Plantigens: best prospect for defeating Malaria?

Australian malaria vaccine researcher Prof Ross Coppel says smallpox was eradicated from the planet because the mass vaccination campaign employed a powdered formulation of dried, attenuated virus.

It was very cheap, and so stable that it did not require refrigeration to prevent it denaturing in tropical heat. But if and when researchers succeed in developing an effective malaria vaccine, it is likely to be expensive to produce in conventional microbial fermentation plants.

And that's just for starters -- the next challenge, says Coppel, is the poor transport infrastructure and lack of refrigeration in malaria's tropical haunts.

An edible vaccine

Coppel's Monash University team hopes to solve these problems with an ingenious, super-cheap production plant: a lettuce, or maybe a carrot.

"This really relates to the logistics of how you deliver a vaccine, cheaply and effectively, to a target population of something like 2 billion people," Coppel said.

A new vaccine is likely to employ at least two to three different antigens and target different stages of the parasite's life cycle to generate a multi-pronged immune response against the parasite. Plasmodium falciparum has a chameleon-like capacity to evade its host's defensive responses by switching antigenic guises.

"We're looking for something hardy," Coppel said. "The original impetus for edible vaccines came from the idea that if we could move production of the proteins away from high-tech, good manufacturing practice (GMP) facilities, then they could be made at lower cost.

"GMP facilities require huge investment in infrastructure like factories and fermentation vessels, and a highly skilled workforce. If we could make the vaccine in a Third-World setting, and dry and powder it, nations could deploy it within their own endemic areas."

Refining an oral vaccine

Despite evidence of successful immunisation against gastrointestinal viruses, bacteria and parasites, it was not clear whether an oral vaccine would work against a blood-borne parasite.

The Monash researchers obtained proof-of-concept by feeding laboratory mice antigens from Plasmodium yoelli, which causes a virulent form of malaria in mice. They used three merozoite surface proteins (MSPs) homologous to the P. falciparum antigens: MSP1, MSP4 and MSP5, alone and in combination.

Coppel said that MSP-1 alone elicited a systemic antibody response, that provided "significant protection" against P. yoelli challenge. Adding MSP-4 and MSP-5 to the mix conferred even stronger protection against infection.

"We had a theoretical demonstration that such an approach was viable, but it's been a much more difficult process to engineer plants so that when mice eat them the antigens will do the same thing -- we have quite a long way to go," he said.

The approach involved cloning the genes of interest from the parasite, and transferring them into tobacco -- an experimental model chosen because it transforms readily, grows rapidly, and delivers high yields for assay purposes. When the system has been proven, lettuce, carrots or tomatoes will be the preferred method of producing the vaccine.

The Monash researchers struck problems with the very different codon bias between the malaria parasite and crop plants -- despite its origins as a marine alga, the parasite's DNA shows a strong A-T bias, whereas plant DNA is G-C rich.

By modifying the parasite's MSP genes to correct this bias, Coppel's team obtained much better expression. However, the degree of antibody protection was strongly dependent on the level of protein ingested.

"It became clear that patients would need a really hearty meal of lettuce or carrot to obtain protection, so we needed to raise protein levels by making the malaria gene more strongly turned on inside the plant.

"We're looking at a variety of ways of increasing the protein expression in plants including using stronger promoter sequences that tell a plant cell to switch on production of proteins.

Strong promoters

The Monash research team has made the move from experimentally expressing the genes into tobacco and has already begun inserting the genes into crops such as lettuce.

They are also collaborating with US researchers to use a chloroplast expression system for malaria antigens.

The chloroplast genes that synthesise chlorophyll and rubisco -- the latter is the world's most abundant protein -- have the very strong promoters needed to maximize expression of malaria antigens while minimising the bulk of material subjects would need to ingest for immunity. These experiments are being performed in carrots.

Coppel said the original notion of having villagers in remote areas growing transgenic vegetables to protect themselves against malaria is not feasible, because of the problem of controlling dosage.

A better idea is to grow the vegetables in a central farm, dry them for extended life during transport and storage, and package them in the correct dose. This will still result in a much cheaper vaccine than is possible by other means.

Another advantage is that the regulatory process for obtaining permission to market a vaccine is much easier for vaccines that are ingested compared to vaccines that are injected.