Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disorder originating from exposure to bovine-spongiform-encephalopathy-like prions. Prion infections are associated with long and clinically silent incubations. The number of asymptomatic individuals with vCJD prion infection is unknown, posing risk to others via blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. We aimed to establish the sensitivity and specificity of a blood-based assay for detection of vCJD prion infection. METHODS: We developed a solid-state binding matrix to capture and concentrate disease-associated prion proteins and coupled this method to direct immunodetection of surface-bound material. Quantitative assay sensitivity was assessed with a serial dilution series of 10⁻7 to 10⁻¹° of vCJD prion-infected brain homogenate into whole human blood, with a baseline control of normal human brain homogenate in whole blood (10⁻6). To establish the sensitivity and specificity of the assay for detection of endogenous vCJD, we analysed a masked panel of 190 whole blood samples from 21 patients with vCJD, 27 with sporadic CJD, 42 with other neurological diseases, and 100 normal controls. Samples were masked and numbered by individuals independent of the assay and analysis. Each sample was tested twice in independent assay runs; only samples that were reactive in both runs were scored as positive overall. FINDINGS: We were able to distinguish a 10⁻¹° dilution of exogenous vCJD prion-infected brain from a 10⁻6 dilution of normal brain (mean chemiluminescent signal, 1·3×105 [SD 1·1×104] for vCJD vs 9·9×104 [4·5×10³] for normal brain; p<0·0001)­an assay sensitivity that was orders of magnitude higher than any previously reported. 15 samples in the masked panel were scored as positive. All 15 samples were from patients with vCJD, showing an assay sensitivity for vCJD of 71·4% (95% CI 47·8­88·7) and a specificity of 100% (95% CIs between 97·8% and 100%). INTERPRETATION: These initial studies provide a prototype blood test for diagnosis of vCJD in symptomatic individuals, which could allow development of large-scale screening tests for asymptomatic vCJD prion infection. FUNDING: UK Medical Research Council.

Uptake and transport of foreign particles in Peyer's patches of both distal ileum and jejunum of calves.

To investigate the uptake and transport patterns of variously sized particles in Peyer's patches (PPs) of calves, intestinal loops were created in four newborn and two 2-month-old calves, and the loops were inoculated with various particles, including carbon black, fluorescein isothiocyanate (FITC)-labeled latex, FITC-labeled dextran, bovine serum, and recombinant mouse prion protein (rMPrP). The intestinal loops were recovered at 3, 6, 9, and 24 h in newborn calves and at 24 h in 2-month-old calves after inoculation, and the transport of the particles was examined by histological and immunohistochemical means. The uptake of the particles was quantified by estimation of signal intensities. A greater intensity was found in newborn calves compared with the 2-month-old calves. The peak uptake of carbon black, FITC-labeled latex, and rMPrP in the PPs of the distal ileum occurred at 6 h after inoculation in newborn calves and then progressively decreased with time. Uptake was also dependent on the site within the small intestine and the size of the particle studied. The transport of carbon black, FITC-labeled latex, and FITC-labeled dextran occurred via the bloodstream, the mesenteric lymph nodes, and the liver of newborn calves. rMPrP was found primarily in the interfollicular regions of the submucosa of the distal ileum of newborn calves. Thus, distal ileal PPs are probably more effective at particle absorption than the jejunal PPs, and the peak uptake of the PPs within the newborn calf occurs at 6 h after inoculation.

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry.

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP(C)) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP binding.

Mucosal immunization with an attenuated Salmonella vaccine partially protects white-tailed deer from chronic wasting disease.

Prion disease is a unique category of illness, affecting both animals and humans, in which the underlying pathogenesis is related to a conformational change of a normal, self-protein called PrP(C) (C for cellular) to a pathological and infectious conformer known as PrP(Sc) (Sc for scrapie). Bovine spongiform encephalopathy (BSE), a prion disease believed to have arisen from feeding cattle with prion contaminated meat and bone meal products, crossed the species barrier to infect humans. Chronic wasting disease (CWD) infects large numbers of deer and elk, with the potential to infect humans. Currently no prionosis has an effective treatment. Previously, we have demonstrated we could prevent transmission of prions in a proportion of susceptible mice with a mucosal vaccine. In the current study, white-tailed deer were orally inoculated with attenuated Salmonella expressing PrP, while control deer were orally inoculated with vehicle attenuated Salmonella. Once a mucosal response was established, the vaccinated animals were boosted orally and locally by application of polymerized recombinant PrP onto the tonsils and rectal mucosa. The vaccinated and control animals were then challenged orally with CWD-infected brain homogenate. Three years post CWD oral challenge all control deer developed clinical CWD (median survival 602 days), while among the vaccinated there was a significant prolongation of the incubation period (median survival 909 days; p=0.012 by Weibull regression analysis) and one deer has remained CWD free both clinically and by RAMALT and tonsil biopsies. This negative vaccinate has the highest titers of IgA in saliva and systemic IgG against PrP. Western blots showed that immunoglobulins from this vaccinate react to PrP(CWD). We document the first partially successful vaccination for a prion disease in a species naturally at risk.

Oriented immobilization of prion protein demonstrated via precise interfacial nanostructure measurements.

Nanopatterning of biomolecules on functionalized surfaces offers an excellent route for ultrasensitive protein immobilization, for interaction measurements, and for the fabrication of devices such as protein nanoarrays. An improved understanding of the physics and chemistry underlying the device properties and the recognition process is necessary for performance optimization. This is especially important for the recognition and immobilization of intrinsically disordered proteins (IDPs), like the prion protein (PrP), a partial IDP, whose folding and stability may be influenced by local environment and confinement. Atomic force microscopy allows for both highly controllable nanolithography and for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of protein interactions in a liquid environment, thus different environmental parameters affecting the biorecognition phenomenon can be investigated in situ. Using nanografting, a tip-induced lithographic technique, and an affinity immobilization strategy based on two different histidine tagged antibodies, with high nM affinity for two different regions of PrP, we successfully demonstrated the immobilization of recombinant mouse PrP onto nanostructured surfaces, in two different orientations. Clear discrimination of the two molecular orientations was shown by differential height (i.e., topographic) measurements, allowing for the estimation of binding parameters and the full characterization of the nanoscale biorecognition process. Our work opens the way to several high sensitivity diagnostic applications and, by controlling PrP orientation, allows for the investigation of unconventional interactions with partially folded proteins, and may serve as a platform for protein misfolding and refolding studies on PrP and other thermodynamically unstable, fibril forming, proteins.

Polylactide-coglycolide microspheres co-encapsulating recombinant tandem prion protein with CpG-oligonucleotide break self-tolerance to prion protein in wild-type mice and induce CD4 and CD8 T cell responses.

Prion diseases are fatal neurodegenerative diseases that are characterized by the conformational conversion of the normal, mainly alpha-helical cellular prion protein (PrP) into the abnormal beta-sheet-rich infectious isoform (PrP(Sc)). The immune system neither shows reaction against cellular PrP nor PrP(Sc), most likely due to profound self-tolerance. In previous studies, we were able to partly overcome self-tolerance using recombinantly expressed dimeric PrP (tandem PrP (tPrP)), in association with different adjuvants. Proof of principle for antiprion efficacy was obtained in vitro and in vivo. In this study, we demonstrate the induction of a specific Th1 T cell response in wild-type mice immunized with tPrP and CpG-oligonucleotide (ODN). Biochemical influences such as refolding conditions, ionic strength, pH, and interaction with CpG-ODN affected antigenic structure and thus improved immunogenicity. Furthermore, s.c. immunization with tPrP and CpG-ODN co-encapsulated in biodegradable polylactide-coglycolide microspheres (PLGA-MS) enhanced CD4 T cell responses and, more prominent, the induction of CD8 T cells. In this vaccination protocol, PLGA-MS function as endosomal delivery device of Ag plus CpG-ODN to macrophages and dendritic cells. In contrast, PLGA-MS-based DNA vaccination approaches with a tPrP construct generated poor humoral and T cell responses. Our data show that prophylactic and therapeutic immunization approaches against prion infections might be feasible using tPrP Ag and CpG-ODN adjuvant without detectable side effects.

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates.

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).