Imunogenicidade do vírus vacina recombinante modificado com o gene do antígeno cárcino-embrionário (CEA) humano em modelo experimental de adenocarcinoma de cólon / Immunogenicity of recombinant vaccinia virus modified with human carcinoembryonic antigen gene in an experimental model of colon adenocarcinoma

RESUMO

Introdução: Os adenocarcinomas do trato gastrointestinal desenvolvem inicialmente a partir do epitélio superficial, e em mais de 90% dos casos, expressam o CEA. Existe compartimentalização e alguma independência entre o sistema imunológico mucoso e sistêmico com tráfego assimétrico de células entre eles. Um melhor conhecimento da imunologia dos tumores tem gerado o desenvolvimento de várias abordagens de vacinas direcionadas contra antígenos tumorais específicos. O vírus vaccinia esta entre os mais potentes para a indução de resposta protetora em animais contra infecções virais e câncer. Este tem sido mostrado como imunogênico em humanos. Imunização com o vírus vaccinia recombinante com o antígeno carcinoembrionário (CEA) pode gerar proteção humoral e celular contra tumores CEA +, dependo da dose e via de imunização... Objetivos: Comparar a imunização intra-retal com a venosa, e a resposta ao nível sistêmico e local apos uma dose e dois reforços por estas duas vias. Desenvolvimento de um animal híbrido que desenvolva tumores espontâneos do trato gastrointestinal com evolução previsível... Material e Métodos: Os animais foram imunizados intra-retal ou intravenoso com 10 milhões de partículas do vírus Vaccinia recombinante com o CEA a cada 15 dias totalizando 3 doses. O grupo controle recebeu a mesma imunização com o mesmo vetor porem, não transduzido com o CEA... Os tumores foram medidos a cada 2 dias. Os animais Apc foram gerados por manipulação genética através da desativação do gene da Polipose colônica adenomatosa (APC) com mutação induzida na posição do aminoácido 1648, gerando proteína instável e não funcional... O camundongo Apc/CEA é um relevante modelo animal para o estudo do desenvolvimento espontâneo do câncer de colón da fase de displasia ao adenocarcinoma invasivo...(AU)

ABSTRACT

Introduction: The adenocarcinomas of the gastrointestinal tract (TGI) develop from superficial epithelium and in more than 90% of cases express CEA. There is compartmentalization with some independence among mucosal versus systemic immune systems and the asymmetric trafficking of cells between them. An improved understanding of tumor immunology has led to the development of several vaccine approaches targeted against specific human tumors antigens. Recombinant vaccinia virus vaccines are among the most potent for inducing protective immunity in animais against many viral infections and cancer. They have been shown to be immunogenic in human. Immunization with recombinant carcinoembryonic antigen (CEA) vaccinia virus can generate humoral and cellular protection against tumors CEA+, depending on the dose and route of administration. One of the major obstacles in vaccine development has been the lack of a relevant animal model for appropriately evaluating vaccine agents and immunization protocols. These results could be more accurate using an animal model that mimics the human situation with development of spontaneous tumors from the gastrointestinal tract with high expression of a specific antigen. Objectives: Comparing intra-rectal and venous immunization, and to evaluate systemic and local immune response after one dose and 2 booster after these routes. Development of a hybrid mouse model that generates spontaneous TGI' s tumors with expected evolution hyperplasia ~ dysplasia ~ adenoma ~ superficial adenocarcinoma ~ invasive adenocarcinoma. The animal model should have high CEA expression in tumors and low at normal tissues as observed in humans. Material e Methods: Animais had intra-rectal (IR) or endovenous immunization with 10 million units of recombinant vaccinia-CEA virus each 15 days in a total of 3 doses. Control had the same immunization routes with vaccinia virus without CEA expression. Half of the animais were sacrificed 7 days after the last immunization to evaluate the induction of an immune response against CEA at the spleen (systemic response) and at the Payer's patches (mucosa! response). Subcutaneous colon tumors, with or without the expression of CEA, were implanted in the other animais. Tumors were measured every 2 days. Animais Apc were generated by genetic manipulation with partia! knock out of the APC (Adenomatous Polyposis coli) gene at the amino acid position 1648. The Apc pro te in was unstable and not functional. The CEA mo use recei ved the complete human CEA DNA. Those 2 transgenic and knock out mice models were crossed generating a hybrid transgenic animal model with spontaneous development of TGI' s tumors with high expression of CEA. Results: The animais immunized with rVac-CEA presented humoral and cellular specific CEA response and some tumor protection for CEA + tumors.The contrai group immunized without CEA had no immune response or protection when challenged with CEA tumor implants. Intra-rectal immunization showed to be similar to intravenous immunization, with systemic and mucosa! responses but low-grade tumor protection. Some immunized animais were tumor free and ali were able to generate cytolytic activity against CEA+ cells. Among those that developed tumor, these were smaller and had decreased growing rate. Immunization did not show protection when animais received CEAtumors indicating specificity. The double transgenic animais presented at 2 months hyperplasic polyps and occult blood in the feces. At 4 months old they showed gastrointestinal polyps that progressed to invasive adenocarcinomas with a similar pattem of dysplasia and CEA expression as observed in human colorectal cancer. Tumoral tissues had high expression of human CEA and tissues like colon, intestine and stomach also expressed CEA but at low levei. These animais exhibited incomplete or partial tolerance to CEA. Conclusions: Intra-rectal immunization with rVac-CEA showed an immune response bigger than 40%. CTL against CEA after intra-rectal and systemic immunization was present at the spleen and Peyer' s patch. Both route of immunizations induce CEA + tumors protection but the EV route was more effective for subcutaneous tumor. The Apc/CEA mouse is an important animal model for the studying of spontaneous cancer colon development from the dysplasia to adenocarcinoma. The human CEA is over expressed in tumors of these animais. This model represents a system to evaluate different treatment options and induction of tolerance and tumor immunity (AU)