State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Diseases Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China.
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, China.
Appl Microbiol Biotechnol. 2019 Oct;103(19):8075-8086. doi: 10.1007/s00253-019-10081-0. Epub 2019 Aug 28.
Foot-and-mouth disease virus (FMDV) has led to serious losses in the farming industry worldwide, particularly in cattle and swine. In developing countries, the control and eradication of FMD rely upon vaccination, in which the inactivated vaccine is predominant. In the preparation of inactivated vaccine, a series of purification methods were used to remove non-structural proteins (NSPs). It is necessary to develop a quantitative detection method of residual NSP and confirm a threshold value for the evaluation of the vaccine. Meanwhile, it is also important to develop a sensitive and rapid diagnostic method to distinguish infected animals from vaccinated animals (DIVA). In this study, three monoclonal antibodies (MAbs) against NSP 3ABC, designated 2G5, 9E2, and 1E10, were used. Subsequently, a series of overlapping peptides were expressed using a prokaryotic expression system to determine the minimal epitopes identified by the MAbs. Three linear B cell epitopes (BCEs), "EYIEKA" "EGPYAGPLE" and "EPHH", were identified by MAbs 2G5, 9E2, and 1E10, respectively. Alanine-scanning mutagenesis analysis confirmed the critical amino acid in these epitopes. The epitope "EYIEKA" is located in 3A, which is deleted in some natural deletion mutants that result in a change in virus tropism. MAb 9E2 that identified the epitope "EGPYAGPLE" reacted with 3B1 and 3B2, but did not react with 3B3. In combination with sequence alignment analysis, the epitope "EGPYAGPLE" is highly conserved among different FMDV isolates. Preliminary screening using the known positive and negative sera indicated the MAb 9E2 has the potential for the development of a diagnostic method for DIVA. The residual NSP in inactivated vaccines can be detected using 9E2-HRP, which indicated the MAb 9E2 is able to evaluate inactivated vaccines. The four-amino acid epitope is the first reported to date that is recognized by 1E10. These results provide valuable insight into the diagnosis of DIVA and the NSP residual evaluation in inactivated vaccines.
口蹄疫病毒（FMDV）在全球范围内给农业产业造成了严重损失，尤其是在牛和猪中。在发展中国家，控制和消灭 FMD 依赖于疫苗接种，其中灭活疫苗占主导地位。在制备灭活疫苗时，使用了一系列纯化方法来去除非结构蛋白（NSPs）。因此，有必要开发一种用于检测残留 NSP 的定量方法，并确定一个用于评估疫苗的阈值。同时，开发一种敏感、快速的诊断方法来区分感染动物和接种动物（DIVA）也很重要。在这项研究中，使用了针对 NSP 3ABC 的三种单克隆抗体（MAbs），分别命名为 2G5、9E2 和 1E10。随后，使用原核表达系统表达了一系列重叠肽，以确定 MAb 识别的最小表位。通过 MAb 2G5、9E2 和 1E10 分别鉴定出三个线性 B 细胞表位（BCEs），“EYIEKA”、“EGPYAGPLE”和“EPHH”。丙氨酸扫描突变分析证实了这些表位中的关键氨基酸。表位“EYIEKA”位于 3A 中，在一些导致病毒嗜性改变的天然缺失突变中缺失。鉴定出表位“EGPYAGPLE”的 MAb 9E2 与 3B1 和 3B2 反应，但与 3B3 不反应。结合序列比对分析，该表位在不同 FMDV 分离株中高度保守。使用已知的阳性和阴性血清进行初步筛选表明，MAb 9E2 有可能开发用于 DIVA 的诊断方法。用 9E2-HRP 可以检测到灭活疫苗中的残留 NSP，这表明 MAb 9E2 能够评估灭活疫苗。这是迄今为止首次报道被 1E10 识别的四个氨基酸表位。这些结果为 DIVA 的诊断和灭活疫苗中 NSP 残留的评估提供了有价值的见解。
