Harmsen Michiel M, Li Haozhou, Sun Shiqi, van der Poel Wim H M, Dekker Aldo
Wageningen Bioveterinary Research, Wageningen University & Research, Lelystad, Netherlands.
Laboratory of Virology, Wageningen University and Research, Wageningen, Netherlands.
Front Vet Sci. 2023 Jan 9;9:1040802. doi: 10.3389/fvets.2022.1040802. eCollection 2022.
Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.
用完整的（146S）口蹄疫病毒（FMDV）颗粒进行疫苗接种可用于控制口蹄疫。然而，146S颗粒很容易解离成免疫原性较低的稳定五聚体12S颗粒。我们之前分离出了几种特异性结合146S或12S颗粒的单域抗体片段（VHH）。这些颗粒特异性VHH是疫苗质量控制的优秀工具。在本研究中，我们通过竞争ELISA、病毒中和以及表位的胰蛋白酶敏感性，绘制了这些VHH识别的抗原位点。我们纳入了两种先前描述的单克隆抗体（mAb），一种是12S特异性的（mAb 13A6），另一种是146S特异性的（mAb 9）。尽管两者都是12S特异性的，但发现VHH M3F和mAb 13A6结合独立的抗原位点。M3F识别一个非中和且对胰蛋白酶不敏感的位点，而mAb 13A6识别对胰蛋白酶敏感的VP2 N端。亚洲1型146S特异性位点对胰蛋白酶敏感、具有中和作用，并且也被VHH M8F识别，这表明它涉及VP1 GH环。A型146S特异性VHH识别两个独立的抗原位点，这两个位点也都具有中和作用但对胰蛋白酶不敏感。通过将两种广泛的毒株反应性146S特异性VHH与FMDV复合进行交联质谱分析（XL-MS），进一步绘制了主要位点。这些表位位于二十面体病毒3D结构的2倍和3倍对称轴附近，主要在VP2和VP3上，与先前鉴定的mAb 9位点重叠。由于这些表位位于单个12S五聚体上，146S特异性不能用表位因12S五聚体解离而分裂来解释。在早期研究中，解析了与O型FMDV复合的146S特异性VHH M170的冷冻电镜结构。据报道，146S特异性是由该表位在12S和146S颗粒中的构象改变引起的。这种机制可能也解释了通过XL-MS绘制的两种A型VHH的146S特异性结合，因为它们的表位与M170识别的表位重叠。令人惊讶的是，146S四级结构内部的残基也与VHH交联。这可能反映了颗粒在溶液中的灵活性。病毒 - 抗体相互作用的分子研究有助于进一步优化疫苗并改善其质量控制。
