Early in the morning, the day after Mardi Gras in New Orleans—an explosion of humanity unimaginable today, though it was only two months ago—a FedEx truck pulled up to a loading dock at Tulane University. Two people stood waiting. The driver pulled a cardboard box out from among the orders of shoes and toilet paper, and delivered it to the woman leading the pair. It contained a frozen vial filled with millions of particles of SARS-CoV-2, the virus that was at the same time silently making its way through the city all around them.
This sample was isolated and propagated from a patient in Seattle, and was bound for the Tulane National Primate Research Center. There, it would be used to infect a group of four Rhesus macaques and four African green monkeys, to see if they could replicate the human disease—one of the first such experiments in the US. The highest-profile vaccine and treatment trials done on animals (including tests of the antiviral drug remdesivir) have been conducted only in Rhesus macaques, one of the most plentiful species of laboratory monkeys. But macaques have so far shown mostly mild symptoms after coronavirus infection. This is still useful—scientists can measure viral replication and see signs of infection. But animals with more severe symptoms make it easier to measure the effect of interventions, like vaccines or antiviral drugs.
Within weeks of the arrival of the virus at the Tulane lab, two of the lab’s African green monkeys had contracted severe lung disease. Skip Bohm, the chief veterinarian at the research center, says these are the first documented cases they’ve seen of severe illness from the new coronavirus in nonhuman primates. “It's an initial study, a few animals. But if it can be reproduced, it would be a very important model for looking at very severe disease in people,” Bohm says. “Before we can study any treatments or vaccines or even just the disease itself, we have to establish that we have an animal model that exhibits the same type of diseases as it occurs in humans.”
An animal model is a proxy that scientists find—or create using gene editing—that experiences a particular disease the way humans do. Studying animals, though controversial, is highly convenient for infectious disease research. It's illegal to deliberately infect human volunteers (though this may change if health authorities allow challenge trials), but laboratory animals are easy to isolate and observe, and they can be subjected to invasive procedures and tissue sampling. Without an animal model that closely replicates what goes on in humans, there’s potential for harm in a fast-moving pandemic response like the one mobilizing now, warns Jay Rappaport, the director of Tulane National Primate Research Center. He points to mistakes made in the response to the SARS epidemic. “During the SARS epidemic, there were some vaccines that were trying to be developed, and they actually caused liver disease and had to be stopped,” Rappaport says.
There’s also potential for vaccines to trigger immune enhancement, meaning people who have been vaccinated actually get sicker when exposed to the disease, which has been an obstacle in trying to create vaccines for other viral illnesses, including dengue fever. “These studies really need to be done first in a model that closely resembles what happens in humans,” Rappaport says.
Yet choosing among our evolutionary cousins to model the human experience of a disease is an exercise in approximation and compromise. Viruses and the living hosts they prey on have been locked in an arms race for millennia, evolving side by side. The battles within each species have been unique, and every animal has a different arsenal of defenses. It may seem like monkeys would always be the best choice to replicate human illnesses, but primate species aren't susceptible to all the same viruses.
“It's not always going to be monkeys,” says Dave O’Connor, a professor of pathology and laboratory medicine at the University of Wisconsin. He works with the Wisconsin National Primate Research Center, which, like Tulane, is part of a network of primate research centers jointly supported by the NIH and university hosts around the country. The centers are now diverting most of their focus to coronavirus research.
As the pandemic became increasingly serious, O’Connor’s team took the sequence of the receptor the virus uses to latch on to human cells, and compared that sequence across a variety of lab animals, including macaques and African green monkeys. They found both of these species and humans have identical sequences at the key place where the virus binds, a reason to believe SARS-CoV-2 might be able to infect nonhuman primates. But this didn’t take into account restriction factors, specialized built-in immune responses—usually proteins—which are released in cells to prevent the replication of different viruses. These can vary widely among species. “You never know ahead of time what sort of restriction factors or other types of blocks might prevent a virus from infecting a particular type of animal,” O’Connor says.
By the time the virus samples arrived at Tulane, a study in China had shown rhesus macaques could be infected, but there was no guarantee the team would be able to replicate their results or successfully infect the African green monkeys. But it was important to try: “Nonhuman primates in general are much more closely related to man than the other species,” Bohm says. “Their organ systems are much more similar to humans than other animal models, so they offer a lot of benefits that other animals don't.” Testing treatments and vaccines in monkeys can say more about their safety and efficacy than tests in other animals might.
The Tulane National Primate Research Center encompasses 500 acres of land, about 40 miles north of Tulane University. Tulane houses over 5,000 monkeys and has a biosafety level three laboratory (the minimum required to study SARS-CoV-2). Their first coronavirus experiment tested two species of monkey, African greens and Rhesus macaques, and two routes of exposure: aerosol exposure (breathing the virus in from the air) and direct contact (the virus was swabbed in the animals’ eyes, noses, and tracheas). The researchers’ goal was to determine how the disease differed between the species. The results would inform their future coronavirus experiments and any trials of vaccines or treatments.
Within days after initial exposure, all the monkeys were shedding the virus from everywhere the scientists sampled: eyes, nose, mouth, rectum, and vagina. But none of the animals had noticeable symptoms. Then, suddenly, the day after an examination in which she had appeared normal, one of the African green monkeys from the direct contact group showed signs of very severe pneumonia. CT scans showed her right lung was full of fluid, and she was so severely ill she met the study's criteria for early euthanasia. Two weeks later, a second African green female, this time from the aerosol group, developed the exact same symptoms in the same abrupt manner. She was also euthanized early, and scans again revealed severe edema in her right lung.
“I think this is a very important step,” says Koen Van Rompay, an infectious disease scientist at the UC Davis National Primate Research Center. Primate researchers from the national centers congregate for weekly calls to discuss their work, and last week Bohm and his colleagues recently shared the news of the severe illness in the African green monkeys, along with some details about the pathology of these animals. “We really have to understand the monkey model,” Van Rompay says. “What are the conditions to develop the best animal model? Which species, which route of inoculation?”
Tulane’s results are one piece in this puzzle, but the study was tiny: only two monkeys of each species in each exposure group, a total of eight animals. Bohm is quick to point out that these results are very preliminary, and need to be repeated to see if more African greens come down with severe illness. The primate centers are working in unusually close collaboration to try and fit this puzzle together more quickly—a group at one of the other centers is also studying the disease in African greens. So far, they have observed illness but not of the same severity as observed at Tulane.
Still, further studies are unlikely to bump up the number of animal subjects by very much. This is one of the major disadvantages of studying primates; scientists are constrained by the expense and the limited supply of animals, and these restrictions are compounded by the novel coronavirus itself. Importation of monkeys for research from China, a major source of macaques, has stopped. Only primate research facilities with appropriate biosafety facilities can conduct coronavirus experiments, and these special facilities have very limited capacity. Tulane has a large biosafety level 3 laboratory and will be able to study up to 112 animals at a time once they begin running vaccine trials, but other facilities are more limited. Rompay, at the Davis Primate Research Center, says his facility can also only enlist eight monkeys in coronavirus experiments at once, and they are also constrained by the limited number of staff with proper training, and a shortage of personal protective equipment for them.
Research on nonhuman primates is out of favor among much of the public in the US and Europe; many people, politicians included, are disturbed by the idea of causing suffering among such highly-conscious animals. US Representative Lucille Roybal-Allard, a Democrat from California who has been a longtime advocate for limits on animal research, worked with several other members of Congress in 2014 to end certain NIH experiments in which baby monkeys were separated from their mothers. The 2020 spending bill requires the Food and Drug Administration and the Department of Veterans Affairs to begin significant reductions in research using nonhuman primates, and the NIH to investigate alternatives to using these animals. Research on great apes, our closest animal relatives, is banned in the European Union and was effectively eliminated in the US after the NIH announced in 2013 that it would phase out research using chimpanzees, and the Fish and Wildlife Service classified all chimps in the US, including those in research facilities, as endangered.
And even researchers who rely on animal models point out that sometimes our close cousins can be poor substitutes for humans. Some of the most important questions being studied for Covid-19 are how comorbidities like diabetes or hypertension affect survival rates. These conditions can't be induced in monkeys overnight, or sometimes ever. (There is a standard diet somewhat degradingly known as the "Americanized diet” which can be used to try to induce diabetes in monkeys, but it can take years to have any effects.)
Because of these limitations, scientists say they will need multiple species of animals that can be infected with the disease for different kinds of experiments. “We need to understand not what animal model, singular, is going to be most useful, but what sort of animal models, plural, are going to be the most useful. Because different types of systems are going to be useful for understanding different aspects of disease,” O’Connor says. For example, hamsters exposed to the virus appear to show some signs of severe pneumonia. This could be useful, as it’s much easier to conduct studies on large numbers of hamsters than monkeys. If Tulane’s results in African green monkeys are replicated, they could be a useful species to study treatments for severe disease, while macaques, with their milder symptoms but available in higher numbers, could be suitable to test vaccines.
Many scientists argue primate research is irreplaceable, at least for now, and that it has been essential to some of the most important advances in modern medicine, including tuberculosis vaccines and many of the best treatments for HIV. Bohm, whose chief role is as the veterinarian to the monkeys at Tulane, has spent a lot of time weighing this calculation. “Anybody who's in research understands that there are sacrifices that the animals make,” Bohm says. He takes this seriously, especially in the case of intelligent animals like nonhuman primates. If scientists conclude the only way to move forward on a particular problem is by studying animals, he says, their next step is to design the experiment to reduce, to the greatest extent possible, the number of animals involved, and the pain and distress they have to endure. In the end, he feels, the benefits to humans justify this kind of work. “I have a daughter who's got type 1 diabetes, and she wouldn't be alive today if it weren't for advances in animal research,” Bohm says.
For now, his team at Tulane is poring over the tissue samples and other results from their first trial. They are planning to replicate their study with additional African greens. Then, within a few short months, they will begin running trials of vaccines and drugs from various external groups, work that couldn’t be more high stakes. “There are a lot of different products coming up through the pipeline, including different treatments,” Bohm says. “[We’re] on the phone almost every day with either biotech groups, Big Pharma, or NIH talking about studies they want to get going.”
Update 5-11-2020 4:30 PM: This article was updated to correct the laboratory capacity at Tulane.
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