Andrew Camilli

		Dr. Andrew Camilli © Mark Morelli

Fortuitous brainstorm may lead to more effective cholera vaccine
by C.W. Wolff

The way to combat cholera, one of the world's most ancient plagues, is with a "designer vaccine" that is carefully focused on proteins expressed by the cholera bacteria at the beginning of the infection process, according to a biomedical researcher at Tufts.

Andrew Camilli, assistant professor of molecular biology and microbiology, has spent the last nine years fine-tuning a method to identify those proteins, a method he thought of in a 10-minute brainstorm when he needed a topic for his postdoctoral fellowship.

It was only later that he learned that two other investigators were pursuing the same idea. One of them was his postdoc adviser, John Mekalanos, chair of Harvard's microbiology department. The Mekalanos lab had been investigating the genetics of cholera for many years.

"He listened to me explain my postdoc project, and I saw his eyes light up," says Camilli. "When I finished, he jumped up and pulled out of his file cabinet the grant proposal he had just written with the exact same idea. It was tremendously fortuitous."

Ineffective vaccine
Vibrio cholerae, waterborne bacteria that exist quite well without humans, can cause severe and often-fatal diarrhea if lodged in sufficient numbers on the walls of the small intestine. Death by dehydration, sometimes within hours of infection, is now limited to about 1 percent of cholera victims, thanks to oral rehydration therapy. But pandemics of cholera continue. The most recentÑthe seventh pandemicÑbegan in 1961 and is still a threat in South Asia, Africa and Latin America.

There is a cholera vaccine, but it is short-lived and fairly ineffective in a major outbreak. Camilli believes that the vaccine targets the wrong proteins, primarily because of the difficulty in identifying the right ones.

But it wasn't a vaccine Camilli had in mind when he began his research. He just wanted to know what happens at the molecular level in V. cholerae, specifically, which genes of the cholera bacteria are switched on during the infection processÑand when.

Just taking the bacteria out of the small intestine at different points of infection doesn't provide those answers. "Within minutes, the bacteria change their gene expression when removed from the host. By the time you isolate the bacteria, it's too late to tell which genes were firing when," says Camilli.

Finding the best reporter
So he turned to recombinant DNA, the backbone of molecular biology for 30 years. Recombinant DNA techniques allow inserting, or splicing, a so-called reporter gene next to the gene under study. The process that switches onÑor firesÑa gene, especially those in bacteria, is usually a bit sloppy, extending beyond the gene in question. So the reporter gene is switched on as well.

The idea is that the reporter endows the bacteria with a new property when switched onÑthe ability to grow in the presence of a particular antibiotic for exampleÑthat lets the scientist know the gene being investigated is also switched on.

This technique was being used with another pathogenic bacteria when Camilli started thinking about his postdoctoral project. "I realized the existing method was limited in scope. It was incapable of allowing detection of certain classes of genes, for example genes that were only switched on transiently," he says.

That's why he came up with the idea of using a reporter gene that coded for a DNA recombinase. The recombinase is a frisky little piece of DNA that, when switched on, makes a recombinase that goes after another specific piece of DNA and cuts it out of the genome. Camilli's idea was to splice in a target for the recombinase that can be cut out of the bacteria's genome.

One more little trick to this process: Into the part of the genome that would be cut out, he inserts a gene that makes the bacteria resistant to the antibiotic tetracycline. So when the bacteria are taken out of the intestine and studied, the scientists don't have to look at their DNA. They just have to put the living bugs in a petri dish with tetracycline, and if they die, they know the gene in question was fired.

Elegant and ingenious, but to what end?

Mapping gene expression
"We've learned that there is an orchestration of virulence factors in Vibrio cholerae," says Camilli, adding that just a few years ago, many scientists believed most cholera genes involved in infection were turned on as soon as they got to the small intestine. "Some genes are turned on immediately when the bacteria arrives in the small intestine; others are turned on hours later. If we can map this orchestration, a 'designer vaccine' targeted for the early virulence factors might be developed."

The advantage of such a vaccine, Camilli hypothesizes, is that it could upset the careful orchestration of the infection process, interrupting it before the cholera bacteria reach the intestine wall where they multiply rapidly andÑagain an educated guess at this pointÑexpress the toxin that causes the life-threatening diarrhea.

Meanwhile, the method of studying gene firing that Camilli developed (he calls it an "ex post facto reporter of gene induction") is being used elsewhere to study the infection process of other virulent bacteria.

The significance of Camilli's research was recently recognized by Eli Lilly and Co. and the American Society of Microbiology, which in May will award him the Eli Lilly and Co. Research Award, considered one of the most prestigious honors a young microbiologist can receive.