Navegando por Palavras-chave "Adjuvantes"

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    Adjuvantes no tratamento da hiperglicemia do diabetes melito tipo 1
    (Sociedade Brasileira de Endocrinologia e Metabologia, 2008-03-01) Gabbay, Monica Andrade Lima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Since Diabetes Control and Complications Trial (DCCT), intensive therapy has been directed at achieving glucose and glycosylated hemoglobin (HbA1c) values as close to normal as possible regarding safety issues. However, hyperglycemia (especially postprandial hyperglycemia) and hypoglicemia continue to be problematic in the management of type 1 diabetes. The objective of associating other drugs to insulin therapy is to achieve better metabolic control lowering postprandial blood glucose levels. Adjunctive therapies can be divided in four categories based on their mechanism of action: enhancement of insulin action (e.g. the biguanides and thiazolidinediones), alteration of gastrointestinal nutrient delivery (e.g. acarbose and amylin) and other targets of action (e.g. pirenzepine, insulin-like growth factor I and glucagon-like peptide-1). Many of these agents have been found to be effective in short-term studies with decreases in HbA1c of 0.5-1%, lowering postprandial blood glucose levels and decreasing daily insulin doses.
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    Análise da resposta imune ZIKA-específica após imunização utilizando vacinas de DNA e proteínas recombinantes do envelope do vírus
    (Universidade Federal de São Paulo, 2022) Rezende, Thaís [UNIFESP]; Rosa, Daniela Santoro [UNIFESP]; http://lattes.cnpq.br/1472798853821902; http://lattes.cnpq.br/7884800233521548
    O vírus Zika (ZIKV) é um arbovírus pertencente à família Flaviviridae cujo genoma codifica uma poliproteína que é clivada em três proteínas estruturais: capsídeo (C), precursor de membrana (prM) e envelope (E). A proteína E é responsável pela ligação e fusão com a membrana da célula hospedeira pode ser dividida em três domínios estruturais e funcionais: domínio central (EDI), domínio II (EDII) e domínio III (EDIII). A proteína E é alvo de anticorpos neutralizantes e por esse motivo, vem sendo avaliada como potente candidato vacinal. Até o momento não há vacina disponível, e a infecção causada pelo ZIKV, bem como as possíveis complicações clínicas associadas salientam a necessidade de uma vacina eficaz. Neste contexto, o presente trabalho teve como objetivo produzir vacinas de DNA que codificam a proteína do envelope (E) do ZIKV e seus domínios (EDI/II e EDIII) separadamente, assim como as respectivas proteínas recombinantes, a fim de avaliar sua imunogenicidade em ensaios pré-clínicos. Inicialmente, comparamos diferentes formulações de adjuvantes quanto à capacidade de induzir anticorpos específicos e neutralizantes após imunização com as proteínas recombinantes. Observamos que as imunizações com as proteínas recombinantes, principalmente EZIKV e EDIIIZIKV, assistidas pelo adjuvante AddaVax induziram resposta humoral de elevada magnitude e com maior capacidade de neutralização. Em seguida, realizamos experimentos com estratégias de imunização conhecida como indução e reforço homóloga e heteróloga. A análise comparativa mostrou que a estratégia homóloga para as proteínas recombinantes assistida pelo AddaVax induziu títulos de anticorpos mais altos e maior porcentagem de neutralização. Entretanto, a análise da resposta celular mostrou que a estratégia de indução e reforço homóloga com as vacinas de DNA induziu o maior número de células produtoras de IFN-. Em resumo, a estratégia homóloga de proteína demonstrou ser a mais eficaz. Assim, com os resultados deste trabalho esperamos contribuir com o desenvolvimento para um possível candidato vacinal contra o ZIKV.
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    Avaliação da imunogenicidade de vacinas de DNA e de proteínas recombinantes do envelope dos vírus chikungunya e zika
    (Universidade Federal de São Paulo (UNIFESP), 2018-05-24) Amaral, Marcelo Pires [UNIFESP]; Rosa, Daniela Santoro [UNIFESP]; http://lattes.cnpq.br/1472798853821902; http://lattes.cnpq.br/1246712263704416; Universidade Federal de São Paulo (UNIFESP)
    Chikungunya (CHIKV) and Zika (ZIKV) viruses’ infection have rapidly enhanced worldwide in the last decades. To date, there is no vaccine available or specific treatment against both viruses. The viral envelope proteins of CHIKV and ZIKV are the main immunodominant antigens of the viruses and have a crucial role during the infection in host cells. Furthermore, E2CHIKV and EZIKV are the main target of neutralizing antibodies against the respective viruses. In this work, we designed and optimized synthetic genes encoding E2CHIKV and EZIKV proteins, subcloned into pVAX1 vector to obtain the DNA vaccines (pVAX-E2CHIKV and pVAX-EZIKV) and purified using Endofree Plasmid Giga kit (Qiagen). The recombinant proteins E2CHIKV and EZIKV were produced as inclusion bodies in BL21(DE3) and BL21(DE3)RIL (respectively), then purified by affinity chromatography. The immunization strategies were performed with the homologous/heterologous prime-boost regimen, 15 days apart, with 100 μg (100 μL final volume) of the DNA vaccines (i.m.) pVAX-E2CHIKV or pVAX-EZIKV or with 10 μg (200 μL final volume) of the recombinant proteins (s.c.) E2CHIKV or EZIKV in the presence of different adjuvants (50 μg poly IC, 20 μg imiquimod or 10 μg CpG) inoculated in 6-8 weeks old female mice, C57BL/6 mice for the vaccines against CHIKV and BALB/c mice for the vaccines against ZIKV. All mice were bled 14 days after each immunization to evaluate specific humoral response, and euthanized 15 days after the last dose to collect spleen and popliteal and inguinal lymph nodes to evaluate specific cellular response. All procedures were performed in accordance to the guidelines of the UNIFESP’s Ethics Committee and approved under protocol number 5759150416. Among all the adjuvants evaluated, poly IC was shown the best candidate to follow the recombinant proteins E2CHIKV and EZIKV vaccines, inducing greater magnitude of specific humoral and cellular responses. C57BL/6 mice immunized with the DNA vaccine pVAX-E2CHIKV in the homologous protocol were not able to induce specific humoral response, unlike the homologous recombinant protein (E2CHIKV + poly IC) and the heterologous (pVAXE2CHIKV / E2CHIKV + poly IC) vaccines that induced high specific antibody titers. Analysis of the cellular response showed that 2 doses of the homologous DNA vaccine pVAX-E2CHIKV was able to induce only interferon γ (IFN-γ) producing cells. The homologous E2CHIKV + poly IC vaccine was able to induce greater magnitude response of IFN-γ producing cells and IgG anti- E2CHIKV, as well as the frequency of germinal center (GC) B cells and T follicular helper (TFH). These cellular responses were also observed in mice immunized with the heterologous vaccine (pVAX-E2CHIKV / E2CHIKV + poly IC), but to a lesser extent. BALB/c mice immunized with the homologous DNA pVAX-EZIKV and the recombinant protein EZIKV + poly IC vaccines had the lowest and the highest magnitude of specific humoral response, respectively. Both heterologous vaccines (pVAX-EZIKV / EZIKV + poly IC, and vice-versa) had a medium magnitude of specific humoral response. Although all immunization groups elicited cellular response after the different immunization strategies, the homologous EZIKV + poly IC vaccine was able to induce higher magnitude of IFN-γ producing cells and IgG anti-EZIKV, higher frequency of GC B cells and TFH, proliferation and production of the intracellular cytokines TNF-α and IFN-γ by CD4+ T lymphocytes (LT). Overall, homologous recombinant protein E2CHIKV or EZIKV vaccines in the presence of the poly IC adjuvant were able to induce greater magnitude of humoral and cellular response, however heterologous vaccine strategy may be another option to induce a better quality and range of the immune response against intracellular pathogens.
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    Imunogenicidade de diferentes candidatos e estratégias vacinais para os vírus Zika e Chikungunya
    (Universidade Federal de São Paulo (UNIFESP), 2021) Oliveira, Fernanda Caroline Coirada [UNIFESP]; Rosa, Daniela Santoro [UNIFESP]; Universidade Federal de São Paulo
    Arbovirus infections such as those caused by Zika virus (ZIKV) and Chikungunya virus (CHIKV) are responsible for a large number of cases with considerable severity and morbidity. So far, there are no vaccines or specific medications available against these arboviruses. The envelope protein (E) of ZIKV and CHIKV is the most abundant on the viral surface and responsible for the binding and fusion process with the target cell membrane. Furthermore, it is the main target of neutralizing antibodies being an important antigen candidate for vaccine development. DNA vaccines are very promising, as they are able to elicit both humoral and cellular immune responses, however, they have limited immunogenicity in humans. In recent years, several strategies have been developed in order to increase the immunogenicity of this vaccine platform, such as antigen targeting to dendritic cells (DCs) subpopulations, association with in vivo electroporation and heterologous prime-boost immunization. In the present work, we produced the recombinant protein E2CHIKVΔTM, and also scFV and pVAX DNA vaccines encoding the same antigen and compared the immunogenicity after homologous or heterologous prime-boost immunization. We observed that heterologous primeboost immunization was able to induce high antibody titers with neutralizing capacity, as well as specific IFN-γ producing cells and polyfunctional CD4+ and CD8+ T cell responses. In addition, we mapped the most immunogenic epitopes along the E2CHIKV protein and identified 14 potential epitopes, with CHIKV (361-374) being the immunodominant. Finally, we evaluated the influence of different adjuvants (poly (I:C), Addavax, Alhydrogel, AS03 and Quil A) on the immune response. We observed that mice immunized with recombinant E2CHIKVDTM combined with AS03 presents the highest humoral response with neutralizing capacity. Regarding ZIKV, we generated different vaccine candidates encoding ZIKV (EZIKV, EDI/IIZIKV and EDIIIZIKV) and CHIKV (E2CHIKV) viral envelope proteins and evaluated different vaccination strategies in a preclinical model. We produced DNA vaccines encoding a single chain variable fragment (scFVs) fused to ZIKV antigens to target it to dendritic cells. In addition, DNA vaccines (pVAX) encoding the same antigens without DC-targeting were produced and the induced specific immune response were compared. We observed that immunization of C57BL/6 mice with the different constructs was able to induce specific antibodies that specifically recognize the ZIKV and neutralize the infection. Immunization with the pVAX-EZIKV DNA vaccine was able to induce higher antibody titers when compared to scFv immunization. Furthermore, immunization with the constructs was able to induce polyfunctional CD4+ and CD8+ T lymphocytes and specific IFN-γ producing cells. The epitope mapping revealed that the peptides ZIKV (E1-20) and ZIKV (E51-70) contained in EDI/II domain and the peptides ZIKV (E351-370) and ZIKV (E361-380) contained in the domain protein EDIII were the most immunogenic. Taking together, our results demonstrated that the different strategies tested were efficient to induce humoral and cellular immune responses against ZIKV and CHIKV, thus opening new perspectives for the development of vaccine candidates.
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    Seleção e desenvolvimento de adjuvantes para uso em imunizações com proteínas recombinantes de Plasmodium
    (Universidade Federal de São Paulo (UNIFESP), 2009-09-30) Bargieri, Daniel Youssef [UNIFESP]; Rodrigues, Mauricio Martins [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The C-terminal region of Plasmodium merozoite surface protein 1 (MSP119) is being studied as one of the main targets for the development of a vaccine against malaria. Several studies have shown high imunogenicity of this region in experimental immunizations when injected in the presence of strong adjuvant formulations. In the present study we evaluate the possibility of using recombinant proteins based on the sequence of MSP119 to immunize mice by a mucosal route. Also, we generate new recombinant proteins consisting in the genetic fusion of the MSP119 to the flagellin FliC (flagellar protein of Salmonella enterica Typhimurium), to increase the immunogenicity of the antigen. Initially, we evaluated the capacity of the molecules cholera toxin (CT), heat-labile E. coli enterotixin (LT) or CpG ODN 1826, to act as adjuvants in mucosal intranasal immunization of mice with the recombinant proteins His6PvMSP119 or His6PvMSP119-PADRE (containing the universal T helper epitope PADRE). When administered in the presence of CT or LT, both recombinant proteins were highly immunogenic by the intranasal route. CpG ODN 1826 was less efficient as adjuvant by this route. The addition of CpG ODN 1826 in CT adjuvanted immunizations increased the specific IgG2c titers. These results showed that CT and LT are potent mucosal adjuvants in immunizations with recombinant malaria antigens and that CpG ODN 1826 can, in this case, be used as a tool to modulate the pattern of the immune response. Subsequently, we expressed a recombinant protein consisting of the sequence of PvMSP119-PADRE genetically fused to FliC (His6FliC-PvMSP119- PADRE). We showed that this fusion protein preserved the antigenic properties of PvMSP119 and the ability of flagellin to activate TLR5. The immunization of mice using this recombinant fusion protein induced high titers of specific antibodies and the presence of antigen-specific IFN-g producing cells in the spleen. The addition of CpG ODN 1826 in the vaccine formulations modulated the immune response by augmenting the specific titers of IgG2c. In addition, sera from immunized mice recognized the parasite in indirect immunofluorescence assay (IFA). Our results provided a new class of malaria vaccine formulation with intrinsic adjuvant property capable of stimulating specific humoral and cellular immune responses when administered alone or in the presence of other adjuvants. Finally, we expressed a recombinant protein containing the sequence of Plamodium falciparum MSP119 (PfMSP119) fused to FliC (His6FliC-PfMSP119). This fusion protein retained the capacity of flagellin to activate TLR5. Immunization of mice with the His6FliC-PfMSP119 alone induced high titers of specific antibodies and IFN-g producing cells. The addition of adjuvants such as CpG ODN 1826 or Quil-A (saponin of Quillaja saponaria) increased the levels of IgG2c and the cellular immune response, measured by the IFN-g secretion by immune spleen cells in culture. In addition, sera from immunized rabbits recognized the parasites in IFA and inhibited parasite growth in vitro. These results provide evidences that the fusion of malaria antigens to flagellin is an inexpensive and viable alternative for the development of a malaria vaccine.