Phages, which are harmless viruses that destroy specific bacteria, are becoming useful tools to protect our food supply.
Every year, one in seven people in America comes down with a foodborne illness, typically caused by a bacterial pathogen, including E.Coli, listeria, salmonella, or campylobacter. That adds up to 48 million people, of which 120,000 are hospitalized and 3000 die, according to the Centers for Disease Control. And the variety of foods that can be contaminated with bacterial pathogens is growing too. In the 20th century, E.Coli and listeria lurked primarily within meat. Now they find their way into lettuce, spinach, and other leafy greens, causing periodic consumer scares and product recalls. Onions are the most recent suspected culprit of a nationwide salmonella outbreak.
Some of these incidents are almost inevitable because of how Mother Nature works, explains Divya Jaroni, associate professor of animal and food sciences at Oklahoma State University. These common foodborne pathogens come from the cattle's intestines when the animals shed them in their manure—and then they get washed into rivers and lakes, especially in heavy rains. When this water is later used to irrigate produce farms, the bugs end up on salad greens. Plus, many small farms do both—herd cattle and grow produce.
"Unfortunately for us, these pathogens are part of the microflora of the cows' intestinal tract," Jaroni says. "Some farmers may have an acre or two of cattle pastures, and an acre of a produce farm nearby, so it's easy for this water to contaminate the crops."
Food producers and packagers fight bacteria by potent chemicals, with chlorine being the go-to disinfectant. Cattle carcasses, for example, are typically washed by chlorine solutions as the animals' intestines are removed. Leafy greens are bathed in water with added chlorine solutions. However, because the same "bath" can be used for multiple veggie batches and chlorine evaporates over time, the later rounds may not kill all of the bacteria, sparing some. The natural and organic producers avoid chlorine, substituting it with lactic acid, a more holistic sanitizer, but even with all these efforts, some pathogens survive, sickening consumers and causing food recalls. As we farm more animals and grow more produce, while also striving to use fewer chemicals and more organic growing methods, it will be harder to control bacteria's spread.
"It took us a long time to convince the FDA phages were safe and efficient alternatives. But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
Luckily, bacteria have their own killers. Called bacteriophages, or phages for short, they are viruses that prey on bacteria only. Under the electron microscope, they look like fantasy spaceships, with oblong bodies, spider-like legs and long tails. Much smaller than a bacterium, phages pierce the microbes' cells with their tails, sneak in and begin multiplying inside, eventually bursting the microbes open—and then proceed to infect more of them.
The best part is that these phages are harmless to humans. Moreover, recent research finds that millions of phages dwell on us and in us—in our nose, throat, skin and gut, protecting us from bacterial infections as part of our healthy microbiome. A recent study suggested that we absorb about 30 billion phages into our bodies on a daily basis. Now, ingeniously, they are starting to be deployed as anti-microbial agents in the food industry.
A Maryland-based phage research company called Intralytix is doing just that. Founded by Alexander Sulakvelidze, a microbiologist and epidemiologist who came to the United States from Tbilisi, the capital of Georgia, Intralytix makes and sells five different FDA-approved phage cocktails that work against some of the most notorious food pathogens: ListShield for Listeria, SalmoFresh for Salmonella, ShigaShield for Shigella, another foodborne bug, and EcoShield for E.coli, including the infamous strain that caused the Jack in the Box outbreak in 1993 that killed four children and sickened 732 people across four states. Last year, the FDA granted its approval to yet another Intralytix phage for managing Campylobacter contamination, named CampyShield. "We call it safety by nature," Sulakvelidze says.
Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder."
Photo credit: Living Radiant Photography
Phage preparations are relatively straightforward to make. In nature, phages thrive in any body of water where bacteria live too, including rivers, lakes and bays. "I can dip a bucket into the Chesapeake Bay, and it will be full of all kinds of phages," Sulakvelidze says. "Sewage is another great place to look for specific phages of interest, because it's teeming with all sorts of bacteria—and therefore the viruses that prey on them."
In lab settings, Intralytix grows phages inside massive 1500-liter fermenters, feeding them bacterial "fodder." Once phages multiply enough, they are harvested, dispensed into containers and shipped to food producers who have adopted this disinfecting practice into their preparation process. Typically, it's done by computer-controlled sprayer systems that disperse mist-like phage preparations onto the food.
Unlike chemicals like chlorine or antibiotics, which kill a wide spectrum of bacteria, phages are more specialized, each feeding on specific microbial species. A phage that targets salmonella will not prey on listeria and vice versa. So food producers may sometimes use a combo of different phage preparations. Intralytix is continuously researching and testing new phages. With a contract from the National Institutes of Health, Intralytix is expanding its automated high-throughput robot that tests which phages work best against which bacteria, speeding up the development of the new phage cocktails.
Phages have other "talents." In her recent study, Jaroni found that phages have the ability to destroy bacterial biofilms—colonies of microorganisms that tend to grow on surfaces of the food processing equipment, surrounding themselves with protective coating that even very harsh chemicals can't crack.
"Phages are very clever," Jaroni says. "They produce enzymes that target the biofilms, and once they break through, they can reach the bacteria."
Convincing the FDA that phages were safe to use on food products was no easy feat, Sulakvelidze says. In his home country of Georgia, phages have been used as antimicrobial remedies for over a century, but the FDA was leery of using viruses as food safety agents. "It took us a long time to convince the FDA phages were safe and efficient alternatives," Sulakvelidze says. "But we had worked with them to gather all the data they needed, and the FDA was very supportive in the end."
The agency had granted Intralytix its first approval in 2006, and over the past 10 years, the company's sales increased by over 15-fold. "We currently sell to about 40 companies and are in discussions with several other large food producers," Sulakvelidze says. One indicator that the industry now understands and appreciates the science of phages was that his company was ranked as Top Food Safety Provider in 2021 by Food and Beverage Technology Review, he adds. Notably, phage sprays are kosher, halal and organic-certified.
Intralytix's phage cocktails to safeguard food from bacteria are approved for consumers in addition to food producers, but currently the company sells to food producers only. Selling retail requires different packaging like easy-to-use spray bottles and different marketing that would inform people about phages' antimicrobial qualities. But ultimately, giving people the ability to remove pathogens from their food with probiotic phage sprays is the goal, Sulakvelidze says.
It's not the company's only goal. Now Intralytix is going a step further, investigating phages' probiotic and therapeutic abilities. Because phages are highly specialized in the bacteria they target, they can be used to treat infections caused by specific pathogens while leaving the beneficial species of our microbiome intact. In an ongoing clinical trial with Mount Sinai, Intralytix is now investigating a potential phage treatment against a certain type of E. coli for patients with Crohn's disease, and is about to start another clinical trial for treating bacterial dysentery.
"Now that we have proved that phages are safe and effective against foodborne bacteria," Sulakvelidze says, "we are going to demonstrate their potential in therapeutic applications."
This article was first published by Leaps.org on October 27, 2021.
Scientists Jason Schrum and Kerry Benenato solved crucial challenges in mRNA vaccine development.
Derrick Rossi, a scientist at MIT, and Noubar Afeyan, a Cambridge-based investor, launched Moderna in September 2010. Their idea was to create mRNA molecules capable of delivering instructions to the body's cells, directing them to make proteins to heal ailments and cure disease. Need a statin, immunosuppressive, or other drug or vaccine? Just use mRNA to send a message to the body's cells to produce it. Rossi and Afeyan were convinced injecting mRNA into the body could turn it into its own laboratory, generating specific medications or vaccines as needed.
At the time, the notion that one might be able to teach the body to make proteins bordered on heresy. Everyone knew mRNA was unstable and set off the body's immune system on its way into cells. But in the late 2000's, two scientists at the University of Pennsylvania, Katalin Karikó and Drew Weissman, had figured out how to modify mRNA's chemical building blocks so the molecule could escape the notice of the immune system and enter the cell. Rossi and Afeyan couldn't convince the University of Pennsylvania to license Karikó and Weissman's patent, however, stymying Moderna's early ambitions. At the same time, the Penn scientists' technique seemed more applicable to an academic lab than a biotech company that needed to produce drugs or shots consistently and in bulk. Rossi and Afeyan's new company needed their own solution to help mRNA evade the body's defenses.
Some of Moderna's founders doubted Schrum could find success and they worried if their venture was doomed from the start.
The Scientist Who Modified mRNA: Jason Schrum
In 2010, Afeyan's firm subleased laboratory space in the basement of another Cambridge biotech company to begin scientific work. Afeyan chose a young scientist on his staff, Jason Schrum, to be Moderna's first employee, charging him with getting mRNA into cells without relying on Karikó and Weissman's solutions.
Schrum seemed well suited for the task. Months earlier, he had received a PhD in biological chemistry at Harvard University, where he had focused on nucleotide chemistry. Schrum even had the look of someone who might do big things. The baby-faced twenty-eight-year-old favored a relaxed, start-up look: khakis, button-downs, and Converse All-Stars.
Schrum felt immediate strain, however. He hadn't told anyone, but he was dealing with intense pain in his hands and joints, a condition that later would be diagnosed as degenerative arthritis. Soon Schrum couldn't bend two fingers on his left hand, making lab work difficult. He joined a drug trial, but the medicine proved useless. Schrum tried corticosteroid injections and anti-inflammatory drugs, but his left hand ached, restricting his experiments.
"It just wasn't useful," Schrum says, referring to his tender hand.*
He persisted, nonetheless. Each day in the fall of 2010, Schrum walked through double air-locked doors into a sterile "clean room" before entering a basement laboratory, in the bowels of an office in Cambridge's Kendall Square neighborhood, where he worked deep into the night. Schrum searched for potential modifications of mRNA nucleosides, hoping they might enable the molecule to produce proteins. Like all such rooms, there were no windows, so Schrum had to check a clock to know if it was day or night. A colleague came to visit once in a while, but most of the time, Schrum was alone.
Some of Moderna's founders doubted Schrum could find success and they worried if their venture was doomed from the start. An established MIT scientist turned down a job with the start-up to join pharmaceutical giant Novartis, dubious of Moderna's approach. Colleagues wondered if mRNA could produce proteins, at least on a consistent basis.
As Schrum began testing the modifications in January 2011, he made an unexpected discovery. Karikó and Weissman saw that by turned one of the building blocks for mRNA, a ribonucleoside called uridine, into a slightly different form called pseudouridine, the cell's immune system ignored the mRNA and the molecule avoided an immune response. After a series of experiments in the basement lab, Schrum discovered that a variant of pseudouridine called N1- methyl-pseudouridine did an even better job reducing the cell's innate immune response. Schrum's nucleoside switch enabled even higher protein production than Karikó and Weissman had generated, and Schrum's mRNAs lasted longer than either unmodified molecules or the modified mRNA the Penn academics had used, startling the young researcher. Working alone in a dreary basement and through intense pain, he had actually improved on the Penn professors' work.
Years later, Karikó and Weissman who would win acclaim. In September 2021, the scientists were awarded the Lasker-DeBakey Clinical Medical Research Award. Some predict they eventually will win a Nobel prize. But it would be Schrum's innovation that would form the backbone of both Moderna and Pfizer-BioNTech's Covid-19 vaccine, not the chemical modifications that Karikó and Weissman developed. For Schrum, necessity had truly been the mother of invention.
The Scientist Who Solved Delivery: Kerry Benenato
For several years, Moderna would make slow progress developing drugs to treat various diseases. Eventually, the company decided that mRNA was likely better suited for vaccines. By 2017, Moderna and the National Institutes of Health were discussing working together to develop mRNA–based vaccines, a partnership that buoyed Moderna's executives. There remained a huge obstacle in Moderna's way, however. It was up to Kerry Benenato to find a solution.
Benenato received an early hint of the hurdle in front of her three years earlier, when the organic chemist was first hired. When a colleague gave her a company tour, she was introduced to Moderna's chief scientific officer, Joseph Bolen, who seemed unusually excited to meet her.
"Oh, great!" Bolen said with a smile. "She's the one who's gonna solve delivery."
Bolen gave a hearty laugh and walked away, but Benenato detected seriousness in his quip.
Solve delivery?
It was a lot to expect from a 37-year-old scientist already dealing with insecurities and self-doubt. Benenato was an accomplished researcher who most recently had worked at AstraZeneca after completing post-doctoral studies at Harvard University. Despite her impressive credentials, Benenato battled a lack of confidence that sometimes got in her way. Performance reviews from past employers had been positive, but they usually produced similar critiques: Be more vocal. Do a better job advocating for your ideas. Give us more, Kerry.
Benenato was petite and soft-spoken. She sometimes stuttered or relied on "ums" and "ahs" when she became nervous, especially in front of groups, part of why she sometimes didn't feel comfortable speaking up.
"I'm an introvert," she says. "Self-confidence is something that's always been an issue."
To Benenato, Moderna's vaccine approach seemed promising—the team was packaging mRNAs in microscopic fatty-acid compounds called lipid nanoparticles, or LNPs, that protected the molecules on their way into cells. Moderna's shots should have been producing ample and long-lasting proteins. But the company's scientists were alarmed—they were injecting shots deep into the muscle of mice, but their immune systems were mounting spirited responses to the foreign components of the LNPs, which had been developed by a Canadian company.
This toxicity was a huge issue: A vaccine or drug that caused sharp pain and awful fevers wasn't going to prove very popular. The Moderna team was in a bind: Its mRNA had to be wrapped in the fatty nanoparticles to have a chance at producing plentiful proteins, but the body wasn't tolerating the microscopic encasements, especially upon repeated dosing.
The company's scientists had done everything they could to try to make the molecule's swathing material disappear soon after entering the cells, in order to avoid the unfortunate side effects, such as chills and headaches, but they weren't making headway. Frustration mounted. Somehow, the researchers had to find a way to get the encasements—made of little balls of fat, cholesterol, and other substances—to deliver their payload mRNA and then quickly vanish, like a parent dropping a teenager off at a party, to avoid setting off the immune system in unpleasant ways, even as the RNA and the proteins the molecule created stuck around.
Benenato wasn't entirely shocked by the challenges Moderna was facing. One of the reasons she had joined the upstart company was to help develop its delivery technology. She just didn't realize how pressing the issue was, or how stymied the researchers had become. Benenato also didn't know that Moderna board members were among those most discouraged by the delivery issue. In meetings, some of them pointed out that pharmaceutical giants like Roche Holding and Novartis had worked on similar issues and hadn't managed to develop lipid nanoparticles that were both effective and well tolerated by the body. Why would Moderna have any more luck?
Stephen Hoge insisted the company could yet find a solution.
"There's no way the only innovations in LNP are going to come from some academics and a small Canadian company," insisted Hoge, who had convinced the executives that hiring Benenato might help deliver an answer.
Benenato realized that while Moderna might have been a hot Boston-area start- up, it wasn't set up to do the chemistry necessary to solve their LNP problem. Much of its equipment was old or secondhand, and it was the kind used to tinker with mRNAs, not lipids.
"It was scary," she says.
When Benenato saw the company had a nuclear magnetic resonance spectrometer, which allows chemists to see the molecular structure of material, she let out a sigh of relief. Then Benenato inspected the machine and realized it was a jalopy. The hulking, aging instrument had been decommissioned and left behind by a previous tenant, too old and banged up to bring with them.
Benenato began experimenting with different chemical changes for Moderna's LNPs, but without a working spectrometer she and her colleagues had to have samples ready by noon each day, so they could be picked up by an outside company that would perform the necessary analysis. After a few weeks, her superiors received an enormous bill for the outsourced work and decided to pay to get the old spectrometer running again.
After months of futility, Benenato became impatient. An overachiever who could be hard on herself, she was eager to impress her new bosses. Benenato felt pressure outside the office, as well. She was married with a preschool-age daughter and an eighteen-month-old son. In her last job, Benenato's commute had been a twenty-minute trip to Astra-Zeneca's office in Waltham, outside Boston; now she was traveling an hour to Moderna's Cambridge offices. She became anxious—how was she going to devote the long hours she realized were necessary to solve their LNP quandary while providing her children proper care? Joining Moderna was beginning to feel like a possible mistake.
She turned to her husband and father for help. They reminded her of the hard work she had devoted to establishing her career and said it would be a shame if she couldn't take on the new challenge. Benenato's husband said he was happy to stay home with the kids, alleviating some of her concerns.
Back in the office, she got to work. She wanted to make lipids that were easier for the body to chop into smaller pieces, so they could be eliminated by the body's enzymes. Until then, Moderna, like most others, relied on all kinds of complicated chemicals to hold its LNP packaging together. They weren't natural, though, so the body was having a hard time breaking them down, causing the toxicity.
Benenato began experimenting with simpler chemicals. She inserted "ester bonds"—compounds referred to in chemical circles as "handles" because the body easily grabs them and breaks them apart. Ester bonds had two things going for them: They were strong enough to help ensure the LNP remained stable, acting much like a drop of oil in water, but they also gave the body's enzymes something to target and break down as soon as the LNP entered the cell, a way to quickly rid the body of the potentially toxic LNP components. Benenato thought the inclusion of these chemicals might speed the elimination of the LNP delivery material.
This idea, Benenato realized, was nothing more than traditional, medicinal chemistry. Most people didn't use ester bonds because they were pretty unsophisticated. But, hey, the tricky stuff wasn't working, so Benenato thought she'd see if the simple stuff worked.
Benenato also wanted to try to replace a group of unnatural chemicals in the LNP that was contributing to the spirited and unwelcome response from the immune system. Benenato set out to build a new and improved chemical combination. She began with ethanolamine, a colorless, natural chemical, an obvious start for any chemist hoping to build a more complex chemical combination. No one relied on ethanolamine on its own.
Benenato was curious, though. What would happen if she used just these two simple modifications to the LNP: ethanolamine with the ester bonds? Right away, Benenato noticed her new, super-simple compound helped mRNA create some protein in animals. It wasn't much, but it was a surprising and positive sign. Benenato spent over a year refining her solution, testing more than one hundred variations, all using ethanolamine and ester bonds, showing improvements with each new version of LNP. After finishing her 102nd version of the lipid molecule, which she named SM102, Benenato was confident enough in her work to show it to Hoge and others.
They immediately got excited. The team kept tweaking the composition of the lipid encasement. In 2017, they wrapped it around mRNA molecules and injected the new combination in mice and then monkeys. They saw plentiful, potent proteins were being produced and the lipids were quickly being eliminated, just as Benenato and her colleagues had hoped. Moderna had its special sauce.
That year, Benenato was asked to deliver a presentation to Stephane Bancel, Moderna's chief executive, Afeyan, and Moderna's executive committee to explain why it made sense to use the new, simpler LNP formulation for all its mRNA vaccines. She still needed approval from the executives to make the change. Ahead of the meeting, she was apprehensive, as some of her earlier anxieties returned. But an unusual calm came over her as she began speaking to the group. Benenato explained how experimenting with basic, overlooked chemicals had led to her discovery.
She said she had merely stumbled onto the company's solution, though her bosses understood the efforts that had been necessary for the breakthrough. The board complimented her work and agreed with the idea of switching to the new LNP. Benenato beamed with pride.
"As a scientist, serendipity has been my best friend," she told the executives.
Over the next few years, Benenato and her colleagues would improve on their methods and develop even more tolerable and potent LNP encasement for mRNA molecules. Their work enabled Moderna to include higher doses of vaccine in its shots. In early 2020, Moderna developed Covid-19 shots that included 100 micrograms of vaccine, compared with 30 micrograms in the Pfizer-BioNTech vaccine. That difference appears to help the Moderna vaccine generate higher titers and provide more protection.
"You set out in a career in drug discovery to want to make a difference," Benenato says. "Seeing it come to reality has been surreal and emotional."
Editor's Note: This essay is excerpted from A SHOT TO SAVE THE WORLD: The Inside Story of the Life-or-Death Race for a COVID-19 Vaccine by Gregory Zuckerman, now on sale from Portfolio/Penguin.
*Jason Schrum's arthritis is now in complete remission, thanks to Humira (adalimumab), a TNF-alpha blocker.
