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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. [ABSTRACT FROM AUTHOR]

• The pyridoxal kinase (PK) protein was evaluated against visceral leishmaniasis. • The protein was administered as a recombinant antigen or DNA vaccine. • Mice receiving both immunization schedules developed a Th1-type response. • Significant reductions in the organic parasitism were found in these animals. • The PK protein was immunogenic in PBMCs from treated patients and healthy subjects. 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. [ABSTRACT FROM AUTHOR]

• A hypothetical protein was evaluated for the serodiagnosis of leishmaniasis. • High sensitivity and specificity values were found for the diagnosis of human disease. • It was used to stimulate PBMC culture from treated mucosal and leishmaniasis patients. • Higher IFN-γ and lower IL-4 and IL-10 levels were found in the cell supernatant. • This protein can be tested as a biomarker for the diagnosis and prognosis of leishmaniasis. 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. [ABSTRACT FROM AUTHOR]

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL. [Display omitted] • Endonuclease III (ENDO) was evaluated as vaccine candidate against L. infantum. • Saponin and polymeric micelles were used as Th1-type adjuvants. • Mice immunized with ENDO plus adjuvants mounted a specific Th1-type response. • Parasitological protection was reached using both vaccine compositions. • Endonuclease III induced in vitro immunogenicity in human cells. [ABSTRACT FROM AUTHOR]

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. • The amastin protein and 1 B cell-specific epitope were tested for the diagnosis of CVL. • Both antigens showed high sensitivity and specificity values to diagnose the disease. • A role of protein in the L. infantum infectivity was showed using murine macrophages. • The immunogenicity of protein was evaluated in PBMCs from VL patients. • The amastin protein showed antigenic and immunogenic role in canine and human VL. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • 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. Abstract 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. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Cloning, expression and purification of Leishmania small myristoylated protein-3. • Bioinformatics assay to identify B cell-specific epitope and its subsequent synthesis. • ELISA experiments using the recombinant protein and synthetic peptide. • Satisfactory performance of both antigens in detects leishmaniasis in dogs and humans. • Employ in other diagnostic platforms to diagnose tegumentary and visceral disease. Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Unlabelled Image Highlights • Cloning, expression, and purification of a conserved Leishmania hypothetical protein. • Bioinformatics assays to evaluate the B cell–specific epitopes in the protein sequence. • ELISA experiments using the recombinant protein against canine and human sera. • High performance of the rLiHyE protein in detecting leishmaniasis in dogs and humans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive serodiagnosis of human VL. [ABSTRACT FROM AUTHOR]