T CELL INDEPENDENT ANTIPSEUDOMONAS VACCINE DEVELOPMENT

这一建议的重点是开发一种疫苗，以防止呼吸道感染假单胞菌，囊性纤维化的主要病原体变成DC和活化CD4+ T细胞的混合体。研究的核心假设是，在体内给药经过基因修饰表达CD40L并以热杀灭假单胞菌为引物的DC将诱导足够强大的抗假单胞菌宿主防御，以防止假单胞菌的呼吸道感染。初步数据表明，CD40L/DC/假单胞菌策略在体内外均可诱导特异性抗假单胞菌免疫。引人注目的是，使用来自CD4-/-小鼠的成分，CD40L基因修饰，假单胞菌引发的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修饰的假单胞菌引物DC体外诱导幼稚B细胞产生假单胞菌特异性免疫球蛋白，保护体内CD4-/-小鼠免受假单胞菌肺攻击。CD40L修饰的假单胞菌通过使用修饰的Ad载体来提高基因转移到DC的效率，以最佳地结合并进入DC，或者通过使用可能允许更长时间表达CD40L转基因的载体来引发DC。具体目标2评估了减少CD40L转基因数量可以实现保护的假设。特异性目标2评估了这样一种假设，即减少CD40L/DC/假单胞菌的数量可以通过修饰DC来表达由活化DC产生的效应介质（例如，IL12）、抑制DC诱导凋亡的分子（crmA， TRANCE）或CD4+共刺激分子（CD40L）的基因编码来实现保护。特异性目的3检验了一个假设，即使用实验室假单胞菌菌株PA01的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个体的各种假单胞菌，并且c57Bl/6小鼠的这种策略对其他小鼠菌株和CFTR-/-小鼠具有普遍的保护作用。特异性目的4评估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是否适用于人体成分的假设。

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.