By David N. Leff
Testing sophisticated defenses against sky-borne enemy missiles is not being pushed solely by the U.S. government. Scientists all over the world are committed to this crash program.
Their rogue enemy is the Anopheles mosquito; its missile is the Plasmodium falciparum malaria parasite. Both are every bit as savvy as the armies of immunologists, parasitologists and vaccinologists engaged in bringing down these identified flying objects and their lethal payloads. Malaria takes the lives of some two million people a year, most of them children, in the tropical regions of the earth. ?It?s the most devastating parasitic disease in humans,? observed pioneer malaria vaccinologist Victor Nussenzweig, at New York University (NYU).
?Once you get bitten by an infected mosquito,? he said, ?the Plasmodium sporozoite-stage it injects into your skin with its saliva goes into your blood stream, then on to your liver ? very quickly. There the sporozoite stage starts to multiply, and releases thousands of merozoite-stage [liver] forms back into your blood stream. This initiates the erythrocytic cycle ? the asexual blood stage ? that causes the disease malaria.
?Antibodies against sporozoites have very little time to act,? Nussenzweig said. ?Therefore, after they enter the liver, they can only be attacked by serum produced by cytotoxic or helper T cells of the immune system?s cellular arm. But, at that point, sporozoites are still alive and producing proteins. Some of those proteins are carried over into the liver, including the main candidate vaccine, the circumsporozoite protein.?
Nussenzweig is senior author of a paper in the Proceedings of the National Academy of Sciences (PNAS) dated Aug. 14, 2001. Its title: ?Exploring the transcriptome of the malaria sporozoite stage.?
?There are many new findings there,? he told BioWorld Today, ?which suggest new candidates for vaccine development. We now have a cDNA [synthetic single-strand gene sequence] library of sporozoites that will give us a representation, probably, of the proteins that are expressed at that liver stage. And this is the first time it?s been done. It was considered impossible to obtain that kind of library because mosquito contamination was thought to be an impediment.
?Nobody knew beforehand what the other proteins are that sporozoites may have carried over, because nobody knew what proteins the sprorozoite makes,? Nussenzweig said. ?Now it is possible, perhaps, to predict some secreted proteins, which would go into the liver and be candidates for vaccine development. The proteins that we did know were very few. Now we have hundreds, and we are pursuing this idea that some of them may be good vaccine candidates.?
Celera Immunologist Counts Plasmodium Genes, Proteins
?Until now, we?ve all been dealing with the same handful of proteins ? say five at the sporozoite stage,? said biochemist Stephen Hoffman, vice president of immunotherapeutics at Celera Genomics, of Rockville, Md. ?The Plasmodium genome,? he added, ?has an estimated 5,000 genes, which express a minimum of 5,000 proteins. Those proteins can come only from the sporozoite stage,? Hoffman observed, ?or potentially at the early liver stage.? Before joining Celera recently, he directed the malaria vaccine program at the U.S. Naval Medical Center, in Bethesda, Md.
Hoffman made the point that this new order of magnitude ?provides a whole new set of targets to be investigated in the lab. ?This requires several more steps: The targets for a vaccine are proteins, not genes. So they might be proteins that are on the surface of sporozoites ? amenable to antibodies. They could be proteins expressed by infected hepatocytes ? targets for T-cell responses. They could be epitopes on the surface of merozoites, which are released from the liver. These would be targets for antibodies.?
Start-Up Girds For Early Clinical Trials
Apovia Inc., a year-old start-up biotechnology company in San Diego, cooperates closely with Nussenzweig. The firm?s director of research, biochemist Ashley Birkett, told BioWorld Today: ?What we?re working on with Victor and other NYU scientists is that we have a unique way of presenting epitopes [antigens] derived from pathogens. We have developed a malaria vaccine candidate, which presents B- and T-cell epitopes from the parasites. These epitopes were identified at NYU,? Birkett said.
?We believe that the new twist by which they?re presented on our particular carrier is optimal for eliciting a protective immune response against malaria,? Birkett said. ?We?re presenting both types of epitopes from the key circumsporozoite coat protein. And we?re looking to elicit a neutralizing antibody response to the parasite, to prevent the sporozoites from invading the liver. The first line of defense is to neutralize the sporozoites before they get to the liver.
?We also have a T cell epitope presented on the vaccine,? he continued. ?If we?re not effective in neutralizing 100 percent of the sporozoites by the antibodies, we believe we have a safety net mechanism, whereby ? as we?ve shown in animal models ? we can target P. falciparum-infected hepatocytes effectively.
?Our unique technology is a modified version of the hepatitis B virus core protein,? Birkett said. ?It?s known to be a very immunogenic protein in humans. We?ve engineered it to be expressed in E. coli. They spontaneously form particulate structures inside the bacteria ? which act as carriers for the target epitopes.
?We?re on the verge of clinical trials,? he said, ?and working on the IND right now. It?s going to be NIH-sponsored. We have some very good collaborative arrangements, funded by NIAID [National Institute of Allergy and Infectious Diseases], through the SBIR [Small-Business Innovative Research] program. Also we have funding from an organization called the Malaria Vaccine Initiative, which gets financing from [computer magnate] Bill Gates. We were actually the first company to receive an award from them.
?The NIH-sponsored Phase I trial will be conducted at the University of Maryland, by the end of this year or early next year,? Birkett said. ?It will enroll 48 healthy volunteers initially. Then, if we get the safety profile we expect, it will progress to an infectious challenge trial next year.
?We have done a lot of preclinical trials in nonhuman primates,? he said, ?and showed our vaccine to be highly immunogenic in both rhesus and cynomolgus monkeys. The Nussenzeig?s epitopes delivered on our platform are orders of magnitude higher than they?ve ever been before.?