RESUMEN
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.
Asunto(s)
Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Adyuvantes Inmunológicos/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Células Cultivadas , Femenino , Humanos , Inmunidad/inmunología , Interferón gamma/inmunología , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/parasitología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/parasitología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Células TH1/parasitologíaRESUMEN
The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Protozoos/administración & dosificación , Leishmania infantum/inmunología , Leishmaniasis Visceral/prevención & control , Animales , Antígenos de Protozoos/inmunología , Femenino , Humanos , Inmunogenicidad Vacunal , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Células TH1/inmunología , VacunaciónRESUMEN
There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed in Leishmania infantum promastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania infantum/fisiología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , ProteómicaRESUMEN
In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection.
Asunto(s)
Leishmania mexicana/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Cutánea/inmunología , Leucocitos Mononucleares/inmunología , Adulto , Animales , Bacteriófagos/inmunología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Linfocitos T/inmunología , Adulto JovenRESUMEN
The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.
Asunto(s)
Antiprotozoarios/farmacología , Cloroquinolinoles/farmacología , Leishmania infantum/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/parasitología , Leishmaniasis Visceral/parasitología , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Anfotericina B/toxicidad , Animales , Antiprotozoarios/uso terapéutico , Antiprotozoarios/toxicidad , Cloroquinolinoles/uso terapéutico , Cloroquinolinoles/toxicidad , Modelos Animales de Enfermedad , Eritrocitos/efectos de los fármacos , Femenino , Concentración 50 Inhibidora , Leishmania infantum/crecimiento & desarrollo , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológico , Hígado/parasitología , Ganglios Linfáticos/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/parasitología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Especies Reactivas de Oxígeno/metabolismo , Bazo/parasitologíaRESUMEN
The present study evaluated the cytokine profile in PBMC supernatants and the humoral response in mucosal leishmaniasis (ML) patients and in healthy subjects living in an endemic area. Four proteins, which had previously proven to be antigenic in the human disease, were tested: LiHyM, enolase, eukaryotic initiation factor 5a, and Beta-tubulin. Results showed that all of the proteins stimulated human cells with higher IFN-γ and lower IL-4 and IL-10 levels. The analysis of antibody isotypes correlated with cell response, since the IgG2 production was higher than IgG1 in both groups. By contrast, a Th2 response was found when an antigenic Leishmania extract was used. Serological analyses revealed high sensitivity and specificity values for the serodiagnosis of the disease, when compared to the data obtained using the antigenic preparation. In conclusion, this study presents new candidates to be evaluated as biomarkers in tegumentary leishmaniasis.
Asunto(s)
Formación de Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Leishmania/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Adulto , Factor 5 Eucariótico de Iniciación/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Células Th2/inmunología , Tubulina (Proteína)/inmunología , Adulto JovenRESUMEN
Visceral leishmaniasis (VL) is a fatal disease when acute and untreated. The treatment against this disease is long and presents toxicity and/or high costs. Moreover, parasite resistance has been increasing. Therefore, alternative control measures to avoid the spread of disease should be considered. It is accepted that the development of the T helper (Th)1 immune response, based on the production of pro-inflammatory cytokines, is required for the control of parasites. Although recombinant protein-based vaccines have been tested against VL, they require supplementation with immune adjuvants. In addition, there is a scarcity of studies that comparatively evaluate the efficacy of the immunogens when administered by different delivery systems in mammalian hosts. In the present study, a Leishmania hypothetical protein, LiHyR, was cloned and evaluated by immunization as a plasmid deoxyribonucleic acid (DNA) vaccine or in a recombinant format plus saponin against Leishmania infantum infection. Results showed that both vaccination regimens induced a Th1 cell-based immunity, since high levels of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α) were found, and were associated with the low production of IL-4, IL-10, and anti-parasite immunoglobulin (IgG)1 isotype. In addition, significant reductions in the parasite load were found in the evaluated organs of the DNA LiHyR or rLiHyR/saponin-vaccinated animals. No significant difference was achieved between groups vaccinated with DNA or the recombinant protein. The antigen proved to be also immunogenic in human peripheral blood mononuclear cells (PBMCs) collected from healthy subjects and from untreated and treated VL patients. A higher IgG2 isotype was also found in sera samples of these subjects, thus demonstrating its possible use as a human vaccine. This study demonstrates the protective efficacy of a new Leishmania protein against VL, when it is administered as a DNA vaccine or a recombinant protein plus saponin, and points out its use as a human vaccine against disease.
Asunto(s)
Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Humanos , Leishmania infantum/efectos de los fármacos , Leishmania infantum/fisiología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Recombinantes/administración & dosificación , Homología de Secuencia de Aminoácido , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.
Asunto(s)
Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Proteínas Represoras/inmunología , Animales , Antígenos de Protozoos/inmunología , Perros , Humanos , Leishmania infantum/inmunología , Leishmania infantum/metabolismo , Leishmaniasis Visceral/metabolismo , Ratones , Ratones Endogámicos BALB C , Prohibitinas , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismo , Células TH1/inmunología , Vacunas/metabolismoRESUMEN
Leishmania proteins have been evaluated as vaccine candidates against leishmaniasis; however, most antigens present low immunogenicity and need to be added with immune adjuvants. A low number of licensed adjuvants exist on the market today; therefore, research conducted to produce new products is desirable. The present study sought to evaluate the immunogenicity and protective efficacy of a recombinant Leishmania hypothetical protein, namely LiHyR, administered with saponin or liposomes in BALB/c mice. Immunological and parasitological parameters were evaluated, and results showed significant protection against Leishmania infantum infection produced by both compositions in the immunized animals; however, this was not identified when the antigen was used alone. In addition, the liposomal formulation was more effective in inducing a polarized Th1 response in the vaccinated animals, which was maintained after challenge and reflected by lower parasitism found in all evaluated organs when the limiting dilution technique and RT-PCR assay were employed. The protected animals showed higher levels of protein and parasite-specific IFN-γ IL-2, IL-12, GM-CSF, and TNF-α, which were evaluated by capture ELISA and flow cytometry, in addition to a higher production of anti-protein and anti-parasite IgG2a antibodies, both before and after challenge. The Lip/rLiHyR combination induced higher IFN-γ production through both CD4+ and CD8+ T cell subtypes. Results indicate the possibility of using the LiHyR, containing a liposomal formulation, as a vaccine candidate against visceral leishmaniasis.
Asunto(s)
Citocinas/inmunología , Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/farmacología , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/patología , Liposomas , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/inmunologíaRESUMEN
In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasite's flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.
Asunto(s)
Antígenos de Protozoos/inmunología , Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/inmunología , Animales , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Citocinas/sangre , Perros , Femenino , Humanos , Inmunoglobulina G/sangre , Interferón gamma/sangre , Interleucina-12/sangre , Vacunas contra la Leishmaniasis/administración & dosificación , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/inmunología , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunologíaRESUMEN
Amphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.
Asunto(s)
Anfotericina B/administración & dosificación , Antiprotozoarios/administración & dosificación , Sistemas de Liberación de Medicamentos/normas , Leishmaniasis Visceral/tratamiento farmacológico , Anfotericina B/toxicidad , Animales , Antiprotozoarios/toxicidad , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Riñón/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Hígado/efectos de los fármacos , Meglumina/administración & dosificación , Antimoniato de Meglumina , Ratones , Ratones Endogámicos BALB C , Micelas , Nitritos/metabolismo , Compuestos Organometálicos/administración & dosificación , Organismos Libres de Patógenos Específicos , Bazo/citología , Bazo/inmunologíaRESUMEN
In a proteomics approach conducted with Leishmania amazonensis, parasite proteins showed either an increase or a decrease in their expression content during extensive in vitro cultivation, and were related to the survival and the infectivity of the parasites, respectively. In the current study, a computational screening was performed to predict virulence factors among these molecules. Three proteins were selected, one of which presented no homology to human proteins. This candidate, namely small myristoylated protein-3 (SMP-3), was cloned, and its recombinant version (rSMP-3) was used to stimulate peripheral blood mononuclear cells (PBMCs) from healthy subjects living in an endemic area of leishmaniasis and from visceral leishmaniasis patients. Results showed high interferon-γ (IFN-γ) production and low levels of interleukin 10 (IL-10) in the cell supernatants. An in vivo experiment was then conducted on BALB/c mice, which were immunized with rSMP-3/saponin and later challenged with Leishmania infantum promastigotes. The rSMP-3/saponin combination induced high production of protein-specific IFN-γ, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the spleen cells of the immunized mice. This pattern was associated with protection, which was characterized by a significant reduction in the parasite load in distinct organs of the animals. Altogether, these results have revealed that this new virulence factor is immunogenic in both mice and humans, and have proven its protective efficacy against visceral leishmaniasis in a murine model.
Asunto(s)
Antígenos de Protozoos/metabolismo , Leishmania/patogenicidad , Leishmaniasis Visceral/prevención & control , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Biología Computacional , Citocinas/metabolismo , Epítopos de Linfocito T/metabolismo , Humanos , Inmunidad Celular , Inmunidad Humoral , Leishmania infantum , Leishmaniasis Visceral/inmunología , Leucocitos Mononucleares/metabolismo , Modelos Lineales , Ratones Endogámicos BALB C , Anotación de Secuencia Molecular , Proteínas Protozoarias/química , Reproducibilidad de los Resultados , Homología Estructural de ProteínaRESUMEN
BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.
RESUMEN
Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are currently no prophylactic vaccines. In this study, we designed in silico a synthetic recombinant vaccine against visceral leishmaniasis (VL) called ChimeraT, which contains specific T-cell epitopes from Leishmania Prohibitin, Eukaryotic Initiation Factor 5a and the hypothetical LiHyp1 and LiHyp2 proteins. Subcutaneous delivery of ChimeraT plus saponin stimulated a Th1 cell-mediated immune response and protected mice against L. infantum infection, significantly reducing the parasite load in distinct organs. ChimeraT/saponin vaccine stimulated significantly higher levels of IFN-γ, IL-12, and GM-CSF cytokines by both murine CD4+ and CD8+ T cells, with correspondingly low levels of IL-4 and IL-10. Induced antibodies were predominantly IgG2a isotype and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide. ChimeraT also induced lymphoproliferative responses in peripheral blood mononuclear cells from VL patients after treatment and healthy subjects, as well as higher IFN-γ and lower IL-10 secretion into cell supernatants. Thus, ChimeraT associated with a Th1 adjuvant could be considered as a potential vaccine candidate to protect against human disease.
RESUMEN
Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.
RESUMEN
New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania, but low toxicity in mammalian hosts. In the present study, a Leishmania proteome mining strategy was developed, in order to select new drug targets with low homology to human proteins, but that are considered relevant for the parasite' survival. Results showed a hypothetical protein, which was functionally annotated as a glucosidase-like protein, as presenting such characteristics. This protein was associated with the metabolic network of the N-Glycan biosynthesis pathway in Leishmania, and two specific inhibitors - acarbose and miglitol - were predicted to be potential targets against it. In this context, miglitol [1-(2-Hydroxyethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol] was tested against stationary promastigotes and axenic amastigotes of the Leishmania amazonensis and L. infantum species, and results showed high values of antileishmanial inhibition against both parasite species. Miglitol showed also efficacy in the treatment of Leishmania-infected macrophages; thus denoting its potential use as an antileishmanial candidate. In conclusion, this work presents a new drug target identified by a proteome mining strategy associated with bioinformatics tools, and suggested its use as a possible candidate to be applied in the treatment against disease.
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Antiprotozoarios/química , Biología Computacional , Minería de Datos , Descubrimiento de Drogas , Leishmania/metabolismo , Proteoma , Proteómica , Animales , Antiprotozoarios/farmacología , Biología Computacional/métodos , Femenino , Humanos , Leishmania/efectos de los fármacos , Leishmania/genética , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Modelos Moleculares , Anotación de Secuencia Molecular , Conformación Proteica , Proteómica/métodos , Relación Estructura-ActividadRESUMEN
BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.
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Anticuerpos Antivirales , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Proteínas Recombinantes , Proteínas Virales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infecciones por Citomegalovirus/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/genética , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
In the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (nâ¯=â¯20) or mucosal (nâ¯=â¯39) leishmaniasis patients, and from Chagas disease (CD) patients (nâ¯=â¯8) and non-infected patients (nâ¯=â¯45). For CVL diagnosis, serum samples from asymptomatic (nâ¯=â¯14), symptomatic (nâ¯=â¯71), non-infected (nâ¯=â¯116), and Leish-Tec®-vaccinated (nâ¯=â¯79) dogs were examined, as well as T. cruzi (nâ¯=â¯11) and Ehrlichia canis (nâ¯=â¯10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminary results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.
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Antígenos de Protozoos/inmunología , Enfermedades de los Perros/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Epítopos de Linfocito B , Leishmania donovani/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/veterinaria , Proteínas Represoras/inmunología , Pruebas Serológicas/veterinaria , Animales , Estudios de Casos y Controles , Enfermedades de los Perros/sangre , Enfermedades de los Perros/inmunología , Perros , Humanos , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Valor Predictivo de las Pruebas , Prohibitinas , Reproducibilidad de los ResultadosRESUMEN
New therapeutics against leishmaniasis are desirable, since the current drugs applied against this disease complex presents problems, such as the toxicity, high cost and/or parasite resistance. In the present study, a new fluoroquinoline derivate, namely 7-chloro-N-(4-fluorophenethyl)quinolin-4-amine or GF1061, was evaluated regarding to its in vitro antileishmanial action against Leishmania infantum and L. amazonensis species, as well as by its toxicity in mammalian cells and efficacy in the treatment of infected macrophages. The mechanism of action of this molecule in L. amazonensis and the therapeutic efficacy in infected BALB/c mice were also evaluated. Results showed that GF1061 was effective against both parasite species, showing selectivity index (SI) of 38.7 and 42.7 against L. infantum and L. amazonensis promastigotes, respectively, and of 45.0 and 48.9 against the amastigotes, respectively. Amphotericin B (AmpB), used as control, showed SI values of 6.6 and 8.8 against L. infantum and L. amazonensis promastigotes, respectively, and of 2.2 and 2.7 against the amastigotes, respectively. The molecule was effective in treat infected macrophages, as well as it induced alterations in the mitochondrial membrane potential, increase in the reactive oxygen species production, and in the cell integrity of the parasites. Regarding to the in vivo experiments, BALB/c mice (n = 8 per group) were subcutaneously infected with 106L. amazonensis stationary promastigotes and, 60 days post-infection, they received saline or were treated during 10 days, once a day, with AmpB (1 mg/kg body weight) or GF1061 (5 mg/kg body weight). One day after the treatment, the infected tissue, spleen, liver, and draining lymph node (dLN) of the animals were collected, and the parasite load was evaluated. GF1061-treated mice, as compared to the saline and AmpB groups, showed significant reductions in the parasitism in the infected tissue (66% and 62%, respectively), liver (69% and 44%, respectively), spleen (71% and 38%, respectively), and dLN (72% and 48%, respectively). In conclusion, results suggested that GF1061 may be considered as a possible therapeutic target to be evaluated against leishmaniasis in other mammalian hosts.
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Anfotericina B/farmacología , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Fluoroquinolonas/uso terapéutico , Leishmania infantum/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Animales , Leishmaniasis/parasitología , Hígado/parasitología , Macrófagos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Carga de Parásitos , Especies Reactivas de Oxígeno , Bazo/parasitologíaRESUMEN
Canine leishmaniosis (CanL) is one of the most important parasitic diseases found in several countries worldwide. Dogs are considered important domestic reservoirs of the parasites, being relevant in the maintenance of transmission cycle of the disease between sandflies and humans. However, the prevalence of asymptomatic infection is considerably higher than that of apparent clinical illness in the infected animals; thus making promptly necessary to diagnose the infection in these animals, which could help to allow to the adoption of more efficient control measures against disease. Parasitological tests, which are considered as gold standard to demonstrate the infection and diagnose the disease, present problems related with their sensitivity. Also, the sample´s collect is considered invasive. As consequence, serological tests could be applied as an additional tool to detect the asymptomatic and symptomatic CanL. For this purpose, distinct recombinant antigens have been studied; however, problems in their sensitivity and/or specificity have been still registered. The present review focus in advances in the identification of new diagnostic targets applied for the CanL diagnose, represented here by recombinant single, combined or chimeric proteins, as well as by peptides that mimic epitopes (mimotopes); which were selected by means of immunoproteomics and phage display.