• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Doctoral Thesis
DOI
10.11606/T.17.2013.tde-22112013-101030
Document
Author
Full name
Wendy Martin Rios
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2013
Supervisor
Committee
Silva, Celio Lopes (President)
Ferreira, Luis Carlos de Souza
Miyoshi, Anderson
Santos, Isabel Kinney Ferreira de Miranda
Soares, Sandro Gomes
Title in Portuguese
Vacinas de DNA codificando antígenos de glioblastoma e proteínas imunomoduladoras: construção e avaliação da imunogenicidade
Keywords in Portuguese
Antígenos de glioblastoma
Glicoproteína D
Glioblastoma
IFN-g
IL-10
Imunoterapia
Proteínas de choque térmico
Vacina de DNA
Abstract in Portuguese
O glioblastoma (GBM) é o tumor cerebral primário mais comum e o mais grave tumor de células da glia. O GBM é um tumor astrocítico de grau IV caracterizado pela proliferação descontrolada, infiltrado difuso, tendência à necrose, angiogênese, resistência a apoptose e grande heterogeneidade genética. Apesar da terapia abranger a remoção cirúrgica máxima, a radioterapia e a quimioterapia, o tumor torna-se resistente à drogas utilizadas no tratamento levando o paciente a recorrência e morte em menos de 15 meses após o diagnóstico. Uma alternativa para o tratamento do GBM é a imunoterapia, a qual é capaz de estimular o sistema imunológico do próprio paciente a gerar uma resposta específica e duradoura que pode proteger contra a recorrência da doença. Uma dessas alternativas envolve o uso de vacinas de DNA codificando antígenos tumorais e proteínas imunomoduladoras capazes de ativar eficientemente linfócitos B e T específicos aos antígenos presentes no tumor. Nesse contexto, o objetivo do presente trabalho foi construir vacinas de DNA utilizando-se os genes dos antígenos EGFRvIII, cERBB2, MAGE e GLEA de GBM e os genes das proteínas imunomoduladoras hsp65, hsp70, gp96 e gD e avaliar suas respectivas imunogenicidades. Os genes foram avaliados in silico, sintetizados in vitro e utilizados na construção das vacinas de DNA. Ferramentas de biologia molecular e o vetor pVAX foram utilizados para obtenção das vacinas. Elas foram caracterizadas por sequenciamento e western blot e utilizadas na imunização de camundongos C57BL/6. As imunizações foram realizadas com três doses em intervalos de 12 dias combinando um antígeno tumoral e uma proteína imunomoduladora na forma de vacina de DNA. A imunogenicidade foi avaliada 20 dias após a última dose. Os ensaios ex vivo foram realizados com o soro dos animais imunizados para dosagem de anticorpos específicos contra os antígenos tumorais e com as células do baço que foram re-estimuladas com as proteínas EGFRvIII, cERBB2, MAGE e GLEA para identificar a presença de células específicas aos antígenos tumorais. Como resultado, a vacina pVAXgDGLEA foi a única capaz de induzir anticorpos do subtipo IgG2a anti-GLEA. As vacinas pVAXgDGLEA, pVAXgDEGFRvIII e pVAXgDMAGE foram capazes de ativar células específicas aos antígenos que após o re-estímulo responderam rapidamente com produção de IFN-g e IL-10. A proteína imunomoduladora gD foi, portanto, capaz de ajudar na indução de um padrão de resposta Th1, específica aos antígenos de GBM, importante no combate ao tumor e a IL-10 pode favorecer e/ou balancear a resposta no cérebro que deve ser eficaz, mas não exacerbada.
Title in English
DNA vaccines codifying glioblastoma antigens and immunomodulating proteins: construction and immunogenicity evaluation
Keywords in English
D glycoprotein
DNA vaccine
Glioblastoma
Glioblastoma antigens
Heat shock proteins
IFN-g
IL-10
Immunotherapy
Abstract in English
Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and the most severe tumour affecting glia cells. GBM is a grade IV astrocytoma known by uncontrolled proliferation, diffused infiltrate, necrosis tendency, angiogenesis, apoptosis resistance and a wide genetic heterogeneity. The standard of care consists of maximal surgical resection, followed by a combination of radiation and chemotherapy. Despite that, tumour becomes resistant to drugs used to treatment, and the patient experiences recurrence followed by death in less than 15 months after diagnosis. An alternative in GBM treatment could be immunotherapy which aims to stimulate patients immunological system in order to obtain a specific and long-term response that can protect against recurrence. One of these alternatives involves the use of DNA vaccines codifying tumoral antigens and immunomodulatory proteins that can effectively activate tumour antigen specific B and T lymphocytes. In this context, the objective of this work was the construction of DNA vaccines using GBM antigen genes (EGFRvIII, cERBB2, MAGE e GLEA) and immunomodulatory proteins (hsp65, hsp70, gp96 e gD), followed by their immunogenicity evaluation. Genes were evaluated in silico, synthesized in vitro and used in DNA vaccines construction. Molecular biology tools and the pVAX vector were used to obtain the vaccine. They were characterized by sequencing, western blot and were used in the immunization of C57BL/6 mice. Immunizations were performed in 3 doses of a DNA vaccine combining a tumoral antigen and an immunomodulatory protein at each 12 days. Immunogenicity was evaluated 20 days after the last dose. The ex vivo assays were performed with the serum of immunized animals for antibody evaluation and spleen cells were stimulated with EGFRvIII, cERBB2, MAGE e GLEA proteins to assess tumoral antigen specific cells. The pVAXgDGLEA vaccine was the only able to induce IgG2a subtype anti-GLEA antibodies. Vaccines pVAXgDGLEA, pVAXgDEGFRvIII e pVAXgDMAGE were able to activate antigen-specific cells that produced IFN-g e IL-10 quickly after reestimulation. The gD immunomodulatory protein was able to induce a Th1 immune response, specific to GBM antigens, which is important in tumor combat while IL-10 could favor and/or balance the response in brain, which should be effective but not exacerbated.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
Publishing Date
2013-12-12
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
Centro de Informática de São Carlos
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2021. All rights reserved.