Demonstration in a real world setting
Following oral delivery of the vaccine to the body, the bacteria will travel down through the GI tract. It is here that they will cross the epithelial layer and encounter M cells in the Peyer’s patches in the MALT (mucosa associated lymphoid tissue). At the peyer’s patches, the bacteria can be phagocytosed by nearby antigen presenting cells (APCs). These APCs will sample the antigen and may present this antigen to B & T cells and therefore cause an immune response.
To mimic these real world conditions, we aimed to use a mouse macrophage cell line (RAW 264.7) to demonstrate that our bacteria can present protein to the cytosol of the macrophage following phagocytosis (where, if it is an antigenic protein, it will be processed through the proteasomal degradation pathway and be presented on the cell surface bound to MHC class II molecules).
In order to visualise the presentation of protein to the cytosol of a macrophage, a reporter protein (iRFP) was utilised. In order for this protein to fluoresce, biliverdin, a cofactor found in the cytosol of mammalian cells, must be present. Thus, if fluorescence was recorded, it could be concluded that the iRFP had been secreted by the L.lactis into the cytosol of the macrophage.
The experiments were designed as in the schematic above. Raw 264.7 macrophages were incubated at 37 Degrees Celsius for four hours in 5% CO2 (1 X 10^5 macrophages per well). L. lactis were then added to these in different wells at three separate multiplicities of infection (MOI): 100:1, 200:1 and 300:1 ratios of L.lactis to macrophages. One set of wells was kept as a negative control and had no L.lactis added to it. These were then incubated for six hours in the conditions described previously. Following this, the DMEM was replaced with DMEM containing penicillin and streptomycin to kill the L.lactis within the cells and in the extracellular medium. The cells were then incubated again for between one and two hours. After this final incubation step, the cells were washed in phosphate buffered saline.
Cells were then analysed using the Odyssey Infrared Imaging System. Plates were photographed (Fig. 1) and fluorescent intensity plotted against MOI (Fig . 2).
Fig. 1: Plates containing Macrophages infected with L.lactis at varying MOI. Left to right: MOI=100, MOI=300, MOI=1,000.
Fig. 2: Bar graph of fluorescent intensity against MOI.
Our results indicate that at all multiplicities of infection, iRFP was present in the cytosol of the macrophages. The negative control showed no fluorescence, verifying that the fluorescence did not come from another source within the macrophages. Thus, when our L.lactis is present in the ECM of macrophages, it can undergo phagocytosis, escape from the phagosome and successfully secrete our intended proteins into the cytosol of the macrophage. In the clinical setting, this could be used to get potential antigens selectively to antigen presenting cells with the aim of immunizing people against diseases. This is how our leishmaniasis antigen could be used in immunizing patients.