Sept. 28, 2005 — A monoclonal antibody that can effectivelytreat mice infected with West Nile virus has an intriguing secret:Contrary to scientists' expectations, it does not block the virus'sability to attach to host cells. Instead, the antibody somehow stopsthe infectious process at a later point.
"This was a completesurprise to us, but it gives us some very useful insights," says seniorauthor Daved Fremont, Ph.D., associate professor of pathology &immunology and of biochemistry & molecular biophysics at WashingtonUniversity School of Medicine in St. Louis. "Based on what we'velearned, we are now developing therapeutic antibodies for relatedviruses that also are effective at stopping the process of infectionafter the virus attaches to host cells."
Detailed study of howthe antibody physically binds to the virus has provided intriguingclues to how it may block infection. Scientists found evidencesuggesting that the antibody prevents the virus from rearranging theprotein envelope that surrounds its genetic material after it enters ahost cell.
To reproduce, a virus must alter its envelope in orderto inject its genetic material inside the cell. After that injection,the virus tricks the host cell into making more copies of the geneticmaterial that can then be assembled into new viral particles or virionsand sent out to infect other host cells and reproduce. But with theviral reproduction process blocked by the antibody, scientists suspectthat the host cell eventually destroys the virion.
Fremont andcolleagues, who publish their results in the Sept. 29 issue of Nature,hope to design a new diagnostic system that can determine whethervaccines for West Nile and related viruses undergoing clinical trialsstimulate production of antibodies that stop infections at a similarpoint.
In 2004, West Nile virus, which is a mosquito-borneflavivirus, reportedly caused 2,470 infections and 88 deaths in theUnited States. First isolated in Africa in 1937, West Nile spread tothe Middle East, Europe, and Asia before arriving in the United Statesin 1999. Most infections with the virus are mild or symptom-free, butinfections in people with weakened immune systems and those over 50sometimes lead to serious complications or death.
Like West Nile,dengue virus is a flavivirus spread by mosquito bites, but only intropical regions of the world. The dengue virus is estimated by Centersfor Disease Control and Prevention epidemiologists to cause100 millioninfections annually worldwide.
"Currently there are no effectiveand safe vaccines for pediatric dengue," says co-author MichaelDiamond, M.D., Ph.D., assistant professor of molecular microbiology, ofpathology & immunology and of medicine. "Thanks to our data fromthe West Nile virus antibody, we believe we now have a much better ideaof how to evaluate vaccines for dengue."
Fremont and Diamond leda team of researchers at Washington University and Macrogenics Inc., aprivate company, that announced the identification of the effectiveWest Nile antibody earlier this year. In a line of mice geneticallyaltered to increase vulnerability to the virus, they found injection ofthe new antibodies could boost survival rates of mice infected with thevirus to greater than 90 percent.
Scientists at Macrogenics areworking on the preliminary studies required before the West Nileantibody can be tested in humans. Meanwhile, researchers at WashingtonUniversity wanted to know why the new antibody was so effective.
Antibodiesnormally work by binding to invaders to flag them for consumption anddestruction by immune system cells known as macrophages. In the priorstudy, which screened several potential West Nile antibodies,scientists found that all the most potent antibodies bound to aparticular section of a protein that makes up the exterior of the viralenvelope. The envelope of a single viral particle or virion iscomprised of 180 copies of this protein.
For the new study,scientists determined the detailed structure of a single antibody boundto its envelope protein target region using the technique of proteincrystallography. Scientists were able to affirm in greater detailearlier observations suggesting that the antibody will be therapeuticfor all strains of West Nile Virus.
Based on this data, they predicted how multiple copies of the successful antibody would bind to a virion.
"Wewere startled to find that the antibody only seemed to be able toattach to 120 of the 180 copies of the target region in the completeviral envelope," says Grant Nybakken, a Washington UniversityM.D./Ph.D. student who was lead author of the study.
Furthertests showed that virions covered in infection-stopping antibodiescould still bind to host cells, while antibodies that were lesseffective at stopping infection could more effectively prevent thevirion from binding to host cells.
How does an antibody that'sbetter at preventing the virus from binding to host cells actually turnout to be worse at treating infection? The key may lie in a theoryknown as antibody-dependent enhancement (ADE) of infection, which hasbeen observed in test tube studies of dengue virus and may be importantto the onset of dengue hemorrhagic fever.
This theory suggeststhat dengue and other viruses may have developed tricks that let themreproduce inside macrophages, the immune cells that normally consumeand destroy any object that they find covered in antibodies. In effect,these tricks turn antibodies that should be death warrants into passesinto cells where invaders can reproduce.
Fremont cautions thatthis phenomenon has not been seen in West Nile virus, but notes thatwhen he and his colleagues tested the ability of several antibodies toprevent West Nile from reproducing inside macrophages, they found thatonly the therapeutic antibodies blocked the virus' reproduction. Thetherapeutic antibodies' ability to stop reproduction in macrophageseven worked when the virions were simultaneously exposed to antibodiesknown to enhance infection.
"Do the therapeutic antibodies alsoprevent the virus from properly injecting its genetic material intomacrophages? It's a tempting possibility, but we don't have theevidence to prove it yet," he says.
Nybakken GE,Oliphant T, Johnson S, Burke S, Diamond MS, and Fremont DH. Structuralbasis of West Nile virus neutralization by a therapeutic antibody.Nature, Sept. 29, 2005.
Funding from the Pediatric Dengue VaccineResearch Institute, the National Institutes of Health, and the MidwestRegional Center of Excellence in Biodefense and Emerging InfectiousDiseases Research supported this research.
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