Researchers exploring 'fusion strategy' against E. coli

The SDSU researchers altered the toxins produced by a form of E. coli and genetically fused the non-poisonous "toxoid" to a protein known to cause an immune reaction. The resulting "fusion protein" could be used to develop a vaccine.

Assistant professor Weiping Zhang in SDSU's Veterinary and Biomedical Sciences Department studies a group of E. coli called enterotoxigenic Escherichia coli, or ETEC. Besides causing diarrheal illness in farm animals such as pigs, ETEC strains are the main source of bacterial-caused diarrhea in human populations in the developing world, and the chief cause of traveler's diarrhea. The World Health Organization estimates that ETEC causes approximately 210 million cases of illness in humans and 380,000 deaths, mostly in children in developing countries.

Enterotoxigenic E. coli produce enterotoxins that affect the tissues lining the intestine and cause the vomiting and diarrhea associated with ETEC.

The research is one of the ongoing projects in SDSU's Center for Infectious Disease Research and Vaccinology, which looks for new ways to diagnose and treat infectious disease in humans and domestic animals.

The ETEC project is innovative in that it uses as vaccine components, the toxins that scientists call "heat-stable enterotoxins," or STs, that are generally harmful to animals and humans and remain active even in a temperature of boiling water.

Zhang said heat-stable enterotoxins can't be used directly as a vaccine component because of their toxicity and because they are poor at causing an immune response unless coupled to a carrier protein. For that reason, many vaccine researchers working with ETEC focus their research on other disease-causing elements -- the so-called heat-labile enterotoxins that are destroyed at high temperatures and the fimbriae, or appendages that help the bacteria hold on to the host and cause disease.

However, Zhang said not including STs as a vaccine component poses a problem because more than two-thirds of human ETEC diarrhea cases and more than one-fourth of ETEC diarrhea cases in pigs are caused by ETEC strains that produce a heat-stabile enterotoxin called STa.

"STa antigens must be included for developing broadly effective vaccines against ETEC infection," Zhang said.

The SDSU research explored an approach for using heat-stable enterotoxins.

"Since they are toxic, we cannot use them directly. So we mutated a gene. We only changed one amino acid for each toxin. And that change shifted them from toxic to non-toxic," Zhang said.

In the same way researchers mutated the gene that produces the heat-labile enterotoxin, which is known to produce an immune response. They then fused the two toxoids to produce a fusion protein.

Importantly, by tweaking only a few amino acids, the researchers left the protein structure of the bacterium largely intact. That is important, Zhang said, because just as the toxin has to bind to a receptor in the small intestine in order to cause the disease, the vaccine component must bind to that same receptor in order to cause an immune response.

Zhang and his colleagues published the study of their "fusion strategy" in January 2010 in the journal Infection and Immunity. Zhang's co-authors were Chengxian Zhang, David H. Francis, Ying Fang, and David Knudsen, all of SDSU; James Nataro of the University of Maryland School of Medicine; and Donald C. Robertson of Kansas State University.

In summer 2010 researchers began studying five possible vaccine components using a pig model. Once they select the best vaccine component, they'll move on to larger lab trials and field trials. The possibility of an improved swine vaccine is important because some estimates say swine producers lose $80 million a year because of illness in pigs in North America alone, Zhang said.

Meanwhile, since the toxins produced by ETEC in pigs and humans are nearly identical, Zhang and his colleagues are using the same system they've developed at SDSU for exploring a swine vaccine to explore a possible human vaccine.

Zhang has received $368,000 in grant funds for vaccine research against enterotoxigenic Escherichia coli-associated diarrhea in humans using a pig model that he and SDSU professor David Francis have developed. The research is sponsored by the U.S. Department of Health and Human Services' National Institutes of Health through its National Institute of Allergy and Infectious Diseases.

Zhang and his colleagues at three other institutions also have received about $1 million in support from the Bill & Melinda Gates Foundation for research into strategies for optimizing the vaccine for humans. Right now they have mutated only a single amino acid. The Gates Foundation wants to know if the vaccine components would be even more effective if researchers mutate other amino acids.

If the research leads to an improved vaccine either for pigs or humans, that entire process of developing the vaccine will take about 10 to 15 years, Zhang said.