New synthetic molecules incorporated into polymeric micelles used for treatment against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems, mainly related to drug toxicity, high cost and/or by emergence of resistant strains. In the present study, two vanillin synthetic derivatives, 3 s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3 t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], were evaluated as therapeutic candidates in a murine model against Leishmania infantum infection. Molecules were used pure (3 s and 3 t) or incorporated into Poloxamer 407-based micelles (3 s/M and 3 t/M) in the infected animals, which also received amphotericin B (AmpB) or Ambisome® as control. Results showed that 3 s/M and 3 t/M compositions induced a Th1-type immune response in treated animals, with higher levels of IFN-γ, IL-2, TNF-α, IL-12, nitrite, and IgG2a antibodies. Animals presented also low toxicity and significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as compared as control groups mice, with the evaluations performed one and 30 days after the application of the therapeutics. In conclusion, preliminary data suggest that 3 s/M and 3 t/M could be considered for future studies as therapeutic agents against VL.

Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Exploring drug repositioning for leishmaniasis treatment: Ivermectin plus polymeric micelles induce immunological response and protection against tegumentary leishmaniasis.

Leishmania amazonensis can cause a wide spectrum of the clinical manifestations of leishmaniasis in humans. The development of new therapeutics is a long and expensive task; in this context, drug repositioning could be considered a strategy to identify new biological actions of known products. In the present study, ivermectin (IVE) was tested against distinct Leishmania species able to cause disease in humans. In vitro experiments showed that IVE was effective to reduce the infection degree and parasite load in Leishmania donovani- and L. amazonensis-infected macrophages that were treated with it. In addition, using the culture supernatant of treated macrophages, higher production of IFN-γ and IL-12 and lower levels of IL-4 and IL-10 were found. Then, IVE was used in a pure form or incorporated into Poloxamer 407-based polymeric micelles (IVE/M) for the treatment of L. amazonensis-infected BALB/c mice. Animals (n = 16 per group) were infected and later received saline, empty micelles, amphotericin B (AmpB), IVE, or IVE/M. They were euthanized at one (n = 8 per group) and 30 (n = 8 per group) days after treatment and, in both endpoints, immunological, parasitological, and biochemical evaluations were performed. Results showed that both IVE and IVE/M induced higher levels of IFN-γ, IL-12, GM-CSF, nitrite, and IgG2a antibodies, as well as higher IFN-γ expression evaluated by RT-qPCR in spleen cell cultures. Such animals showed low organic toxicity, as well as significant reductions in the lesion's average diameter and parasite load in their infected tissue, spleen, liver, and draining lymph node. The efficacy was maintained 30 days post-therapy, while control mice developed a polarized Th2-type response and high parasite load. In this context, IVE could be considered as a new candidate to be applied in future studies for the treatment against distinct Leishmania species.

In vitro evaluation of antileishmanial activity, mode of action and cellular response induced by vanillin synthetic derivatives against Leishmania species able to cause cutaneous and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, mainly due to their toxicity of the drugs, high cost and/or by the emergence of parasite resistant strains. In this context, new therapeutics should be searched. In this study, two novel synthetic derivatives from vanillin: [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] or 3s and [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde] or 3t, were evaluated regarding their antileishmanial activity against distinct parasite species able to cause cutaneous and visceral leishmaniasis. Results showed that compounds 3s and 3t were effective against Leishmania infantum, L. amazonensis and L. braziliensis promastigote and amastigote-like forms, showing selectivity index (SI) of 25.1, 18.2 and 22.9, respectively, when 3s was used against promastigotes, and of 45.2, 7.5 and 15.0, respectively, against amastigote-like stage. Using the compound 3t, SI values were 45.2, 53.0 and 80.0, respectively, against promastigotes, and of 35.9, 46.0 and 58.4, respectively, against amastigote-like forms. Amphotericin B (AmpB) showed SI values of 5.0, 7.5 and 15.0, respectively, against promastigotes, and of 3.8, 5.0 and 7.5, respectively, against amastigote-like stage. The treatment of infected macrophages and inhibition of the infection upon pre-incubation with the molecules showed that they were effective in reducing the infection degree and inhibiting the infection in pre-incubated parasites, respectively, as compared to data obtained using AmpB. The mechanism of action of 3s and 3t was evaluated in L. infantum, revealing that both 3s and 3t altered the parasite mitochondrial membrane potential leading to reactive oxygen species production, increase in lipid corps and changes in the cell cycle, causing the parasite' death. A preliminary assay using the cell culture supernatant from treated and infected macrophages showed that 3s and 3t induced higher IL-12 and lower IL-10 values; suggesting the development of an in vitro Th1-type response in the treated cells. In this context, data indicated that 3s and 3t could be considered therapeutic agents to be tested in future studies against leishmaniasis.

Immunization with recombinant LiHyp1 protein plus adjuvant is protective against tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.

Recombinant guanosine-5'-triphosphate (GTP)-binding protein associated with Poloxamer 407-based polymeric micelles protects against Leishmania infantum infection.

Leishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5'-triphosphate (GTP)-binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lymphoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL.

Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis.

The diagnosis of leishmaniasis presents problems due to the variable sensitivity and/or specificity of tests. In addition, high levels of anti-parasite antibodies can remain after treatment, making it difficult to conduct a prognostic follow-up of patients. In this context, it is necessary to identify new candidates to be examined for the sensitive and specific diagnosis of the disease. In the present study, four Leishmania proteins, previously shown as antigenic for tegumentary leishmaniasis (TL), were evaluated, and their linear specific B-cell epitopes were predicted and used to generate a new gene codifying chimeric protein called ChimB, which was cloned, and the recombinant version was expressed, purified, and evaluated in ELISA (Enzyme-Linked Immunosorbent Assay) to diagnose TL and visceral leishmaniasis (VL). A total of 220 human serum samples were used, and, when ChimB was used, results showed sensitivity, specificity, and positive and negative predictive values of 100% for the diagnosis of both diseases; however, when using peptides, the sensitivity values reached from 28.0% to 57.3% and specificity varied from 16.3% to 83.7%. A soluble Leishmania extract (SLA) showed sensitivity and specificity values of 30.7% and 45.9%, respectively. The area under the curve (AUC) value for ChimB was 1.0, while for synthetic peptides, this value reached between 0.502 and 0.635, whereas for SLA, the value was of 0.589. Serological assays using sera samples collected before and after treatment showed significant reductions in the anti-ChimB antibody levels after therapy, suggesting a prognostic role of this recombinant antigen. In conclusion, preliminary data suggest the use from ChimB as a potential candidate for the diagnosis and prognosis of leishmaniasis.

Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins.

Serological tests used for the diagnosis of tegumentary leishmaniasis (TL) presents problems, mainly related to their variable sensitivity and/or specificity, which can be caused by low levels of antileishmanial antibodies or by presence of cross-reactive diseases, respectively. In this context, the search for new antigenic candidates presenting higher sensitivity and specificity is urgently required. In the present study, the amino acid sequences of the LiHyT, LiHyD, LiHyV, and LiHyP proteins, which were previously showed to be antigenic in the visceral leishmaniasis (VL), were evaluated and eight B-cell epitopes were predicted and used for construction of gene codifying a chimeric protein called ChimLeish. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA (Enzyme-Linked Immunosorbent Assay) for the diagnosis of TL. The own B cell epitopes used to construct the chimera were synthetized and also evaluated as antigens, as well as a soluble Leishmania braziliensis antigenic extract (SLA). Results showed that ChimLeish presented 100% sensitivity and specificity to diagnose TL, while synthetic peptides showed sensitivity varying from 9.1% to 90.9%, while specificity reached from 98.3% to 99.1%. SLA showed sensitivity and specificity of 18.2% and 98.3%, respectively. A preliminary prognostic evaluation showed that anti-ChimLeish IgG antibodies declined in significant levels, when serological reactivity was compared before and six months after treatment, suggesting also a possible prognostic role of this antigen for TL.

Leishmania LiHyC protein is immunogenic and induces protection against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccination could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leishmania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expression evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC associated with adjuvants could be considered for future studies as a vaccine candidate against VL.

Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis: A preliminary study.

Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.

Leishmania eukaryotic elongation Factor-1 beta protein is immunogenic and induces parasitological protection in mice against Leishmania infantum infection.

Treatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.

Diagnostic application of sensitive and specific phage-exposed epitopes for visceral leishmaniasis and human immunodeficiency virus coinfection.

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

Ivermectin presents effective and selective antileishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease of global importance caused by parasites of the genus Leishmania, and coinfection with human immunodeficiency virus (HIV) is common in countries where both diseases are endemic. In particular, widely used immunological tests for VL diagnosis have impaired sensitivity (Se) and specificity (Sp) in VL/HIV coinfected patients and there is also cross-reactivity with other endemic diseases, e.g., Chagas disease, malaria, and tuberculosis. To develop new antigens to improve the diagnosis of VL and VL/HIV coinfection, we predicted eight specific B-cell epitopes of four Leishmania infantum antigens and constructed a recombinant polypeptide chimera antigen called ChimLeish. A serological panel of 195 serum samples was used to compare the diagnostic capabilities of ChimLeish alongside the individual synthetic peptides. ChimLeish reacted with sera from all VL and VL/HIV coinfected patients [Se = 100%; Sp = 100%; area under the curve (AUC) = 1.0]. Peptides showed lower reactivities (Se = 76.8 to 99.2%; Sp = 67.1 to 95.7%; AUC between 0.87 and 0.98) as did a L. infantum antigenic preparation used as an antigen control (Se = 56.8%; Sp = 69.5%: AUC = 0.45). Notably, ChimLeish demonstrated a significant reduction (p < 0.05) of anti-ChimLeish antibodies after treatment and cure of a small number of patients. Although only a limited serological panel was tested, preliminary data suggest that ChimLeish should be evaluated in larger sample studies for the diagnosis of VL and VL/HIV coinfection.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 µg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 µg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum-infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.

A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.

Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Biotechnological applications from a Leishmania amastigote-specific hypothetical protein in the canine and human visceral leishmaniasis.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a rapid and precise diagnosis of the disease should be performed, mainly to treat patients as soon as possible, aiming to reduce the treatment time and the toxicity of the therapeutics. In the present study, the diagnostic role of an amastigote-specific Leishmania protein was evaluated in the canine and human VL. Results showed that the recombinant protein (called rLiHyJ) and one specific B cell epitope (called PeptJ) predicted from protein sequence presented high sensitivity and specificity values to diagnose canine and human disease, showing also a low reactivity against cross-reactive samples. The rA2 protein and a parasite antigenic extract showed variable sensitivity and/or specificity values in the ELISA experiments. A prognostic evaluation of protein and peptide in the human VL indicated that specific IgG antibodies significantly decreased after treatment, when compared to be values obtained before therapy. The in vitro immunogenicity using rLiHyJ in peripheral blood mononuclear cell (PBMC) cultures collected of such patients and healthy subjects suggested that the protein induced lymphoproliferation and high IFN-γ production in the stimulated cells. In conclusion, although preliminary, results suggest that rLiHyJ and PeptJ could present distinct biotechnological applications in the canine and human VL.

Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.

AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.

An immunoproteomics approach to identify Leishmania infantum proteins to be applied for the diagnosis of visceral leishmaniasis and human immunodeficiency virus co-infection.

The co-infection between visceral leishmaniasis (VL) and human immunodeficiency virus (HIV) has increased in several countries in the world. The current serological tests are not suitable since they present low sensitivity to detect the most of VL/HIV cases, and a more precise diagnosis should be performed. In this context, in the present study, an immunoproteomics approach was performed using Leishmania infantum antigenic extracts and VL, HIV and VL/HIV patients sera, besides healthy subjects samples; aiming to identify antigenic markers for these clinical conditions. Results showed that 43 spots were recognized by antibodies in VL and VL/HIV sera, and 26 proteins were identified by mass spectrometry. Between them, ß-tubulin was expressed, purified and tested in ELISA experiments as a proof of concept for validation of our immunoproteomics findings and results showed high sensitivity and specificity values to detect VL and VL/HIV patients. In conclusion, the identified proteins in the present work could be considered as candidates for future studies aiming to improvement of the diagnosis of VL and VL/HIV co-infection.