Development of an antibody against EtpA from enterotoxigenic Escherichia coli and evaluation of its use for bacterial isolation using magnetic beads.

The enterotoxigenic Escherichia coli (ETEC) strain is one of the most frequent causative agents of childhood diarrhea and travelers' diarrhea in low-and middle-income countries. Among the virulence factors secreted by ETEC, the exoprotein EtpA has been described as an important. In the present study, a new detection tool for enterotoxigenic E. coli bacteria using the EtpA protein was developed. Initially, antigenic sequences of the EtpA protein were selected via in silico prediction. A chimeric recombinant protein, corresponding to the selected regions, was expressed in an E. coli host, purified and used for the immunization of mice. The specific recognition of anti-EtpA IgG antibodies generated was evaluated using flow cytometry. The tests demonstrated that the antibodiesdeveloped were able to recognize the native EtpA protein. By coupling these antibodies to magnetic beads for the capture and detection of ETEC isolates, cytometric analyses showed an increase in sensitivity, specificity and the effectiveness of the method of separation and detection of these pathogens. This is the first report of the use of this methodology for ETEC separation. Future trials may indicate their potential use for isolating these and other pathogens in clinical samples, thus accelerating the diagnosis and treatment of diseases.

Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins.

Serological tests used for the diagnosis of tegumentary leishmaniasis (TL) presents problems, mainly related to their variable sensitivity and/or specificity, which can be caused by low levels of antileishmanial antibodies or by presence of cross-reactive diseases, respectively. In this context, the search for new antigenic candidates presenting higher sensitivity and specificity is urgently required. In the present study, the amino acid sequences of the LiHyT, LiHyD, LiHyV, and LiHyP proteins, which were previously showed to be antigenic in the visceral leishmaniasis (VL), were evaluated and eight B-cell epitopes were predicted and used for construction of gene codifying a chimeric protein called ChimLeish. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA (Enzyme-Linked Immunosorbent Assay) for the diagnosis of TL. The own B cell epitopes used to construct the chimera were synthetized and also evaluated as antigens, as well as a soluble Leishmania braziliensis antigenic extract (SLA). Results showed that ChimLeish presented 100% sensitivity and specificity to diagnose TL, while synthetic peptides showed sensitivity varying from 9.1% to 90.9%, while specificity reached from 98.3% to 99.1%. SLA showed sensitivity and specificity of 18.2% and 98.3%, respectively. A preliminary prognostic evaluation showed that anti-ChimLeish IgG antibodies declined in significant levels, when serological reactivity was compared before and six months after treatment, suggesting also a possible prognostic role of this antigen for TL.

Evaluation from a B-cell epitope-based chimeric protein for the serodiagnosis of tegumentary and visceral leishmaniasis.

The diagnosis of leishmaniasis presents problems due to the variable sensitivity and/or specificity of tests. In addition, high levels of anti-parasite antibodies can remain after treatment, making it difficult to conduct a prognostic follow-up of patients. In this context, it is necessary to identify new candidates to be examined for the sensitive and specific diagnosis of the disease. In the present study, four Leishmania proteins, previously shown as antigenic for tegumentary leishmaniasis (TL), were evaluated, and their linear specific B-cell epitopes were predicted and used to generate a new gene codifying chimeric protein called ChimB, which was cloned, and the recombinant version was expressed, purified, and evaluated in ELISA (Enzyme-Linked Immunosorbent Assay) to diagnose TL and visceral leishmaniasis (VL). A total of 220 human serum samples were used, and, when ChimB was used, results showed sensitivity, specificity, and positive and negative predictive values of 100% for the diagnosis of both diseases; however, when using peptides, the sensitivity values reached from 28.0% to 57.3% and specificity varied from 16.3% to 83.7%. A soluble Leishmania extract (SLA) showed sensitivity and specificity values of 30.7% and 45.9%, respectively. The area under the curve (AUC) value for ChimB was 1.0, while for synthetic peptides, this value reached between 0.502 and 0.635, whereas for SLA, the value was of 0.589. Serological assays using sera samples collected before and after treatment showed significant reductions in the anti-ChimB antibody levels after therapy, suggesting a prognostic role of this recombinant antigen. In conclusion, preliminary data suggest the use from ChimB as a potential candidate for the diagnosis and prognosis of leishmaniasis.

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease of global importance caused by parasites of the genus Leishmania, and coinfection with human immunodeficiency virus (HIV) is common in countries where both diseases are endemic. In particular, widely used immunological tests for VL diagnosis have impaired sensitivity (Se) and specificity (Sp) in VL/HIV coinfected patients and there is also cross-reactivity with other endemic diseases, e.g., Chagas disease, malaria, and tuberculosis. To develop new antigens to improve the diagnosis of VL and VL/HIV coinfection, we predicted eight specific B-cell epitopes of four Leishmania infantum antigens and constructed a recombinant polypeptide chimera antigen called ChimLeish. A serological panel of 195 serum samples was used to compare the diagnostic capabilities of ChimLeish alongside the individual synthetic peptides. ChimLeish reacted with sera from all VL and VL/HIV coinfected patients [Se = 100%; Sp = 100%; area under the curve (AUC) = 1.0]. Peptides showed lower reactivities (Se = 76.8 to 99.2%; Sp = 67.1 to 95.7%; AUC between 0.87 and 0.98) as did a L. infantum antigenic preparation used as an antigen control (Se = 56.8%; Sp = 69.5%: AUC = 0.45). Notably, ChimLeish demonstrated a significant reduction (p < 0.05) of anti-ChimLeish antibodies after treatment and cure of a small number of patients. Although only a limited serological panel was tested, preliminary data suggest that ChimLeish should be evaluated in larger sample studies for the diagnosis of VL and VL/HIV coinfection.

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.

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.

Targeting epimastigotes of Trypanosoma cruzi with a peptide isolated from a phage display random library.

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.

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.

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.

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.

Diagnostic application of recombinant Leishmania proteins and evaluation of their in vitro immunogenicity after stimulation of immune cells collected from tegumentary leishmaniasis patients and healthy individuals.

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.

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.

Small Myristoylated Protein-3, Identified as a Potential Virulence Factor in Leishmania amazonensis, Proves to be a Protective Antigen against Visceral Leishmaniasis.

In a proteomics approach conducted with Leishmania amazonensis, parasite proteins showed either an increase or a decrease in their expression content during extensive in vitro cultivation, and were related to the survival and the infectivity of the parasites, respectively. In the current study, a computational screening was performed to predict virulence factors among these molecules. Three proteins were selected, one of which presented no homology to human proteins. This candidate, namely small myristoylated protein-3 (SMP-3), was cloned, and its recombinant version (rSMP-3) was used to stimulate peripheral blood mononuclear cells (PBMCs) from healthy subjects living in an endemic area of leishmaniasis and from visceral leishmaniasis patients. Results showed high interferon-γ (IFN-γ) production and low levels of interleukin 10 (IL-10) in the cell supernatants. An in vivo experiment was then conducted on BALB/c mice, which were immunized with rSMP-3/saponin and later challenged with Leishmania infantum promastigotes. The rSMP-3/saponin combination induced high production of protein-specific IFN-γ, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the spleen cells of the immunized mice. This pattern was associated with protection, which was characterized by a significant reduction in the parasite load in distinct organs of the animals. Altogether, these results have revealed that this new virulence factor is immunogenic in both mice and humans, and have proven its protective efficacy against visceral leishmaniasis in a murine model.

Antifungal activities against toxigenic Fusarium specie and deoxynivalenol adsorption capacity of ion-exchanged zeolites.

Zeolites are often used as adsorbents materials and their loaded cations can be exchanged with metal ions in order to add antimicrobial properties. The aim of this study was to use the 4A zeolite and its derived ion-exchanged forms with Zn2+, Li+, Cu2+ and Co2+ in order to evaluate their antifungal properties against Fusarium graminearum, including their capacity in terms of metal ions release, conidia germination and the deoxynivalenol (DON) adsorption. The zeolites ion-exchanged with Li+, Cu2+, and Co2+ showed an excellent antifungal activity against F. graminearum, using an agar diffusion method, with a zone of inhibition observed around the samples of 45.3 ± 0.6 mm, 25.7 ± 1.5 mm, and 24.7 ± 0.6 mm, respectively. Similar results using agar dilution method were found showing significant growth inhibition of F. graminearum for ion-exchanged zeolites with Zn2+, Li+, Cu2+, and Co2+. The fungi growth inhibition decreased as zeolite-Cu2+>zeolite-Li+>zeolite-Co2+>zeolite-Zn2+. In addition, the conidia germination was strongly affected by ion-exchanged zeolites. With regard to adsorption capacity, results indicate that only zeolite-Li+ were capable of DON adsorption significantly (P < 0.001) with 37% at 2 mg mL-1 concentration. The antifungal effects of the ion-exchanged zeolites can be ascribed to the interactions of the metal ions released from the zeolite structure, especially for zeolite-Li+, which showed to be a promising agent against F. graminearum and its toxin.

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.

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.

An in silico functional annotation and screening of potential drug targets derived from Leishmania spp. hypothetical proteins identified by immunoproteomics.

Leishmaniasis is a parasitic disease caused by the protozoan of the Leishmania genus. While no human vaccine is available, drugs such as pentavalent antimonials, pentamidine and amphotericin B are used for treat the patients. However, the high toxicity of these pharmaceutics, the emergence of parasite resistance and/or their high cost have showed to the urgent need of identify new targets to be employed in the improvement of the treatment against leishmaniasis. In a recent immunoproteomics approach performed in the Leishmania infantum species, 104 antigenic proteins were recognized by antibodies in sera of visceral leishmaniasis (VL) dogs. Some of them were later showed to be effective diagnostic markers and/or vaccine candidates against the disease. Between these proteins, 24 considered as hypothetical were identified in the promastigote and amastigote-like extracts of the parasites. The present study aimed to use bioinformatics tools to select new drug targets between these hypothetical proteins. Their cellular localization was predicted to be seven membrane proteins, as well as eight cytoplasmic, three nuclear, one mitochondrial and five proteins remained unclassified. Their functions were predicted as being two transport proteins, as well as five with metabolic activity, three as cell signaling and fourteen proteins remained unclassified. Ten hypothetical proteins were well-annotated and compared to their homology regarding to human proteins. Two proteins, a calpain-like and clavaminate synthase-like proteins were selected by using Docking analysis as being possible drug targets. In this sense, the present study showed the employ of new strategies to select possible drug candidates, according their localization and biological function in Leishmania parasites, aiming to treat against VL.

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species.

Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb(III)) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3- to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb(III) susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb(III) exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb(III)-sodium nitrate or Sb(III)-potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb(III) alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb(III) and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb(III) susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb(III).

A new Leishmania-specific hypothetical protein and its non-described specific B cell conformational epitope applied in the serodiagnosis of canine visceral leishmaniasis.

The serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for serodiagnosis of CVL.

Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins.

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

A recombinant chimeric antigen constructed with B-cell epitopes from Mycobacterium leprae hypothetical proteins is effective for the diagnosis of leprosy.

The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.