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No One Knows What’s Inside the Smallpox Vaccine

The key ingredient in our oldest vaccine is a mystery that goes back 200 years.

Getty; The Atlantic

At the heart of history’s most successful eradication campaign is a mystery. The smallpox vaccine—now also being deployed against monkeypox—contains a live virus that confers immunity against multiple poxviruses. But it is not smallpox or a weakened version thereof. Nor is it monkeypox. Nor is it cowpox, as suggested by the vaccine’s famous origin story involving pus taken from an infected milkmaid to immunize an 8-year-old boy.

It is something else entirely: a unique poxvirus whose origins have been lost, or perhaps never known at all. Scientists call it vaccinia, and it is pretty much found only in the vaccines. No one knows where vaccinia came from in nature. No one has ever found its animal reservoir. No one knows quite what vaccinia is—even as it has been used to inoculate billions of people and saved hundreds of millions of lives. It is a ghost of a virus that has survived by being turned into a vaccine.

José Esparza first began wondering about vaccinia in the 1980s, when he was assigned an office at the World Health Organization next to the smallpox archives. By then, the disease had already been eradicated, and people had, he says, “little interest in understanding the origins of the vaccine.” He went on to have a long career working on HIV and other viruses at the WHO and the Bill & Melinda Gates Foundation, but in retirement, he has returned to solving the mystery of vaccinia. It is a “hobby,” but also a bit of an obsession. For years now, he has been scouring museums and eBay for old vials of smallpox vaccine, scoring a couple every year. (“EBay—you can find anything you can imagine!” he says.) These vials no longer contain live virus, but the technology now exists to sequence the fragments of viral DNA that remain.

This DNA has revealed tantalizing if perplexing clues. Vaccinia turns out to be most genetically similar to another poxvirus called horsepox. But scientists have only ever sequenced one horsepox sample in the world, and they may never find another; the disease largely disappeared in the early 20th century. If horsepox really is the progenitor of vaccinia, how did that happen? And how did it then become lost to time?

The best-known version of the smallpox-vaccine story goes like this: In 1796, the British doctor Edward Jenner noticed that milkmaids exposed to a mild disease called cowpox were unusually protected from smallpox. He found a young woman with fresh cowpox lesions and scratched material from one into the arm of a boy—the son of Jenner’s gardener, no less—who became mildly ill but survived. He indeed became immune to smallpox. The first vaccine was born.

Jenner was not really the first doctor to make this observation about cowpox. But he did document his experiments in a now seminal book. Intriguingly, he mentions horses in the book’s introduction. On the second page, in fact, he speculates that cowpox originated as “grease,” a horse disease that may have spread from equines to farmworkers to cows to dairy maids. He couldn’t prove this, though; it would take several more decades for scientists to understand that diseases are caused by invisible microbes that spread among people and animals. This brief allusion to horses gets forgotten in favor of a “beautiful tale of the milkmaids,” Esparza says.

Nevertheless, smallpox vaccines soon began spreading around the world. Nineteenth-century vaccines were a far cry from the standardized pharmaceutical products we’re used to today. Preservation of the material on glass or thread was unreliable, so the smallpox vaccine was maintained in the bodies of young children: Liquid from a pox on one child would be transferred into the arm of another, resulting in a pox whose contents could be transferred to another, and so on. And it had to be children, because adults tended to already have immunity to smallpox. In 1803, Spain sent 22 orphan boys on a Royal Philanthropic Vaccine Expedition to bring the smallpox vaccine to its colonies. The number of boys was chosen precisely to span the length of the transatlantic voyage: Every nine or 10 days at sea, doctors would transfer the vaccine to the arms of two new boys—two in case one did not take, so that the ship would arrive in the Americas with the last set of boys still having sores.

The chain of arm-to-arm transfer did sometimes fail, however. When an established source of vaccine wasn’t available, doctors who’d heard about Jenner’s experiment went back to the animals. Cows were used, as were horses. The physician Luigi Sacco, for example, who popularized vaccination in northern Italy, successfully inoculated patients with vaccines derived from grease-infected horses. As doctors such as Sacco experimented with new sources, multiple vaccines probably came into circulation. There was no single canonical vaccine.

Not until the mid-19th century, when scientists figured out how to maintain the smallpox vaccine in calves, did it become a mass-manufactured product. The use of horses faded from living memory. Vaccinia and cowpox eventually became interchangeable names for the virus in the vaccine. In fact, the words vaccine and vaccinia both derive from vacca, which is Latin for cow.

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By the 1930s, though, scientists were realizing that vaccinia and cowpox were not one and the same. When a British microbiologist injected one virus or the other into rabbits, he saw that they induced slightly different immune responses. To the immune system, vaccinia and cowpox are not interchangeable. But the molecular techniques of the time were not sophisticated enough to nail down how they differed.

Meanwhile, other theories flourished: Vaccinia was a domesticated version of cowpox or smallpox, transformed through arm-to-arm passage in the 19th century. Or it was a hybrid of the two. Or perhaps it was a prototype poxvirus that gave rise to all other poxviruses. In the 1970s, poxvirologist Derrick Baxby speculated that horsepox was one possible origin for vaccinia—harkening back to Jenner’s description of grease in horses 200 years earlier. But horsepox had gone extinct, he lamented, with no laboratory experiments having ever been conducted.

Baxby did not know this at the time, but thousands of miles away, a poxvirus had, in fact, been killing horses in Mongolia. Only in the early 2000s, some 25 years later, would scientists rediscover and sequence an archived virus from this forgotten outbreak, which they attributed to horsepox. The results were presented at an international meeting of poxvirologists in 2002. “A lot of their ears perked up,” says Edan Tulman, one of the scientists who sequenced the Mongolia virus at Plum Island Animal Disease Center at the time. The virus it most resembled turned out to be none other than modern vaccinia. Yet, it also has genes that “no other modern-day vaccinia” does, suggesting that it is indeed closer to the wild poxvirus that first gave rise to vaccinia, says Elliot Lefkowitz, who studies microbial genomics at the University of Alabama at Birmingham. The results added new fuel to the horsepox-origin hypothesis.

Old smallpox vaccines have also confirmed a link—they share even more similarities with horsepox. One of the vials Esparza bought off of eBay, manufactured by a Philadelphia company in 1902, contained vaccinia whose core genome was 99.7 percent identical to that of horsepox. Esparza and two close collaborators—Clarissa Damaso at Federal University of Rio de Janeiro and Andreas Nitsche at the Robert Koch Institute in Germany—have since sequenced about 30 old smallpox-vaccine genomes, which have not all been published yet. They have also reanalyzed five published partial genomes from Civil War–era smallpox vaccines at the Mütter Museum. Four looked like 1976 horsepox virus, and a fifth was so similar, Damaso says, that it’s basically “true horsepox virus.”

In fact, all of the vaccinia that the team has found from old vaccines have resembled horsepox, which naturally brings up another question. If doctors in the 19th century were using both cows and horses as sources, how come only horsepox-like viruses survived in vaccines? Perhaps doctors and early manufacturers were inadvertently selecting for horsepox when choosing the best vaccines, suggests David Evans, a poxvirologist at the University of Alberta in Canada. “That’s just a guess,” he says, but “cowpox doesn’t really grow particularly well in humans.” Or perhaps, Damaso muses, cowpox could have been a misnomer this whole time. Just as monkeys are not the natural reservoir of monkeypox, scientists now think that cows are not the natural reservoir of cowpox—it actually seems to be rodents. Poxviruses have historically been named after the animal in which they were first found, but they usually infect a broad range of hosts. Could the virus causing “cowpox” in cows during Jenner’s time actually be what we now classify as “horsepox,” a virus that was possibly infecting cows, humans, and horses? The vaccinia of today is, in fact, capable of infecting multiple species. People who got older versions of the smallpox vaccine sometimes spread it to others. Vaccinia has also spilled over into animals, including buffalorodents, and cattle. And in cattle, the disease manifests a lot like cowpox.

Mysteriously, horsepox seems to have disappeared from the world—perhaps because modern husbandry practices have prevented it from spreading. Mongolia hasn’t recorded any new outbreaks, and Europe hasn’t recorded any in more than a century. A few years ago, Esparza contacted virtually every vet agency in the world looking for any evidence of horsepox, and found none. He’s also tried, unsuccessfully, to hunt for the virus in horses and wild rodents in Mongolia.

Proving conclusively that horsepox is the origin of vaccinia is hard, now that wild horsepox is gone from the world. The questions that linger are unanswerable without more samples: Was the horsepox found in Mongolia in 1976 really the same disease that had afflicted horses thousands of miles away and nearly three centuries earlier in Europe, when the smallpox vaccine was first invented? And even if so, could the Mongolia virus be a very old escaped vaccine virus that spread from inoculated humans to animals, rather than a wild horsepox virus?

This much is clear, though: The 1976 Mongolia virus and vaccinia have a common poxvirus ancestor, which likely circulated among animals in Europe around Edward Jenner’s time. That ancestor, whether horsepox or not, became the progenitor of all viruses in smallpox vaccines today—but has itself disappeared.

Since Jenner’s time, the process of making vaccines has become far more controlled and standardized. The vaccinia in the Jynneos vaccine used against monkeypox today is still live, but it’s no longer capable of replicating in human cells; it lost this ability after being passaged through chicken cells hundreds of times. Vaccinia remains a mystery because it arose before the advent of a modern, regulated pharmaceutical industry. No one kept careful records; even if they did, they wouldn’t have known what to record. It all happened before anyone had an understanding of germ theory. It happened even before the discovery of viruses. Remarkably, the vaccine worked. And it worked so well that we were able to eradicate a human disease for the first and only time.

Sarah Zhang is a staff writer at The Atlantic. In 2021, she was a Livingston Award finalist for her reporting on Down syndrome. Before joining The Atlantic, she was a staff writer at Wired. She covers science and health.

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