Countering Bugs

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BY DEBORAH KOMLOS

Our quest to eliminate “bad” microbes has pushed them to the edge. Through this fight against infectious diseases, we have also created an urgent need for effective, new therapeutics.

A serious concern for health-care strategies targeting infectious diseases, and one that is especially troublesome for industrial nations, is antibiotic resistance, says Julian Davies, PhD, professor emeritus with the department of Microbiology and Immunology at the University of British Columbia (Vancouver, BC). Also a member of the Canadian Bacterial Diseases Network (Calgary, AB), Davies has been involved since the late 1960s/early 1970s with antibiotics, the mechanisms of antibiotic resistance and how it arises in microbes, and the role of antibiotics in nature.

“It’s very depressing to learn that we’ve learned nothing,” Davies says. “And we’ve got the same problems now as we had then, except that they’re worse . . . they’re very serious now.”

Davies is referring to the earliest instances of antibiotic use — dating back to the 1940s — when the first cases of antibiotic resistance were noted, such as the emergence of streptomycin-resistant strains during the course of treating tuberculosis (TB). “You have to realize that bacteria and microbes are the oldest inhabitants of the Earth,” Davies explains. “They’ve been around for more than three billion years. They have survived many crises and due to all kinds of things, they are now facing another crisis, and that is that human beings want to wipe them out.”

Eliminating these microbes destroys a natural and essential part of our existence. “You are covered in bacteria, you know; you can’t live without them. So we don’t want to kill them,” Davies says.

Discussions cropped up during the 1960s, he says, of problems being caused by antibiotic resistance, including the increasing incidence of hospital-borne antibiotic-resistant strains, and there were calls for efforts to curb antibiotic use and to use them properly. These issues have remained, Davies says. One particular concern, he says, is the practice of prescribing antibiotics for the wrong conditions. For instance, giving an antibiotic to treat the flu or other viral infections. “It just won’t work,” Davies says. But there are also other profound troubles, he adds.

“The problem that we face now is that there are many compounds being used which are antibacterial, which I believe are dangerous; they don’t affect humans, but they maintain a selective pressure for antibiotic resistance and this is continuing to happen,” Davies says. “Most antibiotics now have gone beyond the point of no return, since you can’t revert antibiotic resistance.”

Antibacterial agents are everywhere, he says, in thousands of products on the North American market. “You go out and try to see if you can buy a garbage bag that is not impregnated with an antibacterial agent,” Davies says.

Calling for Change

The evolution of drug-resistant strains and the rise in incidence of TB and other infectious diseases have been strong prompts for efforts to understand the immune mechanism of the host response and to develop novel therapeutics.

Dr. Zhou Xing, PhD has provided one such research channel. In 1998, Xing began an infectious diseases program at McMaster University (Hamilton, ON) — where he is head of the division of Infectious Diseases at the Centre for Gene Therapeutics — focusing on intracellular bacterial infections and predominantly mycobacterial infection.

After earning his medical degree in China, Xing worked as an infectious diseases specialist and saw mainly TB and hepatitis patients on a daily basis. “So, in a way, clinically, I knew a lot about TB, how devastating it could be,” he says.

Xing completed a PhD and post-doctoral training at McMaster, together covering the areas of inflammation, immunology and the pathogenesis of asthma, and continued to work on acute bacterial infections. But he realized there was a need for change. He saw a niche to be filled, and the backing of his previous infectious diseases experience further supported his decision to create a program focusing on mycobacterial diseases.

In 1999, Xing secured a grant from the Canada Foundation for Innovation to build the necessary Level 3 biohazard containment facility to work with MTB (Mycobacterium tuberculosis), the organism that causes TB. He immediately started working to produce a vaccine that would be better and safer than BCG, the traditional TB vaccine. McMaster is the second academic institute in Canada (the first was the University of British Columbia in Vancouver, B.C.) to have a Level 3 facility for in vivo animal experimental work, and Xing and his team are the first worldwide to have developed a recombinant adenoviral-based TB vaccine.

Working with their mouse model, Xing says they have shown that vaccine delivery mode significantly influences efficacy. Versus the common intramuscular injection of BCG, intranasal (i.e., mucosal) immunization was more successful for both BCG and the group’s recombinant vaccine.

“The immune protection following intramuscular vaccination itself is very transient,” Xing says. “But with intranasal immunization, not only is it stronger protection but it lasts a lot longer than intramuscular immunization.”

Xing says he is quite excited about his research, but getting movement on the TB research front has been very hard. “Historically, just based upon my own experience, there is something associated with perception as well. People tend to think TB is a very dangerous thing. Therefore, I think in Canada the guidelines for allowing to have such (Level 3) facilities are much, much more strict than those down in the United States,” he says.

As well, Xing adds, while TB is still a health problem worldwide and its importance is recognized in North America, its incidence in the developed world is still low relative to many other diseases. “But potentially it could be a problem because from time to time you do get outbreaks in hospitals or communities,” he says.

Although BCG has been in use for 80 years, it has not been effective enough, Xing says, which explains the current TB epidemics. Bearing this in mind, the team is also working to help enhance the immune-stimulating effect of BCG. Xing says the team has tested various cytokines and has found that GM-CSF — granulocyte-monocyte colony-stimulating factor — works best, when mixed with BCG, to improve immune protection in the group’s MTB challenge model.

The Basics

Despite the progress to date, much more work is needed, Xing says.

“A lot of vaccines currently are being used in human beings, (but) very few have been used mucosally. But we are confronting a great number of mucosal infectious diseases — TB is a mucosal infectious disease, so is HIV, herpes infection,” Xing says. “We know for a fact that the best way to immunize the host against mucosal infectious disease is to immunize the host mucosally — that can generate the best immune protection — although we still understand very little how exactly mucosal immunization differs from systemic immunization. We only have observed the phenomenon, we don’t have a very good grasp of the mechanism yet.

“So, obviously if you want to have better vaccines you must know how you can best target host immune systems,” Xing says. “Whenever we have a better understanding coming out we can immediately apply this to the rational design of novel vaccines.”

Approaching the infectious diseases issue via the mucosal route is also the focus of a $27-million Cdn. collaboration titled Functional Pathogenomics of Mucosal Immunity. Co-led by Lorne Babiuk, PhD, director of the Vaccine & Infectious Disease Organization located at the University of Saskatchewan (Saskatoon, SK), and Robert Hancock, PhD, from the University of British Columbia (Vancouver, BC), the project involves several partners among which are AniGenics Inc. (Chicago, IL), Inimex Pharmaceuticals Inc. (Vancouver, BC), Simon Fraser University (Burnaby, BC), Pyxis Genomics Canada Inc. (Saskatoon, SK) and Genome Prairie (Calgary, AB).

“Ninety per cent of all pathogens enter through mucosal surfaces — that’s where the first encounter between the host and the pathogen is,” Babiuk says. “So, therefore, we want to look at what are the genes that are turned on at mucosal surfaces upon an encounter with a pathogen, an encounter with vaccines or encounters with different immune modulators.”

In addition to the in vivo work, the research is also looking into what genes are regulated when a pathogen is cultured in vitro in chicken, cow and human epithelial cells from the respiratory tract and from the gastrointestinal tract. Doing the comparative biology work will help give the team a level of confidence, Babiuk says, if recurring responses in three different species are observed.

“When a pathogen is grown in culture, they have a different constellation of genes (active) than if they’re grown in vivo or in the lung of an animal or in the gastrointestinal tract of an animal or a human,” Babiuk says. “These new genes that are turned on in vivo probably have a role to play in colonization and in virulence. So then we can again look at those particular genes and predict in a more rational manner which ones might be good targets for vaccines.”

The specific microbes of focus for enteric infections include Rotavirus, Coronavirus, Cryptosporidium, Salmonella and E. coli. Those for respiratory infections involve bovine herpes virus and parainfluenza virus in cattle, metapneumovirus in chickens and Pseudomonas, which Babiuk says is a big problem in cystic fibrosis patients.

Getting into Practice

Babiuk praises the direction in which the field of immunology has moved. “Fifteen years ago, people didn’t pay much attention to the innate immune response at all,” he says. “Even 10 years ago, the true card-carrying immunologist did not really have much time for the innate immune system.”

Today, however, it is recognized that the innate immune response and adaptive immune response are strongly linked. “In fact, the innate system will tell you how good or what the quality of the adaptive immune response will be,” Babiuk says.

The former is the one that has very rapid defences and targets a variety of different pathogens, Babiuk explains, whereas the adaptive immune response is very specific against one pathogen. Part of the alliance’s work, Babiuk says, is to develop potential immune modulators.

“If, for example, you can stimulate the innate immune system and we know specific things will do it, it doesn’t matter whether it’s a virus or a bacterium or a parasite that we encounter,” he says. “So that to me is really what excites me.”

In collaboration with a group led by John Nash, PhD at the Institute for Biological Sciences, Pathogen Genomics Group, National Research Council of Canada (Ottawa, ON), the project researchers have identified a potential target for Campylobacter, which is carried by virtually all chicken flocks, Babiuk says.

“Chickens don’t get disease (caused by Campylobacter), just like E. coli O157:H7 doesn’t cause disease in cattle. But if we can identify what are the targets that can attach and colonize the chicken, we can develop a vaccine to prevent the colonization and then the humans don’t get infected because we reduce environmental contamination,” Babiuk says, adding that Campylobacter diarrhea is as severe and will kill people just as easily as Salmonella. “An outcome would be a more rational approach to vaccine development, a more rational approach to stimulating innate immunity to give rapid defenses. So you combine the rapid resistance with a long-term resistance.”

Babiuk says a milestone of the research is to be able to identify one or two commercially viable immune modulators. A second goal is to identify specific targets for vaccine development against Campylobacter, and a third, to be able to deliver the immune modulators and vaccines together.

“Animals come into a feedlot, they get infected the day they get there. It takes two weeks before the vaccine takes effect. So if you can combine the immune modulator to give them very early protection and give them the vaccine which gives them long-term protection, that again would be a fantastic achievement and we think that we can do that,” he says.

While the mucosal route seems to be a prime direction for targeting culprits of infectious diseases, Xing points out that many challenges still await this form of drug administration.

“Everything you deliver into the lung, for example, you have to worry about inflammatory responses because after all, the lung is our vital organ,” Xing says. “You don’t want to have an overwhelming amount of inflammatory responses going on in there.”

A Long Pathway

Moving novel biotech drugs through the clinical trial process is “a long, arduous, risky, expensive process,” says Dr. David Barnes, PhD with the Chalmers Research Group, based at the Children’s Hospital of Eastern Ontario Research Institute (Ottawa, ON). Barnes and his group offer consultation to startup and medium-sized, more established biotech firms on how to get their products to market, including advice on drug-development strategy, toxicology, designing studies, implementation and data analysis and getting a product through the regulatory process.

Barnes says one difficulty in working with infectious disease agents such as HIV and hepatitis C virus is the challenge of assembling a patient population on which you can test the effects of the vaccine.

“Traditionally, vaccines are used to stimulate the immune system and prevent a disease or moderate the severity of the disease if you should catch it,” Barnes explains. “So, if you can imagine, it’s going to be very tough to test a vaccine on a patient population that is at high risk and possibly young in age and may not have the disease.” These ethical considerations make clinical development of such vaccines very difficult, Barnes says.

“One way around this is to try to work with regulatory authorities to develop parameters that are acceptable to measure the success of the vaccine in a patient population that already has the disease,” he says. “So instead of using it as a preventive, you’re using it as a therapeutic. And then you can get the data that’s used in that context and use it to support regulatory application — that’s as a for instance.”

In Davies’s view, education is the basic requirement to promote the acceptance and proper use of existing and future antibiotics and vaccines. He applauds the efforts of the various physicians and pediatric groups that are already trying to get the message across. “One has to educate the public, physicians, pharmaceutical industry, nurses, everybody about the use of antibiotics and my plan is to put everything on the sports page every day so that every member of the public reads something about the topic,” Davies says. “They have to be told every day about people dying in hospitals, and to stop using antibiotics.”