THE ONLY certain thing about influenza is its unpredictability. Almost 19 years ago, a flu strain which had been around for years flared up and caused 17 000 unexpected deaths in England and Wales. This winter, by contrast, there have been only three mild outbreaks. Good news for most of the population. But if you鈥檙e searching for that most elusive of medical prizes 鈥 a wholly effective flu vaccine 鈥 you may not feel like celebrating. The reason: no flu means no feedback on the various experimental vaccines now being tested in clinics and nursing homes (for it is the elderly who are most at risk). In short, it means no data.
This year that may generate more silent scientific frustration than most. Never before have there been so many prototype flu vaccines to test in laboratories and clinics, or so many waiting in the wings. There are live vaccines, killed vaccines, and wannabe vaccines based on influenza proteins or protein fragments. Indeed research into influenza seems to be entering a bold new phase as scientists finally get to grips with the virus using genetic techniques.
For some researchers the big goal is to bump up the effectiveness of existing vaccines, most of them made from chemically-killed influenza particles or proteins extracted from such particles (see 鈥淎natomy of a killer virus鈥). Other laboratories have set their sights on transforming the flu virus into prototype vaccines for other killer diseases such as HIV and malaria. More ambitious still is a new type of experimental flu vaccine made from DNA rather than the usual proteins or viral particles. The real optimists are even talking of 鈥渁ll-in-one鈥 immunisations 鈥 鈥淒NA vaccines鈥 that in future would protect against several diseases.
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But it would be premature to celebrate. Even after decades of research the 鈥渄ead鈥 flu vaccines available in the West right now are far from perfect. They halve rates of hospitalisation and deaths among elderly people, but the good news stops there. On average, such vaccines prevent only about 70 per cent of infections, sometimes less if the recipients are frail or the vaccine is a poor match for the prevalent influenza strain.
Live issue
Things are different in the former USSR. There doctors can give live flu vaccines based on viruses grown at low temperatures. Such vaccines are not yet licensed in the West despite the fact that they may be just as effective as, and easier to use than, vaccines based on killed or inactivated virus. Millions of people in the former Soviet bloc have received live flu vaccines, says Christopher Potter, a virologist and now director of the University of Sheffield鈥檚 Institute for Cancer Studies. 鈥淚 would have thought we now know as much as we need to know about 鈥榗old-adapted鈥 influenza vaccines. They are safe, they are non-transmissible from person to person and they give protection against the disease.鈥
Robert Betts at the University of Rochester, New York, has reported distinct benefits. He found that live vaccines, when used alongside inactivated vaccines in elderly people, consistently and significantly reduced the incidence of influenza infection. Live vaccines also appeared to reduce the severity of the illness in those who did become infected.
There are other potential advantages, too. While flu vaccines based on inactivated viruses have to be injected, live flu vaccines can be given as nasal sprays or drops. And that鈥檚 not just good news for the needle-shy. A flu vaccine delivered by nasal spray or drops is more likely to stimulate an immune response where it is most needed to fend off future infections 鈥 in the respiratory tract.
A live flu vaccine sprayed into the nose, for instance, can bump up antibody levels in the tissue lining the respiratory tract for months. Injecting an inactivated vaccine, by contrast, raises antibody levels more diffusely in the blood.
Of course, antibodies are not the only immune weapons with which the body can fight off influenza. Immune cells may also swing into action to destroy infected cells. But here again, live influenza vaccines seem to produce the stronger response, triggering the body to produce higher levels of immune cells targeted at infected cells. The reason could be that live flu vaccine, unlike the inactivated kind, replicates in cells of the upper respiratory tract.
So why the delay in licensing live flu vaccines in the West? One reason, says Potter, is that Western drugs companies face more regulatory hurdles than their counterparts in the former Eastern bloc. Government watchdogs like the Food and Drug Administration in the US are renowned for setting stringent guidelines on the safety, transmissibility and genetic stability of modified viruses used as vaccines. 鈥淚f influenza changes every year, and we know it does, the company would have to make a different vaccine every year and satisfy the licensing authorities in a limited period of time,鈥 says Potter. The regulatory procedure in Russia is less drawn out, and as a result, says Potter, 鈥渧accines reach the market quickly鈥.
Another key drawback is that most years, any one of three or more strains could make people ill. Live vaccines work best against a single strain. When two or more strains are mixed in one vaccine, the immune system will tend to mount a response to just one strain in the vaccine. And there is always the fear that a live vaccine could revert to a virulent type, although so far there is no sign of that happening in the former Eastern bloc.
But perhaps the biggest problem with live flu vaccines is that, like their inactivated cousins, they still fall short of offering anything like complete protection. That in itself could be enough to deter drugs companies. Any that do take on the cold-attenuated live vaccine could soon find it eclipsed by other developments. Some researchers are developing prototype flu vaccines based on viruses that have been genetically engineered to make them less virulent. Others are trying to use artificial DNA itself as a vaccine, raising hopes of a new era of so-called 鈥済enetic immunisation鈥.
鈥淎 qualitatively new way of producing vaccination鈥 is how Stephan Johnston, professor of biochemistry and medicine at the South Western Medical Center in Dallas, Texas, describes it. 鈥淲hen we first tried to tell people about genetic immunisation, it wasn鈥檛 well received. People thought it was too simple and too unlikely to succeed. It took us almost a year to get our first publication.鈥
You may have thought that the influenza virus would already have been subjected to relentless genetic tinkering. Not so. In fact, until now influenza has largely escaped the genetic scalpel because it carries its genetic material in the form of RNA, rather than DNA. When it replicates, it is converted from one type of RNA to another, and never passes through a DNA phase. Standard techniques for manipulating virus genes work only on double-stranded DNA and can鈥檛 be used on influenza鈥檚 single strands of RNA.
鈥淎ll the fancy genetic engineering techniques work on DNA,鈥 says Peter Palese, professor of microbiology at Mount Sinai School of Medicine, New York, whose team has been pioneering new methods for genetically engineering influenza. 鈥淎n RNA virus like influenza doesn鈥檛 have a DNA phase in its natural life cycle. It just happens to have the wrong intermediate step.鈥
Artificial DNA
In 1990, Palese came up with the beginnings of a solution to this kind of problem. His approach, now called reverse genetics, was to build a DNA copy of the gene encoding haemagglutinin, the surface protein. In effect, Palese was creating an artificial DNA phase in the virus鈥檚 life cycle, a phase which he hoped would leave its genetic material amenable to manipulation. The strategy paid off. Using reverse genetics, Palese and his colleagues soon developed a recipe for making 鈥渁ttenuated鈥 flu viruses 鈥 viruses designed to be harmless yet stimulate the immune system.
The researchers have similarly manipulated other flu proteins, including neuraminidase. They are now testing viruses containing modified haemagglutinin as a vaccine in animals. The results are promising, says Palese. The vaccine, given as an aerosol, protects mice against infection with a closely matched strain and immunity seems to last longer than with an inactivated vaccine.
If this approach works in humans, the research won鈥檛 necessarily be restricted to influenza. Designer flu viruses may one day form the basis of prototype vaccines against a host of other diseases. Such vaccines will use the flu virus as a convenient carrier for genes and proteins that can protect against other diseases.
Already Palese and his team are looking at malaria. Using reverse genetics, they stitched into the flu virus genes encoding part of a protein found in the malaria parasite. So far, the results of tests on animals have been encouraging. The researchers sprayed mice with the designer flu virus and three weeks later, with a vaccinia virus similarly engineered to produce the entire malaria protein. Two weeks later, when the mice were injected with malaria parasites, those that had been vaccinated were protected from infection.
The aim of current research is to remove the need for vaccinia, which acts as a 鈥渉elper virus鈥, boosting the response to the designer virus. 鈥淲e can鈥檛 do it yet,鈥 admits Palese. 鈥淏ut we do want to get rid of the helper virus. We鈥檇 like to have a single compound.鈥
HIV target
Encouraging findings are emerging also from research aimed at converting the flu virus into a vaccine against AIDS. When HIV invades the body the immune system responds by producing 鈥渘eutralising鈥 antibodies. These antibodies target only certain parts of certain HIV proteins, but fortunately many of these so-called 鈥渁ntigenic鈥 regions have now been identified by immunologists. So too have the genes encoding these HIV proteins 鈥 a fact which Palese and his team have begun to exploit in their efforts to engineer a vaccine. So far they have inserted a gene encoding a segment of a neutralising monoclonal antibody (鈥2F5鈥) into haemagglutinin and given the resulting chimera to mice. It is too early to say whether this approach will prevent infection, but the vaccinated mice did produce antibodies capable of neutralising several strains of HIV.
That might sound promising but isn鈥檛 redesigning flu a rather convoluted route to an HIV vaccine? Why not just engineer HIV itself? The first advantage of influenza, says Palese, is safety. Attenuated HIV strains given as a vaccine by researchers at Harvard to newborn monkeys resulted in deterioration in their immune system. Palese: 鈥淭here鈥檚 some question about the ability to attenuate HIV sufficiently so that it鈥檚 protective but doesn鈥檛 cause disease.鈥
Influenza also produces an extremely strong immune response, stronger than most viruses. 鈥淚t gives long-lasting immunity. People can catch influenza several times only because the virus keeps changing. We want to take that good protective immune response to influenza and graft HIV genes on to it. Another asset, he says, is that influenza infects mucosal cells lining the respiratory tract. Mucosal cells all over the body are linked via the lymphatic system. A local immune response in respiratory mucosa also raises antibody levels in anal and vaginal mucosal cells. As such, it could provide a defence at the site where the HIV virus usually infects first.
Or so the researchers hope. Palese is reluctant to say how long it will be before the fruits of reverse genetics are used in humans. But he believes their scope is immense and may even stretch to cancer.
Genetic techniques may also help researchers to improve existing flu vaccines. The key problem with all of these vaccines is the variability of the virus: a vaccine based on one strain might not work against a different strain, and new strains can appear when least expected. Most changeable of all is the virus鈥檚 spiky coat. Spontaneous mutations, known as antigenic drift, can occur in hemagglutinin or neuraminidase, allowing influenza to slip past immune defences even ones primed by a previous infection. That is why hopes for a more effective inactivated vaccine are pinned on one of influenza鈥檚 core proteins, the nucleoprotein, which varies less from strain to strain than core proteins.
Another new approach to vaccination, so-called 鈥淒NA vaccination鈥 is able to make use of this stability. An experimental DNA vaccine consists of a piece of DNA containing the gene for influenza nucleoprotein. According to Margaret Liu, immunologist with Merck Research Laboratories at West Point, it can protect against different strains of the virus. In her remarkably simple system, a solution of recombinant DNA in saline is simply injected into muscle. Muscle cells soak up the DNA and start using it to make the influenza nucleoproteins 鈥 something the cells must do before they can stimulate an antiflu immune response.
鈥淲e don鈥檛 know exactly what happens in the muscle,鈥 says Liu, 鈥渂ut an immune response is generated against portions of the proteins made by the cells.鈥 Experiments with mice have shown that the DNA vaccine protects not only against the influenza strain in the vaccine, but also against other strains.
One worry is that the DNA vaccine might disrupt the cells鈥 own genes and perhaps increase the risk of cancer. But so far there is little sign of that happening in animal experiments. The injected DNA enters cells, says Liu, but doesn鈥檛 appear to be incorporated with the cells鈥 own DNA. Even so, nobody is being complacent. 鈥淭here is a finite risk that some of the DNA, once in a cell, will integrate with the endogenous DNA in the cell and maybe disrupt a gene important for preventing cancer,鈥 warns Johnston, who is also developing DNA vaccines. 鈥淭here is a finite possibility we could create a cancer cell, and that risk becomes more important depending on the age of the person you are inoculating,鈥 says Johnston.
鈥淚f you鈥檙e giving a flu jab to an 80-year-old person, you might not worry about it so much. But with a two or three-year-old, it鈥檚 more of a concern. We have to do calculations on the probability of that happening. It鈥檚 very very low, but it is measurable.鈥
An advantage of this vaccine is the potential strength of the immune response. Ideally, says Liu, a vaccine should generate a two-pronged attack, by antibodies as well as immune cells. 鈥淚f you can harness both arms of the immune system, you鈥檒l get more protection. Until now, that鈥檚 been difficult to do without using a live vaccine.鈥
Will a DNA vaccine do it? Liu鈥檚 experimental vaccine, based on one of influenza鈥檚 core proteins, certainly stimulates animals to produce killer T cells, something inactivated vaccines do only to a tiny extent. What is more, the T cells stop the virus replicating and seem to give the animals protection against various influenza strains, not just the one in the vaccine.
But so far this vaccine doesn鈥檛 stimulate the production of antibodies. This is because it is the surface proteins rather that the core proteins which produce this response. But DNA vaccines in future could encode both core and surface proteins, and give a 鈥渄ouble-barrelled鈥 approach to vaccination.
Liu is optimistic that potential obstacles will be overcome. 鈥淚t鈥檚 such a simple technology, yet it does appear to give a good immune response. It certainly has the potential to transform the way vaccines are made in future. The big caveats, of course, are that the technology is unproven in humans and we don鈥檛 yet know enough about all of the safety issues.鈥
Dominic Iacuzio, influenza programme officer at the National Institutes of Health near Washington DC, admits to being initially sceptical about the possibilities for DNA vaccination. Now he says: 鈥淗ow things change. I think it might not be too long before this work is being carried out in humans.鈥
But if DNA vaccines do take off, doctors will need a quick and efficient way of administering them. Injecting into the muscle may not be the most effective way of persuading cells to take up DNA. One alternative, being tested by Johnston in Dallas, is to use a 鈥済ene gun鈥. The DNA vaccine is precipitated on the surface of tiny gold beads, less than three microns in diameter.
Between one and ten million of these 鈥渕icroprojectiles鈥 are fired at the skin by the gene gun.
The gun uses high-pressure gas to produce a supersonic shock wave which acts as propellant. The beads lodge in the upper layers of the skin, depositing the DNA in cells and Johnston says accidental firings at experimenters 鈥渘amely myself鈥 have confirmed that the procedure is harmless. The cells start using the DNA to produce influenza protein and the immune system swings into action. But the effects aren鈥檛 permanent. Over the next five days, three weeks at most, the skin cells are sloughed off with normal wear and tear taking the vaccine DNA with them.
The advantage of the gene gun is that a much higher proportion of the vaccine ends up inside cells. And the temporary nature of the DNA provides some reassurance that it will not be permanently integrated into the body鈥檚 own DNA, which again is the major concern with DNA vaccines. Patients need only a hundredth the dose of DNA given in muscle injections. The disadvantage (if there is one) is that delivering vaccines to humans with a special gadget could make commercial and regulatory approval harder to secure.
With or without gene guns, Johnston is optimistic that genetic immunisation will find its way into the clinics 鈥減retty soon鈥. He is working on DNA vaccines against HIV and cancer as well as influenza, and says other innovations based on this technique are still to come. 鈥淲e鈥檝e shown we can incorporate 1000 different genes at once and still get a good immune response,鈥 says Johnston. 鈥淚n future we may get multipotent vaccines.鈥
That could mean vaccines capable of immunising against five influenza strains. It could even mean vaccines capable of immunising against several pathogens at once. In other words, says Johnston, the realisation of one of immunology鈥檚 great dreams 鈥 鈥渁 childhood vaccination protocol all in one shot鈥.
Anatomy of a killer virus
The influenza virus consists of genetic material in the form of nucleic acid wrapped in a spiky coat. The spikes help influenza particles to punch their way into and out of cells, normally those lining the respiratory tract. Most spikes are made of the protein haemagglutinin, which hooks on to receptors on the cells, pulling the virus with it. But some spikes wield a different protein, an enzyme called neuraminidase that helps the virus spread in a more subtle way. Having invaded a cell, influenza will replicate. Neuraminidase alters the surface of such cells so that newly produced virus particles can escape (see Diagram).
Fortunately, this seldom happens without alarm bells rining in the immune system. The virus鈥檚 spiky coat triggers an onslaught of antibodies that lock on to the virus particle. Immune cells swoop in to destroy cells infected with virus. And these responses are all the more vigorous if the immune system has previously encountered influenza.
The core of the virus contains eight separate strands of the genetic material RNA. These encode proteins such as haemagglutinin and neuraminidase which are vital to the life cycle of the virus. Without the RNA, these proteins cannot cause illness, but they can stimulate the immune system.
The 鈥渋nactivated鈥 flu vaccines available in the West are all prepared from viruses grown in hens鈥 eggs and inactivated with chemicals, either formaldehyde hyde or 尾-propiolactone. Some vaccines consist of inactivated whole viruses. In others, the virus has been 鈥渟plit鈥 open with detergent. Still others are prepared by separating the viral coat proteins, haemagglutinin and neuraminidase, from the genetic material found in its core. Each of these components is then purified and given to people in separate injections.
Inactivated vaccines are made from three distinct strains of virus because several strains of influenza circulate simultaneously. The strains are reassessed by the World Health Organization every year, and one or more is usually changed. This means that people at risk from influenza need to be vaccinated every year. Unfortunately, elderly people who are most likely to become seriously ill and die as a result of influenza are least likely to produce a good antibody response to vaccination.
Live flu vaccines are prepared from influenza strains grown for decades at temperatures of around 25 掳C. Such 鈥渃old-adapted鈥 strains lose the ability to replicate at core body temperature (although they can still do so in the upper respiratory tract where temperatures are much lower). To make a vaccine, the strains are 鈥渕ixed鈥 with the prevalent strain of influenza to produce composites. The strains used as live vaccines comprise the genetic core of a cold-adapted virus and the outer coating of the disease-causing strain. In other words, they 鈥渓ook鈥 like a virulent strain of influenza but contain the genetic material of a form of the virus which will only replicate in the cooler superficial lining cells, but not further down the respiratory tract.
Both types of vaccine suffer from the fact that the virus is constantly changing, which presents the immune system with a shifting target. Minor mutations 鈥 antigenic drift 鈥 accumulate in the structures of its surface proteins. Now and then a major change, or reassortment, of the virus results in the emergence of a new strain. Hence the pandemics of 1918, 1957 and 1968. New strains are usually denoted 鈥淗1N1鈥, H3N2鈥 and so on, reflecting the role played by the virus鈥檚 surface proteins in infection.
Another serious problem for vaccine designers is something immunologists jokingly call 鈥渙riginal antigenic sin鈥. This refers to the finding that the immune system tends to react most strongly to the first strain of a virus it encounters. Subsequent strains seem to produce less immunity.
That could spell trouble ahead. A flu virus dubbed H2 disappeared from humans in 1968 but continued to circulate among wild ducks. Its prevalence increased threefold between 1980 and 1988 and more recently it has been detected in domestic poultry. This virus can infect people, as it did at the end of the 19th ceentury, and again in 1957, and the likelihood of it causing a pandemic increases the older the population grows. People born before 1968 still have immunity, but by the first quarter of next century most of the population will have no immunity.
Space: the flu frontier?
Where do new flu strains come from? The conventional hypothesis points to animals. Ducks and other wild aquatic birds harbour many different strains of influenza, which can infect horses, pigs, other birds and humans. If a pig becomes simultaneously infected with both human and avian virus it can act as a 鈥渕ixing vessel鈥, producing a part-human, part-avian virus against which humans have little or no immunity. This may explain why new strains have tended to emerge in Southern China, where pigs live almost as family pets.
But there is a more controversial answer: space. Influenza originates from a pool of viruses and bacteria in the stratosphere, argue Chandra Wickramasinghe, professor of applied mathematics and astronomy at the University of Cardiff, and astrophysicist Fred Hoyle. Too light to be brought down to Earth by gravity, these stratospheric pathogens 鈥 which have yet to be detected 鈥 supposedly remain trapped until blown down by drafts of air. Such downdrafts typically occur during the winter months, which would explain, Wickramasinghe says, why influenza peaks in June or July in the Southern hemisphere and six months later in the North. It also accounts for the patchiness of outbreaks of the disease, he claims.
Working with family doctor John Watkins in Newport, Wales, Wickramasinghe examined the families of 2000 people with confirmed influenza and found that family members were no more likely to become infected than the rest of the population. 鈥淓veryone who was going to get infected tended to become infected on the same day,鈥 says Wickramasinghe, who calls the 鈥渁nimal theory鈥 a 鈥渟cientific myth鈥. Although influenza has not yet been detected in the stratosphere, Wickramasinghe says similar microbes have been found in meteorites. And only a few months ago (New 杏吧原创, 11 June 1994), the amino acid glycine 鈥 an essential ingredient of organic life 鈥 was found in a dust cloud in interstellar space, Sagittarius B2.
But the 鈥渟pace theory鈥 has yet to sway the majority of researchers, who still prefer the more down-to-earth notion that pigs act as mixing vessels for new flu viruses. Pigs are sold at least twice during their lifetime and that movement could be sufficient to account for the spread of influenza around the globe, says Robert Webster, an influenza researcher at St Jude鈥檚 Children鈥檚 Research Hospital in Memphis. Evidence for this from genetics is growing fast, he says.
- 鈥 The bird connection: Webster and his team in Memphis have shown that aquatic birds are infected with all known influenza strains. To do so, the researchers analysed the genetic sequences of influenza viruses isolated from animals, birds and people throughout the world.
- 鈥 Pigs as mixing vessels: Two years ago, Maria Castrucci, a virologist at Instituto Superiore di Sanita in Rome, working with Webster, found evidence of human-like flu viruses and avian-like flu viruses circulating together in Italian pigs. Viruses isolated before 1983 showed no sign of mixing between the two types of virus. But a human-like strain (鈥淗3N2鈥) isolated from pigs after 1985 contained genes of avian-like (鈥淗1N1鈥) viruses. Castrucci concluded that genetic reassortment of human and avian viruses took place in pigs at some time between 1983 and 1985.
- 鈥 Transmission from animals to humans: Last year an outbreak of influenza occurred in a group of Dutch school children. The virus was identical to the part-human, part-avian virus isolated from the Italian pigs. Webster: 鈥淗ere we have transmission from birds to pigs and then to humans. We can see it at a molecular level and follow it round by genetic sequencing.鈥
Even so, the conventional theory doesn鈥檛 explain everything. One awkward observation is the rarity of new flu strains. The last one appeared in 1968.
Why don鈥檛 we get new strains every year, or even every day, given the number of pigs, ducks and people in close contact? asks John Skehel, director of the National Institute of Medical research in Mill Hill, London. Skehel also believes that birds could infect humans without the help of pigs. 鈥淭he next pandemic strain might come directly from birds.鈥
And why do flu outbreaks often seem to strike different areas of the country, or even parts of the world, on the same day? One unproven idea is that the virus may lie dormant in people after an initial attack. Many years later a seasonal trigger 鈥 nobody knows what 鈥 reactivates this latent virus so that people suddenly become highly infectious without becoming ill themselves. Symptomless carriers might live in different parts of the country, even in different countries, but if they are subjected to the same trigger, they might become infectious at the same time.
Yet another puzzle is why flu should be more common in winter. One vague idea points to the influence of sudden changes in temperature, which may be more likely in winter than summer. Another cites seasonal variations in the body鈥檚 hormones. But it could simply be that people are most likely to crowd together indoors during winter months than in summer.