We prepared transgenic Leishmania, which are potentially useful for photodynamic vaccination against leishmaniasis and other diseases. These trypanosomatid protozoa are naturally deficient in heme biosynthesis, thereby rendering them dependent on the exogenous supply of tetrapyrroles for making functional respiratory complexes. We exploit these peculiar defects in Leishmania to produce suicidal mutants. Since these intracellular parasites naturally infect dendritic cells and reside in the phagolysosomes of macrophages, such mutants may serve as effective carriers to deliver pro-drugs or vaccines for their activation or presentation when they are signaled to commit intralysosomal suicidal cytolysis. To engineer such mutants, Leishmania spp. were transfected with mammalian genes encoding the 2nd and 3rd enzymes in heme biosynthesis pathway (Sah et al. 2002. J. Biol. Chem 277, 14902-9). These transfectants were thus rendered porphyric when exposed to an external signal, i.e. delta-aminolevulinate (ALA) - products of the 1st enzyme in this pathway. When macrophages were infected with these transfectants and exposed to ALA, porphyria developed both in the host cells and in their intracellular Leishmania. However, porphyrins formed in macrophages are metabolized rapidly to the background level, while those in Leishmania accumulate and persist due to the absence of heme metabolic pathway. The development of porphyria of these intra- macrophage mutants resulted in their selective cytolysis, which can be regulated and enhanced by light illumination. This selective destruction of intracellular mutants altered the global expression profiles of in vitro infected macrophages, suggestive of enhanced immunogenicity and parasite elimination, as determined by microarray analyses. This is supported by preliminary in vivo data, indicative of protection via this scheme of vaccination against experimental kala-azar in the Syrian Golden hamster model. We propose in this application to further explore the efficacy of this live vaccine model by undertaking the following specific aims: [1] To replicate the positive outcome in the preliminary experiments against kala-azar in the hamster model by paying special attention to the absence of Leishmania persistence and residual pathogenicity; and [2] To extend the findings to additional animal models, e. g. BALB/c mouse, susceptible to cutaneous leishmaniasis by challenging them with the cutaneous species, i.e. Leishmania amazonensis. The results of these exploratory studies help evaluate the potential of the suicidal mutants for use as live vaccines not only against leishmaniasis but also as vaccine purveyors against other infectious and non-infectious diseases. ? ? ?
