Abstract
Foot-and-mouth disease (FMD) affects cloven-hoofed domestic and wildlife animals and an outbreak can cause severe losses in milk production, reduction in meat production and death amongst young animals. Several parts of Asia, most of Africa, and the Middle East remain endemic, thus emphasis on improved FMD vaccines, diagnostic assays, and control measures are key research areas. FMD virus (FMDV) populations are quasispecies, which pose serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent neutralizing epitopes to elicit an adequate immune response. Further investigation of the residues that comprise the antigenic determinants of the virus will allow the identification of mutations in outbreak strains that potentially lessen the efficacy of a vaccine. Additionally, of utmost importance in endemic regions, is the accurate diagnosis of FMDV infection for the control and eradication of the disease. To this end, a phage display library was explored to identify FMDV epitopes for recombinant vaccines and for the generation of reagents for improved diagnostic FMD enzyme-linked immunosorbent assays (ELISAs). A naïve semi-synthetic chicken single chain variable fragment (scFv) phage display library i.e., the Nkuku® library was used for bio-panning against FMD Southern-African Territories (SAT) 1, SAT3, and serotype A viruses. Biopanning yielded one unique scFv against SAT1, two for SAT3, and nine for A22. SAT1 and SAT3 specific scFvs were exploited as capturing and detecting reagents to develop an improved diagnostic ELISA for FMDV. The SAT1 soluble scFv showed potential as a detecting reagent in the liquid phase blocking ELISA (LPBE) as it reacted specifically with a panel of SAT1 viruses, albeit with different ELISA absorbance signals. The SAT1svFv1 had little or no change on its paratope when coated on polystyrene plates whilst the SAT3scFv's paratope may have changed. SAT1 and SAT3 soluble scFvs did not neutralize the SAT1 and SAT3 viruses; however, three of the nine A22 binders i.e., A22scFv1, A22scFv2, and A22scFv8 were able to neutralize A22 virus. Following the generation of virus escape mutants through successive virus passage under scFv pressure, FMDV epitopes were postulated i.e., RGD+3 and +4 positions respectively, proving the epitope mapping potential of scFvs.
Highlights
Diseases caused by RNA viruses are often difficult to control because of the high mutation rate and the continual emergence of novel genetic and antigenic variants that allow escape from immunity [1]
The large semi-synthetic naïve Nkuku R phage display library based on chicken immunoglobulin genes, was panned by exposing the recombinant antibody repertoire to sucrose density gradient (SDG) purified virions of the Foot-and-mouth disease (FMD) SAT1/Kruger National Park (KNP)/196/91, SAT3/KNP/10/90, and A22 viruses
The results revealed that soluble SAT1scFv1 successfully captured the panel of SAT1 viruses tested as ELISA signals of at 450 nm (A450nm) ≥0.48 and ≤1.68 were obtained (Figure 4A)
Summary
Diseases caused by RNA viruses are often difficult to control because of the high mutation rate and the continual emergence of novel genetic and antigenic variants that allow escape from immunity [1]. The high antigenic diversity that exists within the FMDV serotypes hinders FMD control by vaccination, as vaccination against one serotype does not confer protection against another and may only be partially effective against some subtypes within the same serotype [8]. This poses serious implications in vaccine design and efficacy where an effective vaccine should include multiple independent epitopes to elicit an immune response [9]. Four independent antigenic determinants were identified for SAT1 viruses i.e., (i) two occurring in the βG–βH loop of VP1; (ii) two simultaneous residues one in VP3 (position 135 or 71 or 76) and one in VP1 (position 179 or 181); (iii) a conformation dependant site
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