Researchers at The University of California San Francisco report that they have developed a highly sensitive, rapid technique for detecting the infectious agents that cause prion diseases. And they said they expect the assay will ultimately be useful for detecting prions causing "mad cow" disease and Creutzfeldt-Jakob disease in humans.
With automation, they said, the tool could be applied to commercial testing of meat, biological and pharmaceutical products.
"This is an extremely exciting scientific breakthrough," said the lead author of the study, Jiri Safar, MD, an associate adjunct professor of neurology at UCSF. "We still have some scientific aspects of the assay to resolve, but we are moving from a scientific discovery to an engineering challenge."
But the significance of the UCSF study, reported in the October issue of Nature Medicine, extends beyond the hope for an effective screening tool. For the assay has revealed stunning insights into the nature of the novel, inscrutable pathogen that causes "mad cow" disease, Creutzfeldt-Jakob's disease in humans and a variety of other neurodegenerative diseases seen across species and known collectively as spongiform encephalopathies. The findings have given the researchers new direction for exploring the way in which the pathogen, called prion (PREE-on), for proteinaceous infectious particle, functions.
The test tube immunossay, which so far has been used to detect infection in hamsters, identifies extremely low levels of prion protein--the only known component of the infectious prion--and does so within a matter of eight hours. And the researchers said they believe the design can be adapted for large-scale robotic processing.
By contrast, current detection models, called bioassays, involve inserting suspected infectious tissue into the brains of laboratory animals and observing them for development of the disease. The process takes between 60 to 180 days, and cannot be conducted on a large, commercial scale.
The new technique, conducted in plastic plates, is also expected to prove effective for diagnosing new-variant Creutzfeldt-Jakob disease (CJD) in living patients. Scientists fear that some 25 people in Great Britain and France may have developed the disease by eating tainted meat in the 1980s.
But the insights the test offers into the biology of the prion protein are consuming much of the researchers' attention. Previous research has revealed that all mammals examined contain normal, benign prion protein, and it is believed that they only become destructive when the prion protein changes shape, from a coiled structure to a flat sheet. The conversion in the infectious form of the disease (which can also be inherited or occur spontaneously) is believed to occur when already infectious prion protein, or PrPSc, clasps onto the normal prion protein, or PrPC, twisting it down flat in a morbid, fateful dance.
The researchers developed an assay that detects a region of PrPSc protein that, while exposed in normal PrPC protein, becomes tucked, or folded, in the diseased PrPSc molecule. Fluorescently labeled antibody that reacts with the folded region of PrPSc only after the disease protein is unfolded, or denatured, is used in the assay.
The researchers first expose a tissue extract containing infectious prion protein in its natural state to the antibody and measure the reactivity. They then unfold the prion protein by chemical means so that the hidden region will be exposed. Predictably, the antibody's immunoreactivity to the denatured region, as measured by its degree of binding to the molecule, is much higher than it is to the diseased protein in its natural state. The ratio of denatured to native infectious prion protein indicates the amount of PrPSc.
The researchers used the model to test brain tissue taken from hamsters infected with eight different strains of prions. They plotted the results as a function of the concentration of PrPSc for each strain. And their findings were dramatic. Like seemingly insignificant holes cut in paper can create the image of a snowflake, the points on the graph revealed detail about the proteins' unique properties that the molecular biologists couldn't see on their own: specifically, that each of the eight different strains of infectious prions had unique shapes.
Researchers have known that prion diseases, even within species, vary in length of incubation, topology of prion accumulation and distribution of accumulated protein deposits in the brain. But while they have suspected that these variations, or strains, were represented by different protein shapes, they have never had direct evidence. Moreover, it has long been believed that a protein has only a single conformation, as determined by its amino acid sequence, and all eight strains did represent a single molecular sequence.
"We know that PrPC and PrPSc have very distinct shapes. What has become clear is that while all of the strains contain a common molecular sequence, each protein strain has a distinct shape," said Fred E. Cohen, MD, PhD, a professor of pharmacology and medicine at UCSF and a co-senior author of the study.
The assay also revealed that PrPSc protein contains a protease-sensitive fraction, which surprised the researchers. "We always thought PrPSc was strictly protease resistant," said Stanley B. Prusiner, MD, a professor of neurology, biochemistry and biophysics at UCSF, the winner of the 1997 Nobel Prize in Physiology or Medicine, and the other senior author of the study.
In an effort to tease out the component of prion protein that might actually confer the most crucial distinction in strains--the time it takes for the disease to develop--the researchers plotted the protease-sensitive component of the PrPSc versus incubation time and were struck by what Safar called 'a gorgeous straight line.'
"Until now, we believed that once formed in the brain, prions could not be degraded. We now understand that it is the rate at which prions are degraded that explains the differences in the time that it takes a prion strain to cause disease," said Cohen. "Since the body can begin to clear the proteinaceous mess from the brain, treatments are being developed to assist this process."
"The only conclusion," Cohen said, "counterintuitive as it is, can be that the rate-limiting step in prion replication has little to do with PrPsc.
Instead, Cohen and Prusiner suggested, it must have to do with an earlier stage in the development of PrPSc, when normal PrPC protein binds to an as-yet-elusive "protein X." Protein X is believed to act as a molecular chaperone, moving the normal protein out to the dance floor where it presumably is handed off to its deadly suitor.
Needless to say, the researchers are turning their attention to this earlier stage in the conversion cascade, before the protease-resistant fraction is formed.
"While we still can't visualize protein X, we need to see if we can figure out its role," said Safar. The researchers' challenge, which molecular biologist face every day in their explorations, will be developing still more clever techniques that will reveal to them what they can't actually see, in this case the machinations of a deadly protein.
The University of California has filed a patent on the full technology platform for the immunoassay. Centeon Inc. holds a license granting them exclusive rights to the immunoassay technology.
Other co-authors of the UCSF study included Holger Wille, PhD, Vincenza Itri, BS, Darlene Groth, BS, Hana Serban, MS, and Marilyn Torchia, DVM. The study was supported by grants from the National Institutes of Health, as well as well as by gifts from the Leila G. and Harold Mathers Foundation, Sherman Fairchild Foundation and Centeon.
Journal
Nature Medicine