RT-QuIC Assays in Humans … and Animals.

Abstract

Prion diseases are neurodegenerative diseases affecting both humans and animal species. The phenotypic spectrum is broad and includes Creutzfeldt-Jakob disease (CJD) and its variant zoonotic form (vCJD) in humans, while in animals, scrapie of sheep and goats, bovine spongiform encephalopathy and chronic wasting disease of deer, elk and moose are naturally occurring forms. Transmission and pathogenesis appear causally linked to the misfolding of the normal form of the prion protein (PrP^C) into disease associated conformers (PrP^D), the latter enriched in β-strand secondary structure.

Over the past 10 years two protein amplification techniques, the protein misfolding cyclic amplification (PMCA) assay and real-time quaking induced conversion (RT-QuIC) assay have been developed and successfully deployed in prion biology across a range of scientific and clinical applications, including generation of de novo prions, quantitation of prion infectivity and ultra-sensitive detection of PrP^D. While PMCA utilises sonication to facilitate protein amplification, RT-QuIC employs vigorous shaking to achieve this outcome, with both techniques sharing the ability to amplify miniscule quantities of PrP^D seed present in various tissues and body fluids to levels detectable using routine biochemical methods.

The enhanced specificity of the RT-QuIC for detection of PrP^D in cerebrospinal fluid (CSF) has spawned international collaborations to rigorously assess and validate the assay for clinical diagnostic purposes. In parallel with collaborative CSF validation studies have been successful efforts to refine the RT-QuIC allowing its use for more accessible body fluids or tissues such as urine and nasal brushings, as well as promote higher sample throughput, shorten assay times and offer accurate quantification of PrP^D even at levels below those detectable by animal bioassays. Animal studies support the generic capacity of the RT-QuIC for PrP^D detection, underpinning the utility of this assay for studying prion disease and the high likelihood of inter-convertibility of technical refinements for human and animal use.

Introduction

Prion diseases are transmissible neurodegenerative diseases that occur in both humans and animal species. The phenotypic spectrum is broad and includes Creutzfeldt-Jakob disease (CJD) and its variant zoonotic form (vCJD) in humans, as well as Gerstmann-Sträussler-Scheinker syndrome (GSS) and kuru, while in animals, scrapie of sheep and goats, bovine spongiform encephalopathy (“mad cow” disease) and chronic wasting disease of deer, elk and moose are naturally occurring forms¹⁾. Creutzfeldt-Jakob disease (CJD) is the most common human prion disease phenotype, with most cases (85–90%) defined as sporadic (ie unknown aetiology)¹⁾. Transmission and pathogenesis appear causally linked to the misfolding of the normal form of the prion protein (PrP^C) into disease associated conformers (PrP^D)²⁾, the latter enriched in β-strand secondary structure and typically displaying limited solubility in non-ionic detergents and protease resistance³⁾. The centrality of PrP^C in disease occurrence is exemplified by the complete resistance to prion disease transmission of transgenic mice in which the gene encoding expression of this protein (prion protein gene; Prn-p) has been ablated⁴⁾.

Prion protein (PrP^C) is a glycoprotein comprised of a globular C-terminal region containing three alpha-helices and a less structured N-terminus, which can coordinate copper ions through histidine residues within a domain known as the octapeptide repeat region (residues 51–91 − human sequence)⁵⁾. PrP^C is attached to the external leaflet of the cell membrane through a C-terminal glycosylphosphatidylinositol anchor. N-linked glycosylation is possible at two asparagine residues (181 and 197) allowing mature PrP^C to be un-, mono- or di-glycosylated.

The precise composition of the infectious “prion” remains to be determined, although considerable evidence supports that PrP^D is the major, if not exclusive, component (the “protein-only” hypothesis)²⁾. Although mechanistic details are incomplete, conversion of PrP^C to PrP^D is believed to occur through a template-directed, auto-catalytic process, in which generation of a multimeric “nucleus” or “seed” is construed as a critical rate-limiting event, with secondary fragmentation of fibrils modelled as being a critical determinant of the kinetics of fibril growth⁶⁾.

Pre-mortem Prion Disease Diagnostics and The Advent of PrP^D amplification Techniques

The last approximately 20 years has witnessed considerable progress in the development of diagnostic investigations for human prion disease, advancing from electroencephalography⁷⁾ to the routine use of CSF biomarkers⁸⁾ and brain magnetic resonance imaging (MRI)⁹⁾, with both brain MRI and CSF biomarkers now incorporated into diagnostic criteria for sporadic CJD⁹⁾. Although very useful, an important limitation with these diagnostic investigations is their lack of specificity; hence, the considerable interest inspired when a novel technique known as “protein misfolding cyclic amplification (PMCA)”, able to specifically amplify miniscule amounts of PrP^D in biological specimens to detectable levels was reported¹⁰⁾. In addition to its diagnostic potential, the PMCA has enjoyed broad and meritorious application to prion biology, including the provision of further proof of the “protein-only” hypothesis of prion transmissibility being able to generate prions capable of causing typical disease in wild type hamsters, with considerable concordance between the seed PrP^D template and the PMCA reaction product when analysed by western blot after protease and PNGase digestion¹¹⁾.

At least partly relying on the kinetic importance of the secondary fragmentation of fibrils⁶⁾, PMCA centres around the repeated, brief sonication of small volumes of ex vivo samples containing PrP^D seeds immersed in a substantial excess of PrP^C substrate over extended periods (often 24 hours; known as a “round”) to achieve the template direct generation of PrP^D. Cuphorn sonicators are the usual platform equipment for the PMCA, with energy transfer to the tubes within the plate influenced by tube position, tube construction, bath volume and probe age.

Quaking Induced Conversion (QuIC) assay

Shortly after the advent of the PMCA an alternative protein amplification technique was reported in which vigorous shaking (or “quaking”) was employed (alternating with equally short periods of rest) to “energise” the misfolding of exogenous normal PrP^C through templating by an infinitesimal amount of PrP^D contained in biological samples with the new assay known as quaking induced conversion (QuIC)¹²⁾. Beyond just substituting shaking for sonication, the QuIC assay also utilised recombinant PrP^C (recPrP-sen) as the substrate source rather than the PrP^C contained in normal brain homogenate; however, similar to PMCA the earliest iterations of this technique relied on western blot detection of protease-resistant PrP products as the primary read-out¹²⁾. The efficiency of the QuIC amplification was found to be influenced by recPrP-sen concentration, reaction volume, reaction time, NaCl concentration, temperature of the reaction and shaking cycle parameters.

Presaging the future diagnostic utility of this technique, even in the original description of the QuIC assay the authors demonstrated their ability to amplify PrP^D contained in the CSF of hamsters symptomatic from 263 K prion infection¹²⁾. Shortly thereafter, the same group simultaneously reported the superior sensitivity of recombinant full-length hamster PrP (rec-haPrP) as substrate compared to recombinant full-length human PrP (rec-huPrP) for detection of PrP^D in vCJD brain homogenates, while also demonstrating the ability to detect PrP^D in the CSF of sheep manifesting scrapie¹³⁾. Using rec-haPrP, the authors reported their ability to reliably detect 1-10fg of PrP^D from a vCJD brain in a single 12 hour QuIC round.

Building on this success, a landmark publication followed soon after, reporting an important refinement to the assay in which Thioflavin T was added to the reaction to allow “real time” (RT) observation of the generation of nascent PrP^D from rec-huPrP, in parallel with demonstrating the ability of the assay across 238 patients (including 179 non-CJD) to reliably detect PrP^D in human CSF from people manifesting sporadic CJD of various molecular subtypes with >80% sensitivity and very importantly ~100% specificity¹⁴⁾; thereafter the assay has been known as RT-QuIC. Included in this report was a detailed description of method development, including utilization of various concentrations of rec-huPrP as substrate, as well as optimization in regards to guanidine HCl, NaCl concentrations and pH to avoid false positive results¹⁴⁾.

ThT is a benzothiazole dye that displays enhanced fluorescence and a characteristic red shift of its emission spectrum upon binding to peptides/proteins rich in β-strand secondary structure, including amyloids. The use of ThT streamlined the RT-QuIC assay obviating the need for western blot screening of reaction products for detection of PrP^D; however, while quite selective for β-strand secondary structures it is not perfectly sensitive and specific dependent to some extent on the experimental conditions and the particular protein/peptide of interest.

Consequently, the RT-QuIC assay can be simplistically envisaged as a 96-well plate format in which a small volume of CSF (potentially harbouring PrP^D seed) is combined with a buffer containing NaCl, recombinant PrP substrate and ThT with 3 or 4 replicate wells per patient specimen, with the plate located in a heating fluorimeter, which is repeatedly shaken for brief periods (eg 30–60 seconds) alternating with brief rest periods (eg 30–60 seconds) for many hours (10–96 hours), with fluorescence readings taken at regular intervals to monitor generation of nascent PrP^D.

The RT-QuIC as the “next generation” pre-mortem CSF Diagnostic Assay

Following the report by Atarashi and colleagues¹⁴⁾ other groups have reported similar success with the RT-QuIC assay as a pre-mortem diagnostic test using CSF specimens^15,16,17). In the study by McGuire and colleagues¹⁶⁾, a blinded retrospective analysis of CSF samples from 108 patients yielded 91% sensitivity and 98% specificity, while their prospective study of 118 patients delivered 87% sensitivity and 100% specificity, with rec-huPrP evincing greater tendency to spontaneous conversion to PrP^D in non-CJD cases than rec-haPrP. In the study of Cramm and colleagues¹⁵⁾, CSF from 110 prion disease patients (including 39 genetic CJD) and 400 non-CJD controls (including 200 prospective samples) were analysed, delivering an overall 85% sensitivity and 99% specificity. Finally, in the report from Orrú and co-workers¹⁷⁾, they describe significant refinements to the RT-QuIC assay, including use of truncated recombinant hamster PrP 90–231 (rec-thaPrP), addition of 0.002% SDS to CSF samples and an increased temperature from 42 °C to 55 °C, overall providing considerable shortening of the assay to 4–14 hours to observe a positive result. Despite achieving considerable shortening of the assay time, they were able to maintain a sensitivity of 95.8% and specificity of 100% across 87 CSF specimens.

International Collaborative Studies to Validate the CSF RT-QuIC for Clinical Diagnosis

As outlined, virtually every component of the RT-QuIC assay can vary across different laboratories, such as PrP substrate, CSF volumes for analyses, assay temperature, shaking cycles and assay duration. For example, substrate PrP may be full-length hamster, truncated hamster 90–231, full length human, full-length mouse, full-length bank vole or a hamster-sheep chimera. Another important difference across laboratories is what constitutes a “positive” reaction with some laboratories needing 2 or 3 wells with fluorescence above 10,000 relative fluorescence units (RFUs) while others require only 1 of 4 replicate wells to manifest fluorescence above an arbitrary level within a defined time period. Such methodological variations have instilled concerns that results may not be reproducible across different laboratories. To address this issue, international collaborations have formed to assess and validate the RT-QuIC as the potential “next generation” CSF diagnostic biomarker assay for human prion disease, especially sporadic CJD.

In the first collaborative assessment study reported¹⁵⁾ a “blinded” ring trial of 6 CSF samples was undertaken across four participant laboratories with the Fleiss’ kappa of 0.83 supporting very high agreement across the laboratories. Using 12 CJD and 6 control CSF samples, this study additionally assessed the potential influence of storage temperatures (room temperature and +4 °C for up to 8 days; −80 °C for up to 9 years) and repeated freeze-thawing on the RT-QuIC assay with no significant adverse effects. In contrast, this study found that lysis of spiked red blood cells (RBCs) could adversely affect the sensitivity of the RT-QuIC either through spontaneous lysis over time (5,000 RBCs /µl >3 days) or if >1250 RBCs/µl were immediately sonicated¹⁵⁾. In the most recent, larger collaborative study, 11 laboratories from 8 countries participated in a “blinded” ring trial of 15 CSF samples; there was perfect concordance between the laboratories for all CSF samples (McGuire et al. Ann Neurol − in press). This study is additionally remarkable for the fact that there was noteworthy variation in the RT-QuIC techniques employed across substrate (rec-haPrP, rec-huPrP or chimeric sheep-hamster PrP), fluorimeter (BMG OMEGA, BMG OPTIMA, TECAN), volume of CSF tested (5 µL, 15 µL, 20 µL or 30 µL) and shaking conditions (eg 750 rpm 30 sec shake-30 sec rest; 900 rpm 90 sec shake-30 rest). Hence, current understanding aligns with the belief that once individual laboratories rigorously optimise and validate their own RT-QuIC assay then there is a high likelihood of very good inter-laboratory agreement with diagnostic samples.

Emerging Uses of RT-QuIC

Based on studies reported to date, there are at least four areas of prion biology and prion disease diagnostics where RT-QuIC is establishing itself as a useful laboratory tool: human (and animal) clinical diagnosis; monitoring of treatment efficacy; distinguishing between prion strains; and as an alternative to animal bioassay for semi-quantitation of PrP^D load.

Illustrating the utility of RT-QuIC in human diagnostics is when prion disease presents in hitherto unrecognised ways, such as a report of a 73 year old man manifesting hyperekplexia to auditory stimulation in the absence of cognitive impairment¹⁸⁾. Despite negative or inconclusive findings with EEG, CSF 14-3-3 testing and MRI of the brain, RT-QuIC demonstrated a clear positive result, with autopsy eventually confirming CJD. Elaborating this diagnostic capacity for human prion disease and building on previous findings of the presence of substantial amounts of PrP^D in the nasal olfactory epithelium, Orrú and colleagues have described an exciting diagnostic development in which nasal brushings of the olfactory epithelium obtained through a relatively simple outpatient procedure can be used as “seeds” in the RT-QuIC, demonstrating positive results in 30 out of 31 CJD patients (including two E200 K patients) with none of 43 controls yielding positive reactions¹⁹⁾. Adding to the interest in this approach was the apparently enhanced sensitivity compared to CSF as a source of PrP^D seeds from the same patients, with CSF providing positive results in only 23/30 patients with overall lower levels of ThT fluorescence achieved and often taking twice as long to reach the positive threshold cut-off.

In a further illustration of the utility and adaptability of the RT-QuIC, a recent report describes using full-length recombinant mouse PrP (rec-moPrP) as substrate and urine as a source of PrP^D seeds to assess the response to treatment of the aggregation inhibitor anle138b in mice²⁰⁾. Both brain and urine from anle138b treated mice and controls were first treated with proteinase K (PK) before undergoing detergent extraction to generate “PrP^27-30 seed” pellets for RT-QuIC assay with prior PK treatment not affecting the sensitivity of the assay. Urine PrP^27–30 seed levels correlated with those in brain allowing urine to be employed as a surrogate biomarker source for monitoring treatment efficacy; the authors also found that RT-QuIC was up to 10⁶-fold more sensitive than western blot for detection of PrP^27–30 in brain. To allow more accurate estimations of the efficacy of their treatment, these authors developed a quantitative RT-QuIC (qRT-QuIC) analogous to the quantitative reverse transcription polymerase chain reaction (qPCR) for nucleic acid estimation. “Lag time”, which correlated with PrP^27–30 seed load, was the primary metric and was defined as the time to reach “threshold” (3-fold increase in RFU above baseline). Known serial dilutions of brain homogenate were used to generate a calibration curve that could then be used to semi-quantify “PrP^27-30 equivalents” in tissues/fluids with unknown seed loads, such as the urine of treated mice. Applying their qRT-QuIC, the authors were able to demonstrate that the urine of anle138b treated mice did not contain PrP^27–30 seed levels above threshold until 170 days after prion inoculation, while control mice demonstrated threshold levels at 120 days, confirming a substantial delay in treated mice.

Following-on with the theme of semi-quantitative applications of the RT-QuIC, Wilham and colleagues reported using the Spearman-Kärber approach to serial sample (5~10-fold) dilutions of brain homogenates to statistically estimate seeding dose that gives 50% positive reactions (SD₅₀) in 4–8 replicate reactions at a certain dilution as an alternative to formal, quantal, end-point bioassay for determining prion titres²¹⁾. To apply the Spearman-Kärber method a dilution series must have dilutions with 100% of the replicates positive and one with 0% positive replicates. They determined a reaction “positive” if the fluorescence was >200% of the average negative control fluorescence. Applying this approach across four archived stocks of previously bioassayed hamster 263 K brain homogenates, the authors were able to show reproducibility of each stock over three replicate experiments with overall average log SD₅₀ estimates ranging from 11.4–11.9 per g brain compared to the previous bioassay results of 9.8–10.7 LD₅₀ per g brain. Having validated this new technique, this study reported successful application for the quantitation of prion SD₅₀ titres in nasal secretions and CSF from prion infected hamsters. The report also demonstrated the likelihood that most, if not all, tissues and biological fluids most likely harbour “inhibitors” of the RT-QuIC assay and that very frequently concentrated tissue or fluid samples require dilution before the RT-QuIC assay appears to amplify PrP^D seeds. Finally, the study by Shi and colleagues, using Fourier Transform Infrared Spectroscopy (FTIR), confirmed that the RT-QuIC PK-resistant products were rich in β-strands with a very similar profile to the PK-resistant PrP^D seeds²⁰⁾.

In the final, recently described application of the RT-QuIC to be presented, recombinant full-length bank vole PrP (rec-bvPrP) was found to be a “universal” substrate, including for previous strains unable to be amplified by RT-QuIC and for differentiating certain animal and human prion strains²²⁾. Rec-bvPrP allowed RT-QuIC amplification of PrP^D seeds from 28 different prion strains derived from humans, cattle, sheep, cervids and rodents, as well as facilitated discrimination of classical and atypical L-type BSE, classical and atypical Nor98 type scrapie and sporadic and variant CJD. Comparing rec-bvPrP with rec-haPrP, rec-huPrP, rec-moPrP and recombinant hamster-sheep chimeric PrP, rec-bvPrP was able to support amplification in prion diseases previously unable to be amplified by RT-QuIC (using these other substrates) associated with predominantly small (6-14kDa) PrP^D fragments, such as those associated with GSS harbouring P102L, A117V, H187R and F198S mutations, as well as Nor98 atypical scrapie. Illustrating the capacity of rec-bvPrP to assist discrimination of prion strains, rec-bvPrP23-230 in the RT-QuIC supported propagation of both classical BSE and L-type atypical BSE seeds while rec-huPrP and rec-haPrP supported only L-type atypical BSE; rec-bvPrP supported both classical and atypical Nor98 scrapie while hamster-sheep chimeric recPrP only supported classical scrapie propagation at <10⁻⁴ brain homogenate dilutions. Additionally, rec-bvPrP as substrate in RT-QuIC reactions generated products with distinctive western blot banding patterns further assisting differentiation of classical and atypical L-type BSE, as well as classical versus atypical Nor98 type scrapie.

Acknowledgements

S Collins is supported in part by an NHMRC Practitioner Fellowship (#APP1105784). The Australian National Creuzfeldt-Jakob Disease Registry is funded by the Commonwealth Department of Health.

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