Making a malaria vaccine à la Louis Pasteur

Making a malaria vaccine à la Louis Pasteur

Dissecting the salivary glands of a malaria-infected mosquito at the Sanaria lab, whose plan has been called "the best show in town."

The sign on the wall reads "Emergency Response Procedures for a Mosquito Release."

Among them are "Do Not Leave the Room or Open Any Doors!!!" and "Do Not Panic!"

Everything in the room is white, including the lab coats and surgical masks - for sterility, yes, but also the better to see a mosquito. Hanging next to the sign, in vivid orange, is the last line of defense, a brace of fly swatters.

This room, the mosquito dissection lab, in an unassuming biotech park in the Washington suburbs, is at the heart of one of the most controversial ideas in vaccine science.

Sanaria Inc. (meaning "healthy air," a play on the Italian "mal'aria" or "bad air") is making a vaccine the old-fashioned way, more or less as Louis Pasteur did.

Avoiding modern recombinant DNA technology that injects tiny fragments of parasite protein to prime an immune response, Sanaria uses the whole parasite, extracted by hand from the mosquito's salivary glands, and weakened so it cannot multiply.

Pasteur weakened rabies and anthrax bacilli by air-drying them. Sanaria uses gamma rays.

Because the world now fights malaria - ineptly - with nets, insecticides and drugs, a vaccine is desperately needed.

As many as 70 experimental programs exist around the world; eight rival projects are being supported by the Malaria Vaccine Initiative, which was established in 1999 with money from the Gates Foundation.

Sanaria's is the sole vaccine using the whole parasite. Injected into a capillary, it rides the bloodstream to the liver and starts making proteins. But after about three days, it stops, and it never floods the blood with copies of itself.

Radiation-weakened parasites have protected many lab mice and a handful of humans, but making a vaccine that can be mass-produced is a huge challenge.

The lucrative market is made up of tourists and the world's military forces. The larger goal is to protect the 3,000 children who die of malaria each day.

"We've got a lot of work to do, and we're extremely mindful of the fact that the road to success is filled with potholes," said the founder of Sanaria, Dr. Stephen Hoffman, a former chief of malaria research for the U.S. Navy.

Before he died two years ago, Dr. Maurice Hilleman, who invented 40 human and animal vaccines, called the Sanaria plan "the best show in town."

Dr. Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, which gave Sanaria some of its early research money, said he was enthusiastic. "I have as much confidence in this as I do in any candidate," he said, acknowledging that major problems remained to be solved.

Peering through microscopes and wielding tweezers and a fine needle in surgeon-steady hands, six workers snick the heads off anopheles mosquitoes, tease out the transparent salivary glands and pop them into little vials. After the glands and saliva proteins are dissolved, the parasites will be frozen at extremely low temperatures. When enough batches are ready, clinical trials overseen by the navy will begin.

One recent morning, a reporter and photographers were allowed to view the last "engineering run" in the dissection room.

Nearby, in a room protected by five doors air-pressurized to prevent escapes, mosquitoes are being raised from sterilized eggs and fed infected blood.

On harvesting day, they are wheeled to the gamma-ray kiln in red picnic coolers, each batch in a small plastic pipe.

Just before dissection, they are popped into a freezer for a few minutes to stop their flying. The little pile of chilled mosquitoes is shaken into a dish of ethanol and divided into six portions.

The ethanol kills them without killing the parasites, explained B. Kim Lee Sim, the head of manufacturing. The dissectors work rapidly and with little talk, dismembering 80 female mosquitoes an hour.

"We need to get some music in here," Sim said. The males, which do not drink blood, "are just leftovers on the plate, waiting to get incinerated in the biohazard waste," said Richard Stafford, chief of quality control. Anyone with good eyesight and steady hands can be trained. "It's not as difficult as it would seem to a person who doesn't do it," said Sumana Chakravarty, head of the team.

The idea that parasites weakened by radiation could be a vaccine has been around since 1967, when Dr. Ruth Nussenzweig of the New York University Medical School showed that it worked in mice.

In the 1970s, she and others showed that it could work in humans.

Fourteen volunteers were vaccinated with weakened parasites and later exposed to malarial mosquitoes. Thirteen were protected, one so well that he then worked for three years in rural Ghana without taking any anti-malaria drugs.

But the research never proceeded, for the same reason that the number of tests was so small: It was impractical.

The parasites could not survive outside mosquitoes. So the "vaccination" had to be delivered by mosquito bite, not needle. And because just one parasite at a time fits down a proboscis, each bite delivers just a few.

To be sure of receiving enough material, volunteers endured 1,000 bites from irradiated mosquitoes, which took weeks. In the 1980s, said Dr. John McNeil, the scientific director of the vaccine initiative, the decoding of the mosquito genome opened new routes to make vaccines from bits of parasite surface protein that could be brewed quickly.

Early ventures did not work out. In 1987, Hoffman was one of six volunteers to be injected with an experimental vaccine, FSV-1, and then "challenged" by infected mosquitoes. He was so confident that he flew to San Diego for a medical conference.

"In the middle of giving a presentation," he recalled. "I had a rigor, uncontrolled shaking and chills. It took 36 hours to find the parasites, but I had malaria."

That vaccine, however, helped lead to the candidate now farthest along in African trials, RTS,S/AS02A, tentatively named Mosquirix, which has partly protected young children.

But nothing other than the whole parasite yet offers anything like 90 percent protection.

So in 2002 Hoffman founded Sanaria and obtained $15 million in backing from the infectious disease institute, the U.S. Army and the Institute for One World Health. But $29 million from the Gates Foundation last year finally allowed the building of a real assembly line.

There were many hurdles. Sanaria had to work out how to raise sterile mosquitoes, to supercharge them with far more parasites than nature does, to dissolve saliva proteins to avoid allergic reactions and to freeze and thaw parasites without exploding them.

"This is the evolution of the efforts of five years of very dedicated scientists working together," Sim said. "It's a robust, repeatable, reliable process."

Some scientists say the challenges remain insurmountable. At a recent meeting of the American Society for Tropical Medicine and Hygiene, Dr. Zarifah Reed, a parasite specialist in the World Health Organization's vaccine research initiative, said she was troubled that the mosquitoes had to be raised on human blood, which cannot be sterilized.

Even though mosquitoes do not inject one person's blood into another, there is the theoretical risk of passing on a virus, a prion or some other pathogen, she said.

Dr. Pierre Druilhe, a malaria expert at the Pasteur Institute in France and the inventor of a rival vaccine that he said was rejected for a Gates grant, was even more scathing.

"Even calling it a vaccine is a compliment," he said. "It has no chance of offering protection. It is like Captain Ahab in the movie trying to kill Moby Dick with his knife."

Besides the sterilization problem, he said, there was the risk that under-radiated parasites would cause malaria or that weakened parasites would not reach the liver. Cryogenically frozen vaccines, he added, will never be practical at sites with poor refrigeration.Sanaria's defenders said these were legitimate questions, but ones that had answers. Fauci, of the infectious disease institute, said the company had consulted the Food and Drug Administration about every aspect of sterility, "so quite frankly, I'm not impressed by that objection." Dr. Myron Levine, director of the center for vaccine development at the University of Maryland School of Medicine, said that any successful vaccine "would be used in places where kids' chances of dying of malaria are much greater than any theoretical risk of dying from a rare blood problem." How big a dose is needed and where it is injected are questions for the trials, the doctors said. And although cryogenic storage may be impractical in Africa now, such problems can be solved with money, Levine said, adding, "Those are the easiest ones."

Druilhe, in his critique, has said Hoffman was raising millions of dollars with unreal promises to sell Sanaria to private investors and cash out.

Hoffman responded: "Pierre and I are friends and colleagues, but we don't think alike about how to get to the end of the game.

"But let me ask a rhetorical question. If, as he says, this is never going to be a vaccine, who am I going to sell it to?"

Until then, he said, "I do what I do, and if it works, it works."