogous llama antibody (M170 VHH) recognising the intact capsid of FMDV serotype O 18 , 23 , 24 . Supplementary Figure 3 shows the specificity of M377F VHH, as heated samples that were no longer recognised by the llama antibody (Fig.…”

“…SAT2 antigens were incubated at different temperatures (4 °C, 40 °C or 47 °C) for 10 minutes and their stability was analyzed by double antibody sandwich ELISA using llama single-domain antibody fragments (VHH domains), termed M377F VHH, against intact FMD viral particles (146 S infectious virions). M377F VHH was provided by the Central Veterinary Institute of Wageningen UR, AB Lelystad, The Netherlands 18 . The wells of a 96-well microtitre plate were incubated overnight with VHH domains in carbonate/bicarbonate buffer (50 mM, pH 9.6; Sigma Aldrich, UK) at a concentration of 0.5 mg/l.…”