|dc.description.abstract||During the course of this thesis factors mediating successful oral vaccination with lipidencapsulated Mycobacterium bovis BCG were examined. Mice were fed 2x10E07 CFU BCG encapsulated into a lipid matrix to prevent destruction by the gastrointestinal tract and to allow passage through the gut epithelia. In order to trace the vaccine following oral
vaccination, mice were sacrificed at various time points ranging from 6 hours to 8 weeks post vaccination, and macerated lymphatic and non-lymphatic organs plated on solid agar. Initially, BCG was distributed widely in lymphatic and non-lymphatic organs, however, BCG was cleared quickly from most organs and formed small populations of less than 500 CFU/mouse in the mesenteric and cervical lymph nodes, as well as the Peyer’s patches 8 weeks post vaccination. Immuno-histochemistry and confocal microscopy, showed that BCG was absent from the follicles, but instead resided in the T cell containing intra-follicular areas. Very rarely BCG was associated with small CD11b+ cells that did not resemble typical macrophages and lacked peroxidase activity. Instead the majority of BCG could be found forming extracellular groups of 1-4 rods. This was confirmed using cell sorting of leukocytes isolated from alimentary tract lymphatics of orally vaccinated mice and only showed a minority of BCG to be associated with CD11c+ cells. Therefore, BCG is absent from typical antigen presenting cells, but instead might reside in CD11c+CD11b+ myeloid DC.
Additionally, Ziehl-Neelsen staining revealed groups of intracellular coccoid forms of BCG. These were located toward the subcapsular space of draining lymph nodes where they were associated with sub-capsular macrophages. Interestingly, cocci proved to be non-platable using solid agar but instead required resuscitation in liquid media and therefore might resemble a form of dormancy. The presence of extracellular rods and intracellular cocci in on-professional antigen presenting cells might highlight the importance of secreted factors as an antigen source promoting successful activation of the immune system.
Following oral vaccination, IFN-γ producing cells almost exclusively resided in the spleen. In order to characterize these cells, splenocytes of orally vaccinated mice were isolated 6 weeks post vaccination on the basis of surface marker expression using fluorescence activated cell sorting (FACS).
Antigen-specific release of IFN-γ was monitored using ELISA and ELISpot assays and IFN-γ producing T cells were characterized as T effector memory cells expressing CD44,
but not CD62L and lacked the expression of mucosal homing markers such as CD103 or α4β7. In addition, Lincoplex assays revealed the production of IL-17 by splenocytes.
These did not express CD4+ but rather the γδ T cell eceptor.
Together these results show that antigen reservoirs of BCG present in the draining lymphatics contain small numbers
of typical filamentous BCG. A larger population of coccoid forms leaves open the possibility that coccoid forms are a major source of antigen for the stimulation of T cells.
Although typical CD11c+ APC isolated from the lymphatic tissue of immunized mice did not appear capable of stimulating, T cells responses were nevertheless effectively induced by oral vaccination and shown to be IFN-γ producing TEM residing in the spleen but lacking expression of mucosal homing markers.||