Abstract
Photodynamic inactivation of Leishmania has been shown to render them non-viable, but retain their immunological activities. Installation of dual photodynamic mechanisms ensures complete inactivation of species in the Leishmania subgenus, raising the prospect of their safe and effective application as whole-cell vaccines against leishmaniasis. Here, we report the successful extension of this approach to L. braziliensis in the Viannia subgenus, viz. genetic engineering of promastigotes for cytosolic accumulation of UV-sensitive uroporphyrin (URO) and their loading with red light excitable phthalocyanines (PC) that was cationized by chemical engineering. The transgenic strategy used previously produced L. braziliensis transfectants, which gave the same phenotype of aminolevulinate (ALA)-inducible uroporphyria as found in Leishmania subgenus, indicative of pre-subgenus evolutionary origin for similar genetic deficiencies in porphyrin/heme biosynthesis. In the present study, 12 independent clones were obtained and were invariably ALA-responsive, albeit to different extent for uroporphyrinogenesis and UV-inactivation. In a separate study, L. braziliensis was also found, like other Leishmania spp., to take up diamino-PC (PC2) for red light inactivation. In vitro interactions of a highly uroporphyrinogenic clone with primary macrophages were examined with the intervention of URO/PC2-medated double-photodynamic inactivation to ascertain its complete loss of viability. Doubly sensitized L. braziliensis transfectants were photo-inactivated before (Strategy #1) or after (Strategy #2) loading of macrophages. In both cases, macrophages were found to take up L. braziliensis and degrade them rapidly in contrast to live Leishmania infection. The effector functions of macrophages became upregulated following their loading with L. braziliensis photodynamically inactivated by both strategies, including CD86 expression, and IL6 and NO production. This was in contrast to the immunosuppressive infection of macrophages with live parasites, marked by IL10 production. The results provide evidence that photodynamically inactivated L. braziliensis are susceptible to the degradative pathway of macrophages with upregulation of immunity relevant cytokine and co-stimulatory markers. The relative merits of the two loading strategies with reference to previous experimental vaccination were discussed in light of the present findings with L. braziliensis.
Highlights
Photodynamic inactivation of Leishmania has been shown to render them non-viable, but retain their immunological activities
Leishmania braziliensis of the Viannia subgenus was successfully photodynamically inactivated by the dual approach of genetic and chemical engineering, which has proven effective to render members of the Leishmania subgenus non-viable for safe and effective use as experimental whole-cell vaccines[12,13]
All transgenic clones produced in the present work were responsive to delta-aminolevulinate (ALA) for uroporphyrinogenesis, i.e. cytosolic de novo synthesis and accumulation of uroporphyrin 1 (URO), making them sensitive to UV inactivation
Summary
Photodynamic inactivation of Leishmania has been shown to render them non-viable, but retain their immunological activities. Porphyrinogenic Leishmania, e.g. L_amazonensis was previously produced by complementation of such genetic defects in promastigotes with mammalian cDNAs to express the 2nd and 3rd enzymes in the classic heme biosynthetic pathway, i.e. aminolevulinate dehydratase (ALAD) and porphobilinogen deaminase (PBGD), r espectively[18,19] Exposure of these doubly transfected parasites to aminolevulinate (ALA), the product of the 1st enzyme, results in their cytosolic accumulation of uroporphyrin I (URO)—a PS excitable with longwave UV to produce 1O2 and other cytotoxic oxidative metabolites secondarily. Sensitization of L. amazonensis by a combination of endogenously accumulated URO and exogenously supplied cationic PC provided assurance of their complete photo-inactivation without e xception[20] These Leishmania are invariably non-viable, but remain structurally and immunologically intact, as their use to immunize highly susceptible BALB/c mice protected them against a homologous challenge by suppressing lesion development and significantly reducing parasite b urdens[13]
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