Scientists Are Growing an Edible Cholera Vaccine in Rice
The world's attention has been focused on the coronavirus crisis but Yemen, Bangladesh and many others countries in Asia and Africa are also in the grips of another pandemic: cholera. The current cholera pandemic first emerged in the 1970s and has devastated many communities in low-income countries. Each year, cholera is responsible for an estimated 1.3 million to 4 million cases and 21,000 to 143,000 deaths worldwide.
Immunologist Hiroshi Kiyono and his team at the University of Tokyo hope they can be part of the solution: They're making a cholera vaccine out of rice.
"It is much less expensive than a traditional vaccine, by a long shot."
Cholera is caused by eating food or drinking water that's contaminated by the feces of a person infected with the cholera bacteria, Vibrio cholerae. The bacteria produces the cholera toxin in the intestines, leading to vomiting, diarrhea and severe dehydration. Cholera can kill within hours of infection if it if's not treated quickly.
Current cholera vaccines are mainly oral. The most common oral are given in two doses and are made out of animal or insect cells that are infected with killed or weakened cholera bacteria. Dukoral also includes cells infected with CTB, a non-harmful part of the cholera toxin. Scientists grow cells containing the cholera bacteria and the CTB in bioreactors, large tanks in which conditions can be carefully controlled.
These cholera vaccines offer moderate protection but it wears off relatively quickly. Cold storage can also be an issue. The most common oral vaccines can be stored at room temperature but only for 14 days.
"Current vaccines confer around 60% efficacy over five years post-vaccination," says Lucy Breakwell, who leads the U.S. Centers for Disease Control and Prevention's cholera work within Global Immunization Division. Given the limited protection, refrigeration issue, and the fact that current oral vaccines require two disease, delivery of cholera vaccines in a campaign or emergency setting can be challenging. "There is a need to develop and test new vaccines to improve public health response to cholera outbreaks."
A New Kind of Vaccine
Kiyono and scientists at Tokyo University are creating a new, plant-based cholera vaccine dubbed MucoRice-CTB. The researchers genetically modify rice so that it contains CTB, a non-harmful part of the cholera toxin. The rice is crushed into a powder, mixed with saline solution and then drunk. The digestive tract is lined with mucosal membranes which contain the mucosal immune system. The mucosal immune system gets trained to recognize the cholera toxin as the rice passes through the intestines.
The cholera toxin has two main parts: the A subunit, which is harmful, and the B subunit, also known as CTB, which is nontoxic but allows the cholera bacteria to attach to gut cells. By inducing CTB-specific antibodies, "we might be able to block the binding of the vaccine toxin to gut cells, leading to the prevention of the toxin causing diarrhea," Kiyono says.
Kiyono studies the immune responses that occur at mucosal membranes across the body. He chose to focus on cholera because he wanted to replicate the way traditional vaccines work to get mucosal membranes in the digestive tract to produce an immune response. The difference is that his team is creating a food-based vaccine to induce this immune response. They are also solely focusing on getting the vaccine to induce antibodies for the cholera toxin. Since the cholera toxin is responsible for bacteria sticking to gut cells, the hope is that they can stop this process by producing antibodies for the cholera toxin. Current cholera vaccines target the cholera bacteria or both the bacteria and the toxin.
David Pascual, an expert in infectious diseases and immunology at the University of Florida, thinks that the MucoRice vaccine has huge promise. "I truly believe that the development of a food-based vaccine can be effective. CTB has a natural affinity for sampling cells in the gut to adhere, be processed, and then stimulate our immune system, he says. "In addition to vaccinating the gut, MucoRice has the potential to touch other mucosal surfaces in the mouth, which can help generate an immune response locally in the mouth and distally in the gut."
Cost Effectiveness
Kiyono says the MucoRice vaccine is much cheaper to produce than a traditional vaccine. Current vaccines need expensive bioreactors to grow cell cultures under very controlled, sterile conditions. This makes them expensive to manufacture, as different types of cell cultures need to be grown in separate buildings to avoid any chance of contamination. MucoRice doesn't require such an expensive manufacturing process because the rice plants themselves act as bioreactors.
The MucoRice vaccine also doesn't require the high cost of cold storage. It can be stored at room temperature for up to three years unlike traditional vaccines. "Plant-based vaccine development platforms present an exciting tool to reduce vaccine manufacturing costs, expand vaccine shelf life, and remove refrigeration requirements, all of which are factors that can limit vaccine supply and accessibility," Breakwell says.
Kathleen Hefferon, a microbiologist at Cornell University agrees. "It is much less expensive than a traditional vaccine, by a long shot," she says. "The fact that it is made in rice means the vaccine can be stored for long periods on the shelf, without losing its activity."
A plant-based vaccine may even be able to address vaccine hesitancy, which has become a growing problem in recent years. Hefferon suggests that "using well-known food plants may serve to reduce the anxiety of some vaccine hesitant people."
Challenges of Plant Vaccines
Despite their advantages, no plant-based vaccines have been commercialized for human use. There are a number of reasons for this, ranging from the potential for too much variation in plants to the lack of facilities large enough to grow crops that comply with good manufacturing practices. Several plant vaccines for diseases like HIV and COVID-19 are in development, but they're still in early stages.
In developing the MucoRice vaccine, scientists at the University of Tokyo have tried to overcome some of the problems with plant vaccines. They've created a closed facility where they can grow rice plants directly in nutrient-rich water rather than soil. This ensures they can grow crops all year round in a space that satisfies regulations. There's also less chance for variation since the environment is tightly controlled.
Clinical Trials and Beyond
After successfully growing rice plants containing the vaccine, the team carried out their first clinical trial. It was completed early this year. Thirty participants received a placebo and 30 received the vaccine. They were all Japanese men between the ages of 20 and 40 years old. 60 percent produced antibodies against the cholera toxin with no side effects. It was a promising result. However, there are still some issues Kiyono's team need to address.
The vaccine may not provide enough protection on its own. The antigen in any vaccine is the substance it contains to induce an immune response. For the MucoRice vaccine, the antigen is not the cholera bacteria itself but the cholera toxin the bacteria produces.
"The development of the antigen in rice is innovative," says David Sack, a professor at John Hopkins University and expert in cholera vaccine development. "But antibodies against only the toxin have not been very protective. The major protective antigen is thought to be the LPS." LPS, or lipopolysaccharide, is a component of the outer wall of the cholera bacteria that plays an important role in eliciting an immune response.
The Japanese team is considering getting the rice to also express the O antigen, a core part of the LPS. Further investigation and clinical trials will look into improving the vaccine's efficacy.
Beyond cholera, Kiyono hopes that the vaccine platform could one day be used to make cost-effective vaccines for other pathogens, such as norovirus or coronavirus.
"We believe the MucoRice system may become a new generation of vaccine production, storage, and delivery system."
Forty years ago, Joy Milne, a nurse from Perth, Scotland, noticed a musky odor coming from her husband, Les. At first, Milne thought the smell was a result of bad hygiene and badgered her husband to take longer showers. But when the smell persisted, Milne learned to live with it, not wanting to hurt her husband's feelings.
Twelve years after she first noticed the "woodsy" smell, Les was diagnosed at the age of 44 with Parkinson's Disease, a neurodegenerative condition characterized by lack of dopamine production and loss of movement. Parkinson's Disease currently affects more than 10 million people worldwide.
Milne spent the next several years believing the strange smell was exclusive to her husband. But to her surprise, at a local support group meeting in 2012, she caught the familiar scent once again, hanging over the group like a cloud. Stunned, Milne started to wonder if the smell was the result of Parkinson's Disease itself.
Milne's discovery led her to Dr. Tilo Kunath, a neurobiologist at the Centre for Regenerative Medicine at the University of Edinburgh. Together, Milne, Kunath, and a host of other scientists would use Milne's unusual sense of smell to develop a new diagnostic test, now in development and poised to revolutionize the treatment of Parkinson's Disease.
"Joy was in the audience during a talk I was giving on my work, which has to do with Parkinson's and stem cell biology," Kunath says. "During the patient engagement portion of the talk, she asked me if Parkinson's had a smell to it." Confused, Kunath said he had never heard of this – but for months after his talk he continued to turn the question over in his mind.
Kunath knew from his research that the skin's microbiome changes during different disease processes, releasing metabolites that can give off odors. In the medical literature, diseases like melanoma and Type 2 diabetes have been known to carry a specific scent – but no such connection had been made with Parkinson's. If people could smell Parkinson's, he thought, then it stood to reason that those metabolites could be isolated, identified, and used to potentially diagnose Parkinson's by their presence alone.
First, Kunath and his colleagues decided to test Milne's sense of smell. "I got in touch with Joy again and we designed a protocol to test her sense of smell without her having to be around patients," says Kunath, which could have affected the validity of the test. In his spare time, Kunath collected t-shirt samples from people diagnosed with Parkinson's and from others without the diagnosis and gave them to Milne to smell. In 100 percent of the samples, Milne was able to detect whether a person had Parkinson's based on smell alone. Amazingly, Milne was even able to detect the "Parkinson's scent" in a shirt from the control group – someone who did not have a Parkinson's diagnosis, but would go on to be diagnosed nine months later.
From the initial study, the team discovered that Parkinson's did have a smell, that Milne – inexplicably – could detect it, and that she could detect it long before diagnosis like she had with her husband, Les. But the experiments revealed other things that the team hadn't been expecting.
"One surprising thing we learned from that experiment was that the odor was always located in the back of the shirt – never in the armpit, where we expected the smell to be," Kunath says. "I had a chance meeting with a dermatologist and he said the smell was due to the patient's sebum, which are greasy secretions that are really dense on your upper back. We have sweat glands, instead of sebum, in our armpits." Patients with Parkinson's are also known to have increased sebum production.
With the knowledge that a patient's sebum was the source of the unusual smell, researchers could go on to investigate exactly what metabolites were in the sebum and in what amounts. Kunath, along with his associate, Dr. Perdita Barran, collected and analyzed sebum samples from 64 participants across the United Kingdom. Once the samples were collected, Barran and others analyzed it using a method called gas chromatography mass spectrometry, or GS-MC, which separated, weighed and helped identify the individual compounds present in each sebum sample.
Barran's team can now correctly identify Parkinson's in nine out of 10 patients – a much quicker and more accurate way to diagnose than what clinicians do now.
"The compounds we've identified in the sebum are not unique to people with Parkinson's, but they are differently expressed," says Barran, a professor of mass spectrometry at the University of Manchester. "So this test we're developing now is not a black-and-white, do-you-have-something kind of test, but rather how much of these compounds do you have compared to other people and other compounds." The team identified over a dozen compounds that were present in the sebum of Parkinson's patients in much larger amounts than the control group.
Using only the GC-MS and a sebum swab test, Barran's team can now correctly identify Parkinson's in nine out of 10 patients – a much quicker and more accurate way to diagnose than what clinicians do now.
"At the moment, a clinical diagnosis is based on the patient's physical symptoms," Barran says, and determining whether a patient has Parkinson's is often a long and drawn-out process of elimination. "Doctors might say that a group of symptoms looks like Parkinson's, but there are other reasons people might have those symptoms, and it might take another year before they're certain," Barran says. "Some of those symptoms are just signs of aging, and other symptoms like tremor are present in recovering alcoholics or people with other kinds of dementia." People under the age of 40 with Parkinson's symptoms, who present with stiff arms, are often misdiagnosed with carpal tunnel syndrome, she adds.
Additionally, by the time physical symptoms are present, Parkinson's patients have already lost a substantial amount of dopamine receptors – about sixty percent -- in the brain's basal ganglia. Getting a diagnosis before physical symptoms appear would mean earlier interventions that could prevent dopamine loss and preserve regular movement, Barran says.
"Early diagnosis is good if it means there's a chance of early intervention," says Barran. "It stops the process of dopamine loss, which means that motor symptoms potentially will not happen, or the onset of symptoms will be substantially delayed." Barran's team is in the processing of streamlining the sebum test so that definitive results will be ready in just two minutes.
"What we're doing right now will be a very inexpensive test, a rapid-screen test, and that will encourage people to self-sample and test at home," says Barran. In addition to diagnosing Parkinson's, she says, this test could also be potentially useful to determine if medications were at a therapeutic dose in people who have the disease, since the odor is strongest in people whose symptoms are least controlled by medication.
"When symptoms are under control, the odor is lower," Barran says. "Potentially this would allow patients and clinicians to see whether their symptoms are being managed properly with medication, or perhaps if they're being overmedicated." Hypothetically, patients could also use the test to determine if interventions like diet and exercise are effective at keeping Parkinson's controlled.
"We hope within the next two to five years we will have a test available."
Barran is now running another clinical trial – one that determines whether they can diagnose at an earlier stage and whether they can identify a difference in sebum samples between different forms of Parkinson's or diseases that have Parkinson's-like symptoms, such as Lewy Body Dementia.
"Within the next one to two years, we hope to be running a trial in the Manchester area for those people who do not have motor symptoms but are at risk for developing dementia due to symptoms like loss of smell and sleep difficulty," Barran had said in 2019. "If we can establish that, we can roll out a test that determines if you have Parkinson's or not with those first pre-motor symptoms, and then at what stage. We hope within the next two to five years we will have a test available."
In a 2022 study, published in the American Chemical Society, researchers used mass spectrometry to analyze sebum from skin swabs for the presence of the specific molecules. They found that some specific molecules are present only in people who have Parkinson’s. Now they hope that the same method can be used in regular diagnostic labs. The test, many years in the making, is inching its way to the clinic.
"We would likely first give this test to people who are at risk due to a genetic predisposition, or who are at risk based on prodomal symptoms, like people who suffer from a REM sleep disorder who have a 50 to 70 percent chance of developing Parkinson's within a ten year period," Barran says. "Those would be people who would benefit from early therapeutic intervention. For the normal population, it isn't beneficial at the moment to know until we have therapeutic interventions that can be useful."
Milne's husband, Les, passed away from complications of Parkinson's Disease in 2015. But thanks to him and the dedication of his wife, Joy, science may have found a way to someday prolong the lives of others with this devastating disease. Sometimes she can smell people who have Parkinson’s while in the supermarket or walking down the street but has been told by medical ethicists she cannot tell them, Milne said in an interview with the Guardian. But once the test becomes available in the clinics, it will do the job for her.
[Ed. Note: A older version of this hit article originally ran on September 3, 2019.]
Earlier this year, Harvard scientists reported that they used an anti-aging therapy to reverse blindness in elderly mice. Several other studies in the past decade have suggested that the aging process can be modified, at least in lab organisms. Considering mice and humans share virtually the same genetic makeup, what does the rodent-based study mean for the humans?
In truth, we don’t know. Maybe nothing.
What we do know, however, is that a growing number of people are dedicating themselves to defying the aging process, to turning back the clock – the biological clock, that is. Take Bryan Johnson, a man who is less mouse than human guinea pig. A very wealthy guinea pig.
The 45-year-old venture capitalist spends over $2 million per year reversing his biological clock. To do this, he employs a team of 30 medical doctors and other scientists. His goal is to eventually reset his biological clock to age 18, and “have all of his major organs — including his brain, liver, kidneys, teeth, skin, hair, penis and rectum — functioning as they were in his late teens,” according to a story earlier this year in the New York Post.
But his daily routine paints a picture that is far from appealing: for example, rigorously adhering to a sleep schedule of 8 p.m. to 5 a.m. and consuming more than 100 pills and precisely 1,977 calories daily. Considering all of Johnson’s sacrifices, one discovers a paradox:
To live forever, he must die a little every day until he reaches his goal - if he ever reaches his goal.
Less extreme examples seem more helpful for people interested in happy, healthy aging. Enter Chris Mirabile, a New Yorker who says on his website, SlowMyAge.com, that he successfully reversed his biological age by 13.6 years, from the chronological age of 37.2 to a biological age of 23.6. To put this achievement in perspective, Johnson, to date, has reversed his biological clock by 2.5 years.
Mirabile's habits and overall quest to turn back the clock trace back to a harrowing experience at age 16 during a school trip to Manhattan, when he woke up on the floor with his shirt soaked in blood.
Mirabile, who is now 38, supports his claim with blood tests that purport to measure biological age by assessing changes to a person’s epigenome, or the chemical marks that affect how genes are expressed. Mirabile’s tests have been run and verified independently by the same scientific lab that analyzes Johnson’s. (In an email to Leaps.org, the lab, TruDiagnostic, confirmed Mirabile’s claims about his test results.)
There is considerable uncertainty among scientists about the extent to which these tests can accurately measure biological age in individuals. Even so, Mirabile’s results are intriguing. They could reflect his smart lifestyle for healthy aging.
His habits and overall quest to turn back the clock trace back to a harrowing experience at age 16 during a school trip to Manhattan, when Mirabile woke up on the floor with his shirt soaked in blood. He’d severed his tongue after a seizure. He later learned it was caused by a tumor the size of a golf ball. As a result, “I found myself contemplating my life, what I had yet to experience, and mortality – a theme that stuck with me during my year of recovery and beyond,” Mirabile told me.
For the next 15 years, he researched health and biology, integrating his learnings into his lifestyle. Then, in his early 30s, he came across an article in the journal Cell, "The Hallmarks of Aging," that outlined nine mechanisms of the body that define the aging process. Although the paper says there are no known interventions to delay some of these mechanisms, others, such as inflammation, struck Mirabile as actionable. Reading the paper was his “moment of epiphany” when it came to the areas where he could assert control to maximize his longevity.
He also wanted “to create a resource that my family, friends, and community could benefit from in the short term,” he said. He turned this knowledge base into a company called NOVOS dedicated to extending lifespan.
His longevity advice is more accessible than Johnson’s multi-million dollar approach, as Mirabile spends a fraction of that amount. Mirabile takes one epigenetic test per year and has a gym membership at $45 per month. Unlike Johnson, who takes 100 pills per day, Mirabile takes 10, costing another $45 monthly, including a B-complex, fish oil, Vitamins D3 and K2, and two different multivitamin supplements.
Mirabile’s methods may be easier to apply in other ways as well, since they include activities that many people enjoy anyway. He’s passionate about outdoor activities, travels frequently, and has loving relationships with friends and family, including his girlfriend and collie.
Here are a few of daily routines that could, he thinks, contribute to his impressively young bio age:
After waking at 7:45 am, he immediately drinks 16 ounces of water, with 1/4 teaspoon of sodium and potassium to replenish electrolytes. He takes his morning vitamins, brushes and flosses his teeth, puts on a facial moisturizing sunblock and goes for a brisk, two-mile walk in the sun. At 8:30 am on Mondays, Wednesdays, and Fridays he lift weights, focusing on strength and power, especially in large muscle groups.
Tuesdays, Thursdays and Saturdays are intense cardio days. He runs 5-7 miles or bicycles for 60 minutes first thing in the morning at a brisk pace, listening to podcasts. Sunday morning cardio is more leisurely.
After working out each day, he’s back home at 9:20 am, where he makes black coffee, showers, then applies serum and moisturizing sunblock to his face. He works for about three hours on his laptop, then has a protein shake and fruit.
Mirabile is a dedicated intermittent faster, with a six hour eating window in between 18 hours fasts. At 3 pm, he has lunch. The Mediterranean lineup often features salmon, sardines, olive oil, pink Himalayan salt plus potassium salt for balance, and lots of dried herbs and spices. He almost always finishes with 1/3 to 1/2 bar of dark chocolate.
If you are what you eat, Mirabile is made of mostly plants and lean meats. He follows a Mediterranean diet full of vegetables, fruits, fatty fish and other meats full of protein and unsaturated fats. “These may cost more than a meal at an American fast-food joint, but then again, not by much,” he said. Each day, he spends $25 on all his meals combined.
At 6 pm, he takes the dog out for a two-mile walk, taking calls for work or from family members along the way. At 7 pm, he dines with his girlfriend. Like lunch, this meal is heavy on widely available ingredients, including fish, fresh garlic, and fermented food like kimchi. Mirabile finishes this meal with sweets, like coconut milk yogurt with cinnamon and clove, some stevia, a mix of fresh berries and cacao nibs.
If Mirabile's epigenetic tests are accurate, his young biological age could be thanks to his healthy lifestyle, or it could come from a stroke of luck if he inherited genes that protect against aging.
At 8 pm, he wraps up work duties and watches shows with his girlfriend, applies serum and moisturizer yet again, and then meditates with the lights off. This wind-down, he said, improves his sleep quality. Wearing a sleep mask and earplugs, he’s asleep by about 10:30.
“I’ve achieved stellar health outcomes, even after having had the physiological stressors of a brain tumor, without spending a fortune,” Mirabile said. “In fact, even during times when I wasn’t making much money as a startup founder with few savings, I still managed to live a very healthy, pro-longevity lifestyle on a modest budget.”
Mirabile said living a cleaner, healthier existence is a reality that many readers can achieve. It’s certainly true that many people live in food deserts and have limited time for exercise or no access to gyms, but James R. Doty, a clinical professor of neurosurgery at Stanford, thinks many can take more action to stack the odds that they’ll “be happy and live longer.” Many of his recommendations echo aspects of Mirabile’s lifestyle.
Each night, Doty said, it’s vital to get anywhere between 6-8 hours of good quality sleep. Those who sleep less than 6 hours per night are at an increased risk of developing a whole host of medical problems, including high blood pressure, type 2 diabetes, and stroke.
In addition, it’s critical to follow Mirabile’s prescription of exercise for about one hour each day, and intensity levels matter. Doty noted that, in 2017, researchers at Brigham Young University found that people who ran at a fast pace for 30-40 minutes five days per week were, on average, biologically younger by nine years, compared to those who subscribed to more moderate exercise programs, as well as those who rarely exercised.
When it comes to nutrition, one should consider fasting for 16 hours per day, Doty said. This is known as the 16/8 method, where one’s daily calories are consumed within an eight hour window, fasting for the remaining 16 hours, just like Mirabile. Intermittent fasting is associated with cellular repair and less inflammation, though it’s not for everyone, Doty added. Consult with a medical professional before trying a fasting regimen.
Finally, Doty advised to “avoid anger, avoid stress.” Easier said than done, but not impossible. “Between stimulus and response, there is a pause and within that pause lies your freedom,” Doty said. Mirabile’s daily meditation ritual could be key to lower stress for healthy aging. Research has linked regular, long-term meditation to having a lower epigenetic age, compared to control groups.
Many other factors could apply. Having a life purpose, as Mirabile does with his company, has also been associated with healthy aging and lower epigenetic age. Of course, Mirabile is just one person, so it’s hard to know how his experience will apply to others. If his tests are accurate, his young biological age could be thanks to his healthy lifestyle, or it could come from a stroke of luck if he inherited genes that protect against aging. Clearly, though, any such genes did not protect him from cancer at an early age.
The third and perhaps most likely explanation: Mirabile’s very young biological age results from a combination of these factors. Some research shows that genetics account for only 25 percent of longevity. That means environmental factors could be driving the other 75 percent, such as where you live, frequency of exercise, quality of nutrition and social support.
The middle-aged – even Brian Johnson – probably can’t ever be 18 again. But more modest goals are reasonable for many. Control what you can for a longer, healthier life.