Seleção de antígenos de Leishmania infantum por imunoproteômica para emprego no diagnóstico da leishmaniose visceral humana / Selection of Leishmania infantum antigens by immunoproteomics for use in the diagnosis of human visceral leishmaniasis

O controle da leishmaniose visceral (LV) requer um diagnóstico e tratamento adequados, uma vez que o diagnóstico preciso é fundamental para um regime medicamentoso eficaz para os pacientes. Nesse contexto, as ferramentas biotecnológicas devem ser aprimoradas para o manejo clínico e a avaliação epidemiológica da doença. No entanto, existem limitações relacionadas com a sensibilidade e/ou especificidade dos antígenos usados atualmente, mostrando a necessidade de identificação de novas moléculas para serem testadas em um diagnóstico sorológico mais sensível e específico. Neste sentido, no presente estudo, uma abordagem imunoproteômica foi usada para identificar proteínas antigênicas das formas promastigotas e amastigotas da espécie Leishmania infantum, causadora de LV em nosso país, por meio de seu reconhecimento por anticorpos em soros de pacientes com a doença. Amostras de indivíduos saudáveis residentes em região endêmica da doença e de pacientes com Doença de Chagas foram utilizadas com a função de se obter proteínas mais específicas ao parasito Leishmania para serem avaliadas no diagnóstico da LV. Como resultados obtidos, um total de 29 e 21 proteínas foram identificadas nos extratos de formas promastigotas e amastigotas dos parasitos, respectivamente. Para a validação da capacidade diagnóstica, duas proteínas, endonuclease III e GTP-binding protein, foram selecionadas, clonadas, expressas e purificadas para serem testadas em experimentos de ELISA. Os resultados dos testes mostraram valores de sensibilidade e especificidade superiores a 99,0% para a identificação da LV. Os antígenos ainda exibiram um diferencial ao apresentarem baixa reatividade sorológica em pacientes curados e tratados, sugerindo a possibilidade de que as mesmas possam ser aplicadas como marcadores prognósticos da doença. Em conclusão, o estudo imunoproteômico se mostrou eficaz na seleção de proteínas antigênicas de L. infantum e duas delas, endonuclease III e GTP-binding protein, foram bem avaliadas para o diagnóstico da LV frente a um painel sorológico, além de demonstrarem um potencial para monitoramento de pacientes com LV após o tratamento.

The control of visceral leishmaniasis (VL) requires an adequate diagnosis and treatment, since an accurate diagnosis is essential for an effective medication regimen for patients. In this context, biotechnological tools must be improved for the clinical management and epidemiological assessment of the disease. However, there are limitations related to the sensitivity and / or specificity of the antigens currently used, showing the necessity to identify new molecules to be tested in a more sensitive and specific serological diagnosis. In this sense, in the present study, an immunoproteomics approach was used to identify antigenic proteins of the Leishmania infantum promastigote and amastigote forms, which causes VL in our country, through its recognition by antibodies in sera of patients with the disease. Samples from healthy individuals living in an endemic region of the disease and from patients with Chagas disease were used to obtain more specific proteins for the Leishmania parasite, aiming their future application in the VL diagnosis. As results obtained, a total of 29 and 21 proteins were identified in the extracts of parasitic promastigotes and amastigotes, respectively. For validation of the diagnostic capacity, two proteins, endonuclease III and GTP-binding protein, were selected, cloned, expressed and purified to be tested in ELISA experiments. The test results showed sensitivity and specificity values greater than 99.0% for the identification of VL. The antigens also exhibited a differential when presenting low serological reactivity in cured and treated patients, suggesting the possibility that they can be applied as prognostic markers of the disease. In conclusion, the immunoproteomic study proved to be effective in the selection of L. infantum antigenic proteins and two of them, endonuclease III and GTP-binding protein, were well evaluated for the diagnosis of VL against a serological panel, in addition, demonstrating a potential for monitoring patients with VL after treatment.

Antileishmanial Activity, Cytotoxicity and Mechanism of Action of Clioquinol Against Leishmania infantum and Leishmania amazonensis Species.

In this study, a quinoline derivate, clioquinol (5-chloro-7-iodoquinolin-8-ol), was evaluated against Leishmania amazonensis and Leishmania infantum promastigotes and amastigotes. The cytotoxicity in murine macrophages and human red blood cells, as well as the efficacy in treating infected macrophages and the inhibition of infection using pre-treated parasites were also evaluated. Results showed that clioquinol inhibited L. amazonensis and L. infantum promastigotes with effective concentration 50% (EC50 ) values of 2.55 ± 0.25 and 1.44 ± 0.35 µg/mL, respectively, and of 1.88 ± 0.13 and 0.98 ± 0.17 µg/mL against axenic amastigotes, respectively. The cytotoxic EC50 concentrations of clioquinol in murine macrophages and human red blood cells were, respectively, 255 ± 23 and 489 ± 20 µg/mL. With these results, the selectivity index was calculated, showing values of 99.9 and 177.1 against promastigotes, respectively, and of 135.6 and 260.1 against axenic amastigotes, respectively. Significant reductions in the percentage of infected macrophages after treatment using clioquinol were also observed, as well as when parasites were pre-treated with clioquinol and used to infect murine macrophages. The mechanism of action of clioquinol was investigated in L. amazonensis, and results revealed morphological and biochemical alterations in the clioquinol-treated parasites, including reduction in cell volume, loss of mitochondrial membrane potential, increase in the ROS production and rupture of the plasma membrane. The externalization of phosphatidylserine (PS) at the cell surface was evaluated in treated parasites that had been doubly labelled with annexin and propidium iodide (PI). The results showed no significant difference for PS exposure when compared to the untreated control, although a significant increase in the PI/annexin V-labelled cell population was found in the treated parasites. Results suggest that clioquinol induces a discontinuity of the parasite membrane, possibly related to a characteristic event of cell death caused by necrosis. This study demonstrates, for the first time, the antileishmanial activity of clioquinol against two relevant Leishmania species and suggests that the mitochondria of the parasites may be a possible biological target leading to parasite necrosis. Our findings suggest that clioquinol may have a potential application in treatment of leishmaniasis and further studies should be performed in infected mammalian hosts.

Antileishmanial activity of a naphthoquinone derivate against promastigote and amastigote stages of Leishmania infantum and Leishmania amazonensis and its mechanism of action against L. amazonensis species.

Leishmaniasis has become a significant public health issue in several countries in the world. New products have been identified to treat against the disease; however, toxicity and/or high cost is a limitation. The present work evaluated the antileishmanial activity of a new naphthoquinone derivate, Flau-A [2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone], against promastigote and amastigote-like stages of Leishmania amazonensis and L. infantum. In addition, the cytotoxicity in murine macrophages and human red cells was also investigated. The mechanism of action of Flau-A was assessed in L. amazonensis as well as its efficacy in treating infected macrophages and inhibiting infection of pretreated parasites. Results showed that Flau-A was effective against promastigotes and amastigote-like forms of both parasite species, as well as showed low toxicity in mammalian cells. Results also highlighted the morphological and biochemical alterations induced by Flau-A in L. amazonensis, including loss of mitochondrial membrane potential, as well as increased reactive oxygen species production, cell shrinkage, and alteration of the plasma membrane integrity. The present study demonstrates for the first time the antileishmanial activity of Flau-A against two Leishmania species and suggests that the mitochondria of the parasites may be the main target organelle. Data shown here encourages the use of this molecule in new studies concerning treatment against Leishmania infection in mammalian hosts.

Selection strategy of phage-displayed immunogens based on an in vitro evaluation of the Th1 response of PBMCs and their potential use as a vaccine against Leishmania infantum infection.

BACKGROUND: The development of a vaccine for the prevention of visceral leishmaniasis (VL) still represents a significant unmet medical need. A human vaccine can be found if one takes into consideration that many people living in endemic areas of disease are infected but do not develop active VL, including those subjects with subclinical or asymptomatic infection. METHODS: In this study, a phage display was used to select phage-exposed peptides that were specific to immunoglobulin G (IgG) antibodies from asymptomatic and symptomatic VL patients, separating them from non-infected subjects. Phage clones presenting valid peptide sequences were selected and used as stimuli of peripheral blood mononuclear cells (PBMCs) obtained from both patients' groups and controls. Those with higher interferon-gamma (IFN-γ)/interleukin (IL)-10 ratios were further selected for vaccination tests. RESULTS: Among 17 evaluated clones, two were selected, B1 and D11, and used to immunize BALB/c mice in an attempt to further validate their in vivo protective efficacy against Leishmania infantum infection. Both clones induced partial protection against the parasite challenge, which was evidenced by the reduction of parasitism in the evaluated organs, a process mediated by a specific T helper (Th)1 immune response. CONCLUSIONS: To the best of our knowledge, this study is the first to use a rational strategy based on in vitro stimulation of human PBMCs with selected phage-displayed clones to obtain new immunogens against VL.

A vaccine composed of a hypothetical protein and the eukaryotic initiation factor 5a from Leishmania braziliensis cross-protection against Leishmania amazonensis infection.

In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4+ and CD8+ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.

Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

Abstract: Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insufficiently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial efficacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identified using immuno-proteomic techniques.

An 8-hydroxyquinoline-containing polymeric micelle system is effective for the treatment of murine tegumentary leishmaniasis.

The current treatment of leishmaniasis has been hampered due to the high toxicity of the available drugs and long duration protocols, which often lead to its abandonment. In the present study, a poloxamer 407-based delivery system was developed, and a molecule, 8-hydroxyquinoline (8-HQN), was incorporated with it, leading to an 8-HQN/micelle (8-HQN/M) composition. Assays were performed to evaluate the in vitro antileishmanial activity of 8-HQN/M against Leishmania amazonensis stationary promastigotes. The cytotoxicity in murine macrophages and in human red cells, as well as the efficacy of the treatment in macrophages infected by parasites, was also assessed. This product was also evaluated for the treatment of murine tegumentary leishmaniasis, using L. amazonensis-infected BALB/c mice. To evaluate the in vivo efficacy of the treatment, the average lesion diameter (area) in the infected tissue, as well as the parasite load at the site of infection (skin), spleen, liver and draining lymph nodes were examined. Non-incorporated micelle (B-8-HQN/M) and the free molecule (8-HQN) were used as controls, besides animals that received only saline. The parasite burden was evaluated by limiting dilution and quantitative real-time PCR (qPCR) techniques, and immunological parameters associated with the treatments were also investigated. In the results, the 8-HQN/M group, when compared to the others, presented more significant reductions in the average lesion diameter and in the parasite burden in the skin and all evaluated organs. These animals also showed significantly higher levels of parasite-specific IFN-γ, IL-12, and GM-CSF, associated with low levels of IL-4 and IL-10, when compared to the saline, 8-HQN/M, and B-8-HQN groups. A predominant IL-12-driven IFN-γ production, against parasite proteins, mainly produced by CD4+ T cells, was observed in the treated animals, post-infection. In conclusion, 8-HQN/M was highly effective in treating L. amazonensis-infected BALB/c mice and can be considered alone, or combined with other drugs, as an alternative treatment for tegumentary leishmaniasis. Graphical Abstract Therapeutic scheme and immunological and parasitological parameters developed in the present study.

Treatment of murine visceral leishmaniasis using an 8-hydroxyquinoline-containing polymeric micelle system.

New therapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL.

An effective in vitro and in vivo antileishmanial activity and mechanism of action of 8-hydroxyquinoline against Leishmania species causing visceral and tegumentary leishmaniasis.

The development of new therapeutic strategies to treat leishmaniasis has become a priority. In the present study, the antileishmanial activity of 8-hydroxyquinoline (8-HQN) was investigated against in vitro promastigotes and in vivo intra-macrophage amastigotes of three Leishmania species: Leishmania amazonensis, Leishmania infantum and Leishmania braziliensis. Studies were performed to establish the 50% Leishmania inhibitory concentration (IC50) of 8-HQN, as well as its 50% cytotoxic concentration (CC50) on murine macrophages and in human red blood cells. The inhibition of macrophages infection was also evaluated using parasites that were pre-treated with 8-HQN. The effects of this compound on nitric oxide (NO) production and in the mitochondrial membrane potential were also evaluated. Finally, the therapeutic efficacy of 8-HQN was assessed in a known murine model, L. amazonensis-chronically infected BALB/c mice. Our results showed that 8-HQN was effective against promastigote and amastigote stages of all tested Leishmania species, presenting a selectivity index of 328.0, 62.0 and 47.0 for L. amazonensis, L. infantum and L. braziliensis, respectively. It was effective in treating infected macrophages, as well as in preventing the infection of these cells using pre-treated parasites. In addition, 8-HQN caused an alteration in the mitochondrial membrane potential of the parasites. When administered at 10mg/kg body weight/day by subcutaneous route, this product was effective in reducing the lesion diameter, as well as the parasite load in evaluated tissues and organs of infected animals. The results showed the in vitro and in vivo efficacy of 8-HQN against three different Leishmania species causing tegumentary and/or visceral leishmaniasis, and it could well be used for future therapeutic optimization studies to treat leishmaniasis.

Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis.

In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.