A candidate vaccine for human visceral leishmaniasis based on a specific T cell epitope-containing chimeric protein protects mice against Leishmania infantum infection.

Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are currently no prophylactic vaccines. In this study, we designed in silico a synthetic recombinant vaccine against visceral leishmaniasis (VL) called ChimeraT, which contains specific T-cell epitopes from Leishmania Prohibitin, Eukaryotic Initiation Factor 5a and the hypothetical LiHyp1 and LiHyp2 proteins. Subcutaneous delivery of ChimeraT plus saponin stimulated a Th1 cell-mediated immune response and protected mice against L. infantum infection, significantly reducing the parasite load in distinct organs. ChimeraT/saponin vaccine stimulated significantly higher levels of IFN-γ, IL-12, and GM-CSF cytokines by both murine CD4+ and CD8+ T cells, with correspondingly low levels of IL-4 and IL-10. Induced antibodies were predominantly IgG2a isotype and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide. ChimeraT also induced lymphoproliferative responses in peripheral blood mononuclear cells from VL patients after treatment and healthy subjects, as well as higher IFN-γ and lower IL-10 secretion into cell supernatants. Thus, ChimeraT associated with a Th1 adjuvant could be considered as a potential vaccine candidate to protect against human disease.

A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease.

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.

Biotechnological applications from a Leishmania amastigote-specific hypothetical protein in the canine and human visceral leishmaniasis.

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.

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis.

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Evaluation of Leishmania infantum pyridoxal kinase protein for the diagnosis of human and canine visceral leishmaniasis.

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.

A Leishmania infantum hypothetical protein evaluated as a recombinant protein and specific B-cell epitope for the serodiagnosis and prognosis of visceral leishmaniasis.

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.

A biomarker for tegumentary and visceral leishmaniasis based on a recombinant Leishmania hypothetical protein.

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.

Diagnostic evaluation of the amastin protein from Leishmania infantum in canine and human visceral leishmaniasis and immunogenicity in human cells derived from patients and healthy controls.

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.

Potential application of small myristoylated protein-3 evaluated as recombinant antigen and a synthetic peptide containing its linear B-cell epitope for the serodiagnosis of canine visceral and human tegumentary leishmaniasis.

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.

A conserved Leishmania hypothetical protein evaluated for the serodiagnosis of canine and human visceral and tegumentary leishmaniasis, as well as a serological marker for the posttreatment patient follow-up.

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.

Evaluation of a Leishmania hypothetical protein administered as DNA vaccine or recombinant protein against Leishmania infantum infection and its immunogenicity in humans.

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.

Recombinant prohibitin protein of Leishmania infantum acts as a vaccine candidate and diagnostic marker against visceral leishmaniasis.

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals' spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.