Sean Diehl, Ph.D. (left), in his lab with former graduate student Huy Tu, Ph.D., in 2019.
In fall 2022, public health warnings of a possible ”tripledemic” blared across news headlines due to an early-season surge in cases of respiratory syncytial virus (RSV). Marked by cold-like symptoms including cough and runny nose, RSV is reported to be the leading cause of infant hospitalizations in the U.S. and three million hospitalizations per year globally. Annually, the virus causes up to 10,000 deaths in adults 65 and older, according to the Centers for Disease Control and Prevention.
Despite a recent rise in awareness, RSV has been around for a long time. In fact, biomedical scientists have been working to develop vaccines to prevent RSV infection since the 1960s, but it has only been in the past several months since the uptick in cases that several candidate vaccines have reached the regulatory review and approval stage.
In November 2022, the European Commission approved one of the therapies developed for the prevention of RSV in newborns and infants. Called Beyfortus or nirsevimab and developed by AstraZeneca and Sanofi, the medicine is awaiting FDA approval in the U.S. Sean Diehl, Ph.D., associate professor of microbiology and molecular genetics at the University of Vermont, is a co-inventor on the patented technology that led to nirsevimab’s success as a preventative treatment – a process that began through research started in 2003.
“Previously, there had been failed or dangerous RSV vaccines,” said Diehl, who was working as a postdoctoral fellow with immunology research pioneer Hergen Spits, Ph.D., at the University of Amsterdam in the Netherlands from 2003 to 2008. Their team’s research led to the development of a method for discovering antibodies against infectious diseases. Diehl continues – to this day – to use the platform to study antibodies to dengue virus, Zika virus, norovirus, and other pathogens in his research in UVM’s Vaccine Testing Center.
What are Antibodies and B Cells?
RSV is an upper respiratory virus that typically infects infants and makes their breathing difficult. Diehl explains that after age two, people recover from RSV and have antibodies in their immune system. By adulthood, he adds, all healthy adults have antibodies against RSV.
When infected with RSV, people’s immune systems make antibodies that try to control it and prevent it from infecting new cells, Diehl says. “Antibodies are a critical way that the immune system tries to control a virus after it enters your body,” he explains. “While the virus’s job is to replicate, kill some cells, and infect others – which in turn makes you sick – the antibody’s job is to control the spread of the virus,” says Diehl.
According to the National Cancer Institute, the immune system is a complex network of different cells, organs, and tissues throughout the body that help fight infection and diseases. B cells – white blood cells responsible for making antibodies – are part of the immune system and develop from stem cells in the bone marrow. The immune system can be trained to produce antibodies through the process of vaccination, which often introduces a tiny part of the virus to the immune system in order to initiate this antibody production process.
The Science of B Cells and Antibody Production
When they began their research in 2003, Diehl and his colleagues understood that healthy adults have antibodies against RSV and good B cells if they survived RSV. They determined that it might be possible to harvest some of these protective antibodies from adults.
Blood includes a mixture of antibodies and cells that target different viruses, says Diehl, so the researchers’ first step was to grow B cells from people’s blood so they could observe a B cell recognizing a virus and becoming activated.
“We needed to be able to sort through all these cells, [and] we needed a technology to extract antibodies from people,” says Diehl.
He and his research colleagues focused on B cells, because of their role in producing antibodies. “Antibodies are like magnets that attach to viruses and render them unable to replicate; they’re made out of protein, sort of like a lock and key specific to one virus,” he says.
The cell has DNA that encodes for these antibodies, but scientists need to know exactly what they are looking for.
Once a B cell has become activated by a viral infection “[The B cell] starts to copy itself in specialized regions of the immune system called germinal centers and randomly mutates the genes that encode for the antibody,” he explains. Over time, he adds, the antibody gene mutations improve the strength with which the antibody recognizes the virus. “It’s like getting a really sharp eyeglass prescription,” says Diehl. Initially in the response to virus the B cell “can see enough to recognize the virus somewhat, but then it goes to the optometrist (i.e. germinal center) and emerges with a sharper prescription and can see things much more clearly.”
The term to describe this process is “somatic hypermutation.” According to Diehl, after recovery from an infection, the virus-specific B cells in a person’s blood that have these somatic hypermutations that helped the B cell see very clearly calm down and become memory B cells. These cells “remember” the infection so that when the person is re-exposed to that virus, the memory B cells re-activate immediately, and replicate to defend the virus.
To search for protective antibodies against RSV, the researchers recruited a healthy adult daycare worker and drew samples of their blood, from which they extracted memory B cells.
Memory B cells are not very stable outside of the body, so in order to grow the cells, Diehl and his colleagues introduced a process to trick these cells into behaving like they were back in the immune system’s germinal centers, where the B cell activates and divides rapidly. Diehl explains that over the course of a response to a virus, B cells go from a resting state to a phase where they recognize the target, become highly specific regarding the target, and then replicate very quickly.
”Understanding what’s going on in cells helps you understand how to recreate it,” says Diehl.
“Building on work from my colleague Ferenc Scheeren, in the same lab, I discovered that you could take B cells from a healthy person’s blood and infect them with retrovirus to put back two germinal center genes, which makes the cells become reactivated and spit out antibodies,” says Diehl. He adds that he discovered that one gene was not enough; they needed to add a second and also change the ingredients added to the cell culture to mimic the natural help B cells would get from another type of immune cell called a CD4 T cell.
“We figured out that we could put in two genes that are highly expressed during the germinal phase, using a carrier virus – a retrovirus based on HIV that could integrate but not make people sick, which, together with the T cell factors, tricks the cell into behaving the way you want,” he says.
Diehl’s discovery provided the final step in answering the team’s question regarding the identification of the molecules inside the cell that allow a B cell to secrete high-quality, protective antibodies.
Turning an Antibody into a Drug
After developing their unique process for growing B cells, Diehl and his colleagues determined that they could take cells from any adult and use high-throughput sequencing technology to screen for a number of different cellular activities. This process could be used to “immortalize” their special B cells – manipulating them so that they reproduce in culture for up to a year, which was “the longest we tested and more than enough time to find good antibodies,” explains Diehl.
“With this technology, we were able to grow lots of them and find an antibody that was really good at blocking RSV,” says Diehl. The technique they developed allowed them to see how the body naturally produces B cells after successfully fighting off an infection.
The researchers also discovered the significant role of their antibody: “You could directly use the antibody as a preventative treatment,” says Diehl.
There was a precedent for giving a different RSV antibody called palivizumab to a subset of premature babies with congenital heart defects. "Since our antibody was 1000-times more potent than palivizumab in preventing RSV disease in an animal model we dreamed our antibody could be used as a routine, passive immunization for newborns to get them through their first one or two RSV seasons (fall-winter) when they are most vulnerable,” he says.
That drug was called D-25, and then MED18899 when the antibody was licensed to MedImmune, the former global biologics research and development arm of AstraZeneca, which is now an independent biotech company called Viela Bio.
“D-25 in its normal state was potent, but we hadn’t changed anything about it to extend its half-life, the amount of time it would last in the body at high enough levels to be active, which is about a month,” admits Diehl.
But the RSV season is at least three months long, he explains, so MedImmune took the DNA sequence of the antibody and changed parts of it to make it last longer. The company demonstrated that MED18899 has an extended half-life in the bloodstream, and after it was handed off, Sanofi and Astra Zeneca performed clinical trials to show that it was effective in protecting infants from hospitalization for RSV. Now that revised antibody is called nirsevimab.
“Not only did our invention allow the drug to be given as a passive vaccine – so that the immune system can take over when it gets exposed to RSV – but it also enabled other researchers, including Jason McLellan, then at the National Institutes of Health (NIH) and now at University of Texas Austin, to discover a new, better way to make an active RSV vaccine,” Diehl says.
Enabled by their D-25 antibody, NIH researchers were able to visualize the mechanisms involved when the virus infects a cell and now, that model is used as the design for RSV active vaccines.
A Long, Worthwhile Process, with Wide-reaching Impact
The idea of using antibodies against infectious diseases was accelerated during the pandemic, such as monoclonal antibody therapies that were developed for people with COVID-19 infection, explains Diehl.
“[Nirsevimab] is different, as it’s designed to be given preventively, not as a treatment,” he clarifies. “It’s not taxing on an infant’s immune system,” and is designed to provide temporary protection that sets the baby up to eventually establish their own B cell responses to protect themselves when their immune system is stronger.
Diehl has used this technology to look at other viruses and holds a patent on two antibodies against Zika. He’s also been able to use this technique on B cells from babies in research to identify antibodies against norovirus.
While it took a few steps – several journal publications, and multiple studies over 20 years – to become clinically useful, “It all started with trying to understand how immune cells do their job – the molecular mechanisms – and how we made B cells into little ‘factories’ for antibodies,” says Diehl.