RESUMO
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.
RESUMO
Detecting exposure to new or emerging pathogens is a critical challenge to protecting human, domestic animal, and wildlife health. Yet, current techniques to detect infections typically target known pathogens of humans or economically important animals. In the face of the current surge in infectious disease emergence, non-specific disease surveillance tools are urgently needed. Tracking common host immune responses indicative of recent infection may have potential as a non-specific diagnostic approach for disease surveillance. The challenge to immunologists is to identify the most promising markers, which ideally should be highly conserved across pathogens and host species, become upregulated rapidly and consistently in response to pathogen invasion, and remain elevated beyond clearance of infection. This study combined an infection experiment and a longitudinal observational study to evaluate the utility of non-specific markers of inflammation [NSMI; two acute phase proteins (haptoglobin and serum amyloid A), two pro-inflammatory cytokines (IFNγ and TNF-α)] as indicators of pathogen exposure in a wild mammalian species, African buffalo (Syncerus caffer). Specifically, in the experimental study, we asked (1) How quickly do buffalo mount NSMI responses upon challenge with an endemic pathogen, foot-and-mouth disease virus; (2) for how long do NSMI remain elevated after viral clearance and; (3) how pronounced is the difference between peak NSMI concentration and baseline NSMI concentration? In the longitudinal study, we asked (4) Are elevated NSMI associated with recent exposure to a suite of bacterial and viral respiratory pathogens in a wild population? Among the four NSMI that we tested, haptoglobin showed the strongest potential as a surveillance marker in African buffalo: concentrations quickly and consistently reached high levels in response to experimental infection, remaining elevated for almost a month. Moreover, elevated haptoglobin was indicative of recent exposure to two respiratory pathogens assessed in the longitudinal study. We hope this work motivates studies investigating suites of NSMI as indicators for pathogen exposure in a broader range of both pathogen and host species, potentially transforming how we track disease burden in natural populations.
