Search

The diagnosis of visceral leishmaniasis (VL) has improved with the search of novel antigens; however, their performance is limited when samples from VL/human immunodeficiency virus (HIV)-coinfected patients are tested. In this context, studies conducted to identify more suitable antigens to detect both VL and VL/HIC coinfection cases should be performed. In the current study, phage display was performed using serum samples from healthy subjects and VL, HIV-infected and VL/HIV-coinfected patients; aiming to identify novel phage-exposed epitopes to be evaluated with this diagnostic purpose. Nine non-repetitive and valid sequences were identified, synthetized and tested as peptides in enzyme-linked immunosorbent assay experiments. Results showed that three (Pep2, Pep3 and Pep4) peptides showed excellent performance to diagnose VL and VL/HIV coinfection, with 100% sensitivity and specificity values. The other peptides showed sensitivity varying from 50.9 to 80.0%, as well as specificity ranging from 60.0 to 95.6%. Pep2, Pep3 and Pep4 also showed a potential prognostic effect, since specific serological reactivity was significantly decreased after patient treatment. Bioinformatics assays indicated that Leishmania trypanothione reductase protein was predicted to contain these three conformational epitopes. In conclusion, data suggest that Pep2, Pep3 and Pep4 could be tested for the diagnosis of VL and VL/HIV coinfection.

There is no suitable vaccine against human visceral leishmaniasis (VL) and available drugs are toxic and/or present high cost. In this context, diagnostic tools should be improved for clinical management and epidemiological evaluation of disease. However, the variable sensitivity and/or specificity of the used antigens are limitations, showing the necessity to identify new molecules to be tested in a more sensitive and specific serology. In the present study, an immunoproteomics approach was performed inLeishmania infantumpromastigotes and amastigotes employing sera samples from VL patients. Aiming to avoid undesired cross-reactivity in the serological assays, sera from Chagas disease patients and healthy subjects living in the endemic region of disease were also used in immunoblottings. The most reactive spots for VL samples were selected, and 29 and 21 proteins were identified in the promastigote and amastigote extracts, respectively. Two of them, endonuclease III and GTP-binding protein, were cloned, expressed, purified and tested in ELISA experiments against a large serological panel, and results showed high sensitivity and specificity values for the diagnosis of disease. In conclusion, the identified proteins could be considered in future studies as candidate antigens for the serodiagnosis of human VL.

In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection.

Two mimotopes of Leishmania infantum identified by phage display were evaluated as vaccine candidates in BALB/c mice against Leishmania amazonensis infection. The epitope-based immunogens, namely B10 and C01, presented as phagefused peptides; were used without association of a Th1 adjuvant, and they were administered isolated or in combination into animals. Both clones showed a specific production of interferon-gamma (IFN-γ), interleukin-12 (IL-12) and granulocyte/macrophage colony-stimulating factor (GM-CSF) after in vitro spleen cells stimulation, and they were able to induce a partial protection against infection. Significant reductions of parasite load in the infected footpads, liver, spleen, bone marrow and paws’ draining lymph nodes were observed in the immunized mice, in comparison with the control groups (saline, saponin, wild-type and non-relevant clones). Protection was associated with an IL-12-dependent production of IFN-γ, mediated mainly by CD8+ T cells, against parasite proteins. Protected mice also presented low levels of IL-4 and IL-10, as well as increased levels of parasite-specific IgG2a antibodies. The association of both clones resulted in an improved protection in relation to their individual use. More importantly, the absence of adjuvant did not diminish the cross-protective efficacy against Leishmania spp. infection. This study describes for the first time two epitope-based immunogens selected by phage display technology against L. infantum infected dogs sera, which induced a partial protection in BALB/c mice infected with L. amazonensis, Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (PRONEX APQ-0101909, CBB-APQ-00496-11 and CBB-APQ-00819-12), CAPES (Rede anobiotec/Brazil) and CNPq (APQ-472090/2011-9, APQ-482976/2012-8 and APQ-488237/2013-0). M.A.C.F.

In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasite's flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.