Immune Response to LinB13, a Lutzomyia Intermedia Salivary Protein Correlates With Disease Severity in Tegumentary Leishmaniasis.

BACKGROUND: We have previously shown that seropositivity to rLinB-13, a salivary protein from Lutzomyia intermedia, predicted sand fly exposure and was associated with increased risk of developing cutaneous leishmaniasis (CL). METHODS: Here, we investigated the cellular immune response to saliva from Lu. intermedia, using rLinB-13 as a surrogate antigen in naturally exposed individuals presenting positive serology to LinB-13. We also investigated the response to rLinB-13 in leishmaniasis patients, displaying active ulcers and positive PCR for Leishmania braziliensis. RESULTS: Peripheral blood mononuclear cells (PBMCs) stimulated in vitro with rLinB-13 secreted elevated levels of interleukin-10 (IL-10), IL-4, IL-1ß, IL-1α, IL-6, and chemokines (CCL3, CCL4, CCL5, and CXCL5). CL and disseminated leishmaniasis (DL) patients displayed a significantly higher immunoglobulin G (IgG) response to rLinB-13 compared with healthy subjects, and anti-rLinB-13 IgG was positively correlated with the number of lesions in DL patients. Positive serology to rLinB-13 was also associated with chemotherapy failure. PBMCs from DL patients stimulated with rLINB-13 secreted significantly higher levels of IL-10 and IL-1ß compared with CL individuals. CONCLUSIONS: In this study, we observed an association between humoral and cellular immune response to the sand fly salivary protein rLinB-13 and disease severity in tegumentary leishmaniasis. This study brings evidence that immunity to rLinB-13 influences disease outcome in L. braziliensis infection and results indicate that positive serology to rLinB-13 IgG can be used as a marker of DL, an emerging and severe form of disease caused by L. braziliensis.

Immunization with centrin-Deficient Leishmania braziliensis Does Not Protect against Homologous Challenge.

Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen-/-) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen-/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen-/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen-/-) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen-/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.

A pilot and open trial to evaluate topical Bacterial Cellulose bio-curatives in the treatment of cutaneous leishmaniasis caused by L. braziliensis.

The treatment of cutaneous leishmaniasis (CL) in Brazil using pentavalent antimony (Sbv) is associated with a high failure rate and long time to heal. Moreover, standard Sbv treatment cures only 50-60% of the cases. In this pilot clinical trial, we evaluated the topical use of bacterial cellulose (BC) bio-curatives + Sbv in the treatment of CL caused by L. braziliensis, in Bahia, Brazil. A total of 20 patients were randomized in two groups assigned to receive either parenteral Sbv alone or parenteral Sbv plus topically applied BC bio-curatives. CL patients treated with Sbv + topical BC bio-curatives had a significantly higher cure rate at 60 days post initiation of treatment compared to CL patients treated with Sbv alone (P=0.01). At day 90 post initiation of treatment, cure rate was similar in the two groups as was overall healing time. Adverse effects or local reactions to topical BC application were not observed. This pilot trial shows that the potential use of a combined therapy consisting of topical BC bio-curatives and parenteral Sbv in favoring healing of CL lesions caused by L. braziliensis, at an early time point.

Serodiagnosis and therapeutic monitoring of New-World tegumentary leishmaniasis using synthetic type-2 glycoinositolphospholipid-based neoglycoproteins.

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 (Galfß1,3Manα), GIPL-2 (Galα1,3Galfß1,3Manα), and GIPL-3 (Galα1,6Galα1,3Galfß) 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 Galα1,6Galα1,3Galfß glycotope for the serological diagnosis of American TL, particularly of the subclinical form.

Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing.

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.

Photodynamic inactivation of Leishmania braziliensis doubly sensitized with uroporphyrin and diamino-phthalocyanine activates effector functions of macrophages in vitro.

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.

The Long Pentraxin 3 (PTX3) Suppresses Immunity to Cutaneous Leishmaniasis by Regulating CD4+ T Helper Cell Response.

The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3-/-) mice are highly resistant to L. major and L. braziliensis infections. This enhanced resistance is associated with increases in Th17 and IL-17A responses. The neutralization of IL-17A abolishes this enhanced resistance, while rPTX3 treatment results in decrease in Th17 and IL-17A responses and increases susceptibility. PTX3-/- CD4+ T cells display increased differentiation to Th17 and expression of Th17-specific transcription factors. The addition of rPTX3 suppresses the expression of Th17 transcription factors, Th17 differentiation, and IL-17A production by CD4+ T cells from PTX3-/- mice. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by negatively regulating Th17 and IL-17A responses.

Insecticidal action of sodium anacardate from Brazilian cashew nut shell liquid against Aedes aegypti.

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.

Generation and Characterization of a Dual-Reporter Transgenic Leishmania braziliensis Line Expressing eGFP and Luciferase.

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.

Photodynamic Vaccination of BALB/c Mice for Prophylaxis of Cutaneous Leishmaniasis Caused by Leishmania amazonensis.

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 Leishmaniaamazonensis 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.