Table 1_Bioinformatic and experimental identification and characterization of Clostridioides difficile lipoproteins as potential vaccine candidates.docx

IntroductionTreatment options for C. difficile infection are limited, with very high rates of recurrence. Active vaccination provides an attractive opportunity to prevent C. difficile infection (CDI) and recurrence. In a search for potential surface-exposed antigens involved in C. difficile colonization, two putative lipoproteins, designated LP1 and LP2, were identified from C. difficile R20291.
MethodsLipoprotein sequences were aligned, analyzed, and evaluated for their immune properties. The antigenic characteristics of both LP1 and LP2 were assessed in silico and in a mouse model of immunization and CDI.
ResultsMultiple sequence alignments showed that the lipoprotein sequences were highly conserved among various ribotypes. In silico analysis predicted that LP1 and LP2 possess cytotoxic T-lymphocyte, helper T-lymphocyte, and B-cell epitopes with antigenic and immunogenic properties. Immune simulation provided insights into the ability of LP1 and LP2 to stimulate humoral and cellular immune responses. These properties were further examined in a mouse model of immunization and CDI. After three immunizations at 12-day intervals, significant amounts of IgG and IgA antibodies were detected in sera and feces. LP1 and LP2 immunizations provided mice with intermediate and higher levels of protection, respectively, against R20291 infection, and significantly reduced C. difficile spore and toxin levels in feces. Furthermore, anti-LP1 and anti-LP2 sera significantly inhibited adhesion of R20291 vegetative cells to HCT-8 gut epithelial cells.
DiscussionThese results indicate that both lipoproteins play a significant role in C. difficile adhesion and that LP1 and LP2 are promising immunogens for preventing C. difficile colonization.

引言
艰难梭菌（C. difficile）感染的治疗选择十分有限，且复发率极高。主动疫苗接种为预防艰难梭菌感染（CDI）及其复发提供了极具吸引力的途径。本研究在筛选参与艰难梭菌定植的潜在表面暴露抗原过程中，从艰难梭菌R20291菌株中鉴定出两种推定脂蛋白，分别命名为LP1与LP2。

方法
对脂蛋白序列进行比对、分析，并评估其免疫特性。针对LP1与LP2的抗原特征，分别通过计算机模拟（in silico）实验及艰难梭菌感染免疫小鼠模型开展评估。

结果
多序列比对结果显示，不同核糖体分型（ribotypes）的艰难梭菌脂蛋白序列具有高度保守性。计算机模拟预测表明，LP1与LP2均携带有具备抗原性与免疫原性的细胞毒性T淋巴细胞、辅助T淋巴细胞及B细胞表位。免疫模拟分析揭示了LP1与LP2刺激体液免疫与细胞免疫应答的能力。上述特性进一步在免疫感染小鼠模型中得到验证。以12天为间隔完成三次免疫接种后，可在小鼠血清与粪便中检测到大量IgG与IgA抗体。LP1与LP2免疫分别为小鼠提供了中等及更高水平的R20291菌株感染防护能力，并显著降低了粪便中的艰难梭菌芽孢与毒素水平。此外，抗LP1与抗LP2血清可显著抑制R20291营养体细胞对HCT-8肠道上皮细胞的黏附作用。

讨论
本研究结果表明，两种脂蛋白均在艰难梭菌黏附过程中发挥重要作用，且LP1与LP2均为极具潜力的免疫原，可用于预防艰难梭菌定植。