Harmsen M M, van Solt C B, Fijten H P D, van Keulen L, Rosalia R A, Weerdmeester K, Cornelissen A H M, De Bruin M G M, Eblé P L, Dekker A
Animal Sciences Group of Wageningen University and Research Centre, 8200 AB Lelystad, The Netherlands.
Vet Microbiol. 2007 Mar 10;120(3-4):193-206. doi: 10.1016/j.vetmic.2006.10.029. Epub 2006 Oct 28.
Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.
口蹄疫（FMD）是一种极具传染性的疾病，偶尔会在欧洲爆发。在疫情爆发时，需要有能提供快速口蹄疫防护的疗法。我们旨在通过用羊驼单域抗体片段（VHHs）进行被动免疫来提供这种快速防护。通过噬菌体展示从免疫的羊驼中分离出24种在体外结合口蹄疫病毒O型血清型的VHHs，并在面包酵母中表达以进行进一步表征。它们识别四个功能独立的抗原位点。六种强口蹄疫病毒中和VHHs与代表病毒蛋白1的GH环的肽结合，已知该肽参与口蹄疫病毒与细胞受体的结合。识别该抗原位点的克隆M8和识别另一个抗原位点的克隆M23在体外病毒中和中表现出协同作用。对三种口蹄疫病毒特异性VHHs进行聚乙二醇化，以减少其在血液中的快速清除，从而能够进行体内豚鼠保护实验。用单个VHHs进行被动免疫未显示出保护作用，但M8和M23的混合物显示出部分短暂保护作用。然而，与恢复期豚鼠血清提供的完全保护相比，这些VHHs提供的保护作用较低。相比之下，这些VHHs在体外对口蹄疫病毒的中和作用比恢复期豚鼠血清更有效。体外中和与体内保护之间缺乏相关性，这进一步支持了口蹄疫病毒的调理吞噬作用对体内保护很重要这一观点。
