Worldwide, more than a million deaths occur each year due to diarrheal diseases that lead to dehydration and malnutrition. Yet no vaccine currently exists to fight or prevent these diseases, which are caused by bacteria, such as certain strains of Escherichia coli and, in mice, the mouse-specific pathogen Citrobacter rodentium. A new study in mice by Salk Institute for Biological Sciences scientists has now revealed a potential vaccination strategy, with the finding that pairing specific diets with disease-causing bacteria can create lasting immunity without causing sickness. The results could pave the way for development of vaccines that promote immunity for people with diarrheal diseases and possibly other infections.

“We discovered that immunization against diarrheal infections is possible if we allow the bacteria to retain some of its disease-causing behavior,” said Professor Janelle Ayres, PhD, Salk Institute Legacy Chair and head of the Molecular and Systems Physiology Laboratory. “This insight could lead to the development of vaccines that could reduce symptoms and mortality, as well as protect against future infections.” Ayres is senior author of the team’s published paper in Science Advances, which is titled “Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.”

For many infections there is substantial variation in host susceptibility to developing disease, the authors explained. Susceptible individuals will develop the disease as a result of infection, and then, with time, will either recover, or eventually succumb and die, the team pointed out. In contrast, some people who become infected will remain asymptomatic, and can carry the disease-causing pathogen without becoming ill, but still transmit the pathogen to other people. “For asymptomatic carriers, a pathogen can infect, replicate, and transmit without causing clinical signs or symptoms of disease in the primary host,” the investigator noted. “We have relatively little understanding of how asymptomatic infections occur mechanistically or how they contribute to host defense and susceptibility to future infections.”

From left: Janelle Ayres and Natalia Thorup. [Salk Institute]
From left: Janelle Ayres and Natalia Thorup. [Salk Institute]

In 2018, Ayres’ lab looked at how dietary interventions can create an asymptomatic infection, which Ayres calls a cooperative relationship between bacteria and the human or animal host that the bacteria have infected. They discovered that an iron-rich diet enabled mice to survive a normally lethal bacterial infection without ever developing signs of sickness or disease. The high-iron diet increased unabsorbed sugar (glucose) in the animals’ intestines, which the bacteria could feast on. This excess sugar served as a “bribe” for the bacteria, keeping them incentivized to not attack the host.

This process produced long-term asymptomatic infection with the bacteria, leading the researchers to believe that the adaptive immune system (cells and proteins that “remember” infections) may be involved. In their newly releases paper in Science Advances, the team commented, “Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens.”

The paper’s first author Grischa Chen, PhD, a former postdoctoral researcher in Ayres’ lab, added, “Being able to generate lasting immunity against bacteria like C. rodentium or E. coli has not been possible using established vaccination strategies. We wanted to figure out what mechanism was sustaining this lasting immunity, so we could use that mechanism to create an impactful solution to these diarrheal diseases.”

Grischa Chen [Salk Institute]
Grischa Chen [Salk Institute]

The researchers set out to investigate how the body suppresses infection symptoms, and to determine whether infection without symptoms can create long-term immunity. If so, they wanted to know whether that immunity might be reproducible as a vaccination strategy.

The team compared mice given iron-rich, or normal diets after C. rodentium infection, to find out whether the diet impacted on symptomless infection. They found that immediately after infection, mice fed an iron-rich diet had no symptoms, whereas mice fed a normal diet did have symptoms. All the mice were then put on a normal diet to see whether the asymptomatic infection would last.

The scientists showed that mice with nonfunctional adaptive immune systems (SCID-/- animals, regardless of whether they had ever been on an iron-rich diet, could not continue to maintain a cooperative relationship with the bacteria. Although the iron-rich diet suppressed symptoms immediately after infection, the adaptive immune system was required for lasting cooperation.

Importantly, infected animals that did have functional adaptive immune systems (SCID+/- or SCID+/+ mice) showed no symptoms. “SCID−/− mice given dietary iron for 2 weeks had an initial survival advantage compared to both SCID+/+ or +/− and SCID−/− infected mice fed a control chow diet,” the investigators noted. “However, 100% of the iron fed SCID−/− mice eventually died ~3 weeks postinfection (1 week post iron diet withdrawal), while iron fed SCID+/+ or +/− with functional adaptive immunity survived and were protected from infection-induced weight loss.” Immunity in these animals was long lasting, as demonstrated by their survival upon reinfection after a month.

Ayres and team concluded that while an iron-rich diet alone can prevent bacteria from creating deadly symptoms in mice during active infection, a functional adaptive immune system is required for immunity against future infection in the absence of dietary supplementation. “Thus, the adaptive immune response is not necessary for iron-mediated protection from infection but is required for long-term survival during the iron independent phase,” they noted.

Some bacterial strains, if mutated enough, don’t cause symptoms. To test whether such bacteria could also produce lasting immunity, the team repeated their iron diet versus normal diet experiment in mice, but this time infecting some mice with bacteria that could cause disease, and others with bacteria that could not cause disease. The results showed that only mice that received disease-causing, unmutated bacteria were able to support immunity upon reinfection. “… asymptomatic carriage of genetically virulent but not genetically attenuated C. rodentium confers protection from subsequent challenges with the parental virulent strain of C. rodentium,”  the team stated. “Our work reveals previously unknown insight into how asymptomatic infections can arise mechanistically and that physiological defenses cooperate with immune resistance to confer protection against lethal infections.”

The scientists note that their results should not prompt people to consume large amounts of iron. The findings are preliminary and will need to be confirmed in human subjects. The researchers do hope their insights will provide a basis for future research in humans and the creation of a vaccination strategy that could protect and prevent against diarrheal illness.

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