Sep. 29, 2005
Sept. 28, 2005 — A monoclonal antibody that can effectively treat mice infected with West Nile virus has an intriguing secret: Contrary to scientists' expectations, it does not block the virus's ability to attach to host cells. Instead, the antibody somehow stops the infectious process at a later point.
"This was a complete surprise to us, but it gives us some very useful insights," says senior author Daved Fremont, Ph.D., associate professor of pathology & immunology and of biochemistry & molecular biophysics at Washington University School of Medicine in St. Louis. "Based on what we've learned, we are now developing therapeutic antibodies for related viruses that also are effective at stopping the process of infection after the virus attaches to host cells."
Detailed study of how the antibody physically binds to the virus has provided intriguing clues to how it may block infection. Scientists found evidence suggesting that the antibody prevents the virus from rearranging the protein envelope that surrounds its genetic material after it enters a host cell.
To reproduce, a virus must alter its envelope in order to inject its genetic material inside the cell. After that injection, the virus tricks the host cell into making more copies of the genetic material that can then be assembled into new viral particles or virions and sent out to infect other host cells and reproduce. But with the viral reproduction process blocked by the antibody, scientists suspect that the host cell eventually destroys the virion.
Fremont and colleagues, who publish their results in the Sept. 29 issue of Nature, hope to design a new diagnostic system that can determine whether vaccines for West Nile and related viruses undergoing clinical trials stimulate production of antibodies that stop infections at a similar point.
In 2004, West Nile virus, which is a mosquito-borne flavivirus, reportedly caused 2,470 infections and 88 deaths in the United States. First isolated in Africa in 1937, West Nile spread to the Middle East, Europe, and Asia before arriving in the United States in 1999. Most infections with the virus are mild or symptom-free, but infections in people with weakened immune systems and those over 50 sometimes lead to serious complications or death.
Like West Nile, dengue virus is a flavivirus spread by mosquito bites, but only in tropical regions of the world. The dengue virus is estimated by Centers for Disease Control and Prevention epidemiologists to cause100 million infections annually worldwide.
"Currently there are no effective and safe vaccines for pediatric dengue," says co-author Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, of pathology & immunology and of medicine. "Thanks to our data from the West Nile virus antibody, we believe we now have a much better idea of how to evaluate vaccines for dengue."
Fremont and Diamond led a team of researchers at Washington University and Macrogenics Inc., a private company, that announced the identification of the effective West Nile antibody earlier this year. In a line of mice genetically altered to increase vulnerability to the virus, they found injection of the new antibodies could boost survival rates of mice infected with the virus to greater than 90 percent.
Scientists at Macrogenics are working on the preliminary studies required before the West Nile antibody can be tested in humans. Meanwhile, researchers at Washington University wanted to know why the new antibody was so effective.
Antibodies normally work by binding to invaders to flag them for consumption and destruction by immune system cells known as macrophages. In the prior study, which screened several potential West Nile antibodies, scientists found that all the most potent antibodies bound to a particular section of a protein that makes up the exterior of the viral envelope. The envelope of a single viral particle or virion is comprised of 180 copies of this protein.
For the new study, scientists determined the detailed structure of a single antibody bound to its envelope protein target region using the technique of protein crystallography. Scientists were able to affirm in greater detail earlier observations suggesting that the antibody will be therapeutic for all strains of West Nile Virus.
Based on this data, they predicted how multiple copies of the successful antibody would bind to a virion.
"We were startled to find that the antibody only seemed to be able to attach to 120 of the 180 copies of the target region in the complete viral envelope," says Grant Nybakken, a Washington University M.D./Ph.D. student who was lead author of the study.
Further tests showed that virions covered in infection-stopping antibodies could still bind to host cells, while antibodies that were less effective at stopping infection could more effectively prevent the virion from binding to host cells.
How does an antibody that's better at preventing the virus from binding to host cells actually turn out to be worse at treating infection? The key may lie in a theory known as antibody-dependent enhancement (ADE) of infection, which has been observed in test tube studies of dengue virus and may be important to the onset of dengue hemorrhagic fever.
This theory suggests that dengue and other viruses may have developed tricks that let them reproduce inside macrophages, the immune cells that normally consume and destroy any object that they find covered in antibodies. In effect, these tricks turn antibodies that should be death warrants into passes into cells where invaders can reproduce.
Fremont cautions that this phenomenon has not been seen in West Nile virus, but notes that when he and his colleagues tested the ability of several antibodies to prevent West Nile from reproducing inside macrophages, they found that only the therapeutic antibodies blocked the virus' reproduction. The therapeutic antibodies' ability to stop reproduction in macrophages even worked when the virions were simultaneously exposed to antibodies known to enhance infection.
"Do the therapeutic antibodies also prevent the virus from properly injecting its genetic material into macrophages? It's a tempting possibility, but we don't have the evidence to prove it yet," he says.
Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, and Fremont DH. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature, Sept. 29, 2005.
Funding from the Pediatric Dengue Vaccine Research Institute, the National Institutes of Health, and the Midwest Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Research supported this research.
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