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Item Canine visceral leishmaniasis : detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real-time polymerase chain reaction (rt-PCR).(2019) Monteiro, Fabricio Moreira; Machado, Amanda Sanchez; Silva, Fabiana Rocha da; Assunção, Cláudia Barbosa; Melo, Cidiane Graciele da Silva; Costa, Lourena Emanuele; Portela, Aquila Serbate Borges; Coelho, Eduardo Antônio Ferraz; Figueiredo, Sônia Maria de; Caligiorne, Rachel BasquesVisceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150–152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.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 High-through identification of T cell-specific phage-exposed mimotopes using PBMCs from tegumentary leishmaniasis patients and their use as vaccine candidates against Leishmania amazonensis infection.(2019) Carvalho, Gerusa Brandão de; Costa, Lourena Emanuele; Lage, Daniela Pagliara; Ramos, Fernanda Fonseca; Santos, Thaís Teodoro de Oliveira; Ribeiro, Patrícia Aparecida Fernandes; Dias, Daniel Silva; Salles, Beatriz Cristina Silveira; Lima, Mariana Pedrosa; Carvalho, Lívia Mendes; Dias, Ana Carolina Silva; Alves, Patrícia Terra; Franklin, Michelle Lucrécio; Silva, Renata A. M.; Duarte, Mariana Costa; Souza, Daniel Menezes; Roatt, Bruno Mendes; Chávez Fumagalli, Miguel Angel; Goulart Filho, Luiz Ricardo; Teixeira Junior, Antonio Lucio; Coelho, Eduardo Antônio FerrazIn the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infectionItem 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 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 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 Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs using peptides selected from hypothetical proteins identified by an immunoproteomic approach.(2013) Chávez Fumagalli, Miguel Angel; Martins, Vívian Tamietti; Testasicca, Miriam Conceição de Souza; Lage, Daniela Pagliara; Costa, Lourena Emanuele; Lage, Paula Souza; Duarte, Mariana Costa; Ker, Henrique Gama; Ribeiro, Tatiana Gomes; Carvalho, Fernando Aécio de Amorim; Régis, Wiliam César Bento; Reis, Alexandre Barbosa; Tavares, Carlos Alberto Pereira; Soto, Manuel; Fernandes, Ana Paula Salles Moura; Coelho, Eduardo Antônio FerrazIn Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study’s findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogs.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.