Safe mucosal vaccines

 描述（由申请方提供）：口腔、鼻咽、胃肠道、呼吸道和阴道的粘膜是大多数感染性微生物的进入点。为了防止这些病原体的感染，包括多种抗生素耐药微生物（例如，金黄色葡萄球菌、链球菌spp.，等等）。以及新出现或重新出现的病原体（例如，埃博拉病毒、肠道病毒、结核病、艰难梭菌、格特隐球菌、SARS、禽流感等），迫切需要能诱导强烈和保护性粘膜应答的疫苗。不幸的是，抑制对外来抗原的免疫应答的内源性调节系统使得在粘膜上引发对疫苗的强烈免疫应答具有挑战性。为了避开这些抑制系统，疫苗必须在粘膜佐剂存在下施用。迄今为止，已经描述的最有效的粘膜佐剂属于细菌不耐热肠毒素（HLT）的超家族。最近，确定LT（I型HLT亚家族的原型HLT）在经由鼻内途径引入时具有经由嗅觉神经从鼻腔运输至脑的倾向，其中HLT诱导炎性细胞因子的表达，该效应可能通过结合其特异性神经节苷脂受体而引发。这 不希望的副作用阻碍了HLT作为粘膜佐剂的临床应用。为了消除这种不良副作用并恢复HLT作为临床粘膜佐剂的潜在用途，我们的实验室评估了LT-IIb（T13 I）的免疫特性，LT-IIb（T13 I）是一种非人II型HLT的单点取代突变体，具有：（i）对其神经节苷脂受体的亲和力改变，（ii）没有可检测的毒性，（iii）强的粘膜佐剂性质，但（v）不诱导神经炎症。另外两种无毒的HLT已经被工程化，表现出类似的受体和免疫特性。我们假设鼻内给予LT-IIb（T13 I）、LT-IIb（T14 I）和LT-IIb（T13 I/T14 I），由于其受体结合特性改变，不会动员到脑中，或者不会诱导神经炎性细胞因子的表达或将炎性细胞募集到脑中。我们将使用放射性碘标记的HLT、免疫组织化学和体内表达成像（IVET）来检验这一假设。将采用流式细胞术评价鼻内施用突变型和wt HLT的小鼠脑中的细胞因子环境和炎性细胞的存在或不存在。虽然我们认识到目前在临床试验中评估HLT作为佐剂的阻力，但这些临床前实验将证明这种新的O类突变体HLT佐剂的安全性和有效性。在这项原理验证研究成功完成后，我们将提交R01申请，以研究非人灵长类动物中突变HLT的特性，这将提供将这些有价值的免疫制剂投入临床试验并最终投入临床使用所需的数据。

 DESCRIPTION (provided by applicant): The mucosae of the oral, nasopharyngeal, gastrointestinal, respiratory, and vaginal tracts are the points of entry for most infectious microorganisms. To preclude infection by those agents, which include multiple antibiotic resistant microorganisms (e.g., Staph aureus, Strep. spp., etc.) and pathogens that are newly emerging or reemerging (e.g., Ebola virus, enterovirus, tuberculosis, Clostridium difficile, Cryptococcus gattii, SARS, avian flu, etc.), vaccines that will induce strong and protective mucosal responses are desperately needed. Unfortunately, endogenous regulatory systems that suppress immune responses to foreign antigens make it challenging to elicit strong immune responses to vaccines on the mucosae. To circumvent these suppressive systems, vaccines must be administered in the presence of mucosal adjuvants. To date, the most potent mucosal adjuvants that have been described belong to the superfamily of bacterial heat-labile enterotoxins (HLT). Recently, it was determined that LT, a prototypical HLT of the Type I HLT subfamily, when introduced via the intranasal route, has a propensity to traffic from the nasal cavity to the brain via the olfactory nerve where the HLT induces expression of inflammatory cytokines, an effect that is likely initiated by binding to its specific ganglioside receptor. This undesirable side-effect has hindered the clinical use of HLTs as mucosal adjuvants. To abrogate this undesirable side-effect and to restore the potential use of HLTs as mucosal adjuvants for clinical use, our laboratory has evaluated the immune properties of LT-IIb(T13I), a single point substitution mutant of a non-human Type II HLT that has: (i) altered affinity for its ganglioside receptors, (ii) no detectible toxicity, (iii) strong mucosal adjuvant properties, but (v) DOES NOT induce neuroinflammation. Two additional non-toxic HLTs have been engineered that exhibit similar receptor and immune properties. We HYPOTHESIZE that intranasally administered LT-IIb(T13I), LT-IIb(T14I), and LT- IIb(T13I/T14I), due to their altered receptor-binding properties, do not mobilize into the brain, or alternatively, do not induce expression of neuroinflammatory cytokines or recruit inflammatory cells into the brain. We will test that hypothesis using radioiodinated HLTs, immunohistochemistry, and In Vivo Expression Imaging (IVET). Flow cytometry will be employed to evaluate the cytokine environment and the presence or absence of inflammatory cells in the brains of mice intranasally administered mutant and wt HLTs. While we recognize the current resistance to evaluating HLTs as adjuvants in clinical trials, these pre-clinical experiments will demonstrate the safely and efficacy of this new class o mutant HLT adjuvants. At the successful completion of this proof-of-principle research, we will submit an R01 application to investigate the properties of the mutant HLTs in non-human primates, which will provide the data needed to move these valuable immune agents into clinical trials and, ultimately, into clinical use.