With the flu season about at its peak, people most at risk of influenza — the very young, the elderly, the debilitated — should have had their flu shots by now.

Polio immunization for young children, and hepatitis for those exposed to that viral infection, are among other vaccines available and recommended. HIV, despite years of research, still resists the best efforts of vaccinologists.

Now the quickest-killing virus of all — Ebola — is up at bat for a vaccine. "The news," University of Michigan molecular biologist Gary Nabel told BioWorld Today, "is that it is possible to generate immunity to Ebola."

His research report in the just-published January issue of Nature Medicine matches action to those words. Its title: "Immunization for Ebola virus infection."

It was 21 years ago, in 1976, that an unknown pathogen along Central Africa's Ebola river quickly laid low 430 (80 percent) of its 550 infected victims. Less than two decades later, in 1994 and 1995, the virus reemerged to take another 245 lives (77 percent) of 316 new cases.

Most recently, last year, an Ebola virus scare hit the small Texas town of Alice, when a shipment of 100 research primates from the Philippines tested positive. (See BioWorld Today, April 17, 1996.)

"Since their discovery in 1967 in Marburg, Germany," wrote Thomas Folks, in a commentary accompanying Nabel's Nature Medicine article, "nearly 1,000 cases of Ebola or Marburg hemorrhagic fever have been reported, with greater than 50 percent of patients dying within days."

The Ebola virus announces its arrival with diarrhea, vomiting, skin rashes and dropping blood pressure, then strikes inward to wreak uncontrolled bleeding in liver, kidneys and other internal organs.

One glance at the viral particle under the electron microscope shows, by its weird and varied shapes, that it's not like other viruses. A large RNA virus studded with spikes, it has seven genes, which encode eight proteins. Among these is a sizable surface glycoprotein (GP) that pokes its spikes through the viral membrane, and a smaller, secreted GP (sGP) that gets exported beyond the viral envelope into the surrounding liquid medium.

To Trigger Immune Response, Naked DNA Into Muscle

Among Ebola's other gene products is an internal nucleoprotein (NP). Nabel and his co-authors chose these GP, sGP and NP as three presumably immunogenic epitopes to arm their experimental vaccine.

They packaged naked cDNA encoding these proteins into a plasmid, and injected the genetic vaccine intramuscularly into both legs of guinea pigs. Guinea pigs are sitting ducks for Ebola virus, which attacks these animals and humans in much the same manner, causing similar symptoms and outcomes.

By seeding their nonviral vector directly into muscle tissue, the co-authors hoped to provoke an immune response, when the genes expressed their putatively immunogenic viral proteins. (See BioWorld Today, Oct. 9, 1996.)

Once vaccinated, the animals were transported from the university, at Ann Arbor, Michigan, to the level-4 containment lab at the Centers for Disease Control (CDC), in Atlanta. There, they were lethally challenged with infectious Ebola virus. One cohort of guinea pigs got those deadly shots two months after vaccination, when their antibody levels were high. They showed strong immunity with both glycoprotein and nucleoprotein immunization.

A second group of animals was challenged at four months, after the guinea pigs' initial immune reaction had had a chance to subside. Nevertheless, they still displayed good immunity from the glycoprotein vaccines, but no longer from the nucleoprotein.

Neither group succumbed to the infection.

Nabel said, "Our next step is to study the DNA vaccine in non-human primates, followed by controlled toxicity trials in humans. Such clinical trials in human subjects — who would not be exposed to the Ebola virus — could begin within one to two years, if the next phase of studies is promising.

"In addition to vaccine development," he concluded, "these same molecular tools will help us develop strategies to intervene in the viral infection process."

Handling Ebola With Recombinant Kid Gloves

As CDC's Folks pointed out in his editorial, "The extensive safety measures necessary for working with Ebola virus in vivo preclude attempting large-scale virus preparation. Clearly, new vaccine approaches are needed."

One such approach is reported in the current Proceedings of the National Academy of Sciences (PNAS), dated Dec. 23, 1997, and titled: "A system for functional analysis of Ebola virus glycoprotein." Among the paper's co-authors is virologist Anthony Sanchez, of CDC's Special Pathogens Branch. He is also a co-author of the Nature Medicine report.

"With mortality rates of up to 90 percent," the PNAS paper begins, "studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment."

The article's senior author, virologist and molecular biologist Yoshihiro Kawaoka, told BioWorld Today how the research reported in PNAS aims to circumvent this safety problem:

"First of all," he explained, "we are not using the Ebola virus. Instead, we are using a vesicular stomatitis virus (VSV), engineered to be defective." (Vesicular stomatis causes mouth sores in horse and cattle, but rarely in humans.)

That defect in their VSV, Kawaoka explained, is lack of a gene encoding the protein by which the virus can bind to its receptor. "We are interested," he added, "in studying the receptor-binding protein of the Ebola virus."

Accordingly, Kawaoka — who moved recently from St. Jude Children's Research Hospital, in Memphis, Tenn., to the University of Wisconsin, in Madison — continued: "Once we had that defective virus, we infected cells with it. At the same time, we transfected those same cells with plasmid expressing the Ebola glycoprotein. So when the VSV replicates and buds out it acquires that Ebola GP on its cell surface. It doesn't have either the Ebola GP gene or the VSV receptor-binding gene.

"This virus particle," he concluded, "infects the next cell, but only one time. There's no further transmission. So it's very safe, but at the same time it makes it possible to analyze the function of Ebola GP." *

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