In deciding when to freeze the WI-38 cells, Hayflick had to strike a balance. He wanted to produce enough cells to fill plenty of ampules for future needs. He was keenly aware that two hundred ampules of WI-26 had not been enough. On the other hand, he didn’t want the cells to get too old—to divide too many times—before he froze them. Vaccine makers wanted youthful cells that they could expand through many more divisions before they reached the end of their usefulness. They were also wary of older cells because each cell division increased the chance of chromosomal aberrations and thus, they feared, of the cells becoming cancerous.
And in fact one year after Hayflick launched WI-38, it would emerge that the chromosomes in WI-38 cells developed spontaneous abnormalities when the cells aged beyond forty divisions. These were not cancerous changes, according to the paper published in the Proceedings of the National Academy of Sciences by Moorhead and Eero Saksela, a young Finnish scientist then working at the Wistar Institute. “On no occasion,” they wrote, did any of the cells with altered chromosomes develop abnormal shapes or start to divide abnormally quickly or open-endedly, all classic signs of cancer.29
But the abnormalities that Saksela and Moorhead documented would push vaccine makers and regulators alike to steer clear of WI-38 cells at high population doubling levels. By the late 1960s companies in the United Kingdom, one of the first countries to embrace human diploid cells for making vaccines, were beginning vaccine-making campaigns with cells no older than the thirtieth population doubling level.30
Hayflick decided to freeze the cells when they had been split into new bottles eight times. Eighth passage cells—so called because they had been “passed” into new bottles eight times—would be plenty youthful, and there would be plenty of them. Just one small Blake bottle of cells, split eight times, produced 256 bottles, each containing roughly ten million cells at confluence. And Hayflick typically placed the cells from a pair of lungs into four small Blake bottles at the outset.
Some bottles, Hayflick conjectured in 2013, were probably lost to contamination, perhaps early in the splitting process, eating into the total number of bottles available when the time came to freeze the cells. At any rate, there is no record of exactly how many bottles accumulated before Hayflick gave the order that the cells in them should be distributed into tiny ampules and frozen. What is certain is that the task at hand was huge.
Hayflick was not present in his lab on the day in late July 1962 that a number of Wistar technicians, some of them borrowed from other labs, assembled to do the job. Hayflick had traveled two weeks earlier to a World Health Organization meeting being held in Geneva to discuss the potential vaccine-making uses of his human diploid cell strains. Possibly he was still on the road.
The crew of lab techs faced long hours of monotonous work. They had first to loosen the cells from where they lay in single, sticky layers on the bottoms of the Blake bottles, using lung power and the pipette technology invented by Louis Pasteur nearly one hundred years earlier. They sucked up a mix of culture medium and cells, then blasted the fluid back into the bottle and repeated this over and over again, until the fluid turned milky white with floating cells.
Then they went to work with syringes, sucking up tiny portions of the fluid and injecting these into steam-sterilized gas ampules. It was delicate work. Each little wine bottle–shaped vial was about two inches tall and had an open neck roughly one sixteenth of an inch wide. Once loaded with cells, it needed sealing. The workers—some with more finesse than others—sealed the neck of each ampule by melting it with a pass through the flame of a Bunsen burner. Using tweezers, they pulled on the string of melting glass to work it into a blunt closure—all the while trying not to kill the cells in the ampule with too much heat. They were executing what would, with hindsight, become a critically important operation: getting the cells into the ampules without letting bacteria contaminate them.
Sterilizing procedure was nowhere remotely as good in the early 1960s as it is today, in large part because technologies now taken for granted didn’t exist. For instance, today’s ubiquitous laminar flow hoods, which prevent microbial contamination of the air over a scientist’s work space, were just being developed. The ultraviolet lights then used at night had serious limits: the light they emitted killed organisms on surfaces but not in things—like cell cultures.
True, penicillin and other antibiotics were in wide use by 1962, and many labs used them liberally to protect cell cultures from bacterial contamination. But, Hayflick says, vaccine manufacturers were wary of antibiotics because of their potential for provoking allergic reactions in vaccinees. He decided to take the calculated risk of not using antibiotics. That decision would come back to bite him.
By the end of the day on July 31, 1962, the Wistar work crew had produced more than eight hundred ampules of WI-38. Each tiny vial held 1.5 million to 2 million cells. They were placed in a big, communal dry ice chest. A couple of months later Hayflick transferred them to more permanent digs in a liquid nitrogen tank in the Wistar basement. There they remained, tucked away at –320 degrees Fahrenheit.31
In October of 1962 a seasoned cell culturist named Robert Stevenson paid Hayflick a visit at the Wistar. The straight-shooting Stevenson was Hayflick’s project officer at the National Cancer Institute, charged with overseeing the smooth and proper execution of the contract under which Hayflick was producing, storing, and distributing human diploid cell strains. Perhaps Stevenson learned during this visit that Hayflick had, ten days earlier, handed one hundred ampules of the new WI-38 cells to a visitor, Frank Perkins of Britain’s Medical Research Council, the UK’s top vaccine regulator. Perkins had taken them back to London on a transatlantic flight.32
On October 18, 1962, two days after he visited Hayflick’s Wistar operation, Stevenson wrote a memo summarizing his visit and noting that he had made it clear to Hayflick that the cells were U.S. government property.
While the cells, he wrote, couldn’t be subcontracted out to a non-U.S.-government agency for distribution, the Medical Research Council, or MRC, could function as a “distribution depot.”33 This the British agency promptly became, shipping the cells to scientists in Berlin, Madrid, Milan, Tehran, and Uppsala.34
At the end of the eventful year of 1962, Hayflick filed a progress report to Stevenson at the National Cancer Institute—a routine report owed to the government agency under the contract. It included a section entitled “Characterization of a New Human Diploid Cell Strain WI-38.”35
• • •
As soon as the WI-38 ampules were first frozen, Hayflick went back to Sven Gard in Stockholm for the documentation that he knew that regulators would require. For the purposes of the Division of Biologics Standards, the unit at the National Institutes of Health that in those days licensed new vaccines, Hayflick’s word would not be enough. He needed papers proving that Mrs. X, the mother of the WI-38 fetus, was as healthy as a horse and that no cancers, hereditary diseases, or infections were lurking in her or in the fetus’s father.
Getting that information was a delicate task. Two months after the abortion, Mrs. X had no idea that her fetus had ended up anywhere but in a medical waste incinerator. In 1962 in Sweden, as in the United States, tissue from aborted fetuses was routinely used by scientists without the knowledge of the women who had the abortions. The stern Gard, aged fifty-six and childless, was not inclined to take up this job. He delegated it to someone he may have considered better suited to approach Mrs. X: a thirty-five-year-old physician named Margareta Böttiger. Böttiger was earning her PhD in his lab while running human studies monitoring the effects of the Swedish polio vaccine.
A dark-haired beauty with an oval face who hailed from a storied and powerful Swedish family, Böttiger was reserved, mannerly, and unthreatening. Like the gynecologist Ernholm, she confronted a medical profession in which just 14 percent of physicians were women. Unlike Ernholm, she had small children—her girls were three and six
years old that August—and a physician husband who did no housework. She had survived by embracing the regular hours of a scientist in Gard’s lab, rather than the pediatrics she had trained for immediately after medical school. Paying her invaluable nanny, Harriet, consumed almost her entire salary—but it never occurred to her to give up the work she loved.
The task at hand didn’t quite fit that description. But Gard had asked her to do it, and she was not one to buck authority. Böttiger telephoned Mrs. X’s primary-care doctor. Working with that doctor and the doctor’s nurse, she pieced together as much as she could and got hold of the operative record from the hospital where the abortion was performed. The whole enterprise took some doing, and it wasn’t until more than a year later—October 1963—that she wrote to Hayflick, attaching Mrs. X’s medical record.
It cataloged her childhood bouts with measles and scarlet fever and whooping cough; her freedom from other infectious diseases since then; her several healthy children; and the absence of known hereditary diseases or tumors in her family.
In her accompanying cover letter Böttiger wrote that she believed Mrs. X to be perfectly fine. But, she added, the father appeared to be subpar mentally. Böttiger also warned Hayflick, who had apparently asked for blood samples from the couple, that when Mr. X got back from his out-of-town labor, it might be tough to get his blood drawn. She did not elaborate on why.
Asked about this in 2015, Hayflick could not recall why he would have requested a blood sample from Mr. X. He added that Mr. X’s reported mental deficiency did not trouble him. Mental deficiency, he wrote, is not infectious and would not have been relevant to the cells’ safety for vaccine making.36
CHAPTER SEVEN
Polio Vaccine “Passengers”
Bethesda, Maryland; Philadelphia; and Clinton Farms, New Jersey, 1959–62
Oh, they kept taking rooms away from me, and help. But I—my best people stayed with me.
—Bernice Eddy, former NIH vaccine safety scientist, 19861
In the spring of 1960, at the National Institutes of Health in Bethesda, Maryland, just outside Washington, DC, a feisty fifty-seven-year-old PhD scientist with an open, square face and neatly coiffed dark brown hair was worrying about her alarming findings in a group of newborn hamsters.
Bernice Eddy had grown up in a family of physicians in Auburn, a town of 199 in rural West Virginia, and earned her PhD in microbiology from the University of Cincinnati. After a stint researching leprosy in Louisiana, she came to the NIH in 1937.2 By the late 1950s she had been working for more than twenty years in the NIH division that was then the gatekeeper of the U.S. vaccine market. The Division of Biologics Standards (DBS) filled the role played by a key branch of today’s Food and Drug Administration, assessing new vaccines and issuing licenses when it deemed a product ready for market. Eddy had done well in her work scrutinizing vaccine safety and effectiveness: in 1953 she won a “superior accomplishment” award and a pay raise from the NIH director.
The next year Eddy was put in charge of the unit’s polio vaccine safety tests. It was a high-stakes job. On the heels of a 1952 polio epidemic that had infected nearly 58,000 Americans and paralyzed more than 21,000 of them—the country’s worst polio epidemic ever—virologists were in the midst of an all-out sprint to get a polio vaccine licensed. That first vaccine, newly invented by Jonas Salk, was made from polio virus that multiplied in monkey kidney cells and was then killed with formaldehyde. The Salk vaccine was entering human trials in 1954 when Eddy isolated live polio virus from three lots of the vaccine made by the California-based Cutter company. She injected monkeys with the suspect vaccine and found that it paralyzed some of them. She reported the findings to her bosses and sent them photos of the afflicted animals.3 They were ignored, and in April 1955 the Salk vaccine was licensed.4 Mass vaccinations began, and the Cutter vaccine was among those distributed. It paralyzed 192 people, many of them children.5 Ten people died.6 The government was forced to temporarily recall all polio vaccine, sowing public panic. Manufacturing changes were mandated, and the vaccine returned to the market. But public confidence took months to rebuild, a fact that was responsible for many of the more than 28,000 cases of polio in the United States that year.7
Despite her earlier efforts to warn her bosses about the flawed vaccine, and over her protestations, Eddy was pulled off polio vaccine work as part of the Cutter-episode fallout.8 It was worse for the higher-ups: senior officials including the director of the NIH, William Sebrell, and the secretary of health, education and welfare, Oveta Culp Hobby, lost their jobs. So did William Workman, Eddy’s boss.
Eddy, who would show amazing staying power over the years, persisted at the DBS and in 1959 landed in Time magazine with her friend and NIH colleague physician/scientist Sarah Stewart. The pair had discovered a mouse virus that was now named “SE polyoma”—“SE” for Stewart and Eddy, and “polyoma” meaning “many tumors.” Stewart isolated the virus from tumors in three laboratory mice. Then the two scientists conducted experiments showing that fluid from the mouse tumors caused new malignant tumors not just when injected into other mice but also when injected into hamsters and rats.9 The notion that viruses might cause cancer was resurfacing and gaining traction, and their discovery got the attention of other scientists in a big way.
But beginning shortly before the Time article was published that July, Eddy launched another experiment—one born of a nagging worry that her polyoma studies had prompted and that now wouldn’t leave her alone.
Her concern was this: if virus-bearing fluid from a mouse tumor could so easily cross species lines to cause cancer in hamsters and rats, why couldn’t a virus from, say, a monkey—a species much closer to Homo sapiens—cause cancer in humans? Her question wasn’t academic. Salk’s polio vaccine had been injected into more than 69 million Americans since its launch in 1955. The vaccine was grown in monkey kidney cells. And Eddy well knew, as did any vaccinologist who worked with them, that those kidney cells harbored plenty of viruses—“simian” viruses, in scientific parlance—from simia, the Latin word for ape.
These were viruses that lurked in apparently healthy monkeys, and especially in their kidneys. However normal the animals seemed, the viruses regularly killed their kidney cells in the lab, forcing scientists to jettison spoiled cultures. The Wistar’s chief Koprowski, who had a keen interest in the matter because he was developing his own polio vaccine, noted that the viruses “are more often than not dormant in the intact [monkey], but go on a rampage when infected tissues are removed soon after the animal’s death.”10
A scientist at the drug company Eli Lilly, Robert Hull, had begun cataloging each new simian virus that was discovered, classifying it according to the cellular damage that it caused in monkey kidney cell cultures. In 1958 he reported that eighteen new simian viruses had been discovered in just the previous two years. “As long as primary monkey kidney cultures are used in the production and testing of virus vaccines,” his paper concluded, “the problem of simian virus contamination will remain.”*11
The assumption about these simian viruses, made by everyone from Salk to the NIH’s top vaccine overseers, was that while they might be an annoyance in the lab or on the production line, they were not a danger to human beings, because they were killed by the same formaldehyde that killed the polio virus in the Salk vaccine. What was more, the reasoning went, even if simian viruses occasionally managed to survive the manufacturing process, they were clearly innocuous in humans: weren’t there tens of millions of healthy Salk vaccinees walking the streets to prove the point?
Bernice Eddy couldn’t so casually accept those assumptions. In June of 1959, without her NIH boss’s knowledge, she launched a bold experiment. She took monkey kidney cultures prepared by the DBS—these were from rhesus monkeys, the species widely used in polio vaccine making, which will be important. She froze them, ground them up, put them through a very fine filter that strained out bacteria but not vir
uses, and injected a fraction of an ounce of the resulting fluid under the skin of newborn hamsters. Fully 70 percent of the 154 animals that she injected developed tumors, and every animal that did so died. What was worse, it wasn’t just one or two of the ground-up cultures that were at fault: she had prepared twelve lots of ground-up monkey kidney cells, each derived from between eight and thirty-two monkeys. Nine of the twelve turned out to be cancer causing. The virus appeared to be common in the monkeys.12
Eddy then went a step further, taking tumors from two of the sick hamsters, mincing them finely with scissors, and injecting tiny bits of tumor under the skin of forty more newborn hamsters. All but two of those hamsters got cancer and were dead in less than three months.13
It was by now the early summer of 1960, and Eddy was anxiously pondering her findings when she got word of a speech that had been given at a major polio vaccine conference in nearby Washington, DC. In the world of polio vaccinologists and public-health people, the talk, by Maurice Hilleman—a highly respected, tough-talking Montanan who headed vaccine research at Merck—landed with the explosive force of a hand grenade. It soon caused Eddy to gather her courage and approach her boss with her findings.
The Vaccine Race Page 13