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Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease. [ABSTRACT FROM AUTHOR]

[Display omitted] • An urine-based ELISA assay was developed to diagnose TL. • The rLiHyA protein, a synthetic peptide and L. braziliensis SLA were used as antigens. • Two hundred and five paired urine and serum samples were used in the assays. • Protein and peptide presented the better accuracy results in the urine-based assays. • Both urine and serum-based ELISA showed a drop in antibody levels. Laboratorial diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests, which are still hampered by blood́ invasive collection. In this context, in the present study, we develop a serum- and urine-based ELISA to TL diagnoses. A recombinant protein (rLiHyA), which was previously showed to be antigenic for the disease, as well as a B-cell epitope produced as synthetic peptide and a Leishmania antigenic extract (SLA), were used as antigens. A total of paired 205 urine and serum samples were used, which were comprised by samples from cutaneous (n = 30) and mucosal (n = 30) leishmaniasis patients, as well as from healthy individuals living in endemic region of disease (n = 45), of patients with Chagas disease (n = 30), leprosy (n = 35), malaria (n = 15) or HIV-infected (n = 20). Results showed that serum-based ELISA presented sensitivity of 24.0 %, 100 % and 41.0 %, when SLA, rLiHyA and synthetic peptide were used as antigens, and specificity of 98.4 %, 98.4 % and 98.4 %, respectively. The area under the curve (AUC) was calculated and results were 0.74, 1.0, and 0.71, respectively, when SLA, rLiHyA and synthetic peptide were used as antigens. Performing an urine-based ELISA, sensitivity was 28.0 %, 100 % and 75.0 %, respectively, when SLA, rLiHyA, and synthetic peptide were used, while specificity values were of 98.4 %, 98.4 % and 98.4 %, respectively. In addition, the AUC values were 0.82, 1.0, and 0.94, respectively. A significant drop in specific antibodies levels in both patientś serum and urine samples was found six months after treatment, suggesting a prognostic role of rLiHyA for TL. In conclusion, preliminary data suggest the potential of use patient urine to TL diagnoses. [ABSTRACT FROM AUTHOR]

• The rLiHyV protein, synthetic peptides and SLA were used as antigens in ELISA. • They were tested against paired urine and serum samples from 215 patients to diagnose TL. • The recombinant protein presented better sensitivity and specificity values. • Patients' urine showed better performance as biological analyte as compared to the use of serum. • An urine-based ELISA using rLiHyV could be considered for the diagnosis of TL. The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases. [Display omitted] [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]

• B-cell epitopes from specific M. leprae hypothetical proteins were predicted. • They were used to construct a novel recombinant chimeric protein, M1. • M1 antigen was tested in ELISA experiments for the leprosy diagnosis. • High sensitivity and specificity was found to both pauci- and multibacillary forms. • This new antigen could be considered as a candidate for the diagnosis of disease. 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. [Display omitted] [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 new leishmania amastigote protein, LiHyG, was evaluated against L. infantum. • It was associated with saponin or poloxamer 407-based polymeric micelles. • Both compositions induced the development of specific Th1-type response. • Both vaccines reduced organ parasitism significantly. • LiHyG/micelles afforded better protection than lihyg/saponin. Vaccination against visceral leishmaniasis (VL) should be considered as a control measure to protect against disease, and amastigote-specific proteins could help to develop such vaccines, since this parasite form is in contact with the host immune system during the active disease. In this study, a Leishmania amastigote-specific protein, LiHyG, was evaluated as recombinant protein (rLiHyG) as vaccine candidate against Leishmania infantum infection in BALB/c mice. The protein was associated with saponin (rLiHyG/Sap) or Poloxamer 407-based polymeric micelles (rLiHyG/Mic) as adjuvants, and animals receiving saline, saponin or micelle as controls. Immunological and parasitological analyses were performed before (n = 8 per group; as primary endpoint) and after (n = 8 per group; as secondary endpoint) infection. Results showed that, in both endpoints, rLiHyG/Sap and rLiHyG/Mic induced higher levels of IFN-γ, IL-12 and GM-CSF in spleen cell cultures from vaccinated animals, besides elevated presence of IgG2a isotype antibodies. Decreased hepatotoxicity and 'positive lymphoproliferative response were also found after challenge. Such findings reflected in significantly lower levels of parasite load found in their spleens, livers, bone marrows and draining lymph nodes. In conclusion, rLiHyG associated with Th1-type adjuvant could be considered for future studies as vaccine candidate to protect against VL. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • A GTP-binding protein was used as recombinant antigen against L. infantum. • It was associated with saponin or in polymeric micelles as adjuvants. • Results showed that rGTP/Sap and rGTP/Mic induced the Th1-type immune response. • Immunized mice showed also higher lymphoproliferation and lower parasitism. • The rGTP/Mic composition was more immunogenic and protective than rGTP/Sap. Leishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5′-triphosphate (GTP)-binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lymphoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL. [ABSTRACT FROM AUTHOR]

• A Leishmania amastigote-specific protein was evaluated as vaccine against VL. • The protein was administered as a recombinant antigen or as DNA vaccine. • Mice receiving both immunization schedules mounted a Th1-type response. • Significant reductions in the parasite load were found in all evaluated organs. • LiHyJ was immunogenic in PBMCs from treated patients and healthy subjects. Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL. [ABSTRACT FROM AUTHOR]