Safe mucosal vaccines

 描述(申请人提供)：口腔、鼻咽、胃肠、呼吸道和阴道的粘膜是大多数感染性微生物的入口点。以防止感染这些病原体，包括多种耐药微生物(如金黄色葡萄球菌、链球菌。SPP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、隐球菌、SARS、禽流感等)，迫切需要能够诱导强烈和保护性粘膜反应的疫苗。不幸的是，抑制对外来抗原的免疫反应的内源性调节系统使其对粘膜上的疫苗产生强烈的免疫反应具有挑战性。为了绕过这些抑制系统，疫苗必须在黏膜佐剂存在的情况下接种。到目前为止，已描述的最有效的粘膜佐剂属于细菌不稳定肠毒素(HLT)超家族。最近，人们发现LT是I型HLT亚家族的一个典型的HLT，当通过鼻腔途径引入时，它倾向于通过嗅神经从鼻腔进入大脑，在那里HLT诱导炎性细胞因子的表达，这一效应可能是通过与其特定的神经节苷脂受体结合而开始的。这 不良的副作用阻碍了HLTS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HLTS作为粘膜佐剂的潜在临床使用，我们的实验室评估了LT-IIb(T13I)的免疫特性，它是非人类II型HLT的单点替代突变体，具有：(I)改变其神经节苷脂受体的亲和力，(Ii)无可检测到的毒性，(Iii)强大的粘膜佐剂特性，但(V)不会引起神经炎症。另外两种无毒的HLT已经被设计出来，表现出类似的受体和免疫特性。我们假设，鼻腔给药LT-IIb(T13I)、LT-IIb(T14I)和LT-IIb(T13I/T14I)由于其受体结合特性的改变，不会动员到脑内，或者不诱导神经炎性细胞因子的表达或向脑内招募炎性细胞。我们将使用放射性碘标记的HLTS、免疫组织化学和活体表达成像(IVET)来验证这一假说。流式细胞术将用于评估鼻腔注射突变和wt HLTS的小鼠脑内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作为佐剂的抵抗力，但这些临床前实验将证明这种新型突变HLT佐剂的安全性和有效性。在这项原则验证研究成功完成后，我们将提交一份R01申请，以调查非人类灵长类动物中突变的HLT的性质，这将提供将这些有价值的免疫制剂转移到临床试验并最终进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.