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Navy Wages Biotech War On Malaria
The U.S. Navy's warships are as smart as they come: Thanks to cutting-edge information technologies, these ocean-going fighters are outfitted with networking operating systems, relational databases, and all the other high-tech systems and equipment that can be found in any information-intensive facility or business. The war-fighting network is vast, indeed, linking together all U.S. forces -- and eventually even America's allies.
But the Navy's also using high-tech to fight its non-human enemies -- the infectious diseases and parasites that end up killing or seriously injuring more troops than any sort of weapon. Chief among those is malaria: According to Daniel Carucci, the new director of the Navy's malaria program, "In every [military] campaign in the 20th century, more people were lost to malaria than to bullets."
Today, the Navy's as deeply involved in cutting-edge research in genetics, genomics and immunology as any biotech company. Moreover, to aid its internal malaria programs, it's established a wide network of alliances with biotech firms and non-profit organizations. Together, these scientists finally have the weapons they need to bring down their formidable opponent.
Of course, it's not only military personnel who are dying from malaria. According to the World Health Organization (WHO), malaria kills more than one million people every year -- 90 percent of them children living in sub-Saharan Africa. Moreover, almost 300 million clinical cases of malaria occur annually and 40 percent of the world's population lives where there is a risk of contracting the disease.
Thus, despite the best efforts of hundreds of dedicated scientists over the decades -- even centuries -- mankind is still afflicted by this most ancient of scourges. Treatments come and go, but the disease remains as fearful -- and feared -- as ever. That's because the enemy is extraordinarily clever: Not only does the parasite quickly learn to resist whatever drugs it encounters, but also the female mosquito (Anopheles gambiae) that transmits the parasite to humans quickly adapts to insecticides, making it virtually impossible to wipe out.
Moreover, there are four strains of the parasite that infect humans -- although Plasmodium falciparum is the most dangerous. This genetic diversity means it's even harder to devise a drug or vaccine that might affect them all. To top it all off, the parasite has an extremely complicated life cycle: It spends most of its life in its human host hiding from the immune system, either in liver cells (where sporozoites mature into merozoites) or red blood cells (where merozoites multiply and mature into sexual forms which mate once they're back in the mosquito's gut, forming sporozoites once again).
Interestingly, many individuals who live in endemic areas and are infected time after time do eventually develop immunity to disease. "Children who make it to the age of 10 and don't die of malaria and have no serious disease" have acquired immunity, according to Carucci.
As well, researchers performed one clinical trial in which healthy volunteers were injected with irradiated sporozoites (delivered via mosquito) and were then challenged with bites from infected mosquitoes. These individuals were protected -- showing once again that an immune response is not only possible, but that it can protect against the disease. The trick is figuring out how to create an appropriate vaccine -- one that will stimulate both humoral and cellular immunity as well as one that will be active against all life-cycle stages.
The Navy's malaria program, which is part of the Naval Medical Research Center in Silver Spring, MD, is "structured like a small biotech company," Carucci explained. Research conducted by the military and civilian personnel spans the spectrum -- from genomics, applied genomics and immunology to regulatory affairs and clinical trials, he continued. And most of this R&D is now focused on devising DNA-based vaccines.
In fact, the Navy's research group -- led by Stephen Hoffman until January 2001, when he left to join Celera Genomics as its senior VP of immunotherapeutics -- was the first to publish the results of a single-gene DNA vaccine trial in 20 healthy human volunteers. Those results, published in Science in October 1998, demonstrated that a DNA vaccine is safe and well tolerated. Moreover, the majority of the test subjects developed a killer T-cell response, indicating that such a vaccine can generate an immune response. That's critical, because without the cellular immune response, it's unlikely that any vaccine will prove very effective.
This first vaccine resulted from a long-standing collaborative effort involving the Navy, Aventis Pasteur and Vical Inc. -- an alliance that centers on the use of Vical's naked DNA technology for making vaccines. In brief, the technology involves the construction of plasmids that contain the gene of interest, as well as short sequences that control protein production. These plasmids can be directly delivered into cells, without the need for delivery vehicles or viral vectors.
By August 2000, Phase II clinical trials of a multi-gene DNA vaccine were underway. This new vaccine, designated MuStDO 5, incorporates five genes that are designed to cause production of P. falciparum immunogens and trigger an immune response against both the liver and sporozoite stages of the parasite's life cycle. The trial tested not only the multi-gene vaccine, but also a naked DNA agent encoding granulocyte macrophage-colony stimulating factor (GM-CSF) to stimulate the immune system. According to Vical spokesman Alan Engbring, the vaccination and challenge aspects of the Phase II trial are now complete, and results are expected in the second half of 2001.
Once the results of the Phase II multi-gene trial are analyzed, it should become evident whether GM-CSF acts as an effective immune stimulant. Meanwhile, the Navy's team has been investigating other ways to boost the immune system as well as alternate delivery methods which might improve the vaccine's effectiveness. These efforts stem largely from its numerous agreements with biotech companies, some of which are highlighted below.
For instance, the researchers have already found that a needle-free jet injection device developed by Bioject Medical Technologies Inc. "seems to improve the immune response," according to Vical's Engbring. That device, the Biojector 2000, was used in both malaria vaccine trials.
But the Navy's also investigating alternate delivery methods. For instance, it's testing a non-viral gene transfer method developed by Cleveland-based Copernicus Therapeutics Inc. under a Cooperative Research and Development Agreement (CRADA) signed in December 2000. Copernicus Therapeutics' PLASmin Complexes consist of single molecules of plasmid DNA condensed with polycations to achieve the smallest possible size. These particles end up being 18 to 25 nanometers in diameter, according to Mark Cooper, Copernicus Therapeutics' senior VP of science and medical affairs. The starting size of the plasmid is not a limiting factor, either: "Most plasmids we work with are 4 to 8 kilobases, but we've compacted plasmids as large as 100 kilobases," Cooper said. The complexes are readily taken up by cells, he said, and because they are able to cross the nuclear membrane, they can be expressed even in non-dividing cells. It's also possible to selectively target the complexes by attaching them to cell-specific ligands.
This approach could prove very useful, especially because the DNA vaccine candidate currently in trials contains five genes -- and there are plans to increase that number to eight in the near future.
There might be other ways to affect the immune system, and the Navy's investigating those as well. Through its ongoing CRADA with Cel-Sci Corp., for instance, it's looking into the feasibility of applying the company's LEAPS technology to modulate T cells. LEAPS (ligand epitope antigen presentation system) compounds consist of a peptide epitope associated with a disease-causing agent linked to a T-cell binding peptide ligand. Together they induce the immune system to mount either a cellular, humoral or mixed immune response. According to Daniel Zimmerman, Cel-Sci's senior VP of research, cellular immunology, in malaria the focus is on directing the immune system towards a cell-mediated response. "We know that T cells are involved in malaria and that an antibody response is not enough."
Experimental results in a mouse model system have shown that a peptide based on the LEAPS technology was able to induce protective immune responses against challenge from malarial sporozoites. The next step is to develop this approach for use in humans, Zimmerman said. "We've worked with peptide sequences common to mouse and human, but we haven't started working with the human peptide sequence yet." (Rodents are infected with a different strain of the parasite, Plasmodium yoelii.).
The Navy's also got a collaboration with Epimmune Inc., under which the company is applying its functional genomics technology, ImmunoSense, to the malaria parasite's genome. ImmunoSense will be used to identify antigens and antigen fragments from primary DNA sequences to formulate a vaccine.
First the genomic data are analyzed via computer algorithms to predict which genomic sequences will code for antigens that can be recognized by T cells. From there, researchers will run assays to generate a database of biochemical and immunological information that can be used to narrow the choice of targets for vaccine development.
Identifying promising malarial epitopes is no small undertaking, either, for each stage of the parasite's life cycle exhibits different antigens -- all of which, theoretically, could be used in vaccines. The trick, once again, is finding out which particular antigens are recognized when a human host does mount an effective immune response -- as in the irradiated sporozoite trial described earlier, where the test subjects were protected from challenge. "If we can recreate this immune reaction, then we have a vaccine," explained Alessandro Sette, VP and chief scientific officer at Epimmune. "But the sporozoite is made up of many different antigens, and nobody knows which antigens are recognized." Moreover, the basis of the protective mechanism is not understood.
Making sense of it all is where Epimmune comes in, Sette said. Epimmune will "scan the sequences of all predicted proteins, pull out the fragments that have a chance of being recognized and screen them against the blood of the individuals who participated in the irradiated sporozoite trial. This way, we can determine which proteins are being recognized in the people who are protected." From there, it might be possible to "reconstruct" a vaccine. "We're linking genomic data with biological information."
This approach would be infinitely harder if the malaria genome sequencing project weren't already well underway. According to Sette, "There's still a lot of sequence to be analyzed, but we can start working with what's already available."
Indeed, researchers are not only sequencing the genome of the malaria parasite, but also that of its insect vector. The International Malaria Genome Sequencing Consortium was formed in 1996 to sequence the genome of P. falciparum (25 million base pairs). Sequencing is being performed by The Institute for Genomic Research (TIGR), the Navy's malaria program, The Sanger Centre and Stanford University. As well, in April 2001 researchers and genome sequencing centers from around the world convened at the Institut Pasteur to organize an international network for sequencing the genome of A. gambiae (260 million base pairs). The organizations -- which include TIGR and Celera Genomics, among many others -- anticipate completing the first version of the mosquito sequence this year.
The malarial parasite has 14 chromosomes and an estimated 6,000 - 7,000 genes. To date, only chromosomes 2 and 3 have been completely sequenced and annotated, but preliminary sequence data exist for greater than 90 percent of the genome. The consortium anticipates that the entire P. falciparum genome will be sequenced by the end of 2002.
Although the sequence is far from complete, "we've already identified some key technologies for using the genome sequence in vaccine development," Carucci explained. For instance, the parasite's DNA content is "very rich in A+T [adenine and thymine], about 82 percent, whereas human DNA is about 45 percent A+T," he continued. Not only did this difference make it difficult to get good clones in bacteria, Carucci said, but also it might have contributed to less-than-optimal immune responses. Consequently, researchers have been able to construct synthetic P. falciparum genes that are codon-optimized (so each amino-acid-coding triplet matches the preferred codon usage in humans) and they've found that the immune response in cultured human cells, and in mice, is substantially improved.
The Navy's malaria program is about 12 years old -- and it's been working on vaccines for the past five years. But it's only one of the many organizations involved in malaria research, a worldwide effort that's intensified in recent years. In fact, another sort of vaccine candidate is entering clinical trials this month: GlaxoSmithKline Biologicals, in partnership with the Malaria Vaccine Initiative, is about to start trials of its RTS,S vaccine (a recombinant polypeptide consisting of part of the sporozoite protein fused to hepatitis B surface antigen) in children in The Gambia, West Africa.
Cover image: Female Anopheline mosquito, courtesy Stephen L. Hoffman
originally published 05/11/2001 |