By David N. Leff
The term ¿germ warfare¿ evokes thoughts of secret laboratories run by evil dictators in faraway countries. But the clear and present danger for humanity ¿ the actual perpetrators of germ warfare ¿ are the germs themselves.
Among the countless bacterial war criminals that infect and decimate human populations, a top candidate for public enemy No. 1 is Pseudomonas aeruginosa.
¿About 16 percent of all nosocomial [in-hospital] pneumonias,¿ observed bacterial geneticist Dara Frank, at Wisconsin Medical College, in Milwaukee, ¿are due to P. aeruginosa. The problem with that statistic is that for 40 percent of those people it will be a fatal infection. It can colonize almost anything in the hospital, because it¿s a soil-and-water-dwelling organism. So, it¿s brought in with water and soil. And it¿s very difficult to eradicate, plus P. aeruginosa¿s resistance to antibiotics makes it very difficult to treat.¿
Not that that particular pathogen has its knives out for Homo sapiens in particular. ¿This bacterium is an opportunistic pathogen,¿ Frank said, ¿which means it¿s only an accidental inhabitant of human beings. It doesn¿t really care about us people.¿ (See BioWorld Today, March 3, 1999, p. 1.)
¿Because this bug lives in the soil and the water,¿ she said, ¿we hypothesize that its whole system has been maintained for long periods of time to fight off the eukaryotic cells that compete with it in that environment. For example, if the germ lives in pond water, it¿s going to come in contact with amoeba, which probably want to eat it. The germ¿s virulence protein could be a means of paralyzing or blocking the amoeba, so that the bacteria can live on. And it eats many other things besides amoeba.¿
Frank is senior author of a paper in the April issue of Nature Medicine titled: ¿Active and passive immunization with the Pseudomonas V [virulence] antigen protects against type III intoxication and lung injury.¿
Lung Tissue Is Favorite Target
Lung tissue is the bacterium¿s favorite target. It strikes hospitalized patients with acute pneumonia, and cystic fibrosis (CF) sufferers with lifelong chronic pulmonary infection. Just how it wreaks this life-threatening havoc is the germ¿s closely held secret, which Frank and her co-authors are beginning to crack.
¿Our overall finding so far,¿ she told BioWorld Today, ¿is that we can prevent lung injury with a vaccine or a blocking antibody directed against this particular bacterial cytotoxic protein. This protein has homologs ¿ DNA sequence similarities ¿ in other bacteria. We discovered in our laboratory that its closest relative is Yersinia pestis, the plague pathogen. This same protein in Y. pestis does serve as a protective antigen. You can raise antibodies to it in Yersinia to protect against the plague. So, once we discovered this antigenic protein in Pseudomonas, and saw that the Yersinia homology was as high as it was ¿ 40 percent, in both protein and in function ¿ it seemed to us a logical extension to go ahead and try this as a vaccine, to see if it would work in P. aeruginosa infections. And, sure enough, it did.¿
¿We then needed to work out the mechanism of how it operated,¿ Frank said. ¿Because, even in Yersinia, [we] didn¿t know how exactly the vaccine is protected. In our system, it looks as if the antibodies block the ability of the bacterium to intoxicate macrophages in particular, thus tipping the balance toward bacterial replication. What happens then is that the immune system can react to the bacterium, and get rid of the infective inoculum very quickly. Macrophages are apparently very susceptible to these toxins, which also intoxicate the lung¿s epithelial cells.
¿The particular toxin that seems to do a number on the macrophages in our system,¿ she continued, ¿is called ExoU. We don¿t know how it works, but it is a very lethal toxin. So, this might be one of the toxins responsible for infections that are fatal very quickly. It¿s only in a certain percentage of the strains, so if you¿re unlucky enough to get a bacterium that encodes this ExoU toxin, then you might incur a higher probability of having a fatal outcome.¿
Frank described the in vivo experiments she and her co-authors reported in their journal article.
¿What we ended up doing,¿ she said, ¿was to make antibodies to this protein in rabbits, and these enabled us to passively transfer protection into mice challenged with lethal doses of P. aeruginosa. We also showed that those animals did not have the type of lung injury that affected control mice unprotected by the antibodies.¿
Biotech Start-Up Tackles Vaccine Development
Frank¿s goal is to ¿treat patients with the antibody, perhaps those in the intensive care unit, where they¿re likely to come down with a nosocomial pneumonia, because they¿re being mechanically ventilated. We¿d like to develop this into a therapeutic that we can use on those types of patients, to prevent them from getting lung damage and coming down with a fatal sepsis or organ failure. We¿re on an aggressive schedule, but haven¿t even started Phase I clinical trials. Right now, we¿re in the process of preparing toxicity testing in animals.
The medical college is applying for a patent covering Frank¿s antibody as a protective therapeutic and as a preventative vaccine. About two weeks ago, it signed a license agreement with Intermune Inc., a start-up biotechnology company in Palo Alto, Calif.
That firm¿s president and CEO, Scott Harkonen, told BioWorld Today, ¿The status is that [Frank] has identified and cloned the target protein. The next steps involve two pathways: First, development of an active vaccine, using this protein with an appropriate adjuvant. We¿re just beginning that work. And the second pathway is to develop a monoclonal antibody for passive protection. She¿s got the monoclonals, and we¿re just beginning to look at humanization, what would be the appropriate strategy as a pharmaceutical.
¿Our three initial targets,¿ Harkonen said, ¿would certainly be in children with [CF], chronically infected with Pseudomonas; also in burn patients, and the third [target] would be people in intensive-care units [ICUs], on long-term respirators ¿ which means about three days.
He concluded: ¿What this bacterium does is take advantage of an immunocompromised state. In the case of a burn patient, just the loss of the barrier function of the skin. In the ICU, where patients are intubated, their lungs become susceptible to Pseudomonas. And in CF patients, there seems to be a localized immune defect in the lungs.¿