Navegando por Autor "Martins, Vivian Tamietti"
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Item A candidate vaccine for human visceral leishmaniasis based on a specific T cell epitope-containing chimeric protein protects mice against Leishmania infantum infection.(2020) Lage, Daniela Pagliara; Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Mendonça, Débora Vasconcelos Costa; Ramos, Fernanda Fonseca; Carvalho, Lívia Mendes; Oliveira, Daysiane de; Steiner, Bethina Trevisol; Martins, Vivian Tamietti; Melo, Luísa Helena Perin de; Machado, Amanda Sanchez; Santos, Thaís Teodoro de Oliveira; Tavares, Grasiele de Sousa Vieira; Humbert, Maria Victoria; Coelho, Eduardo Antônio Ferraz; Christodoulides, MyronLeishmaniases are neglected diseases caused by infection with Leishmania parasites and there are currently no prophylactic vaccines. In this study, we designed in silico a synthetic recombinant vaccine against visceral leishmaniasis (VL) called ChimeraT, which contains specific T-cell epitopes from Leishmania Prohibitin, Eukaryotic Initiation Factor 5a and the hypothetical LiHyp1 and LiHyp2 proteins. Subcutaneous delivery of ChimeraT plus saponin stimulated a Th1 cell-mediated immune response and protected mice against L. infantum infection, significantly reducing the parasite load in distinct organs. ChimeraT/saponin vaccine stimulated significantly higher levels of IFN-γ, IL-12, and GM-CSF cytokines by both murine CD4+ and CD8+ T cells, with correspondingly low levels of IL-4 and IL-10. Induced antibodies were predominantly IgG2a isotype and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide. ChimeraT also induced lymphoproliferative responses in peripheral blood mononuclear cells from VL patients after treatment and healthy subjects, as well as higher IFN-γ and lower IL-10 secretion into cell supernatants. Thus, ChimeraT associated with a Th1 adjuvant could be considered as a potential vaccine candidate to protect against human disease.Item Comparing the therapeutic efficacy of different amphotericin Bcarrying delivery systems against visceral leishmaniasis.(2018) Mendonça, Débora Vasconcelos Costa; Martins, Vivian Tamietti; Lage, Daniela Pagliara; Ribeiro, Patrícia Aparecida Fernandes; Carvalho, Ana Maria Ravena Severino; Dias, Anna Leticia Teotonio; Miyazaki, Carolina Kei; Souza, Daniel Menezes; Roatt, Bruno Mendes; Tavares, Carlos Alberto Pereira; Duarte, Mariana Costa; Coelho, Eduardo Antônio FerrazAmphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.Item Development of an immunogen containing CD4+/CD8+ T‐cell epitopes for the prophylaxis of tegumentary leishmaniasis.(2022) Ferraz, Isabela de Andrade; Carvalho, Ana Maria Ravena Severino; Brito, Rory Cristiane Fortes de; Roatt, Bruno Mendes; Martins, Vivian Tamietti; Lage, Daniela Pagliara; Cruz, Luiza dos Reis; Medeiros, Fernanda Alvarenga Cardoso; Gonçalves, Denise Utsch; Rocha, Manoel Otávio da Costa; Coelho, Eduardo Antônio Ferraz; Mendes, Tiago Antônio de Oliveira; Duarte, Mariana Costa; Souza, Daniel MenezesTegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefciency of control measures, such as high toxicity and costs of current treatments and the lack of efective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL+MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and efector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These fndings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an efective tool to prevent TL.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 Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis : a preliminary study.(2022) Mendonça, Débora Vasconcelos Costa; Tavares, Grasiele de Sousa Vieira; Pereira, Isabela Amorim Gonçalves; Silva, João Augusto Oliveira da; Ramos, Fernanda Fonseca; Lage, Daniela Pagliara; Machado, Amanda Sanchez; Carvalho, Lívia Mendes; Reis, Thiago Alves Rosa dos; Carvalho, Ana Maria Ravena Severino; Ottoni, Flaviano Melo; Ribeiro, Fernanda Ludolf; Freitas, Camila Simões de; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Duarte, Mariana Costa; Humbertf, Maria V.; Roatt, Bruno Mendes; Souza, Daniel Menezes; Alves, Ricardo José; Coelho, Eduardo Antônio FerrazVisceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6- deoxy-β-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/ Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological eval- uations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite- specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, pre- liminary data suggest that this composition could be considered in future studies as promising therapeutic 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 Immunogenicity and protective efficacy of a new Leishmania hypothetical protein applied as a DNA vaccine or in a recombinant form against Leishmania infantum infection.(2019) Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Lage, Daniela Pagliara; Martins, Vivian Tamietti; Costa, Lourena Emanuele; Santos, Thaís Teodoro de Oliveira; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Mendonça, Débora Vasconcelos Costa; Ribeiro, Fernanda Ludolf; Gomes, Dawidson Assis; Rodrigues, Michele Angela; Chávez Fumagalli, Miguel Angel; Silva, Eduardo Sergio da; Galdino, Alexsandro Sobreira; Duarte, Mariana Costa; Roatt, Bruno Mendes; Souza, Daniel Menezes; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazVaccination is one the most important strategies for the prevention of visceral leishmaniasis (VL). In the current study, a new Leishmania hypothetical protein, LiHyP, which was previously showed as antigenic in an immunoproteomic search in canine VL, was evaluated regarding its immunogenicity and protective efficacy against Leishmania infantum infection. The effects of the immunization using LiHyP were evaluated when administered as a DNA plasmid (DNA LiHyP) or recombinant protein (rLiHyP) associated with saponin. The immunity elicited by both vaccination regimens reduced the parasitism in liver, spleen, bone marrow and draining lymph nodes, being associated with 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. CD4+ T cells contributed more significantly to IFN-γ production in the rLiHyP/saponin group, while CD8+ T cells were more important in the production of this cytokine in the DNA LiHyP group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from treated VL subject and healthy individuals were stimulated with the recombinant protein. In conclusion, when administered either as a DNA plasmid or recombinant protein, LiHyP can direct the immune response towards a Th1 immune profile, protecting animals against L. infantum infection; therefore, it can be seen as a promising immunogen against human VL.Item Immunotherapy using immunogenic mimotopes selected by phage display plus amphotericin B inducing a therapeutic response in mice infected with Leishmania amazonenses.(2023) Soyer, Tauane Gonçalves; Ramos, Fernanda Fonseca; Pereira, Isabela Amorim Gonçalves; Lage, Daniela Pagliara; Bandeira, Raquel Soares; Jesus, Marcelo Moreira de; Costa, Guilherme de Paula; Machado, Amanda Sanchez; Freitas, Camila Simões de; Vale, Danniele Luciana; Martins, Vivian Tamietti; Galdino, Alexsandro Sobreira; Chávez Fumagalli, Miguel Angel; Souza, Daniel Menezes; Duarte, Mariana Costa; Roatt, Bruno Mendes; Coelho, Eduardo Antônio Ferraz; Tavares, Grasiele de Sousa VieiraLeishmania amazonensis can cause cutaneous and visceral clinical manifestations of leish- maniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion’s average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against leishmaniasis.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 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 LiHyC protein is immunogenic and induces protection against visceral leishmaniasis.(2022) Machado, Amanda Sanchez; Lage, Daniela Pagliara; Vale, Danniele Luciana; Freitas, Camila Simões de; Linhares, Flávia Prata; Cardoso, Jamille Mirelle de Oliveira; Silva, João Augusto Oliveira da; Pereira, Isabela Amorim Gonçalves; Ramos, Fernanda Fonseca; Tavares, Grasiele de Sousa Vieira; Ribeiro, Fernanda Ludolf; Bandeira, Raquel Soares; Maia, Luiz Gustavo Nobre; Souza, Daniel Menezes; Duarte, Mariana Costa; Chávez Fumagalli, Miguel Angel; Roatt, Bruno Mendes; Christodoulides, Myron; Martins, Vivian Tamietti; Coelho, Eduardo Antônio FerrazTreatment against visceral leishmaniasis (VL) presents problems by the toxicity of drugs, high cost and/or emergence of resistant strains. The diagnosis is hampered by variable sensitivity and/or specificity of tests. In this context, prophylactic vaccina- tion could represent a control measure against disease. In this study, the protective efficacy of Leishmania LiHyC protein was evaluated in a murine model against Leish- mania infantum infection. LiHyC was used as recombinant protein (rLiHyC) associated with saponin (rLiHyC/S) or Poloxamer 407-based polymeric micelles (rLiHyC/M) to immunize mice. Animals received also saline, saponin or empty micelles as controls. The immunogenicity was evaluated before and after the challenge, and results showed that vaccination with rLiHyC/S or rLiHyC/M induced the production of high levels of interferon-gamma (IFN-γ), interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor in cell culture supernatants, as well as higher IFN-γ expres- sion evaluated by RT-qPCR and involvement from CD4+ and CD8+ T-cell subtypes producing IFN-γ, tumor necrosis factor-α and IL-2. A positive lymphoproliferative response was also found in cell cultures from vaccinated animals, besides high levels of rLiHyC- and parasite-specific nitrite and IgG2a antibodies. Immunological assays correlated with significant reductions in the parasite load in the spleens, livers, bone marrows and draining lymph nodes from vaccinated mice, when compared to values found in the controls. The micellar composition showed slightly better immunological and parasitological data, as compared to rLiHyC/S. Results suggest that rLiHyC asso- ciated with adjuvants could be considered for future studies as a vaccine candidate against 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 Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.(2020) Pereira, Isabela Amorim Gonçalves; Mendonça, Débora Vasconcelos Costa; Tavares, Grasiele de Sousa Vieira; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Silva, João Augusto Oliveira da; Antinarelli, Luciana Maria Ribeiro; Machado, Amanda Sanchez; Carvalho, Lívia Mendes; Carvalho, Ana Maria Ravena Severino; Salustiano, Iorrana Vieira; Reis, Thiago Alves Rosa dos; Bandeira, Raquel Soares; Silva, Alessandra M.; Martins, Vivian Tamietti; Chávez Fumagalli, Miguel Angel; Humbert, Maria Victoria; Roatt, Bruno Mendes; Duarte, Mariana Costa; Souza, Daniel Menezes; Coimbra, Elaine Soares; Leite, João Paulo Viana; Coelho, Eduardo Antônio Ferraz; Gonçalves, Denise UtschAims: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. Methods and Results: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4’-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. Conclusion: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent 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 of murine visceral leishmaniasis using an 8-hydroxyquinoline-containing polymeric micelle system.(2016) Duarte, Mariana Costa; Lage, Letícia Martins dos Reis; Lage, Daniela Pagliara; Martins, Vivian Tamietti; Carvalho, Ana Maria Ravena Severino; Roatt, Bruno Mendes; Souza, Daniel Menezes; Tavares, Carlos Alberto Pereira; Alves, Ricardo José; Barichello, José Mario; Coelho, Eduardo Antônio FerrazNewtherapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL.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.