Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador

Abstract

The use of bacteria as vehicles for the delivery of vaccine plasmids by oral route constitutes a promising strategy for vaccination against various infectious diseases. Attenuated pathogenic bacteria such as Shigella, Listeria and Salmonella have been widely used for such purposes, although presenting potential risk to revert into their wild-type phenotype. In this regard, the use of non-pathogenic bacteria, such as lactic acid bacteria (LAB), constitutes a more attractive and safe alternative. Lactococcus lactis, which belongs to the LAB group, obtained the GRAS (Generally Recognized As Safe) status due to their lack of pathogenicity and has been extensively used for the production and delivery of antigens and cytokines to the mucosal level. As such, L. lactis represents an alternative to attenuated pathogenic bacteria for the delivery of vaccine plasmids although not having invasive capacity; hence, invasive L. lactis strains (L. lactis FnBPA, Innocentin et al., 2009) as well as a plasmid that replicates in L. lactis and contains a eukaryotic expression cassette (pValac, Guimarães et al., 2009) were constructed. It is therefore believed that the use of invasive L. lactis containing the pValac vector, for eukaryotic expression of the ESAT-6 antigen (6-kDa Early Secreted Antigenic Target) of Mycobacterium tuberculosis, could represent a newstrategy for controlling Tuberculosis, an infectious disease that affects, in latent form, 1/3 of the worlds population. Thus, the goal of this project was to construct the plasmid vaccine pValac:ESAT-6, verify its functionality in vitro, its cloning capacity into the invasive strain of L. lactis and its use as delivery vehicle of an oral DNA vaccine. For this purpose, the coding sequence of ESAT-6 was isolated by PCR from the genomic DNA of M. tuberculosis strain H37Rv, cloned into the vector Zero Blunt ® TOPO ® and subsequently into pValac. The final construct pValac:ESAT-6 was first obtained in Escherichia coli TG1. In order to evaluate the functionality of the plasmid, CHO (Chinese Hamster Ovary) cell lines were transfected with the plasmid pValac:ESAT-6 and the ESAT-6 expression evaluated by RT-PCR, Western blotting and immunocytochemistry; in doing so, the functionality of the pValac:ESAT-6 plasmid was confirmed. Finally, the plasmid pValac:ESAT-6 was transformed into the invasive strain of L. lactis generating the strain L. lactis FnBPA(pValac:ESAT-6). This workconstitutes a first step towards validation of the efficiency and effectiveness of a new genetic vaccine based on genetically modified LAB and oral administration through mucous membranes, which may provide valuable information for research and for the development of vaccines against other pathogens.