Bob Johnston’s work with mutant versions of a virus started off as basic research but lead to a vaccine that can save Latin American children from encephalitis. And now, products from the vaccine development are helping him answer more of the basic questions he posed twenty years ago.

Bob Johnston didn’t set out to make a vaccine. Twenty years ago, he began studying the life cycles of viruses - basic research.

Johnston, professor of microbiology and immunology, wanted to learn how viruses cause disease. He pictured fatal infections as a pathway, a sequence of unknown steps - each an opportunity to block the virus. One way to identify those steps was to study mutant viruses that had stopped prematurely. By determining how their structures and RNA sequences had changed, Johnston thought he could specify what the virus needed to continue.

He and his co-workers had begun by working with a mutant version of Sindbis, a “model” virus that infects mice yet causes little disease. Several years later, they could describe critical steps in the virus’ life cycle. That’s when a colleague half-jokingly challenged Johnston to work with a “real” virus - one that causes illness in humans.

“I asked him, ‘Just what real virus do you have in mind?’” Johnston says. “He suggested the one he was working on, Venezuelan equine encephalitis (VEE).”

VEE can reproduce inside mosquitoes, which periodically bring it out of its secluded home in Central and South American forests to infect livestock and people. One hundred thousand people were infected, and hundreds, mainly children, died during an outbreak in Venezuela and Columbia that ended about a year ago. Thousands of horses also died. In 1971, a similar outbreak reached southern Texas.

Early symptoms of VEE infection are flu-like: headache, chills, fever, muscle pain, and nausea. Severe infections can lead to encephalitis, an infection of the brain, causing convulsions, paralysis, and death. Although some South American livestock are immunized against VEE, the current vaccine is risky for humans, sometimes causing flu-like symptoms.

Johnston decided to switch to VEE, once more studying crippled mutants. The approach worked again, and this time success came with a bonus - a new vaccine.

“When we did the work with a ‘real’ virus, we anticipated that the experiments would hand us a vaccine,” says Nancy Davis, a research associate professor in Johnston’s lab. The mutants had all the right properties for a vaccine: They would grow inside a mouse and induce an immune response, but they would not cause disease. And the animal was protected from the naturally occurring virus, even a year after inoculation.

The final VEE vaccine has been tested successfully in monkeys. If all goes well and if the FDA approves the vaccine, it should be ready for human testing by the end of this year.

Meanwhile, Johnston is making good use of other VEE mutants. He turned some into “expression vectors” - shuttles that carry foreign genes. Genes from virtually any organism can be inserted to produce or “express” a protein.

Johnston realized the expression vectors made from VEE could be vaccines, too. If the inserted gene belongs to another infectious organism, then the vector induces immunity to it and to VEE. Johnston, Davis, and other lab members developed a version of this “double-promoter” vaccine to protect mice against VEE and influenza.

And they, and their collaborator, Dr. Jonathan F. Smith, took the idea one step further, creating a second type of vector that immunizes against the inserted gene but not VEE. This vector, called a “replicon,” does not carry the gene for the coat protein that encapsulates VEE, making VEE virtually invisible to the immune system and allowing the vector to be re-used for multiple vaccines or booster shots. The lack of a coat protein gene also prevents the virus from reproducing on its own, making it a very safe vaccine.

“The replicon is a ‘suicide’ particle,” Johnston says. “After the initial infection, the virus hits a dead end because it can’t reproduce without a helper. The virus can’t spread.”

A collaborative effort among Johnston, Davis, and the labs of Drs. Jeff Frelinger and Ron Swanstrom combined the replicon with the double-promoter vector to make a combination vaccine for SIV, a monkey virus related to HIV. If these trials - conducted by collaborator Phil Johnson - are successful, the next step will be to test an HIV vaccine. Johnston says he can’t predict how long it might take to reach that stage.

In the meantime, he is putting together a company to explore other uses for the expression vectors, such as vaccines for influenza or the virus that causes croup. Later, the vectors might be adapted to boost a person’s immunity to cancer-causing viruses or to target specific cells with gene therapy.

Turning these projects over to the company should let his lab focus exclusively on basic research again. “There are a lot of fundamental issues left,” Johnston says. “How does the virus spread in the body? How does it invade the brain? How does it grow in neurons?”

Johnston’s expression vectors might help him find the answers. The lab now has a version that produces a green fluorescent protein, normally found in jellyfish. This protein highlights the virus, literally letting the researchers trace its path through a mouse’s body.

Products like this are bringing Johnston back to basics. Or maybe he never really left.