Multivalent vaccines against Clostridium difficile infection

项目摘要/摘要 艰难梭菌感染(Cdi)是医疗保健环境中感染性腹泻的最常见原因，约有 据美国疾病控制与预防中心2015年报告，美国每年有45.3万例病例和2.9万人死亡。严重程度的持续上升 CDI已经在世界范围内得到了观察。目前，CDI的标准治疗方法是使用抗生素。 虽然有效，但这些治疗方法并不能预防疾病复发，而且可能会导致15%-35%的疾病复发。 预防复发是该领域最具挑战性的方面之一。CDI症状范围从 腹泻到肠道炎症/病变和死亡。症状主要由两种外毒素TcdA和TcdA引起 Tcdb。这两种毒素具有非常相似的结构域结构，包括N-末端催化葡萄糖基转移酶 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 C末端受体结合域(RBD)。目前还没有针对CDI的疫苗获得许可。 该项目的目标是开发针对艰难梭菌毒素和 定植/黏附因素。我们之前已经展示了有效的保护与早期 免疫基因mTcd138，它包含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mTcd138：1)将TcdB的RBD与mTcd138相加，得到融合蛋白mTcd169；2)融合 MTcd169与鼠伤寒沙门氏菌鞭毛蛋白(SFliC)的融合蛋白Tcd169F1；3)表达 非产毒艰难梭菌的mTcd138，命名为NTCD_Tcd138。口服免疫接种 NTCD_Tcd138孢子对小鼠有完全保护作用，对仓鼠有明显的保护作用 使用超强毒力艰难梭菌菌株。此外，我们还表达和纯化了艰难梭菌Cwp84蛋白。 是一种半胱氨酸蛋白酶，在表层蛋白质的成熟过程中起着关键作用。据报道， Cwp84免疫通过延缓艰难梭菌的定植，为仓鼠提供了显著的保护作用。 在本项目中，我们将：1)在无毒艰难梭菌85(NTCD)中进一步表达mTcd169和Tcd169F1 分别生成菌株NTCD_Tcd169和NTCD_Tcd169FL；2)评估以下四个 免疫原作为CDI小鼠和仓鼠模型的潜在候选疫苗：Tcd169F1，一种 Tcd169F1和Cwp84(指定为Tcd169F1/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.