Spillover

by David Quammen

  The result of that flawed vaccinating, certain people have argued, was iatrogenic infection (disease caused by medical treatment) of an unknown number of Central Africans with what later became recognized as HIV-1. By this notion, known for short as the OPV (oral polio vaccine) theory, a single reckless researcher had seeded the continent—and the world—with AIDS.

  The OPV theory has been around and notorious since 1992, when a freelance journalist named Tom Curtis described it in a long article for Rolling Stone. Curtis’s piece ran under the headline: THE ORIGIN OF AIDS: A STARTLING NEW THEORY ATTEMPTS TO ANSWER THE QUESTION, ‘WAS IT AN ACT OF GOD OR AN ACT OF MAN?’ Several other researchers had mooted the idea earlier, more obscurely, and one of them had put Tom Curtis onto the story. When Curtis started looking into it, some eminent scientists responded with defensive dismissals, which served only to suggest that maybe the theory did merit consideration. Curtis even drew a brusque comment from the head of research for WHO’s Global Programme on AIDS, Dr. David Heymann: “The origin of the AIDS virus is of no importance to science today.” He quoted another expert, William Haseltine of Harvard, as saying: “It’s distracting, it’s nonproductive, it’s confusing to the public, and I think it’s grossly misleading in terms of getting to the solution of the problem.” After publication of the piece, lawyers for Hilary Koprowski filed a lawsuit against Curtis and Rolling Stone, charging defamation, and the magazine ran a “clarification,” admitting that the OPV theory and Koprowski’s role represented just an unsupported hypothesis. But as the dust settled at Rolling Stone, an English journalist named Edward Hooper took hold of the OPV theory as a personal obsession and an investigative crusade, giving it a second life.

  Hooper spent years researching the subject with formidable tenaciousness (though not always critical good sense) and in 1999 made his case in a thousand-page book titled The River: A Journey to the Source of HIV and AIDS. Hooper’s river was a metaphorical one: the flow of history, the stream of cause-and-effect, from a very small beginning to an ocean of consequences. In the book’s prologue, he alluded to the quest by Victorian explorers for the source of the Nile. Does that river begin from Lake Victoria, pouring out at Ripon Falls, or is there another and more obscure source upstream from the lake? “The controversy surrounding the source of the Nile,” Hooper wrote, “is strangely echoed by another controversy of a century and a half later, the long-running debate about the origins of AIDS.” The Victorian explorers had been wrong about the Nile and, according to Hooper, so were the modern experts wrong about the starting point of the AIDS pandemic.

  Hooper’s book was massive, overwhelmingly detailed, seemingly reasonable, exhausting to plod through but mesmerizing in its claims, and successful at bringing the OPV theory to broader public attention. Some AIDS researchers (including Phyllis Kanki and Max Essex) had long been aware that vaccine contamination, with SIV from monkey cells, was at least a theoretical possibility; they had even conducted screening efforts on vaccine lines, and found no evidence of such a problem. Hooper, following Tom Curtis, raised the idea from a concern to an accusation. His vast river of information and his steamboat of argument didn’t prove the essential thesis—that Koprowski’s vaccine had been made from chimp cells contaminated with HIV. But his work did seem to raise the possibility that the vaccine could have been made from chimp cells that might have been contaminated.

  The issue of possibility then gave way to the issue of fact. What had actually happened? Where was the evidence? At the urging of an eminent evolutionary biologist named William Hamilton, who believed that the OPV theory deserved investigation, the Royal Society convened a special meeting in September 2000 to discuss the subject within its broader context. Hamilton was a senior figure, liked and respected, whose early work in evolutionary theory helped inform Edward O. Wilson’s Sociobiology and Richard Dawkins’s The Selfish Gene. He swung the Royal Society into giving the OPV theory a fair hearing. Edward Hooper, though not a scientist himself, was invited to speak. Hilary Koprowski also came, as well as a roster of leading AIDS researchers. By the time that meeting convened, though, William Hamilton was dead.

  He died suddenly in March 2000, of intestinal bleeding, after an attack of malaria contracted during a research trip to DRC. In his absence, his colleagues at the Royal Society discussed a wide range of matters related to the origins of HIV and AIDS. The OPV theory was just one topic among many, though implicitly it drove the agenda of the whole meeting. Did the available data from molecular biology and epidemiology tend to support, or to refute, the vaccine-contamination scenario? A corollary to that question was: When had HIV-1 first entered the human population? If the earliest infections occurred before 1957, those infections couldn’t have resulted from Koprowski’s OPV trials. Archival HIV-positives might be decisive.

  This is the context that brought DRC60 out of Kinshasa. After the Royal Society meeting, a Belgian physician named Dirk Teuwen, who had taken part, recollected some references to early pathology work in the Congo that he had seen in archival reports of the colonial medical laboratories. Teuwen conceived the idea—and raised it with other attendees—that HIV-1 might be detected in some of the tissues preserved within those old paraffin blocks. He met skepticism; the others doubted that any useful traces of virus could have survived through the decades—decades of tropical heat, simple storage, administrative upheaval, and revolution. But Teuwen was stubborn. He enlisted an ally, a senior Congolese bacteriologist named Jean-Jacques Muyembe, and, with approval from the Ministry of Health, Muyembe started looking. He went up to the University of Kinshasa, rifled through the pantry behind the blue curtain, packed 813 paraffin-embedded specimens into an ordinary suitcase, and carried it with him on his next professional visit to Belgium. There he handed the trove to Dirk Teuwen. Teuwen, in accord with a prior agreement for collaborative study, sent the samples to Michael Worobey in Tucson.

  These two lines of narrative fold back into each other. Worobey, as a grad student, knew both Bill Hamilton at Oxford and some of the disease biologists in Belgium. Impelled by his own interest in the origins of HIV, Worobey accompanied Hamilton to DRC on that last fatal fieldtrip. They went in January 2000, during the chaotic aftermath of the civil war, which had replaced President Mobutu Sese Seko with President Laurent Kabila. Hamilton wanted to collect fecal and urine samples from wild chimpanzees; those specimens, he hoped, might help confirm or refute the OPV theory. Worobey, for his part, put little stock in the OPV theory but wanted more data from which to chart the origin and evolution of HIV. It was a crazy time in DRC, more crazy than usual, because two rebel armies opposed to Laurent Kabila still controlled much of the eastern half of the country. Hamilton and Worobey flew into Kisangani (formerly Stanleyville), a regional capital along the upper Congo River, the same city where Koprowski had begun his vaccinating enterprise. Now it was occupied by Rwanda-backed forces on one riverbank and Uganda-backed forces on the other. Commercial airlines weren’t flying, because of the war, so the two biologists shared a small, chartered plane with a diamond dealer. In Kisangani they paid their respects to the Rwanda-backed commander, whose ambit included most of the city, and as quickly as possible got out into the forest, where they would be safer among the leopards and snakes. They spent a month collecting fecal and urine samples from wild chimpanzees, with help from local guides, and by the time they left, Hamilton was sick.

  Neither he nor Worobey knew how sick, but they caught the next exit flight they could, which took them to Rwanda. From there they bounced to Entebbe in Uganda, where Hamilton got a confirmed diagnosis of falciparum malaria and some treatment, then onward to Nairobi, and from Nairobi up to London Heathrow. By now Hamilton seemed past the worst of his illness; he was feeling much better. They had accomplished their mission and life was good. An American field biologist once expressed to me how he felt in such moments. “That’s the name of the game: getting home with the data.” This man’s research too involved dangers—shipwreck, starvation, drowning, snakebite,
though not malaria and AK rifles. “If you take too many risks, you don’t get home,” he said. “If you take too few, you don’t get the data.” Hamilton and Worobey got the data, got home, and then learned that the ice cooler containing their precious chimp specimens had gone astray in luggage handling somewhere between Nairobi and London.

  I visited Michael Worobey in Tucson to hear about all this. “Everything was fine,” he told me, “except we checked six bags, including the cooler that had samples, and five of our bags came through the carousel and the one with the samples disappeared.” His friend Hamilton, feeling ill again the next morning, went to a hospital—and hemorrhaged catastrophically, perhaps due to anti-inflammatory drugs he’d been taking against the malarial fever. Worobey phoned and got the news from Hamilton’s sister: Who are you why are you calling Bill is in extremis. Worobey meanwhile had been hassling by long-distance phone with a luggage handler in Nairobi, who assured him that the cooler had been found and would arrive on the next flight. What arrived was someone else’s cooler, full of sandwiches. “So that was an extra bit of drama that unfolded as Bill was dying in the hospital,” Worobey told me. The correct cooler arrived two days later but Hamilton was in no shape to celebrate. He went through a series of surgeries and transfusions and then, after weeks of struggle, he died.

  The fecal samples from Congolese chimps, for which Hamilton had given his life, yielded no SIV-positives. A couple of urine samples registered in the borderline zone for antibodies. Those results weren’t clear or dramatic enough to merit publication. Good data are where you find them, not always where you look. Several years later, when the human pathology samples from Kinshasa reached Tucson—those 813 little blocks of tissue in paraffin, the ones J. J. Muyembe had carried to Belgium in a suitcase—Michael Worobey was ready. He found DRC60 among them, and it told an unexpected story.

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  Screening paraffin-embedded hunks of old organ samples to find viral RNA isn’t easy, not even for an expert. Those little blocks, Worobey said, turned out to be “some of the nastiest kinds of tissues to do molecular biology with.” The problem wasn’t forty-three years at room temperature in a dusty equatorial pantry. The problem was the chemicals used in fixing the tissues—the 1960 equivalent of the beakers of methanol and xylol that Professor Kabongo had shown me. Back in those days, pathologists favored something called Bouin’s fixative, a potent little mixture containing mostly formalin and picric acid. It worked well for preserving the cellular structure of tissues, like salmon in aspic, so that samples could be sliced thin and examined under a microscope; but it was hell on the long molecules of life. It tended to break up DNA and RNA into tiny fragments, Worobey explained, and form new chemical bonds, leaving “sort of a big, tangled mess rather than a nice string of beads that you can do molecular biology on.” Because the process was so laborious, he screened just 27 of the 813 tissue blocks from Kinshasa. Among those twenty-seven, he found one containing RNA fragments that unmistakably signaled HIV-1. Worobey persisted adeptly, untangling the mess and fitting the fragments to assemble the sequence of nucleotide bases he named DRC60.

  That was the wet work. The dry work, done largely by computer, entailed base-by-base comparisons between DRC60 and ZR59. It also involved broader comparisons, placing those two within a family tree of known sequences of HIV-1 group M. The point of such comparisons was to see how much evolutionary divergence had occurred. How far had these strains of virus grown apart? Evolutionary divergence accumulates by mutation at the base-by-base level (other ways too, but those aren’t relevant here), and among RNA viruses such as HIV, as I’ve explained, the mutation rate is relatively fast. Equally important, the average rate of HIV-1 mutation is known—or anyway, it can be carefully estimated from the study of many strains. That rate of mutation is considered the “molecular clock” for the virus. Every virus has its own rate, and therefore its own clock measuring the ticktock of change. The amount of difference between two viral strains can therefore reveal how much time has passed since they diverged from a common ancestor. Degree of difference factored against clock equals elapsed time. This is how molecular biologists calculate an important parameter they call TMRCA: time to most recent common ancestor.

  Okay so far? You’re doing great. Take a breath. Now those bits of understanding will boost us across a deep gulf of molecular arcana to an important scientific insight. Here we go.

  Michael Worobey found that DRC60 and ZR59, sampled from people in Kinshasa during almost the same year, were very different. They both fell within the range of what was unmistakably HIV-1 group M; neither could be confused with group N or group O, nor with the chimp virus, SIVcpz. But within M, they had diverged far. How far? Well, one section of genome differed by 12 percent between the two versions. And how different was that, measured in time? About fifty years’ worth, Worobey figured. More precisely, he placed the most recent common ancestor of DRC60 and ZR59 in the year 1908, give or take a margin of error.

  So the spillover had occurred by 1908? That’s much earlier than anyone suspected, and therefore the sort of discovery that gets into an august journal such as Nature. Publishing in 2008, a century after the fact, with a list of coauthors that included Jean-Jacques Muyembe, Jean-Marie Kabongo, and Dirk Teuwen, Worobey wrote:

  Our estimation of divergence times, with an evolutionary timescale spanning several decades, together with the extensive genetic distance between DRC60 and ZR59 indicate that these viruses evolved from a common ancestor circulating in the African population near the beginning of the twentieth century.

  To me he said: “This wasn’t a new virus in humans.”

  Worobey’s work directly refuted the OPV hypothesis. If HIV-1 existed in humans as early as 1908, then obviously it hadn’t been introduced via vaccine trials beginning in 1958. Clarity on that point was valuable—but it was only part of Worobey’s contribution. Placing the crucial spillover in time represented a big step toward understanding how the AIDS pandemic may have started and grown.

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  Placing the spillover in space was equally important, and achieved by a different laboratory. Beatrice Hahn is somewhat older than Worobey and had begun her work on the origin of AIDS long before he found DRC60.

  Born in Germany, Hahn got a medical degree in Munich, then came to the United States in 1982 and spent three years as a postdoc in Robert Gallo’s lab, studying retroviruses. She moved next to the University of Alabama at Birmingham, where she became Professor of Medicine and Microbiology and codirector of a center for AIDS research, with a group of bright postdocs and grad students working under her aegis. (She remained at Alabama from 1985 to 2011, a period encompassing most of the work described here, and then joined the Perelman School of Medicine at the University of Pennsylvania, in Philadelphia.) The broader purpose of Hahn’s various projects, and the goal she shares with Worobey, is to understand the evolutionary history of HIV-1 and its relatives and antecedents. The fittest label for that sort of research is the one Worobey mentioned when I asked him to describe his field: molecular phylogenetics. A molecular phylogeneticist scrutinizes the nucleotide sequences in the DNA or RNA of different organisms, comparing and contrasting, for the same reason a paleontologist scrutinizes fragments of petrified bone from extinct giant saurians—to learn the shape of lineages and the story of evolutionary descent. But for Beatrice Hahn especially, as a medical doctor, there’s an additional purpose: to detect how the genes of HIV-1 function in causing disease, toward the prospects of better treatment, prevention, and maybe even a cure.

  Some very interesting papers have come out of Hahn’s laboratory in the past two decades, many of them published with a junior researcher as first author and Hahn in the mentorship position, last. That was the case in 1999, when Feng Gao produced a phylogenetic study of SIVcpz and its relationship to HIV-1. At the time there were only three known strains of SIVcpz, all drawn from captive chimps, with Gao’s paper adding a fourth. The work appeared in Nature, highlighted by a commentary c
alling it “the most persuasive evidence yet that HIV-1 came to humans from the chimpanzee, Pan troglodytes.” In fact, Gao and his colleagues did more than trace HIV-1 to the chimp; their analysis of viral strains linked it to individuals of a particular subspecies known as the central chimpanzee (Pan troglodytes troglodytes), whose SIV had spilled over to become HIV-1 group M. That chimpanzee lives only in western Central Africa, north of the Congo River and west of the Oubangui. So the Gao study effectively identified both the reservoir and also the geographical area from which AIDS must have arisen. It was a huge discovery, as reflected in the headline of Nature’s commentary: FROM PAN TO PANDEMIC. Feng Gao at the time was a postdoc in Hahn’s lab.

  But because Gao based his genetic comparisons (as Martine Peeters had done earlier) on viruses drawn from captive chimps, the soupçon of uncertainty about infection among wild chimpanzees remained, at least for a few more years. Then, in 2002, Mario L. Santiago led a list of coauthors announcing in Science their discovery of SIVcpz in the wild. Santiago was a PhD student of Beatrice Hahn’s.

  The most significant aspect of Santiago’s work, for which he got his richly deserved doctorate, was that on the way toward detecting SIV in a single wild chimpanzee (just one animal, of fifty-eight tested), he invented methods by which such detections could be made. The methods were “noninvasive,” meaning that a researcher didn’t need to capture a chimp and draw its blood. The researcher needed only to follow animals through the forest, get under them when they pissed (or, better still, send a field assistant into that yellow shower), collect samples in little tubes, and then screen the samples for antibodies. Turns out that urine can be almost as telling as blood.