RxPG News : West Nile Virus

How West Nile virus evades immune defenses

Thu, 05 Oct 2006 01:04:37 PST

( from http://www.rxpgnews.com ) West Nile virus evades the body's immune defenses by blocking immune signaling by a protein receptor, a finding that could pave the way for a vaccine to protect against North American strains of the virus, UT Southwestern Medical Center researchers report.

Researchers discovered the receptor's key role in controlling West Nile infection by conducting a study, described in October's Journal of Virology, that compares the genetics of an illness-causing Texas strain of the virus to a harmless African strain.

The Texas strain can inflict illness because it blocks the signaling activity of a protein receptor called the interferon alpha/beta receptor, or IFNAR, disrupting a cell's ability to direct the immune system to fight off the virus.

The African strain does not block IFNAR activity, so the immune system renders it harmless. The strain is harmful, however, in mice with dysfunctional receptors.

"We now hope to harness the African strain as the basis for West Nile vaccine studies. The virus has spread across the country and infected more than 2,100 U.S. residents 180 in Texas this year alone, so we have to learn how to deal with it," said Dr. Michael Gale, associate professor of microbiology at UT Southwestern and director of the study. Brian Keller, a student in the Medical Scientist Training Program at UT Southwestern, is the first author of the study.

West Nile virus, which is transmitted by mosquito bite, arrived in the United States in 1999 and has become an epidemic that flares up in the summer and lasts into fall.

Infection causes mild flu-like symptoms in most people, but about one in every 150 develop serious illness, that can include high fever, coma, seizures and encephalitis and meningitis. Children, the elderly or people with weak immune systems are most at risk.

There is no vaccine. Doctors can only treat symptoms of the disease.

Searching for clues that might allow development of a vaccine, Dr. Gale and his research team compared one strain from each of West Nile's two basic categories: the harmful strains associated with outbreaks of encephalitis and meningitis in North America, and non-harmful strains from Madagascar and Cyprus.

They studied a harmful strain isolated from an infected grackle from Hall County, Texas, in 2002, and a harmless strain isolated from an infected parrot from Madagascar in 1978.

They mapped the genetic makeup of each strain, and then tested the viruses in mice.

West Nile infection triggers production of interferon, a group of proteins that are crucial in immune defense. Interferon, which binds to IFNAR, subsequently signals the JAK-STAT molecular pathway, a series of biochemical reactions essential for turning on immune-defense genes, allowing the body to clear out the virus. This process occurs normally in the African strain.

Infection by the Texas strain, however, blocked IFNAR signaling activity, allowing the virus to replicate and spread.

This highlights the integral role of interferon and IFNAR signaling in innate immunity.

Dr. Gale said the mechanisms at work in the African strain could be used as a basis for a vaccine, perhaps mutating North American strains so they no longer disrupt immune signaling. The remaining key is figuring out the exact mechanics of how the strains block signaling, a project Dr. Gale's team is already at work on.

Fortunately, North American strains are extremely similar in fact, the one that appeared in the United States in 1999 and the Texas strain used in this study are 99 percent identical. One vaccine could, in theory, prevent illness from many of the harmful strains, Dr. Gale said.

"We feel a vaccine could be highly effective in preventing infection," said Dr. Gale.

Secrets to monoclonal antibody's success against West Nile Virus

Thu, 29 Sep 2005 20:53:38 PST

( from http://www.rxpgnews.com ) 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.

Monoclonal antibody cures West Nile Virus

Mon, 25 Apr 2005 19:58:38 PST

( from http://www.rxpgnews.com ) A newly developed monoclonal antibody can cure mice infected with the West Nile virus, scientists at Washington University School of Medicine in St. Louis report. If further studies confirm the effectiveness and safety of the antibody, it could become one of the first monoclonal antibodies used as a treatment for an infectious disease.

In a strain of mice that normally only has about a 10 percent survival rate after West Nile infection, scientists found that single doses of the antibodies given soon after infection could boost survival rates to 90 percent or higher.

"To our knowledge, these experiments are the first successful demonstration of the use of a humanized antibody as a post-exposure therapy against a viral disease," says senior investigator Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, pathology and immunology and of medicine. "They also suggest antibody-based therapeutics may have a broader utility against other infectious diseases."

Diamond points out that Macrogenics Inc., of Rockville, Md., a company that contributed to the study and licensed the antibody from Washington University, must complete other preliminary studies before the antibody can be tested in humans. But he and his colleagues are excited both by the apparent potency of the antibody and its potential to help them explore new possibilities for treating related viruses that are more prolific causes of human disease and death.

"We could give a single dose of this antibody to mice as long as five days after infection, when West Nile virus had entered the brain, and it could still cure them," says Diamond. "It also completely protected against death from the disease."

Diamond and his colleagues will report their results in the May issue of Nature Medicine.

In 2004, West Nile virus reportedly caused 2,470 infections and 88 deaths in the United States. The mosquito-borne virus, first isolated in Africa in 1937, 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.

Scientists initially produced a panel of many West Nile virus antibodies from mouse cells. The human immune system would clear out these foreign antibodies quickly, so when they had identified a potent antibody, scientists at Macrogenics clipped out the genetic material that controls the antibody's targeting and cloned it into a human antibody. The "humanized" antibody should be less likely to induce an adverse human immune system response. A second round of tests in mice confirmed that the new antibodies retained their ability to stop West Nile virus.

Other monoclonal antibodies are currently in development or use as anti-cancer and anti-inflammatory treatments. An antibody against respiratory syncytial virus (RSV) is approved for use as a prophylactic treatment in children at risk of the disease in hospitals. Unlike the West Nile virus antibody, though, the RSV antibody has to be given prior to infection.

West Nile virus belongs to a family of viruses known as flaviviruses, several of which are spread by mosquito bites. Other flaviviruses include the virus that causes dengue fever, a potentially life-threatening infection prevalent in tropical cities. Centers for Disease Control and Prevention epidemiologists estimate that there are100 million cases of dengue worldwide every year.

"A lot of what we're learning from the West Nile virus antibody will be of consequence for the development of a pediatric dengue vaccine," says co-author Daved Fremont, Ph.D., associate professor of biochemistry and molecular biophysics and of pathology and immunology. "Currently there are no safe vaccines for dengue infections."

Important insights from the production and selection of the new antibody include a close fix on where the antibody binds to West Nile virus. Antibodies typically work by attaching to a piece of a foreign cell or substance, which causes immune system cells known as macrophages to pick up the substance and clear it from the body.

Binding to the invader is just the beginning of the battle, though. Some antibodies can bind to an invader but do so in a way that fails to slow the invader down or trigger a response from macrophages. In one rare case that involves the dengue fever virus, antibodies can adhere to the virus in a way that accelerates the infection.

From their initial pool of West Nile virus antibodies, researchers identified 46 that could bind to the West Nile virus' envelope (E) protein. Further testing showed that 12 could bind to the virus in a way that consistently neutralized it, shutting down infections in cell cultures and in mice.

To determine where these potently neutralizing antibodies were binding to the envelope protein, a task known as epitope mapping, researchers modified a yeast-based screening system. The system let them test individual antibodies for their ability to bind to many versions of the E protein, each with slight alterations. By analyzing the changes in the versions of the protein that antibodies had difficulty binding to, they isolated first a region of the E protein, known as domain III, and then a group of amino acids in that domain.

"The big surprise for us was that all of the potently neutralizing antibodies appear to recognize the same general region of this domain," says Fremont. "It was very consistentall the neutralizing antibodies that bind this domain adhere to that area; all the non-neutralizing antibodies that bind this domain adhere to different areas."

Fremont notes that while the E proteins of various flaviviruses are generally very similar, domain III can vary significantly. He and others are working to detail the precise mechanisms that allow the new West Nile antibody to block viral infection.

Diamond and Fremont are looking for other areas of the West Nile virus E protein that antibodies can bind to and neutralize the virus. Diamond is also using the yeast screening system to epitope map the sites on the dengue fever virus where antibodies can bind and inadvertently enhance infection instead of fighting it.

"We don't really understand on a molecular level what's happening in these cases, which are called enhancing antibodies," Diamond explains. "Epitope mapping may help us better understand this potentially dangerous interaction."