PLGA nanoparticles loaded with KMP-11 stimulate innate immunity and induce the killing of Leishmania.

We recently demonstrated that immunization with polyester poly(lactide-co-glycolide acid) (PLGA) nanoparticles loaded with the 11-kDa Leishmania vaccine candidate kinetoplastid membrane protein 11 (KMP-11) significantly reduced parasite load in vivo. Presently, we explored the ability of the recombinant PLGA nanoparticles to stimulate innate responses in macrophages and the outcome of infection with Leishmania braziliensis in vitro. Incubation of macrophages with KMP-11-loaded PLGA nanoparticles significantly decreased parasite load. In parallel, we observed the augmented production of nitric oxide, superoxide, TNF-α and IL-6. An increased release of CCL2/MCP-1 and CXCL1/KC was also observed, resulting in macrophage and neutrophil recruitment in vitro. Lastly, the incubation of macrophages with KMP-11-loaded PLGA nanoparticles triggered the activation of caspase-1 and the secretion of IL-1ß and IL-18, suggesting inflammasome participation. Inhibition of caspase-1 significantly increased the parasite load. We conclude that KMP-11-loaded PLGA nanoparticles promote the killing of intracellular Leishmania parasites through the induction of potent innate responses. FROM THE CLINICAL EDITOR: In this novel study, KMP-11-loaded PLGA nanoparticles are demonstrated to promote the killing of intracellular Leishmania parasites through enhanced innate immune responses by multiple mechanisms. Future clinical applications would have a major effect on our efforts to address parasitic infections.

Preparation and characterization of a bacterial cellulose/silk fibroin sponge scaffold for tissue regeneration.

Bacterial cellulose (BC) and silk fibroin (SF) are natural biopolymers successfully applied in tissue engineering and biomedical fields. In this work nanocomposites based on BC and SF were prepared and characterized by scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). In addition, the investigation of cytocompatibility was done by MTT, XTT and Trypan Blue dye technique. Cellular adhesion and proliferation were detected additionally. The evaluation of genotoxicity was realized by micronucleus assay. In vitro tests showed that the material is non-cytotoxic or genotoxic. SEM images revealed a greater number of cells attached at the BC/SF:50% scaffold surface than the pure BC one, suggesting that the presence of fibroin improved cell attachment. This could be related to the SF amino acid sequence that acts as cell receptors facilitating cell adhesion and growth. Consequently, BC/SF:50% scaffolds configured an excellent option in bioengineering depicting its potential for tissue regeneration and cultivation of cells on nanocomposites.

Chemotherapeutic potential of 17-AAG against cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

BACKGROUND: Leishmaniasis remains a worldwide public health problem. The limited therapeutic options, drug toxicity and reports of resistance, reinforce the need for the development of new treatment options. Previously, we showed that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), a Heat Shock Protein 90 (HSP90)-specific inhibitor, reduces L. (L.) amazonensis infection in vitro. Herein, we expand the current knowledge on the leishmanicidal activity of 17-AAG against cutaneous leishmaniasis, employing an experimental model of infection with L. (V.) braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of axenic L. (V.) braziliensis promastigotes to 17-AAG resulted in direct dose-dependent parasite killing. These results were extended to L. (V.) braziliensis-infected macrophages, an effect that was dissociated from the production of nitric oxide (NO), superoxide (O(-2)) or inflammatory mediators such as TNF-α, IL-6 and MCP-1. The leishmanicidal effect was then demonstrated in vivo, employing BALB/c mice infected with L. braziliensis. In this model, 17-AAG treatment resulted in smaller skin lesions and parasite counts were also significantly reduced. Lastly, 17-AAG showed a similar effect to amphotericin B regarding the ability to reduce parasite viability. CONCLUSION/SIGNIFICANCE: 17-AAG effectively inhibited the growth of L. braziliensis, both in vitro and in vivo. Given the chronicity of L. (V.) braziliensis infection and its association with mucocutaneous leishmaniasis, 17-AAG can be envisaged as a new chemotherapeutic alternative for cutaneous Leishmaniasis.

Cross-protective effect of a combined L5 plus L3 Leishmania major ribosomal protein based vaccine combined with a Th1 adjuvant in murine cutaneous and visceral leishmaniasis.

BACKGROUND: Two Leishmania major ribosomal proteins L3 (LmL3) and L5 (LmL5) have been described as protective molecules against cutaneous leishmaniasis due to infection with L. major and Leishmania braziliensis in BALB/c mice when immunized with a Th1 adjuvant (non-methylated CpG-oligodeoxynucleotides; CpG-ODN). In the present study we analyzed the cross-protective efficacy of an LmL3-LmL5-CpG ODN combined vaccine against infection with Leishmania amazonensis and Leishmania chagasi (syn. Leishmania infantum) the etiologic agents of different clinical forms of human leishmaniasis in South America. METHODS: The combined vaccine was administered subcutaneously to BALB/c mice. After immunization the cellular and humoral responses elicited were analyzed. Mice were independently challenged with L. amazonensis and L. chagasi. The size of the cutaneous lesions caused by the infection with the first species, the parasite loads and the immune response in both infection models were analyzed nine weeks after challenge. RESULTS: Mice vaccinated with the combined vaccine showed a Th1-like response against LmL3 and LmL5. Vaccinated mice were able to delay lesion development due to L. amazonensis infection and to control parasite loads in the site of infection. A reduction of the parasite burden in the lymph nodes draining the site of infection and in the liver and spleen was observed in the vaccinated mice after a subcutaneous infection with L. chagasi. In both models of infection, protection was correlated to parasite antigen-specific production of IFN-γ and down-regulation of parasite-mediated IL-4 and IL-10 responses. CONCLUSIONS: The data presented here demonstrate the potential use of L. major L3 and L5 recombinant ribosomal proteins for the development of vaccines against various Leishmania species.

Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis.

Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.

Vaccination with L. infantum chagasi nucleosomal histones confers protection against new world cutaneous leishmaniasis caused by Leishmania braziliensis.

BACKGROUND: Nucleosomal histones are intracellular proteins that are highly conserved among Leishmania species. After parasite destruction or spontaneous lysis, exposure to these proteins elicits a strong host immune response. In the present study, we analyzed the protective capability of Leishmania infantum chagasi nucleosomal histones against L. braziliensis infection using different immunization strategies. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were immunized with either a plasmid DNA cocktail (DNA) containing four Leishmania nucleosomal histones or with the DNA cocktail followed by the corresponding recombinant proteins plus CpG (DNA/Protein). Mice were later challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development, parasite load and the cellular immune response were analyzed five weeks after challenge. Immunization with either DNA alone or with DNA/Protein was able to inhibit lesion development. This finding was highlighted by the absence of infected macrophages in tissue sections. Further, parasite load at the infection site and in the draining lymph nodes was also significantly lower in vaccinated animals. This outcome was associated with increased expression of IFN-γ and down regulation of IL-4 at the infection site. CONCLUSION: The data presented here demonstrate the potential use of L. infantum chagasi nucleosomal histones as targets for the development of vaccines against infection with L. braziliensis, as shown by the significant inhibition of disease development following a live challenge.

Towards development of novel immunization strategies against leishmaniasis using PLGA nanoparticles loaded with kinetoplastid membrane protein-11.

BACKGROUND: Vaccine development has been a priority in the fight against leishmaniases, which are vector-borne diseases caused by Leishmania protozoa. Among the different immunization strategies employed to date is inoculation of plasmid DNA coding for parasite antigens, which has a demonstrated ability to induce humoral and cellular immune responses. In this sense, inoculation of plasmid DNA encoding Leishmania kinetoplasmid membrane protein-11 (KMP-11) was able to confer protection against visceral leishmaniasis. However, recently the use of antigen delivery systems such as poly(lactic-co-glycolic acid) (PLGA) nanoparticles has also proven effective for eliciting protective immune responses. METHODS: In the present work, we tested two immunization strategies with the goal of obtaining protection, in terms of lesion development and parasite load, against cutaneous leishmaniasis caused by L. braziliensis. One strategy involved immunization with plasmid DNA encoding L. infantum chagasi KMP-11. Alternatively, mice were primed with PLGA nanoparticles loaded with the recombinant plasmid DNA and boosted using PLGA nanoparticles loaded with recombinant KMP-11. RESULTS: Both immunization strategies elicited detectable cellular immune responses with the presence of both proinflammatory and anti-inflammatory cytokines; mice receiving the recombinant PLGA nanoparticle formulations also demonstrated anti-KMP-11 IgG1 and IgG2a. Mice were then challenged with L. braziliensis, in the presence of sand fly saliva. Lesion development was not inhibited following either immunization strategy. However, immunization with PLGA nanoparticles resulted in a more prominent reduction in parasite load at the infection site when compared with immunization using plasmid DNA alone. This effect was associated with a local increase in interferon-gamma and in tumor necrosis factor-alpha. Both immunization strategies also resulted in a lower parasite load in the draining lymph nodes, albeit not significantly. CONCLUSION: Our results encourage the pursuit of immunization strategies employing nanobased delivery systems for vaccine development against cutaneous leishmaniasis caused by L. braziliensis infection.