Construction of foot-and-mouth disease (FMD) virus-specific phage-display libraries and epitope identification for improved FMD vaccines generation

Project summary

Foot-and-mouth disease (FMD) ranks as one of the most economically important infectious diseases of cloven-hoofed animals and is endemic in large regions of Africa, Asia and the Northern part of South America. The majority of FMD virus (FMDV)-neutralizing antibodies are directed against epitopes located on the surface-exposed capsid proteins. However, the location of these antigenic sites and their antigenic features may vary between the different strains. Antigenic variation results from changes to the viral capsid because of the high mutation rate of the virus such that each FMDV isolate is antigenically unique in its fine epitopic composition. This process leads to the generation of new variants circulating in the field, which may be different from those included in the vaccines. Therefore, vaccine efficacy and effectiveness of vaccination programs may be dramatically affected. Knowledge of the amino acid residues that comprise the antigenic determinants of FMD viruses is essential for the rational design of vaccine virus seeds that antigenically match circulating emerging or re-emerging strains, as well as induce a broad immunological response. Monoclonal antibodies, traditionally used for mapping of viral epitopes, have several limitations such as low throughput and high cost, which renders this technology unappealing. This project aims to identify critical antigenic determinants within the FMDV capsid from strains that circulate in different parts of the world, using newly developed assays. It will join the complementary expertise of four FMDV research institutes: ARC-OVR (South Africa), the Pirbright Institute (UK), INTA (Argentina) and the University of Glasgow (UK). We aim to use a library of recombinant “mini”-antibodies generated from FMDV-immune buffalos and cattle by making use of a phage-display library technology, which has been extensively and successfully used by ARC-OVR. This joint-project will provide important information by identifying critical FMDV epitopes, unique or shared among different strains, which can be implemented to produce improved vaccines.

Project outcomes

Foot-and-mouth disease (FMD) is caused by the foot-and-mouth disease virus (FMDV). It is the most wide-spread transboundary disease due to its highly infectious nature and is endemic in many African countries including, South Africa. There are seven serotypes of FMDV i.e., SAT1, SAT2, SAT3, A, O, C, and Asia1. The disease affects cloven-hoofed animals such as cattle, sheep, pigs and goats. Furthermore, it causes significant economic losses due to high morbidity in infected animals and stringent trade restrictions imposed on animal products from affected countries. An important control measure is vaccination, however, the FMDV high mutation rate results in antigenic variation rendering vaccines less effective. Knowledge of FMDV epitopes is advantageous and can be included in FMDV recombinant vaccine development resulting in vaccines that induce a broad immunological response and thus offer improved protection. In this regard, a bovine immune recombinant phage antibody library was constructed, using tissue samples taken from the (right) prescapular lymph nodes of six Jersey steers vaccinated with a commercial tetravalent FMD vaccine used in Argentina. Following RNA extraction, variable heavy (VH) and light chain (VL) regions of the immunoglobulin G antibody (IgG) genes were amplified by RT-PCR, joined by a flexible linker and cloned into a phagemid vector. The construct was transformed into electro-competent E.coli TG1 cells and repeated until an immune library was constructed consisting of 1.8 X 107 different clones 2 of small recombinant single chain variable fragments (scFvs). Affinity selection was undertaken using the constructed bovine phage library against an inactivated and sucrose density purified A24 Cruzeiro FMD virus to identify FMDV-specific single-chain variable fragments (scFvs). Enrichment of the four rounds of affinity selection performed was confirmed using a polyclonal ELISA whilst a monoclonal ELISA was used to identify individual possible scFv binders. A total of 96 clones were screened and 13 of those were possible positive scFv binders based on the ELISA A450nm values. Phagemid DNA was then isolated from these 13 virus-specific phagemids and the nucleotide sequence determined to identify unique clones. The sequence results revealed 3 unique scFv binders. These scFvs will be further characterised and utilised to identify FMDV epitopes and investigate its potential as reagents in a diagnostic ELISA. This is the first report of a FMDV-specific bovine phage library construction.