Researchers at the Biodesign Institute at Arizona State University have made
a major step forward in their work to develop a biologically engineered
organism that can effectively deliver an antigen in the body.
The researchers report that they have been able to use live salmonella
bacterium as the containment/delivery method for an antigen.

The work is a major step forward in development of a new means of biological
containment that would be a key component to a new way to deliver vaccines
in animals and humans. If fully developed, the new method could be used to
administer vaccines to many of those who do not benefit from traditional
vaccines because of their cost, because of drug resistance or because of
limited effects on children.

Outlined in the paper, "Regulated programmed lysis of recombinant Salmonella
in host tissues to release protective antigens and confer biological
containment," published on the online version (July 7) of the Proceedings of
the National Academy of Sciences, the researchers describe a new, novel and
effective means of biological containment for antigen delivery. The method
not only effectively delivers the antigen in the body, but does so in a way
that does not infect the body with salmonella and does not leave any vaccine
cells in the environment.

The research team includes scientists formally at Washington University, St.
Louis, and Megan Health Inc., St. Louis, who are now at ASU's Biodesign
Institute and the School of Life Sciences.

"Our goal is to design, engineer and evaluate a live bacterial (using
salmonella) antigen delivery system that would display regulated delayed
lysis in vivo after invasion into and colonizing internal lymphoid tissues
in an immunized individual," said Roy Curtiss, director of the Center for
Infectious Diseases and Vaccinology at the Biodesign Institute and a
professor in ASU's School of Life Sciences. Curtiss was part of the research
team that made the discovery.

"We wanted to do this in a way so that no disease symptoms due to salmonella
would arise, a protective immune response would be induced to the pathogen
whose protective antigen was delivered by the vaccine construction (in this
case against S. pneumoniae due to an immune response to PspA), and there
would be no ability for live bacterial vaccine cells to either persist in
vivo or to survive if shed into the environment," Curtiss added.

"The biological containment system we developed is sufficient by itself on
conferring attenuation, the inability to cause disease symptoms, and ability
to deliver an antigen to induce protective immunity," Curtiss said. "We have
high expectations that this delivery system will be safe and effective when
administered to animals and humans."

A key to the project, according to Curtiss, is "turning a foe into a
friend." That foe is the salmonella bacterium?the leading cause of human
food-borne illness and which is currently in the news due to contaminated
tomatoes and other food crops. Curtiss' team, through genetic know-how, has
developed a variety of ways to tame salmonella in the lab and use it as a
delivery vector for vaccines.

"We try to genetically modify the salmonella bacterium to eliminate its
harmful effects -- the diarrhea, gut inflammation and fluid secretion --
while keeping the wherewithal to induce immunity against the bacteria
causing pneumonia or other infectious diseases," Curtiss said. Several in
his research team attack the problem from different angles, with some
focusing on weakening salmonella, others boosting the immune response and
others optimizing the self-destruct mechanism.

Speaking about the application of a pneumonia antigen, team leader Wei Kong,
of the Biodesign Institute, said: "If we tried to use live Streptococcus
pneumoniae causing pneumonia for a vaccine, we would obviously kill the
patient. The benefit of a live vaccine that uses a weakened form of
salmonella, is that the salmonella can be taken up through the intestinal
lining and stimulate an immune response by using just a portion of the
bacteria causing pneumonia that itself is not deadly."

In experiments, the genetically modified Salmonella enterica bacterium
colonizes the lymph tissues of the host and manufactures a protein from the
S. pneumoniae bacterium, which then triggers a strong antibody response.
Unlike most vaccines that are entirely manufactured by a vaccine company,
the attenuated recombinant salmonella vaccine after entry into the immunized
individual serves as its own factory to produce (manufacture) the protective
antigens (proteins) from the S. pneumoniae pathogen. This ability to cause
manufacture in the immunized individual dramatically decreases the cost of
such vaccines to make them affordable for use in the developing world,
Curtiss said.

An important factor for the research team was to genetically program the S.
enterica bacterium to destroy itself so that it is not released into the
environment, Curtiss said.

"Biological containment systems are important to address the potential risk
posed by any unintentional release of the modified salmonella into the
environment," he explained. The salmonella life cycle is balanced to allow
enough time to enter the body and build an immune response, while leading to
cell death by bursting the cells and preventing the vaccine strain from
spreading into the environment.

"The data show that the system we have devised results in cell lysis in the
absence of arabinose and clearance of the strain from host tissues," the
researchers state in the PNAS article.

"More importantly, our strain was fully capable of delivering a test antigen
and inducing a robust immune response comparable to that of a vaccine strain
without this containment system, thereby demonstrating that this system has
all of the features required for biological containment of a recombinant
attenuated salmonella vaccine," they added.

The research was funded by the U.S. Department of Agriculture and the
National Institutes of Health.
www.asu.edu