A promising vaccine
for West Nile virus?

Engineered protein gives mice complete protection, could lead to a diagnostic test.

A vaccine developed by Yale scientists has protected mice from the West Nile virus, a mosquito-borne infection that has been linked to about 10 deaths in the United States since the summer of 1999.

The virus, first identified in Uganda in 1937, surfaced in the New York City area in 1999 and has subsequently appeared in the South and Midwest. It is considered an emerging disease, said Erol Fikrig, M.D., associate professor of medicine and of epidemiology and public health. “Its seriousness as a public health threat is not fully known yet,” said Fikrig, who directed the development of the vaccine. “That should become apparent over the next two to three years. If the vaccine proves necessary, its development will be valuable.”

The virus, which infects birds as well as humans, spreads through mosquito bites primarily in warm-weather months. There is currently no cure, although infection does not generally cause serious consequences. Elderly patients, however, can develop fatal encephalitis, a central nervous system infection.

Fikrig and colleagues, including Tian Wang, Ph.D., a postdoctoral fellow in his laboratory, and John F. Anderson, Ph.D., research affiliate in epidemiology, and associates from the Connecticut Agricultural Experiment Station in New Haven, isolated a sample of the virus found in an infected bird. They genetically engineered a protein in the virus, which they then injected into uninfected mice. Immunization with the vaccine provided complete protection for the mice against West Nile virus.

Because diagnosis of West Nile virus can be difficult using current methods, the protein used to make the vaccine could also be employed to develop a diagnostic test, Fikrig said.

Results of the study were published online in the Journal of Immunology on October 23 and appeared in the November 1 print issue.

Molecule that targets tissue factor is found to thwart tumors in mice

Yale researchers have developed a molecule that, when injected into tumors in mice, destroyed blood vessels in tumors and left normal tissue unharmed. Their findings, published in the October 9 issue of Proceedings of the National Academy of Sciences, hold the promise of a new therapy for metastatic cancer.

The molecule, developed by Alan Garen, Ph.D., and Zhiwei Hu, Ph.D., in the Department of Molecular Biophysics and Biochemistry, is an immunoconjugate, or icon, which joins elements of different molecules. The team’s strategy—targeting a tumor’s blood vessels without harming normal cells—led them to the molecule tissue factor (TF), which is expressed on the inner surface of the tumor blood vessels and initiates blood clotting.

Garen and Hu’s icon is made up of two elements, one that draws it to TF and another that initiates an immune response. The first element is factor VII, a molecule that circulates in the blood and binds to TF. Once factor VII has drawn the icon to TF in the tumor, the icon triggers its second element, the Fc region of a human antibody, which activates the immune system against cells that bind to the icon. A replication-incompetent adenoviral vector was used to deliver the gene encoding the icon to the tumor cells. The tumor, once infected, produces and secretes the icon into the blood, where it circulates throughout the body seeking tumor blood cells.

“The result,” said Garen, “is that the tumor’s blood vessels are destroyed by the immune system, and consequently, the tumor cells die because they lack a blood supply. Normal blood vessels survive because they do not express tissue factor and therefore do not bind to the icon.”

Garen and Hu first generated human prostatic and melanoma tumors in mice, then injected the vector into one of the tumors. A control group of mice received a blank vector; those mice died within 63 days after tumors appeared on the skin.

In the mice that received the icon molecule, tumor cells were eliminated and the mice remained free of the disease for at least 194 days. They received their last injection of the vector on the experiment’s 53rd day, suggesting that the molecule’s effects are long-lasting. In addition, the icon acted against tumors that had not been injected with the vector, offering the possibility of a treatment for metastatic cancer patients. “This icon should work against all types of tumors that contain blood vessels,” Garen said.

Knock-out study shows how some white blood cells regulate skin cancer

A type of white blood cell that is found in the skin and assists in the body’s immune response also helps prevent skin cancer, Yale researchers have found.

Gamma-delta T cells play a major role in local immunity and “are likely to be crucial to an early defense against skin cells that have recently transformed to a premalignant or malignant state,” said Michael Girardi, M.D., assistant professor of dermatology. Girardi, primary author of a paper on the findings published in Science in September, was part of a team that included colleagues at Guy’s King’s St. Thomas Medical College in London. The team genetically engineered mice that were incapable of producing gamma-delta T cells, then exposed the knock-out mice to three different models of skin cancer.

In one model, tumor cells were injected into the skin. In another, a carcinogen was injected into the skin. For the third, carcinogens were repeatedly painted onto the skin. This last model most closely resembles cancer development in humans because it mimics repeated exposures that progress from a benign thickening of tissue to premalignant papilloma to carcinoma formation.

Girardi and his team found that, in all three models, the absence of gamma-delta T cells resulted in a higher level of skin cancer formation. In the third model, however, another type of T cell, alpha-beta, contributed to skin cancer development and progression. “There appears to be a yin-yang contribution by alpha-beta T cells to skin cancer, in that they can act in both the defense against and the promotion of carcinoma,” Girardi said.

Gamma-delta T cells work by expressing a protein, NKG2d, which binds to a molecule that is expressed by tumor cells. Once the molecule, Rae-1, is engaged by the NKG2d protein, gamma-delta T cells can kill the tumor cell. Rae-1 is expressed only in skin cells that have been exposed to chemical carcinogens that stimulate the transition to cancer. “This is an initial and important distress signal to the local T cells, and to some other cells of the immune system, that things are wrong,” Girardi said.

Route of infection

Investigators in microbial pathogenesis have described a secretion system that many bacteria—including those that cause plague, dysentery and typhoid—use to infect other cells. The type III secretion system found in Salmonella is a hollow, needle-like structure that delivers bacterial proteins into a host cell. “Many pathogens use a similar mechanism,” said principal investigator Jorge E. Galan, Ph.D., chair of the Section of Microbial Pathogenesis, of the findings published in the November 1 issue of Nature. “Insight into any of them gives you insight into all of them. From this fundamental information we can begin to develop completely new therapeutic strategies to halt or prevent infections by these pathogens.”

Crossing over

Two Yale neuroscientists have discovered a pathway, apparently unique to humans, that guides neurons between different brain regions. “Disregarding boundaries between major brain divisions is unusual,” said principal investigator Pasko Rakic, M.D., Ph.D., chair of the Department of Neurobiology and the Dorys McConnell Duberg Professor of Neuroscience, “and could explain how parts of the cerebral cortex associated with the highest cognitive functions may have coordinated their growth with subcortical structures during human brain evolution.” Rakic and graduate student Kresimir Letinic found that attractive and repulsive molecules directed neuronal stem cells from the ganglionic eminence to the diencephalon. They published their findings in the September issue of Nature Neuroscience.

Clues to how a cell moves

Using X-ray crystallography, investigators at Yale and the Salk Institute have solved the structure of Arp2/3, a complex of seven proteins that helps cells move. “Knowledge of the three-dimensional structure not only provides key insights about the Arp2/3 complex, but it will also elevate the level of research on cellular movements for years to come,” said principal investigator Thomas D. Pollard, Ph.D., the Eugene Higgins Professor of Molecular, Cellular and Developmental Biology. The Arp2/3 complex initiates the assembly of the protein actin into filaments at the front end of a cell, which pushes the cell forward. The findings were published in the November 23 issue of Science.

Also in Findings:

West Nile virus vaccine?  |  Thwarting tumors in mice  |  How some white blood cells regulate skin cancer  |  Route of infection  |  Crossing over  |  Clues to how a cell moves

Chronicle  |  Rounds

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Originally published in Yale Medicine, Winter 2002.
Copyright © 2002 Yale University School of Medicine. All rights reserved.