While many of us try not to consider whether another pandemic is imminent, some members of the Bloom Lab in the Basic Sciences Division are busy thinking of nothing else. They’re hard at work doing crucial research to get ahead of the next pandemic, wherever it may come from. The latest contribution in this vein was recently published in PNAS.
This study was led by Dr. Brendan Larsen, a postdoctoral researcher who is specializing in bat- and rodent-borne viruses that can spill over into humans. These zoonotic pathogens are some of the most likely candidates for the next pandemic—and the most frightening.
One of these is Nipah virus, which “regularly spills over from bats into humans, particularly in India and Bangladesh,” says Dr. Larsen. “Human infections have a fatality rate of 50-90%, and there are no approved vaccines or therapeutics available for treatment.” The most recent outbreak was earlier this year, which prompted contact tracing and airport screenings in Asia per the BBC. However, widespread human transmission has not yet been reported.
“Virus entry proteins need to accomplish two key steps to infect host cells: bind host receptors and then fuse the viral and host cell membrane,” explains Dr. Larsen. He has previously probed the evolutionary and antigenic landscape of Nipah’s receptor binding protein (RBP) to determine how mutations can impact human infectivity and immune escape (you can find that study here).
However, there is another vital component that impacts viral entry. “For this work, we focused on the Nipah virus fusion protein,” Dr. Larsen reports. Also called F, the fusion protein is triggered by RBP to fuse membranes via a dramatic structural change.
This structural reorganization is what makes F tricky to study in vitro: its post-fusion conformation is much more stable than the pre-fusion version, despite pre-fusion F being the dominant form displayed on viral particles. But it’s worth characterizing F despite the technical hurdles, as it is an important target for neutralizing antibodies.
In fact, it’s thought that F may be a better target for therapeutics and vaccines than RBP. One of the reasons for this is that F seems to be quite evolutionarily stable; the F in today’s strains of Nipah differs in only 10 amino acids from the F in 1999 strains. F is broadly conserved among Nipah and related viruses, like Nipah’s cousin Hendra virus, which also is being watched for pandemic potential.
However, we don’t want to spend a lot of effort making therapeutics against the F protein if it can easily escape by mutating key residues. To understand how F evolves under evolutionary pressure, Dr. Larsen and his team used deep mutational scanning to systematically test every possible mutation in the F protein. “We used non-replicative pseudoviruses to safely measure the effects of nearly all amino acid mutations on the fusion protein,” he reassures us.
Large libraries of viruses expressing different versions of F in concert with RPB enter target cells engineered to express Nipah virus receptors from bats. Their lack of replication ensures that no infectious particles are produced from the initial infection. This allows safe profiling of the mutational profile of deadly human pathogens, and the team has used it for in other studies (you can read about their work on SARS-Cov-2 here).
Through this, the lab tested the fitness impact of every single amino acid substitution at every site on F, a 500+ amino acid protein. They found that the impact of each mutation ranged from highly deleterious to virus entry to only slightly beneficial. This indicates that F is structurally constrained, a comforting finding that suggests Nipah virus cannot constantly evolve F to evade immune recognition.
“Our mutation data shows that the fusion process is much more constrained than receptor binding,” explains Dr. Larsen. “This is likely because fusion requires the protein to undergo dramatic changes that must be precisely controlled.”
Their deep mutational scanning data revealed key vulnerabilities of the protein. “We identified specific regions of the fusion protein where single mutations completely disrupt entry into cells,” Dr. Larsen says. They also “found mutations that likely lock the protein in the prefusion conformation, which can be used as stabilized vaccine antigens to elicit potent neutralizing antibodies,” he adds.
When the team focused on the 10 residues that differ between current and ancestral Nipah strains, they found that each one seems to have negative impacts on viral entry.
“We also measured how mutations affect neutralization by a panel of monoclonal antibodies,” Dr. Larsen says. They found that single mutations in F could escape neutralization by four of the six antibodies tested. However, the two remaining antibodies in the panel were less easy to evade and retained neutralization activity.
Finally, Dr. Larsen wondered if their work could be generalized to Hendra virus. They were able to use Nipah F deep mutational scanning data to predict which antibodies might neutralize Hendra virus as well as Nipah. This allowed them to find “antibodies [that] neutralize all known circulating variants of Nipah and the closely related Hendra virus,” he reports.
“This work reveals the function and antigenicity of the Nipah virus fusion protein, which will hopefully assist in vaccine design and therapeutic development,” Dr. Larsen summarizes. “This finding helps explain how viruses evolve and suggests new strategies for designing treatments that target the fusion process.”
In the future, the Bloom lab is interested in better understanding the interplay between RBP and F. How does Nipah virus coordinate “the attachment and fusion processes across two separate proteins?” asks Dr. Larsen. This is an important question not just for Nipah virus, but for many other viruses that use similar receptor binding and fusion to enter cells.
This work is just a part of a widespread effort to be prepared for the next pandemic. Hopefully, we’ll never need most of the knowledge learned in studies like this – but it’s better to be safe than sorry.
The spotlighted research was funded by the National Institute of Allergy and Infectious Disease and the Howard Hughes Medical Institute.
Larsen BB, Harari S, Gen R, Stewart C, Veesler D, Bloom JD. Functional and antigenic constraints on the Nipah virus fusion protein. 2026. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.2529505123.
Hannah Lewis is a postdoctoral research fellow with Jim Boonyaratanakornkit’s group in the Vaccine and Infectious Disease Division (VIDD). She is developing screens to find rare B cells that produce protective antibodies against human herpesviruses. She obtained her PhD in molecular and cellular biology from the University of Washington.
For the Media