Molecular Pathogenicity Studies of Enteric Bacteria 
EPEC produce bundle-forming pili and coalesce 
as infectious units. In contrast, when in inani- 
mate environmental reservoirs such as water or 
sewage, their production is repressed and they 
exist as single, well-separated organisms. This 
phenomenon, together with the findings de- 
scribed above, shows that EPEC exemplify two 
features of bacterial pathogenesis: first, that 
pathogenicity is often the consequence of several 
distinct virulence determinants of the organism 
acting together and, second, that the expression 
of these determinants may be regulated by local 
features of the organism's environment. 
We are now attempting to use these findings to 
design an EPEC vaccine for the prevention of diar- 
rheal illnesses in children. We hope to delete 
from the EPEC chromosome the gene that causes 
functional abnormalities of the host cell while 
retaining the genes that direct the cell attachment 
capacity and the production of bundle-forming 
pili. A genetically modified strain of this kind 
should be attenuated with respect to its viru- 
lence, yet fully capable of colonizing the small 
intestine, where it would be expected to stimu- 
late a protective immune response. 
Using Attenuated Strains of the Typhoid 
Fever Bacillus as Vaccines 
Fully virulent strains of Salmonella typhi 
cause typhoid fever, whereas an attenuated strain 
of the same species, administered as a living oral 
vaccine, is now FDA approved and widely used 
for the prevention of typhoid fever. Attenuated 
strains of this kind have been proposed as vehi- 
cles that might be able to carry other vaccine sub- 
stances — termed "passenger antigens" — de- 
rived from a variety of microorganisms. Such a 
vaccine would stimulate immunity not only to S. 
typhi, but to other infectious agents as well. We 
have been studying this system for the delivery of 
T cell epitopes. 
For the purposes of this study, T cell epitopes 
are considered to be localized regions of a pro- 
tein that stimulate a cellular immune response 
and are required for the elimination of intracellu- 
lar pathogens, including some bacteria and many 
viruses and protozoan parasites. We have found 
that T cell epitopes can be effectively processed 
and presented to the immune system when ex- 
pressed in the center of the flagellin protein of S. 
typhi. Flagellin is the protein building block of a 
whip-like extracellular filament that acts like a 
motor to propel the bacterium through liquids. 
Because these vaccine strains would express a fla- 
gellin protein that would also contain a foreign T 
cell epitope, they are referred to as "chimeric" 
flagellins. 
We have taken advantage of recent information 
about how epitopes of this kind are processed by 
cellular components of the immune system. It is 
now clear that one common pathway would en- 
tail the presence of the chimeric flagellin in the 
endosome of a macrophage. In that specialized 
compartment of the cell, it would be digested by 
endosomal proteolytic enzymes, resulting in the 
release of the epitope as a small peptide prior to 
its presentation to other components of the im- 
mune system. A T cell epitope was placed in the 
flagellin protein flanked at either end by amino 
acids known to be sites of cleavage by endosomal 
proteolytic enzymes. This has yielded a five- to 
eight-fold increase in the magnitude of the result- 
ing immune response. 
Further modifications have entailed placing 
the expression of these chimeric flagellins under 
the control of the heat-shock protein promoter, 
which is known to be activated in Salmonella 
growing in macrophages. Other modifications in- 
clude the use of multiple tandem copies of these 
epitopes and the use of flagellins that are secreted 
as soluble proteins into the macrophage endo- 
some. Taken together, these studies have demon- 
strated that it is possible to use attenuated strains 
of S. typhi to deliver T cell epitopes and that the 
effectiveness of this delivery system can be en- 
hanced by the design features described above. 
We are now working in collaboration with sci- 
entists at the National Institutes of Health to place 
T cell epitopes of a human immunodeficiency 
virus (HIV) protein in Salmonella flagellins, 
with a view to preparing useful vaccines for the 
prevention of AIDS. 
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