AIDS has become the death-dealing scourge of Southern Africa. In Central Africa - and also India - it's malaria. That infection kills an estimated 700,000 to 2.7 million victims a year, most of them children under age 5, and infects 300 million to 500 million surviving sufferers. The true figures are likely much higher.
Unless some new strategies for controlling malaria come along, the malaria death toll is likely to double in the next 20 years. Human efforts to reduce the grim and growing grip of malaria are up against two enemies - a specialized mosquito and its parasite payload.
Of all the thousands of mosquito types tormenting vertebrates, only a handful, named Anopheles, transmit malarial parasites in their bite. The deadly microorganism they inject is Plasmodium falciparum - a protozoan - which enjoys a weirdly complex life cycle between the mosquito's gut and mouth, and on into the liver and blood of its human victims.
Its point of departure is when Anopheles bites an infected human and takes up its blood.
"When a mosquito ingests blood from an infected host," observed molecular geneticist Marcelo Jacobs-Lorena at Case Western Reserve University in Cleveland, "Plasmodium enters the insect's body and goes through several transformations before taking a form that the mosquito can pass on by biting another person."
Jacobs-Lorena is senior author of an article in Nature, dated May 23, 2002. Its title: "Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite."
"Related to the significance of our paper," Jacobs-Lorena told BioWorld Today, "is that unlike AIDS or TB, for instance, malaria can be transmitted only by an anopheline mosquito. Because people have not made much progress in fighting the disease with insecticides, drugs or vaccines, we needed to find different approaches. Our objective was to genetically modify the mosquitoes in such a way as to make them a poor host for the parasite, but still leave them as fit as the wild-type Anopheles.
"To do so," he explained, "we needed a gene that interfered with parasite development. Previously, we had identified a small protein that can attach itself to both the insect's midgut and its salivary gland. This peptide attaches to a putative receptor on the surface of the midgut wall. That receptor is also a molecule that has to be recognized by the parasite. So if we occupy all those sites with our peptide, when the parasite arrives it's unable to cross the gut and proceed with its development in the mosquito - so it dies off. That's the basis for our genetically engineered gene that encodes such a peptide.
"When we tested the genetically engineered mosquitoes," Jacobs-Lorena continued, "we found that indeed parasite development is inhibited by about 80 percent. And we found that the transgenic mosquitoes are also heavily impaired in their ability to pass the parasite to the next vertebrate host.
"This proof of principle showed that it is possible to genetically engineer a mosquito to make it resistant to the parasite."
Inhibition Was Quite Significant
Jacobs-Lorena and his co-authors constructed a completely synthetic gene in vitro. "To determine the sequence of this gene," he recounted, "we used a phage display library. Phages are essentially viruses that infect bacteria. They are made up of a DNA core and a protein coat. Other people modified the gene encoding the coat protein to extend it by 12 amino acids. The sequence that encoded those 12 amino acids was random, so each phage particle gene expressed a protein with a different peptide extension.
"Our library had an estimated 1 billion different peptides in it," Jacobs-Lorena went on, "phages displaying 109 different peptides. That was our starting material. Then we prepared a concentrated solution or suspension of the phages, and made the mosquitoes feed on it. The phage collection contained in the mosquito's midgut, in contact with the midgut wall, was the surface recognized and invaded by the parasite. After we injected the library," he continued, "we dissected the gut and opened it up in a sheet that exposed its inner wall. Next, we amplified it and fed it to mosquitoes again. We kept selecting for phages that bound tightly to the wall. So, after four rounds of selection, we saw that it was highly enriched for one specific peptide that we called SM-1 - for salivary gland and midgut. The gene that encoded SM-1 we inserted in mosquitoes encoding four units of the 12-amino-acid string."
Next: In Vivo Experiments With Mice
"They were just normal mice," Jacobs-Lorena said. "For measuring parasite transmission, we fed an equal number of transgenic and nontransgenic mosquitoes onto the same infected mouse. So both types of mosquitoes ingested the same number of parasites. Then we waited about 25 days, to allow the parasite to completely develop in the mosquito, after which we fed individual mosquitoes on individual na ve mice - that did not carry the parasite.
"We had 10 transgenic mosquitoes and 10 nontransgenic, each one feeding on a single mouse. Then we asked: How many of the animals that were bitten by the transgenic mosquitoes acquired the parasite? And then we compared them to the proportion bitten by the nontransgenic mosquitoes.
"In two of three such experiments, the block was complete, meaning none of the mice bitten by the transgenic mosquitoes were infected, while the nontransgenics did infect a significant proportion of the animals. So these experiments showed that the mosquitoes are impaired in transmission of the parasite.
"In continuation of this work," Jacobs-Lorena went on, "one important question is that our experiments all use the mouse parasite. So it will be important to determine whether the SM-1 peptide also blocks development of the human parasite, P. falciparum. That's one high priority, and experiments are in progress, using monkeys. The human parasite also infects certain primates.
"The major unanswered question is: How would we go about spreading the gene into the natural environment and control the transmission there of malaria?' One approach is population replacement - wiping out as many mosquitoes as possible in a target area with insecticides, then releasing the transgenic mosquitoes to occupy that biological niche. This is, I think, feasible, but not on a continent-wide scale. However," he concluded, "it will be very useful to verify the effectiveness of this approach in the field situation."