Type II enterotoxins as mucosal immunomodulators

描述（由申请人提供）：迫切需要开发新的方法来增强针对感染口腔、胃和泌尿生殖道粘膜的病原体的保护性免疫应答。本提案的目的是评价两种大肠杆菌II型肠毒素LT-IIa和LT-IIb的粘膜佐剂活性。LT-IIa和LT-IIb诱导增强免疫应答的独特模式，其与霍乱毒素（CT）诱导的免疫应答截然不同。尽管CT通常基于抗体同种型和细胞因子模式诱导主要的Th 2型应答，但给予II型肠毒素作为佐剂的小鼠表现出更平衡的Th 1/Th 2应答。我们还证明了LT-IIa、LT-IIb和CT与不同的淋巴细胞群体相互作用，并在这些群体中诱导独特的细胞反应（细胞凋亡、细胞因子产生、增殖等）。总的来说，这些数据提供了强有力的证据表明，LT-IIa和LT-IIb（和CT）利用不同的细胞和分子机制进行免疫调节。我们的假设是，LT-Ⅱ a和LT-Ⅱ b（和CT）的独特的佐剂活性是由它们对免疫活性细胞上不同受体的结合亲和力引起的，这些受体是触发特定信号转导事件所必需的。为了检验这一假设，LT-IIa和LT-IIb的佐剂活性，以及受体结合活性改变的突变肠毒素的集合，将在粘膜小鼠模型中使用口腔病原体变形链球菌的Agl/II作为模型抗原进行分析。将对其他LT-IIa和LT-IIb突变体进行工程改造，以确定ADP-核糖基化和细胞运输在免疫调节中的重要性。先前的研究已经揭示了淋巴细胞上的受体，其可能是LT-IIb的佐剂活性的触发器。使用相关淋巴细胞的消融和阻断实验将用于表征受体。将研究肠毒素对树突状细胞（粘膜的主要前哨抗原呈递细胞）的细胞和分子应答的影响，作为将LT-IIa和LT-IIb的佐剂活性与特定淋巴细胞相关联的进一步手段。突变肠毒素作为保护性粘膜佐剂的效力也将使用已建立的S.变形菌鼠定殖模型。本文获得的基本信息对于确定II型肠毒素或其无毒突变体作为随后疫苗使用的粘膜佐剂的潜力至关重要。

DESCRIPTION (provided by applicant): There is an urgent need to develop new means for potentiating protective immune responses against pathogens that infect the oral, gastric and urogenital mucosae. The objective of this proposal is to evaluate the mucosal adjuvant activities of LT-IIa and LT-IIb, two Escherichia coli Type II enterotoxins. LT-IIa and LT-IIb induce distinctive patterns of enhanced immune responses which are profoundly different from those induced by cholera toxin (CT). Whereas CT commonly induces a predominant Th2-type response based on antibody isotype and cytokine patterns, mice administered with Type II enterotoxins as adjuvants exhibit a more balanced Th1/Th2 response. We have also demonstrated that LT-IIa, LT-IIb, and CT interact with different populations of lymphocytes and induce in those populations distinctive cellular responses (apoptosis, cytokine production, proliferation, etc.). Collectively, these data provide strong evidence that LT-IIa and LT-IIb (and CT) utilize different cellular and molecular mechanisms for immunomodulation. Our hypothesis is that the distinctive adjuvant activities of LT-IIa and LT-IIb (and CT) are elicited by their binding affinity for different receptors on immunocompetent cells which are required to trigger specific signal transduction events. To test this hypothesis, the adjuvant activities of the LT-IIa and LT-IIb, and a collection of mutant enterotoxins with altered receptor-binding activities, will be analyzed in a mucosal mouse model using Agl/II of the oral pathogen Streptococcus mutans as a model antigen. Other LT-IIa and LT-IIb mutants will be engineered to establish the importance of ADP- ribosylation and cellular trafficking in immunomodulation. Prior investigations have revealed a receptor on lymphocytes which is likely the trigger for the adjuvant activities of LT-IIb. Ablation and blocking experiments using relevant lymphocytes will be used to characterize the receptor. The affect of the enterotoxins on the cellular and molecular responses of dendritic cells, the major sentinel antigen- presenting cells of the mucosa, will be investigated as a further means to correlate the adjuvant activities of LT-IIa and LT-IIb with particular lymphocytes. Efficacy of the mutant enterotoxins as protective mucosal adjuvants will also be determined using an established S. mutans murine colonization model. The fundamental information obtained herein will be essential for establishing the potential of Type II enterotoxins, or their non-toxic mutants, as mucosal adjuvants for subsequent vaccine use.