Evaluation of Bacillus subtilis spores as a probiotic and a delivery system for heterologous antigens
Bacillus spores are widely used as commercial oral probiotic preparations, both
for human and farm animals use. Nevertheless, the mechanisms underlying the
probiotic effects are not yet known, although they are likely to be the result of
a complex network of interconnected microbiological and immunological
The broad aim of the research work carried out for this thesis was to gain a
better understanding of the immunological and probiotic properties of spores,
exploring the application of these findings in the design of spore-based
mucosal vaccine vehicles and assessing their potential for novel probiotic
applications. Characterization studies on the immunomodulatory activity of B.
subtilis spores at the mucosal and systemic level in a mouse and a rabbit model
as well as studies on the immune recognition and surface display of spore coat
proteins are described.
Heterologous antigen delivery was achieved both at the level of the spore
surface and spore interior. The OG, 6H loop of the VP1 protein of Foot-and-
Mouth Disease virus (FMDV) and the recombinant adjuvant molecule CTA1-
OVA-DD were chosen as model heterologous antigens. Immunization studies
showed a specific anti-FMDV mucosal immune response upon intranasal
The use of B. subtilis as a competitive exclusion agent in an E. coli 078: K80
chick model of infection was explored. Results showed that a single oral dose
of B. subtilis spores could suppress all aspects of E. coli 078: K80 infection,
along with a substantial reduction of dissemination and colonization.
Finally, in order to determine the life stage of the Bacillus life cycle
responsible for its probiotic action, an RT-PCR study of spore germination was
carried out in a mouse model to identify loci and timing of this response at the
intestinal level. Results showed that Bacillus subtilis spores are able to
germinate in the jejunum and the ileum.
These findings provide relevant insights for future studies on the probiotic
activity of Bacillus subtilis as well as for the development of improved
Bacillus mucosal delivery systems for heterologous antigens