Last year, following the publication of his latest research in the journal Vaccine, dozens of emails flooded polysaccharide chemist Mario Monteiro’s inbox. Monteiro’s paper, about a potential vaccine for gastrointestinal bacterial infection in autism spectrum children, struck a chord with parents, who cope daily with their youngster’s chronic constipation and diarrhea, self-destructive behaviours and cognitive and social delays. “They want to volunteer their child for study groups, that’s how desperate they are,” says Monteiro, a professor in the Department of Chemistry at the University of Guelph and a global leader in microbial polysaccharide immunochemistry. “If you are willing to do that you are really at the end of your rope — and they were talking just about the diarrhea, not the autism.”
Mario Monteiro holds a model of one of the structural key markers of Campylobacter polysaccharides, a rare sugar called 6-deoxy-heptose. Different types of Campylobacter express polysaccharides with their own characteristic 6-deoxy-heptose. Photo credit: Trina K Photography
In the past decade, researchers have focused on a possible connection between the high bacterial loads found in the gastrointestinal tract of autistic children and their neural development disorders, a link commonly called the ‘gut-brain’ balance. So far, the main culprits include microbes from Clostridia, Desulfovibrio and Sutterella species. The hypothesis, says Monteiro, is supported statistically. Researchers are aware that about 90 percent of autistic kids have severe gastrointestinal upsets while 75 percent of those have chronic diarrhea, forcing them to remain diapered at an age when other youngsters are mastering their ABCs at school. Monteiro says that some studies suggest that metabolites released by these bacteria, such as hydrogen sulphide and propionic acid, not only irritate the gut but may also indirectly affect the brain at a young age. Studies show that mice, for example, exhibit autism-type behavior when injected with propionic acid, Monteiro says. Such studies have helped strengthen the ‘gut-brain’ theory, for when autistic children in a study were put on a regimen of strong antibiotics, not only did the gastrointestinal problems disappear but so did many of the bewildering behaviors. “If you cure the diarrhea, you may also indirectly lessen some of the symptoms of autism, so we’re developing an anti-diarrheal vaccine against the pathogens associated with autistic children,” says Monteiro.
This molecular model of a prototype polysaccharide vaccine against Campylobacter jejuni, one of the causes of traveller’s diarrhea, shows its helical conformation. Photo credit: Monteiro Lab, University of Guelph.
Although Monteiro is not an autism expert, he is in an excellent position to develop a vaccine against autism-linked pathogens. During his PhD studies in the early 1990s, Monteiro, a Portuguese native, delved into the study of bacterial polysaccharides, the long, sugar-based polymers that cocoon bacteria in little carbohydrate ‘jackets.’ Conventional vaccines work by injecting the inactivated pathogen or their proteins into the bloodstream, triggering the development of antibodies. Polysaccharide vaccines operate the same way but they use a different class of target molecules, allowing researchers to go after organisms for which the conventional approach has failed. For his graduate studies, Monteiro focused on the polysaccharides of Helicobacter pylori (H. pylori), a “very hot” area of research at the time. H. pylori is linked to gastric ulcers and stomach cancer and is especially prevalent in Canada’s aboriginal communities as well as the developing world, where it can infect up to 50 percent of a population. “I discovered that H. pylori used its polysaccharides to hide from the immune system through molecular mimicry, allowing for a long undetected life in the human stomach,” says Monteiro, who is presenting the paper, “An efficacious polyvalent Clostridium difficile vaccine,” at the May 31-June 1 Asia-Canada Glycoscience Satellite Meeting, immediately preceding the 97th Canadian Chemistry Conference and Exhibition in Vancouver.
Upon completion of his PhD, Monteiro was snapped up by, first, the National Research Council’s (NRC) Canadian Bacterial Diseases Network in Ottawa and later Wyeth Pharmaceuticals in the United States. Wyeth (now part of Pfizer) wanted Monteiro’s polysaccharide expertise to develop a vaccine against H. pylori and other nasty bugs, such as those responsible for meningitidis and pneumococcal infections.
Polysaccharide vaccine research has roots in the early 20th century, when American immunologists Michael Heidelberger and Oswald Avery discovered that Streptococcus pneumoniae polysaccharides act as antigens, which spark an immune response in the body just as proteins do. Unfortunately, the antibodies spawned by T-cell independent polysaccharides generally don’t last as long as those raised against T-cell dependent proteins, in that polysaccharides raise mostly short-lived IgM antibodies while charged proteins generate long-lived IgG antibodies. (T-cells are produced by the thymus gland and are a key part of the immune response.)
Further advances in the field were stymied when Scottish Nobel laureate Alexander Fleming discovered penicillin a few years later, allowing many bacteria to be treated by antibiotics rather than vaccines. It took half a century for polysaccharide vaccine research to be revived; among the first success stories was a pneumococcal vaccine developed for adults, partially based on structural work by Monteiro’s mentor, the late Malcolm Perry of the NRC. The elderly are especially susceptible to Streptococcus pneumoniae, which causes pneumonia, meningitis and septicemia. Like many bacterial diseases, it is increasingly antibiotic resistant, one of the factors that has pushed polysaccharide vaccines back into vogue.
Clostridium difficile molecule.
One of Monteiro’s goals is to overcome the need for frequent booster shots by making the antibodies raised against polysaccharides last longer. A popular approach is called the “conjugation technique,” which links the polysaccharide molecule to a protein carrier molecule. It’s also in this area that Monteiro’s skills as a chemist come to the fore. “Our first step is the discovery of the polysaccharide structure and its immunogenicity and then, if needed, we devise special chemical strategies to covalently join the polysaccharide to an immunostimulatory protein.” Recently, Monteiro has developed a method that in some cases may bypass the need for conjugation — a chemical manipulation in which polysaccharides become T-cell dependent by making them zwitterionic. “I think that one of the reasons why T-cells are involved in protein uptake is because proteins have charges in their amino acids. So, by giving my polysaccharides some charge, I make them T-cell dependent just like proteins.”
Another reason polysaccharide vaccine research languished for so long was the inherent complexity of the molecules. Although bacteria are primitive and have inhabited the earth far longer than mammals or even plants, their carbohydrate structures “are a million times more complex,” says Monteiro. “They have the genetic ability to make really, really strange polysaccharide structures, so it takes a long time to figure out what these structures are and use them in a vaccine.” So, why are polysaccharides better targets than proteins for some organisms? Protein-based vaccines with their peptide backbone — while relatively easy to produce and construct — are capricious. When protein is removed from a bacterial cell and purified to make the vaccine, they sometimes lose their functional shape. This means that they have low immunogenicity — antibodies will not recognize the live invader due to this difference. Polysaccharides, however, keep their main conformation when removed from a bacterium, giving them high immunogenicity, says Monteiro. A polysaccharide-based vaccine has an additional advantage. Because the molecules are repeating blocks of oligosaccharides, antibodies recognize many parts of the polysaccharide. “You have a better chance of having a greater number of effective antibodies,” Monteiro says.
Vaccine development is a long process, taking anywhere from 10 to 30 years to bring to market. But Monteiro is already seeing the fruits of his labour with Campylobacter jejuni (C. jejuni). Referred to as travellers’ diarrhea by those who visit the developing world, C. jejuni can be contracted while swimming or by eating undercooked meat or unpasteurized milk products. For young children, it can spell long-term health problems or even death. “Children die from Campylobacter diarrhea in the developing world,” Monteiro says. For the US Navy, C. jejuni is also a bane, striking naval service members on ship, shore and submarine. Phase 1 human trials — funded by the American National Institute of Health and the US Navy — are poised to begin shortly to test the efficacy of the C. jejuni vaccine that Monteiro has developed. (It triggered full protection against diarrhea in monkeys during pre-clinical trials.) “It is our first major vaccine and the first from the University of Guelph to enter human trials,” Monteiro says.
The navy first contacted Monteiro a decade ago to inquire about collaborating on a C. jejuni vaccine to inoculate personnel. Navy microbiologists supplied Monteiro with the bacteria he needed to work with and undertook the pre-clinical trials. The intellectual property for the vaccine is being shared equally between the navy and the University of Guelph, Monteiro says.
Another vaccine, this one against C. difficile, is also poised for human trials, says Monteiro. This growing scourge is more dangerous than C. jejuni and is spread in hospitals, attacking the weak and sometimes causing death. To date, says Monteiro, most C. difficile research has focused on finding a way to nullify the toxins excreted by the bacteria. Unfortunately the pathogen is unharmed; the patient survives the infection, but can become an asymptomatic C. difficile carrier. The vaccine that Monteiro developed triggers an attack against the bacteria’s surface polysaccharide antigens. Monteiro has since sold the C. difficile patent to a California company, which is currently in the midst of animal studies with the vaccine. “It controls infection in mice — mind you mice aren’t humans,” says Monteiro, who is still involved in refining the vaccine and estimates human trials will begin in one to three years.
Monteiro’s unique approach to anti-diarrheal vaccine development began, he says, with “a vision. You have to ask yourself, ‘what diseases are really important that we have to save people from? That I could use a polysaccharide vaccine against?’ ” Just as it takes a village to raise a child, it takes an extensive network of collaborators to create a vaccine, including coteries of dedicated undergraduate and grad students as well as microbiologists, who grow and supply the bacteria needed for countless experiments.
Monteiro is proud of the vaccines that he has created to date. “It’s very rare one gets a chance to give someone your discovery and say, ‘inject this into a human being.’ ” As the father of two young, active boys, Monteiro is especially determined to create a vaccine to help autistic children and their desperate parents, who must plumb bottomless wells of patience and tolerance. “Because I have kids, this one is really in my heart.”