Chitray Melanie, Opperman Pamela Anne, Rotherham Lia, Fehrsen Jeanni, van Wyngaardt Wouter, Frischmuth Janine, Rieder Elizabeth, Maree Francois Frederick
Agricultural Research Council, Onderstepoort Veterinary Research, Vaccines and Diagnostic Development, Onderstepoort, Pretoria, South Africa.
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa.
Front Vet Sci. 2020 Aug 11;7:475. doi: 10.3389/fvets.2020.00475. eCollection 2020.
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 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.
口蹄疫（FMD）会感染偶蹄类家畜和野生动物，疫情爆发会导致牛奶产量严重损失、肉类产量下降以及幼畜死亡。亚洲的几个地区、非洲大部分地区和中东地区仍然是口蹄疫的流行区，因此，改进口蹄疫疫苗、诊断检测方法和控制措施是关键研究领域。口蹄疫病毒（FMDV）群体为准种，这在疫苗设计和效力方面带来了严重影响，因为有效的疫苗应包含多个独立的中和表位，以引发足够的免疫反应。进一步研究构成病毒抗原决定簇的残基，将有助于识别疫情毒株中可能降低疫苗效力的突变。此外，在流行地区，准确诊断FMDV感染对于控制和根除该疾病至关重要。为此，研究了一个噬菌体展示文库，以鉴定用于重组疫苗的FMDV表位，并生成用于改进FMD酶联免疫吸附测定（ELISA）的试剂。一个未经免疫的半合成鸡单链可变片段（scFv）噬菌体展示文库，即文库，用于针对口蹄疫南非领土（SAT）1、SAT3和A血清型病毒进行生物淘选。生物淘选产生了一个针对SAT1的独特scFv、两个针对SAT3的scFv和九个针对A22的scFv。利用SAT1和SAT3特异性scFv作为捕获和检测试剂，开发了一种改进的FMDV诊断ELISA。SAT1可溶性scFv在液相阻断ELISA（LPBE）中显示出作为检测试剂的潜力，因为它与一组SAT1病毒发生特异性反应，尽管ELISA吸光度信号不同。当包被在聚苯乙烯板上时，SAT1svFv1的互补决定区几乎没有变化，而SAT3scFv的互补决定区可能发生了变化。SAT1和SAT3可溶性scFv不能中和SAT1和SAT3病毒；然而，九个A22结合物中的三个，即A22scFv1、A22scFv2和A22scFv8能够中和A22病毒。通过在scFv压力下连续传代产生病毒逃逸突变体后，推测了FMDV表位，即分别为RGD +3和+4位，证明了scFv的表位作图潜力。
