Edward Jenner was not the first to notice the beauty of milkmaids. But he thought he knew what kept their faces smooth and clear-skinned in an age when much of the population bore the scars and pocks that marked a survivor of the dreaded and deadly smallpox.

As an English country doctor, Jenner had heard the local superstition that milkmaids who caught cowpox, a mild disease transmitted from cattle to humans, never got cowpox again—and never got smallpox either. He thought there might be something to it. He knew that people lucky enough to suffer a mild case of smallpox never came down with the more ferocious form of the disease.

Jenner decided to test the idea. In 1796, he found a milkmaid named Sarah with an active case of cowpox (which she caught from a cow named Blossom). He took fluid from a cowpox blister on her hand and injected it into an eight-year-old boy named James. As expected, James developed cowpox. Later Jenner inoculated the boy with smallpox itself. The boy escaped a single symptom of the dread disease.

Jenner called his discovery a “vaccine” after the Latin word vaccinus, meaning “of or from cows.” Within a short time, vaccination against smallpox became commonplace and fear of the disease began to fade.

Today, smallpox is considered eradicated from the planet. After Louis Pasteur put forth the germ theory of disease in 1865, vaccines were developed against a number of other diseases, culminating in the spectacularly successful Salk and Sabin vaccines against polio in the 1950s.

Vaccines work (we know today) by stimulating the body's natural defenses. When the immune system senses a foreign substance (an antigen) in the body, it creates a chemical known as an antibody that is tailor-made to latch onto and neutralize the intruder. The next time that substance shows up, the antibodies are ready to pounce.

So why is it that the world is now entering its third decade of the AIDS epidemic with no sure-fire vaccine still in sight? Each year, the U.S. government spends $200 million in pursuit of this biomedical Holy Grail.

“No one's been able to come up with an effective vaccine to AIDS because of two things,” said Ken Roux, a biochemist who studies the curious physical shape of the AIDS virus.

First, the disease is caused by a virus (human immunodeficiency virus, or HIV) that mutates rapidly after it enters the body, Roux said. “This virus is constantly mutating and changing its surface coat to try to outsmart the immune system. So the immune system is always playing catch-up,” Roux said.

Second, antibodies typically target proteins. But HIV is covered with a fatty membrane that antibodies ignore. The few proteins on the surface are mostly covered with carbohydrates, which antibodies usually don't bind to, Roux said. Furthermore, the carbohydrates are a form of sugar normally found in the body, so the immune system pays little attention to them.

These factors combine to make HIV a truly sinister invader. Before the development of an effective AIDS-killing vaccine is possible, scientists are going to have to understand a lot more about the virus and the immune system's response to it than they do today.

Frankly, finding a good AIDS vaccine ranks as one of the most formidable challenges in biomedical research. But recently, Roux and his research associate Ping Zhu have been recognized for their remarkable success in discovering new clues about how the AIDS virus works.

Nature's Own Vaccine

Amazingly, a few people infected with HIV never become sick. Why is that? Some of these lucky few have immune systems that build a half dozen or so antibodies that effectively neutralize the AIDS virus.

Not surprisingly, these extraordinary antibodies have drawn lots of attention from scientists, who know that if a way could be found to induce a person's immune system to make these compounds, a true AIDS vaccine might be within reach.

One such antibody, called 2G12, was cultured from a single cell of an HIV-infected individual who, after many years of being infected with HIV, never came down with AIDS. The antibody was discovered about a decade ago by Hermann Katinger, an Austrian doctor.

Studies showed the compound to be effective in neutralizing a number of HIV strains, so Roux decided to take a closer look at the antibody with an electron microscope.

All antibodies have molecular skeletons shaped like a “Y,” Roux explained. The two arms making up the “V” part of the “Y” are tipped with sites, unique to each antibody, where the antibody binds with a foreign substance. These arms are free to twist around, so each antibody can bind to two antigens, if they are in reach.

But when Roux took electron microscope photographs of 2G12 bound to its antigen, he saw something puzzling.

“The antibody's arms were always parallel to each other,” he said.

At first he thought that the antibody was binding to two adjacent antigens, thus holding the arms together.

“That turned out to be incorrect,” he said. “When we went back and looked at the antibody by itself, it looked the same. The two arms were always associated with each other. That suggested there was something really peculiar about this molecule.”

To investigate further, Roux used the computer to average a number of images together. “Then we could see a little bit more detail. It looked like the arms were criss-crossed, which was unusual,” he said.

Meanwhile, researchers at Scripps Research Institute out in California were well along in their work to figure out the precise atomic structure of 2G12. Roux was soon able to use Scripps' detailed atomic blueprint of the antibody to confirm his suspicions about the molecule's unusual shape.

Molecular chains in the two arms were twisted together, fusing the arms. The twisting was caused by several mutations in the region where the criss-cross occurs, Roux said.

“About 250 antibody molecules have been studied at the atomic level,” Roux said, “and a vast number of others by other techniques. None of them have this structure.”

For some time, researchers had known that the 2G12 antibody possessed another odd feature that explains how it is able to neutralize HIV when other antibodies can't.

Since antibodies typically bind only to proteins, the AIDS virus forms a shield (made out of carbohydrates) over itself to mask most of its protein surface. The only proteins on the surface of the virus are found in “spikes” that stick up like mushrooms above the virus's fatty membrane.

When HIV infects a cell, it's these spikes that attach themselves to the cell membrane and allow the virus to inject its genetic material into the cell.

In theory, scientists say that if a way could be found to block the spikes from doing their job, the AIDS virus would be rendered harmless.

“This 2G12 antibody is unusual in that it binds very strongly to the carbohydrate coat,” Roux said. “And in doing so it covers up the surface of the virus so the virus can't approach the target cell and latch on to it.”

Even though the carbohydrates HIV uses to disguise itself are the same ones normally found in the body, their molecular arrangement is different-they are very tightly packed together. The 2G12 recognizes this arrangement as foreign and so latches onto the virus.

Furthermore, the mutations that cause 2G12's arms to fuse also create two additional binding sites, the research team found.

“So this molecule can bind to four carbohydrates instead of two. That tremendously increases the strength of the binding. It actually binds thousands of times stronger than it would if it had only two binding sites,” Roux said.

Along with other antibodies, 2G12 (grown in culture) is being used in clinical trials to treat patients who have recently been exposed to HIV, in hopes of slowing or preventing infection. Modified versions of the antibody may someday prove useful against other diseases as well.

“It's been suggested that 2G12 might be re-engineered to bind to carbohydrates on other organisms,” Roux said. “It may be possible to change 2G12 so that it would be specific to a different set of carbohydrates.”

Spike Science

But such uses are not the same as a vaccine.

“If you could induce the immune system to make this particular kind of antibody, you might have a decent AIDS vaccine candidate,” Roux said. “But nobody knows if this kind of molecule can be induced because it's so rare. Nobody's ever seen one like this before.”

Since 2G12 resulted from several mutations, Roux doubts it could be generated by a vaccine. At present, there's no way to reproduce those mutations in a patient.

Still, to prevent infection, any potential vaccine would have to create antibodies that attack those spikes dotting HIV's surface, since the spikes are the only exposed part of the virus and are essential for the virus to invade cells.

Roux, in collaboration with scientists at the National Cancer Institute, decided to take a closer look at the surface of the virus to see what he could learn about the spikes.

Others had made electron microscope pictures of the AIDS virus before, but the nature of the techniques used led to inevitable distortions in the images. Roux used advanced staining and computer analysis to obtain significantly clearer images

With an electron microscope, he then created a tomogram, a picture similar ito a hospital's CAT scan. The electron microscope took dozens of pictures of the virus from different angles. Then a computer put the pictures together to create a three-dimensional image that showed how the virus looks in its natural form.

Each spike on the virus's surface consists of two parts: a stalk and a head. Roux's team obtained the first detailed pictures of the virus heads which were found to be composed of three segments arranged like the blades on a propeller.

Previously, scientists have believed that the virus is initially covered with 70 to 80 of these stalk-and-head outgrowths but that the majority of the heads soon fell off.

Roux's team found that this wasn't the case at all. Instead, each AIDS virus contains about eight spikes, the researchers discovered.

“The virus is actually formed with complete spikes, but few of them, and they are very stable and not prone to falling off,” Roux said. “These findings have fundamentally changed our view of the virus morphology and stability from what we used to think just a few years ago.”

Roux did find an exception-a mutant form of the virus called short-tailed SIV which infects monkeys. This virus is covered with about 80 spikes-apparently an effect of its mutation.

Still, Roux believes that the discovery that HIV wears only a few spikes may help solve a longstanding puzzle.

“Only a tiny percentage of the virus particles are actually infectious,” Roux said. “So maybe the infectious ones need a cluster of these spikes to be in one particular area. With only a few spikes on the surface, that may not happen very frequently.”

Does this new knowledge put us closer to the day when AIDS will go the way of polio?

When it comes to fighting a many-headed beast like HIV, any knowledge is surely power. Despite the enormous challenge, Roux is optimistic that such a vaccine will turn up some day. Some scientists scoffed that effective vaccines for polio would ever be found-the search took half a century and hundreds of millions of dimes collected by schoolchildren.

Now, thanks to Roux's research, scientists now have a much clearer picture of one of nature's most formidable foes.

SHIELDING THE AIDS VIRUS is a cleaver scheme that repels the body's own efforts to produce effective antibodies to kill or suppress the killer once it's unleashed. Normal antibodies attach to exposed protein found on invading particles. All of the protein on the surface of the HIV virus is found on spike-like protuberances scattered around its body. But a simple carbohydrate (sugar) masks much of each spike, preventing normal antibodies from attaching.
A rare antibody discovered in humans—named 2G12— prevented one lucky HIV-infected patients from developing full-blown AIDS. The reason, FSU scientists have confirmed, is that 2G12 fuses its "arms" into a single unit that overwhelms the sugars covering HIV's spikes.

The AIDS virus bristles with propeller-like spikes that clamp onto healthy cells as a means of doing thier deadly work. These images, called tomograms (similar to a hospital's CAT scan), show marked differences in the prevalence of spikes on the virus that infects monkeys (top) and the HIV virus (bottom) blamed for killing an more than 22 million people worldwide since 1970.