Disrupting viral packaging may be key to pandemic preparedness, Stanford researchers find

Like savvy international travelers, viruses know exactly how to pack. With the genetic instructions for the next generation folded just so, snuggled into a custom-made outer covering studded with cell-grabbing proteins, these tiny invaders quest for new digs on the reg. When they find them, they get to work. Each newly infected cell soon releases thousands of perfectly packed, fresh-faced viral particles — fueling an infection’s exponential growth.

Now a Stanford Medicine study of influenza and of SARS-CoV-2 — the virus that causes COVID-19 — shows that antiviral drugs that disrupt this game of genomic Tetris can bring infections to a screeching halt. At the same time, these drugs allow just enough exposure to the virus to jumpstart a natural immune response that confers lasting protection.

Because tried-and-true packing strategies are shared among viral family members, one antiviral drug can be effective against several closely related viruses, such as seasonal influenza A, swine flu and bird flu. And because it’s difficult to rejigger a three-dimensional puzzle, viruses are unlikely to become resistant to a treatment that harnesses this tactic.

“These antivirals can be tailored to nearly any virus,” Jeffrey Glenn, MD, PhD, professor of microbiology and immunology, said. “They provide immediate protection when administered either prior to or after exposure, and they stimulate a lasting immune response that neutralizes a subsequent challenge of even a tenfold lethal dose of influenza. This is really exciting.”

The study, which was conducted in mice, hamsters and human cells grown in the laboratory, was published online in Nature Medicine Aug. 18. Glenn, the Joseph D. Grant Professor II, is the senior author of the study. Research scientist Rachel Hagey, PhD, is the lead author of the paper.

A tool for the next pandemic

The discovery suggests the possibility of quickly dampening the spread of some of humankind’s deadliest viruses with off-the-shelf custom antivirals designed, manufactured and stockpiled before the next outbreak occurs.

The findings are the first to come out of Stanford’s newly formed SyneRx, which is one of nine Antiviral Drug Discovery Centers for Pathogens of Pandemic Concern funded by the National Institute for Allergy and Infectious Diseases, and ViRx@Stanford, which is Stanford Medicine’s Biosecurity and Pandemic Preparedness Initiative. Glenn leads the center, which received $69 million in May to help design antivirals to combat COVID-19 and other diseases with the potential to cause future pandemics.

Vaccines that fight viruses typically encourage the body’s immune system to recognize and react to critical viral proteins, such as the spike protein of SARS-CoV-2. But, as has become obvious during the ongoing pandemic, proteins can mutate in subtle ways to evade the immune system, leading to breakthrough infections in vaccinated people.

Glenn and Hagey wondered if antiviral drugs targeting more mundane, but equally important, steps in the viral life cycle could stop or slow infections — genome packaging, for example. After all, packing may be boring, but it’s key to a successful trip, whether you’re going to Paris or to the cell next door. And it can be surprisingly complex.

The genome of influenza A virus, for instance, consists of eight separate, single-stranded RNA segments. Each segment folds on itself in a bespoke combination of stems and loops dictated by the sequence of their building blocks, called nucleotides. Viral protein butlers then collaborate to tidily tuck one of each of the eight segments into an outer shell, much the way parents fit a college-bound child’s belongings into the back of the car. The new viral particle is then ready for its maiden voyage to continue the cycle of infection. Other viruses have other packing requirements. But once a successful blueprint has been committed to memory, it is unlikely to be jettisoned, and it is shared among family members like a favorite recipe.

The researchers used a type of analysis called SHAPE and computational modeling to identify a segment of RNA called PSL2 with a predicted three-dimensional structure that is nearly 100% identical among various seasonal and pandemic influenza A strains. When they mutated the sequence of PSL2, the virus was unable to infect cells grown in the laboratory — confirming the importance of the region to viral replication.

Hagey and her colleagues then designed several short stretches of single-stranded DNA that would recognize and bind to matching sequences on PSL2 — latching onto and interfering with its ability to contort itself into the precisely defined shape necessary for successful packing. They designed the DNA pieces to resist degradation and last longer in the body using a technique called locked nucleic acids, or LNAs.

Nowhere to go

The researchers found that treating the laboratory-grown cells with certain LNAs either before or after infection with an influenza strain called H1N1, or swine flu, dramatically reduced the ability of the virus to make new infectious particles. Additionally, they saw no signs of the virus mutating to escape the effects of the LNA even after several generations. In contrast, when they treated the cells with Tamiflu, the virus quickly altered its genome to sidestep the anti-influenza drug, which inhibits the activity of a viral protein that facilitates the release of new viral particles from an infected cell.

“We found that if we design drugs against these highly conserved structures necessary for viral genome packaging, there are very few ways the virus can escape,” Glenn said. “This structure is shared in every known isolate of influenza A, including bird flu and swine flu. So, if we are able to translate these findings into humans, it’s possible we could see universal protection with just one dose.”

The researchers then tested whether laboratory mice would be protected against influenza infection by the most effective LNA. They found that a single dose of the antiviral administered in the animals’ noses one week before exposure to a lethal dose of the virus protected 100% of the treated animals from death. In contrast, the control animals became severely ill and were all humanely sacrificed by day six. They then increased the dose of the LNA and administered it two weeks before virus exposure. Again, none of the mice died, and most were only mildly ill.

Finally, the researchers explored whether the low-level viral replication experienced by the mice in the first series of experiments could protect them from future infection. Sixty-five days after their initial infection, the researchers exposed the same mice again, but with 10 times the normal lethal dose.

“They didn’t bat an eye,” Glenn said. “They didn’t lose weight or appear sick at all. This indicates their initial exposure led to enough residual viral replication to stimulate a broad immune response that remains protective months later.”

A check on COVID-19

The researchers conducted a similar series of experiments with the SARS-CoV-2 virus. As with influenza, they found that LNAs targeting highly conserved structured regions important in the viral genome inhibited the replication of the virus in human cells grown in the laboratory. Even a highly mutated version of the SARS-CoV-2 virus isolated from a chronically infected cancer patient was unable to replicate in the presence of the antiviral.

The protective effect extended to Syrian hamsters, an animal commonly used to study SARS-CoV-2 infection. Hamsters given two single daily sniffs of the LNA before romping with infected peers remained healthy and, after four days, had dramatically less virus in their lungs than control animals.

The researchers are now testing the approach in pigs, using the same dose and intra-nasal applicators they hope to one day use in humans. They foresee a time during the next pandemic when strategically designed LNAs, which they term programmable antivirals, could be used to treat already infected people, to protect people during the lag between vaccination and the development of protective immunity, or to provide prophylactic protection if there is no available vaccine.

“COVID-19 caught us with our pants down,” Glenn said. “But what really keeps me up at night is the likelihood of a highly pathogenic virus like the 1918 influenza pandemic or a new, weaponized version of influenza. Today we have nothing to stop its spread, and a virus like that could kill hundreds of millions of people. But our approach could be enacted ahead of time and provide protection against a wide range of viruses.”

Researchers from the University of North Carolina at Chapel Hill; Utah State University; the University of Cincinnati College of Medicine; the University of Texas Medical Branch; the National Institute of Allergy and Infectious Diseases; the Chan Zuckerberg Biohub; and the Veterans Administration Medical Center, Palo Alto, also contributed to the study.

The study was supported by the National Institutes of Health (grants 5T32AI007328-24, 5T32DK007056, R56A1111460, U19A1109662, RO1AI132191 and U19AI171421), the Department of Defense, an influenza Harrington Scholar Innovator grant, a COVID-19 Harrington Scholar Innovator grant, a Mona M. Burgess Stanford Bio-X Interdisciplinary graduate fellowship, Fastgrants and the Dr. Tri Cao Nguyen Fund for Pandemic Preparedness.