|dc.description.abstract||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.||nb_NO