Pick Your Antipoison

On a warm, sunny afternoon last June, emergency room physician Sean Bush got a call on his pager that made his blood run cold. The number was his wife’s, followed by three digits: 9-1-1. Whatever the page concerned, Bush knew that it was a serious emergency—he and his wife don’t take those numbers lightly.

A quick call from the hospital where he was on duty to his home 22 miles away brought terrifying news. Through panicked tears, Bush’s wife related that a small rattlesnake had just bitten the couple’s 2-year-old son, Jude, as he played with another child in the couple’s backyard. When the curious boy had reached down to pick up the snake, the reptile sank its fangs between Jude’s right thumb and forefinger.

“It’s not every parent’s nightmare, but it’s certainly mine,” says Bush, who specializes in treating venomous snakebites at Loma Linda University Medical Center in California.

Bush had seen some frightening snakebite scenarios: victims twitching, hemorrhaging throughout their bodies, or unconscious from venom’s effects. He knew that a 2-year-old boy could die from a rattlesnake bite that might only wound an adult.

A rescue team to air lifted Jude to meet him and a team of other doctors at the emergency room, where a complex cocktail of antibodies, known collectively as antivenom (also antivenin or antivenene), awaited the boy. The intravenous treatment neutralized the toxin in time to save Jude’s life—a testament to antivenom’s powers.

Although treatment with antivenom in the United States is usually successful, for most people around the world who are bitten by snakes, the story doesn’t have a happy ending. Because the treatment is costly, it isn’t available to many people in developing countries. Even among people who do receive antivenom, problems such as life-threatening allergic reactions, can arise. New research using several strategies is improving on methods of producing antivenom and may soon make treatment of venomous bites and stings less expensive, less risky, and more effective.

Synthetic solution

In most places around the world, antivenom is still made in much the same way as it was in the late 1800s. Experienced handlers milk venom from poisonous snakes, spiders, and scorpions. Workers then inject small amounts of that venom into large animals, such as a horses, cows, or sheep.

The tiny quantities of venom don’t harm the animals but spur them to produce floods of antibodies, the immune molecules that attach to venom’s toxins and tag them for destruction by other parts of the immune system. After a few weeks, a worker collects an injected animal’s blood and removes the blood cells to isolate the serum, which is swimming with antibodies against the venom. Those antibodies are purified from the serum and administered to patients.

A dose of antivenom is specific for the poisons produced by a single species of snake or other poisonous animal. To make an antivenom that fights venoms of several species, vaccine-farm workers inject a large animal with several venoms at once. Poison-control centers typically keep such polyvalent antivenoms on hand rather than stocking vials of antivenoms geared toward each of the poisonous species in the area. In the United States, the most common treatment uses portions of antibodies to neutralize the venom of several snakes native to this country.

In developing countries, tens of thousands of people die from snakebites each year. One reason is that the current method of antivenom production isn’t practical in many of the places where treatment is needed most, notes Simon Wagstaff, a researcher at the Liverpool School of Tropical Medicine in England.

For example, the treatment can cost thousands of dollars per dose. To make each batch of antivenom, a manufacturer must pay for housing and handling of both the poisonous creatures and the host animals that produce the antibodies.

Moreover, antivenom can have its own dangerous effects. About a quarter of patients treated develop extreme allergic reactions to antibodies and other substances present in the host animal’s serum. This allergic reaction, called anaphylaxis, is a danger for a snakebite victim being treated even in the most sophisticated hospital, and it’s all the more threatening in a village clinic in rural Africa.

Researchers have speculated that antivenom might spur fewer allergic reactions if it contained only antibodies targeted to the most damaging toxins in venom. However, Wagstaff notes that researchers would have to isolate and purify individual poisons before injecting them into antibody-producing animals. “That’s a difficult order, since venom has so many components,” he explains.

Wagstaff and his colleagues published a study in the June PLoS Medicine that provides a clue to alleviating both the cost and safety problems of snake antivenom. Rather than injecting large animals with milked venom to make serum, Wagstaff says, antivenom producers might inject the animals with snake DNA.

The researchers worked with DNA isolated from the venom glands of saw-scaled vipers, the species responsible for most of Africa’s snakebite deaths. Wagstaff and his colleagues determined the sequence of about a dozen genes that code for metalloproteases. In a snakebite victim, these enzymes break down tissues, including the proteins that line blood vessels, and cause profuse bleeding.

The researchers took snippets of seven metalloprotease genes and strung them together to create a synthetic gene that’s easy to generate in large quantities in the lab. They then inserted this manufactured gene into mice, whose cells produced pieces of snake-venom proteins. The rodents, playing the part of the typical horse, cow, or sheep in today’s antivenom operation, produced antibodies to counter these proteins.

In lab-dish tests, the researchers found that those mouse antibodies recognized and latched on to venom much as conventional antivenoms do. The mouse antibodies attached to venom not only from saw-scaled vipers but also from several other African viper species, such as horned vipers and puff adders, that share many metalloprotease genes, says Wagstaff.

He and his colleagues also found that antibodies from the DNA-treated mice could neutralize bleeding in other mice that had been injected just under the skin with saw-scaled viper venom.

Wagstaff notes that injecting antibody-production animals with snake DNA instead of venom could eliminate the expensive and dangerous task of maintaining and milking venomous snakes. The synthetic gene can be produced in a laboratory.

By selecting DNA for specific venom toxins, manufacturers could also reduce the variety of antibodies that the large animals produce. That would lessen the chance of allergic reactions, Wagstaff adds.

“We’re very encouraged by this proof-of-principle study,” he says. He and his team are currently supplementing their synthetic gene with pieces of venom-producing DNA from a variety of snakes.

Looking to nature

Though snakebites can be fatal for people, certain species of mammals, including mongooses, opossums, and ground squirrels, can resist some venoms’ effects. Scientists are studying this resistance for clues to developing new types of antivenom, says chemist Jim Biardi of the University of California, Davis.

Decades ago, scientists nailed down the basic mechanism for how these mammals avoid damage by venom. Proteins that circulate in a resistant animal’s blood neutralize venom’s toxins. These protective proteins belong to a class of molecules called protease inhibitors. People have many types of protease inhibitors in their blood, but not the ones that break down certain venoms.

Injecting people with protease inhibitors collected from venom-resistant animals would be expected to cause allergic reactions at least on par with those associated with traditional antivenom, so most scientists don’t consider that a viable strategy. Instead, some researchers have proposed modeling synthetic-antivenom drugs on the venom-fighting enzymes, says Biardi.

However, he adds, several questions first need to be answered. For example, can resistant animals neutralize a variety of venoms or just those to which they’re likely to be exposed? Drugs based on protease inhibitors from animals resistant to multiple venoms would be more useful than those that can neutralize the venom from just a single species.

Biardi and his colleagues are currently examining California ground squirrels, a species that’s resistant to rattlesnake bites. These small mammals adopt menacing postures, throw dirt, and even bite snakes that encroach on their burrows (SN: 10/9/99, p. 237). In a study published in the November 2005 Journal of Chemical Ecology, Biardi’s team described its geographic survey of venom neutralizing among hundreds of ground squirrels in California.

They surveyed squirrel populations in four locations in the state, ranging from as far north as Winters, near Sacramento, to as far south as Santa Barbara. Squirrels in three of the locations lived near Northern Pacific rattlesnakes, while those in the southernmost location lived near a second species, the Southern Pacific rattlesnake.

When Biardi and his colleagues tested the squirrels’ blood serum against venoms from both those snake species and several others, they found that all the squirrels could counteract venom from both the northern and southern California rattlers. However, the animals’ serum had little effect on the venom of related rattlesnake species, such as the western diamondback, whose range is distinct from that of California ground squirrels.

Although California ground squirrels seem to resist only the venom from local rattlesnakes, further research might turn up animals that can counteract several types of venom. “Finding a prey species that’s resistant to a bunch of venom variants could be great for developing new therapeutics,” says Biardi.

Right here, right now

Although snake DNA or antibodies from mammals naturally resistant to bites could someday yield new snakebite treatments, some scientists make their first priority improving antivenoms now in use.

Six years ago, U. S. doctors started using the first new antivenom approved by the Food and Drug Administration in more than 50 years. The drug, CroFab, neutralizes venom from a group of snake species that includes pit vipers, rattlesnakes, and coral snakes.

Old-school antivenom formulations contain Y-shaped antibody molecules known as immunoglobulin Gs (IgGs). The two short arms of an IgG molecule neutralize venom, while the long tail prompts most allergic reactions. CroFab’s manufacturer, Protherics of Brentwood, Tenn., makes its antivenom using IgG fragments representing only the arms.

Switching to the new antivenom has reduced the proportion of people who have problematic reactions to treatment for poisonous bites. Up to 75 percent of people getting whole-IgG antivenoms have at least minor allergic reactions, whereas less than 5 percent of bite victims receiving IgG fragments do, says Jude McNally of the University of Arizona in Tucson.

However, cleaving the antibodies introduces a treatment challenge. Unlike the larger IgG antibodies, which can neutralize toxins for weeks, the smaller pieces that make up CroFab are often taken up by the kidneys before they’ve done their jobs. After a large initial dose of antivenom, patients may need to receive maintenance doses for several days, McNally explains.

He and his colleagues intend to apply a strategy that researchers in Europe and some Latin American countries have used to create a new generation of antivenoms not yet in use in the United States. Sophisticated biochemical techniques retain an IgG’s hinge portion while eliminating its tail. This keeps the Y’s arms connected, thereby postponing their removal.

McNally and his colleagues, led by University of Arizona researcher Leslie Boyer, are conducting a trial of a hinged-fragment antivenom against scorpion stings in people. The drug is produced by a company called Bioclon Institute in Mexico City and has been used for 5 years in Mexico. Boyer, McNally, and their colleagues intend for the drug to be available for the 200 or so cases of serious scorpion stings that occur each year in the United States, mostly in Arizona.

Although the trial isn’t complete, Boyer says that she’s “very excited” about the preliminary findings. The drug appears to be safe, effective, and long lasting, she says.

Bush says that such improvements to antivenoms could make treating venomous bites and stings cheaper and more effective by the time his son grows up. But in the meantime, Bush says, he and his wife are trying to reduce the chance that their son will be bitten by another rattlesnake.

“I think we’re all a little more the wiser from the experience,” he says. “I don’t take my eyes off him in the backyard, even for a moment.”