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.
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