Fourth stage larva of Aedes aegypti, CDC/Dr. Pratt.

The virus that causes epidemics of dengue fever in south-east Asia and central America, and is threatening Australia and the US, evolves new strains so rapidly that an effective vaccine is many years away, today warned Vincent Chow, associate professor in molecular genetics at the National University of Singapore.

Dengue fever infects as many as 60 million people each year, resulting in around 500,000 hospitalizations and 30,000 deaths worldwide. Two of the most recent outbreaks persist in Rio de Janeiro and in Cuba, where urban overcrowding encourages vector transmission by the mosquito Aedes aegypti.

The disease has recently begun to encroach upon the Northern Territories and Queensland states of Australia, while Florida's proximity to Cuba has US authorities worried.

"To me, it's only a matter of time before dengue will go into the US", Chow told BioMedNet News.

While four main serotypes of the virus are found in all parts of the world, there are dozens of different strains within each serotype, which show more regional variation.

Chow's team is now revealing the impact that such variations might be having on the immune defences of infected individuals, and for vaccine design.

Until now, the only vaccines to enter clinical trials have been live vaccines containing a mix of four strains, representing each of the four main serotypes. But tests so far have been hampered by the reversion of one of the strains back into a more pathogenic organism, according to Chow.

Chows findings now challenge the usefulness of limiting these "tetravalent" vaccines to only four strains. Strain variation is so wide that the antibody response after infection with one strain fails to protect against challenge with a second strain of the same serotype, he says.

This is the picture at least as far as experimental mice are concerned, infected first with a local variant of serotype 2, called Den 2 Sin, and then infected again with the main reference strain of serotype 2, called Den 2 NGC.

Serotype 2 is the one most associated with the serious manifestations of disease, hemorrhagic fever and dengue shock syndrome, and therefore highest on the hit list for vaccines.

Taking their analysis a step further, the team used gene sequencing to identify no fewer than 16 mutations which appear to account for the antigenic differences between the two strains. Many mutations occur in the region of the viral envelope protein that forms the binding site for entry into human monocytes, the white blood cells that are thought to be the targets of infection.

"If that's true, then you've got not four serotypes, but n serotypes, which could become a huge problem," agreed Tom August, a vaccine researcher at John's Hopkins University in Baltimore.

Chow's team are now placing their bets for the best vaccine candidate on a more conserved protein of the virus, the protease known as NS3, which normally splices the virus's initial polyprotein chain. They are now testing this as a construct in a DNA vaccine, together with a second target, NS1, for its ability to protect mice against viral challenge.

His concern is that the tetravalent vaccines currently in clinical trials will be inadequate. "A vaccine that works against certain strains may not be useful against new strains that may emerge in the future. From the molecular work they are emerging very quickly," he said. In addition, the team has conducted an epidemiological study on healthy student volunteers attending the National University of Singapore.

They found that as many as 40% of the group have antibodies to dengue viruses, suggesting some past exposure to the virus. The students were unaware of ever having been infected. But in only half of these cases were the antibodies detected by ELISA tests able to neutralize serotype 2 virus.

This means that even after natural exposure, many people are still vulnerable to a second infection. Furthermore, those with non-neutralizing antibodies face the risk of even worse disease, through a phenomenon known as "antibody enhancement" in which antibodies bind to the virus, and enhance its uptake into monocytes through Fc receptors.

"We need a tetravalent vaccine, given early in life. But the bad news is that there is not going to be a vaccine anytime soon," concluded Chow.