Development of a Mucosal Vaccine Against Francisella tularensis

描述（由申请人提供）：大多数传染性病原体通过我们的粘膜表面引起疾病，这也适用于生物战剂。因此，在开发针对传染性/生物战剂的疫苗时，重要的是要诱导在粘膜表面以及系统隔室中起作用的反应。土拉菌是一种革兰氏阴性病原体，是土拉菌病的病因。这种微生物是一种“a类病原体和生物战剂”。该项目的总体目标是开发一种安全的粘膜疫苗，有效地诱导对土拉菌感染的保护性反应。具体而言，我们将1)确定土拉菌热休克蛋白的免疫原性，以及皂苷类似物GPI-0100和蓖麻毒素B在小鼠全身或鼻内免疫后调节宿主免疫反应的作用。将收集血清和分泌物，并通过ELISA测定抗原特异性抗体活性的性质和水平。细胞将被培养并评估抗原特异性T细胞增殖反应和细胞因子产生（通过ELISA）。在兔拉菌感染全身或粘膜后，将评估对保护反应的有效性。2)确定土拉菌及其LPS和佐剂调节宿主反应的细胞机制。toll样受体（TLRs）和B7共刺激系统在介导宿主反应和感染中的作用将在体外和体内进行评估。将在体外刺激正常小鼠和TLR缺陷小鼠的抗原呈递细胞，并通过流式细胞术评估MHC和B7表达的变化，通过ELISA评估细胞因子的产生。参与细胞激活的细胞信号通路也将被确定。TLR和B7敲除小鼠将被用来确定TLR和B7在土拉菌及其LPS应答中的作用。体液和细胞反应将如上所述进行评估。土拉菌的清除将通过微生物学分析来测量。3)获得并鉴定具有苜蓿酸盐和/或嘌呤代谢途径突变的土拉菌LVS的遗传定义减毒菌株，用于活疫苗。将衍生出突变体，并在小鼠中测试其安全性、通过微生物学分析在宿主组织中的持久性、通过诱导细胞（细胞增殖和细胞因子产生）和体液（通过ELISA检测抗体活性的性质和水平）反应的免疫原性，以及诱导保护性免疫的有效性。这些研究将确定先天免疫系统和适应性免疫系统在诱导对土拉菌病的保护性反应中的作用，并将确定一种安全有效的疫苗，以对抗土拉菌病的粘膜或全身攻击。

DESCRIPTION (provided by applicant): Most infectious agents cause disease via our mucosal surfaces, which also applies to biological warfare agents. Therefore, in developing a vaccine against infectious/biological warfare agents, it is important to induce responses that would act at mucosal surfaces, as well as in the systemic compartment. Francisella tularensis is a gram-negative pathogen and cause of tularemia. This microorganism is a "category A pathogen and biological warfare agent". The overall goal of this project is to develop a safe mucosal vaccine effective in inducing protective responses against infection by F. tularensis. Specifically, we will 1) Determine the immunogenicity of heat shock proteins of F. tularensis and the effect of the saponin analog GPI-0100 and of ricin B in modulating host immune responses following systemic or intranasal immunization of mice. Serum and secretions will be collected and assayed for the nature and level of antigen-specific antibody activity by ELISA. Cells will be cultured and assessed for antigen-specific T cell proliferative responses and cytokine production (by ELISA). The effectiveness of the response on protection will be assessed following systemic or mucosal challenge with F. tularensis. 2) Determine the cellular mechanism(s) by which F. tularensis and its LPS, and the adjuvants modulate host responses. The role of Toll-like receptors (TLRs) and the B7 costimulatory system in mediating host responses and infection will be assessed in vitro and in vivo. Antigen-presenting cells from normal and TLR deficient mice will be stimulated in vitro and assessed for changes in the expression of MHC and B7 by flow cytometry and for cytokine production by ELISA. The cell signaling pathways involved in cell activation will also be determined. TLR- and B7-knockout mice will be used to determine the role of TLRs and B7 in responses to F. tularensis and its LPS. Humoral and cellular responses will be assessed as indicated above. Clearance of F. tularensis will be measured by microbiologic analysis. 3) Derive and characterize genetically defined attenuated strains of F. tularensis LVS with mutations in the shikimate and/or purine metabolic pathways for use as a live vaccine. Mutants will be derived and tested in mice for their safety, persistence in host tissue by microbiologic analysis, immunogenicity by inducing cellular (cell proliferation and cytokine production) and humoral (nature and level of antibody activity by ELISA) responses, and effectiveness in inducing protective immunity. These studies will define the role of the innate and adaptive immune systems in inducing protective responses to F. tularensis and will define a safe and efficacious vaccine against mucosal or systemic challenge with F. tularensis.