Determining vaccine candidacy of LT and STa toxoid fusions against enterotoxigeni

说明(申请人提供)：产肠毒素大肠埃希氏菌(ETEC)菌株被NIAID归类为B优先病原体。ETEC相关性腹泻每年在世界各地造成近100万人死亡，此外还导致免疫受损的个人和旅行者，包括部署的军事人员患病。ETEC相关性腹泻的关键毒力因子是细菌黏附素或定植因子抗原(CFAs)和肠毒素。CFAs介导细菌附着到宿主上皮细胞，但刺激液体高分泌和引起腹泻的是肠毒素，即热不稳定(LT)和热稳定(STA)。早期使用LT和StA抗原开发抗毒素疫苗的研究成果有限，因为这两种毒素的毒性都很高，StA抗原不具有免疫原性，而且缺乏合适的动物研究模型。分子生物学的最新进展使消除或降低毒性和增强STA的免疫原性成为可能。在本研究中，我们将对表达无毒或低毒的LT和STA类毒素的LT和STA基因进行基因突变，然后将这两个突变的基因融合到LT和STA类毒素融合抗原中，以提高STA的免疫原性。这些融合抗原有望诱导产生抗LT和抗StA抗体，这些抗体将中和本地LT和StA毒素，从而对ETEC感染提供保护。在我们的初步研究中，我们证明了猪LT类毒素与STA类毒素或STB(另一种非免疫原性热稳定毒素)的基因融合可诱导抗LT和抗ST抗体，这些抗体在仔猪攻击模型中对ETEC感染具有保护作用。仔猪模型，特别是用表达K88ac受体的仔猪和表达K88ac菌毛的ETEC菌株组成的系统，可能是研究人类ETEC相关性腹泻的最佳模型。K88ac受体阳性仔猪对表达K88ac菌毛的ETEC菌株具有中性易感性，感染ETEC菌株后与人类腹泻患者发生相同的临床疾病。此外，使用猪模型可以检查肠道免疫，这对提供预防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利用猪模型进行ETEC腹泻研究和预防的最佳人选。确定针对ETEC腹泻的LT和STA类毒素融合疫苗的候选状态将为抗毒素疫苗的开发提供必要的信息，并将对制定预防和治疗策略以保护人类和动物免受腹泻疾病的影响非常有指导意义。公共卫生相关性：产肠毒素大肠杆菌(ETEC)是腹泻疾病的主要原因。ETEC腹泻每年造成近100万人死亡，并给免疫受损个人(特别是五岁以下儿童)、国际旅行者和军事人员带来重大健康问题和经济后果。ETEC菌株产生的热不稳定性(LT)和耐热性(STA)肠毒素是腹泻感染的毒力决定因素。它和STA会刺激肠道内液体的过度分泌，导致腹泻。然而，目前还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都有足够的毒性，因此它们不能直接用作抗原来开发抗毒素疫苗。本研究旨在构建LT类毒素和STA类毒素，并在仔猪模型中确定全长LT类毒素和STA类毒素融合抗原对抗ETEC感染的候选疫苗。确定LT和STA类毒素融合疫苗的候选状态将为开发有效的抗毒素疫苗以预防或控制该病提供必要的信息。

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.