2020: The Human Immunome: Small Moves Become a Movement

AWARDEE: James E. Crowe, Jr.

FEDERAL FUNDING AGENCIES: Department of Defense, National Institutes of Health

The Human Immunome: Small Moves Become a Movement

James E. Crowe, Jr.

“We’re basic scientists. We did not set out initially just to make drugs. That’s not what it was about when we started. I’ve just always been interested in how the human immune system even recognizes a virus,” says James E. Crowe, Jr., a well-known infectious disease expert who directs the Vanderbilt Vaccine Center in Nashville, Tennessee. “Now we can study how the body works and have curiosity about that, but also have drugs in the pipeline. It’s the unexpected benefit we get by delving deeply into the human immune system.” Crowe is one of the recipients of the 2020 Golden Goose COVID-19 Recognition for his decades-long pursuit of a better understanding of the human immune system, which is now paying dividends in the fight against COVID-19.

Investigating the Immune System

A pediatrician by training, Crowe spent part of his time in medical school caring for children in developing countries, where he saw the difference that access to health care could make. Vaccines held a particular interest. He was inspired by the advent of the life-changing polio vaccine, so much that he decided to join the lab of the late Robert Chanock, who led vaccine research at the NIH campus in Bethesda, Maryland. “Because of this deep immersion,” he says, “I became interested in research and its potential to save the world from the consequences of many diseases.” In 1995, Crowe joined the Vanderbilt faculty, where he soon won an award for young investigators from the American Society for Microbiology (ASM). At the same ASM meeting was innovator Craig Venter, famous for his role in the Human Genome Project. Sitting in the audience, listening to Venter’s speech, Crowe began thinking about small moves in science that can lead to a movement. If Venter could do it with the genome, why couldn’t Crowe do it with the immune system?

Well, for one thing: The genes that encode antibodies, proteins that function in the body as soldiers against disease, are more complicated. “Antibodies are formed by a combination of genes. You can make over ten thousand combinations, almost to the scale of the genome, but the three genes used are not stitched end to end,” Crowe explains. The spaces between them, where the antibody genes are joined, are filled with random DNA sequences that lead to many times more combinations. Even more challenging? The antibody genes can mutate. Factoring in those mutations, the scale of trying to characterize the human immunome, the “parts list” of the human immune system, is immense—billions of times larger than the human genome.

The focus on antibodies appealed to Crowe’s sense of wonder at the complex forms and patterns to be found in a biodiverse world. “My fascination with the pattern in nature touches back to my childhood,” Crowe says. “I was a collector of rocks and seashells and stamps, and the idea was to get one of everything. The idea of sequencing every possible antibody gene of the billions possible spoke to me.”

At the time Crowe was beginning to launch the idea, another epidemic was tearing through the globe: AIDS. HIV, the virus that causes AIDS, was garnering significant scientific interest. Crowe received a grant from the National Institutes of Health (NIH) to study B cells, the type of immune cell that makes antibodies, and he began to sequence antibodies in patients who tested positive or negative for HIV. Since then, the field has advanced in areas such as genome sequencing, synthetic biology, and data analysis, and this advancement has enabled the groundbreaking work that Crowe and his colleagues are now doing to understand the human immune system. Despite the potential, “sometimes big science can get criticized for scope relative to return on investment,” says Crowe’s colleague Jennifer Pietenpol, who oversees research at the Vanderbilt University Medical Center. It was a perception that Crowe and his team had to consider as they ramped up their antibody work over the years. However, as Pietenpol shares, “the techniques developed and learnings from big projects typically benefit the entire discovery continuum.”

The Human Genome Project, for example, took 13 years and cost around $3 billion, and it raised similar questions. Today, next-generation DNA sequencers can do the same job in a day for less than $1,000. Motivated by such advances, in 2016 a public-private partnership called The Human Vaccines Project launched the Human Immunome Program, with Crowe as its scientific director. The decade-long, multi-million-dollar effort brings together the expertise of many different disciplines and partners—a clear example of “team science.” A stronger understanding of the immunome could offer the potential to tailor human immune response and better ward off illness.

Advances in immunology have also led to a focus on monoclonal antibodies, lab-produced proteins that can bind to substances in the body that cause disease. Crowe’s lab has been able to generate such antibodies to target viruses that include dengue, Ebola, HIV, influenza, norovirus, respiratory syncytial virus (RSV), rotavirus, Zika virus and others. Antibodies the lab made for Marburg virus and chikungunya virus are now being explored in clinical trials. As with genome sequencing, the isolation and production of such antibodies is becoming easier and cheaper for scientists.

A Serendipitous Delivery

Crowe knew his lab’s approach to antibodies could help when the first U.S. cases of COVID-19 emerged in Seattle—but before his lab could engage, he needed to get blood samples from the patients to his teammates. “We had to get a blood sample from the first U.S. case from Seattle to Nashville on a Saturday night,” Crowe recalls. What followed was a series of calls—and, like many Golden Goose stories, a little serendipity.

“I called my CEO and asked him to call the CEO of FedEx, because I figured they knew each other,” he says, noting that FedEx is also based in Tennessee. “They got it done! Someone in a Lincoln town car drove the sample to my home on Sunday morning, I walked it into work, and we were up and running.”

Crowe and his wife in Italy

Problem solving is a necessity, and not only in collecting viable samples. In Crowe’s case, this work often means devising new strategies on short notice. Case in point: He and his wife departed for a long-planned three-month sabbatical to Italy in February–just before the novel coronavirus swept that country. After 25 fast-paced years at Vanderbilt, Crowe had been excited to have time and space to do some strategic thinking about high impact work he wanted to do over the next decade. So he made preparations to manage his lab from afar. These plans quickly changed as COVID-19 spread and Italy became the next major hot spot; Crowe and his wife made it onto one of the last available flights home.

By then, he and his team had worked out the techniques they wanted to use to do the coronavirus antibody work. Fortuitously, the best blood samples arrived the day he got back to the lab. “You have to let the immune response mature over time to get the best antibodies,” he says. “We tracked down some people who had been infected in Wuhan in December and got their samples in March.”

Just how does a scientist track down blood samples in situations like this? One has to be part news junkie, part detective. Crowe and his colleagues would scan the headlines for cases, distribute flyers, and get in touch with research and public health contacts in other cities who could share publicly available information and spread the news of the lab’s efforts. Often, the patients who learned of the project were eager to help. One patient even came twice when called for a blood draw—the second time on the way to the airport at the crack of dawn.

Accelerated Work

As the team collected samples, they were also conducting the science at a vastly accelerated pace. Though Crowe’s work on the Human Immunome Program was the basis for the work his lab is doing to help solve COVID-19, it wasn’t a straight line from one to the other. Crowe also credits work funded by the Defense Advanced Research Projects Agency (DARPA) at the Department of Defense for enabling the lab to respond so quickly and efficiently to the pandemic.

Running in the Alps

In late 2017, the team received a grant from DARPA to take on a challenge: find a way to produce antibodies for any virus within 60 days of collecting a blood sample from a survivor. DARPA focuses on cutting-edge defense-related research and is often know for outside-the-box thinking; in this case, the aim was to protect American troops if they were to confront a deadly virus in the field. “We thought it sounded crazy—this type of work usually takes years—but that’s what DARPA does. It funds the crazy,” Crowe says.

The team decided to attempt the rapid production of Zika virus antibodies after a flare-up of the mosquito-borne disease that caused birth defects in several countries. “We took a sample, started the clock, and made Zika antibodies and fully tested them in 78 days. We had a lot of semi-disastrous stuff happen like instruments breaking, so we had already learned how to make mistakes, face unexpected accidents, and move on.” The team had begun planning a second simulation of an avian influenza outbreak for 2020 but pivoted in January to handling COVID-19 as it became clear how serious the new virus had become. “With COVID-19, we jumped right in. We worked at a scale that was unprecedented because we knew how to do this already. … Staying stuck or quitting is not acceptable. You have to move forward—like in improv comedy. It was bootstrapping with very few assets to start with, and this type of work is very exciting if the team works together well. Each person has to enjoy the freedom of curiosity and extemporaneous ideas, while working up to 20 hours a day with urgent goals and timelines.”

With the blood samples they received from the Wuhan patients, Crowe and his team made thousands of monoclonal antibodies. After selecting the most promising ones and rapidly testing them against the virus in animal models, they sent the leading candidates for antibody tests and treatments to pharmaceutical companies. The first antibody sequences, which went to Astra Zeneca, have now led to potential treatments being tested in five different Phase 3 clinical trials. Some of the antibodies also are being readied for clinical trials by the U.S. government’s Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense (JPEO-CBRND). “In the context of this pandemic,” says Pietenpol, “the Crowe team’s pursuit of large-scale antibody science is returning milestone advances for the prevention and treatment of COVID-19.”

Crowe points out the importance of having multiple federal agencies with different missions and approaches, like NIH, DARPA, and the Biomedical Advanced Research and Development Authority (BARDA) supporting scientific research. “[COVID-19] is the best case I’ve ever seen where the agencies were all working together” toward the same goal, he says. He sees it as a testament to what federal R&D can accomplish to help solve pressing public health problems—now and in the future.

 By Erin Heath

2020: A Llama Named Winter

AWARDEES: Jason McLellan and Daniel Wrapp

FEDERAL FUNDING AGENCIES: National Institutes of Health, Department of Energy

A Llama Named Winter: An Unlikely Partner in the Fight Against COVID-19

Winter the llama

Winter the llama currently spends her days grazing in peaceful retirement on a research farm in Ghent, Belgium, with about 130 of her llama and alpaca friends. But although you wouldn’t know it to look at her, this cocoa-colored, long-legged camelid is already playing a pivotal role in the hunt for effective treatments for COVID-19.

In partnership with researchers at Ghent University, Dr. Jason McLellan, a structural virologist at the University of Texas at Austin, and Daniel Wrapp, a doctoral student in McLellan’s lab, linked a special antibody produced by Winter to a human antibody to create a new antibody that binds to a protein on the coronavirus that causes COVID-19, thus inhibiting the virus from infecting human cells.

The Spike of the Virus

To fully grasp Winter’s unlikely contribution to the battle against COVID-19, it helps to understand the way coronaviruses spread throughout the body.

Scientists have identified hundreds of unique coronaviruses, although most of them circulate in non-human mammals and birds. Seven coronaviruses are known to cause sickness in humans; of these, four cause only mild-to-moderate illness, including some variations of the common cold. The other three have been the cause of more serious diseases: SARS, MERS, and COVID-19.

Coronaviruses get their name from a group of specialized proteins, called spike proteins, which dot the surface of each viral envelope and give the virus the appearance of a crown-like ring, similar to a solar corona. These proteins are what allow coronaviruses to break into human cells, after which they begin to use the machinery of those cells to start replicating. If this break-in is interrupted, the virus can’t infect the host.

Here’s Where the Llama Comes In

Scientists in the 1990s discovered something special about the antibodies produced by camelids, the family of animals that includes llamas, alpacas, and camels, among others. While humans only make one kind of antibody, made up of heavy and light protein chains arranged in a Y shape, camelids produce two. One of these is very similar to a human antibody. The other, called a single-domain antibody, VHH, or nanobody, does not have any light-chain proteins. This makes it much smaller, which is a boon to researchers.

A rendering of a camelid antibody

One of the ways an antibody can disrupt a coronavirus is by binding to key areas on the spike protein. Because they are smaller, the nanobodies produced by camelids can sneak into nooks and crannies on the spike protein while larger antibodies are blocked. These nanobodies also have the advantage of being stable and easy to manipulate. They can be linked with other antibodies, including human antibodies, to increase their effectiveness. Furthermore, nanobodies can be nebulized and used in an inhaler, which is good news for a respiratory illness such as COVID-19.  

Camelids are not the only animals that produce nanobodies: sharks produce them, as well, although biologists suspect that those nanobodies evolved through different processes.

Since the discovery of nanobodies, scientists have worked with camelids (and even some sharks) to develop promising therapies to treat various diseases. McLellan and Wrapp are among those scientists.

Building Relationships

A first-generation college student from a suburb outside of Detroit, McLellan fell in love with structural biology in college before training in the field in graduate school. “In structural biology, we’re trying to determine the first structures of proteins and molecules,” he says. “We’re answering basic science questions about molecules, their structure, their function, but then also trying to take some shots on goal and actually generate some products—some vaccines, some antibodies—that could have an impact on human health.”

As a post-doc, he joined a lab run by Dr. Peter Kwong, who was working on the possibility of a structure-based vaccine for HIV. A quirk of fate led to another fruitful partnership. According to McLellan, there was no room for him on Dr. Kwong’s lab on the fourth floor, so he moved to the second floor. It was there that he met Dr. Barney Graham—another 2020 Golden Goose honoree—who would become a close friend and collaborator.

“He’s a really generous, warm person,” McLellan says about Graham. “He’s just one of the really good people in science.”

McLellan recalls describing his frustrations with structure-based vaccine design for HIV to Graham, who suggested that he try out his ideas on respiratory syncytial virus (RSV), a respiratory illness that can be serious in infants and older adults. McLellan’s work on the structure of the RSV F protein (a very similar protein to coronavirus spikes) caught the attention of a group of researchers in Ghent led by Dr. Xavier Saelens. They reached out about a potential collaboration. Their goal? Identifying and isolating camelid nanobodies that could neutralize RSV.

Along with Graham, McLellan and Saelens signed an agreement to work together on RSV in July 2013. Over the next couple years, the labs traded material. McLellan’s lab sent over stabilized RSV F proteins, which Saelens’ group then used to immunize a llama from their herd (Winter wasn’t born yet). When that llama produced antibodies, Saelens’ lab sent those back to McLellan’s group, where a first-year graduate student named Daniel Wrapp helped to map their structures.

“That was actually the first crystal structure I ever solved,” says Wrapp, “that first little camelid nanobody that neutralized RSV.”

Prior to this first big solve, back when he was still a senior in college, Wrapp started reading the work on RSV that McLellan was publishing. “This is the coolest thing in the world,” he recalls thinking. “He’s found a way to look at these proteins on an atomic level. He’s figured out how to manipulate them, and make them function—or not function—exactly the way that he wants, and he’s figured out how to manipulate that and leverage it to elicit a particular immune response that will be effective at neutralizing a virus. And I thought that that was incredible.” Wrapp applied to Dartmouth, where McLellan was at the time, specifically because he wanted to join his lab.

It was a charmed partnership from the start. During Wrapp’s first year of graduate school, the lab received a National Institutes of Health R01 grant to look at the structure and function of coronaviruses. He remembers the date they received the good news with absolute clarity: it was his birthday.

Though the NIH funding was critical, the team’s investigation of protein structures, including those related to SARS-CoV-2, also received a critical assist from the Department of Energy’s Argonne National Laboratory. “For structural determination, we hit the protein crystals with x-rays,” McLellan explains. Argonne allocate time to the team to use its synchrotron facilities for this purpose. 

McLellan and Wrapp in lab

Laying the Groundwork

After the success of their work on RSV, McLellan and Graham decided to apply what they had learned about structure-based vaccine design to coronaviruses. It was a natural leap: the RSV F protein McLellan and his team had mapped out belongs to the same family of proteins as the coronavirus spike. And again, they reached out to Saelens’ lab—and herd—in Ghent.

Winter was just nine months old when she was chosen at random to participate in the coronavirus study. This was in 2016. Just as they had with another llama and RSV, scientists at Winter’s facility in Ghent injected her with stabilized spike proteins from SARS-CoV-1 and MERS-CoV. Inspired by work in the influenza field, their hope was to isolate a single antibody that could neutralize all coronaviruses.

“We wanted to get the one antibody or nanobody to rule them all,” McLellan says. That didn’t work. But the team was able to isolate some potent MERS-specific and SARS-specific nanobodies. They were writing up their findings when everything started to change in January 2020.

The Call

McLellan was on a snowboarding trip with his family in Park City, UT, when he got the call that changed everything. It was Dr. Graham, calling to tell him that he had been in contact with the CDC, and that it looked like the new pathogen that was making headlines was a coronavirus. McLellan recalled Graham wanting to know if he wanted to rush to make a vaccine together. “I said, ‘sure, let’s do it. This is what we’ve been preparing for,’” McLellan says. “I immediately messaged Daniel, told him, ‘Be ready. We’re going all-in as soon as we get the sequence.’”

Jason McLellan

Because of their past work understanding and manipulating the spike proteins of SARS-CoV and MERS-CoV, McLellan’s team, which included colleague Nianshuang Wang as well as Wrapp, was able to rapidly map the structure of the SARS-CoV-2 spike protein and develop a stabilized version of it that could be used as a COVID-19 vaccine antigen. Antigens, which are molecules or molecular structures that trigger a hoped-for immune response, are crucial elements of vaccine development. Shortly after that, the team filed a joint patent application on their stabilized spike protein along with fellow 2020 Golden Goose Award winners Graham and Kizzmekia Corbett. (For more on Graham and Corbett, read A Spike in Momentum.) Genetic information from this stabilized protein has been incorporated in the vaccines currently under development at Moderna, Pfizer/BioMTech, Novavax, and Johnson & Johnson.

 At the same time, Wrapp and McLellan still had Winter’s MERS- and SARS-reactive nanobodies and decided to put together an experiment to see if either of them bound to SARS-CoV-2. They discovered that one of them, the SARS-CoV-1 nanobody, did. They then linked this to a fragment from a human antibody to produce an antibody that tightly binds to a key area of the SARS-CoV-2 spike protein, effectively blocking it from infecting human cells. 

Daniel Wrapp

Wrapp recalls that the experiment “was kind of almost an afterthought, which sounds ridiculous now, because it ended up being so fruitful.” The hope is that Winter’s nanobodies might be used to develop prophylactic treatments. Unlike a vaccine, which must be administered before a person is infected to provide protection, antibody therapies can be used to treat someone who is already sick. The team published their findings earlier this year, and a company has formed with the hopes of shepherding the research through the testing process to a viable product. In the meantime, Wrapp plans to finish his Ph.D. Winter will continue to enjoy her retirement.

Reflecting on the discovery, McLellan stressed the importance of funding basic research. When they first immunized Winter, he pointed out, SARS no longer in the human population, and MERS was only popping up sporadically. Their funding “wasn’t in response to a pandemic: it was just good science on an important class of pathogens, and I think it’s really paid off.”

By Haylie Swenson