Soon after that false lead from Manchester was debunked, another lead emerged in New York. By now it was 1998. A team of researchers including Tuofu Zhu, based at the Rockefeller University, obtained an archival specimen from Africa dating back to the same year as the sailor’s, 1959. This time it wasn’t tissues; it was a small tube of blood plasma, drawn from a Bantu man in what had been Léopoldville, capital of the Belgian Congo (nowadays Kinshasa, capital of the Democratic Republic of the Congo), and stored for decades in a freezer. The man’s name and his cause of death weren’t reported. His sample had been screened during an earlier study, in 1986, along with 1,212 other plasmas—some archival, others new—from various locations in Africa. This man’s was the only one that tested unambiguously positive for HIV. Tuofu Zhu and some colleagues probed further, working with what little remained of the original sample and using polymerase chain reaction (PCR, a biochemical technique that involves heating and cooling a sample in the presence of a certain enzyme) to amplify fragments of the viral genome. Then they sequenced the fragments to assemble a genetic portrait of the man’s virus. In their paper, published in February 1998, they called the sequence ZR59, referencing Zaire (as the country had long been known) and the year 1959. Comparative analysis showed that ZR59 was quite similar to both subtype B and subtype D (finer divisions within the HIV-1 group M lineage) but fell about halfway between, which suggested that it must closely resemble their common ancestor. In other words, ZR59 was a glimpse back in time, a genuinely old form of HIV-1, not a recent contamination. ZR59 proved that HIV-1 had been present—simmering, evolving, diversifying—in the population of Léopoldville by 1959. In fact it proved more. Further analysis of ZR59 and other sequences, led by Bette Korber of the Los Alamos National Laboratory, yielded a calculation that HIV-1 group M might have entered the human population around 1931.
For a decade, from the Zhu publication in 1998 until 2008, that landmark stood alone. ZR59 was the only known version of HIV-1 from a sample taken earlier than 1976. Then someone found another. This one became known as DRC60, and by now you can probably decode the label yourself: It came from the Democratic Republic of the Congo (same nation as Zaire, latest name, often abbreviated as DRC) and had been collected in 1960.
DRC60 was a biopsy specimen, a piece of lymph node snipped from a living woman. Like the Manchester sailor’s bits of kidney and spleen, it had been locked away in a little pat of paraffin. Thus preserved, it needed no refrigeration, let alone freezing. It was as inert as a dead butterfly and less fragile. It could be stored and ignored on a dusty shelf—as it had been. After more than four decades, it emerged from a specimen cabinet at the University of Kinshasa and offered a new jolt of insight to AIDS researchers.
7
The University of Kinshasa sits on a hilltop near the edge of the city, reachable by an hour’s taxi ride through the broken streets, the smoggy sprawl, the snarled traffic of vans and buses and pushcarts, past the street-side vendors of funerary wreaths, the cell-phone-recharge kiosks, the fruit markets, the meat markets, the open-air hardware stores, the tire-repair shops and cement brokers, the piles of sand and gravel and garbage, the awesome decrepitude of a postcolonial metropolis shaped by eight decades of Belgian opportunism, three decades of dictatorial misrule and egregious theft, then a decade of war, but filled with 10 million striving people, some of whom are dangerous thugs (as in all cities) and most of whom are amiable, hopeful, and friendly. The university campus, on its hill, loosely called “the mountain,” presents a relatively verdant and halcyon contrast to the city below. Students go there, climbing by foot from a crowded bus stop, to learn and to escape.
Professor Jean-Marie M. Kabongo is head of pathology in the university’s Department of Anatomic Pathology. He’s a small, natty man with a huge graying handlebar mustache and full muttonchops, making a forceful visual impression that’s vitiated by his gentle manner. He wears heavy brown glasses and a white lab coat over his shirt and tie. When I met the professor in his office, on the second floor of a building that overlooks a grassy concourse shaded with acacias, he pleaded imperfect knowledge of DRC60 and the patient from whom that specimen came. An old case, after all, going back long before his time. Yes, a woman, he believed. His memory was vague but he could check the records. He began taking notes as I questioned him and suggested I come back in a couple days, when he might be better prepared with answers. But then I asked about the room where DRC60 had been stored, and he brightened. Oh, of course, he said, I can show you that.
He fetched a key. Down a corridor beyond his office, he unlocked a blue door. Swinging it open, he welcomed me into a large sunlit laboratory with walls of white tile and two long, low tables down the middle. Against one wall stood an old GE refrigerator. On one of the low tables rested an old-fashioned folio ledger, with curling pages, like something from Chancery in the time of Dickens. On the far windowsill stood a row of beakers containing liquids in increments of color, beaker by beaker, from piss-yellow to vodka-clear. The yellowest, Professor Kabongo told me, was methanol. The clearest was xylol. We use these in preparing a tissue sample, he said. The point of such organic solvents is to extract the water; desiccation is prerequisite to fixing tissues for the long term. The methanol was darkened from processing many samples.
He showed me a small orange plastic basket, with a hinged lid, about the size and shape of a matchbook. This is a “cassette,” Professor Kabongo explained. You take a lump of tissue from a lymph node or some other organ and enclose it in such a cassette; you soak the whole thing in the beaker of methanol; from the methanol, it goes through the intermediate baths in sequence; finally you dunk it in the xylol. Methanol draws out the water; xylol draws out the methanol, preparing your specimen for preservation in paraffin. And this device, Professor Kabongo said, indicating a large machine on one of the tables, delivers the paraffin. You take a leached tissue sample from its cassette, he explained, and place it on a little silver tray, here. From that spigot, you dribble out a stream of warm, liquid paraffin. It cools on the sample like a pat of butter. Now you remove the cassette lid and label the base with an individual code—for instance, A90 or B71. That’s your archival specimen, he said. “A” means that it came from an autopsy. “B” indicates a biopsy. So the paraffin-caked bit of lymph node that yielded DRC60 would have been labeled B-something, as a biopsy specimen. Each coded specimen gets recorded in the big ledger. Then the specimens go into storage.
Storage. Storage where? I asked.
At the far end of the lab was another doorway, this one hung with a blue curtain. Professor Kabongo pushed the curtain aside and I followed him into a specimen pantry, narrow and tight, lined with shelves and cabinets along one side. The shelves and cabinets contained thousands of dusty paraffin blocks and old microscope slides. The paraffin blocks were in stacks and cartons, some of the cartons dated, some not. It appeared to be organized chaos. A wooden stool awaited use by any curious, tireless soul wishing to rummage through the samples. Although I didn’t plan to rummage, my tour had suddenly come to its crescendo. Here? Yes, just here, said the professor. This is where DRC60 sat for decades. He could have added, with local pride: before becoming a Rosetta stone in the study of AIDS.
8
From the pantry behind the blue curtain, that sample and hundreds of others had traveled a circuitous route, to Belgium and then the United States, ending up in the laboratory of a young biologist at the University of Arizona. Michael Worobey is a Canadian, originally from British Columbia, whose specialty is molecular phylogenetics. After his undergraduate work he went to Oxford on a Rhodes scholarship, which ordinarily means two years of mildly strenuous academic work plus lots of tea, sherry, tennis on grass, and genteel anglophilia before the “scholar” returns to professional school or a career. Worobey put Oxford to more serious use, staying on, finishing a doctorate and then a postdoc fellowship in evolutionary biology at the molecular level. From there he returned to North America in 2003, accepting an assistant prof
essorship at Arizona and building himself a BSL-3 lab (Biosafety Level 3, the second most secure category) for work on the genomes of dangerous viruses. Several years later, it was Worobey who detected evidence of HIV in a certain Congolese biopsy specimen from 1960.
Worobey amplified fragments of the viral genome, pieced the fragments together, recognized them as an early version of HIV-1, and named the sequence DRC60. Comparing his sequence with ZR59, the other earliest known strain, he reached a dramatic conclusion: that the AIDS virus has been present in humans for decades longer than anyone thought. The pandemic may have gotten its start with a spillover as early as 1908.
To appreciate Worobey’s discovery and how it splashed down amid previous ideas, you need a little context. That context involves a heated dispute over just how HIV-1 entered the human population. The prevailing notion as of the early 1990s, based on what had been learned about HIV-2 and the sooty mangabey, among other factors, was that HIV-1 also came from an African primate, and that it had probably gotten into humans by way of two separate instances (for groups M and O, the ones then recognized) of butchering bushmeat. This became known as the cut-hunter hypothesis. In each instance, a man or a woman had presumably butchered the carcass of an SIV-positive primate and suffered exposure through an open wound—maybe a cut on the hand, or a scratch on the arm, or a raw spot on any skin surface that got smeared with the animal’s blood. A wound on the back might have sufficed, if the carcass were draped over shoulders for carrying home. A wound in the mouth, if some of the meat were consumed raw. All that mattered was blood-to-blood contact. The cut-hunter hypothesis was speculative but plausible. It was parsimonious, requiring few complications and no unlikelihoods. It fit the known facts, though the known facts were fragmentary. And then in 1992 a contrary theory arose.
This one was heterodox and highly controversial: that HIV-1 first got into humans by way of a contaminated polio vaccine tested on a million unsuspecting Africans. The vaccine itself, by this theory, had been an unintended delivery system for AIDS. Someone, according to the theory, had monumentally goofed. Someone was culpable. Scientific hubris had overridden caution, with horrendous results. The scariest thing about the polio-vaccine theory was that it also seemed plausible.
Viruses are subtle. They get in where they shouldn’t. Laboratory contaminations occur. Even viral or bacterial contamination of a vaccine at the production level—it has happened. Back in 1861, a group of Italian children vaccinated against smallpox, with material direct from a “vaccinal sore,” came down with syphilis. Smallpox vaccine administered to kids in Camden, New Jersey, at the start of the twentieth century, seems to have been contaminated with tetanus bacillus, resulting in the death of nine vaccinated children from tetanus. Around the same time, a batch of diphtheria antitoxin prepared in St. Louis, using blood serum from a horse, also turned out to carry tetanus, which killed another seven children. Producers then began filtering vaccines, an effective precaution against bacterial contamination; but viruses passed through the filters. Formaldehyde was sometimes added to inactivate a target virus, and that supposedly killed unwanted viruses too, but the supposition wasn’t always correct. As late as the midcentury, some of the early batches of the Salk polio vaccine were adulterated with a virus known as SV40, endemic in rhesus macaques. SV40 in vaccine became a hot issue, several years later, when suspicions arose that the virus causes cancer. It’s important to note that vaccine purification methods and safety precautions have improved vastly since the time of the Salk vaccine, and that adamant resistance to vaccination by some activists in the present era is misguided, unjustified by scientific data, and costly to public health. But a half century ago, the possibility of vaccine contamination did exist.
Whether vaccine contamination happened with HIV-1, around the same time as the SV40 problem and far more consequentially, is another matter. That the vaccine in question had been given to Africans was not in dispute. Between 1957 and 1960, a Polish-born American researcher named Hilary Koprowski—a lesser-known competitor in the same vaccine-development race that engaged Jonas Salk and Albert Sabin—arranged for his oral polio vaccine to be widely administered in areas of the eastern Belgian Congo and adjacent colonial holdings. These were parts of what would eventually be DRC, Rwanda, and Burundi. Koprowski himself visited Stanleyville, in 1957, and made contacts who later oversaw the trials. Children and adults lined up trustingly, in places like the Ruzizi Valley north of Lake Tanganyika, to receive doses of liquid vaccine from a tablespoon or a squirting pipette. Spritz, you’re good. Next! The numbers are uncertain. By one account, roughly seventy-five thousand kids were vaccinated just in Léopoldville. The heterodox theory argued two additional points about this enterprise: First, that Koprowski’s vaccine was produced by growing the virus in chimpanzee kidney cells (rather than in monkey kidney cells, the standard technique); second, that at least some batches of that vaccine were produced from chimpanzee kidneys drawn from animals infected with SIVcpz.
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 and ill-considered comment from the head of research for the World Health Organization’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 vacci
ne 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 the Democratic Republic of the Congo. 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 therefore be decisive.
The Chimp and the River: How AIDS Emerged from an African Forest Page 4
