Prions are infectious pathogens that cause invariably fatal prion diseases (spongiform encephalopathies) of the central nervous system in humans and animals. Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that no nucleic acid is necessary to allow for the infectivity of a prion protein to proceed.
A major step in the study of prions and the diseases they cause was the discovery and purification of a protein designated prion protein [Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al. (1983) Cell 35:57-62]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP.sup.c is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrP.sup.c is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases results from the transformation of the normal form of prion protein (PrP.sup.c) into the abnormal form (PrP.sup.Sc). There is no detectable difference in the amino acid sequence of the two forms. However, PrP.sup.Sc when compared with PrP.sup.c has a conformation with higher .beta.-sheet and lower .alpha.-helix content [Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) J Biol Chem 268:20276-20284]. The presence of the abnormal PrP.sup.Sc form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrP.sup.Sc plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration [Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103-143]. The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172:21-38]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Streussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) N Engl J Med 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene [Hsiao and Prusiner (1990) Neurology 40:1820-1827; Goldfarb, Petersen et al. (1992) Science 258:806-808; Kitamoto and Tateishi (1994) Philos Trans R Soc Lond B 343:391-398]. However, the human prion diseases are also infectious; the first recognized example being kuru which is believed to spread in New Guinea highlands by ritualistic cannibalism. Another example of human-to-human transmission are cases of iatrogenic CJD, caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown, Preece et al. (1992) Lancet 340:24-27]. A newly perceived threat of human infection arises in the recent cases of variant CJD with the possible transmission of prions from BSE-infected cows. The seriousness of the health risk resulting from the lack of a direct prion assays in different body fluids, tissue samples or human- and animal-derived pharmaceuticals is exemplified below.
More than 75 young adults who were previously treated with (HGH) human growth hormone derived from human pituitaries have developed CJD [Koch, Berg et al. (1985) N Engl J Med 313:731-733; Buchanan, Preece et al. (1991) Br Med J 302:824-828; Fradkin, Schonberger et al. (1991) JAMA 265:880-884; Brown, Preece et al. (1992) Lancet 340:24-27]. Fortunately, recombinant HGH is now used, although the seemingly remote possibility has been raised that increased expression of wild-type PrP.sup.c stimulated by high HGH might induce prion disease [Lasmezas, Deslys et al. (1993) Biochem Biophys Res Commun 196:1163-1169]. The conclusion that the HGH prepared from pituitaries was contaminated with prions, is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH [Gibbs, Asher et al. (1993) N Engl J Med 328:358-359]. Because of the long incubation times associated with prion diseases it will not be possible to determine the full extent of iatrogenic CJD in thousand of people treated with HGH worldwide for decades. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotropin hormone [Cochius, Mack et al. (1990) Aust N Z J Med 20:592-593; Cochius, Hyman et al. (1992) J Neurol Neurosurg Psychiatry 55:1094-1095; Healy and Evans (1993) Br J Med 307:517-518] as well as at least 11 patients receiving dura mater grafts [Thadani, Penar et al. (1988) J Neurosurg 69:766-769; Nisbet, MacDonaldson et al. (1989) J Am Med Assoc 261:1118; Willison, Gale et al. (1991) J Neurol Neurosurg Psychiatry 54:940; Brown, Preece et al. (1992) Lancet 340:24-27]. These cases of iatrogenic CJD underscore the need to screen pharmaceuticals that might possibly be contaminated with prions.
Recently, two physicians in France were charged with involuntary manslaughter of a child who had been treated with growth hormones extracted from corpses. The child developed Creutzfeldt-Jakob Disease (see New Scientist, Jul. 31, 1993, page 4). According to the Pasteur Institute, since 1989 there have been 24 reported cases of CJD in young people who were treated with human growth hormone between 1983 and mid-1985. Fifteen of these children have died. It appears that hundreds of children in France have been treated with growth hormone extracted from dead bodies that were at risk for developing CJD (see New Scientist, Nov. 20, 1993, page 10).
Another major concern is the epidemic of BSE in Great Britain and additional cases in some other countries of European Community [Wilesmith (1996) Methods in Molecular Medicine: Prion Diseases: 155-173]. The epidemic spread in the early 80s was probably due to the recycling of prion-infected animals in the rendering process and the feeding of cattle with prion-contaminated protein supplement. The enormous economic cost of eradication of BSE, if ever completely possible [Anderson, Donnelly et al. (1996) Nature 382:779-788], is now outweighed by the discovery of new variant CJD in young people in Great Britain which was probably transmitted by consumption of BSE-contaminated beef [Collinge, Beck et al. (1996) Lancet 348:56; Collinge, Sidle et al. (1996) Nature 383:685-690; Will, Ironside et al. (1996) Lancet 347:921-925]. Because of the long incubation time of CJD, it is too early to estimate the true extent of threat to the general population in Great Britain and the rest of the Europe from the available epidemiology. The BSE epidemic in cows, the "new variant" CJD and all the cases of iatrogenic CJD in young people underscore the need for screening food sources and pharmaceuticals that might possibly be contaminated with prions.
The most sensitive method today to detect and measure prions is bioassay in transgenic animals overexpressing the cellular prion protein PrP.sup.c. The current prion titrations are performed in two steps: (1) the sample material is first injected into susceptible experimental animals to amplify prions and PrP.sup.Sc protein to detectable levels; (2) the clinically symptomatic animals are euthanized and the disease is verified by detecting disease-specific PrP.sup.Sc and pathology. Since the discovery of protease resistance of PrP.sup.Sc more than 15 years ago, the PrP.sup.Sc detection is exclusively based on protease treatment of brain samples with proteinase K; the residual C-terminal protease-resistant fragment PrP 27-30 is then detected in denatured form by polyclonal or monoclonal antibodies recognizing prion protein on Western blots. More recent modifications of the same principle are semiquantitative dot blots or qualitative histoblots [Serban, Taraboulos et al. (1990) Neurology 40:110-117; Taraboulos, Jendroska et al. (1992) Proc Natl Acad Sci USA 89:7620-7624].
Despite the dramatic shortening of incubation time of human prions in transgenic mice overexpressing chimeric or human PrP genes, in some cases to less than 120 days, the potential for broad and high flow-through application of such prion bioassays is still limited. One possibility further shortening the assay time is to increase the sensitivity of PrP.sup.Sc detection. This would shorten the necessary observation time, increase the flow-through and as a result, make assays less expensive and broadly applicable.
A system for detecting PrP.sup.Sc by enhancing immunoreactivity after denaturation is provided in Serban, et al., Neurology, Vol. 40, No. 1, Ja 1990. Sufficiently sensitive and specific direct assay for infectious PrP.sup.Sc in biological samples could potentially abolish the need for animal inoculations completely. Unfortunately, such does not appear to be possible with current PrP.sup.Sc assays--it is estimated that the current sensitivity limit of proteinase-K and Western blot-based PrP.sup.Sc detection is in a range of 1 .mu.g/ml which corresponds to 10.sup.4 -10.sup.5 prion infectious units. Additionally, the specificity of the traditional proteinase-K-based assays for PrP.sup.Sc is in question in light of recent findings of only relative or no proteinase-K resistance of undoubtedly infectious prion preparations [Hsiao, Groth et al. (1994) Proc Natl Acad Sci USA 91:9126-9130] Telling, et al. (1996) Genes & Dev.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly .beta.-sheet structured units, and serves as a transporter of hormone thyroxin. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) [Kelly (1996) Curr Opin Strut Biol 6(1):11-7]. The cause of amyloid formation in FAP are point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in bioptic material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear .beta.-sheet structured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxin or triiodophenol [Miroy, Lai et al. (I1996) Proc Natl Acad Sci USA 93(26):15051-6].
In view of the above points, there is clearly a need for a specific, high flow-through, and cost-effective assay for testing sample materials for the presence of infectious form of prion protein, PrP.sup.Sc, which is believed to be the cause of prion diseases, such as BSE, CJD and scrapie. The presented invention offers a method of improving sensitivity of a range of different assays.