T CELL INDEPENDENT ANTIPSEUDOMONAS VACCINE DEVELOPMENT

这项提议的重点是开发一种疫苗，以预防呼吸道感染假单胞菌，假单胞菌是囊性纤维化的主要病原体，它是DC和活化的CD4+T细胞的杂交。中心假设是，在体内给予表达CD40L的转基因DC，并用热灭活假单胞菌启动，将诱导足够强大的抗假单胞菌宿主防御，以防止假单胞菌呼吸道感染。初步数据表明，CD40L/DC/假单胞菌策略在体内外均能诱导特异性抗假单胞菌免疫。值得注意的是，利用来自CD4-/-小鼠的成分，CD40L基因修饰的假单胞菌诱导的DC在体外诱导幼稚的B细胞产生假单胞菌特异性免疫球蛋白，并在体内保护CD4-/-小鼠免受假单胞菌的肺部攻击。CD40L修饰的假单胞菌致敏的DC在体外诱导幼稚B细胞产生假单胞菌特异性免疫球蛋白，并在体内保护CD4-/-小鼠免受假单胞菌的肺部攻击。CD40L修饰，假单胞菌引发的DC，通过使用修改为最佳地结合和进入DC的Ad载体或通过使用允许CD40L转基因表达更长时间的载体来提高向DC的基因转移的效率。特定目的2评估了通过减少CD40L转基因数量就可以实现保护的假设。特殊目的2评估了这样的假设，即减少CD40L/DC/假单胞菌的数量可以通过修改DC来表达编码由激活的DC产生的效应介质(例如IL12)的基因，这些分子将起到抑制DC(CrmA，TRANCE)或除CD40L(CD28)之外的CD4+共刺激分子的诱导凋亡的作用。特异目的3检验了这样一种假设，即使用实验室假单胞菌株PA01用CD40L/DC/假单胞菌策略诱导的抗假单胞菌免疫足以保护来自CR患者的各种假单胞菌分离株，并且该策略在C57BL/6小鼠中提供的保护对其他品系的小鼠和CFTR-/-小鼠是普遍的。特异性目标4评估了在小鼠系统中建立的CD40L/DC/假单胞菌策略诱导的抗假单胞菌免疫适用于人成分的假设。

The focus of this proposal is to develop a vaccine to prevent respiratory infection with Pseudomonas, the major pathogen in cystic fibrosis into a hybrid of a DC and activated CD4+ T cell. The central hypothesis is that in vivo administration of DC that have been genetically modified to express CD40L and primed with heat killed Pseudomonas will induce sufficiently robust anti-Pseudomonas host defenses to prevent respiratory infection with Pseudomonas. The preliminary data demonstrates that the CD40L/DC/Pseudomonas strategy works to induce specific anti- Pseudomonas immunity in vitro and in vivo. Strikingly, using components from CD4-/- mice, the CD40L genetically modified, Pseudomonas primed DC induce naive B cell to produce Pseudomonas- specific immunoglobins in vitro and protect CD4-/- mice in vivo from pulmonary challenge with Pseudomonas. CD40L modified, Pseudomonas primed DC induce naive B cell to produce Pseudomonas specific immunoglobulins in vitro and protect CD4-/- mice in vivo from pulmonary challenge with Pseudomonas. CD40L modified, Pseudomonas primed DC by enhancing the efficiency of gene transfer to the DC using Ad vectors modified to optimally bind to, and enter, DC, or by using vectors that may permit longer duration of expression of the CD40L transgene. Specific aim 2 evaluates the hypothesis that protection can be accomplished with reduced numbers of CD40L transgene. Specific aim 2 evaluates the hypothesis that protection can be accomplished with reduced numbers of CD40L/DC/Pseudomonas by also modifying the DC to express genes coding for effector mediators produced by activated DC (e.g., IL12), molecules that will function to suppress the induction of apoptosis in DC (crmA, TRANCE), or CD4+ co-stimulatory molecules in addition to CD40L (CD28). Specific aim 3 examines the hypothesis that the anti-Pseudomonas immunity induced with the CD40L/DC/Pseudomonas strategy using the laboratory Pseudomonas strain PA01 is sufficient to protect against a variety of isolates of Pseudomonas from individuals with CR, and that the protection afforded by this strategy in c57Bl/6 mice is universal for other strains of mice, and for CFTR-/- mice. Specific aim 4 assesses the hypothesis that the anti- Pseudomonas immunity induced with the CD40L/DC/Pseudomonas strategy established in the murine system is applicable to human components.