Development of mAb immunotherapy for genetically modified plague

鼠疫耶尔森氏菌是一种A类病原菌，是肺鼠疫的病原体。AIII型分泌物 鼠疫杆菌使用对毒力至关重要的系统(T3SS)输出一组被称为YOPs的蛋白质。 Yop效应物(如YopE)被运送到宿主细胞内，扰乱细胞内的信号通路。是的 效应器由转运子YopD和YopB跨宿主细胞质膜运送。这个 定位于T3SS顶端的LcrV蛋白需要将YopD和YopB插入质膜 是一种特性良好的保护性抗原。鼠疫杆菌还在其表面组装蛋白质纤维，这些纤维是 由F1蛋白组成。F1是一种保护性抗原，正在与LcrV一起作为一种 亚单位疫苗。针对LcrV或F1的小鼠免疫球蛋白单抗已被证明具有被动保护作用 对抗老鼠的鼠疫。然而，对鼠疫杆菌进行基因改造使其对LcrV具有抵抗力是可能的 基于F1的疫苗或免疫疗法，因为F1突变株保持完全毒力和保护性 LcrV内的表位是可以改变的。因此，需要识别额外的保护性抗原，并 发展成为疫苗或免疫疗法的靶标，以对抗转基因Y病毒的威胁。 鼠疫。本研究将鉴定和鉴定针对YopD和YopB的保护性抗体 可以被用来被动地免疫人类对抗转基因鼠疫。在目标1中，我们将 用Yop转运子特异性单抗展示对F1-Y型鼠疫的被动保护作用 蛋白质。这些实验将建立一个原理证明，肺鼠疫是由基因引起的 改良的鼠疫杆菌可以通过单抗免疫疗法来预防。在目标2中，我们将确定YOP中的表位 保护性单抗识别的转位蛋白。这些研究将提供有关性质的信息 保护性表位，并允许开发单抗鸡尾酒作为优化的免疫疗法。在……里面 目的3，我们将确定恒重(CH)区在保护性MAb功能中的作用。F(ab‘)2碎片 将产生保护性抗YopB和抗YopD单抗，并测试其保护活性。嵌合体 (CH)含有保护性单抗的可变区和不同同种类型的人CH区的单抗将是 构建、表征亲和力和特异性，并检测其保护功能。小鼠表达 人Fc受体将用于检测鼠-人ch单抗的保护活性。结果将 增加这些单抗作为人类免疫疗法的有效性，以及提供关于 恒定区在单抗功能中的作用。

Yersinia pestis is a Category A pathogen and the causative agent of pneumonic plague. A type III secretion system (T3SS) that is essential for virulence is used by Y. pestis to export a set of proteins known as Yops. Yop effectors (e.g. YopE) are delivered into host cells to disrupt intracellular signaling pathways. Yop effectors are delivered across the host cell plasma membrane by the translocators YopD and YopB. The LcrV protein, localized to the tip of the T3SS, is required to insert YopD and YopB into the plasma membrane and is a well-characterized protective antigen. Y. pestis also assembles protein fibers on its surface that are composed of the F1 protein. F1 is a protective antigen and is being developed together with LcrV as a subunit vaccine. Murine IgG MAbs specific for LcrV or F1 have been shown to confer passive protection against plague in mice. However, it is possible to genetically modify Y. pestis to make it resistant to LcrVand F1-based vaccines or immunotherapeutics, because F1- mutants remain fully virulent, and protective epitopes within LcrV can be altered. Therefore, additional protective antigens need to be identified and developed into targets for vaccines or immunotherapeutics to counteract the threat of genetically modified Y. pestis. This research will identify and characterize protective IgG MAbs specific for YopD and YopB that could be used to passively immunize humans against genetically modified plague. In Aim 1, we will demonstrate passive protection of mice against F1- Y. pestis with MAbs specific for Yop translocator proteins. These experiments will establish a proof-of-principle that pneumonic plague caused by genetically modified Y. pestis can be prevented by MAb immunotherapy. In Aim 2, we will identify epitopes in Yop translocators proteins recognized by protective MAbs. These studies will provide information on the nature of the protective epitopes, and allow for development of a MAb cocktail as an optimized immunotherapy. In Aim 3, we will determine role of constant heavy (CH) regions in protective MAb function. F(ab')2 fragments of protective anti-YopB and anti-YopD MAbs will be generated and tested for protective activity. Chimeric (ch) MAbs containing variable regions of protective MAbs and human CH regions of different isotypes will be constructed, characterized for affinity and specificity, and assayed for protective function. Mice expressing human Fc receptor will be used to measure protective activity of murine-human ch MAbs. Results will increase the usefulness of these MAbs as immunotherapeutics in humans, as well as provide knowledge on the role of constant region in MAb function.