T CELL INDEPENDENT ANTIPSEUDOMONAS VACCINE DEVELOPMENT

该提案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转化为DC和活化的CD 4 + T细胞的杂交体。中心假设是，体内施用已被遗传修饰以表达CD 40 L并用热灭活的假单胞菌引发的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，CD 40 L/DC/假单胞菌策略在体外和体内诱导特异性抗假单胞菌免疫。引人注目的是，使用来自CD 4-/-小鼠的组分，CD 40 L基因修饰的假单胞菌致敏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CD 4-/-小鼠免受假单胞菌的肺攻击。CD 40 L修饰的假单胞菌致敏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CD 4-/-小鼠免受假单胞菌肺攻击。通过使用经修饰以最佳地结合并进入DC的Ad载体或通过使用可允许CD 40 L转基因表达的更长持续时间的载体来增强基因转移至DC的效率，来增强CD 40 L修饰的假单胞菌致敏的DC。具体目标2评估了可以用减少数量的CD 40 L转基因实现保护的假设。具体目标2评估了这样的假设，即通过还修饰DC以表达编码由活化的DC产生的效应介质的基因（例如，IL 12）、将起到抑制DC中细胞凋亡诱导作用的分子（crmA，TRANCE）或除CD 40 L之外的CD 4+共刺激分子（CD 28）。具体目标3检查了以下假设：使用实验室假单胞菌菌株PA 01，用CD 40 L/DC/假单胞菌策略诱导的抗假单胞菌免疫足以保护免受来自CR个体的各种假单胞菌分离株的侵害，并且该策略在c57 Bl/6小鼠中提供的保护对于其他小鼠品系和CFTR-/-小鼠是通用的。具体目标4评估了以下假设：在鼠系统中建立的CD 40 L/DC/假单胞菌策略诱导的抗假单胞菌免疫适用于人组分。

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.