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Protein conformation and diagnostic tests: the prion protein.

Protein Conformation and Dynamics in Protein-based Diagnostic Assays

Most proteins assume a distinct three-dimensional structure in vivo and in vitro, and this native structure is necessary for function. A proteins structure is determined by its amino acid sequence and the surrounding environment. There are various levels of structure: primary structure refers to the amino acid sequence; secondary structure refers to the fold of the peptide, i.e., a-helices, [beta]-strands, and turns; and tertiary structure describes the three-dimensional structure and is determined by the packing of the secondary structural elements and the amino acid side chains. As a protein or peptide unfolds, interactions are disrupted and both secondary and tertiary structure can be lost. In addition, the folded and unfolded states of proteins are in equilibrium. Even under conditions that favor the folded state, a protein is not locked into a single conformation. The amino acids are free to interact and move according to the forces placed on them by neighboring atoms and the solvent, producing many conformations that may differ only very slightly (conformers).

Protein motion occurs on a wide spectrum with respect to time. Bond vibration and rotations occur on a femtosecond timescale ([10.sup.-15] s), whereas amino acid side chains move on a picosecond to nanosecond timescale ([10.sup.-12]-[10.sup.-9] s). Small segments of the peptide backbone can reposition themselves in a matter of nanoseconds to microseconds ([10.sup.-9]-[10.sup.-6] s). The arrangement of surface amino acids can be affected by such dynamic behavior. Such changes in the surface of a protein can alter potential binding sites for other molecules. Complete folding of a protein can occur in microseconds to milliseconds or hours, depending on the sequence and solvent environment. Furthermore, proteins and peptides can adopt different, folded structures under different conditions (1-3). Even slight changes in binding epitopes attributable to either localized or more dramatic conformational changes of a protein can have profound effects on antibody-based diagnostic tests. This issue is particularly pertinent and potentially problematic in the case of the development of diagnostic tests for transmissible spongiform encephalopathies, or prion diseases.

Conformational Change and Disease: Transmissible Spongiiform Encephalopathies

There is currently much interest in transmissible spongiform encephalopathies (TSEs) [1] because of outbreaks of bovine spongiform encephalopathy (BSE, or mad cow disease) in the United Kingdom and elsewhere (4,5). BSE began to receive a great deal of media attention in the mid to late 1990s when massive herd culls occurred in Great Britain and the incidence of Creutzfeldt-Jakob disease (CJD) increased dramatically among young people (4, 6, 7). Recent reports of chronic wasting disease (CWD) in elk and deer from the Western United States and Canada, as well as scrapie in sheep in the Northeast, continue to keep TSEs in the news headlines and a priority among government agencies (8, 9).

Prion diseases affect the central nervous system and are a result of a conformational change in the prion protein, often leading to prion protein-based plaques. In humans, these diseases can be sporadic, inherited, or infectious disorders. This protein is responsible for the following diseases in humans: Kuru, Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI), CJD, and the BSE-linked, variant CJD (vCJD). These diseases are always fatal. Inherited disease maps exclusively to mutations in the prion protein. The infectious nature of this disease differs from that of other infectious diseases in that the pathogen is a proteinaceous particle lacking nucleic acids; thus, the "protein-only" hypothesis was proposed (6).

The essential component of prions is the scrapie prion protein ([PrP.sup.Sc]; or [PrP.sup.res], for the protease-resistant form). [PrP.sup.Sc] is chemically indistinguishable from the normal, cellular prion protein ([PrP.sup.C]; or [PrP.sup.sen], as it is sensitive to proteases) (10); however, their secondary and tertiary structures differ (11-17). [PrP.sup.C] is a monomeric, glycosylated, glycosylphosphatidylinositol (GPI)-linked extracellular protein that appears to play a role in signal transduction (18) and/or in the maintenance of the proper copper ion concentration (19-21). In contrast, [PrP.sup.Sc] adopts an oligomeric arrangement and has no known function. Fourier transform infrared (FTIR) and circular dichroism spectroscopy studies indicate that [PrP.sup.C] is highly helical (42%) with little [beta]-sheet structure (3%) (15). In contrast, [PrP.sup.Sc] contains less helical structure (30%) and a large amount of [beta]-structure (43%). [PrP.sup.C] can be converted to the lethal [PrP.sup.Sc] conformation on contact with [PrP.sup.Sc] (22-24). These results, and others, suggest that a conversion of [alpha]-helices to [beta]-sheets is a critical feature in the formation of [PrP.sup.Sc] from [PrP.sup.C] and that the protein can aid in its own conversion (24). Several mechanisms have been proposed for the spontaneous and/or assisted conversion of endogenous [PrP.sup.C] to [PrP.sup.Sc] (24). Residues in close proximity to the C-terminal end of [PrP.sup.C] have been associated with [PrP.sup.Sc] binding, which is subsequently followed by conversion of [PrP.sup.C] (25). A confounding factor in conversion is that [PrP.sup.Sc] is conformationally heterogeneous, which appears to be important in determining strain differences and perhaps species barriers (23,26-28). Unfortunately, high-resolution structures exist only for portions of [PrP.sup.C] (Fig. 1). There are many other fascinating aspects of this system, but unfortunately they cannot be described here; the reader is directed to an excellent series of reviews edited by B. Caughey for more details (29).


Given the lack of detailed structural information regarding the conversion process of the prion protein as well as [PrP.sup.Sc] and its many possible conformers, we have been performing molecular dynamics simulations, beginning with [PrP.sup.C], in which we destabilize the protein by lowering the pH (21, 30, 31) as is believed to occur in vivo. Such an approach can provide atomic-resolution information about the putative conversion process as well as possible [PrP.sup.Sc] models. However, these models must be carefully compared with experimental data to ensure that they are realistic. In any case, to illustrate some of the complexities involved in the conversion of PrP and in developing diagnostics for [PrP.sup.Sc], we provide some of these preliminary models for [PrP.sup.Sc], along with nuclear magnetic resonance (NMR)-derived models for [PrP.sup.C], in Fig. 2. Structures for the hamster, bovine, and human forms of the prion protein are shown in the cellular and putative disease-causing conformations, illustrating how the same amino acid sequence can adopt different conformations. Several antigenic sites for [PrP.sup.C] are highlighted (Fig. 2 and Table 1), as these are the sites exploited in the antibody-based diagnostics described below. In extreme examples such as these, the surface of the protein can change dramatically so that epitopes found in [PrP.sup.C] are unavailable for binding in [PrP.sup.Sc]'. For example, the 3F4 epitope (red patch on the structures in Fig. 2) is less accessible in the simulated [PrP.sup.Sc] form, which is consistent with experimental findings (32).


Rationale for Development of Diagnostics for the Prion Protein

The protein-only hypothesis is important from a scientific point of view because it challenges the dogma regarding the transfer of biological information. From a public health point of view, a new route of disease transmission exists, and the ramifications are potentially enormous. Important sectors of the economy are susceptible to problems posed by transmissible prion diseases, with agriculture being one of the most vulnerable for several reasons. The first reason is the possibility of contamination of the food supply; the second reason is that the resulting loss incurred by agricultural producers when infection is discovered and eradicated can be enormous. Contamination of the food supply can occur in various ways, with transmission by meat products being only one possible route. Animal-based food supplements are found throughout the processed food and health supplements industries (33). Cosmetics may also contain oils, fats, or other rendered animal products (34). Furthermore, traditional medicines made from animal parts pose a risk (35). Individuals infected by ingesting contaminated food pose a risk to blood supplies and tissue banks. The American Red Cross and US Food and Drug Administration (FDA) have acknowledged this particular risk recently, and they have established guidelines to restrict possible contamination (36). Other sources of potential infection include pharmaceuticals that rely on animal remains, either in the manufacturing process or in the final product. The pharmaceutical industry was warned recently by the US FDA to restrict the use of animal-derived materials in the production of vaccines and other therapeutics (37). Several recent studies have shown the great difficulties in estimating the future risk of infection in humans and animals, further necessitating the development and widespread use of sensitive, reliable, and inexpensive TSE detection methods (38-41).

Rapid, specific, ultrasensitive, robust, and noninvasive diagnostic tools are desired for the prevention of transmission of prion diseases. These diagnostics are needed, especially in areas at high risk of transmission to large numbers, such as cattle herds and human patients reliant on chronic therapy regimens that include animal or even human-derived pharmaceuticals. Until recently, the only definitive diagnosis of BSE was by autopsy. Prion detection with the new antibody-based assays is now relatively rapid (8-24 h) (42) compared with past tests, which were long-term projects (>260 days for mouse infectivity assays). However, brain tissue samples are still required for these faster assays. Tonsil biopsies have been proposed as a less invasive alternative to brain biopsies (43), and a patent application has been filed. In addition, the detection of surrogate proteins (tau, 14-3-3, S100, and neuron-specific enolase) in cerebrospinal fluid has been proposed (44). These studies are promising, but an invasive lumbar puncture is required. Other methods for determining [PrP.sup.Sc] in central nervous system tissues include fluorescence correlation spectroscopy (45) and FTIR spectroscopy (46). The fluorescence correlation spectroscopy method has been shown to have 100% specificity and a sensitivity 20 times that of current Western blot techniques. Kneipp et al. (45) have shown that disease-specific changes in several parts of the brain can be detected by FTIR microspectroscopy. This method does not rely completely on the presence of [PrP.sup.Sc] for detection but rather monitors the change in infrared absorbances in carbohydrates and lipids. Data for other tissues and double-blind studies have not been published.

No absolute lethal prion dose or incubation time has been determined for cattle or humans (38, 39), which places rigorous requirements on diagnostic sensitivity. Lack of specificity could allow real infections to proceed undetected (false-negative results), whereas false-positive outcomes could lead to unnecessary expense and disrupted lives. An example of the havoc created by a false-positive result was experienced by a patient at our institution who was diagnosed with an extremely aggressive form of cancer (gestational trophoblastic tumors) (47). The diagnosis was apparently based on the AxSYM [beta]-Human Chorionic Gonadotropin test manufactured by Abbott Laboratories. Unnecessary chemotherapy sessions and surgeries were performed as a result of a diagnostic test that purportedly gave a false-positive result 44 times, highlighting the general need for multiple diagnostic tests for a particular condition. Ethical questions aside, several biochemical problems exist in the development of prion diagnostic assays.

Standard immunometric diagnostics rely on binding events predicated on the target protein adopting a single conformation. However, in the case of infectious prions, [PrP.sup.Sc] appears to adopt a variety of conformations (23, 26, 27), possibly requiring separate antibodies for detection. The sensitivity must be such that it is possible to detect prion protein in body tissues more accessible than the central nervous system (e.g., blood and urine). However, it is not known whether all conformations of [PrP.sup.Sc] cause disease (16). Here we review several diagnostic assays in use or in development for prion diseases; we also examine emerging technologies that address some of the concerns outlined above. Although diagnostic tests that detect the infectious form of the protein before the onset of clinical symptoms are of paramount importance for the detection and disposal of contaminated material, correct diagnosis of prion diseases is important, even after clinical signs appear, to provide appropriate treatment when such agents become available, especially because prion diseases may be misdiagnosed as Alzheimer disease (48). Therefore, in addition to discussing potential diagnostic tests, we also briefly summarize recent studies focusing on the development of therapeutic agents against prion diseases.

Diagnostic Tests in Use and under Development for the Prion Protein


In July 1999, the European Commission requested an evaluation of four promising prion diagnostic assays. These four candidates were selected from a total of 30 applications. Each assay was tested under double-blind conditions in four categories: specificity, sensitivity, limit of detection, and reproducibility. A brief description of each assay (42) is given below:

Test A: Diagnostic by E.G. & G Wallace Ltd. This test is under development and uses a two-site noncompetitive immunometric procedure with two different monoclonal antibodies. A small sample of brainstem (100 mg) is taken from the animal, cell membranes are disrupted, and proteins are extracted by use of a chaotropic agent. Extracts are then digested with two different concentrations of proteinase K, and the analyte is concentrated by centrifugation. After resuspension in 6 mol/L guanidinium hydrochloride, the extract is diluted 1:50 and subjected to a time-resolved fluorescence immunoassay, DELFIA, for detection of [PrP.sup.Sc] (specifics regarding the antibodies used and their respective PrP epitopes were not provided). In its current state of development, the test requires <24 h, although this could be reduced with automation.

Test B: Diagnostic by Prionics AG. "Prionics Check" is an immunoblotting test based on a Western blotting procedure for the detection of [PrP.sup.Sc] that uses the 6H4 mouse-derived monoclonal antibody (epitope, human residues 144-152; recognition sequence, DYEDRYYRE; Table 1 and Fig. 2). The test procedure can be summarized briefly as follows: A piece of brain stem or cervical spinal cord (typically 0.5 g of tissue) is homogenized in a plastic, single-use container. The homogenate is then digested with proteinase K, boiled in sodium dodecyl sulfate sample buffer, and loaded on a sodium dodecyl sulfate-polyacrylamide gel. After electrophoretic separation, proteins are transferred to a membrane, and [PrP.sup.Sc] is detected with the antibody coupled to a chemiluminescent marker. The minimum time to complete the test is 7-8 h.

Test C: Diagnostic by Enfer Scientific Ltd. (Protherics). The Enfer test is a novel, high-throughput chemiluminescent ELISA that can be completed in <4 h; it uses a polyclonal anti-PrP antibody (no specific information regarding the antibody was provided) for detection. The test itself includes a rapid sample extraction procedure, coupled to a simple ELISA technique. For detection, a polyclonal primary antibody and a horseradish peroxidase-conjugated secondary antibody are used with an enhanced chemiluminescent reagent.

Test D: Diagnostic by CEA-Bio-Rad. The CEA-Bio-Rad test, originally developed by the Commissariat a l'Energie Atomique, is a sandwich immunoassay for [PrP.sup.Sc] carried out after denaturation and concentration steps. Two monoclonal antibodies are used (details regarding the antibodies are not available). In its current state of development, the test takes <24 h to complete; this could be reduced with automation.

Summary of test results. Each of the previous assays was tested under double-blind conditions in four categories: specificity, sensitivity, limits of detection, and reproducibility. Results reported to the European Commission are summarized in Table 2. The Bio-Rad product (Test D) performed well. Subsequent studies using a commercial form of the Bio-Rad assay have been conducted with similar results (49). As stated previously, these tests are used for deceased cattle. The European Commission has started evaluating another set of "rapid" postmortem diagnostics, with field trials imminent (50). No commercially available human test exists at this time.


Progress has been made recently in detecting prion protein from peripheral tissues as well as increasing the sensitivity of the current assays. In addition, several studies have reported progress in methods to increase the concentration of prion protein to enhance sensitivity. We report on four of these areas below. Detection of prion protein in blood. RNA aptamer technology has been proposed as a diagnostic tool (51) and as a method to disinfect donated blood (52,53). Patent applications have been filed for this technology (52,54) with at least one company, V.I. Technologies (Watertown, NIA), solidly promoting their blood disinfectant abilities (53). In the study by Weiss et al. (51), two RNA aptamers highly specific for [PrP.sup.C] were generated. RNA oligomers that recognized [PrP.sup.C] contained four sets of three-guanine base repeats that formed a "G quartet scaffold". The RNA binding site mapped to the N[H.sub.2], terminus of [PrP.sup.C] (residues 23-52). In studies with scrapie-infected brain homogenates, only [PrP.sup.C] was detected by the RNA aptamers. Although the Weiss group has filed a patent application for a blood-based diagnostic (53), no further studies have been published addressing this use. V.I. Technologies use proprietary nucleotide aptamers licensed from William James at Oxford University in their blood disinfectant protocol. Like the Weiss study, the aptamers in the V.I. protocol have an affinity for [PrP.sup.C]; however, the V.I. protocol does suggest that aptamer-dependent clearance of [PrP.sup.Sc] is possible, although few data have been published to support this assertion (53).

Schmerr et al. (55) are also working on a prion diagnostic protocol that uses readily accessible blood samples. The method, which uses capillary electrophoresis and fluorescently labeled markers, has detected very small amounts of [PrP.sup.Sc] in blood from sheep and elk. A peptide consisting of residues 218-232 was labeled with fluorescein at its N[H.sub.2] terminus during synthesis. Antibodies to this peptide were raised in rabbits and isolated by affinity chromatography. These antibodies also reacted against scrapie-infected brain samples. In control studies, capillary electrophoresis led to two fluorescence peaks, one for the free peptide and another for the peptide-antibodyprion complex. A positive result for scrapie is indicated by a dramatic reduction in the free peptide peak and a corresponding increase in the immunocomplex peak. Infected and healthy sheep and elk were tested at several time intervals. In one case, an elk in the wild tested negative in the first screen and then was found to be positive for CWD several months later, before the onset of clinical symptoms.

The sensitivity of this diagnostic technique was tremendous; only 50 [micro]mol/L ([10.sup.-18] mol/L) fluorescent marker was used. However, the extraction of prion protein from the blood samples was not detailed for "proprietary" reasons. [PrP.sup.Sc] concentration enhancement is directly linked to the extraction procedure; therefore, it is not clear what effect the extraction step had on the detection of the peptide-antibody-prion complex peak. Specificity was not addressed explicitly in this study, nor were immunocomplex affinity data reported. Because the detection of scrapie consists of peak differences, nonspecific binding may contribute to the results. Double-blind studies using this method have not been reported. Another confounding element is the use of fetal bovine serum in the assay, which could be contaminated. This method is promising because it uses only very small quantities of material and each assay can be completed in several minutes. However, a recent report by Brown et al. (56) describes their difficulties in reproducing the results of Schmerr et al. (55)

Detection of prion protein in urine. The first potential prion diagnostic to test urine samples was described recently by Shaked et al. (57). The protocol relies on the prion protein being small enough to be filtered by the kidneys, such that it is concentrated several hundred times over its concentration in blood. Dialysis was also performed during purification, which appeared to increase sensitivity, although rigorous testing by serial dilutions was not reported. Although not double-blind, the protocol did show some specificity and all controls were correctly identified. A larger, double-blind study that includes serial dilutions is needed to address specificity, sensitivity, and reproducibility. The purification portion of the protocol was not dependent on prion strain per se, but the quantification depended on the conformation of the prion protein through use of the 3F4 murine-derived hamster antibody (epitope, hamster sequence residues 109-112; recognition sequence, MKHM; Fig. 2 and Table 1) and 6H4 (epitope, human sequence residues 144-152; recognition sequence, DYEDRYYRE; Fig. 2 and Table 1). This diagnostic exploits the fact that the binding epitope is the same in the hamster and human proteins. Infection of Syrian hamsters with enriched urine prion protein (denoted by the authors as U[PrP.sup.Sc]) appeared to alter the time course of infection. Prion protein was observed in urine from animals near day 60. Additionally, one apparently healthy U[PrP.sup.Sc]-infected hamster was sacrificed at 120 days after infection, and [PrP.sup.Sc] was found in the brain of this animal. However, no fatalities had occurred at the time of publication (>270 days after infection). The apparent lack of clinical symptoms in [PrP.sup.Sc]-positive hamsters was thought to be the result of a subclinical prion infection. No double-blind studies were reported.

Enrichment of prion protein in biological samples. Because it is theoretically possible that a small number of [PrP.sup.Sc] molecules can cause infection, highly sensitive assays are necessary. In this regard, Saborio et al. (58) recently developed a PCR-like prion-protein amplification method that might serve as a front end to the various tests described above. In this study, potentially infectious brain homogenate was mixed with fresh, prion-free brain matter, and the conversion process was allowed to proceed, as originally pioneered by Caughey's group (16). Because the conversion tends to be inefficient and the newly converted protein is often hidden in the background of the assay, Saborio et al. (58) introduced a cyclical protocol involving incubation and sonication. After the initial incubation, the sample is sonicated to break up the newly formed prion aggregate and more normal brain matter is added. Five cycles of incubation/ sonication produced 97% conversion. The authors addressed sensitivity by serial dilution studies. Although the evidence is impressive, with [PrP.sup.Sc] detection at >10 000-fold dilution, it is somewhat hampered by low-resolution immunoblotting. Other than controls, double-blind studies were not reported. The throughput is limited by the time of each cycle, currently reported to be 1 h. However, the trade-off in cycle time may be balanced by increased sensitivity. The protocol, as published, appears to be independent of prion strain, but the authors suggest that this protocol may not work when the samples to be amplified are not from brain tissue. Additionally, they note that a catalyst in brain homogenate may be required for proper [PrP.sup.Sc] amplification.

Detection of prion disease by altered gene expression. It is natural to assume that most diagnostics for prion diseases would use methods to detect [PrP.sup.Sc]. However, other organs and tissues may be affected during TSE infection before the onset of clinical symptoms. For example, the lymphoreticular system, and the spleen in particular, has been implicated in the pathology of TSEs (59). Consequently, Miele et al. (59) investigated altered gene expression in spleens from prion-infected mice by differential-display reverse transcription-PCR. In so doing, they identified a 0.5-kb gene corresponding to the erythroid differentiation-related factor (EDRF) from 10 000 transcripts. EDRF expression was reduced in prion-infected mice compared with control animals. EDRF is expressed in spleen, bone marrow, and blood in several lines of mice (59). However, EDRF is expressed only in blood and bone marrow in healthy sheep, cattle, and humans. EDRF expression decreases 20% by 40 days postinfection in mice, and it is <10% at the final stage of the disease (162 days postinfection). Studies with TSE-infected sheep and cattle also show a decrease in EDRF expression in both blood and bone marrow. The authors demonstrated that erythroid cells are solely responsible for the expression of EDRF, but it is not clear why infection causes a decrease in the expression of this protein.

In light of this method being a potential diagnostic tool, several issues should be addressed. One issue is that sensitivity was shown qualitatively by comparing EDRF in TSE-infected animals and controls. Although a substantial decrease in EDRF expression was seen 40 days postinfection, clinical symptoms seemed to be present at this time point as well. More precise data are needed earlier in the course of the disease. Time course data were not presented for other animals or for humans, which have longer incubation periods. Application of this method as a diagnostic tool depends on observing down-regulation of EDRF expression before the onset of clinical symptoms. Nonetheless, a nice feature of this protocol is that it could potentially circumvent the problems associated with multiple conformations of [PrP.sup.Sc] and the possible need for [PrP.sup.Sc] conformation-specific probes.


As research progresses on the cause, transmission, and detection of TSEs, studies focusing on treatment and prevention are appearing in greater numbers. Promising small-molecule-based treatments have been described in the last several years, primarily from the research groups of Caughey and Prusiner. As early as 1992, Congo red and various sulfonated glycans were shown to inhibit formation of [PrP.sup.Sc] (60, 61). Protection was obtained if these and other, more toxic drug classes [polyene antibiotics and anthracycline (62)] were administered very near to the actual time of infection. Caughey's group (62, 63) also examined other, less toxic, actively transported compounds containing derivatized porphyrins and phthalocyanines, with ICso values in the range of 0.5-1 [micro]mol/L.

In addition, Prusiner's group (64) have explored the use of branched polyamines for the treatment of TSEs with some success, as well as the synthesis of molecules to mimic [PrP.sup.C] regions containing mutations that prevent the proposed binding of protein X (65). Caughey's group described lysosomotropic and cysteine protease inhibitor compounds that inhibit [PrP.sup.Sc] accumulation in cell cultures (66). CJD therapy using flupirtine, which blocks apoptosis induced by [PrP.sup.Sc], has been reported (67, 68). More recently, Korth et al. (69) have shown that two FDA-approved drugs, quinacrine and chlorpromazine, inhibit [PrP.sup.Sc] formation in cellular assays. At least one study has linked the protective effects of quinacrine to inhibition of 5-lipooxygenase and reduction of neurotoxicity in cells inoculated with PrP106-126 (70). The main difference between these two compounds and other isosteric molecules is, for example, the rotational freedom of the aliphatic chain in chlorpromazine vs the fixed configuration of chlorprothixen. Consequently, further research into the role of the aliphatic chain in prohibiting prion conversion appears to be warranted. Given that these drugs are already approved and side-effect profiles are well documented, Korth et al. (69) advocate the use of these compounds in individuals currently suffering from prion diseases. In a preliminary trial with quinacrine at the University of California-San Francisco,

the progression of CJD was slowed, at least in one of two CJD-afflicted volunteers (71). Unfortunately, both patients subsequently died, but the disease had already progressed very far when treatment began. The death of one patient may have been linked to the high doses of quinacrine that were administered (72). Larger trials involving quinacrine are currently being pursued in the United Kingdom (71) and at the University of California-San Francisco. Korth et al. (69) also suggest that quinacrine should be used in animals to treat and possibly halt transmission in contaminated herds, thereby hopefully removing the need for massive herd culls to contain outbreaks of the disease.

Other possible therapeutic interventions targeting different pathways in prion infection have been reviewed (73). One promising postexposure treatment involves blockade of lymphotoxin [beta]-receptor (LTBR) in the lymphoreticular system. A recent study by Oldstone et al. (74) has reported on the lack of prion infection in LTBR knockout mice that were fed [PrP.sup.Sc]. Progression of prion infection occurred more rapidly in lymphotoxin a-receptor-null mice, and suppression of follicular dendritic cells and CD11c(+) did nothing to alter the kinetics of neuro-invasion of prions. The authors propose that LTBR blockade is a viable intervention and should be pursued (74).

Both the Caughey and Prusiner research groups are also pursuing peptide- and protein-based inhibition of [PrP.sup.C]-[PrP.sup.Sc] binding. Following up on their previous studies, Caughey's group has shown that peptide fragments from the C-terminal portion of the Syrian hamster [PrP.sup.C] protein inhibit [PrP.sup.Sc] binding and consequently conversion of endogenous [PrP.sup.C] to the protease-resistant form (25). Two peptides (residues 166-179 and 200-223) in particular show promise. Data from cell-free conversion assays indicate that complete inhibition of conversion is obtained at a peptide concentration of 50 [micro]mol/L, with ICSO values of 10-15 [micro]mol/L. Random mutations to these peptides severely reduce their ability to inhibit conversion. Inhibition occurs when the peptides adopt [beta]-sheet structure. The sensitivity of this method was addressed in several experiments involving many peptides. Encouragingly, their data show complete inhibition of [PrP.sup.C][right arrow][PrP.sup.Sc] conversion for several of the peptides.

One of the latest advances in the possible treatment of prion diseases with protein-based molecules involves antibody-based inhibition of prion propagation with clearance of infectivity (75). Several antibodies that bind to a variety of epitopes on [PrP.sup.C] were investigated, and two in particular, D13 and D18, displayed dose-dependent clearance of [PrP.sup.Sc] with [IC.sub.50] values of 12 and 9 nmol/L, respectively (32, 75-77). Experiments were also conducted to determine whether infectious prion protein reappeared when the antibodies were removed. The D18 antibody (Fig. 2 and Table 1) effectively prevented reemergence of [PrP.sup.Sc] when cells were first grown in antibody culture for at least 2 weeks before the antibody was removed from the medium. The antibody concentration used in these studies was 10 mg/L, and the [IC.sub.50] for D18 was 0.45 mg/L. The authors propose that the antibody binds to the nascent prion protein before the binding of template [PrP.sup.Sc] or other unknown factors. Animal studies were also conducted in which mice were inoculated with mouse ScN2a cells treated with various antibodies. Those animals that received D18 (binds residues 132-156), D13 (binds residues 95-104), or R2 (binds residues 225-231 and 29-37) antibodies were free from disease after 265 days (75). Because the antibodies bind to [PrP.sup.C], the strain issue should not be a concern; however, antibody-related loss of native [PrP.sup.C] function may be an issue if long-term therapy is necessary.


Protein motion can have profound effects on protein-protein interactions, specifically antibody-epitope recognition. For the prion protein, the same amino acid sequence can produce different folded structures, which can hide or create new epitopes. This phenomenon contributes to the already difficult and critically important task of detecting prions in TSEs such as BSE and CJD.

Fortunately, progress has been made in the last 2 years in the field of TSE detection in animals. Current assays provide fairly sensitive and specific results in <24 h (42), which represents a substantial improvement over the previous biological assays (>260 days). However, major limiting factors to these commercial diagnostics include dependence on brain samples from deceased animals as well as issues of sensitivity and specificity. Furthermore, we still lack a commercial test for TSEs in humans.

Several promising technologies have recently been reported that deal with improved TSE detection in animals and proposed diagnostics for humans. Tissue sample accessibility is very important for a mainstream diagnostic tool, and in this regard, the blood and urine assays hold great promise. Sensitivity is crucial for catching presymptomatic cases of TSE disease; methods for amplification and enrichment of infectious prion protein may be very useful to achieve this goal. Advances in analytical methods will also contribute greatly to development of sensitive diagnostics. In addition, there are several promising therapeutic avenues being explored that involve small molecules, peptides, and proteins.

We thank Darwin Alonso for critical reading of the text and contributions to the figures. This work was supported by the National Institutes of Health (Grant GM 50789 to V.D.) and a National Institute of General Medical Sciences National Research Service Award (Grant GM-07750 to B.J.B.).


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Department of Medicinal Chemistry, Box 357610, University of Washington, Seattle, WA 98195-7610.

* Author for correspondence. E-mail Received May 9, 2002; accepted September 19, 2002.

[1] Nonstandard abbreviations: TSE, transmissible spongiform encephalopathy; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; [PrP.sup.C], cellular prion protein; [PrP.sup.Sc], scrapie prion protein; GPI, glycosylphosphatidylinositol; FTIR, Fourier transform infrared; NMR, nuclear magnetic resonance; FDA, Food and Drug Administration; EDRF, erythroid differentiation-related factor; and LTBR, lymphotoxin [beta]-receptor.
Table 1. Antibody detection of PrP: Epitopes, conformation
recognized, and current or proposed applications. (a)

Antibody Epitope, Conformation Diagnostic Therapeutic
 residues recognized agent agent

3F4 (b) 109-112 [PrP.sup.C]
D13 (b) 95-103 [PrP.sup.C] X (c)
6H4 (d) 144-152 [PrP.sup.C] + X (e)
D18 (f) 132-156 [PrP.sup.C] X (c)

(a) Current information about diagnostics and therapeutics development
can be found at

(b) Data from Peretz et al. (32). Also note that both 3F4 and D13 can
recognize denatured [PrP.sup.Sc].

(c) Data from Peretz et al. (75).

(d) Data from Korth et al. (78).

(e) "Prionics Check" by Prionics AG (

(f) Data from Williamson et al. (76).

Table 2. Test results of PrP diagnostic tests from the
European Commission. (a)

 Test A Test B (b) Test C (c) Test D (d)

Sensitivity, % 70 100 100 100
Specificity, % 90 100 100 100
 [10.sup.3.1] 6/6 6/6 6/6 6/6
 [10.sup.-1] 0/20 15/20 20/20 20/20
 [10.sup.-1.5] 0/20 20/20 20/20
 [10.sup.-2.0] 0/20 20/20
 [10.sup.-2.5] 18/20
 [10.sup.-3.0] 1/20
 [10.sup.-3.5] 0/20

(a) Tests B, C, and D correctly identified all duplicate samples. No
analysis of the relationship between test quantification and tissue
location was reported. Data from Valleron et al. (42).



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Date:Dec 1, 2002
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