Determining vaccine candidacy of LT and STa toxoid fusions against enterotoxigeni

描述（由申请人提供）：产肠毒素大肠杆菌 (ETEC) 菌株被 NIAID 归类为优先 B 病原体。 ETEC 相关腹泻每年导致全世界近百万人死亡，此外还导致免疫功能低下的个人和旅行者（包括部署的军事人员）患病。 ETEC 相关腹泻的关键毒力因子是细菌粘附素或定植因子抗原 (CFA) 和肠毒素。 CFA 介导细菌与宿主上皮细胞的附着，但肠毒素、不耐热 (LT) 和耐热 (STa) 会刺激液体过度分泌并引起腹泻。早期使用 LT 和 STa 抗原开发抗毒素疫苗的研究取得的成功有限，因为这两种毒素均具有高毒性，STa 抗原无免疫原性，并且缺乏合适的动物研究模型。当前分子生物学的进步使得消除或减少毒性以及增强 STa 免疫原性成为可能。在本研究中，我们将对LT和STa基因进行基因突变，以表达无毒或低毒的LT和STa类毒素，然后将这两个突变基因进行基因融合，得到LT和STa类毒素融合抗原，以增强STa免疫原性。这些融合抗原预计会诱导抗 LT 和抗 STa 抗体，这些抗体将中和天然 LT 和 STa 毒素，从而提供针对 ETEC 感染的保护。在我们的初步研究中，我们证明猪 LT 类毒素与 STa 类毒素或 STb（另一种非免疫原性热稳定毒素）的基因融合诱导了抗 LT 和抗 ST 抗体，并且这些抗体在仔猪攻击模型中具有针对 ETEC 感染的保护作用。仔猪模型，特别是使用表达 K88ac 受体的仔猪和表达 K88ac 菌毛的 ETEC 菌株的系统，可能是研究人类 ETEC 相关腹泻的最佳模型。 K88ac受体阳性仔猪对表达K88ac菌毛的ETEC菌株呈中性敏感，并且在感染ETEC菌株后发展出与人类腹泻患者相同的临床疾病。此外，使用猪模型使我们能够检查肠道免疫力，这对于提供针对这种疾病的保护至关重要。我们在应用猪模型研究 ETEC 方面的专业知识使我们的研究团队成为使用猪模型进行 ETEC 腹泻研究和预防的最佳人选。确定 LT 和 STa 类毒素融合体对抗 ETEC 腹泻的疫苗候选资格将为抗毒素疫苗的开发提供重要信息，并且对于制定保护人类和动物免受腹泻病的预防和治疗策略非常有指导意义。公共卫生相关性：产肠毒素大肠杆菌 (ETEC) 是腹泻病的主要原因。 ETEC 腹泻每年导致近一百万人死亡，并给免疫功能低下的个人（特别是五岁以下儿童）、国际旅行者和军事人员带来严重的健康问题和经济后果。 ETEC 菌株产生的热不稳定（LT）和热稳定（STa）肠毒素是腹泻感染的毒力决定因素。 LT 和 STa 会刺激肠道内液体过度分泌，从而导致腹泻。然而，没有疫苗可以控制或预防这种疾病。开发抗毒素疫苗可能是预防腹泻的有效策略。然而，LT和STa均具有足够的毒性，因此它们不能直接用作开发抗毒素疫苗的抗原。这项拟议的研究是构建 LT 和 STa 类毒素，并确定全长 LT 类毒素和全长 STa 类毒素融合抗原在仔猪模型中针对 ETEC 感染的疫苗候选资格。确定 LT 和 STa 类毒素融合体抗腹泻疾病的疫苗候选资格将为开发有效的抗毒素疫苗来预防或控制这种疾病提供重要信息。

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) strains are categorized as priority B pathogens by NIAID. ETEC-associated diarrhea is responsible for nearly one million deaths each year around the world in addition to causing of illness to immunocompromised individuals and travelers including deployed military personnel. The key virulence factors in ETEC- associated diarrhea are bacterial adhesins or colonization factor antigens (CFAs) and enterotoxins. CFAs mediate bacterial attachment to the host epithelium cells, but it is the enterotoxin, the heat-labile (LT) and heat-stable (STa), that stimulate fluid hyper-secretion and cause diarrhea. Early studies using LT and STa antigens for developing antitoxin vaccines had limited success because of the high toxicity of both toxins, non-immunogenicity of STa antigen, and lack of a suitable animal study model. Current advance in molecular biology makes it feasible to eliminate or reduce toxicity and to enhance STa immunogenicity. In this study, we will genetically mutate both LT and STa genes for expressing non- or low toxic LT and STa toxoids, and then genetically fuse these two mutated genes for LT and STa toxoid fusion antigens to enhance STa immunogenicity. These fusion antigens are expected to induce anti-LT and anti-STa antibodies, and those antibodies will neutralize native LT and STa toxins thus provide protection against ETEC infection. In our preliminary studies, we demonstrated that a genetic fusion of a porcine LT toxoid with a STa toxoid or STb (another non-immunogenic heat stable toxin) induced anti-LT and anti-ST antibodies, and these antibodies are protective against ETEC infection in a piglet challenge model. Piglet model, especially the system using piglets expressing K88ac receptors and ETEC strains expressing K88ac fimbriae, is perhaps the best model to study human ETEC associated diarrhea. K88ac receptor positive piglets are neutrally susceptible to ETEC strains expressing K88ac fimbriae, and develop identical clinical disease after infection of ETEC strains as human diarrheal patients. Furthermore, using pig model allows us to examine intestinal immunity, which is critical for providing protection against this disease. Our expertise in applying pig model in studying ETEC makes our research team the best candidate to conduct ETEC diarrhea research and prevention using the pig model. Determination vaccine candidacy of LT and STa toxoid fusions against ETEC diarrhea will provide essential information for antitoxin vaccine development, and will be very instructive in developing prevention and treatment strategies to protect humans and animals from diarrhea disease. PUBLIC HEALTH RELEVANCE: Enterotoxigenic Escherichia coli (ETEC) are the primary cause of diarrheal disease. ETEC diarrhea is responsible for near one million deaths each year, and brings major health problems and economic consequences in immunocompromised individuals (especially children under age of five years), international travelers and military personnel. Heat labile (LT) and heat-stable (STa) enterotoxins produced by ETEC strains are the virulence determinants in diarrheal infection. LT and STa stimulate fluid hyper-secretion in guts that results in diarrhea. Yet, there are no vaccines available to control or prevent this disease. Development of antitoxin vaccines could be an effective strategy to prevent diarrhea. However, both LT and STa are sufficiently toxic, thereby they cannot be used directly as antigens for developing antitoxin vaccines. This proposed research is to construct LT and STa toxoids, and to determine vaccine candidacy of fusion antigens from a full-length LT toxoid and a full-length STa toxoid against ETEC infection in a piglet model. The determination of vaccine candidacy of LT and STa toxoid fusions against diarrheal disease will provide essential information for developing effective antitoxin vaccines to prevent or control this disease.