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.

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American tegumentary leishmaniasis (TL) caused by Leishmania braziliensis is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of Leishmania -specific anti-α-Gal antibodies. However, the native parasite α-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the L. major- derived type-2 glycoinositolphospholipid (GIPL)-1 (Gal f β1,3Manα), GIPL-2 (Galα1,3Gal f β1,3Manα), and GIPL-3 (Galα1,6Galα1,3Gal f β) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that L. braziliensis -specific anti-α-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of L. major type-2 GIPL-3-derived Galα1,6Galα1,3Gal f β glycotope for the serological diagnosis of American TL, particularly of the subclinical form.

In this study, we generated a transgenic strain of Leishmania braziliensis, an etiological agent associated with a diversity of clinical manifestations of leishmaniasis ranging from localized cutaneous to mucocutaneous to disseminated disease. Transgenic parasites expressing reporter proteins are valuable tools for studies of parasite biology, host-pathogen interactions, and anti-parasitic drug development. To this end, we constructed an L. braziliensis line stably expressing the reporters eGFP and luciferase (eGFP-LUC L. braziliensis). The integration cassette co-expressing the two reporters was targeted to the ribosomal locus (SSU) of the parasite genome. Transgenic parasites were characterized for their infectivity and stability both in vitro and in vivo. Parasite maintenance in axenic long-term culture in the absence of selective drugs did not alter expression of the two reporters or infection of BALB/c mice, indicating stability of the integrated cassette. Infectivity of eGFP-LUC, L. braziliensis, both in vivo and in vitro was similar to that obtained with the parental wild type strain. The possibility of L. braziliensis tracking and quantification using fluorescence and luminescence broadens the scope of research involving this neglected species, despite its importance in terms of public health concerning the leishmaniasis burden.

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.

25 26 2 27 Abstract:28 The long Pentraxin 3 (PTX3), a soluble pattern recognition molecule, plays a critical role 29 in inflammation, tissue repair and wound healing. Here, we show that PTX3 regulates 30 disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression is increased in 31 active skin lesions in patients and mice during CL, with higher levels being expressed in 32 individuals with severe disease. PTX3 deficient (PTX3 -/-) mice were highly resistant to L.33 major infection and the enhanced resistance was associated with increased IL-17 34 response. Neutralization of IL-17A abolished this enhanced resistance while treatment 35 with recombinant PTX3 resulted in reduced IL-17A response and increased susceptibility 36 to L. major infection. Naïve CD4 + T cells from PTX3 -/mice displayed increased 37 differentiation into Th17 cells, which was reversed in the presence of recombinant PTX3. 38 The enhanced Th17 response observed in PTX3 -/cells was associated with increased 39 Leishmania specific IL-6 production from dendritic cells along with enhanced expression 40 of Th17-specific transcription factors including RORt, AhR and STAT3. Addition of 41 recombinant PTX3 significantly inhibited the expression of Th17-specific transcription 42 factors and dramatically reduced the frequency of Th17 cells in Th17-polarizing cultures 43 of PTX3 -/-CD4 + T cells. Collectively, our results show that PTX3 contributes to the 44 pathogenesis of CL by suppressing Th17 differentiation and IL-17A production. 45 46 Author Summary:47 Cutaneous leishmaniasis (CL) is caused by several species of Leishmania. Currently, 48 there is no approved vaccine against human CL because of the poor understanding of 49 the mechanisms that regulate disease pathogenesis and correlates of protective 50 immunity. Because the long pentraxin 3 (PTX3, a soluble pattern recognition molecule 51 that forms an integral part of the host innate immunity), regulates inflammation and 3 52 tissue repair, which are critical physiological events associated with resolution of skin 53 lesions during CL, we investigated its role in disease pathogenesis.54 Here, we show that PTX3 levels were elevated in skin-lesions in patients and mice 55 during CL. Using a loss of function approach, we showed that PTX3 contributes to 56 pathogenesis, and this was associated with increased IL-17A responses. Neutralization 57 and recombinant cytokine treatment studies showed that the increased resistance of 58 PTX3 deficient mice to L. major is due to enhanced Th17 response in these mice. We 59 further show that PTX3 negatively regulates IL-6 production by dendritic cells and the 60 expression of IL-17A-specific transcription factors (including RORT, STAT3, IRF4, 61 BATF and AhR) in CD4 + T cells. Collectively, these findings show that PTX3 is a 62 negative regulator of Th17 response and protective immunity during L. major infection. 4 64 Introduction: 65 66 Cutaneous leishmaniasis (CL) is caused by several species of protozoan parasites that 67 belong to the genus Leishman...