RESUMEN
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.
Asunto(s)
Leishmania/inmunología , Vacunas contra la Leishmaniasis/inmunología , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos T CD8-positivos/inmunología , ADN/inmunología , Femenino , Humanos , Leishmania/patogenicidad , Leishmania infantum/inmunología , Leishmaniasis Visceral/inmunología , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunologíaRESUMEN
Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.
Asunto(s)
Antígenos de Protozoos/aislamiento & purificación , Enfermedades de los Perros/parasitología , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Médula Ósea/parasitología , Biología Computacional , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Enfermedades de los Perros/diagnóstico , Perros , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/química , Femenino , Humanos , Inmunoglobulina G/sangre , Leishmania infantum/genética , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/veterinaria , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Protozoarias/química , Sensibilidad y Especificidad , Alineación de Secuencia , Pruebas Serológicas , Bazo/parasitología , Adulto JovenRESUMEN
Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Piridoxal Quinasa/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Humanos , Leishmania infantum/inmunología , Ratones , Proteínas Recombinantes/inmunología , Vacunas de ADN/inmunologíaRESUMEN
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.
Asunto(s)
Enfermedades de los Perros , Leishmania infantum , Leishmaniasis Visceral , Animales , Antígenos de Protozoos , Enfermedades de los Perros/diagnóstico , Perros , Epítopos de Linfocito B , Humanos , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/veterinaria , Leucocitos MononuclearesRESUMEN
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.
Asunto(s)
Coinfección/diagnóstico , Infecciones por VIH/diagnóstico , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Proteómica/métodos , Proteínas Protozoarias/análisis , Adulto , Brasil , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Visceral leishmaniasis (VL) is a highly neglected disease that is present in several countries worldwide. Present-day treatments against this disease are unsuitable, mainly due to the toxicity and/or high cost of drugs. In addition, the development of vaccines is still insufficient. In this scenario, a prompt VL diagnosis was deemed necessary, although sensitivity and/or specificity values of the tests have been. In this context, new antigenic candidates should be identified to be employed in a more precise diagnosis of canine and human VL. In this light, the present study evaluated the diagnostic efficacy of the Leishmania infantum pyridoxal kinase (PK) protein, applied in its recombinant version (rPK). In addition, one specific B-cell epitope derived of the PK sequence was predicted, synthetized, and evaluated as diagnostic marker. Results in ELISA tests showed that the antigens were highly sensitive to VL identification in dogs and human sera, presenting a low reactivity with VL-related disease samples. The recombinant A2 (rA2) protein and L. infantum antigenic preparation (SLA), used as controls, also proved to be highly sensitive in detecting symptomatic cases, although a low sensitivity was found when asymptomatic sera were analyzed. High cross-reactivity was also found when these antigens were evaluated against VL-related disease samples. The post-therapeutic serological follow-up showed that anti-rPK and anti-peptide IgG antibody levels decreased in significant levels after treatment. By contrast, the presence of high levels of the anti-rA2 and anti-SLA antibodies was still detected after therapy. In conclusion, rPK and its specific B-cell epitope should be considered for future studies as a diagnostic marker for canine and human VL.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Perros/diagnóstico , Leishmania infantum/enzimología , Leishmaniasis Visceral/diagnóstico , Enfermedades Desatendidas/diagnóstico , Proteínas Protozoarias/inmunología , Piridoxal Quinasa/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Reacciones Cruzadas , Enfermedades de los Perros/parasitología , Perros , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Humanos , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/veterinaria , Enfermedades Desatendidas/parasitología , Enfermedades Desatendidas/veterinaria , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Piridoxal Quinasa/química , Piridoxal Quinasa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
The serodiagnosis of visceral leishmaniasis (VL) presents problems related to the sensitivity and/or specificity of the tests. In this context, more refined antigens should be identified and applied for the improvement of disease diagnosis. In the present study, DNA with an encoding of a Leishmania infantum hypothetical protein, LiHyC, was cloned, and the recombinant protein was expressed, purified, and evaluated for the serodiagnosis of canine and human VL. In addition, a specific B-cell epitope present in the LiHyC sequence was predicted; the peptide was both synthetized and evaluated in the ELISA experiments. For comparison, commercial diagnostic kits were used against positive (VL hosts) and negative (healthy hosts) samples. Results showed that the recombinant protein (rLiHyC) and synthetic peptide (PeptC) were highly sensitive and specific to diagnose canine and human VL, with 100% sensitivity and specificity, while no false-positive or false-negative result was detected. When the DPP® CVL kit was used to identify canine samples, 44 and 52 of the 60 L. infantum-infected animals, without or with clinical signals of disease, respectively, were identified, while eight and four samples were considered as false-negatives, respectively. For human VL, an IT LEISH® kit was used, and 33 of the 40 VL patients were identified, while seven samples were considered to be false-negatives. Post-therapeutic serological follow-up testing sera samples from treated and untreated VL patients showed a significant drop in the anti-PeptC and anti-rLiHyC antibody levels, thus suggesting the feasibility to use the recombinant protein and/or synthetic peptide in future studies as diagnostic and/or prognostic markers for VL.
Asunto(s)
Epítopos de Linfocito B/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Adulto , Animales , Antígenos de Protozoos/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Pruebas Serológicas/métodosRESUMEN
The laboratorial diagnosis of leishmaniasis is based on parasitological methods, which are invasive, present high cost, require laboratorial infrastructure and/or trained professionals; as well as by immunological methods, which usually present variable sensitivity and/or specificity, such as when they are applied to identify asymptomatic cases and/or mammalian hosts presenting low levels of antileishmanial antibodies. As consequence, new studies aiming to identify more refined antigens to diagnose visceral (VL) and tegumentary (TL) leishmaniasis are urgently necessary. In the present work, the Leishmania eukaryotic elongation factor-1 beta (EF1b) protein, which was identified in L. infantum protein extracts by antibodies in VL patients' sera, was cloned and its recombinant version (rEF1b) was expressed, purified and tested as a diagnostic marker for VL and TL. The post-therapeutic serological follow-up was also evaluated in treated and untreated VL and TL patients, when anti-rEF1b antibody levels were measured before and after treatment. Results showed that rEF1b was highly sensitive and specific to diagnose symptomatic and asymptomatic canine VL, as well as human TL and VL. In addition, low cross-reactivity was observed when sera from healthy subjects or leishmaniasis-related diseases patients were tested. The serological follow-up showed also that rEF1b-specific antibodies declined significantly after treatment, suggesting that this protein could be also evaluated as a prognostic marker for human leishmaniasis.
Asunto(s)
Enfermedades de los Perros/parasitología , Factor 1 Eucariótico de Iniciación/inmunología , Leishmania infantum/inmunología , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/inmunología , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Reacciones Cruzadas , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/inmunología , Perros , Ensayo de Inmunoadsorción Enzimática , Factor 1 Eucariótico de Iniciación/genética , Femenino , Humanos , Leishmania infantum/genética , Leishmania infantum/aislamiento & purificación , Leishmaniasis/diagnóstico , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Leishmaniasis/veterinaria , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/inmunología , Masculino , Persona de Mediana Edad , Proteínas Protozoarias/genética , Pruebas SerológicasRESUMEN
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.
Asunto(s)
Enfermedades de los Perros/diagnóstico , Leishmania infantum/inmunología , Leishmaniasis/veterinaria , Pruebas Serológicas/veterinaria , Animales , Bacteriófagos , Enfermedades de los Perros/parasitología , Perros , Epítopos , Humanos , Leishmaniasis/diagnóstico , Péptidos , Proteínas Recombinantes , Sensibilidad y EspecificidadRESUMEN
The measures for leishmaniasis control include the precise diagnosis of disease. However, although several recombinant antigens have been tested with this biotechnological purpose, no effective product exists, which could detects patients with the active disease, as well as differentiates them from cured and treated patients. In this study, a conserved Leishmania hypothetical protein, which was identified in Leishmania infantum parasites, but evaluated to presents high homology in the amino acid sequences between distinct parasite species, was evaluated for the diagnosis of tegumentary and visceral leishmaniasis. In addition, PBMCs collected from treated and untreated mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) patients, as well as in healthy subjects living in endemic region of disease, were in vitro stimulated, when IFN-γ, IL-4 and IL-10 levels were evaluated in the cell supernatant. Regarding the serological analyses, ELISA experiments using the recombinant protein (rLiHyL) and a human serological panel revealed high sensitivity and specificity values to detect both diseases, while control antigens showed worst results. Regarding the cellular response, results showed that rLiHyL-stimulated cells produced higher IFN-γ and lower IL-4 and IL-10 levels in the supernatants. Also, the anti-protein antibody production was evaluated in these patients, and data showed higher IgG2 and lower IgG1 levels found in the treated patients and healthy controls, demonstrating the stimulation of a Th1-type response induced by the rLiHyL protein. In conclusion, this hypothetical protein can be considered as antigenic in TL and VL, as well as a vaccine candidate to be tested in future studies to protect against disease.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmaniasis Visceral/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes , Adulto , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/genética , Biomarcadores , Estudios de Casos y Controles , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Masculino , Persona de Mediana Edad , Proteínas Protozoarias/genética , Pruebas SerológicasRESUMEN
The diagnosis of visceral leishmaniasis (VL) presents problems due to the toxicity and/or high cost of drugs. In addition, no vaccine exists to protect against human disease. In this study, the antigenicity and immunogenicity of amastin protein were evaluated in L. infantum-infected dogs and humans. For the diagnosis, besides the recombinant protein, 1 linear B-cell epitope was synthetized and evaluated in serological assays. Results showed high sensitivity and specificity values to detect the disease when both antigens were employed against a canine and human serological panel. By contrast, when using rA2 and a soluble Leishmania antigenic preparation, sensitivity and specificity values proved to be lower. A preliminary immunogenicity study showed that the amastin protein induced high IFN-γ and low IL-10 production in stimulated PBMC derived from treated VL patients and healthy subjects, thus suggesting a potential use of this protein as an immunogen to protect against human disease.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Células Cultivadas , Citocinas/metabolismo , Enfermedades de los Perros/sangre , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Perros , Epítopos de Linfocito B/inmunología , Humanos , Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Leishmaniasis Visceral/sangre , Leishmaniasis Visceral/parasitología , Leucocitos Mononucleares/inmunología , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas SerológicasRESUMEN
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
Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.
Asunto(s)
Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniasis Visceral/parasitología , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Anticuerpos Antiprotozoarios/química , Anticuerpos Antiprotozoarios/inmunología , Técnicas de Visualización de Superficie Celular , Citocinas/metabolismo , Humanos , Leishmania infantum/efectos de los fármacos , Leishmania infantum/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/tratamiento farmacológico , Modelos Moleculares , Conformación Proteica , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunología , Nanomedicina Teranóstica , Tubulina (Proteína)/genética , Tubulina (Proteína)/inmunologíaRESUMEN
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.
Asunto(s)
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
Vaccination is one the most important strategies for the prevention of visceral leishmaniasis (VL). In the current study, a new Leishmania hypothetical protein, LiHyP, which was previously showed as antigenic in an immunoproteomic search in canine VL, was evaluated regarding its immunogenicity and protective efficacy against Leishmania infantum infection. The effects of the immunization using LiHyP were evaluated when administered as a DNA plasmid (DNA LiHyP) or recombinant protein (rLiHyP) associated with saponin. The immunity elicited by both vaccination regimens reduced the parasitism in liver, spleen, bone marrow and draining lymph nodes, being associated with high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD4+ T cells contributed more significantly to IFN-γ production in the rLiHyP/saponin group, while CD8+ T cells were more important in the production of this cytokine in the DNA LiHyP group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from treated VL subject and healthy individuals were stimulated with the recombinant protein. In conclusion, when administered either as a DNA plasmid or recombinant protein, LiHyP can direct the immune response towards a Th1 immune profile, protecting animals against L. infantum infection; therefore, it can be seen as a promising immunogen against human VL.
Asunto(s)
Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/inmunología , Vacunas de ADN , Adulto , Animales , Anticuerpos Antiprotozoarios/inmunología , Citocinas/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Vacunas contra la Leishmaniasis/inmunología , Vacunas contra la Leishmaniasis/farmacología , Leishmaniasis Visceral/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Vacunas de ADN/inmunología , Vacunas de ADN/farmacologíaRESUMEN
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
Serological tests are important tools for the diagnosis of Leishmania infection. However, they are not effective markers to diagnose asymptomatic cases of visceral leishmaniasis (VL) and patients developing tegumentary leishmaniasis (TL), since antileishmanial antibodies can be encountered in low levels resulting in false-negative results in the serological trials. In this context, antigens able to be recognized by antibodies in sera from both VL and TL patients will be desirable to be employed in a more sensitivity and specific diagnosis of disease. In the present study, a conserved Leishmania protein, small myristoylated protein-3 (SMP-3), which was showed to be conserved in different Leishmania species and an effective vaccine candidate against Leishmania infantum infection in a murine model, was cloned and the recombinant protein was evaluated as a serological marker for the diagnosis of human TL and canine VL. In addition, a linear B cell-specific epitope (MQKDEESGEFKCEL) was identified, synthetized and also investigated as a serological marker. As antigen controls, rA2 protein and antigenic Leishmania extracts (SLA) were used. Results showed that ELISA-rSMP-3 and ELISA-Peptide presented sensitivity and specificity values higher than 90% in both diseases in humans and canids, having identified all asymptomatic cases and did not present cross-reaction with cross-reactivity diseases in both mammalian hosts. On the other hand, sensitivity and specificity values were worst when rA2 or SLA were used as antigens in humans and dogs. In conclusion, results showed the efficacy and Leishmania SMP-3 protein, employed as a recombinant antigen or a B cell epitope, for the improvement of the serodiagnosis of human TL and canine VL. This candidate can be tested in other diagnostic platforms, such as rapid immunochromatographic dipstick tests, aiming its use in epidemiological studies in remote areas where laboratories are not readily accessible for conventional assays.
Asunto(s)
Antígenos de Protozoos/inmunología , Enfermedades de los Perros/diagnóstico , Epítopos de Linfocito B/inmunología , Leishmania/fisiología , Leishmaniasis Visceral/diagnóstico , Proteínas Recombinantes/inmunología , Zoonosis/diagnóstico , Adulto , Anciano , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Reacciones Cruzadas , Perros , Epítopos de Linfocito B/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/genética , Sensibilidad y Especificidad , Pruebas Serológicas , Adulto JovenRESUMEN
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
In the present study, a conserved Leishmania hypothetical protein, LiHyE, was evaluated for the serodiagnosis of leishmaniasis. Results showed that it presented high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) to serologically identify visceral leishmaniasis (VL) dogs when 40 positive sera and 95 cross-reactive samples were used. rLiHyE also showed the best results of sensitivity, specificity, PPV, and NPV to identify tegumentary leishmaniasis (TL) and VL patients when 45 leishmaniasis patients' sera and 90 cross-reactive samples were used. Results were better in comparison to those obtained when rA2 or Leishmania antigenic extract was employed as controls. The posttreatment follow-up showed that rLiHyE-specific antibodies declined significantly after the end of treatments, and a predominance of the IgG2 subclass was found in comparison to IgG1 levels in both TL and VL patients. In conclusion, rLiHyE can be considered a candidate for the serodiagnosis of canine and human leishmaniasis.