By Merrill Goozner

Beija-Flor, Brazil- It took a four-wheel-drive vehicle a kidney-jarring hour to traverse five miles of rutted, rocky road leading to the one-room church serving the agricultural settlement. Once inside, immunologist Jeff Bethony of George Washington University (GWU) and physician David Diemert of Washington, DC-based Sabin Vaccine Institute, pushed aside the wooden benches to set up a scale, an examination space, and a makeshift interview room.

The researchers were at a critical juncture. Today, they were going to register the first volunteers for a clinical trial that would test the safety of a hookworm vaccine in people routinely exposed to the parasite, Necator americanus.

The researchers hovered nervously nearby as a local physician and several assistants interviewed the first three candidates. Azizo Alves Dos Santos, 36, one of the first to arrive, was eager to volunteer. He previously had several serious hookworm infections, which sapped his strength and kept him away from the hillside fields where he grew beans and manioc, the local staples. An active member of the local agricultural laborers association, Santos was familiar with the toll that the disease took on farm workers like him. When they can't work, they don't get enough to eat. "This vaccine will benefit this region, Brazil, and all the world," Santos says through a Portuguese translator.

Unlike developing world pandemics such as HIV/AIDS and malaria, mortality among the 500 million people worldwide infected with hookworms is not so high - an estimated 65,000 people a year. Studies have shown, however, that hookworm infestations reduce agricultural productivity by 40% and lead to a 25% reduction in childhood educational performance among those chronically infected. An estimated 44 million hookworm-infected women give birth each year. They deliver low-weight babies, have impaired milk production, and have higher-than-expected infant and maternal mortality.

Infection requires only a few dozen larvae migrating from the lungs to the bronchial tract, where they are coughed up and swallowed. The surviving larvae eventually latch onto the intestinal wall where they grow to maturity while sucking the victim's blood. They also mate, with the females' eggs exiting the host in feces. After hatching in the surrounding environment, the larvae can live as long as a year in warm, wet climates before infecting other people, thus completing the cycle.

Though a three-day course of benzimidazoles (albendazole or mebendazole are most commonly used) effectively eliminates the worms, in poorer precincts where clean water and sanitary facilities are rare, reinfection within less than a year is common. Even in areas with annual deworming campaigns - the World Health Organization's Partners in Parasite Control program estimates that 55.6 million children were dewormed in 2005 - infection rates remain above 50% of the local population. In this section of Brazil, for instance, the infection rate is above 60%.

Even in relatively prosperous Brazil, deworming campaigns can pay only sporadic visits to isolated rural communities. It would take four years to get to every pocket of the Texas-sized state of Minas Gerais, where hookworm is endemic, according to Rodrigo Corrêa Oliveira, chief of the immunology laboratory at René Rachou FIOCRUZ, the Ministry of Health's research arm in Belo Horizonte. "The Gates Foundation is funding a massive deworming program in Africa for which we're producing the drugs," he says, "but it is not sustainable."

The extent to which a person gets sick is directly proportional to the number of worms inhabiting his or her intestinal tract. Reducing that number can substantially reduce the overall disease burden, which argues for a vaccine. The principal developer of the vaccine, Peter Hotez of GWU, knows that creating a vaccine that would make an individual totally immune to hookworm infection is not possible. "The goal," he says, "is to elicit an antibody response that reduces the number of larvae that will migrate and become adult hookworms."

Vaccines against multicellular parasites such as N. americanus, however, have proven almost impossible to produce. That's because most vaccines work by mobilizing the immune system to generate a massive response to the presence of a pathogen, usually a bacteria or a virus. This sterilizing immunity kills off the pathogen shortly after exposure. The natural immune reaction to the presence of a large and complex organism simply isn't adequate to provide protection.

This inadequacy occurs in part because hookworms, which can grow to one centimeter in length once they lodge in the intestines, have their own immune systems. After larvae enter humans through the skin, the worms deploy defenses that may cloak their presence in the blood stream during their early stages of life. Moreover, its extensive genome - the Sanger Institute estimates 25,000-plus genes - produces many proteins that interact in complex ways. So even when the human immune system produces antibodies, it is usually only in minute quantities and often aimed at the wrong targets. The ineffective reaction may kill off some larvae, but it allows others to escape.

So while a successful vaccine would help, "the likelihood of success is not very high," says Oliveira. "This is a complex parasite that has evolved over a million years." Unlike water-borne schistosomiasis, he explains, to which people build up immunity after repeated exposures, many people's immune systems never learn how to resist hookworm. For some, the older they get, the worse the infections become. "We have to show that the immune response generated by a vaccine is quite distinct from the immune response of the actual infection," he says. "We're hoping it will be."

It took decades for the Sabin research team to reach a point at which it had a vaccine worth trying in Beija-Flor. Hotez, who received his medical and biochemistry training at Rockefeller University, began his parasitology studies in 1980. Short in stature but long in optimism, Hotez wears bow ties and round, gold-framed glasses. His brown curly hair has only a hint of gray and his perpetual smile is infectious. He stumbled onto his life's calling after reading a 1962 paper by Norman Stoll, the pathbreaking Rockefeller parasitologist who called hookworms "the great infection of mankind."

In 1989, Hotez moved to Yale University, where he conducted most of the early scientific work aimed at identifying a vaccine target. His early epidemiologic studies in China had revealed a wide variation in the number of worms among people who were routinely exposed to the parasite. He also knew from his early days as a graduate student that an attenuated larvae vaccine for canine hookworm had been developed in the 1960s and 1970s. Its manufacturer discontinued making it in 1975 after only a few years on the market, citing poor sales. Veterinarians, it turned out, made more money from routine deworming than from a one-time vaccine. Plus, some pet owners complained because they still saw worm eggs in their dogs' feces. To Hotez, though, the partial response suggested that some naturally occurring antibody, albeit stronger in some hosts than in others, was offering partial immunity.

Hotez began hunting for the antigen that was generating the response. If reproduced in large quantities through recombinant engineering and administered as a vaccine, he hypothesized, the body might build up a large store of the immune system's memory cells that could generate an overwhelming antibody response against future invaders.

Working with canine hookworm, Hotez ran experiments to isolate potential antigens from the larvae that could form the basis of a recombinant vaccine. Using a method that a postdoc, John Hawdon, had established for getting larvae to resume development, the two men were stymied initially by the activated larvae's failure to produce proteins that might be potential vaccine targets. "There were no secretions," Hotez recalled. "You had to trick them to think they were in the host."

So Hotez and colleagues placed the larvae in media containing either host blood serum, glutathione, or both, and they managed to identify three major proteins emitted from the invading larvae. Two were Ancyclostoma-secreted proteins (ASP-1 and ASP-2), which belong to a family of signaling proteins that all animals and some plants secrete in the presence of pathogens; the third was MTP-?1, which also plays a role in the worm's own immune system.2 The next steps, however, required serious funding, which Hotez set out to procure.

Moises Gonzales de Chagas, a 54-year-old agricultural laborer, is typical of the rural populations around the world that would benefit from a hookworm vaccine. The day in early May that I visited his small, cinderblock home, he told me he had begun feeling ill last December. As the months went by, he grew gradually weaker. Diemert, the project's lead field physician, later speculated the man had probably harbored some worms for years - they can live up to five years in the intestines. It was only when the population of blood-sucking visitors reached a critical mass (between 40 and 120 worms) that he developed iron-deficiency anemia, the primary symptom of the disease.

Emerging from the shadows of his three-room house, Gonzales' eyes slowly adjust to the late afternoon sunlight. He sticks out his dry tongue to reveal white spots. Two days earlier, he had walked to a government-run traveling clinic where he was treated with albendazole, a deworming drug whose three-day course kills the parasites to provide temporary relief. When he returns to his job, however, the moist fields where he works will be filled with hookworm larvae, deposited there in human waste. He will probably get infected again, since humans do not naturally develop immunity, even with repeated exposures.

An effective vaccine would be a godsend to the estimated half billion people around the globe who are infected with hookworms. Although the parasite usually doesn't kill, chronic hookworm infections sap the strength and productivity of adults. "Before I was sick, I worked on the farm," Gonzales says. "Now I don't work."

Chronic infection also retards the mental and physical development of children - perhaps its most pernicious and socially debilitating effect. At the cabin next door, a farmworker cradles his son in his arms. For months, 6-year-old Renilson dos Santos had been tired and listless, doing poorly in school and not wanting to play football with his friends. After being treated by the traveling clinic (it comes by only around twice a year), his mother Maria notices the difference. "He plays. He has more energy now," she says.

By the 1990s, foundations were becoming interested in helping Renilson Santos and others in similar straits. In 1998, shortly after hearing about the Bill and Melinda Gates Foundation from Jeffrey Sachs at Harvard University, whose growing interest in the issue of global poverty had led him to the hookworm project, Hotez applied for and received one of the first Gates grants for research in neglected diseases. The Sabin Institute has received $54 million since 1999.

"I always say, be careful what you wish for," Hotez says. He moved his operation to GWU in 2000 to be near the vaccine development infrastructure that surrounds the nation's capital. "I had to set up a biotech company," he says. "We recruited an extraordinary bunch of PhD scientists who were interested in becoming vaccine manufacturers."

The researchers needed to isolate and identify the human hookworm equivalents of the protein targets they had identified in canine hookworm, clone those proteins, combine them with an adjuvant, and begin injecting experimental animals to determine the protein that generated the best immune response. They developed a scoring system based on measurements of adult worm reduction, host blood loss, and eggs per gram of feces. The animal tests all pointed to ASP-2 as the best lead candidate.

Bethony's crackerjack epidemiologic field work supplemented these animal experiments. The immunologist, who spends most of his time in Brazil, created a blood test to check for antibodies. He discovered that 15% of the population had measurable levels of antibodies to ASP-2. "We found that (those) people ... did not have heavy infections, so we deduced they were at lower risk," Bethony says.

At this point, an academic researcher with a lead candidate will usually license his or her discovery to a pharmaceutical company to begin scaling up production for clinical trials. But no company was interested in developing a vaccine for a disease that affects only the globe's poorest citizens.

Maria Elena Bottazzi, the newly hired product development officer at the Hookorm Vaccine Initiative, instead turned to the Walter Reed Army Institute of Research (WRAIR) for help in setting up a small facility to produce large quantities of the protein for an experimental vaccine. WRAIR used recipes that Bottazzi's team had developed in a pilot plant on the GWU campus. Next, a trial in 36 healthy US volunteers showed that the injection was safe and generated a substantial immunologic response, at least in people who had never been exposed to the parasite.

The same technology is now being transferred to the government-run Butantan Institute in Sao Paolo, Brazil, which will eventually produce the vaccine for the large trials needed to prove efficacy. The Butantan Institute, Sao Paolo's public health research arm, is best known for its research into antivenoms and antitoxins for rabies, diphtheria, and botulina. It also houses one of the world's largest vaccine manufacturing complexes, producing nearly 200 million vaccine doses per year, including tetanus, pertussis, hepatitis B, and the latest influenza strain.

Using money from the Gates Foundation, Butantan recently purchased the equipment needed to produce a sufficient number of vaccine doses for the final efficacy trials, which should be underway by the end of the decade. The plant, which uses a 60-liter fermenter, may be large enough to supply the entire country once ANVISA (Agencia Nacional Vigilancia Sanitario), the Brazilian equivalent of the US Food and Drug Administration, approves the vaccine. "Our bottom line is the right to produce it here for Brazil," says 80-year-old Isaias Raw, president of Butantan. "We would like to provide it if possible to organizations like UNICEF and the Pan American Health Organization."

Raw, whose excellent English skills date from his time in exile teaching at City College in New York, says his experience at government-run Butantan defies the conventional wisdom that "governments are incompetent and companies are competent. Our problem is that whatever doesn't make profit isn't their concern. You're never going to vaccinate unless the price is low. If you show up with a vaccine that costs even $50 a person, in a country like ours, it might as well not exist," he says.

The Sabin Institute's Hotez, who holds the patent to the hookworm vaccine, follows an intellectual property model adopted by most nonprofits that the Gates Foundation funds. It has agreed to transfer the cell line, the manufacturing technology, and a royalty-free license to any country able and willing to produce it at standards set by the FDA and other regulatory agencies. It is already exploring partnerships with India, China, Indonesia, and Cuba, in addition to Brazil. "We will send it to anyone," says Hotez.

"I feel like I'm the conductor of a symphony orchestra," Hotez says. "I'm organizing the labs, the science, the production for delivery to the field - all to solve a common problem among the world's poorest people."

The night before the team registered their first patients, Bethony and Diemert described the difficulties of adhering to "good clinical practices" while conducting clinical trials in developing countries. They have built a small, three-bedroom house in nearby Americaninhas where the research team bunks four to a room. A long picnic table fills the dining area of the communal kitchen. Their work shed, complete with a small microscopy lab, has satellite-driven Internet access to the outside world, that is, when surges or outages don't crash the computers.

The logistic challenges of conducting a clinical trial in this resource-poor setting are extraordinary. The three-shot vaccine has to be administered over three months since the goal is to build a large store of immune-system memory cells that will produce a high volume of anti-ASP-?2 antibodies when the person is exposed to hookworm larvae. Each dose has to be maintained between 3°C and 9°C while being transported from the United States, and the temperature must be monitored and documented every step of the way.

Moreover, while stool samples collected in the field can be analyzed in the local microscopy lab for their hookworm egg count (a primary measure of efficacy, but a secondary endpoint in this safety trial), the blood samples for measuring antibody response must be sent to a more sophisticated lab in the regional capital, 12 hours away by car. Detailed case report forms must be filled out for every patient. "A properly designed study will be a failure if the data are not collected in a fashion that can be verified, which in turn compromises the ethics of conducting such a trial, because participants will have been exposed to risk needlessly," Diemert and Bethony wrote recently in response to an editorial questioning the applicability of First World regulatory standards in tropical disease trials.

The forests that once blanketed the hills in this equatorial climate have long since been stripped away for cattle grazing and subsistence farming of sugar cane, corn, beans, and manioc. The people who eke out a subsistence living from this rugged terrain understand perfectly well how hookworms and the other infectious parasites such as water-borne schistosomiasis are ruining their lives and robbing their children's future. Bethony, Diemert, and their colleagues must explain to these volunteers that the vaccine is only an early safety test and that there are risks with no guarantee that it will work. "You don't want them [to participate] because they think they're going to get something in return," Bethony says. "You want them to understand the benefits and risks. That's the challenge."

Most of the people in the area are illiterate, Bethony says. "So many of our participants have witnesses appointed by the community to make sure they've heard the whole consent form and presentation, and that they comprehend." Long before today's final interview, social scientists working on Sabin's Hookworm Vaccine Initiative had traveled throughout the region showing a video to educate the public about the life cycle of the disease and the purpose of the experimental vaccine program. Potential volunteers, before signing up, had to attend community lectures.

Now that sign-up day has finally arrived, the volunteers, accompanied by a community ombudsman who serves as their advocate, must take a final test showing that they understand the risks associated with being among the first people living in an endemic setting to receive the vaccine. Institutional review boards at both the state and national levels in Brazil approved both the experimental protocol and the informed consent process.

Erleide Pereira Viana, a 27-year-old mother with one preschool child, shows up early, her red cap turned jauntily backwards. She looks on pensively as project officials tally her responses to a test of whether she understands the risks of the trial. Told she has passed, she smiles. "It's good for the health of the people," she says.

Even as this trial gets underway, the Sabin team is laying the groundwork for the next phase of the project: identifying a second target in mature hookworms that may escape the initial immune system onslaught. "You want to hit them a second time after they become adults," Hotez says. With Alex Loukas of Australia's Queensland Institute of Medical Research in the lead, the Sabin team, again working with canine hookworm, identified an aspartic protease (APR-1) in the worm that is key to digesting hemoglobin once the parasite is in the intestines. A vaccine using a recombinant version of APR-1 significantly reduced hookworm burdens in dogs.

However, the protein is proving very costly and difficult to manufacture in bulk. The search is now on for a second candidate. "If you can't make it cheaply, you might as well not make it at all," Hotez said. "We have to build into our design process the ability to deliver this vaccine at less than $1 a dose."

Even if they finally do come up with a vaccine that can be made cheaply, it will be of little use unless it can be delivered to the world's poor. Hotez and the Sabin Vaccine Institute recently launched Global Network for Neglected Tropical Disease Control, whose partners include the World Health Organization and nonprofits fighting schistosomiasis, trachoma, and the soil-transmitted worms known as helminths (hookworm, ascaris, and trichuris). The partnership's goal is to set up healthcare delivery systems, especially in sub-Saharan Africa where the need is greatest, for rapidly delivering low-cost drugs that are effective in treating these diseases. The same networks will later be able to administer vaccines if they become available.

That's still a big if. If the safety trial that started in June is successful, the efficacy trials that will be needed for regulatory approval will take at least another half decade, maybe longer. The entire process will then have to be repeated for a vaccine that includes the second antigen attacking adult hookworms that escape the initial immune system reaction.

"A lot of academics think that if you isolate a protein in a lab, you've done your job," Hotez says. "Then all you have to do is turn it over to a manufacturer and it appears in a bottle. This takes a long time. Vaccine development takes a lifetime of refining."

Lucimar Medina de Souza Gomes, a 29-year-old mother whose young child clings to her skirt, is hopeful as she inks her thumbprint to the consent form. "It will help develop a vaccine that will put an end to the worm."