Thaís T.O. Santos

Mapping B-Cell Epitopes for the Peroxidoxin of Leishmania (Viannia) braziliensis and Its Potential for the Clinical Diagnosis of Tegumentary and Visceral Leishmaniasis

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

A vaccine combining two Leishmania braziliensis proteins offers heterologous protection against Leishmania infantum infection.

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.

Selection strategy of phage-displayed immunogens based on an in vitro evaluation of the Th1 response of PBMCs and their potential use as a vaccine against Leishmania infantum infection

BackgroundThe development of a vaccine for the prevention of visceral leishmaniasis (VL) still represents a significant unmet medical need. A human vaccine can be found if one takes into consideration that many people living in endemic areas of disease are infected but do not develop active VL, including those subjects with subclinical or asymptomatic infection.MethodsIn this study, a phage display was used to select phage-exposed peptides that were specific to immunoglobulin G (IgG) antibodies from asymptomatic and symptomatic VL patients, separating them from non-infected subjects. Phage clones presenting valid peptide sequences were selected and used as stimuli of peripheral blood mononuclear cells (PBMCs) obtained from both patients’ groups and controls. Those with higher interferon-gamma (IFN-γ)/interleukin (IL)-10 ratios were further selected for vaccination tests.ResultsAmong 17 evaluated clones, two were selected, B1 and D11, and used to immunize BALB/c mice in an attempt to further validate their in vivo protective efficacy against Leishmania infantum infection. Both clones induced partial protection against the parasite challenge, which was evidenced by the reduction of parasitism in the evaluated organs, a process mediated by a specific T helper (Th)1 immune response.ConclusionsTo the best of our knowledge, this study is the first to use a rational strategy based on in vitro stimulation of human PBMCs with selected phage-displayed clones to obtain new immunogens against VL.

Identification of immune biomarkers related to disease progression and treatment efficacy in human visceral leishmaniasis.

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.

A vaccine composed of a hypothetical protein and the eukaryotic initiation factor 5a from Leishmania braziliensis cross-protection against Leishmania amazonensis infection.

In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4+ and CD8+ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.

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.

An ELISA immunoassay employing a conserved Leishmania hypothetical protein for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans

In the present study, a conserved Leishmania hypothetical protein, namely LiHypA, was evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans. This protein showed a high amino acid sequence homology between viscerotropic and cutaneotropic Leishmania species. An enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant antigen (rLiHypA), in addition to the A2 protein and two parasite antigenic preparations, which were used as controls. Regarding human diagnosis, results showed that rLiHypA was more sensitive and specific than ELISA-L. braziliensis SLA in detecting both cutaneous or mucosal leishmaniasis patients, but not those from Chagas disease patients or healthy subjects. Regarding canine diagnosis, this recombinant antigen showed higher sensitivity and specificity values, as well as a perfect accuracy to identify asymptomatic and symptomatic visceral leishmaniasis (VL) in dogs, but not those from vaccinated animals or those developing babesiosis, ehrlichiosis, or Chagas disease. However, using the rA2 protein or L. braziliensis SLA as controls, significant cross-reactivity was found when these samples were used, hampering their sensitivity and specificity values for the diagnosis. In this context, LiHypA could be considered a candidate to be evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis in dogs and humans.

Recombinant small glutamine-rich tetratricopeptide repeat-containing protein of Leishmania infantum: Potential vaccine and diagnostic application against visceral leishmaniasis.

Graphical abstract Figure. No caption available. HighlightsUse of the Leishmania SGT protein against visceral leishmaniasis.Serological marker to identify VL patients, but without presents cross‐reactivity.Partial protection induced in BALB/c mice against L. infantum infection.Immunogenicity in PBMCs from recovered and treated VL patients with IFN‐&ggr; production.A new candidate to studies as vaccine or serological marker against human VL. &NA; 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‐&ggr;, 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‐&ggr; 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.

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis

&NA; 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‐&ggr; (IFN‐&ggr;), interleukin‐12 (IL‐12), granulocyte‐macrophage‐colony‐stimulating factor (GM‐CSF), and tumor necrosis factor‐&agr; (TNF‐&agr;), 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‐&ggr; 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‐&ggr;, 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‐&ggr; 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.

Serological diagnosis and prognostic of tegumentary and visceral leishmaniasis using a conserved Leishmania hypothetical protein.

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.