Project 3: The Immune Response to Bacterial Spores
It
is clear that the marginal
zone of the spleen is particularly involved in the antibody response to
bacteria and bacterial products. We have chosen to study the immune
response
to the inert metabolically inactive spores of gram positive bacilli as
model antigens since these are trapped very efficiently in the marginal
zone and they do not express the various mitogenic components expressed
by the vegetative forms.
Approaches
Generation of Monoclonal Antibodies
Panels
of mouse monoclonal antibodies
were made against B. subtilis, B.
thuringiensis B. cereus
and B. anthracis spores by immunization with intact
native spores
(or a mixture of 40kGy irradiated DAmes and Sterne strains in the case
of B. anthracis). These antibodies were purified,
conjugated with
fluorochromes, and screened for specificity by ELISA and by flow
cytometry.
In each case multiple monoclonal antibodies were obtained which were
specific
for the immunizing spore species and did not cross-react with spores of
other closely related organisms or vegetative forms of these bacilli.
These
antibodies also discriminate between mixtures of spores (Fig. 7).
Antibodies to Spores Are Relatively Restricted in Their Repertoire
The
immunoglobulin heavy and light
chain genes from the panels of spore-specific hybridomas, when
subjected
to nucleotide sequence analysis, revealed highly restricted antibody
repertoires.
In the case of B. subtilis antibodies one VH gene,
VH7183.6, was
used predominantly in conjunction with l1 light chains. Because a
peptide
made from this VH sequence could also be shown to bind to the spores,
the
framework 3 region of this gene appears to be involved in the binding
to
spores. Of 13 B. anthracis spore-specific
antibodies analyzed, these
also showed restriction in diversity with only three VH gene families,
J558, VH7183 and Q52, used. In contrast to B. subtilis
specific
antibodies, this panel used k light chains. However half of the
antibodies
reactive with B. anthracis and thuringiensis
used the same
Vk23 light chain gene again an indication of a restricted antibody
response.
In the Future
Preliminary
immunochemical analyses
by PAGE and Western blot showed distinct but specific patterns of
proteins
extracted from the spore coats that were reactive with these monoclonal
antibodies. Future efforts will be made to extend the biochemical
analyses
of the spore coats using immunological methods.
Significance of our studies
These are the first antibodies that can discriminate between the spore of non-pathogenic and pathogenic Bacilli and will be useful in diagnostic uses and as tools in the analysis of the antibody response.
These
results suggest that the
repertoire of germline genes in mice may contain highly conserved
antibody
V gene sequences, which encode for antibodies, which do not need
extensive
somatic diversification for their ability to bind to Bacillus
spores.
Some of these antibodies are highly specific and can discriminate
between
spores of the Bacillus family, including the
strategically important
B. anthracis.