Sub-clonagem em vetor de expressão procarioto de uma cisteíno proteinase de Boophilus microplus

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Antigen Targeting to Dendritic Cells Allows the Identification of a CD4 T-Cell Epitope within an Immunodominant Trypanosoma cruzi Antigen

Targeting antigens to dendritic cells (DCs) by using hybrid monoclonal antibodies (mAbs) directed against DC receptors is known to improve activation and support long-lasting T cell responses. in the present work, we used the mAb alpha DEC205 fused to the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) to identify a region of this protein recognized by specific T cells. the hybrid alpha DEC-ASP2 mAb was successfully generated and preserved its ability to bind the DEC205 receptor. Immunization of BALB/c mice with the recombinant mAb in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)) specifically enhanced the number of IFN-gamma producing cells and CD4+ T cell proliferation when compared to mice immunized with a mAb without receptor affinity or with the non-targeted ASP-2 protein. the strong immune response induced in mice immunized with the hybrid alpha DEC-ASP2 mAb allowed us to identify an ASP-2-specific CD4+ T cell epitope recognized by the BALB/c MHCII haplotype. We conclude that targeting parasite antigens to DCs is a useful strategy to enhance T cell mediated immune responses facilitating the identification of new T-cell epitopes.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)BNP-Paribas Bankcommercial source BNP-Paribas BankUniv São Paulo, Inst Biomed Sci, Dept Parasitol, Lab Antigen Targeting Dendrit Cells, São Paulo, BrazilUniversidade Federal de São Paulo, CTCMol, São Paulo, BrazilNatl Inst Sci & Technol Vaccines, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, CTCMol, São Paulo, BrazilCNPq: 15203*12FAPESP: 2007/08648-9Web of Scienc

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.Brazilian National Research Council (CNPq)National Institute of Science and Technology Research Funding Agency (INCTV) - 15203*12São Paulo State Research Funding Agency (FAPESP) - 2007/08648-9, 2011/51761-6BNP-Paribas BankRio de Janeiro State Research Funding Agency (FAPERJ

Characterization of the immune response when targeting a protein from Plasmodium to dendritic cells.

Imunidade protetora depende da geração e manutenção do repertório de linfócitos T de memória. A geração dessas células está correlacionada com a apresentação de antígenos pelas células dendríticas (DCs). O direcionamento de antígenos tem sido estudado como um novo método vacinal que consiste em entregar antígenos diretamente para DCs usando anticorpos monoclonais. O principal objetivo desse trabalho foi direcionar uma proteína de Plasmodium para a subpopulação DEC205+ de DCs. Camundongos foram imunizados e então desafiados dias depois, com esporozoítos de P. yoelii. A proteína direcionada não protegeu camundongos do desafio, mas a proteína não direcionada protegeu, alcançando níveis de proteção estéril em torno de 100% em alguns casos. Observamos correlação entre a quantidade dos anticorpos e a proteção relativa dos animais imunizados com a proteína não direcionada. Além disso, utilizando anticorpos monoclonais demonstramos que a região conhecida como major repeat pode ser utilizada como alvo direto de pesquisas em vacinas contra malária.In general, protective immunity against many pathogens depends on the generation of memory T cells, and the survival of cells for a long period of time after initial contact with pathogens. We know that the generation of these cells is correlated with the activation of parasite-specific immune cells and the presentation of antigens for dendritic cell (DCs). Targeting antigens to DCs has been studied as a new vaccination method, delivering antigens directly to DCs using monoclonal antibodies. The goal was target circunsporozoite protein (CSP), from Plasmodium, to a DEC205+ (DC) subset. Mice were immunized and challenged days later using P. yoelii sporozoites. Targeting protein did not protect mice from challenge, but non-targeting CS lead to 100% of protection. We found correlation between levels of antibody with protection, and high levels of anti-CS IgG in mice immunized with non-targeting protein. Using monoclonal antibodies we were able to map major repeat as a potential target for new researches in malaria vaccine

Production and characterization of a monoclonal antibody aDEC205 coupled to MSP-1(19) protein from Plasmodium chabaudi.

Apesar da forte ativação do sistema imune que ocorre durante a infecção pelo Plasmodium, a memória imunológica à infecção é restrita a pacientes residentes em áreas endêmicas. Dessa forma é importante a geração de métodos capazes de induzir uma resposta imune eficaz e duradoura contra o parasito. Nesse contexto o direcionamento de antígenos para células centrais do sistema imune tem se apresentado como uma alternativa promissora. Produzimos e caracterizamos um anticorpo híbrido específico para a molécula DEC205, um receptor endocítico presente nas células dendríticas, acoplado à proteína MSP-1(19) de P. chabaudi, para fins de imunização e análise da resposta imune celular e humoral. Ensaios de imunização mostraram a indução de resposta humoral em camundongos imunizados com anticorpo híbrido e seu controle isotípico, caracterizada pela produção de IgM. Nossos resultados prévios indicam que o direcionamento de antígenos aliado a outras estratégias de imunizações podem resultar na ativação da resposta imune específica ao parasita.Despite the strong activation of the immune system that occurs during infection by Plasmodium, the immunological memory to infection is restricted to patients residing in endemic areas. Thus it is important to the generation of methods to induce an effective immune response against the parasite. In this context, the targeting of antigens to the central cells of the immune system has emerged as a promising alternative. We produce and characterize a hybrid antibody molecule specific for DEC205, an endocytic receptor present on dendritic cells, coupled to protein MSP-1(19) of P. chabaudi, for immunization and analysis of cellular and humoral immune response. Immunization tests showed the induction of humoral response in mice immunized with hybrid antibody and isotype control, characterized by production of IgM. Our previous results indicate that targeting antigens combined with other strategies for immunization may result in the activation of specific immune response to the parasite

Sub-clonagem da BMCL1 em PCDNA3

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Clonagem em vetor de expressão em eucarioto de uma Catepsina-L de Boophilus microplus

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Clonagem em vetor de expressão em eucarioto de uma Catepsina-L de Boophilus microplus

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Sub-clonagem em vetor de expressão procarioto de uma cisteíno proteinase de Boophilus microplus

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Immunization with the hybrid DEC-ASP2 mAb induces IFN-γ production and CD4⁺ T cell proliferation.

<p>BALB/c mice were immunized with two doses of the hybrid mAbs or the rec. ASP2 protein as described in materials and methods. Fifteen days after the administration of the second dose, mice were euthanized and their splenocytes were plated in the presence or absence of rec. ASP2 or ovalbumin, as a control. (A) The number of IFN-γ producing cells was detected by ELISPOT and each bar represents the mean ± SD of triplicates of pooled splenocytes. Numbers of IFN-γ producing cells in the absence of any stimulus were subtracted from the stimulated samples. (B) The percentage of CD3⁺CD4⁺ T cells that proliferated after 5 days of re-stimulation with the rec. ASP2 or ovalbumin is shown. Bars represent the mean ± SD of pooled animals performed in triplicates. Percentages of proliferating cells in the absence of any stimulus were subtracted from the stimulated samples. Experiment was analyzed by one-way ANOVA followed by the post-test HSD Tukey. * refers to p<0.05. The graph is representative of 2 (A) or 3 (B) independent experiments.</p