Dispersion of Leishmania (Leishmania) infantum in central-southern Brazil : evidence from an integrative approach

Leishmania infantum is the zoonotic agent of visceral leishmaniasis (VL). The epidemiological cycle in Brazil has dispersed from the Northeast to other regions of the country. Hypotheses regarding possible entries of Leishmania (L.) infantum into the area of the triple border are presented and discussed. The State of Parana´ borders the states of São Paulo and Mato Grosso, which have experienced VL epidemics over the past 20 years. The study used genetic markers to understand the dispersion of Leishmania infantum throughout central-southern Brazil. Results show two possible agent inputs in the Parana´ state, one coming from Paraguay, the other from Santa Catarina state.Conselho Nacional de Desenvolvimento Científico e TecnológicoFundação Araucária de Apoio ao Desenvolvimento Científico e ecnológico do Paran

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures.We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptidemix generated the IFN-g, IL-12, IL-4 and TGF-b that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that themix of peptides derived fromhistone and parasitesmembranemolecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Development of new biomoleculess as promissing antigen candidates for Leishmanioses diagnosis and vacinnes

Orientadora: Profª Drª Vanete Thomaz Soccol.Tese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 18/04/2019.Inclui referências: p. 79-107.Resumo: As doenças tropicais negligenciadas (DTNs) são um grupo de doenças infecciosas que predominam em 149 países de regiões tropicais e subtropicais, afetando mais de um bilhão de pessoas. No Brasil, as DTNs como a leishmaniose, doença de Chagas, dengue e tuberculose estão presentes em quase todo o território, principalmente nas regiões com menor índice de desenvolvimento humano (IDH). As DTNs são responsáveis por consequências sociais e econômicas que as definem como doenças prioritárias na prevenção e no controle. Inserido neste cenário, o nosso grupo de pesquisa vem trabalhando no campo do desenvolvimento e aperfeiçoamento de técnicas de diagnóstico e profilaxia para as diferentes DTNs. Alguns destes trabalhos realizados demonstraram importantes e promissores resultados na avaliação de peptídeos miméticos como antígenos para o diagnóstico e vacina de leishmaniose. Algumas hipóteses foram levantadas e as quais serão respondidas neste trabalho. Em um primeiro momento, este trabalho convergiu para o diagnóstico de leishmaniose propondo o aperfeiçoamento do teste cutâneo. Estabeleceu-se um novo modelo animal para ser utilizado no controle de qualidade da produção dos antígenos do teste cutâneo, uma vez que nem sempre há coincidência entre o estágio das infecções em pacientes e a etapa de produção. O modelo animal proposto foi Cavia porcellus (porquinho da índia) devido à sua habilidade em reproduzir resposta imune similar a dos humanos. Para validar os resultados obtidos com os porquinhos da índia, o mesmo desenho experimental foi realizado em hamsters. Os resultados dos testes de imunização com as duas espécies animais validaram o modelo C. porcellus para o teste cutâneo, e demonstraram que eles possuem forte potencial como modelo in vivo para o controle de qualidade de produção dos antígenos do teste cutâneo. Em seguida, com base em bons resultados previamente obtidos com três peptídeos miméticos selecionados por phage display, e quimicamente sintetizados como moléculas solúveis, avaliou-se o potencial dos mesmos como antígenos no teste cutâneo. Os peptídeos, individualmente (PA1, PA2 e PA3) ou em conjunto (PAMix), foram testados em modelo animal (C. porcellus) imunizado com duas espécies de Leishmania (Leishmania amazonensis ou Leishmania braziliensis) e comparados com o antígeno referência do teste cutâneo. Os resultados mostraram que os peptídeos, individualmente ou em conjunto, foram capazes de promover reações de induração em 48 e 72 h após a realização do teste. Nos animais imunizados com L. amazonensis o melhor resultado foi alcançado pelo antígeno PA3, o qual foi superior ao do antígeno referência. Nos animais imunizados com L. braziliensis dois antígenos peptídicos apresentaram resultados satisfatórios, o antígeno PA2 e o PAMix, promoveram reações de induração com efeito mais prolongado do que o antígeno padrão. Estes resultados reforçam a hipótese de que os peptídeos podem ser utilizados como antígenos no teste cutâneo, e poderiam substituir o antígeno atualmente utilizado no diagnóstico de leishmaniose cutânea. Nova hipótese é levantada acerca da habilidade destes peptídeos em promoverem resposta imune celular e o seu potencial para serem aplicados como antígenos vacinais para a leishmaniose. Alinhado a isso, sugere-se que a capacidade imunogênica do antígeno poderia ser aperfeiçoada misturando os peptídeos já sintetizados e testados (PA1, PA2 e PA3), os quais são epítopos gerados a partir de proteínas da membrana do parasita, com peptídeos miméticos derivados de proteínas intracelulares do parasito. Com base nisso, a segunda etapa deste trabalho objetivou a pesquisa de epítopos derivados de histonas e sua avaliação in silico e in vitro. Pela análise in silico foi possível selecionar 11 peptídeos miméticos como moléculas imunogênicas. Destes, três (PH31, PH202 e PH293) foram selecionados pelo ensaio de ELISA indireto e encapsulados em 7 lipossomos em conjunto com os peptídeos PA1, PA2 e PA3. O conjunto destes peptídeos foi analisado em testes biológicos como candidatos antigênicos. Primeiramente foram imunizados coelhos para avaliar a produção de anticorpos para confirmar a antigenicidade. Uma vez confirmada a imunogenicidade da quimera dos antígenos foi realizada infecção in vitro com macrófagos tratados com o conjunto de peptídeos, a qual demonstrou que os peptídeos foram capazes de induzir a síntese de IL-12, IFN-y e IL-4. Os resultados demonstraram que para IL-12 os peptpideos aumentaram a taxa de produção em 5,64 (48 h) e 1,6 (72 h) em relação ao grupo controle negativo e quando infectados com L. braziliensis. Para L. amazonensis as taxas foram de 3,08 (48 h) e 4,34 (72 h). No caso IFN-y as taxas foram de 0,27 (48 h) e 0,95 (72 h) nas infecções com L. braziliensis, sendo as taxas nas infecções com L. amazonensis de 1,81 (48 h) e 1,94 (72 h). Para IL-4 as taxas aumentaram em 1,46 (48 h) e 2,38 (72 h) para infecções com L. braziliensis, e 0,60 (48 h) e 2,38 (72 h) para L. amazonenses. Estes resultados demonstram que os peptídeos foram capazes de induzir a produção de um perfil de citocinas que conduz a uma resposta imune Th1 e Th2. Estes resultados também sugerem que os peptídeos são capazes de mimetizar proteínas do parasito que apresentam importante função na interação parasito-hospedeiro, e consequentemente poderiam ser usados para vacinas e para imunoterapia.Abstract: Negleted tropical diseases (NTDs) are a group of infectious illnesses that prevail in 149 tropical and subtropical countries affecting more than one billion of people. In Brazil, NTDs such as Leishmaniasis, Chagas diseases, dengue fever and turberculosis are present in over almost the entire territory, mainly in the regions with lowest human development indices. NTDs are responsible for social and economic consequences defining them as priorities for prevention and control. On this scenario, our research group has been working on the fields to improve and develop diagnosis and prophylaxis techniques for the different NTDs. Some of these previous works demonstrated great and promising results on the evaluation of mimetic peptides as antigens for leishmaniasis diagnosis and vaccines. Their results raised some hypotheses, which concern the focus of this work. At a first moment, this work focused on leishmaniasis diagnosis by proposing improvements for skin tests. The search looked to stablish a new animal model to be used on the quality control of skin test antigen production. The animal model proposed was Cavia porcellus (guinea pigs) due to its ability on reproducing a similar immune response to humans. To validate the results obtained with guinea pigs the same experimental design were tested in hamsters. The results from immunization tests with the two animal specie validated C. porcellus as a good model for skin test. The results also showed that they have strong potential as an in vivo model for quality control of skin test antigen production. Afterwards, based on good results previously obtained with three mimetic peptides selected by phage display and chemically synthesized as soluble molecules, it was evaluated their potential as antigens on skin test. The peptides, individually (PA1, PA2 and PA3) or in a mix (PAMix), were tested on animal model (Cavia porcellus) immunized with Leishmania amazonensis or Leishmania braziliensis and compared to the standard skin test antigen. The results demonstrated that the peptides, individually or in a mix, were able to promote induration reactions at 48 and 72 h after the test was performed. In animals immunized with L. amazonensis the best result was achieved by PA3 antigen, which were higher than the reference antigen. In animals immunized with L. braziliensis two peptides antigens, PA2 and PAMix, presented satisfactory results. These peptides promoted induration reactions for longer period of time than the standard antigen. These results reinforced the hypothesis that peptides can be used as antigens in skin test and could replace the current antigen used for cutaneous leishmaniasis diagnosis. The results from these works raised new hypothesis concerning about the ability of these peptides to promote cellular immune response and their potential to be applied as antigens on leishmaniasis vaccines. Align to this, another hypothesis suggests that the antigenicity capacity of the antigen could be improved by mixing the already synthesized and tested peptides (P1, P2 and P3), which are epitopes generated by parasites membrane proteins, with mimetic peptides from parasites intracellular proteins. Based on these, the second part of this work focused on search for epitopes from histone proteins and the in silico and in vitro evaluation of them. By the in silico analysis was possible to select 11 mimetic peptides as immunogenic molecules. From that, three (PH31, PH202 and PH293) were selected by indirect ELISA and were entrapped in liposome structures in a mix with peptides P1, P2 and P3. This chimeric molecule was analyzed in biological assays as antigens candidates. The in vitro infection analysis with peptides mix-treated macrophages demonstrated that the peptides mix is able to induce the synthesis of IL-12, IFN-y and IL-4. The results showed that for IL-12 the peptides stimulated an increase fold of 5.64 (48 h) and 1.6 (72 h) in relation to negative control group in infections with L. braziliensis. For L. amazonensis folds were 3.08 (48 h) and 4.34 (72 h). In the case of IFN-y the folds were 9 0.27 (48 h) and 0.95 (72 h) in infections with L. braziliensis, and in infections with L. amazonensis were 1.81 (48 h) and 1.94 (72 h). For IL-4 the increase folds were 1.46 (48 h) and 2.38 (72 h) in infections with L. braziliensis, and 0.60 (48 h) and 2.38 (72 h) for L. amazonensis infections. The peptides mix was able to induce a cytokine profile production that lead to a Th1 and Th2 immune response. These results suggest that they are able to mimic parasites proteins that present important role in host-parasite interaction, and consequently could be used for vaccines and immunoprophilaxies

New strategy to improve quality control of Montenegro skin test at the production level

International audienceIntroduction: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production.Methods: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis, and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis, and, after 45 days, they were skin tested with standard Montenegro antigen.Results: Cavia porcellus immunized with L. amazonensis or L. braziliensis, and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test.Conclusions: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen

New strategy to improve quality control of Montenegro skin test at the production level

Abstract INTRODUCTION: The production of the Montenegro antigen for skin test poses difficulties regarding quality control. Here, we propose that certain animal models reproducing a similar immune response to humans may be used in the quality control of Montenegro antigen production. METHODS: Fifteen Cavia porcellus (guinea pigs) were immunized with Leishmania amazonensis or Leishmania braziliensis , and, after 30 days, they were skin tested with standard Montenegro antigen. To validate C. porcellus as an animal model for skin tests, eighteen Mesocricetus auratus (hamsters) were infected with L. amazonensis or L. braziliensis , and, after 45 days, they were skin tested with standard Montenegro antigen. RESULTS: Cavia porcellus immunized with L. amazonensis or L. braziliensis , and hamsters infected with the same species presented induration reactions when skin tested with standard Montenegro antigen 48-72h after the test. CONCLUSIONS: The comparison between immunization methods and immune response from the two animal species validated C. porcellus as a good model for Montenegro skin test, and the model showed strong potential as an in vivo model in the quality control of the production of Montenegro antigen

Biological evaluation of mimetic peptides as active molecules for a new and simple skin test in an animal model

International audienceA skin test is a widely used tool in diagnostic evaluations to investigate cutaneous leishmaniases (CL). The actual antigen (Montenegro skin test [MST] antigen) presents some difficulties that pertain to its manufacturing and validation. To contribute to overcoming this problem, we propose the application of new-generation molecules that are based on skin antigen tests. These antigens were obtained through biotechnology pathways by manufacturing synthetic mimetic peptides. Three peptides, which were selected by phage display, were tested as skin test antigens in an animal model (Cavia porcellus) that was immunized with Leishmania amazonensis or Leishmania braziliensis. The peptide antigens, individually (PA1, PA2, PA3) or in a mix (PAMix), promoted induration reactions at 48 and 72 h after the test was performed. The indurations varied from 0.5 to 0.7 cm. In the animals immunized with L. amazonensis, the PA3 antigen showed better results than the standard MST antigen. In animals immunized with L. braziliensis, two peptide antigens (PA2 and PAMix) promoted induration reactions for a longer period of time than the standard MST antigen. These results validate our hypothesis that peptides could be used as antigens in skin tests and may replace the current antigen for CL diagnosis

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures. We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptide mix generated the IFN-γ, IL-12, IL-4 and TGF-β that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that the mix of peptides derived from histone and parasites membrane molecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.Fil: Guedes, Deborah Carbonera. Universidade Federal do Paraná; BrasilFil: Santiani, Manuel Hospinal. Universidade Federal do Paraná; BrasilFil: Carvalho, Joyce. Universidade Federal do Paraná; BrasilFil: Soccol, Carlos Ricardo. Universidade Federal do Paraná; BrasilFil: Minozzo, João Carlos. Universidade Federal do Paraná; Brasil. Secretaria de Saúde Do Estado Do Paraná; BrasilFil: Machado de Ávila, Ricardo Andrez. Universidade Do Extremo Sul Catarinense; BrasilFil: de Moura, Juliana Ferreira. Universidade Federal do Paraná; BrasilFil: Ramos, Eliezer Lucas Pires. Universidade Federal do Paraná; BrasilFil: Castro, Guillermo Raul. Laboratorio Max Planck de Biología Estructural, Química y Biofísica Molecular de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Chávez Olórtegi, Carlos. Universidade Federal de Minas Gerais; BrasilFil: Thomaz Soccol, Vanete. Universidade Federal do Paraná; Brasi