The Biodefenders (Part 2 of 2):
Red blood cells help the body resist infection by adhering to invading microbes; the cells perform this function with the assistance of antibodies and complement. Then, when these encumbered red cells pass through the liver, their foreign cargo is ingested by Kupffer cells and destroyed. The red cells themselves, relieved from temporary duty as dump trucks, return to the circulation unharmed.
EluSys was founded in 1998 on the work of Ronald Taylor of the University of Virginia, who developed a way to use bi-specific monoclonal antibodies to speed up antigen clearance. Taylor arranges two different monoclonal antibodies into what he calls a heteropolymer by crosslinking their constant (Fc) regions. One antibody in the heteropolymer takes the job of binding an antigen (an anthrax toxin, for instance); the other always binds CR1 (complement receptor 1), a protein found in greatest abundance on the surface of red blood cells.
A heteropolymer injected into a monkey (no human experiments have been done yet) quickly binds CR1 on a red blood cell. Once the other end of the heteropolymer binds its antigen, the red cell is ready for its trip to the liver. A key point is that the heteropolymer represents a short cut in preparing the red cell for its journey, for it dispenses with the need to activate the set of antigen clearance proteins collectively known as complement. Normally, the intricate complement cascade binds complement proteins to an antibody-antigen complex, and in the final step, anchors the complex to the red cell by attaching it to CR1. Taylor ingeniously bypasses complement by binding CR1 directly with one end of his heteropolymer.
Graphic courtesy EluSys Therapeutics
Antigen clearance using heteropolymers is very fast, says Linda Nardone, vice president of clinical and regulatory affairs. Taylor's proof-of-principle experiments showed that heteropolymers can reduce the concentration of a virus one million-fold in one hour; after two days, the virus drops below detectable levels. Injected before exposure, heteropolymers can protect mice against otherwise lethal infections.
The company's vision is that soldiers threatened by anthrax attack would receive injections of anthrax heteropolymers. "Instant immunity," Nardone calls it, lasting as long as the heteropolymers circulate, which she estimates could be three or four weeks. Consider that it takes over a year to develop immunity with the current anthrax vaccine, and you see the appeal of heteropolymers.
EluSys develops heteropolymers with USAMRIID under a CRADA (Cooperative Research and Development Agreement). EluSys is testing the heteropolymer potential of about 150 monoclonal antibodies created at USAMRIID for their anti-anthrax potential. The antibodies are made against anthrax toxins. EluSys' evaluation is unfinished, so no candidates have been chosen for further development. But USAMRIID is interested enough in heteropolymers that EluSys has been asked to work on a similar project against plague.
Biodefense is far from the only application for heteropolymers. The company's most advanced civilian project adapts heteropolymers to clear DNA-antibody complexes found with systemic lupus erythematosus; there are also projects to clear leukemias and lymphomas.
EluSys' contribution to biodefense won't be a vaccine, admits George Spitalny, vice president of R&D, but that doesn't mean it is second best: "A vaccine implies memory immunity. You don't get that here; you get immediate response and elimination." But if heteropolymers work well, he says, an anthrax vaccine might not be needed.
Besides working with antibody enhancer EluSys, USAMRIID works with antibody maker Abgenix Inc., of Fremont, CA. USAMRIID is in good company; Abgenix has a host of customers, A-list pharma companies eagerly placing orders for monoclonal antibodies. Abgenix earned its success with a tour de force of genetic engineering that gave mice the ability to generate the bulk of the human antibody repertoire. "There's no shortage of business," confesses Geoff Davis, the company's chief scientific officer.
It took Abgenix six years to place most of the human genes that generate antibodies into mice. Since the original mouse antibody genes no longer function in these animals, there's no question that any antibody they produce will be fully tolerated by humans. While xenomice, as they are known, can't make all the antibodies that humans can, they come "very close," Davis says. "We've been in business for five years and we've never found an antigen we couldn't make an antibody for."
Start to finish, Davis says it takes three months to make a human monoclonal antibody with one of these mice; from there to Phase I clinical trials would take about two years. Three Abgenix monoclonals are now in clinical trials, two of which it made for itself.
The aim of the collaboration with USAMRIID is to make human monoclonal antibodies against smallpox, Ebola and Marburg. USAMRIID makes and tests the antibodies using xenomice kept in its Ft. Detrick facilities. Abgenix will eventually manufacture and stockpile the antibodies USAMRIID chooses to produce. Discussions are underway for an anthrax project.
A hemorrhagic filovirus like Ebola, Marburg deserves to be better known. (Click here for electron micrographs of these viruses.) The name comes from Marburg, Germany, where it was first isolated in 1967. In his book, Alibek tells what happened when a researcher named Nikolai Ustinov accidentally injected Marburg into his thumb. Severe headache and nausea the next day showed infection had taken hold. Not until the fourth day did the virus truly begin to show its face, when small bruises splotched Ustinov's body as capillaries beneath his skin began to break. By the 15th day Ustinov's deepening hemorrhage could be read from his dark blue skin. Marburg forbids normal coagulation; Ustinov oozed blood. Trails of blood-black diarrhea fouled his bed sheets. He died soon after, as Marburg proceeded to "liquefy" his organs.
Normal burial was inconceivable. The ravaged corpse was soaked in disinfectant and then enclosed in a metal box, which was welded shut before being set inside a wooden casket. Virus sampled from Ustinov's body proved more stable and virulent that the strain that killed him, and naturally became the new favorite for weapons development. In 1989, about a year after the accident, a cryptogram passed across Alibek's desk informing him that Marburg Variant U had been successfully weaponized.
Abgenix's Davis is confident that human monoclonal antibodies can give excellent protection against these viruses. As "the response to these viruses is more in the nature of antibodies," he suspects that cell mediated immunity won't be needed. "If you're sending soldiers into battle where you know that the enemy has these weapons, the half-life of antibodies in circulation [about three weeks] is such that a single injection would be expected to last up to two months. If you give a higher-than-effective dose, it can go through a couple of half-lives and still leave an effective dose in the bloodstream."
Besides better prevention and treatment, the military needs ways to monitor the environment for an attack and identify the agent. A solution might be a device using DNA chips to analyze field samples for pathogens. DNA hybridization assays with a well-designed biodefense chip could identify which of a multitude of pathogens has been used for an attack.
Of course, the faster the pathogen's presence and identity are known, the more valuable the information. The need for speed is why DARPA and USAMRIID support development of prototype biowarfare agent detectors by San Diego-based Nanogen Inc. Nanogen's genetic diagnostic systems, which are used by clinical and research labs, are based on its NanoChip technology.
Image of NanoChip courtesy Nanogen
A pathogen detector for biodefense will be similar to Nanogen's current tabletop system, but smaller ("like a laptop computer," says Mike Heller, the company's chief technical officer; and maybe someday, "like a cell phone"). Just like the NanoChip system, it will use fluidics to bring reagents on and off the chip, and use laser scanning to detect hybridization. Its design will depend in part on where it is mounted and on the type of sample; helicopter or humvee; air, urine, or blood.
The military's device will also be more complex than Nanogen's commercial ware because it must prepare samples without human intervention. In the lab, someone prepares the samples; a soldier in the field won't do that. The machine must separate a field sample from surrounding dirt and grit unaided, and for genetic analysis, extract microbial DNA.
"What's completely different about our technology," says Heller, "is that we use electrical fields to greatly enhance the reaction kinetics and specificity." Nanogen applies an electrical field to the surface of the chip during the hybridization reaction. As a result, the hybridization probes for identifying pathogens become concentrated near the target DNA on the chip, and the reaction speeds up. "It's not science fiction," Heller comments, to believe that a biodefense chip could complete its work in less than 30 minutes. Handling immunodiagnostics assays would be possible, too, and take even less time.
With a DARPA grant, Nanogen is working on the sample preparation problem. "The problem in virtually all DNA-based diagnostic assays is sample preparation," explains Bruce Wallace, Nanogen's vice president of molecular biology. "What we're trying to do is to separate the organisms from everything else first, and then separate the DNA from the organisms." The company's researchers have adapted the electrical fields they use to move DNA to also move microbes. A technology called dielectrophoresis can isolate organisms on the chip surface. "The samples are all different and the organisms are all different, so we have taken this novel approach of using electronic devices," says Wallace.
Nanogen has been asked by USAMRIID to develop a chip to detect B. anthracis, Y. pestis, vaccinia, and Staphylococcus aureus. "Here it's a question of sensitivity," Wallace says. "Can you detect multiple organisms; can you work with samples containing multiple organisms?"
Before hybridization, the researchers will use DNA amplification to boost the concentration of sequences unique to each species. This is the only way to get a strong enough hybridization signal and reduce background. ("In diagnostic assays, the whole issue is signal-to-noise," according to Wallace.) PCR is less suitable for a biodefense chip than another amplification technique called SDA (strand displacement amplification) which Nanogen licensed from Becton, Dickinson and Company. The interesting thing about SDA is that it allows DNA amplification on the surface of a chip.
The stage is set for SDA after oligonucleotide primers (attached to designated chip locations) hybridize electronically with microbial DNA from the field sample. SDA yields amplified DNA just as PCR does, but with the difference that SDA-amplified DNA remains anchored to the chip at one strand. By anchoring reaction products to assigned positions on the chip, a huge number of amplification reactions pack into a tiny area without losing the ability to interpret the results.
Heller and Wallace are enthusiastic that these projects could not only help the armed forces, but also yield a commercial spin-off. Success could usher in a new product, "point-of-care" diagnostic chip systems that might go into doctors' office and ambulances, Heller says.
The Pentagon isn't the only government agency stockpiling biodefense vaccines. The Centers for Disease Control and Prevention (CDC) in Atlanta have a civilian defense project that calls for a national stockpile of 40 million doses of smallpox vaccine. The stockpile isn't just for young people born in the last twenty years: Many baby boomers' vaccines may have worn off, leaving them just as vulnerable. The 20-year contract for building and maintaining the stockpile belongs to Acambis plc, of Cambridge, MA and Cambridge, England. "It's about the longest government contract anyone's ever heard of," comments Thomas Monath, the company's vice president of research and medical affairs.
Acambis specializes in manufacturing live viral vaccines, "all the way from inception to R&D to manufacture," as Monath puts it. Its projects target diseases of the tropics that concern travelers -- yellow fever, West Nile virus, typhoid, traveler's diarrhea and others. The "millions of people who are traveling to endemic areas" will be Acambis' primary market.
Acambis will take the manufacturing method for smallpox vaccine though its greatest change since Edward Jenner's day, says Monath. "The smallpox vaccine goes back nearly 200 years, and over that time making it changed from a cottage industry to more formal manufacturing by pharmaceutical companies. But the method was principally the same."
Principally, that method called for growing vaccinia virus on a calf's belly. Later, you harvest the virus by scraping pox off the calf, get rid of chunks of skin and so forth, and there you are -- vaccine. The drug companies refined this of course, but according to Monath they still left bacteria in the licensed, finished product. Acambis will produce vaccinia in culture, harvesting it from human primary lung fibroblasts, and apply rigorous quality controls to assure a pure product. DynPort, in charge of the military's smallpox vaccine, will follow a similar process.
To secure its contract, Acambis took the unusual step of putting together a consortium of insurers to indemnify the government against claims for damages due to adverse vaccination events. Unlike the DOD, the Department of Health and Human Services, of which CDC is part, does not have the power to indemnify. The vaccine is not eligible for liability protection under the National Vaccines Injury Compensation Act, which concerns only children's vaccines. "It was thought simply more expedient for the vaccine manufacturer to indemnify the government," Monath says.
He projects that the new vaccine will be available in 2004. Acambis will have the right to sell smallpox vaccines to other countries. Monath mentions that several are interested. But first Acambis will bring the national stockpile to 40 million doses, ready to aid the country in time of attack. Smallpox warfare is a prospect the government has to take seriously, just as, he agrees, it must take seriously reports that smallpox stocks may have been smuggled out of Russia. (The world's two official repositories of the smallpox virus are Russia's Vector Institute and the CDC.) Monath, who was formerly chief of virology at USAMRIID, is unwilling to say whether he knows more about smallpox smuggling than what has been said in scattered news bulletins.
In BioHazard, Alibek lists more than a dozen nations with biological weapons programs, including some, like Iraq, that were signatories to the 1972 Biological Weapons Convention treaty banning their production. Some names will surprise no one -- Libya, Iran, Syria -- but the list itself is "just an approximation," he confides, these things being easy to hide. Look how well Biopreparat hid the massive scale of Soviet activities.
As advances in microbiology and immunology tell us more about the interplay between pathogen and immune response, some of these nations may use this knowledge for making weapons. With plenty of bacterial genomics data to draw upon for inspiration, bioweapons design could be headed into a new, harder-to-defend, even deadlier era. Passing a laboratory-cultivated pathogen through a man will not be the only way to enhance killing power.
Designer bioweapons aside, it is lamentable that weapons made with the old standbys like anthrax are so cheap -- taking "a thousand times less money to maintain than maintaining a nuclear capability," Alibek declares. A few hundred thousand dollars is enough to make a start on an anthrax weapon, although not of the kind Alibek was used to dealing with. Soviet bioweapons required a sophisticated industrial process, solving myriad problems of pathogen growth, concentration, stability, packaging, delivery and dispersal. But it is a mistake to take comfort in the technical hurdles before our enemies, he says: "Some people overestimate how difficult the technology is to apply. Unfortunately, they are wrong. All in all, a biologist knowledgeable in bioweaponry has a better chance of making a weapon than a physicist has in making a nuclear bomb." |
