Otimização de um plasmídeo vacinal por introdução de elementos de direcionamento nuclear a ser veiculado por Lactococcus lactis

Abstract

Lactococcus lactis, the Lactic Acid Bacteria model, is considered to be safe and has been widely used for the production and delivery of antigens and cytokines to the mucosal level. More recently some studies have focused on their use as delivery vehicles of DNA vaccines, an innovating vaccine platform that involves administration of a plasmid vector of eukaryotic expression capable of coding imunogenic or imunomodulatory proteins with high potential as profilaxic and/or therapeutic agent. DNA vaccine delivery methodologies developed till today present relative efficacy in delivering the vaccine plasmids to host cells, although they are failures in their delivery to the cell nucleus, which is the crucial event for expression of the ORF of interest and DNA vaccine action. Considering that the nuclear envelope constitutes the major obstacle for nuclear entry of vaccine plasmids into target cells, optimizing the delivery of these to the nucleus, as well as transpositioning this barrier, is of fundamental importance for the efficiency of a DNA vaccine. With the objective of improve nuclear import levels and gene expression of a vaccine plasmid developed by our research group (pValac; Vaccination using lactic acid bacteria), to be delivered specifically by an invasive strain of L. lactis, this works goal was to optimize this plasmid by inserting a nucleotide sequence from the simian virus 40 (SV40), characterized by its capacity to deliver exogenous DNA to the nucleus of eukaryotic cells and increase gene expression of the plasmids that contain it. This sequence, called DNA nuclear Targeting Sequence (DTS) was cloned into the pValac vector and in order to evaluate the functionality of this construction, the ORF of the Green Fluorescent Protein (GFP) was used as reporter. The functionality of the final construction, pValac::DTS::gfp, was assessed by transfection of mammalian cells and analysed through fluorescent microscopy and RT-PCR. In order to compare GFP expression between the pValac::DTS::gfp plasmid and the control plasmid, pValac::gfp, flow cytometry experiments were also performed. The obtained results showed that the presence of the DTS in the pValac::gfp plasmid was not able to improve the expression of the reporter protein, disagreeing with most of the results reported in the literature.