Targeting of lactobacillus to M-cells to improve its potential as vaccine delivery vector
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- Master's theses (KBM) 
This thesis describes a part of a larger project aimed at developing oral mucosal vaccines based on lactic acid bacteria (LAB). LAB are considered attractive candidates as vaccine delivery vectors because of their Generally Regarded As Safe (GRAS) status. Furthermore, many LAB are natural inhabitants of the gastrointestinal tract, where some are further thought to have probiotic effects on the host. Lactobacillus plantarum is of particular interest because of its ability to persist in the gastrointestinal tract of humans for up to seven days, and also because of its resistance to bile and low pH. Different delivery routes and targets may be considered to obtain efficient LAB-based mucosal vaccines, and this thesis describes the development of one such route, where L. plantarum is targeted to M-cells in the gastrointestinal tract through expression and surface-display of M-cell binding proteins. The idea behind this strategy is that M-cells may transcytose the bound bacteria to underlying Peyer’s patches, where they are exposed to high concentrations of immune cells. The proteins FimH from Salmonella typhimurium and enteropathogenic Escherichia coli and Invasin from Yersinia enterocolitica were selected for their ability to bind M-cell receptors. Plasmids were constructed for the expression, secretion and subsequent anchoring of FimH and Invasin in L. plantarum, using both cell wall- and lipoprotein anchors. Using western blotting and flow cytometry, it was shown that the proteins were produced and displayed on the surface of L. plantarum. Caco-2 cells were used as an in vitro model to investigate the ability of the proteins to promote internalization, and the recombinant L. plantarum strains all showed increased internalization compared to the strain harbouring an empty vector (pEV). However, the experiment did not distinguish between bound and internalized bacteria. Additional in vitro studies, using M-cell-like cells, showed signs of FimHand Invasin-promoted transcytosis. In one of two individual experiments, promising results were obtained for several strains, in particular for the one expressing Invasin with an N-terminal lipoprotein anchor. The work described in this thesis gives promising indications that FimH and Invasin are involved in promoting transcytosis of L. plantarum via M-cells. It thus seems reasonable to further pursue this strategy, starting with further analyses to evaluate the efficiency of the transcytosis process and to verify the possibility to deliver vaccine antigens to Peyer’s patches.