By David N. Leff

What with all the millennium-mindedness raging these days, which 2Oth century event has been called "the most mobile and lethal pandemic the world has ever seen?"

That's how a contemporary medical historian describes the global outbreak of "Spanish influenza" that took some 25 million lives around the world in six months of 1918 and 1919. That was three times the 8,528,831 battlefield deaths suffered during the 4.5 years (1914-18) of World War I.

People at the time blamed the origin of the deadly flu virus on Spain, because that country suffered the largest proportion of victims. Ever since that epochal pandemic died out in 1919 as suddenly as it had flared up in 1917, people have wishfully thought that its likes would never rise again. And sure enough, despite frequent local epidemics - such as the recent one in Hong Kong - nothing in the eight decades since the end of World War I has come anywhere near that influenza pandemic.

Given the unpredictable, ever-shifty behavior of that viral serial killer, virologists can't let down their guard. Every year, they consult the tea leaves and entrails of possibly pathogenic influenza virus mutations among swine and poultry in China, which historically constitutes the reservoir of any new flu virus uprising. "In fact," observed virologist Yoshihiro Kawaoka, "influenza remains a major public health menace, and infects up to 40 million people a year in the U.S. alone, of whom 20,000 die."

So every year in "flu season," young children and the elderly - the most susceptible groups for viral infection - are urged to get their annual anti-flu shots. "Current human influenza vaccines," Kawaoka pointed out, "consist of inactivated influenza A and B viruses, in the same injection." (Influenza viral strain A is somewhat more virulent than B. It is more apt to change its antigenic spots by mutating, and gives rise to more lethal outbreaks.)

"The existing human vaccines are good," Kawaoka allowed, "but can be improved upon by using live, attenuated-virus vaccines. These could be advantageous," he explained, "because they induce both cellular and antibody immune responses. They also produce immunity where it needs to be," he added, "such as the nasal cavity and respiratory tract."

To create such live designer vaccines, Kawaoka related, "The obvious thing is to introduce mutations that make your influenza virus less pathogenic. So such a vaccine infects humans or animals, but does not cause disease, while still inducing immunity. There is one live attenuated virus currently in clinical trials," he continued, "conducted by the Aviron company, of Mountain View, Calif. That attenuated live-virus vaccine was generated by passaging the virus at low temperature - essentially asking nature to introduce mutations. So the number of mutations is not very high."

Kawaoka, a professor of virology at the University of Wisconsin's School of Veterinary Medicine in Madison, is senior author of an article in the current Proceedings of the National Academy of Sciences (PNAS), dated Aug. 3, 1999. Its title is, "Generation of influenza A viruses entirely from cloned cDNAs."

"Our goal," he told BioWorld Today, "was to make an influenza virus from plasmid DNA, so that we could introduce mutations into the viral genome any way we want. So we can generate a virus that is not pathogenic, which the normal influenza virus is."

Vaulting Previous Hurdles

The obstacle he and his co-authors faced in this endeavor, Kawaoka recalled, "was that the previous technique didn't allow us that complete control. It enabled one to make virus containing a portion of the gene, derived from DNA plasmid, but the rest had to come from normal influenza viruses. That method was very complicated. But with this new technique of ours, scientists can generate any influenza virus."

Their novel system involved three major innovations, he said. "First, we used a human kidney cell line that can accept many plasmids. Second, we used a method that allows one to make viral RNA within the cell from plasmids. Third, I think most people didn't realize that cells can take that many different plasmids.

"The influenza virus genome consists of eight RNA segments," Kawaoka pointed out. "So we had to make these eight different molecules from plasmids, which are independent DNA segments that can replicate on their own. In addition," he went on, "we needed to have nine more of those plasmids, to express the influenza virus proteins. Some of them are polymerase; others, surface proteins; some internal proteins." In this entirely gene-cloned influenza virus construction, their system yielded 1 in 1,000 cells producing infectious virus particles.

Potentials: Flu Vaccine, Viral Carrier, Gene Vehicle

The university has applied to patent Kawaoka's invention, and he is now in the market for one or more investment-minded biotech partners.

"My message is this," he stated: "They can do three things: one, make influenza virus vaccines; two, use our influenza virus as a vaccine vector for other pathogens, because one can introduce a gene encoding, say, other viruses, and make influenza virus to express their proteins; and three, use this influenza virus as a gene therapy vector. So we can introduce a gene of interest and let the virus deliver that gene to humans or animals. Here, a typical application might be for cancer treatment, delivering, say, the p53 tumor suppressor.

"People have been testing adenovirus [AV] as a vector for delivering p53 to cancer cells," Kawaoka pointed out. "One of the problems with AV is that the human and animal recipients make an immune response to the viral vector itself. Influenza virus can infect the same individual many times, because the virus changes. There are many variants that are known to reinfect the same person. So with influenza virus," he concluded, "we can change the virus surface proteins and treat the same patient many times."

Meanwhile, he and his colleagues are busy "working now to do the same for the influenza virus's B strain."