The reports from Dhaka are hopeful. It is 2005, and Dr. Firdausi Qadri and colleagues at the International Center for Diarrheal Disease Research, Bangladesh, are testing a new cholera vaccine on children. In their study, a single dose of live, crippled bacteria goes down easily in a fizzy drink. Within days, most of the children are showing exactly the kind of robust immune response that should enable them to fend off an attack from the deadly pathogen, with no notable side effects.
The study follows a similar success immunizing adults in the same city. The stage is now set for a large-scale trial to test the vaccine, known as Peru-15, to prevent the seasonal flare-ups of life-threatening diarrhea that dog the poor in Bangladesh and other regions of the developing world.
In the developed world, too, Peru-15 is close to final testing as a rapidly acting traveler’s vaccine.
These ongoing clinical trials cap a decades-long quest by John Mekalanos of Harvard Medical School (HMS) for a safe and widely effective cholera vaccine. As the Adele Lehman professor of microbiology and molecular genetics and chairman of that department, Mekalanos is on the verge of seeing 20-plus years of his work transformed into a life-saving preventive against one of humankind’s most wretched enemies.
On one hand, the testing of Peru-15 in Bangladesh sprang from one scientist’s vision and commitment to bettering global health. On the other hand, vision means little without the resources to do the work, and that’s where the National Institutes of Health enters the story. Since 1981, the NIH has provided an unbroken string of grant support to Mekalanos that has enabled him to carry out the basic research necessary to invent Peru-15 and to refine it. With a backbone of reliable, steady funding, the vaccine project’s success attracted additional money from the Department of Defense, the biotech company AVANT Immunotherapeutics, and finally, the Bill and Melinda Gates Foundation. If studies continue as expected over the next few years, the NIH investment in Peru-15 could pay off with the first oral, single-dose cholera vaccine licensed in the United States and a valuable new weapon against cholera in the developing world.
The Toll of CholeraDespite the absence of cholera from richer nations, the bacteria are alive and thriving in poor countries on all continents, where they are particularly deadly to children. Cholera showed up in 56 countries in 2005, and estimates put the number of cases at 1 million per year worldwide, with more than 20,000 deaths. In the last few decades, an aggressive new V. cholerae strain, El Tor, has made its way across Asia, the Middle East, Africa, and South America. In addition to the steady assault of endemic cholera, epidemics often break out during wars or natural disasters that disperse refugees and spread the disease.
What gives V. cholerae its sting? That question was answered in the 1960s, when researchers identified cholera toxin, a protein product of the bacteria that causes massive diarrhea and vomiting. Normally, V. cholerae enters its victims through contaminated food or water and swims until it reaches the cells lining the gut. There, it adheres, multiplies, and cranks up production of the two proteins that make up cholera toxin. The B subunit serves to deliver the poisonous A to intestinal cells, where it acts to throw open ion channels, causing chloride and other ions to flood the intestine, followed by massive amounts of water. The resulting rapid loss of gallons of fluid in a characteristic watery diarrhea leads to dehydration and, often, death—sometimes within hours.
Surviving a bout of cholera produces strong and long-lasting immunity, and early on, researchers arrived at an ideal strategy for making a vaccine: produce a harmless form of Vibrio by stripping the bacteria of their ability to make cholera toxin, then administer the attenuated bacteria orally to mimic natural infection. Experience with smallpox and polio proved the value of live, crippled organisms as vaccines, and so the goal was to play the same trick of attenuation in V. cholerae.
In the days before molecular biology, researchers were limited to attempts at selecting, from nature or a flask of bacteria in the lab, attenuated organisms as they arose by chance. But Mekalanos’s entry into research as a graduate student in the 1970s coincided with the beginning of the molecular biology revolution, and he immediately understood the power of applying genetic engineering techniques to the problem of attenuation. In 1981, Mekalanos joined the HMS faculty and, with the support of his very first NIH grant (which continues to fund his lab today), started unraveling the molecular genetics of Vibrio cholerae.
In just two years, Mekalanos and colleagues had identified and completely sequenced for the first time the genes that encode cholera toxin. With that DNA in hand, they were able to quickly create a new V. cholerae strain identical to the pathological classic strain Ogawa 395, which provokes a strong immunity, except that it lacked the gene for the toxic A subunit. (The B subunit was retained, since it triggers a helpful immune response upon infection). Their work, published in the journal Nature in 1983, marked a watershed for cholera vaccine research by introducing to the world the first genetically defined Vibrio strains with the potential to become vaccines.
Trial and ErrorTesting of those strains followed quickly, but with disappointing results. Evaluating cholera vaccines requires human volunteers—there are no animal models that exactly replicate the interaction between the bug and the gut or that can prove vaccine safety. To vet the vaccine, researchers must feed it to people, monitor for side effects, and measure production of blood-borne antibodies. When the first toxin-deleted strains were given to volunteers (in a collaboration with investigators at the University of Maryland), the vibrios stimulated the hoped-for immune response. But some of the volunteers did not do so well, developing vomiting and diarrhea. The effects were not of the magnitude seen with cholera itself, but they were unpleasant enough to send researchers back to the drawing board with the realization that simply deleting the gene for cholera toxin A was not going to result in a safe and tolerable vaccine.
At that stage, Mekalanos, by then a professor at HMS, launched an intensive search for other genes that contribute to the pathogenicity of V. cholerae. Taking advantage of the explosive growth of molecular biology techniques and continued NIH support through the 1980s, the lab made a series of key discoveries about how the bug uses regulatory proteins to turn on toxin expression. They also identified a host of other genes regulated in concert with the toxin. One of these makes a cell surface protein, the toxin coregulated pili (TCP) that enables the vibrio to attach to the human intestine long enough to replicate and do its damage. These studies led to a second, ongoing NIH grant to study gene regulation in V. cholerae.
All along, the researchers continued to tinker with V. cholerae’s genes in hopes of weakening the bacteria, thus making it safer without affecting its recognition by the immune system. Even though it might have appeared that the vaccine was not moving forward, the insights gained at this time were filling in a blueprint for vaccine development that would be in demand soon enough.
Bringing In the ArmyFor Mekalanos, the inspiration that sparked the Peru-15 vaccine came in 1991. During that year, disaster struck close to home when the new El Tor strain hit South America, the first time cholera had appeared in the western hemisphere in more than a hundred years. After popping up first in Peru, the epidemic sickened a million people and killed 10,000 across Latin America. Mekalanos, who had been working on El Tor, felt an acute need for a vaccine against this new and deadly strain.
As luck would have it, Mekalanos was in Geneva at a meeting of the World Health Organization and happened to meet Colonel Jerald Sadoff from Walter Reed Army Hospital. After the first Gulf War, cholera broke out among the Kurdish refugees crowded into camps on the Turkish border. Was the infection natural or a bioweapon? the Army wondered. In either case, the military was taking a new interest in infectious diseases. Mekalanos’s NIH funding could not support clinical trials, but the Army could. That meeting began a fruitful collaboration in which Mekalanos started producing live vaccines from crippled versions of El Tor, and Sadoff with the Army helped test the vaccines in human volunteers.
The initial recipe was this: first, delete the cholera toxin A and B subunit genes. Because the scientists had learned that the toxin genes are mobile, jumping in and out of the genome like many bacterial genes do, the team built in safety features to prevent the vaccine from recapturing these genes. This involved deleting the insertion site and a DNA recombinase enzyme that might catalyze re-insertion. In place of this enzyme (recombinase A), they put back the toxin B subunit gene, because of that protein’s ability to raise a good immune response without harmful effects.
With tweaking and testing, the vaccine improved; the engineered organisms remained highly immunogenic and side effects continued to decrease. But the vaccine in its best form was still causing some residual diarrhea and intestinal upset, so the scientists searched for one more thing to knock out. The idea came up to select bacteria that did not swim well. By slowing down the bug, the researchers thought they might be able to knock back its invasiveness, but still have enough activity to stimulate the intestinal immune response.
When this last modification was made—by selecting from cultures the slowest swimming bacteria to make the vaccine—the preparation stopped causing diarrhea in human volunteers. In a human trial, 10 of 11 subjects developed vibriocidal antibodies after one oral dose. One month later, when five of those vaccinated subjects received a dose of real cholera bacteria, three showed no symptoms and two showed diarrhea that was much less severe than the symptoms of unvaccinated controls. In 1995, after trying 15 versions, the researchers had their vaccine, which they named Peru-15 after the country of origin of the parental El Tor––type V. cholerae strain.
In the developing world, cholera is a moving target, with new forms like El Tor bursting on the scene periodically. For this reason, Mekalanos hoped that the attenuation blueprint could be replicated in new strains, and he did not have to wait long to test this idea. Even before Peru-15 was completed, another V. cholerae strain emerged in India. Genetic studies showed that on the inside, the new strain, Bengal O139, is quite similar to El Tor. The big difference lies in its outer coat. Because it presented a new face to the immune system, immunity against El Tor did not protect against Bengal—the new strain would require its own vaccine. Reassuringly, the Peru-15 blueprint transferred flawlessly to Bengal, and a similar set of genetic alterations resulted in a vaccine that has successfully passed initial trials in people.
The Rise of Peru-15When the uptick in vaccine research occurred in 1991, Mekalanos was busy figuring out how to quickly bring the El Tor vaccine to the clinic. That year, he cofounded a biotech company, which licensed the cholera vaccine technology from Harvard with an eye toward commercial development. The company, now AVANT Immunotherapeutics, worked with the Army in the early studies and since then has made steady progress in the clinical development of Peru-15 in both North America and Bangladesh.
In North America, and with the help of NIH funding, AVANT has shown the safety and effectiveness of the vaccine in more than 300 people. Now, the company is gearing up for an expanded safety trial, dosing 3,000 to 4,000 people. There are currently no cholera vaccines sold in the United States, but AVANT plans to use the trial results to apply for an FDA license to market Peru-15 as a traveler’s vaccine.
Although the trials in North America are promising, protecting patients from a controlled exposure to cholera is no guarantee that the vaccine will give the required long-lasting protection in an entirely different population and in a setting where cholera is all around, all the time. Using the vaccine in developing countries requires field trials in those countries. Fortunately, the Bill and Melinda Gates Foundation, via its support of the International Vaccine Institute, has assumed the role of white knight by providing the money for such trials, with vaccine provided by AVANT. Based on the encouraging results from Bangladesh in 2005, the International Vaccine Institute received additional funding from Gates in 2006 and is working with AVANT, the International Center for Diarrheal Disease Research, and the Indian Institute in Kolkata to carry out phase II and III trials.
Beyond biology, getting Peru-15 and other live attenuated vaccines to developing countries presents a daunting practical challenge. In its original form, the vaccine had to be stored frozen, a serious impediment to its distribution in poor countries. In a major advance, AVANT has pioneered a method for turning live bacterial vaccines into a glasslike solid that stays stable for months at temperatures close to 100 degrees F. The dried preparation can in theory be stockpiled, shipped, and stored anywhere, to be mixed into a drink just before use. In the summer of 2005, HMS and AVANT received half a million dollars from the NIH to produce the thermostable Peru-15 vaccine, just the first of what should be many vaccines with the shelf life required for use all over the world.
Not content to rest on his laurels, Mekalanos continues to build on his laboratory’s success by pushing into new territory. In 2003, the last year of the five-year doubling period for the NIH budget, the lab captured funding for two additional projects. In one study, American and Bangladeshi investigators are collaborating to determine the genetic evolution of new pathogenic cholera strains in that country. Insights from this four-year, international effort will help to deploy the vaccine effectively and to head off future epidemics before they occur. The second study, funded for five years with biodefense funds, aims to develop derivatives of Peru-15 as delivery vehicles for oral vaccines against a variety of organisms, including anthrax and West Nile Virus.
Sitting in his office at the Medical School recently, Mekalanos recalled the long path to Peru-15, a journey made possible by 25 years of steadfast grant support from the NIH. “There’s no question that the continuous funding of my cholera grants have been an absolutely essential part of this success story,” he said. “Those grants help maintain an infrastructure of engaged and motivated researchers, both here and in Bangladesh, who are trying to solve the cholera problem.” Their vision and efforts will pay off some day soon, Mekalanos believes, when cholera joins the ranks of small pox and polio as another affliction largely eradicated from the world by an effective vaccine.
