BioWorld International Correspondent

LONDON - A vaccine to protect humans against malaria using live, genetically modified parasites might one day be possible, a study carried out in rodents suggests.

Deletion of a single gene from the malaria parasite Plasmodium berghei resulted in live parasites that were able to infect rodent liver cells but unable to spread into blood cells. An equivalent gene is present in Plasmodium falciparum, the species of parasite that causes most deaths in humans.

"This is a big step forward toward a vaccine against malaria," Ann-Kristin Mueller, of Heidelberg University School of Medicine, told BioWorld International. "It provides proof of principle that if you knock out only one gene in this parasite, you get an attenuated, live, whole-organism vaccine. It is really quite amazing."

Mueller, who carried out the study for her doctorate thesis, cautioned that more research would need to be completed before trials in humans could begin. In particular, scientists have not yet successfully deleted genes at will from the genome of P. falciparum, although work is under way.

The work on P. berghei is reported in the Dec. 6, 2004, issue of Nature in a paper titled "Genetically modified Plasmodium parasites as a protective experimental malaria vaccine."

Malaria is transmitted when an infected mosquito bites one of the parasite's vertebrate hosts, such as a human. The mosquito injects a form of the parasite called a sporozoite. Those migrate in the blood to the liver, where the parasite invades liver cells, transforms into trophozoites and replicates, eventually releasing thousands of liver merozoites into the bloodstream. They invade red blood cells, multiplying and eventually releasing blood merozoites, which repeat the cycle of infecting red blood cells. It is the cyclical, synchronized release of merozoites from red blood cells that causes the periodic fever and other symptoms typical of malaria.

When another mosquito bites the infected person, that insect becomes infected. The parasite invades its mid-gut, developing into what is called an ookinete. The ookinete then invades the mosquito gut lining and develops into an oocyst, which produces hundreds of sporozoites that migrate to the salivary glands, ready to be injected when the mosquito next takes a blood meal.

Kai Matuschewski, team leader on the project in the department of parasitology at Heidelberg, already had identified genes that were up-regulated by the sporozoites in the salivary glands of the mosquito, but not by those found in the mid-gut of the insect. Mueller then began to characterise those genes, with the aim of determining which were important for the parasite's survival in the host, for example, or for allowing it to establish itself in the liver.

A gene called uis3 was one of the most strongly expressed in the salivary sporozoites, so the team decided to knock its function out in P. berghei.

The researchers found that the genetically modified parasite was still capable of infecting mosquitoes. Further experiments in the laboratory showed that although the sporozoites with uis3 knocked out were able to infect cultured hepatocytes and transform into liver-stage trophozoites, their development ended there; they did not go on to produce blood-stage merozoites.

Using a rodent model of P. berghei infection, the team then discovered that when mice were infected with the genetically modified parasites, those animals, too, never developed blood-stage infection.

But would animals treated in that way be immune to wild-type parasites? Mueller and her colleagues embarked on a project to evaluate immunization protocols and ways of challenging the immunized animals. They found that a regimen involving a priming injection followed by two boosts protected the animals from infection with wild-type sporozoites.

"It was really exciting when I saw that all the immunized animals were blood-stage negative," Mueller recalled.

The researchers showed that the immune response protected against the sporozoite phase of the mosquito's life cycle: When they directly inoculated mice with blood-stage parasites, all developed high levels of blood-stage infection within two days.

In the long run, the team wants to do human trials using a similar protocol with P. falciparum as they used for P. berghei in mice. "But first we have to knock out the uis3 gene in P. falciparum," Mueller said. "We also want to analyze the immune response that is responsible for the protection."

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