Navegando por Autor "Oliveira, Jamil Silvano de"
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Item Evaluation of a Leishmania hypothetical protein administered as DNA vaccine or recombinant protein against Leishmania infantum infection and its immunogenicity in humans.(2018) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Costa, Lourena Emanuele; Martins, Vivian Tamietti; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Lima, Mariana Pedrosa; Oliveira, Jamil Silvano de; Steiner, Bethina Trevisol; Ávila, Ricardo Andrez Machado de; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Souza, Daniel Menezes; Duarte, Mariana Costa; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazVisceral 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.Item Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.(2020) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Vale, Danniele Luciana; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Steiner, Bethina Trevisol; Roque, Marjorie Coimbra; Silva, João Augusto Oliveira da; Oliveira, Jamil Silvano de; Tavares, Grasiele de Sousa Vieira; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Moreira, Ricardo Luiz Fontes; Souza, Daniel Menezes; Duarte, Mariana Costa; Oliveira, Mônica Cristina de; Ávila, Ricardo Andrez Machado de; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazThe 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..Item Immunodiagnosis of human and canine visceral leishmaniasis using recombinant Leishmania infantum Prohibitin protein and a synthetic peptide containing its conformational B-cell epitope.(2019) Rodrigues, Marcella Rezende; Santos, Lucas Magno Oliveira; Miyazaki, Carolina Kei; Martins, Vivian Tamietti; Ribeiro, Fernanda Ludolf; Kursancew, Amanda Christine da Silva; Ramos, Fernanda Fonseca; Dias, Daniel Silva; Oliveira, Jamil Silvano de; Vieira, Paula Melo de Abreu; Roatt, Bruno Mendes; Ávila, Ricardo Andrez Machado de; Gonçalves, Denise Utsch; Souza, Daniel Menezes; Coelho, Eduardo Antônio Ferraz; Duarte, Mariana CostaIn the present study, Leishmania infantum's Prohibitin was cloned and, alongside a synthetic peptide, evaluated for the serodiagnosis of visceral and tegumentary leishmaniasis (CVL and TL, respectively) in dogs and humans. For TL diagnosis, this study analyzed serum samples from cutaneous (n=20) or mucosal (n=39) leishmaniasis patients, and from Chagas disease (CD) patients (n=8) and non-infected patients (n=45). For CVL diagnosis, serum samples from asymptomatic (n=14), symptomatic (n=71), non-infected (n=116), and Leish-Tec®- vaccinated (n=79) dogs were examined, as well as T. cruzi (n=11) and Ehrlichia canis (n=10) infected animals. An indirect ELISA method using rProhibitin showed diagnostic sensitivity and specificity values of 91.76% and 89.91%, respectively. L. infantum SLA showed 86.11% and 48.24% of specificity and sensitivity, respectively, for CVL serodiagnosis, and 98.31% and 84.91% sensitivity and specificity, respectively for TL diagnosis. L. braziliensis SLA showed 75.47% and 83.05% of specificity and sensitivity, respectively, for TL diagnosis. The synthetic peptide showed a better result in TL than in CVL diagnosis. In conclusion, preliminar results suggest that the detection of antibodies against the rProhibitin protein and the synthetic peptide improves the serodiagnosis of TL and CVL.Item A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.(2020) Silva, João Augusto Oliveira da; Machado, Amanda Sanchez; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Pereira, Isabela Amorim Gonçalves; Santos, Thaís Teodoro de Oliveira; Martins, Vivian Tamietti; Carvalho, Lívia Mendes; Freitas, Camila Simões de; Ribeiro, Fernanda Ludolf; Reis, Thiago Alves Rosa dos; Bandeira, Raquel Soares; Silva, Alessandra M.; Costa, Lourena Emanuele; Oliveira, Jamil Silvano de; Duarte, Mariana Costa; Roatt, Bruno Mendes; Teixeira, Antônio Lúcio; Coelho, Eduardo Antônio FerrazMost studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigoteexpressed 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.Item Leishmania eukaryotic elongation Factor-1 beta protein is immunogenic and induces parasitological protection in mice against Leishmania infantum infection.(2021) Santos, Thaís Teodoro de Oliveira; Machado, Amanda Sanchez; Ramos, Fernanda Fonseca; Silva, João Augusto Oliveira da; Lage, Daniela Pagliara; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Cardoso, Mariana Santos; Siqueira, Williane Fernanda; Martins, Vívian Tamietti; Ribeiro, Fernanda Ludolf; Reis, Thiago Alves Rosa dos; Carvalho, Lívia Mendes; Freitas, Camila Simões de; Bandeira, Raquel Soares; Silva, Alessandra M.; Oliveira, Jamil Silvano de; Moreira, Ricardo Luiz Fontes; Fujiwara, Ricardo Toshio; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Humbert, Maria Victoria; Teixeira, Antônio Lúcio; Coelho, Eduardo Antônio FerrazTreatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.Item A Leishmania hypothetical protein-containing liposome-based formulation is highly immunogenic and induces protection against visceral leishmaniasis.(2018) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Novais, Marcus V. M.; Lage, Daniela Pagliara; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Oliveira, Jamil Silvano de; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Duarte, Mariana Costa; Souza, Daniel Menezes; Ribeiro, Fernanda Ludolf; Tavares, Carlos Alberto Pereira; Oliveira, Mônica Cristina de; Coelho, Eduardo Antônio FerrazLeishmania proteins have been evaluated as vaccine candidates against leishmaniasis; however, most antigens present low immunogenicity and need to be added with immune adjuvants. A low number of licensed adjuvants exist on the market today; therefore, research conducted to produce new products is desirable. The present study sought to evaluate the immunogenicity and protective efficacy of a recombinant Leishmania hypothetical protein, namely LiHyR, administered with saponin or liposomes in BALB/c mice. Immunological and parasitological parameters were evaluated, and results showed significant protection against Leishmania infantum infection produced by both compositions in the immunized animals; however, this was not identified when the antigen was used alone. In addition, the liposomal formulation was more effective in inducing a polarized Th1 response in the vaccinated animals, which was maintained after challenge and reflected by lower parasitism found in all evaluated organs when the limiting dilution technique and RT-PCR assay were employed. The protected animals showed higher levels of protein and parasite-specific IFN-γ IL-2, IL-12, GM-CSF, and TNF-α, which were evaluated by capture ELISA and flow cytometry, in addition to a higher production of anti-protein and anti-parasite IgG2a antibodies, both before and after challenge. The Lip/rLiHyR combination induced higher IFN-γ production through both CD4+ and CD8+ T cell subtypes. Results indicate the possibility of using the LiHyR, containing a liposomal formulation, as a vaccine candidate against visceral leishmaniasis.Item Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.(2020) Ribeiro, Patrícia Aparecida Fernandes; Vale, Danniele Luciana; Dias, Daniel Silva; Lage, Daniela Pagliara; Mendonça, Débora Vasconcelos Costa; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Steiner, Bethina Trevisol; Roque, Marjorie Coimbra; Silva, João Augusto Oliveira da; Oliveira, Jamil Silvano de; Tavares, Grasiele de Sousa Vieira; Galvani, Nathália Coral; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Moreira, Ricardo Luiz Fontes; Souza, Daniel Menezes; Oliveira, Mônica Cristina de; Ávila, Ricardo Andrez Machado de; Teixeira, Antonio Lucio; Coelho, Eduardo Antônio FerrazThe 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.Item Leishmania infantum pyridoxal kinase evaluated in a recombinant protein and DNA vaccine to protects against visceral leishmaniasis.(2020) Silva, João Augusto Oliveira da; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Machado, Amanda Sanchez; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Pereira, Isabela Amorim Gonçalves; Martins, Vívian Tamietti; Carvalho, Lívia Mendes; Ribeiro, Fernanda Ludolf; Santos, Thaís Teodoro de Oliveira; Reis, Thiago Alves Rosa dos; Oliveira, Camila S.; Bandeira, Raquel Soares; Silva, Alessandra M.; Costa, Lourena Emanuele; Oliveira, Jamil Silvano de; Duarte, Mariana Costa; Souza, Daniel Menezes; Roatt, Bruno Mendes; Teixeira, Antonio Lucio; Coelho, Eduardo Antônio FerrazLeishmania 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.Item Liposomal formulation of ChimeraT, a multiple T-cell epitope-containing recombinant protein, is a candidate vaccine for human Visceral Leishmaniasis.(2020) Lage, Daniela Pagliara; Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Mendonça, Débora Vasconcelos Costa; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Steiner, Bethina Trevisol; Tavares, Grasiele de Sousa Vieira; Martins, Vivian Tamietti; Machado, Amanda Sanchez; Silva, João Augusto Oliveira da; Santos, Thaís Teodoro de Oliveira; Freitas, Camila Simões de; Oliveira, Jamil Silvano de; Roatt, Bruno Mendes; Ávila, Ricardo Andrez Machado de; Humbert, Maria Victoria; Christodoulides, Myron; Coelho, Eduardo Antônio FerrazBackground: 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.Item A novel and efficient and low-cost methodology for purification of Macrotyloma axillare (Leguminosae) seed lectin.(2008) Santana, Marcos Aurélio de; Santos, Alexandre Martins Costa; Oliveira, Marcelo Eurípedes; Oliveira, Jamil Silvano de; Babá, Élio Hideo; Santoro, Marcelo Matos; Andrade, Milton Hércules Guerra deThe N-acetyl-galactosamine specific lectin fromMacrotyloma axillare seeds (LMA)was purified by precipitation and ion exchange chromatography. The LMA 0.2 mol L−1 fraction showed hemagglutinating activity on erythrocytes A1. The results for molecular mass determinations were about 28 kDa. The LMA pHdependent assays showed best hemagglutinating activity at pH 6.0–8.0; being decreased at acidic/alkaline conditions and by EDTA treatment. LMA is a tetramer at pH 8.2 and a dimer at pH 4.0. Human erythrocytes from ABO system confirmed the A1 specificity for LMA. This new methodology is useful and easy, with low costs, for lectin purification in large amounts.Item Recombinant endonuclease III protein from Leishmania infantum associated with Th1-type adjuvants is immunogenic and induces protection against visceral leishmaniasis.(2023) Lage, Daniela Pagliara; Machado, Amanda Sanchez; Freitas, Camila Simões de; Vale, Danniele Luciana; Linhares, Flávia Prata; Cardoso, Jamille Mirelle de Oliveira; Silva, João Augusto Oliveira da; Ramos, Fernanda Fonseca; Pereira, Isabela Amorim Gonçalves; Ribeiro, Fernanda Ludolf; Tavares, Grasiele de Sousa Vieira; Bandeira, Raquel Soares; Oliveira, Jamil Silvano de; Souza, Daniel Menezes; Duarte, Mariana Costa; Galdino, Alexsandro Sobreira; Christodoulides, Myron; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Martins, Vívian Tamietti; Coelho, Eduardo Antônio FerrazVaccination 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.Item Recombinant guanosine-5--triphosphate (GTP)-binding protein associated with Poloxamer 407-based polymeric micelles protects against Leishmania infantum infection.(2022) Lage, Daniela Pagliara; Machado, Amanda Sanchez; Vale, Danniele Luciana; Freitas, Camila Simões de; Linhares, Flávia Prata; Cardoso, Jamille Mirelle de Oliveira; Pereira, Isabela Amorim Gonçalves; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Ribeiro, Fernanda Ludolf; Silva, João Augusto Oliveira da; Bandeira, Raquel Soares; Silva, Alessandra M.; Simões, Luciana C.; Reis, Thiago Alves Rosa dos; Oliveira, Jamil Silvano de; Christodoulides, Myron; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Martins, Vivian Tamietti; Coelho, Eduardo Antônio FerrazLeishmania 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 lym- phoproliferation, 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.Item A recombinant Leishmania amastigote-specific protein, rLiHyG, with adjuvants, protects against infection with Leishmania infantum.(2022) Machado, Amanda Sanchez; Lage, Daniela Pagliara; Vale, Danniele Luciana; Freitas, Camila Simões de; Linhares, Flávia Prata; Cardoso, Jamille Mirelle de Oliveira; Pereira, Isabela Amorim Gonçalves; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Ribeiro, Fernanda Ludolf; Silva, João Augusto Oliveira da; Bandeira, Raquel Soares; Simoes, Aratti Cãndido; Duarte, Mariana Costa; Oliveira, Jamil Silvano de; Christodoulides, Myron; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Martins, Vivian Tamietti; Coelho, Eduardo Antônio FerrazVaccination 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 infan- tum 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 lymphoprolifer- ative 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.Item Treatment with distinct antibiotic classes causes different pulmonary outcomes on allergic airway inflammation associated with modulation of symbiotic microbiota.(2022) Cavalcante, Gregório Grama; Guimarães, Anna Gabriella; Glauss, Camila Pereira Queiroz; Pereira, Marcela Helena Gonçalves; Dias, Angélica Samer Lallo; Horta, Laila Sampaio; Oliveira, Jamil Silvano de; Cangussú, Silvia Dantas; Magalhães, Paula Prazeres; Russo, Remo de Castro; Santiago, Helton da CostaBackground. Asthma is a chronic pulmonary disease that affects about 300 million people worldwide. Previous studies have associated antimicrobial use with allergies, but the real impact of antibiotics on asthma is still elusive. We investigated the potential impact of amoxicillin (Amox), trimethoprim/sulfamethoxazole (TMP/SMX), and metronidazole (Metro) in a murine model of OVA-induced allergic airway inflammation. Methods. BALB/c mice received three cycles of 7 days of antibiotics in drinking water followed by 7 days washout and were sensitized i.p. with OVA/Alum at days 0 and 14. After the end of the last antibiotic washout, the mice were challenged with aerosolized OVA. Pulmonary parameters were evaluated, and serum, BAL, and feces were collected for analysis. Results. Amox- and TMP/SMX-treated animals displayed more severe allergic airway inflammation parameters with increased airway hyperresponsiveness, reduced lung alveolar volume, and increased levels in BAL of IL-4 and IL-6. In contrast, Metro-treated mice showed preserved FEV-50, decreased lung inflammation, and higher levels of butyrate and propionate in their feces. Metro treatment was associated with increased OVA-specific IgA in serum. BAL microbiota was abundant in allergic groups but not in nonallergic controls with the Amox-treated group displaying the increased frequency of Proteobacteria, while Metro and TMP/SMX showed increased levels of Firmicutes. In the gut, we observed the enrichment of Akkermansia muciniphila associated with reduced airway inflammation phenotype in the Metro group, even after the recovery period. Conclusion. Our data suggest that different antibiotic treatments may impact the course of experimental allergic airway inflammation in diverse ways by several mechanisms, including modulation of short-chain fat acids production by intestinal microbiota.Item Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.(2018) Dias, Daniel Silva; Ribeiro, Patrícia Aparecida Fernandes; Martins, Vivian Tamietti; Lage, Daniela Pagliara; Costa, Lourena Emanuele; Chávez Fumagalli, Miguel Angel; Ramos, Fernanda Fonseca; Santos, Thaís Teodoro de Oliveira; Ribeiro, Fernanda Ludolf; Oliveira, Jamil Silvano de; Mendes, Tiago Antônio de Oliveira; Silva, Eduardo Sergio da; Galdino, Alexsandro Sobreira; Duarte, Mariana Costa; Roatt, Bruno Mendes; Souza, Daniel Menezes; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazVaccination 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-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), 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-γ 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-γ, 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-γ 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.