Table_1_Immunoinformatics Approach Toward the Introduction of a Novel Multi-Epitope Vaccine Against Clostridium difficile.xlsx

Clostridium difficile (C.difficile) is an exclusively anaerobic, spore-forming, and Gram-positive pathogen that is the most common cause of nosocomial diarrhea and is becoming increasingly prevalent in the community. Because C. difficile is strictly anaerobic, spores that can survive for months in the external environment contribute to the persistence and diffusion of C. difficile within the healthcare environment and community. Antimicrobial therapy disrupts the natural intestinal flora, allowing spores to develop into propagules that colonize the colon and produce toxins, thus leading to antibiotic-associated diarrhea and pseudomembranous enteritis. However, there is no licensed vaccine to prevent Clostridium difficile infection (CDI). In this study, a multi-epitope vaccine was designed using modern computer methods. Two target proteins, CdeC, affecting spore germination, and fliD, affecting propagule colonization, were chosen to construct the vaccine so that it could simultaneously induce the immune response against two different forms (spore and propagule) of C. difficile. We obtained the protein sequences from the National Center for Biotechnology Information (NCBI) database. After the layers of filtration, 5 cytotoxic T-cell lymphocyte (CTL) epitopes, 5 helper T lymphocyte (HTL) epitopes, and 7 B-cell linear epitopes were finally selected for vaccine construction. Then, to enhance the immunogenicity of the designed vaccine, an adjuvant was added to construct the vaccine. The Prabi and RaptorX servers were used to predict the vaccine’s two- and three-dimensional (3D) structures, respectively. Additionally, we refined and validated the structures of the vaccine construct. Molecular docking and molecular dynamics (MD) simulation were performed to check the interaction model of the vaccine–Toll-like receptor (TLR) complexes, vaccine–major histocompatibility complex (MHC) complexes, and vaccine–B-cell receptor (BCR) complex. Furthermore, immune stimulation, population coverage, and in silico molecular cloning were also conducted. The foregoing findings suggest that the final formulated vaccine is promising against the pathogen, but more researchers are needed to verify it.

艰难梭菌（Clostridium difficile）是一类专性厌氧、产芽孢的革兰氏阳性（Gram-positive）病原菌，为医院获得性腹泻（nosocomial diarrhea）的最常见致病菌，且在社区中的流行率正逐年升高。由于艰难梭菌为严格厌氧菌，可在外界环境中存活数月的芽孢是其在医疗环境与社区中持续存续与扩散的关键因素。抗菌治疗（antimicrobial therapy）会破坏肠道原生菌群（natural intestinal flora），使芽孢萌发为可定植（colonization）于结肠并产生毒素的增殖体（propagules），进而引发抗菌药物相关性腹泻（antibiotic-associated diarrhea）与假膜性肠炎（pseudomembranous enteritis）。然而目前尚无获批用于预防艰难梭菌感染（Clostridium difficile infection, CDI）的疫苗。本研究借助现代计算机辅助技术设计了一款多表位疫苗（multi-epitope vaccine），选取分别影响孢子萌发（spore germination）的靶蛋白CdeC与影响增殖体定植的靶蛋白fliD，以此构建可同时诱导针对艰难梭菌两种存在形式（芽孢与增殖体）的免疫应答的疫苗。研究序列从美国国家生物技术信息中心（National Center for Biotechnology Information, NCBI）数据库获取。经过多轮筛选流程，最终选取5个细胞毒性T淋巴细胞（cytotoxic T-cell lymphocyte, CTL）表位、5个辅助性T淋巴细胞（helper T lymphocyte, HTL）表位与7个B细胞线性表位（B-cell linear epitopes）用于疫苗构建。为提升所设计疫苗的免疫原性（immunogenicity），研究还添加了佐剂（adjuvant）以完成疫苗构建体的组装。分别使用Prabi与RaptorX服务器预测了该疫苗的二维（two-dimensional）与三维（three-dimensional）结构。此外，研究对该疫苗构建体的结构进行了优化与验证。通过分子对接（molecular docking）与分子动力学（molecular dynamics, MD）模拟，验证了疫苗-Toll样受体（Toll-like receptor, TLR）复合物、疫苗-主要组织相容性复合体（major histocompatibility complex, MHC）复合物以及疫苗-B细胞受体（B-cell receptor, BCR）复合物的相互作用模型。此外，本研究还开展了免疫刺激（immune stimulation）分析、人群覆盖度（population coverage）评估与计算机分子克隆（in silico molecular cloning）实验。综上研究结果表明，最终开发的这款疫苗对该病原菌具有良好的防控潜力，但仍需更多后续研究予以验证。