May 27, 2021
As we’ve seen over the past year-plus, research and development on COVID-19 moved at an astonishingly rapid pace. From the first identification of the novel virus causing the pandemic, to emergency authorization of multiple protective vaccines and discoveries in between, scientists have worked feverishly to understand the SARS-CoV-2 virus and the disease and develop therapies to combat it.
Dr. Daria Mochly-Rosen was no exception. As she focused on keeping SPARK afloat during the pandemic, she also turned her attention to researching SARS-CoV-2 variants and developing a passive vaccine to block the virus. As part of her team studying variants, two first year graduate students were quickly pulled into fast-paced work alongside Mochly-Rosen.
Here, we take a look at the team’s variant research from the perspective of the two graduate students. Born and raised in Nepal, Suman Pokhrel graduated from University of New Orleans with degrees in biology and chemistry and worked at Bayer Crop Science before coming to Stanford University for graduate school. Ben Kraemer has a degree in neuroscience from University of St. Thomas in Minnesota, with minors in chemistry and Spanish, and focused on organic chemistry research while at the university. Both are Ph.D. students in the department of Chemical and Systems Biology.
Both were doing rotations in Mochly-Rosen’s lab when she approached them about helping with some research she had in mind. They eagerly accepted and got to work.
Pokhrel said the team was concerned about reports of mutations in SARS-CoV-2. “We thought it might be useful to analyze those [DNA sequences from patients] and see which regions [of the spike protein] are more conserved and which regions are more variable, and that would help inform other antibody or drug discovery work.”
Kraemer said, “In the news it seemed every day a new variant was a cause for concern, but there wasn’t really any consensus as to where these variants were popping up, which parts of the spike protein, were they even relevant” to therapies or prophylactics.
“Part of our analysis was to find out what was happening in those regions of the protein,” Kraemer added, with the hope that their work could aid development of vaccines and therapeutics to stop COVID-19 transmission.
The team first examined mutations in SARS-CoV-2 that arose naturally as the virus spread around the world. The virus goes through a natural evolution as it spreads between people and small changes occur in the viral genome. The mutations can cause the virus to spread more easily, or to evade therapies and vaccines. The team sought to determine if there were regions of the virus’ genome that had fewer changes, or were more “conserved,” and could be better targeted with therapies and vaccines.
The team used data from an open-source database, called the Global Initiative on Sharing Avian Influenza Data (GISAID), in which scientists around the world deposited hundreds of thousands of coronavirus genome sequences.
Kraemer first took data on the SARS-CoV-2 spike protein, the part of the virus that binds to the human ACE2 receptor to enter the body, and looked at the frequency of mutations in specific positions of the viral protein. The team color-coded those changes “to pick out hotspots that don’t undergo a lot of variation,” Kraemer said. Some positions of the spike protein had up to 10 amino acid substitutions, whereas other positions had none. Those positions were considered “hotspots” that were well conserved.
Kraemer then gave that data to Pokhrel, who mapped the data into 3D structures to show where those frequencies of mutations occurred on the virus’ surface.
“Our analysis highlighted some really important regions that should be targeted for prophylactics, and some that should be abandoned as potential targets,” Kraemer said.
That study was published on bioRxiv and has been accepted for publication in a peer-reviewed journal.
The team’s next study came about as the so-called U.K. variant entered the news cycle, Kraemer said. It appeared to be more transmissible, and the team wanted to find out why.
The group took a close look at the B.1.1.7, or 501Y.V1 variant, first identified in the U.K., and the B.1.351 or 501Y.V2 variant, identified by SPARK colleagues in South Africa. They used computational methods and predictive software to examine how the variants became more infective.
The team found that certain mutations in the SARS-CoV-2 spike protein increased the sites at which enzymes called proteases can cut and activate the spike protein, which exposes the receptor-binding domain (the part of the spike protein that sticks to the human ACE2 receptor on cells), thereby increasing ACE2 binding and viral entry into human cells.
The team focused on proteases in the nose, where the virus enters the body, including one called neutrophil elastase. The new variants had new elastase cleavage sites, which the team thinks contribute to increased transmission as they increase the ability of neutrophil elastase to activate the virus and increase binding to ACE2. In that case, Pokhrel said, targeting neutrophil elastase in the human could be a way to curb spread of the virus.
Kraemer said, “We hope these results inspire someone to do some wet lab work, and test this in cell culture or lab animals, and see if neutrophil elastase is sufficient to cause increased cleavage of the virus, and allow it to open and bind to ACE2, and if that leads to increased transmission.” If that’s true, Kraemer added, existing FDA approved elastase inhibitors could be used as a treatment to stop the spread of the virus.
The study was published on bioRxiv and has been accepted for publication in a peer-reviewed journal.
An additional study looked at how the virus moved between minks and humans. Mochly-Rosen, Pokhrel and Kraemer teamed up with a group from Flow Pharma Inc., who was investigating transmission between humans and animals, and had found some variants that developed in mink populations and appeared to be jumping back into humans.
The team did the same work they’d performed in previous studies to compare genetic differences between spike protein sequences of viruses isolated from minks and humans, including predictive software and structural modeling to examine the variants.
The results suggested that the animals were hosts for the virus and allowed them to accumulate mutations, and “made it just that much more easy to jump back into humans,” Kraemer said. When the virus comes back into humans, the mutations it gained in the animals could potentially allow increased transmissibility and/or increased disease severity. It’s like playing with fire, Kraemer said.
Kraemer said the study shows “we need to be careful with introducing infectious diseases to” animals that live and interact closely with humans, in particular minks, who are raised on farms for fur. Minks are very aggressive – they will scratch and bite and leave their saliva on you, Kraemer added, increasing chances for transmission.
The mink study first appeared on bioRxiv and was published in Infection, Genetics and Evolution in May.
Pohkrel and Kraemer described their first Stanford experience as surreal, as they started graduate school in the middle of a pandemic. “It’s been a wild ride,” Kraemer said. They both live on campus, in small cohorts, and feel “a little bit isolated,” Kraemer added.
Their work on this computer-based project and in the lab has helped them stay busy and productive, to get out more and break up the routine, Pokhrel said.
“Having the chance to work on this stuff with Daria has been pretty rewarding, and it feels like we’ve been using our time well and making a difference in this pandemic,” said Kraemer.
Mochly-Rosen said the students researched the variants on top of the projects they worked on in their rotation in her lab. “They basically spent their Christmas break and all their evenings to get this done,” she said. “What an incredible achievement!”