Saturday, March 7, 2009

New Target Against Flu Virus May Extend Vaccine Potency

Antibody Uncovers Vulnerability of Protein Stem

HMS researchers have found an Achilles heel in the influenza virus that may someday make annual flu shots a thing of the past. By targeting a hidden pocket in the microbe with a newly discovered antibody, they disabled a wide range of viruses, including those that cause the avian flu and the virulent 1918 Spanish flu.

While this research could lead to clinical trials of a new antiviral as soon as 2012 and may eventually lead to a more durable influenza vaccine, its influence may extend even further. The work, described in the March Nature Structural and Molecular Biology, validates a novel approach to finding such viral vulnerabilities and reveals what may be a more general principle for defeating a variety of pathogens.

Photo by Graham Ramsay

Wayne Marasco and Jianhua Sui have discovered influenza’s Achilles heel and devised a method to attack it.

Striking Gold
The story begins in the lab of Wayne Marasco, HMS associate professor of medicine at the Dana–Farber Cancer Institute. Twelve years ago, Marasco collected blood from 57 healthy Bostonians and used the samples to create a library of 27 billion different human antibodies.

Researchers “pan” the library by presenting it with an antigen, such as a whole virus or a protein on the viral surface. Panning unearths antibodies that bind to that antigen. Marasco used his library to isolate an antibody against SARS in 2004.
When the avian flu appeared, Marasco and first author Jianhua Sui put the library to work again. But instead of panning with the whole H5N1 influenza virus, they focused on a single protein. They isolated the H5 version of hemagglutinin, a surface protein on influenza that allows the virus to invade a cell and replicate. (The N portion is a different surface protein called neuraminidase, which allows the virus to exit the cell.) The effort uncovered 10 potential antibodies.

Sui and Marasco, in partnership with co-author Rubin Donis, chief of the molecular virology and vaccines branch of the Centers for Disease Control and Prevention, tested three of these antibodies in mice infected with a lethal dose of avian flu. The antibodies neutralized between 80 and 100 percent of the infections.

Unexpectedly, the antibodies also neutralized other strains. They knocked down H1N1 (the 1918 Spanish flu), H2N2, H6N1 and more. “It became apparent very quickly that the target they were recognizing was highly conserved,” said Marasco.

Not only were these antibodies more broadly effective than expected, they also worked differently. Most antibodies stick to the round top of the lollipop-shaped hemagglutinin protein and interfere with the protein’s ability to bind to the cell membrane. But Marasco and Sui’s antibodies were not blocking the membrane binding. “That told us right there that the antibody wasn’t working against the globular head,” said Marasco.

Stemming the flu. Each year, scientists develop new influenza vaccines to target the ever-mutating globular heads (light red) of the hemagglutinin proteins that coat the virus. A newly discovered antibody binds to the much less variable and much less accessible stem of the protein (red). Work is under way to turn this antibody into an antiviral that can be used to contain a pandemic and to protect immunosuppressed individuals during flu season. Since the machinery of the stem evolves more slowly than the head, the discovery may lead to a broadly effective influenza vaccine that lasts for longer than a single season.

At this point, a third part of the team became critically important. Robert Liddington and his team at the Burnham Institute for Medical Research crystallized one of the antibodies bound together with hemagglutinin. “That’s when the revelations started coming,” said Marasco.

The crystal confirmed that the antibody was not bound to the enticing top of the protein, but rather to a pocket in the stem. That pocket contains complex machinery. It houses three entangled moving parts that allow the virus to infect the cell (see video). The crystal revealed that the antibody grabs onto all three and prevents that machinery from working. “In the past, people didn’t even know to look in that pocket,” said Sui.

Sui took this information and used it to search a database of more than 6,000 (and growing) known genetic variants of the flu. She found that only two versions of this complex stem-based machinery have evolved. An examination by Liddington’s team of crystal structures of known variants found the same. The antibodies Sui and Marasco found work against one version. They are now running the other version of the stem through the same panning process to find an antibody against it.

Enduring Weakness
The contrast between the slow evolution of the stem and the impossible-to-keep-up-with evolution of the head is stunning. But it is not surprising. The part of the headpiece that binds to the cell membrane is very small, said Sui. So the rest of the headpiece can change dramatically without compromising the function.

But in the stem, “the delicate and complex machinery is highly conserved,” said Donis. “The virus cannot mutate it because by doing so, it would commit suicide.” Indeed, Donis’s team attempted to force mutations in the stem, but none emerged.

In discovering this new, hidden vulnerability, the researchers have realized that the virus has been fooling them, and our bodies, all along. “The virus has very cleverly developed an area on the top of its coat protein that creates a molecular decoy,” said Marasco. He speculates that the immune system mounts a full-scale attack against the easy-to-spot decoy while it simultaneously suppresses any efforts to target the elusive stem. Similarly, new vaccines chase the decoy each season hoping to hit it just right.


Courtesy Dana–Farber Cancer Institute

HMS researchers are targeting a common weakness to tackle influenza.

Courtesy Dana–Farber Cancer Institute

HMS researchers are targeting a common weakness to tackle influenza.

But now, with the new insights from this work, “a pan-therapy for all kinds of influenza may be within our grasp,” said Liddington. Further, Marasco suspects that the influenza virus’s means of protecting its most vulnerable machinery may be a more general strategy. He has observed almost the exact same system in corona viruses, such as SARS.

Assuming that approval for human testing proceeds without a hitch, the new influenza antibody will likely be used as an antiviral first. Since it is unlikely that a mutant will evolve to defeat it, the hope is that this antiviral can be stockpiled and stored for years. Marasco also speculates that it may be possible to develop a vaccine that both masks the decoy and allows the immune system to attack the less flexible stem.

In the future, Marasco plans to apply these same methods to other viruses. The team’s approach not only allows them to find novel antibodies and hidden targets, it also helps researchers respond nimbly as resistant strains evolve.

Students may contact Wayne Marasco at for more information.

Conflict Disclosure: The authors report no conflicts of interest.

Funding Sources: National Institutes of Health