RESUMEN
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.
Asunto(s)
Biomarcadores/sangre , Enfermedades de los Perros/diagnóstico , Leishmaniasis Visceral/diagnóstico , Leishmaniasis/diagnóstico , Proteínas Protozoarias , Pruebas Serológicas , Animales , Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/parasitología , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Seguimiento , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas/métodosRESUMEN
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) is a potentially fatal disease, in which the treatment based on chemotherapy is considered toxic. The cure of disease is associated with the life-long Th1-type immunity against the infection. The Th1-related cytokines production by peripheral blood mononuclear cells (PBMCs) seems to be crucial for host control of parasite load and clinical cure. In the current study, we used five proteins (IgE-dependent histamine-releasing factor [HRF], LiHyD, LiHyV, LiHyT and LiHyp6) recently shown to be antigenic and/or immunogenic in the canine VL, aiming to evaluate the antigen-specific antibody levels and cytokine production in PBMCs culture supernatants collected from VL patients before and after anti-VL treatment. In the results, when PBMCs were exposed to rHRF, rLiHyD and rLiHyT, higher IFN-γ and lower IL-10 levels were observed in all patients that were treated and clinically cured. Analysis of specific antibody subclasses was in line with in vitro cellular response, since a higher IgG2 production was found in the treated and cured patients, when compared to the IgG1 subclass levels. In addition, evaluating the diagnostic efficacy of the recombinant molecules, the rHRF, rLiHyD and rLiHyT proteins showed the best results in the serology assays to identify all VL patients, as well as these antigens were not recognized by antibodies in sera from non-infected subjects or those with leishmaniasis-related diseases. Our results corroborate the view that clinical cure of VL is associated with a sustained Th1-related response, and indicate the potential use of rHRF, rLiHyD and rLiHyT as immune biomarkers of VL treatment.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Biomarcadores Farmacológicos/sangre , Leishmania infantum/fisiología , Leishmaniasis Visceral/diagnóstico , Leucocitos Mononucleares/inmunología , Células TH1/inmunología , Adulto , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Células Cultivadas , Progresión de la Enfermedad , Perros , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Leishmaniasis Visceral/terapia , Leucocitos Mononucleares/parasitología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Adulto JovenRESUMEN
Different Leishmania proteins have been evaluated in order to find a potential vaccine candidate or diagnostic marker capable of providing long lasting protection against infection or helping to identify infected mammalian hosts, respectively. However, just few molecules have fulfilled all the requirements to be evaluated. In the current study, we evaluated the prophylactic and diagnostic value against visceral leishmaniasis (VL) of a small glutamine-rich tetratricopeptide repeat-containing (SGT) protein from Leishmania infantum species. In a first step, the immune response elicited by the immunization using the recombinant protein (rSGT) plus saponin was evaluated in BALB/c mice. Immunized animals had a low parasitism in all evaluated organs. They developed a specific Th1 immune response, which was based on protein-specific production of IFN-γ, IL-12 and GM-CSF, and a humoral response dominated by antibodies of the IgG2a isotype. Both CD4+ and CD8+ T cells contributed to the IFN-γ production, showing that both T cell subtypes contribute to the resistance against infection. Regarding its value as a diagnostic marker, rSGT showed maximum sensitivity and specificity to serologically identify L. infantum-infected dog and human sera. No cross-reactivity with sera from humans or dogs that had other diseases was found. Although further studies are necessary to validate these findings, data showed here suggest immunogenicity of rSGT and its protective effect against murine VL, as well as its potential for the serodiagnosis of human and canine VL.
Asunto(s)
Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Reacciones Cruzadas , Citocinas/inmunología , Perros , Femenino , Humanos , Inmunoglobulina G/inmunología , Leishmania infantum/genética , Vacunas contra la Leishmaniasis/genética , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Protozoarias/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Células TH1/inmunología , Células TH1/patologíaRESUMEN
In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.
Asunto(s)
Antígenos de Protozoos/inmunología , Técnicas de Visualización de Superficie Celular/métodos , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/veterinaria , Péptidos/inmunología , Proteínas Protozoarias/inmunología , Pruebas Serológicas/métodos , Animales , Anticuerpos Antiprotozoarios/inmunología , Bacteriófagos , Biomarcadores/sangre , Brasil , Enfermedades de los Perros/diagnóstico , Perros , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Leishmania infantum/inmunología , Masculino , Péptidos/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.