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Imunizao Nasal Em Coelhos Com Neisseria Lactamica

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Imunizao Nasal Em Coelhos Com Neisseria Lactamica IMUNIZAÇÃO NASAL EM COELHOS COM NEISSERIA LACTAMICA: IMPORTÂNCIA DOS ANTÍGENOS DE REATIVIDADE CRUZADA CLAUDIA FERIOTTI TUNES Dissertação de Mestrado apresentada ao Programa de Pós-Graduação Interunidades em Biotecnologia USP/ Instituto Butantan / IPT, para a obtenção do Título de Mestre em Biotecnologia. São Paulo 2006 IMUNIZAÇÃO NASAL EM COELHOS COM NEISSERIA LACTAMICA: IMPORTÂNCIA DOS ANTÍGENOS DE REATIVIDADE CRUZADA CLAUDIA FERIOTTI TUNES Dissertação de Mestrado apresentada ao Programa de Pós-Graduação Interunidades em Biotecnologia USP/ Instituto Butantan / IPT, para a obtenção do Título de Mestre em Biotecnologia. Área de concentração: Biotecnologia Orientadora: Profª. Dra. Elizabeth Natal De Gaspari São Paulo 2006 DADOS DE CATALOGAÇÃO NA PUBLICAÇÃO (CIP) Serviço de Biblioteca e Informação Biomédica do Instituto de Ciências Biomédicas da Universidade de São Paulo reprodução não autorizada pelo autor Tunes, Claudia Feriotti. Imunização nasal em coelhos com Neisseria lactamica: importância dos antígenos de reatividade cruzada / Claudia Feriotti Tunes. -- São Paulo, 2006. Orientador: Elizabeth Natal De Gaspari. Dissertação (Mestrado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT. Área de concentração: Biotecnologia. Linha de pesquisa: Imunologia - estudo de vacinas. Versão do título para o inglês: Nasal immunization in rabbits with Neisseria lactamica: the importance of cross reactive antigens. Descritores: 1. Neisseria lactamica 2. Neisseria meningitidis 3. Coelhos 4. Resposta imune humoral 5. Anticorpo monoclonal 6. Vacinas I. De Gaspari, Elizabeth Natal II. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação em Biotecnologia III. Título. ICB/SBIB205/2005 UNIVERSIDADE DE SÃO PAULO Programa de Pós-Graduação Interunidades em Biotecnologia Universidade de São Paulo, Instituto Butantan, Instituto de Pesquisas Tecnológicas _________________________________________________________________________________________________________ Candidato (a): Claudia Feriotti Tunes. Título da Dissertação: Imunização nasal em coelhos com Neisseria lactamica: importância dos antígenos de reatividade cruzada. Orientador (a): Elizabeth Natal De Gaspari. A Comissão Julgadora dos trabalhos de Defesa da Dissertação de Mestrado, em sessão pública realizada a ................./................./................., considerou ( ) Aprovado(a) ( ) Reprovado(a) Examinador (a): Assinatura:........................................................................................ Nome: .............................................................................................. Instituição: ....................................................................................... Examinador (a): Assinatura:........................................................................................ Nome: .............................................................................................. Instituição: ....................................................................................... Examinador (a): Assinatura:........................................................................................ Nome: .............................................................................................. Instituição: ....................................................................................... SECRETARIA DE ESTADO DA SAÚDE COORDENAÇÃO DOS INSTITUTOS DE PESQUISA INSTITUTO ADOLFO LUTZ CTC/IAL APROVAÇÃO DE PROJETO DE PESQUISA IAL/CCD/CEPIAL/CTC PROJETO CCD BM N° 03/2003 Coordenador: Elizabeth Natal de Gaspari Projeto : Imunização Nasal em Coelhos com Neisseria lactamica: Importância dos Antígenos de Reatividade Cruzada Inicio: Março 2003 Término: Dezembro 2004 Sra. Coordenadora: Comunicamos que o projeto supra citado foi cadastrado no Conselho Técnico Científico do Instituto Adolfo Lutz, após aprovação na CCD/BM e CEPIAL. Parecer favorável recebido pelo CTC em 22/4/2004 Myrna Sabino Presidente do CTC – 2003/2004 Via 1: Coordenador Via 2: CCD/BM Via 3: CTC Este trabalho foi realizado na Seção de Imunologia do Instituto Adolfo Lutz, sendo o projeto aprovado pelo Comitê Técnico e Científico desta Instituição. Apoio Financeiro FAPESP Processo 99/005576 DEDICATÓRIA Às minhas queridas, filhas Bruna e Aline que sempre me incentivaram. Ao Jason, pelo apoio e colaboração, sempre estando ao meu lado em todos os momentos. AGRADECIMENTOS À Prof. Dra. Elizabeth Natal De Gaspari pelo incentivo e estímulo para a realização deste trabalho. À seção de Imunologia do Instituto Adolfo Lutz, onde foram realizados os experimentos, à todos que direta ou indiretamente contribuíram para realização deste trabalho. Aos meus colegas Andréia, Simone, André e Verônica pela amizade, apoio e contribuição. "Comece fazendo o que é necessário, depois o que é possível, e de repente você estará fazendo o impossível". (São Francisco de Assis) RESUMO RESUMO Neisseria lactamica, uma bactéria comensal não patogênica, predominantemente humana e usualmente encontrada no trato respiratório superior de crianças, está intimamente relacionada a Neisseria meningitidis patogênica. A colonização com N. lactamica pode ser responsável pelo envolvimento da imunidade natural contra a infecção pelos meningococos em crianças pequenas, quando as taxas de portadores de meningococos são baixas. Estas características levam a sugerir que os componentes de N. lactamica possam ser um elemento-chave para a produção de uma nova vacina para N. meningitidis. Devido ao pouco conhecimento sobre a dinâmica dos portadores e sobre a diversidade da população de N. lactamica em crianças, tem sido difícil escolher um isolado representativo para preparar um adequado produto imunogênico. Em nosso estudo, foi proposto um protocolo para estudar a imunogenicidade de whole cells de N. lactamica, N. meningitidis, N. sicca ou N. meningitidis c (isoladas de portadores), através da imunização intranasal em coelhos, considerando a via de entrada natural do patógeno. Isolados da orofaringe de N. lactamica, N. meningitidis, N. sicca ou N. meningitidis c, foram inoculados em coelhos adultos pela via intranasal, numa concentração de densidade ótica 1.0 a 650nm, num volume de 1000 µL. Os coelhos foram imunizados por quatro vezes em intervalos de sete dias. Também foram usadas cepas como N. subflava, N. elongata, N. sicca, N. perflava, N. mucosa isoladas do líquido cérebro-espinhal ou sangue de pacientes. Os coelhos desenvolveram níveis de anticorpos IgG específicos no soro, como foi determinado por ELISA usando whole cells de cepas homólogas e heterólogas. O soro dos coelhos imunizados com N. lactamica, N. meningitidis, N.sicca ou N. meningitidis c, apresentaram anticorpos IgG que reagiram com antígenos numa faixa de 5 a 130 kDa por meio de immunoblot. Os anticorpos presentes nos soros dos coelhos imunizados com N. lactamica não induziram altos títulos de anticorpos com atividade bactericida contra as cepas de N. meningitidis, no entanto, esta atividade pode ser observada com anticorpos produzidos pelos coelhos imunizados intranasalmente com N. meningitidis. Anticorpos IgG de alta avidez foram produzidos, embora não tenha sido determinada uma significativa correlação entre atividade bactericida e a indução de anticorpos IgG de alta avidez, principalmente nos coelhos imunizados com N. lactamica. A imunização RESUMO intranasal usando whole cells de N. lactamica foi adequada para sensibilizar eficientemente o sistema imune de mucosa no modelo coelho. ABSTRACT ABSTRACT Neisseria lactamica, a commensal bacterium non-pathogenic to human beings and usually found in the upper respiratory tract of children, is closely related to pathogenic Neisseria meningitides. Colonization with N. lactamica can be responsible for evolving natural immunity to meningococcal infection in childhood, when rates of meningococcus carriers are low. These features lead to suggest that N. lactamica components can be key-elements in the production of a new vaccine for N. meningitides. As little is known about dynamic carriers and N. lactamica population diversity in children, it has been difficult choosing a representative for preparing an adequate immunogenic product. A protocol was proposed to study immunogenicity of whole cells of N. lactamica, N. meningitidis, N. sicca or N. meningitides c (carrier-isolated) by i.n. immunization in rabbits considering the natural pathogen entry route. Oropharinx-isolated N. lactamica, N. meningitidis, N. sicca, or N. meningitides c were i.n. inoculated into adult rabbits, in a concentration of optical density 1.0 at 650nm in a volume of 1000 µL. The rabbits were immunized four times at seven-day intervals. N. subflava, N. elongata, N. sicca, N. perflava, N. mucosa strains isolated from CSF and blood from patients were also used. The rabbits developed levels of specific lgG antibodies in serum, as determined by ELISA using whole cells of homologous and heterologous strains. Serum from rabbits immunized with N. lactamica, N. meningitidis, and N. sicca or N. meningitides c, presented lgG antibodies reactive to 5 to 130 kDa antigens on immunoblot. Antibodies in serum from rabbits immunized with N. lactamica failed to induce high concentration of antibodies with bactericidal activity against N. meningitidis; however, this activity could be observed with antibodies produced by rabbits i.n. immunized with N. meningitidis. High avidity lgG antibodies were produced, although a significant correlation between bactericidal
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