Two
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 β-tubulin protein as the target antigen in
. 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.
New candidates for serological markers against leishmaniasis are required to be identified, since the presence of high titers of anti-Leishmania antibodies remain detected in sera of treated and ...cured patients, when current antigens have being employed. In this study, the diagnostic performance of a conserved Leishmania hypothetical protein was evaluated against a human and canine serological panel. The serological follow-up of the patients was also evaluated, using this recombinant antigen (rLiHyS) in ELISA assays. In the results, high sensitivity and specificity values were found when rLiHyS was used in the serological tests, while when the recombinant A2 (rA2) protein or an antigenic Leishmania preparation were used as controls, low sensitivity and specificity were found. Regarding the serological follow-up of the patients, significant reductions in the anti-rLiHyS antibody levels were found and, one year after the treatments, the anti-protein IgG production was similar to this found in the non-infected groups, reflecting a drop of the anti-rLiHyS antibody production. In conclusion, the present study shows for the first time a new recombinant antigen used to identify tegumentary and visceral leishmaniasis, as well as being able to serologically distinguish treated and cured patients from those developing active disease.
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•A conserved Leishmania protein, LiHyS, was evaluated in serological assays.•High sensitivity and specificity was reached when canine and human sera were tested.•The anti-LiHyS antibody presence was investigated before and after the treatment.•The specific-protein antibodies decrease after the end of the treatment.•The humoral response one year after was similar to this found in control subjects.
The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in ...subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationary-phase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL.
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, β-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.
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.
Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of ...interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.
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•Two antigenic L. braziliensis proteins were selected.•They present high amino acid sequence homology between different Leishmania species.•A polyproteins vaccine was developed based ...on these two recombinant proteins.•BALB/c mice were immunized and challenged with L. infantum promastigotes.•The protection was based on low parasite levels and a parasite-specific Th1 immunity.
In the present study, two Leishmania braziliensis proteins, one hypothetical and the eukaryotic initiation factor 5a (EiF5a), were cloned and used as a polyproteins vaccine for the heterologous protection of BALB/c mice against infantum infection. Animals were immunized with the antigens separately or in association, and in both cases saponin was used as an adjuvant. In the results, spleen cells from mice inoculated with the individual or polyproteins vaccine and lately challenged produced significantly higher levels of protein- and parasite-specific IFN-γ, IL-12, and GM-CSF, when both a capture ELISA and flow cytometry assays were performed. Evaluating the parasite load by a limiting dilution as well as by RT-PCR, these animals presented significant reductions in the parasite number in all evaluated organs, when compared to the control (saline and saponin) groups. The best protection was reached when the polyproteins vaccine was employed. Protection was associated with the IFN-γ production against parasite extracts, which was mediated by both CD4+ and CD8+ T cells and correlated with the antileishmanial nitrite production. In this context, this vaccine combining two L. braziliensis proteins was able to induce a heterologous protection against VL, and could be considered in future studies to be tested against other Leishmania species or in other mammalian hosts.
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•A Leishmania hypothetical protein, LiHyP, was evaluated as a vaccine candidate in mice.•It was administered as a recombinant protein plus saponin or in a DNA plasmid.•Both ...immunization strategies induced Th1 response in the vaccinated animals.•DNA LiHyP and rLiHyP/saponin induced partial protection in L. infantum-infected mice.•LiHyP was immunogenic in human PBMC from healthy subjects and VL patients.
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.
Stryphnodendron obovatum Benth. is a Brazilian tree used to treat skin ulceration, promote wound healing, and inhibit the growth of protozoa, including Trypanosoma and Leishmania species. Bioguided ...fractionation of the ethanol extract of S. obovatum stem bark was performed, and antileishmanial and antioxidant activities of the standardized fractions were analyzed.
Stationary-phase Leishmania amazonensis promastigotes, murine macrophages, and human red blood cells (RBCs) were exposed to plant extract, standardized fractions or isolated compounds for 48h at 37°C to evaluate their antiparasitic activity and cytotoxicity. The 2,2-diphenyl-1-picryl-hidrazyl assay was used to evaluate antioxidant activity.
The S. obovatum extract and fractions showed antileishmanial and antioxidant activity; however, the organic fraction (OF) showed the best efficacy. We identified gallic acid, gallocatechin, epigallocatechin, catechin, and epigallocatechin gallate in the OF fraction. These compounds effectively inhibited L. amazonensis activity, with gallic acid, gallocatechin, and epigallocatechin gallate showing the highest selectivity. Furthermore, the evaluated compounds had no significant effect on murine macrophages and human RBCs.
The compounds present in the S. obovatum plant bark ethanol extract may provide an alternative therapeutic approach for L. amazonensis treatment.
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•Proteins showed antigenic in the canine VL were applied in the human disease.•Specific antibody levels and cytokine production in PBMCs culture were evaluated.•Visceral leishmaniasis ...patients before and after their treatment were investigated.•Three of them showed higher Th1 immune response before and after treatment.•These antigens can be applied as biomarkers associated with the clinical cure of VL.
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.
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