Utilização de uma linhagem invasiva de Lactococcus lactis como veículo para a entrega de um plasmídeo vacinal codificando o antígeno Ag85A de Mycobacterium tuberculosis em células mamíferas e avaliação do perfil de resposta imunológica gerado em modelo murino

• Ano de defesa: 2014
• Autor(a) principal: Pamela Del Carmen Mancha Agresti
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Genética; Mycobacterium tuberculosis; Plasmideos; Vacinas de DNA
• Link de acesso: http://hdl.handle.net/1843/BUOS-9N6KWZ

Using bacteria as a vehicle for orally delivering vaccines plasmids is considered a promissing vaccination strategy. Thus, attenuated pathogenic bacteria, such as Shigella, Yersinia, Listeria and Salmonella are being used for delivering plasmids of vaccine type to mammal cells. However, there is a risk of reversing to its wild phenotype. In order to counteract this problem, we propose the use of non pathogenic bacteria, as lactic bacteria (BL). Within the group of lactic bacteria, the Lactococcus lactis is deemed as a model microorganism, which is being extensively used for antigen and cytokines production and delivery to the mucosal level. Recently studies about this bacteria have focused on their usage as vehicles for the delivery of genic vaccines. For this, invasive strains of L. lactis have been developed in order to increase the delivery efficiency of these vaccines to host cells. Furthermore, our investigation team developed a plasmid that replicates in L. lactis and contains a eukaryotic expression cassette named pValac (Vaccination using lactic acid bactéria). In this way, the use of invasive strains of L. lactis for the delivery of vector pValac expressing the antigen Ag85A of Mycobacterium tuberculosis could represent a new strategy for controlling tuberculose. The aim of this investigation was not only the development of plasmid vaccine pValac:Ag85A and its functional evaluation in vitro but also its clonation in its L. lactis FnBPA+ invasive strain for using this system as a potential genic vaccine. The highly functional performance of the eukaryotic expression cassette when coding antigen Ag85A was confirmed through the detection of Ag85A protein through of immunocytochemistry; and flow cytometry after tansfecting pValac within mammalian cells. Finally, plasmid pValac:Ag85A was transformed into invasive L. lactis strain, originating L. lactis FnBPA+ (pValac:Ag85A) strain. This strain was used in C57BL/6 mice intranasal immunization. The humoral and cellular immune responses were evaluated. We observed a polarization of the immune response towards a Th1, in immunized mice with it strain. In the respiratory mucosal, we detected production of both, IgG and sIgA, antibody. In this way, this project is the first step towards the effective validation of new genetic vaccines based on lactic bacteria genetically modified administered in mucosal level.