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.

Aedes aegypti is the major vector of 1 of the most concerning arboviruses of the world, the dengue fever. The only effective way of reducing the incidence of dengue fever is to control the vector mosquito, mainly by application of insecticides to its breeding places. This study was aimed at assessing the insecticidal activity of sodium anacardate, isolated from Brazilian cashew nut shell liquid (CNSL), against the eggs, 3rd instars or pupae of Ae. aegypti. In addition, the acute toxicity of sodium anacardate to mice was also investigated. Sodium anacardate showed toxicity against Ae. aegypti eggs (median effective concentration [EC50] = 162.93 +/- 29.93 microg/ml), larvae (median lethal concentration [LC50] = 55.47 +/- 3.0 microg/ml) and pupae (LC50 = 369.78 - 52.30 microg/ml). On the other hand, even at high dose (0.3 g/kg body weight), this compound did not cause any adverse effects on mice, suggesting that this compound is safe to mammals. Therefore, sodium anacardate may be a viable low-cost alternative to help combat Ae. aegypti.

Background: Photosensitizers (PS), like porphyrins and phthalocyanines (PC) are excitable by light to generate cytotoxic singlet oxygen and other reactive oxygen species in the presence of atmospheric O2. Photodynamic inactivation of Leishmania by this means renders them non-viable, but preserves their effective use as vaccines. Leishmania can be photo-inactivated after PS-sensitization by loading via their endocytic uptake of PC or endogenous induction of transgenic mutants with delta-aminolevulinate (ALA) to accumulate cytosolic uroporphyrin I (URO). Here, PS-sensitization and photo-inactivation of Leishmania amazonensis was further examined in vitro and in vivo for vaccination against cutaneous leishmaniasis (CL).Methods and Results: Leishmania amazonensis promastigotes were photodynamically inactivated in vitro by PC-loading followed by exposure to red light (1–2 J/cm2) or ALA-induction of uroporphyrinogenic transfectants to accumulate cytosolic URO followed by longwave UV exposure. When applied individually, both strategies of photodynamic inactivation were found to significantly, albeit incompletely abolish the MTT reduction activities of the promastigotes, their uptake by mouse bone marrow-derived macrophages in vitro and their infectivity to mouse ear dermis in vivo. Inactivation of Leishmania to completion by using a combination of both strategies was thus used for the sake of safety as whole-cell vaccines for immunization of BALB/c mice. Different cutaneous sites were assessed for the efficacy of such photodynamic vaccination in vivo. Each site was inoculated first with in vitro doubly PS-sensitized promastigotes and then spot-illuminated with white light (50 J/cm2) for their photo-inactivation in situ. Only in ear dermis parasites were photo-inactivated beyond detection. Mice were thus immunized once in the ear and challenged 3 weeks later at the tail base with virulent L. amazonensis. Prophylaxis was noted in mice photodynamically vaccinated with doubly photo-inactivated parasites, as indicated by a significant delay in the onset of lesion development and a substantial decrease in the parasite loads.Conclusion: Leishmania doubly PS-sensitized and in situ photo-inactivated as described proved to be safe and effective when used for one-time immunization of ear dermis, as indicated by its significant protection of the inherently very susceptible BALB/c mice against CL.

Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen−/−) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen−/− and wild-type promastigotes were similar, but axenic and intracellular LbCen−/− amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen−/− were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen−/−, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen−/− can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.