Multivalent vaccines against Clostridium difficile infection

项目总结/摘要 C.艰难梭菌感染（CDI）是医疗机构中感染性腹泻的最常见原因， 根据CDC在2015年的报告，美国每年有453，000例病例和29，000例死亡。持续上升的严重 CDI在世界范围内都有出现。目前，CDI的标准治疗方法是使用抗生素。 虽然有效，但这些治疗不能预防，并可能导致15- 35%的疾病复发率。 预防复发是该领域最具挑战性的方面之一。CDI症状包括 腹泻至肠道炎症/病变和死亡。症状主要由两种外毒素TcdA和 TcdB。这两种毒素具有非常相似的结构域结构，包括N-末端催化葡糖基转移酶 结构域（GT）、自溶半胱氨酸蛋白酶结构域（CPD）、中央易位结构域（TM）和半胱氨酸蛋白酶结构域（T-P）。 C-末端受体结合结构域（RBD）。目前没有针对CDI的疫苗获得许可。 本项目的目标是开发新的粘膜疫苗，靶向C。艰难梭菌毒素和 定植/粘附因子。我们以前已经证明了有效的保护， 免疫原mTcd 138，含有TcdB的GT和CPD结构域以及TcdA的RBD。我们进一步 通过以下方式增强mTcd 138：1）将TcdB的RBD添加到mTcd 138，产生融合蛋白mTcd 169; 2）融合 mTcd 169与鼠伤寒沙门氏菌鞭毛蛋白（sFliC），产生融合蛋白Tcd 169 F1;和3）表达 mTcd 138在非致突变C.艰难梭菌菌株，命名为NTCD_Tcd138。口服免疫， NTCD_Tcd138孢子在小鼠中提供完全保护，在仓鼠中提供显著保护以抵抗攻击 用高毒力C.艰难菌株此外，我们还表达并纯化了C.艰难梭菌蛋白Cwp 84， 是一种半胱氨酸蛋白酶，在表层蛋白的成熟过程中起着关键作用。有消息称 Cwp 84免疫通过延迟C.艰难的殖民 本课题的主要目的是：1）进一步在非转基因的大肠杆菌中表达mTcd 169和Tcd 169 F1。艰难梭菌85（NTCD）至 分别产生菌株NTCD_Tcd169和NTCD_Tcd169FL; 2）评估以下四种 在CDI的小鼠和仓鼠模型中作为潜在疫苗候选物的免疫原：Tcd 169 F1， Tcd 169 Fl和Cwp 84（指定为Tcd 169 Fl/Cw）、NTCD_Tcd169和NTCD_Tcd169Fl。

Project Summary/Abstract C. difficile infection (CDI) is the most common cause of infectious diarrhea in the healthcare setting with about 453,000 cases and 29,000 deaths yearly in the U.S. as reported by CDC in 2015. A continual rise in severe CDI has been observed worldwide. Currently, standard treatment for CDI is the administration of antibiotics. While effective, these treatments do not prevent and may contribute to a disease recurrence rate of 15-35%. Prevention of recurrence is one of the most challenging aspects in the field. CDI symptoms range from diarrhea to intestinal inflammation/lesion and death. Symptoms are mainly caused by two exotoxins TcdA and TcdB. Both toxins share a very similar domain structure including the N-terminal catalytic glucosyltransferase domain (GT), the autolytic cysteine proteinase domain (CPD), the central translocation domain (TM), and the C-terminal receptor-binding domain (RBD). No vaccine against CDI is currently licensed. The objective of this project is to develop novel mucosal vaccines that target both C. difficile toxins and colonization/adhesion factors. We have previously demonstrated effective protection with an earlier immunogen mTcd138, which contains the GT and CPD domains of TcdB and the RBD of TcdA. We further enhanced mTcd138 by: 1) adding the RBD of TcdB to mTcd138, resulting in fusion protein mTcd169; 2) fusing mTcd169 with Salmonella typhimurium flagellin (sFliC), resulting in fusion protein Tcd169Fl; and 3) expressing mTcd138 in non-toxigenic C. difficile strains, designated as NTCD_Tcd138. Oral immunizations with NTCD_Tcd138 spores provided full protection in mice and significant protection in hamsters against challenge with hyper-virulent C. difficile strains. In addition, we expressed and purified C. difficile protein Cwp84, which is a cysteine protease and plays a critical role in maturation of surface-layer proteins. It was reported that Immunization with Cwp84 provides significant protection in hamsters by delaying C. difficile colonization. In this project, we will: 1) further express mTcd169 and Tcd169Fl in non-toxigenic C. difficile 85 (NTCD) to generate strains NTCD_Tcd169 and NTCD_Tcd169FL, respectively; 2) evaluate the following four immunogens as potential vaccine candidates in mice and hamster models of CDI: Tcd169Fl, a mixture of Tcd169Fl and Cwp84 (designated as Tcd169Fl/Cw), NTCD_Tcd169, and NTCD_Tcd169Fl.