Development of mAb immunotherapy for genetically modified plague

鼠疫耶尔森氏菌是A类病原体，是肺鼠疫的病原体。A型III分泌物 系统（T3 SS）是致病所必需的。鼠疫杆菌输出一组被称为Yops的蛋白质。 Yop效应物（例如YopE）被递送到宿主细胞中以破坏细胞内信号传导途径。Yop 效应子通过易位子YopD和YopB穿过宿主细胞质膜传递。的 定位于T3 SS尖端的LcrV蛋白是将YopD和YopB插入质膜所必需的 并且是一种充分表征的保护性抗原。Y.鼠疫菌还在其表面组装蛋白质纤维， 由F1蛋白组成。F1是一种保护性抗原，目前正在与LcrV一起开发， 亚单位疫苗对LcrV或F1具有特异性的鼠IgG单克隆抗体已显示可提供被动保护 在老鼠身上对抗鼠疫。然而，有可能对Y进行遗传修饰。使其对LcrV和 基于F1的疫苗或免疫治疗剂，因为F1突变体仍然具有完全毒性和保护性 可以改变LcrV内的表位。因此，需要鉴定另外的保护性抗原， 开发成疫苗或免疫治疗剂的靶点，以对抗转基因Y。 鼠疫本研究将鉴定和表征特异性针对YopD和YopB的保护性IgG单克隆抗体， 可以用来被动免疫人类对抗转基因鼠疫。在目标1中，我们 证明小鼠对F1- Y的被动保护。具有Yop易位蛋白特异性单克隆抗体的鼠疫 proteins.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Y.单克隆抗体免疫疗法可以预防鼠疫。在目标2中，我们将鉴定Yop 保护性单克隆抗体识别的转运蛋白。这些研究将提供关于 保护性表位，并允许开发MAb混合物作为优化的免疫疗法。在 目的3，我们将确定恒定重链（CH）区在保护性单抗功能中的作用。F（ab '）2片段 将产生100 μ g的保护性抗YopB和抗YopD MAb，并测试其保护活性。嵌合 (ch)含有保护性MAb的可变区和不同同种型的人CH区的MAb将被用于免疫原性检测。 构建，表征亲和力和特异性，并测定保护功能。小鼠表达 人Fc受体将用于测量鼠-人ChMAb保护活性。结果将 增加这些单克隆抗体作为人类免疫治疗剂的有用性，并提供有关 恒定区在单克隆抗体功能中的作用。

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.