UNIVERSITY OF CALGARY

Timing recombinant prion protein conversion as a measure of prion activity in

chronic wasting disease

by

John Geoffrey Gray

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN VETERINARY MEDICAL SCIENCES

CALGARY, ALBERTA

MAY, 2014

© John Geoffrey Gray 2014

ii

Abstract

Chronic Wasting Disease (CWD) is a fatal neurological disease affecting cervids caused

by prions. Infected cervids shed the CWD prion in bodily fluids and excrement,

contaminating the environment and creating an agricultural and ecological calamity.

Preclinical antemortem CWD testing method is demanded by CWD risk management

programs. In vitro PrP-conversion assays have been developed as potential tools for such

an approach with increasing sensitivity for prion detection. However, no method has been

routinely employed thus far. Timing recombinant-PrP conversion into amyloid fibrils,

seeded by elk CWD prion, as a diagnostic method is presented herein. The assay, termed

“RePLICA”, is at least as sensitive for detecting elk CWD in brain tissues as Tg(CerPrP-

M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay models. The assay performs

within a period of 35 hours, is consistently reproducible, and functions on elk brain and

tonsil tissues. There are indications RePLICA has the potential to titre CWD infectivity.

iii

Acknowledgements

First, I wish to collectively thank my supervisory committee: Dr. Markus Czub (my

academic supervisor), Dr. Stefanie Czub, Dr. Sabine Gilch, and Dr. Hans-Joachim

Wieden. Thank you for your insight and guidance throughout this project, and shaping

my future in science. I feel privileged to have had such an accolade-loaded committee put

their support behind me.

I need to specifically thank Stefanie and H-J, who have been mentors to me since 2007.

H-J, the mentorship and confidence I received in your lab as a young disciple is the

reason I choose pursue science. Stefanie, your trust and confidence in me is—beyond a

doubt—the reason for any of this work to come to fruition.

To the TSE unit (headed by Stefanie) at the Canadian Food Inspection Agency’s

Lethbridge Laboratory (Notably: Dr. Catherine Graham, Mr. Sandor Dudas, Ms. Renée

Clark, Ms. Keri Colwell, Ms. Tammy Pickles), your scientific and logistic support was

instrumental to the success of this project. Dr. Pam Gale (Director, CFIA Lethbridge

Laboratory), thank you…

To my closest friends, Brandon Hisey and Alexandra Cookson, you both run the best

B&B in YYC. Your generosity was indispensable. Pamela Lussier, your cheer made life

easy.

Finally, I would like to thank the Alberta Livestock and Meat Agency (ALMA), and the

Canadian Food Inspection Agency for financially supporting this work.

iv

Dedication

To the most important people in my life:

My parents, John A. and Susan

, and my sister, Kimmy

If there’s one page you should understand in this book

, it’s this one.

v

Table of Contents

Abstract ............................................................................................................................... ii

Acknowledgements ............................................................................................................ iii

Dedication .......................................................................................................................... iv

Table of Contents .................................................................................................................v

List of Tables ................................................................................................................... viii

List of Figures and Illustrations ......................................................................................... ix

List of Symbols, Abbreviations and Nomenclature ........................................................... xi

Epigraph ........................................................................................................................... xiv

LITERATURE REVIEW .......................................................................1 CHAPTER ONE:

1.1 Prions: Disease History and Discovery .....................................................................1

1.2 Prions: Propagation, Pathogensis, and Disease Concept ...........................................4

1.3 Prions: Significance ...................................................................................................5

1.4 Chronic Wasting Disease (CWD) ..............................................................................7

1.4.1 CWD Epizoology ..............................................................................................7

1.4.2 CWD Pathology .................................................................................................9

1.4.3 CWD Genetic Susceptibility ...........................................................................10

1.4.4 CWD Detection and Diagnostic Testing Methods ..........................................10

1.5 Cellular PrP (PrPc) ...................................................................................................13

1.6 In Vitro Recombinant PrPc Conversion Methods ....................................................15

1.6.1 Autocatalytic Conversion ................................................................................16

1.6.2 Amyloid Seeding Assay (ASA) .......................................................................17

1.6.3 Real-Time Quaking Induced Conversion (RT-QuIC) .....................................18

1.7 Research Foci and Objectives ..................................................................................21

1.8 Hypothesis ...............................................................................................................23

GENERAL METHODS AND MATERIALS.....................................24 CHAPTER TWO:

2.1 Tissue Solubilization and In Vitro Conversion Detergent System ..........................24

2.2 Tissue Preparation and Homogenization .................................................................26

2.3 Preparation of Recombinant PrPc (rPrP

c) ................................................................27

2.3.1 Molecular Biology and Vector Construction ..................................................27

vi

2.3.2 Escherichia coli (Rosetta™ II DE3) Transformations ....................................29

2.3.3 rPrPc Overexpression and Purification ............................................................30

2.3.4 Inclusion Body Isolation ..................................................................................30

2.3.5 rPrPc Purification .............................................................................................31

2.4 In Vitro Conversion Assay Setup ............................................................................33

2.4.1 Solutions and Reaction Mixtures .....................................................................33

2.4.2 Seeding rPrP-conversion .................................................................................35

2.4.3 In Vitro rPrP-Conversion Conditions ..............................................................36

2.5 Data Interpretation and Statistical Analysis .............................................................36

2.5.1 Defining rPrP-conversion Time ......................................................................36

2.5.2 Diagnostic Criteria: ROC curves .....................................................................37

OPTIMIZATION OF THE REPLICA CONVERSION CHAPTER THREE:

BUFFER SYSTEM ...................................................................................................40

3.1 Introduction ..............................................................................................................40

3.2 Methods and Materials .............................................................................................41

3.3 Results and Discussion ............................................................................................42

3.3.1 Brain Tissue Optimizations .............................................................................43

3.3.3 Lymphoid Tissue Optimization .......................................................................50

PERFORMANCE OF THE REPLICA CONVERSION SYSTEM CHAPTER FOUR:

ON A MULTIANIMAL ELK PANELS. .................................................................61

4.1 Introduction ..............................................................................................................61

4.2 Methods and Materials. ............................................................................................61

4.3 Results and Discussion ............................................................................................63

4.3.1 Multianimal Brain Panel ..................................................................................63

4.3.2 Multianimal Tonsil Panel ................................................................................69

EVALUATING THE SENSITIVITY OF THE REPLICA CHAPTER FIVE:

CONVERSION SYSTEM VERSUS BIOASSAY ...................................................74

5.1 Introduction ..............................................................................................................74

5.2 Methods and Materials .............................................................................................75

vii

5.3 Results and Discussion ............................................................................................78

5.3.1 Sensitivity of RePLICA versus Bioassay ........................................................78

GENERAL CONCLUSIONS ................................................................86 CHAPTER SIX:

REFERENCES ..................................................................................................................88

viii

List of Tables

Table 1. Mean conversion times for RePLICA reactions seeded by dilutions of

CWD+ elk cerebral cortex. ....................................................................................... 45

Table 2. Mean conversion times for RePLICA reactions seeded by dilutions of

CWD+ elk ileocecal lymph node. ............................................................................. 52

ix

List of Figures and Illustrations

Figure 1. RePLICA ThT fluorescence curves generated by CWD+ and CWD- elk

cerebral cortex seed-homogenates. ........................................................................... 43

Figure 2. The RePLICA CT-plot for CWD+ Rxns and CWD- Rxns shown in Figure 1

for elk cerebral cortex seed-homogenates. ................................................................ 44

Figure 3. RePLICA’s change in sensitivity versus specificity as the assay duration

progresses for the elk cerebral cortex seed-homogenate dilution-sets in Figure 2. .. 46

Figure 4. RePLICA ThT fluorescence curves generated by CWD+ and CWD- elk

ileocecal lymph node seed-homogenates. ................................................................. 50

Figure 5. The ReLICA CT-plot for CWD+ Rxns and CWD- Rxns shown in Figure 4

for elk ileocecal lymph node seed-homogenates. ..................................................... 51

Figure 6. RePLICA’s change in sensitivity versus specificity as the assay duration

progresses for the elk ileocecal lymph node dilution-sets in Figure 5. ..................... 53

Figure 7. The effect of dilution on CWD+ and CWD- Rxns’ kinetic profiles and

conversion time, seeded by ileocecal lymph node, in the RePLICA buffer

system. ...................................................................................................................... 54

Figure 8. A RePLICA CT-plot of elk cerebral cortex seed-homogenates from multiple

animals. ..................................................................................................................... 64

Figure 9. A RePLICA CT-plot depicting how sensitivity and specificity can be

interpreted in the cut-off time zone as the assay’s duration progresses. ................... 65

Figure 10. A RePLICA CT-plot of elk tonsil seed-homogenates from multiple

animals. ..................................................................................................................... 70

x

Figure 11. The RePLICA CT-plot for elk CWD+ Rxns seeded by a Tg(CerPrP-

M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay titred CWD+ brain

homogenate. .............................................................................................................. 79

Figure 12. A dose-response curve of the CWD+ Rxns seeded by dilutions of a

Tg(CerPrP-M132)1536+/-

and Tg(CerPrP-E226)5037+/-

titred CWD+ elk brain

homogenate. .............................................................................................................. 80

xi

List of Symbols, Abbreviations and Nomenclature

%(v/v) Percent volume per unit volume

%(w/v) Percent weight per unit volume

132LL Elk PRNP polymorphism, homozygous for leucine

at PrPc amino acid position 132

132ML Elk PRNP polymorphism, heterozygous for

methionine and leucine at PrPc amino acid position

132.

132MM Elk PRNP polymorphism, homozygous for

methionine at PrPc amino acid position 132.

225SS Deer PRNP polymorphism: homozygous for serine

at PrPc position 225.

225SF Deer PRNP polymorphism: heterozygous for serine

and phenylalanine at PrPc position 225.

ANOVA Analysis of Variance

ASA Amyloid Seeding Assay

BSE Bovine spongiform encephalopathy

CHAPS 3-[(3-Cholamidopropyl) dimethylammonio]-1-

propanesulfonate

CJD Creutzfeldt-Jakob Disease

CNS Central Nervous System

CSF Cerebral Spinal Fluid

Cu2+

Copper (II)

CWD Chronic Wasting Disease

CWD- Uninfected with CWD

CWD+ Infected with CWD

CWD- Rxns Seeded fibrilizing of rPrPc not attributed to CWD+

seeds.

CWD+ Rxns Seeded fibrilizing of rPrPc attributed to CWD+

prion seeding activity.

DNA Deoxyribonucleic acid

DRM Detergent resistant membrane

EC50 Half-maximum effective concentration (dose-

response)

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme Linked Immunosorbant Assay

ER Endoplasmic Reticulum

FAE Follicle-Associated Epithelium

xii

fCJD Familial Creutzfeldt-Jakob Disease

FDC Follicular-Dendritic Cells

FFI Fatal Familial Insomnia

fg Femtogram

G G-force (relative centrifugal force)

GPI-anchor Glycophosphatidylinositol anchor

GSS Gerstmann–Sträussler–Scheinker syndrome

GuHCl Guanidinium hydrochloride

Ha90-231 N-terminally truncated Hamster PrP

HE Hematoxylin and Eosin stain

IHC Immunohistochemistry

kDa kilodaltons

KH2PO4 Potassium phosphate monobasic

LD50 Half-lethal dose (bioassay)

LR Likelihood Ratio

M cell Microfold cell

mAb Monoclonal antibody

MBM Meat and Bone Meal

Milli-Q H2O Ultra-pure water

mL Millilitre

Mo89-231 N-terminally truncated Mouse PrP

MWCO Molecular Weight Cut-Off value

Na2HPO4 Sodium phosphate dibasic

NaCl Sodium chloride

ng Nanogram

nm Nanometer

NaN3 Sodium azide

NDSB Non-Detergent Sulfobetaine

NDSB-201 Non-Detergent Sulfobetaine "201"; (3-(1-Pyridinio)

-1-propanesulfonate)

Ni2+

-NTA Nickle-charged Nitrilotriacetic acid resin

OD600 Optical Density (absorbance 600nm)

Partial-ETA Effect-size (statistics)

PBS Phosphate Buffered Saline

PBSk Phosphate Buffered Saline (Na+ and K

+)

pET41b Bacterial expression vector

PK Proteinase K

Prion Proteinacious infectious particle

PRNP Gene coding for PrPc

xiii

PRNP-/-

A PRNP gene knock-out

PrP Prion-related Protein (general terms)

PrPc Cellular PrP (non-diseased)

PrPCWD

PrPd of CWD

PrPd Disease-associate Prion Protein (infectious)

PrPsc

PrPd of Scrapie

PTA Phosphotungstic Acid

RAMALT Rectoanal Mucosa-Associated Lymphoid Tissue

RePLICA Recombinant PrP Latent Infectivity Conversion

Assay

RFU Relative Fluorescence Units

RNA Ribonucleic Acid

ROC curve Receiver Operating Characteristic curve

RPLN Retropharyngeal Lymph Node

rpm Revolution Per Minute

rPrPc Recombinant PrPc (bacterially expressed)

rPrP-conversion Process of rPrPc conversion into fibrils/amyloid

RT-QuIC Real-Time Quaking Induced Conversion

SDS Sodium Dodecyl Sulfate

SOC Super Optimal broth (w catabolite repression)

medium

sQuIC Standard Quaking Induced Conversion

TE Tris-EDTA buffer

Tg(CerPrP E226)5037+/-

Transgenic mouse model (cervid PrPc with

glutamate at position 226).

Tg(CerPrP M132)1536+/-

Transgenic mouse model (cervid PrPc, methionine

at position 132).

ThT Thioflavin T

Tris Cl Tris(hydroxymethyl)aminomethane) chloride

TSE Transmissible Spongiform Encephalopathy

µg Microgram

UK United Kingdom

µL Microlitre

uM Micromolar

vCJD Variant Creutzfeldt-Jakob Disease

xiv

Epigraph

One of the most remarkable features of slow infections is the clockwork

precision with which the replication of prions occurs…The molecular mechanisms

controlling this extraordinarily precise process are unknown.

─S.B. Prusiner, M.R. Scott, S.J. DeArmond, and G. Carlson

"Transmission and Replication of Prions." 2004. Prion Biology and Diseases. 2nd

ed.

Cold Spring Harbor Laboratory Press, New York.

1

Literature Review Chapter One:

1.1 Prions: Disease History and Discovery

Prion-diseases, or transmissible spongiform encephalopathies (TSEs), are an invariably

fatal class of neurological diseases, caused by “prions”. Early origins of prion science

began in agriculture, where ovines and caprines with the disease “Scrapie” were first

formally published by Johann Leopoldt in 1750. Leopoldt described excessive nibbling,

scratching, and wasting of affected sheep, noting they would never recover. He had even

suggested Scrapie was contagious, and culling infected animals was recommended

(reviewed by Schneider et al [146]).

Scrapie transmissibility had not been proven until mid-1930s: Cuille and Chelle had

shown Scrapie, or “tremblant” (trembling), to be inoculable in 1936 [42, 43, 98]. In 1937,

sheep haphazardly developed Scrapie two years after receiving a sub-cutaneous louping-

ill vaccine made of ovine brain and spleen homogenate, which was unknowingly

contaminated by the perceived “filter-able virus” responsible Scrapie [43, 62]. Since the

vaccine material had been previously treated with 0.35%(v/v) formalin, it was also the

first observation of Scrapie’s resistance to chemical inactivation, which was unusual for a

virus [1, 62, 98].

Scrapie, or “rida” (Icelandic for “trembling”), had been categorized as a “slow-virus” by

Bjorn Sigurdsson (1954). The long incubation period and clinical symptoms emulated

that of the “visna” (wasting) virus [96, 149]. However, pathological lesions of “visna”

and “rida” were quite dissimilar: “rida” showed vacuolar neurodegeneration and gliosis

2

in central nervous system (CNS) tissues without the expected immune responses of

inflammation or infiltration of leukocytes, characteristic of a viral infection [96].

Around the same time, “Kuru”, a neurodegenerative disease affecting the Fore people of

Papua New Guinea, was described by Carleton Gadjusek (1957) [53]. Kuru had

eventually been linked to a cannibalistic mourning practice, as infections declined after

the cessation of such practices [52]. Gadjusek described symptoms of ataxia, tremors,

exaggerated voluntary motor functions, temperament changes, a flexed posture, and

eventual wasting [53], comparatively reflected in sheep with Scrapie. A seminal

inference by William Hadlow in 1959 likened the histological spongiform brain lesions

of Kuru to that of Scrapie [69]. With experimental evidence showing Scrapie to be

transmissible to sheep by intracerebral/subcutaneous inoculation, Hadlow suggested Kuru

be inoculated into a primate model to determine if the same transmissible component was

present [69].

Kuru transmission studies during the 1960s, showed brain homogenates from deceased

Kuru patients caused disease in chimpanzees, creating identical pathological features of

Kuru [19]. Creutzfeldt-Jakob Disease (CJD), described in the 1920s by Creutzfeldt [40]

and Jakob [84], shared similar pathological profiles to Kuru, and had also been

transmitted to primates [1, 55, 89, 98]. CJD, Kuru, and Scrapie had been interrelated as

the same form of “unconventional-virus” infection [52, 56]. The incubation period of the

“slow-virus” would be months, upwards of decades prior to clinical signs. Various

inoculation studies confirmed the agent could be transmitted and “adapt” to new animal

hosts, resulting in shortened latent periods after successive passages [52].

3

The consensus at the time was either a causative “slow-virus”, or potentially a viroid of

sorts. However, the Scrapie agent’s resistance to ionizing radiation, nucleases, and

chemical hydrolysis of RNA and DNA suggested the pathogen did not require an intact

genome to establish infection [48, 128]. Additionally, a projected molecular weight for

the agent was ~50,000 Daltons, physically too small to be a viroid [128]. The absence of

immune response from the affected host raised suspicion about the validity of a causative

virus [52, 56, 128, 166].

Unique, unconventional alternatives had been suggested (reviewed in [146]): Pattison and

Jones (1967) suggested Scrapie could be caused by a small protein [126]. Gibbons and

Hunter (1967) proposed an infectious membrane fragment [54], supported by a

suggestion of Alper et al, where the replicative unit was lipid associated [3]. Griffith

(1967) proposed a model for aggregation of a small protein, driven by template-directed

conformational changes [67]. He theorized the protein might be host-encoded, self-

replicative, and the aggregation process could be passed between animals, or perhaps

arise spontaneously [67].

The paradigm-shift to the “protein-hypothesis” became predominantly accepted when

Prusiner (1982) coined the term “prion”, and identified an essential, protease-resistant

protein associated with the infectivity of Scrapie [128, 132]. As surmised by Griffith, the

“prion-related-protein” (PrP) was indeed found to be host-encoded [117]. Sporadic

human prion-diseases such as Gerstmann–Sträussler–Scheinker syndrome (GSS), familial

CJD (fCJD), and Fatal Familial Insomnia (FFI) became linked to certain human PrP gene

4

polymorphisms (reviewed in [1, 38], [60]). By the early 1980s, prions were emerging as a

new concept of disease.

1.2 Prions: Propagation, Pathogensis, and Disease Concept

The current understanding is PrP exists in two states: the host-encoded cellular form

(PrPc), and the disease-associated form (PrP

d). Prion-disease could be contracted by

either acquiring PrPd, or PrP

d forming spontaneously. PrP

c is encoded by the PRNP gene,

and is expressed predominantly on neurons and glial cells, and in decreasing amounts in

lymphoid and T-cells, respectively [28, 83, 92, 109].

The initiating, or sporadic, event how PrPd comes into existence is undefined. The

presence of PrPd purportedly coaxes endogenous host PrP

c to convert to additional PrP

d

within the host. PrPc undergoes a series of conformational changes via transition-state

intermediates, changing from its native α-fold to a β-fold, leading to the accumulation of

PrPd aggregates [1, 34, 131, 167]. The process is continuously propagative, so long as

PrPc is present to convert.

PrPd propagation is dependent on host PrP

c expression. Mice devoid of PrP

c (PRNP

-/-)

mice do not propagate PrPd related amyloid, nor are susceptible to prion disease [30,

168]. Therefore, endogenous PrPc must be present in order for PrP

d to exert its

cytotoxicity. Mallucci et al further highlighted the importance of endogenous PrPc by

using a transgenic mouse model showing that Cre-recombinase/MloxP-mediated post-

5

natal excision of the PRNP gene stopped the progression of Scrapie within infected mice

[101]. The cytotoxic mechanism of PrPd remains enigmatic: It may disrupt PrP

c

localization by cytosolic PrPd-like aggregates and cause ER-stress, or initiate apoptotic

signal transduction pathways [2, 27, 29, 33, 81, 100, 151].

PrPd is characterized by its high β-sheet content (~45%) and amyloidogenic tendencies

[123]. It is the principle protein component of TSE-related amyloid from infected tissues.

PrPd was first identified as a residual PrP species denoted as “PrP27-30” is seen on

immunoblots (western blot), after TSE-infected tissue homogenates were treated with

Proteinase K (PK) [117, 128, 129]. When TSE-negative tissue homogenates are treated

with PK, endogenous PrPc is degraded and no western-blot signal is generated. The “27-

30” notation is ascribed to the 27-30 kDa molecular weight of the protease resistant core

remaining after PK treatment, as the N-terminal strand is cleaved-off [129]. The

resilience of PrPd to PK is likely attributable to its amyloidogenic properties [136]. This

protease resistant property is the foremost aspect of PrPd which is exploited by

biochemical detection methods, later discussed.

1.3 Prions: Significance

The natural route for prion-transmission is considered to be oral exposure to the agent,

and was thought as more-or-less intraspecies specific: For instance, Scrapie could be

passed orally between ovines by infected brain homogenate [70], and Kuru was passed by

anthropophagy [52]. Regional CJD prevalence studies with Scrapie as a risk factor had

6

not shown any correlation [1, 35, 39]. The zoonotic importance of animal TSEs on

human health was not fully realized for some time. The occurrence of Bovine

Spongiform Encephalopathy (BSE) in Europe during the 1980s profoundly changed the

perception concerning the modes and ease of cross-species prion-transmission.

BSE had been propagated in cattle by recycling offal from slaughtered livestock (cattle,

sheep, etc.) in the form of processed meat and bone-meal (MBM), used as high-protein

feed [1, 4, 170]. An imposed MBM feed ban saw the decline in British BSE, much like

the cessation of a cannibalistic mourning ritual ended Kuru [1]. A change to a

hydrocarbon-free rendering process of offal presumably led to an amassing of intact

Scrapie and/or bovine prions in MBM feed to an infectious capacity [1, 170].

Circumstantially synonymous to the Scrapie-containing looping-ill vaccine, BSE became

a tragic, unplanned experiment: orally passaged prions in cattle became casually linked

with ensuing cases of variant CJD (vCJD) in predominantly young adults, supporting the

zoonotic threat of agricultural prion-diseases [37, 172]. Only 228 vCJD cases worldwide

have been reported since 1991, which is relatively small considering it is estimated one

million BSE-infected cows between 1974 and 1995 in the UK alone [4]. However,

nascent evidence of abnormal PrP in human appendixes questions whether the full extent

of the vCJD epidemic has been realized. Latent vCJD infections may be as high as 1 in

2000 in the United Kingdom [58, 140]. Furthermore, asymptomatic vCJD carriers pose

risks to the national blood supplies: iatrogenic CJD can be contracted by receiving vCJD

contaminated blood (reviewed in [13]). The devastating social and economic toll of BSE

7

has put prions at the forefront of animal health and food safety, highlighting the need for

various TSE surveillance programs [21, 78, 130].

BSE and Scrapie are largely under control: BSE declined through MBM feed bans, and

Scrapie eradication measures are underway through selective breeding programs [99].

However, Chronic Wasting Disease (CWD) is a TSE affecting cervids [68, 175], and is

spreading rapidly across North America. CWD poses various agricultural and ecological

threats [143]. Ultrasensitive detection of CWD in cervid tissue is the focus of this study.

Improving detection capabilities will hopefully contribute to CWD control in North

America.

1.4 Chronic Wasting Disease (CWD)

1.4.1 CWD Epizoology

First noticed in Colorado in the 1960s, CWD is now found across the American West,

Midwest, and Canadian prairies [1, 143, 174]. CWD is most similar to Scrapie [173], in

that CWD can be horizontally transmitted to mule-deer, white-tailed deer, black-tailed

deer, elk and moose with relative ease [148]. Infected cervids contaminate the

environment: The CWD prion is shed through saliva, urine, blood, and feces, which in

turn remains infectious in the soil for extended periods of time [106, 144, 145, 157, 158].

CWD is further propagated when non-infected herds graze on contaminated land. Clinical

8

signs of infected cervids include the following: wasting, self-alienation from the herd,

pacing, hypersalavation, and eventual death [1, 173]. Because CWD is well-established

in wild cervid populations [1, 143], co-grazing activities with farmed-livestock are a

paramount concern. Elk and deer farms are at risk of contracting CWD through contact

with infected cervids, contaminated soil/water and/or co-grazing.

Mule-deer CWD can be transmitted to Suffolk sheep and cattle by intracranial

inoculation, though transmission is inefficient [75, 76]. Interestingly, cattle are far more

susceptible to white-tailed deer CWD, than that of mule-deer [77]. Cattle directly

exposed to CWD and cattle co-gazing with CWD infected mule-deer have not developed

disease, despite a six year incubation period [148]. Nonetheless, co-grazing of CWD

infected cervids with domestic livestock is a concern. Ecological concerns pertain to

possible transmission to caribou, bighorn sheep, and mountain goats [108].

CWD infectivity has been found in cervid skeletal muscle and antler velvet which

suggests that aboriginal communities and hunters are likely to have been exposed [5, 6,

148]. Transmission models suggest the CWD risk to humans is low. New evidence shows

squirrel monkeys appear susceptible to elk and deer CWD by oral inoculation (after a ~68

month incubation period), however cynomolgus macaques fail to produce disease [133].

In addition, transgenic mice expressing human PrPc also prove resistant to CWD [90,

141, 176]. This suggests a species barrier likely protects humans from CWD

transmission. At present, CWD remains an agricultural, ecological, and trade concern

[150]. For instance, between 1994 and 1997, Canadian elk imported by South Korea

developed CWD.

9

1.4.2 CWD Pathology

Any contact of CWD prion with mucosal membranes of an uninfected cervid poses a

horizontal transmission risk: Prion entry can be via ingestion, gingival scarification, and

potentially spread through aerosols [46, 47, 105]. Oral transmission and uptake of CWD

and Scrapie prions follow a general mechanism [74]. Prion uptake in the gut is facilitated

by large endosomes of microfold cells (M cells) [80] and follicle-associated epithelium

(FAE) enterocytes of Peyer’s patches [95]. Macrophages and FAE enterocytes release

prions in exosomes into the sub-epithelial dome, then are transported to follicular-

dendritic cells (FDC) by tingible body macrophages [95].

FDCs are the first site of prion propagation, attributable to their elevate PrPc expression

[28, 95]. The prion spreads to submucosal and myenteric plexuses [70, 95, 97, 163],

subsequently infecting the enteric nervous system and spreading to vagus nerve and CNS

[20, 174]. CWD PrPd (PrP

CWD) is usually detected earlier in tonsil and lymph nodes,

followed by brain tissues [10, 51, 152, 169]. Later stages of disease show a more

ubiquitous presence of PrPd throughout the body in various organs including adrenal

glands, heart, pancreas, and spleen [10, 51]. Histopathological lesions comprise of

spongiform lesions (vacuolization) in the medulla oblongata (obex), thalamus, and

parasympathetic vagal nucleus [175].

10

1.4.3 CWD Genetic Susceptibility

Cervid species and PRNP genotype also influences CWD susceptibility and incubation

period [51, 65, 85, 115, 116, 137]. Free-ranging mule-deer homozygous for serine at

codon 225 propagate CWD more rapidly than those with the 225 serine/phenylalanine

allele. PrPCWD

can be detected in lymph nodes ~100 days earlier in 225SS deer than in

225SF heterozygotes, although PrPCWD

distribution in lymphatic tissues is consistent

between alleles [51]. Deer which are 225SS show widespread, heavy PrPCWD

deposition

in CNS, gut, and endocrine tissues ≥630 days post-inoculation. The 225SF deer display

less PrPCWD

, linked to a slower incubation period [51].

In elk, the PRNP polymorphism at codon 132 for methionine or leucine profoundly

influences CWD progression, in addition to biochemical properties of PrPCWD

[74, 116].

Methionine homozygotes exhibit the shortest CWD incubation period. Heterozygous

132ML elk are as susceptible to CWD as 132MM elk, though the incubation period is

significantly longer. Elk which are 132LL demonstrate the highest resistance to CWD

[74].

1.4.4 CWD Detection and Diagnostic Testing Methods

Post-mortem histopathology of hematoxylin and eosin (HE) stained CWD elk brain

shows pale, fibrillar eosinophilic areas of neuropil, typically surrounded by vacuoles,

referred to as “florid plaques” [1, 174]. Visualization is significantly enhanced using

11

immunohistochemistry (IHC), staining specifically for PrPCWD

. IHC is more sensitive as

it also detects widespread deposition of PrPCWD

in brain areas in the absence of

vacuolization. IHC also detects PrPCWD

deposits in lymphoid tissues, which do not show

classic TSE histopathological lesions (reviewed in [174]). Retropharyngeal lymph nodes

(RPLN) are a reliable target for early PrPCWD

detection in deer, however a study of

captive elk showed 10-15% of infected animals only had detectable PrPCWD

in brain

tissues [152, 174]. Therefore, testing of both brain (obex) and RPLN are required in elk

CWD surveillance programs [174].

Samples for live animal testing are available for CWD and Scrapie, though options are

quite restrictive. Testing the nictitating membrane (3rd

eyelid) is an approved option for

Scrapie, but is not as effective in deer and elk [174]. Tonsil biopsy testing could be

viable, though the invasive nature and anesthetic requirement make it unsuited for routine

testing [88, 174]. In addition, elk show less PrPCWD

deposits in tonsil compared to deer

[134], which may be related to the greater variability of peripheral PrPCWD

distribution in

elk [152, 153, 174].

Antemortem detection of CWD in elk is possible by testing rectoanal mucosa-associated

lymphoid tissue (RAMALT). PrPCWD

can be detected in an average of 6/10 mucosa-

associated lymphoid follicles in clinical and preclinical elk [153]. However, variability of

IHC results can be high, and age-related involution of mucosa-associated lymph follicles

may compromise sensitivity [153]. Accuracy of RAMALT testing is subject to elk age (≤

5 years is preferred), an individual’s sampling familiarity, and potential inconsistencies of

12

PrPCWD

distribution in peripheral tissues of elk (particularly in early disease stages), as

compared to deer [152, 153].

Alternatives to IHC for CWD detection are biochemical rapid-test platforms: a CWD Dot

Blot, ELISAs (BioRad and IDEXX Laboratories), western blot (BioRad Laboratories)

and a lateral flow immunoassay (Prion Development Laboratories (PDL), Inc.) [24, 174].

Like IHC, these tests are dependent on the immunological detection of PrPCWD

in the

brain (post-mortem collection) and lymphoid tissues (post-mortem or invasive

antemortem collection). Rapid-tests offer a lower cost and shorter testing time alternative

to IHC, albeit at the cost of test sensitivity.

TSE rapid-test protocols require the tissue sample to be homogenized. In most rapid tests,

the homogenate is then treated with a protease, typically PK [41, 64, 110, 118, 122]. PrPc

is degraded, leaving the protease resistant PrPd for detection. The use of aggressive

proteases like PK can be problematic. Protease-sensitive PrPd isoforms of Scrapie,

sporadic CJD, and atypical PrPBSE

have been identified, and protease cut-sites are not

consistent on all PrPd isoforms [31, 41, 49, 125, 127, 138, 161].

Elk PRNP polymorphisms appear to affect some aspect regarding the conformation of the

PrPCWD

monomer or amyloid fibril structure. The result is an altered protease processing

of the PrPCWD

, risking a false-negative test result. PrPCWD

from 132LL elk is not reactive

with the commonly used CWD/Scrapie sensitive monoclonal antibody (mAb) P4, but

rather mAb 8G8 [116]. PrPCWD

from 132MM and 132 LM elk are reactive with both

mAbs. In addition, when mAb 8G8 is used, PK-processed PrPCWD

from 132LL elk

13

displays a lower molecular weight profile on a western-blot compared to PrPCWD

from

132MM and 132LM elk [116]. The 132LL PrPCWD

molecular weight downshift and loss

of the mAb P4 epitope, indicates a change in PK-accessible cleavage-sites, and detection

was missed [66].

No rapid-test is certified for use on bodily fluids such as urine, saliva, or blood, despite

the apparent presence of CWD infectivity, determined by transgenic mouse bioassay

models [63, 72, 79, 103]. There is a demand for a sensitive, reliable, and non-invasive

testing method for CWD—and all other animal and human TSEs for that matter. In

addition, the distribution of detectable quantities of elk PrPCWD

in tissues is not as

predictable or consistent as that in deer [148, 152, 174]. To compensate, test sensitivity

must be increased. Research has sought to exploit the replicative property of PrPd as a

novel means to increase diagnostic sensitivity. This has put in vitro PrP-conversion

methods at the forefront of TSE-diagnostic research.

Prior to introducing in vitro PrP-conversion methodology, a brief overview of the

cellular-PrP (PrPc) isoform’s cell-biology is provided as support for the technical aspect

of this study. Notable features of the protein which play a role in PrP-conversion are

presented.

1.5 Cellular PrP (PrPc)

Synthesis and translocation of the protein is directed to the endoplasmic reticulum by the

signal sequence, residues 1-22. PrPc (23-253) is trafficked to the cell-surface via transit

14

through the Golgi body where it acquires complex, branched glycosyl groups on

asparagine residues ~181 and ~197 [129]. Two cysteine resides, ~172 and ~214, form a

disulfide, while C-terminal amino acids 232-253 are replaced with a glycosyl

phosphatidylinositol (GPI)-anchor [129, 154]. The final PrPc product is mostly found on

detergent-insoluble membranes/lipid rafts [136]. PrPc exists largely in an α-fold,

stabilized by lipid-raft components and the heavily glycosylated sphingolipids within

[155]. There are three main regions to the protein: the N-terminal region, a hydrophobic

region, and the C-terminal domain (~120- ~230) which is comprised of three α-helices

and a short, two-stranded antiparallel β-sheet [44, 135].

The N-termini region of PrPc is characterized as a strand from residues 23 through 93,

typically containing 5 conserved octapeptide repeats of P(Q/H)GGGWGQ from residues

~51-91 [129]. Certain single amino acid insertions and deletions are species-specific,

though the overall physicochemical properties of the region are maintained [177]. The

region is distinguished by the high glycine content found adjacent to histidine residues,

which is well-known to coordinate divalent metal ions [91]. Glycylglycylglycine peptides

form uniquely stable complexes with Cu2+

ions [111]. Bound Cu2+

ions bound in the

octapeptide region contribute to the overall α-helical fold and stability of the protein

[107, 123]. This region is conformationally altered in the PrPd isoform, and may be

important in the PrPc conversion to PrP

d process and disease propagation [178].

Though raft-associated, PrPc is also seen leaving rafts and being internalized and recycled

in endosomes via clathrin-coated pits [155]. Internalization of PrPc is facilitated in part by

a motif of basic residues—KKRPKP—on the N-terminus [155]. The motif is also

15

subsequently apart of the peptide fragment 23-35 (KKRPKPGGWNTGG) known to

specifically bind glycosaminoglycans (GAGs), where binding is further enhanced when

Cu2+

is bound in the octapeptide region [61, 124]. Substitutions of charged residues in the

KKRPKP motif abolishes PrPc internalization, likely by preventing binding of heparin to

PrPc [155]. A recent finding considers this motif to be essential for PrP

c binding to PrP

d,

and subsequent PrPd propagation [160, 179].

The hydrophobic region possesses a unique palindromic sequence: AGAAAAGA. This

sequence has been identified as a major contributor of PrPc-PrP

d association and

aggregation, is implicated in transmembrane forms of PrPc, and has cytotoxic

associations [23, 26, 86, 113].

1.6 In Vitro Recombinant PrPc Conversion Methods

The principle of in vitro PrP-conversion as a TSE detection method is to exploit the

propagative nature of PrPd. If a tissue homogenate or biological fluid from a suspect TSE

case contains PrPd (a “seed”), it is expected the PrP

d would act upon a PrP

c source (a

“substrate”) and convert the PrPc to more PrP

d (as previously described). Therefore, a

previously undetectable presence of PrPd should show itself by converting PrP

c into a

form of PrPd aggregate, fibril, or amyloid which is detectable.

The following describes how PrPd-seeded conversion of bacterially expressed

recombinant PrPc (rPrP

c) has evolved into a TSE detection technique. In this thesis, this

process will be referred to as “rPrP-conversion”.

16

1.6.1 Autocatalytic Conversion

Bacterially expressed recombinant PrPc (rPrP

c) has been used to study the spontaneous

formation of rPrPc β-oligomers and amyloid fibrils [14, 15, 17, 18]. Baskakov et al

(2002) have examined folding pathways of truncated versions hamster and mouse rPrPc

(Ha90-231 and Mo89-231) in partially denaturing conditions using chaotropes (urea and

GuHCl). They determined that more acid pHs, as found in endocytic vesicles, favour the

formation of β-oligomers, whereas neutral pHs favoured amyloid fibril formation [18].

Seeding Autocatalytic Conversion reactions with pre-formed rPrP-fibrils markedly

reduces the lag-phase for amyloid fibril formation, allowing the system to by-pass the

initial rate-limiting nucleation step: α-monomers (rPrPc) becoming β-oligomeric nuclei

[16]. Even under conditions where β-oligomers were not favoured to form, pre-formed

human rPrP-fibrils effectively seeded the remaining human rPrPc into amyloid [14].

Amyloid formation would readily occur under continuous agitation at 37oC, while β-

oligomer formation occurs regardless of any mechanical disturbances [14]. Oligomeric

formation was regarded as “off-pathway” to amyloid formation, as β-oligomers could be

switched into amyloid under continuous agitation.

The same pH and chaotropic effects could be applied to full-length murine rPrPc (23-231)

[25]. Only under constant agitation and at pHs > 5.5 does fibril/amyloid formation occur.

Full-length rPrPc exhibits a much higher propensity for fibrillating compared to N-

terminally truncated (~90-231) rPrPc [16]. As opposed to truncated rPrP

c, full-length

17

rPrPc maintains its fibril forming activity independent of concentration, even displaying

short-lag periods with protein concentration as little as 0.22µM [16].

Rather than seed Autocatalytic Conversion reactions with pre-formed rPrPc fibrils,

attempts have been made to seed rPrP-conversion with PrPd from TSE-positive tissues.

The Amyloid Seeding Assay (ASA) is described below.

1.6.2 Amyloid Seeding Assay (ASA)

The ASA was derived from Autocatalytic Conversion. The key difference is seeding

rPrP-conversion with PrPd, rather than by pre-formed fibrils made from rPrP

c [36]. Colby

et al have seeded N-terminally truncated hamster, mouse, and human rPrPc (~88-230), as

well as full-length mouse rPrPc (23-230) with a variety of PrP

d sources [36]. PrP

d from

tissues had been semi-purified from tissues by homogenization in sarkosyl, followed by

phosphotungstic acid (PTA) precipitation. The PTA-precipitated material was

resuspended in water and used to seed the formation of rPrPc fibrils/amyloid. A dye,

Thioflavin T (ThT), was used to monitor amyloid fibril growth, as its fluorescence

properties change when bound to amyloid [11]. The conversions reactions were

performed in PBS with ~500mM GuHCl, 10µM ThT, and 50µg/mL of rPrPc. GuHCl is

traditionally used to slightly destabilize the rPrPc tertiary structure, close to a transitory

state, priming it for PrPd-seeded conversion (reviewed in [1]).

18

The rPrP-conversion reactions were continuously shaken and incubated at 37oC, though a

glass bead was added to each reaction well to increase agitation [36]. PrPd from tissues

was as efficient at seeding conversion reactions as pre-formed fibrils made using solely

rPrPc [36]. The ASA displayed a very high sensitivity for detecting PrP

d, extrapolated

down to ~1fg [36]. The mean rPrP-conversion time for reactions seeded by PrPd and pre-

formed fibrils (controls) was ~2 hours, compared to spontaneous rPrP-conversion

occurring around 12 hours [36].

ASA demonstrated the novel possibility to take a kinetic approach to decipher between

PrPd (TSE-positive) and non-specifically seeded (TSE-negative) rPrP-conversion by

timing the duration of the lag-phase. However, PTA-precipitated material from non-

diseased tissues would regularly seed rPrP-conversion. The promiscuous non-specific, or

spontaneously occurring, rPrP-conversion activity in ASA is problematic for test

specificity. This issued had to be addressed for further consideration regarding diagnostic

use [121]. The ASA methodology was improved upon by another in vitro rPrPc

conversion assay, “RT-QuIC”.

1.6.3 Real-Time Quaking Induced Conversion (RT-QuIC)

The RT-QuIC has evolved from its predecessor, “Standard-QuIC” (sQuIC), and features

of ASA. RT-QuIC has been successful for detecting hamster-adapted Scrapie,

sCJD/vCJD, and white-tailed deer CWD [50, 79, 119, 171]. Initial sQuIC trials consisted

19

of 100µL aliquots of Syrian hamster rPrPc (23-231) substrate in a conversion buffer

consisting of PBS, 0.05%(v/v) Triton X-100, 0.05%(w/v) SDS [9]. The assay could be

seeded with as little as 10fg of hamster-adapted PrPsc

from hamster brain homogenate [9].

Conversion reaction components would vigorously shake (or quake) for 1 minute,

followed by a 1 minute rest period, while incubating at temperatures between 42oC and

65oC. The formation of rPrP-fibrils was determined by PK-digestion of the rPrP-

conversion mixture, post shaking/incubation. Any PK-resistant rPrP species would be

revealed by western-blot, just as any current TSE rapid-test would show [9]. The

presence PK-resistant rPrP species would correspond to the presence of PrPsc

from the

seed material, with much less spontaneous rPrP-conversion.

The assay evolved to a microplate format and adopted the ThT fluorescence read-out

method used in ASA. Instead of an end-point analysis by western blot, PrPd-seeded

amyloid fibril growth could be monitored in “real-time”, hence RT-QuIC [171]. To

reduce non-specific/spontaneous rPrP-conversion, Wilham et al optimized their RT-QuIC

parameters from ASA by replacing the chaotrope (GuHCl) in the conversion reaction

with a Triton X-100 and SDS, and using intermittent shaking rather than continuous

[171]. An RT-QuIC protocol by Atarashi et al for CJD detection in brain and CSF does

not use any detergent in homogenization buffer or diluent [7, 8]. It was specified that the

omission of chaotropes from the rPrP-conversion system was preferred in order to keep

rPrP-conversion more specific to being seeded by PrPd rather than forming spontaneously

[7, 8]. In effect, RT-QuIC shows an rPrP-conversion assay may perform better under

non-denaturing conditions.

20

RT-QuIC assays have proven as sensitive as a bioassay, in terms of being able to detect

an infectious concentration of hamster adapted PrPsc

and deer PrPCWD

from brain [50,

171]. However, RT-QuIC results are customarily reported based on a dichotomous

outcome: whether or not a fluorescence signal is generated by any given seeded reaction

over the assay’s 48 to 60 hour duration [7-9, 119, 120, 142, 171]. The lag-phase

preceding PrPd-seeded rPrP-conversion is recognized as being synonymous to the latent-

period of a TSE prior to clinical symptoms appearing [171]. However, rPrP-conversion

time in RT-QuIC is not formally considered in the data analysis, as in ASA.

Incorporating rPrP-conversion time as a scalar outcome creates a new dimension for

analysis. End-point titrations of PrPd in bioassay have the dichotomous outcome of

survival or death. However, the incubation-time until clinical disease has also been used

to titre PrPd infectivity (reviewed in [1]).

To summarize, the ASA can amplify the presence of as little as 1fg of PrPd from semi-

purified from TSE-diseased tissues. However, its specificity is tarnished by a high

abundance of spontaneous rPrP-fibril formation, seeded by PTA-precipitated TSE-

negative material and unseeded control reactions. Ideally, only PrPd-seeded rPrP-

conversion reactions would produce a ThT positive signal. Most RT-QuIC protocols

replace the use of chaotropic agents in the rPrP-conversion system with detergents. This

has limited the occurrence of spontaneous rPrP-conversion, maintaining the assay’s

specificity for detecting PrPd.

21

Alternatively, the notion of ASA being able to time PrPd-seeded rPrP-conversion as a

measure of prion converting activity is intriguing. It provides an additional quantitative

dimension for analysis, perhaps used in a similar manner to incubation-time analysis of

bioassay. RT-QuIC protocols focus on a dichotomous outcome of whether or not a ThT

signal is generated from a seeded rPrP-conversion reaction. Conversion time is not

formally incorporated into any analysis [7, 8, 50, 73, 79, 87, 121, 142, 171].

The rPrP-conversion time should be considered in an analysis, particularly in a diagnostic

setting. Time can be used as a second measure to validate how likely any given rPrP-

conversion event, witnessed by ThT fluorescence, is actually seeded by a prion (PrPd)

versus an erroneous spontaneous event. Combining aspects of both ASA and RT-QuIC

may lead to more informative and standardized in vitro PrP-conversion assay suited for

diagnostic use.

1.7 Research Foci and Objectives

In vitro PrP-conversion methods are at the forefront of TSE-diagnostic research to

achieve a reliable ultra-sensitive diagnostic tool for less invasive antemortem testing. The

goal of this study is to identify and differentiate tissues from CWD infected (CWD+) elk

from uninfected (CWD-) elk using in vitro PrP-conversion methodology.

The first objective is to optimize the in vitro PrP-conversion assay seeding-conditions

(tissue input) for use in a diagnostic setting. Brain and lymphoid tissues are used, as these

22

are target tissues for CWD surveillance (Chapter 3). The aim is to achieve the highest

CWD+ seeding activity on rPrPc, with the lowest amount of non-specific/spontaneous

rPrP-conversion events. The diagnostic read-out or cut-off criteria of the assay will be

based on the time required for seeding material (tissue homogenates) from CWD+ versus

CWD- to elicit an rPrP-conversion event.

The second objective is to perform the assay with the optimized in vitro PrP-conversion

seeding-conditions on panels of multiple elk brain and lymphoid tissues, mimicking a

diagnostic screening setup (Chapter 4). The validity of using rPrP-conversion time as a

diagnostic parameter and the associated diagnostic criteria established within the first

objective will be scrutinized. Panels will consist of tissues from elk per orally challenged

with CWD+ and CWD- material. Animals had been euthanized at different time-points

post-inoculation. This will be used to evaluate the in vitro PrP-conversion assay’s ability

to detect pre-clinical CWD+ elk. Note: no animal experiments took place during this

work, and all elk tissues had been donated (detailed in Chapter 2).

The third objective is to evaluate the developed in vitro PrP-conversion assay’s

sensitivity for elk CWD infectivity, using the diagnostic cut-off criteria of rPrP-

conversion time determined in the first objective. Sensitivity will be compared to a

Tg(CerPrP-M132)1536+/-

and Tg(CerPrP-E226)5037+/-

bioassayed (titred) pool of CWD+

elk brain homogenates [22]. Timing seeded rPrP-conversion will be analyzed as a

possible method to titre prion-activity.

23

1.8 Hypothesis

It is hypothesized that timing in vitro conversion of rPrPc amyloid formation can be used

to quantify prion-activity in a brain homogenate as effectively as a bioassay model, thus

supporting the usefulness of this assay and criterion for early, sensitive and specific

CWD detection.

24

General Methods and Materials Chapter Two:

2.1 Tissue Solubilization and In Vitro Conversion Detergent System

A CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and NDSB

(non-detergent sulfobetaine) combination was selected for a non-denaturing tissue

solubilization and subsequent in vitro PrP-conversion detergent system. CHAPS possess

unique solubilizing characteristics that are of particular interest concerning prion

biochemistry. Specific purposes for selecting a CHAPS-NDSB combination are placed in

this section to complement the methodology described below.

CHAPS is a zwitterionic form of cholate, and is used as a non-denaturing detergent for

solubilizing membrane proteins and complexes [12, 82, 139, 147]. CHAPS detergent-

resistant membranes (DRMs) are quite different than those from classic Triton X-100

DRMs, in terms of PrPc content [139]. Nycodenz® gradient flotation studies showed

CHAPS created an array of differently sized—potentially novel—DRMs/microdomains.

PrPc is predominantly found in medium-sized CHAPS DRMs [139].

Medium and small sized CHAPS-DRM complexes are rich in cholesterol, ganglioside

(GM1), and PrPc [139]. PrP

c conversion to PrP

d is known to occur via cholesterol-

dependant pathways [57], since PrPd is found primarily within saponin-sensitive domains

[112]. PrPc interaction with rafts is essential for efficient conversion to PrP

d, suggesting

PrPd is likely attached to small cholesterol and glycolipid-rich microdomains on

organelles and late endosomes [112, 159, 162]. Intact microdomains may contain

additional unrecognized cofactors aiding conversion [112], and may act as more efficient

25

seeds for in vitro PrPd-seeded rPrP-conversion. This highlights the potential that a

CHAPS based solubilization system may maintain the integrity of various sized PrPd rich

microdomains and any conversion cofactors, enhancing in vitro PrP-conversion and thus

prion detection.

In an unrelated study evaluating the efficacy of different detergents to solubilize the

serotonin 5-HT1A receptor, 40% of the total CHAPS-solubilized lipid was

phosphatidylethanolamine (PE) [12]. PE is suggested as major cofactor in PrPd

replication and infectivity [45]. CHAPS solubilizes more lipid than SDS, and twice as

much PE as SDS [12]. The heightened amount of solubilized PE might boost PrPd-seeded

conversion in the in vitro PrP-conversion assay.

A recent PrPc solubilisation study had shown different detergents’ PrP

c solubilizing

abilities in terms of quantity and suspended glycoforms [93]. The study might also be

partially reflective of the detergents’ abilities to solubilize PrPd as well. SDS proved to be

the most efficient solubilizer of PrPc [94]. CHAPS’ solubilizing efficacy of PrP

c was

approximately 75% that of SDS, while Triton X-100 only solubilized ~50% that of SDS.

However, the protein solubilizing efficacy of CHAPS can be pointedly increased with the

addition of non-detergent sulfobetaines (NDSB) [164, 165]. In a comparison of

membrane-protein extraction from microsomes between SDS and CHAPS, a 1%(w/v)

CHAPS solution has approximately ~53% the total protein solubilizing efficacy as 1%

w/v SDS [164]. However, a combination of 1%(w/v) CHAPS/ 1M NDSB-201

significantly raised CHAPS’ protein solubilizing efficacy to ~71%, respective to 1%(w/v)

26

SDS [164]. Incorporating NDSBs into CHAPS-based homogenization buffers may

enhance PrPd solubilization, performing similarly to SDS.

In addition, NDSBs are effective protein folding agents. NDSBs assist proteins through

early metastable folding intermediates by coordinating transient, non-native hydrophobic

patches that would otherwise lead to non-specific protein aggregation [165].

Concentrations of NDSBs in the 1M-2M range can effectively refold and restore

enzymatic activity of previously denatured enzymes [59]. Such molecular properties

make NDSBs interesting candidates to incorporate into homogenization buffers as

restorative agents for proteins, post-homogenization and sonication. Because NDSBs

mediate a balance between protein folding and aggregation, yet prevent non-specific

protein interactions, they are enticing candidates to include in rPrP-conversion assays.

For the aforementioned reasons, a CHAPS-NDSB solubilizing agent combination was

selected due to some unique properties that pertain to prion molecular biology. The high

lipid solubilization characteristics of CHAPS is of particular interest due to the apparent

roles lipids may play in PrPc conversion to PrP

d [45, 104, 156, 160, 179].

2.2 Tissue Preparation and Homogenization

Tissues from experimentally inoculated elk were kindly donated by Dr. Catherine

Graham at the Canadian Food Inspection Agency (CFIA), Lethbridge Laboratory. All

tissues from CWD+ and CWD- control animals were trimmed in a class II biosafety

27

cabinet, in a biosafety-level 2+ laboratory. Disposable scalpels (single use) were used to

trim each and any tissue sample (CWD+ or CWD-), along with replacements of cutting

board liners to eliminate cross-contamination. Tissue samples of ~0.2g were

homogenized in homogenization buffer (1%(w/v) CHAPS, 1M NDSB-201, PBSk

[10mM Na2HPO4, 2mM KH2PO4, 137mM NaCl, 2mM KCl, pH 7.4], 1mM EDTA,

cOmplete™ Protease Inhibitors (Roche)) using a ribolyser with grinding beads (BioRad

Precess®). Homogenates were stored at -80oC in 100µL aliquots until further use for

seeding rPrP-conversion reactions.

2.3 Preparation of Recombinant PrPc (rPrP

c)

2.3.1 Molecular Biology and Vector Construction

The gene insert for full-length rPrPc of elk (Cervus elaphus nelsoni; Accession #:

P67986) [114] was ordered from Integrated DNA Technologies, shipped lyophilized in

pIDTSmart plasmids. The pIDTSmart plasmid was resuspended in 100µL TE buffer

(10mM Tris∙Cl, 1mM EDTA, pH 7.8) to ~4ng/µL. Twenty five microliters of chemically

competent OneShot® Top10 cells (Life Technologies) were heat-shock transformed at

42oC for 30 seconds with 2µL of the pIDTSmart vector. Cells were then incubated on ice

for 60 seconds. SOC medium was added (150µL), and cells were incubated at 37oC for 1

hour. Fifty microliters of cells were plated on LB agar containing 50µg/mL kanamycin,

then left to incubate at 37oC overnight.

28

Single colonies were selected to grow minicultures to amplify the vector. Minicultures

were grown in 5mL of LB broth with 50µg/mL kanamycin overnight at 37oC. Cells were

pelleted at 3000G for 5 minutes from 1mL of culture for subsequent vector purification.

Cells were lysed and the amplified pIDTSmart vector was purified using a QiaPrep

Miniprep kit (Qiagen) and QIAcube, and conducted as per the manufacture’s protocol. To

extract the insert coding for elk rPrPc, ~1µg of purified vector was treated with NdeI and

HindIII endonucleases (20 units each) in CutSmart™ buffer (New England Biolabs). The

excised insert was separated from the vector on a 1%(w/v) agarose gel in TE running

buffer at 100V for 40 minutes. The gel was stained with ethidium bromide, and the insert

band was excised from the gel. The insert was purified from the agarose gel using a

QIAquick Gel Extraction kit and QIAcube, as per the manufacture’s protocol (Qiagen).

The purified insert was ligated into pET41b overnight at room temperature, using 400

units of T4 ligase. The insert to pET41b ratio was 3.5:1 (300ng of insert to 85ng of

pET41b). Chemically competent OneShot® Top10 cells were transformed and plated, as

previously described, with 10µL of the ligation reaction product. Individual colonies

were selected for inoculating 5mL minicultures to increase the pET41b copy number for

subsequent colony polymerase chain reaction (PCR), to verify for the correct insert size.

Vectors showing the correct insert size were also sent for T7 promoter/terminator

sequencing of the multiple cloning site to doubly verify the insert correctly coded for elk

rPrPc (Eurofins MWG|Operon). The correct elk rPrP

c coding pET41b vectors were grown

and purified using a QiaPrep Miniprep kit (Qiagen) and QIAcube, and stored at -20oC

until subsequent transformation of Rosetta™ II DE3 cells, used as the expressing cell line

29

(The above preparation of the elk rPrPc pET41b construct was conducted with the kind

help of Mr. Sandor Dudas, Ms. Renée Clark, and summer students Mr. Jace James and

Mr. Ben Vuong).

2.3.2 Escherichia coli (Rosetta™ II DE3) Transformations

Transformations of Rosetta™ II DE3 competent cells were conducted as per the supplier

directives (Life Technologies). Competent cells were thawed on ice for 5 minutes, and

then gently dispensed in 20µL aliquots into chilled microcentrifuge tubes. One microliter

of elk rPrPc coding pET41b vector was added to each tube. The cell/pET41b mixtures

were gently mixed by pipette and incubated on ice for 5 minutes. Cells were heat-

shocked for 30 seconds at 42oC, and then placed back on ice for 2 minutes. Cells were

incubated for 1 hour at 37oC in 250µL SOC medium, with gentle agitation. The cell

suspension was plated on LB agar, supplemented with 50µg/mL kanamycin and

34µg/mL chloramphenicol. Plates were left to incubate overnight at 37oC.

Individual colonies were selected, and used to inoculate 5mL minicultures grown in LB

broth with 50µg/mL kanamycin and 34µg/mL chloramphenicol. Cultures were grown

overnight at 37oC, under agitation. Glycerol stocks for each pET41b transformed

Rosetta™ II DE3 culture were made by mixing 500µL of the overnight culture (OD600

~2.5) with 1mL of sterile 60%(v/v) glycerol in Milli-Q water. Cells were stored at -80oC

until subsequent use for rPrPc overexpression and purification.

30

2.3.3 rPrPc Overexpression and Purification

The E. coli glycerol-stock containing the pET41b coding for elk rPrPc construct was used

to inoculate 5mL of LB broth, containing 50µg/mL kanamycin and 34µg/mL

chloramphenicol. A disposable 10µL inoculating loop was used to capture a small piece

of the glycerol-stock, which was then stirred into the LB broth. The miniculture was left

to grow overnight (~16 hours) at 37oC, with gentle shaking. Two millilitres of

miniculture was used to inoculate each 1L of LB broth for the rPrPc overexpressing

culture. The LB broth was supplemented Overnight Express™ Autoinduction System 1

(Millipore), 50µg/mL kanamycin, and 34µg/mL chloramphenicol. The rPrPc

overexpressing culture was incubated at 37oC for 24 hours, swirling at 100rpm on a

platform shaker.

Cells were pelleted from 250mL aliquots of the culture by centrifuging at 2500G for 20

minutes at 4oC. The supernatant was discarded and the cell pastes were collected, pooled,

and weighed. The cell pastes were frozen at -80oC in 50mL polypropylene tubes prior to

inclusion body isolation, and subsequent rPrPc purification.

2.3.4 Inclusion Body Isolation

31

Cell pastes were thawed at 37oC for 15 minutes. Cells were lysed using BugBuster®

MasterMix, and inclusion body preparation conducted as per the manufacturer’s protocol

(Millipore). For each 1g of thawed cell paste, 5mL of BugBuster® MasterMix was

added. Cell lysis was facilitated using a mechanical homogenizer on a low speed setting,

careful to avoid frothing. The lysate was incubated at room temperature for 30 minutes,

and then centrifuged at 16,000G at 4oC for 20 minutes.

The supernatant was discarded and the insoluble pellet was resuspended in BugBuster®

MasterMix using the mechanical homogenizer (5mL of reagent per 1g of original cell

paste mass). The suspension was placed on a rocking platform (low setting) for 5 minutes

at room temperature. An equal volume of 1:10 BugBuster® MasterMix was added to the

suspension; the suspension was centrifuged at 5000G at 4oC to collect the rPrP

c inclusion

bodies. The supernatant was discarded and the inclusion body was washed twice more

using 1:10 BugBuster® MasterMix as per the previous step. The inclusion body was

stored at -80oC until subsequent purification.

2.3.5 rPrPc Purification

Purification reagents and procedures were adapted from the rPrPc purification protocol

outlined in Wilham et al [171]. Inclusion bodies isolated from ~5g of cell paste were

thawed at room temperature, and then solubilized in 30mL of inclusion body

solubilisation buffer (8M GuHCl, 100mM NaH2PO4, pH 8) for 40-60 minutes under

32

gentle agitation. The suspension was centrifuged at 16,000G for 20 minutes at 22oC, and

then the supernatant was immediately decanted into a clean 50mL polypropylene tube.

The pelleted debris was discarded. The clarified supernatant was doubled in volume with

denaturing buffer (6M GuHCl, 100mM NaH2PO4, 10mM Tris∙Cl, pH 8).

A 25mL bed-volume of Superflow® Ni2+

-nitrilotriacetic acid agarose resin (Ni2+

-NTA)

(Qiagen®) had been pre-washed and incubated in denaturing buffer for 20 minutes, prior

to incubation with the solubilized inclusion body. The inclusion body suspension was

incubated with the resin for 40 minutes in a low-pressure chromatography column

(BioRad™ catalogue#: 7372522) under gentle agitation. During this time, all

chromatography buffers were vacuum-filtered through a 0.2µm filter, and degassed.

After the inclusion body suspension/resin incubation, the suspension was removed by

gently drawing it out from the bottom of the column using a 60mL syringe. This action

also lightly packed the Ni2+

-NTA resin. A small amount of suspension material was left

behind to not dry-out the resin, and to provide a submerged interface between the resin

and flow-adaptor to prevent air in the system (described in the following paragraph). The

suspension in the 60mL syringe was saved until the rPrPc purification was verified to be

successful.

All air was purged from chromatography system lines with denaturing buffer prior to

connecting the column. The flow-adaptor (BioRad™ catalogue#: 7380017) was

connected to a low-pressure BioRad™ EconoSystem. The adaptor was inserted and

fastened into the column, taking care that no bubbles were present at the adaptor/resin-

33

bed interface. The resin was washed with ~2 bed volumes of denaturing buffer at

2mL/min for 30 minutes to remove loosely and/or unbound molecules. To renature the

resin-bound rPrPc, GuHCl was gradually removed over a 4 hour linear gradient from

denaturing buffer into refolding buffer (100mM NaH2PO4, 10mM Tris∙Cl, pH 8). The

resin was equilibrated for an additional 30 minutes with refolding buffer prior to elution.

To elute the rPrPc from the resin, a 2mL/min linear gradient was applied over 1 hour,

moving from refolding buffer into elution buffer (500mM imidazole, 100mM NaH2PO4,

10mM Tris∙Cl, pH 5.6). Once the monitored A280nm value began to rise, indicating the

start of the elution peak, the eluate was collected into 50mL polypropylene tubes until the

gradient reached 100% elution buffer. The tubes with the entire elution peak were gently

mixed between tubes for a homogeneously buffered eluate. The eluate was loaded into

7kDa MWCO SnakeSkin® dialysis tubing (Thermo Scientific), and was dialyzed against

two changes of 3.6L of dialysis buffer (10mM sodium phosphate, pH 5.6) at 4oC.

Between dialysis buffer changes, the eluate was passed through a 0.2µm HT Tuffryn®

syringe filter membrane (Pall) to remove any precipitate. After the final dialysis, the

eluate was filtered once more before being stored in aliquots at -80oC.

2.4 In Vitro Conversion Assay Setup

2.4.1 Solutions and Reaction Mixtures

34

Concentrated stock solutions, working reagents, and rPrPc-conversion mixtures were

prepared in a dust-free AirClean® combination workstation. CHAPS and NDSB-201

powders were dissolved in Milli-Q water and heated in 55oC in a waterbath for 10

minutes to ensure complete dissolution of the detergent, avoiding undissolved colloids.

Concentrated stock solutions of PBSk, ThT, EDTA, CHAPS and NDSB-201 were filter

sterilized using a 0.2µm syringe filter.

All rPrPc substrate mixtures were prepared as follows: 50µg/mL rPrP

c, 1mM EDTA,

5µM ThT, and 310µM NaN3, in PBSk (pH 7.5). When making rPrPc mixtures, buffer

components were always mixed prior to the addition of rPrPc. All rPrP

c substrates were

thawed at room temperature and filtered through a 100kDa MWCO Nanosep® Omega

spin column (Pall), by centrifugation at 7500G at room-temperature, in order to remove

any impurities [171]. Concentrations of rPrPc were determined by measuring A280nm from

a 5µL sample using a Nanodrop® (Thermo). Protein concentration was determined over

an average of serial dilutions from 1:1 through 1:8. Addition of the rPrPc to the buffer

components were calculated based on the determined concentration after filtration.

The rPrPc substrate mixtures were never vortexed, and mixed only immediately prior to

adding to the fluorescence microplate. Substrate mixtures were gently rocked side-to-side

in the polypropylene tube in which they were made, and then poured into a 50mL reagent

reservoir to ensure a complete homogenous dispersion of rPrPc substrate. The mixture

was then dispensed by reverse-pipetting 98uL/well on a black 96 well optical-bottom

Nunc™ microplate (Thermo Cat#: 265301). Reverse-pipetting was preferred for two

35

reasons: precise dispensing of the rPrPc substrate mixture, and avoiding bubbles. Wells

were then “seeded” with the tissue homogenates.

2.4.2 Seeding rPrP-conversion

All conversion reactions were setup in a class II biosafety cabinet. Homogenates for

seeding rPrP-conversion reactions were thawed in 37oC water within an incubated

sonicator cup-horn, followed by sonication at high power for 20 seconds. Homogenates

were then diluted through seed-diluent (1%(w/v) CHAPS, 1M NDSB-201, PBSk),

accordingly to the experimental setup. At this point, the homogenate diluted in seed-

diluent is referred to as the seed, or seed-homogenate. This was then spiked into the rPrPc

substrate mixture to initiate rPrP-conversion into fibrils/amyloid. Two microliters of

seed-homogenate was reverse-pipetted into each well, ensuring the pipette tips were

completely submerged in the substrate, delivering precisely 2µL. Graduated microtips

were specifically used to verify 2µL were delivered to each well. Microplates were sealed

with optical sealing film and loaded into the FLUOstar Omega microplate readers (BMG

Labtech).

The final concentration of CHAPS and NDSB-201 in each conversion reaction was

0.33mM and 20mM, respectively. The CMC of CHAPS is between 8mM and 10mM

[82], so at 0.33mM and not expected to play a solubilizing role once in the conversion

reaction, nor interfere with rPrP-conversion processes. However, NDSBs are known to

inhibit spontaneous amyloid formation. For example, NDSB-201 decreases amylin

36

aggregation by ~40% at 100mM [71]. At a 1M concentration in the tissue

homogenization buffer and seed-diluent, NDSB-201 should maximize CHAPS

solubilizing efficacy [164]. However, once seeded into the conversion assay, NDSB-201

is diluted by 1:50, and the final 20mM concentration is not expected to interfere with

PrPd-seeded rPrP-conversion, using the previously published amylin data as a guide [71].

2.4.3 In Vitro rPrP-Conversion Conditions

The FLUOstar Omega plate-readers were preset to 37oC, and held at temperature for the

duration of the rPrP-conversion reaction. Fluorescence from amyloid-bound ThT was

measured using excitation and emission wavelength filters of 450±10nm and 480±10nm,

respectively (20 flashes per well, and the gain set manually to 2000) [11, 171]. Readings

were taken every 20 minutes. Throughout the assay, the microplate was subject to

constant shaking in a double-orbital pattern at 600rpm. No cyclical pauses during the

conversion reaction were imposed, as consistent shaking promotes fibril formation, as

demonstrated by Autocatalytic Conversion studies [14].

2.5 Data Interpretation and Statistical Analysis

2.5.1 Defining rPrP-conversion Time

37

The rPrP-conversion time represents the end of the lag-phase prior to fibril/amyloid

formation. This time was recorded when the ThT fluorescence signal of any given rPrP-

conversion reaction (any seeded microplate well) exceeded a statistically determined

fluorescence threshold value. Ideally, only CWD+ seed-homogenates will seed rPrP-

conversions. However, any non-specific rPrP-conversion reaction may occur, regardless

of having received a CWD+ or CWD- seed. Therefore, the primary measure to determine

the probability any given ThT signal was associated with a CWD+ or CWD- seed status

was the rPrP-conversion time. The reasoning is as follows: the longer the duration of the

assay, the more likely a spontaneous or non-specific rPrP-conversion will occur, as

previously demonstrated by Autocatalytic Conversion and ASA experiments. The rPrP-

conversion times were used to establish the diagnostic cut-off criteria the assay.

2.5.2 Diagnostic Criteria: ROC curves

Receiver-Operating Characteristic (ROC) curve analysis was used to determine the

diagnostic cut-off time for the assay. Sensitivity, specificity, and likelihood ratios were

calculated with respect to different time points throughout the assay’s duration (hours).

“Sensitivity” is a ratio determining the ability to detect true CWD+ cases, and is

calculated as follows: [The number of ThT signals generated by CWD+ seeded reaction

wells / actual number of CWD+ seeded reaction wells].

38

“Specificity” is a ratio defining the ability to correctly identify CWD- cases; it is

calculated as follows: [The total number of CWD- seeded reaction wells / (the number of

CWD- wells producing a ThT signal (false positive) + the total number of CWD- seeded

reaction wells)].

The “likelihood ratio” is calculated as: [sensitivity / 1-specificity]. This ratio describes

how many times more likely is a ThT fluorescence signal is CWD+ associated, when

specificity is not 100%. As rPrP-conversion is a time-dependant process, sensitivity,

specificity, and likelihood ratios will change as the assay progresses.

Cut-off times were established where 100% sensitivity and 100% specificity was

achieved. Likelihood ratios were reported where 100% sensitivity was achieved, but

specificity was < 100%. ROC curves were calculated using PRISM v5.0 (GraphPad).

In order to calculate ROC curve cut-off times, a value must be associated with every

reaction well on the microplate. Therefore, wells that did not produce a ThT signal were

assigned an rPrP-conversion time equal to the assay’s duration. It should be noted that

conversion data for CWD- seeded reactions are skewed, in the sense that reaction wells

that had not produced a ThT signal (no conversion) are recorded as though they had at the

end of the assay’s duration.

Univariate multifactor ANOVAs were conducted to identify the major sources of the

variance in the assay: factor interactions, their respective effect-size, and significance (p)

were recorded. Effect-sizes for each factor are reported as partial-ETA squared ( ). The

following factors were considered for their effects on rPrP-conversion time: CWD status

39

(CWD+ or CWD-) of the seed-homogenates, dilution factors, mechanical inconsistencies

between the two FLUOstar plate readers (FLUOstars), and run to run variability, where

applicable. Univariate multifactorial ANOVAs were performed using SPSS v20.0.0.1

(IBM).

In Chapters 3 to 5, an assay is presented that is a partial replica of ASA and RT-QuIC

protocols. ASA and RT-QuIC are published protocols, used frequently in the research

community. Therefore, to distinguish from other protocols, the assay herein is termed

“RePLICA” (recombinant prion-protein latent-infectivity conversion assay) to

differentiate the assay’s buffer and detergent conditions, in addition to emphasize the

assay’s method and criteria for deciphering between TSE-positive TSE-negative seeded

rPrP-conversion. By high replicate numbers of rPrP-conversion reactions, the assay

evaluates the time required for a seed-homogenate to convert rPrPc into fibrils and/or

amyloid, as indicated by amyloid-specific ThT fluorescence, in order to quantify prion-

converting activity.

40

Optimization of the RePLICA conversion buffer system Chapter Three:

3.1 Introduction

The following experiment addresses the first objective of the study. The primary goal is

to use in vitro rPrP-conversion as a method to differentiate between CWD+ and CWD-

elk tissues. The first objective is to optimize the in vitro rPrP-conversion assay seeding-

conditions (tissue input) for use in a diagnostic setting, including brain and lymphoid

tissues in the CHAPS-NDSB detergent system of RePLICA. RT-QuIC publications

mention unidentified factors in seeding material which can be inhibitory to rPrP-

conversion, and need to be removed through dilution [50, 121, 171]. As brain and

lymphoid tissue are target CWD surveillance tissues, the optimum input quantity for

these tissues needs to be determined prior to running the multianimal panel (Chapter 4).

In a diagnostic surveillance setting, a sample is usually given only one chance to test

either TSE-positive or TSE-negative on the assay platform. Therefore, the most efficient

rPrP-conversion conditions must be present so the weakest TSE-positive seeding-

homogenate elicits conversion of rPrPc into detectable amyloid. An optimal seed-

homogenate dilution will provide the shortest rPrP-conversion time for CWD+ seeded

reactions, and the best differentiation between rPrP-conversions seeded by CWD+ and

CWD- seed-homogenates. Optimal seeding-conditions will dictate the RePLICA cut-off

criteria.

41

3.2 Methods and Materials

Cerebral cortex and ileocecal lymph node tissues were homogenized from one clinically

symptomatic CWD+ elk, and one CWD- elk. Homogenization and assay setup was

conducted as described in Chapter 2. Homogenates began as 10%w/v, and were serially

diluted through seed-diluent by a ~1/3 gradient, 33.3µL into 66.7µL (100µL Vf). The

seed dilutions ranged from 1.00E-01 down to 4.54E-05. ROC curve cut-offs were

determined for each seed-homogenate dilution-set. Each dilution-set consists of one

CWD+ seed-homogenate and a corresponding CWD- seed-homogenate, at the same

dilution.

Data is presented in two ways within this chapter: First, ThT fluorescence curves are

plotted to show the raw RePLICA data output. This data illustrates the kinetic profiles of

rPrPc amyloid generated by CWD+ seed-homogenates (CWD+ Rxns), and any amyloid

which happened to form in CWD- seeded reactions (CWD- Rxns). The second data

presentation scheme is referred to as an “rPrP-conversion time-plot” (CT-plot). The

objective of the CT-plot is to illustrate the difference in rPrP-conversion times—being the

length of the lag-phase prior to amyloid formation—between CWD+ Rxns and any

occurring CWD- Rxns. It is from the CT-plot data that ROC curve cut-off times were

determined for each dilution-set.

42

3.3 Results and Discussion

RePLICA successfully demonstrates that timing of rPrP-conversion is a valid diagnostic

criterion for detecting CWD in both elk brain and lymphoid tissues. The mean baseline

reading for all RePLICA reactions was ~250 relative fluorescence units (RFU), and was

especially consistent. A 50 RFU deviation from the mean represented 10 standard

deviations from the mean baseline. To keep a consistent standard fluorescence threshold

that applied to every RePLICA run, a threshold of 500 RFU (double the baseline) was

selected.

ThT signals generated by CWD+ seed-homogenates (CWD+ Rxns) versus ThT signals

generated by CWD- seed-homogenates (CWD- Rxns) showed a clear partitioning in

rPrP-conversion time.

43

3.3.1 Brain Tissue Optimizations

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85100

1000

10000

100000CWD+ (3.33E-02)

CWD+ (1.11E-02)

CWD+ (3.69E-03)

CWD+ (1.23E-03)

CWD+ (1.36E-04)

CWD+ (4.54E-05)

CWD- (3.33E-02)

CWD- (1.11E-02)

CWD- (3.69E-03)

CWD- (4.09E-04)

CWD- (1.36E-04)

CWD- (4.54E-05)

CWD- (1.23E-03)

CWD+ (4.09E-04)

Hours

Flu

ore

scen

ce U

nit

s

Figure 1. RePLICA ThT fluorescence curves generated by CWD+ and CWD- elk

cerebral cortex seed-homogenates. Red-shaded data points (squares) represent ThT

fluorescence curves of CWD+ Rxns. Blue-shaded data points (circles) represent ThT

fluorescence curves of CWD- Rxns. The dashed-horizontal line indicates the ThT

fluorescence threshold (500 RFU), at which point the rPrP-conversion times were

recorded. Data points decrease in shading according to seed-dilution factors, listed to the

right (n = 12 per dilution).

44

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

4.54E-05

1.36E-04

4.09E-04

1.23E-03

3.69E-03

1.11E-02

3.33E-02

1.00E-01

< 42.02 hrs

< 45.21 hrs

< 32.98 hrs

< 36.15 hrs

< 41.34 hrs

< 31.52 hrs

< N/AR

OC

Cu

toff T

imes

< 59.23 hrs100% Sens

92% Spec (LR:12)

Hours (Fluorescence 500 RFU)

Tis

su

e H

om

og

en

ate

/Seed

Dilu

tio

n

Figure 2. The RePLICA CT-plot for CWD+ Rxns and CWD- Rxns shown in Figure 1

for elk cerebral cortex seed-homogenates. Each data point represents the time (hours) an

rPrP-conversion event occurred (ThT signal), defined by exceeding the 500RFU

threshold. Data points are arranged in rows, respective to the dilution-set labeled on the

left axis. Red-shaded data points (squares) represent CWD+ Rxns. Blue-shaded data

points (circles) represent CWD- Rxns. Data points at 85 hours did not produce a ThT

signal (no rPrP-conversion). Double-headed arrows illustrate the ROC curve cut-off time

determined for each dilution-set. Exact cut-off times are listed on the right axis. Assay

duration cut-off times represent 100% sensitivity and 100% specificity, other than the

4.54E-05 dilution-set. No cut-off time could be calculated for the 1.00E-01 dilution-set

due to inhibitory factors. The grey-shaded region depicts the cut-off time zone. The

vertical dashed line represents where the RePLICA was 100% specific (~45 hours) for

the dilution-sets combined; the solid vertical line represents 100% sensitive (~56 hours)

for the dilution-sets combined. Error bars represent the mean and 1 standard deviation.

45

Table 1. Mean conversion times for RePLICA reactions seeded by dilutions of CWD+

elk cerebral cortex.

Mean conversion time (Fluorescence ≥500 RFU)

Optimum dilution†

( Sub-optimum dilutions

‡

( Mean difference

*

3.69E-03 ( = 7.30)

3.33E-02 ( = 23.01) -15.71* (< 0.001)

1.11E-02 ( = 11.14) -3.84 (= 0.808)

1.23E-03 ( = 7.51) -0.22 (= 1.000)

4.09E-04 ( = 9.07) -1.76 (= 0.995)

1.36E-04 ( = 10.43) -3.12 (= 0.917)

4.54E-05 ( = 22.44) -15.14* (< 0.001)

† The “Optimum dilution” represents the dilution factor of a 10%(w/v) CWD+ seed-

homogenate (elk cerebral cortex) providing the shortest mean rPrP-conversion time (ThT

signal).

‡ “Sub-optimum dilutions” are dilutions of a 10%(w/v) CWD+ seed-homogenate (elk

cerebral cortex) which had lengthier mean rPrP-conversion times. “Mean difference”

indicates how many hours, on average, the “Optimum dilution” of a 10%(w/v) CWD+

seed-homogenate (elk cerebral cortex) produced an rPrP-conversion event sooner than

the respective “Sub-optimum dilution”.

*Mean differences and significance (p-values) were determined using univariate ANOVA

with Tukey HSD post-hoc testing.

46

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850

10

20

30

40

50

60

70

80

90

100Sensitivity%

Specificity%

LR

Hours

%an

d L

ikelih

oo

d R

ati

o (L

R)

Figure 3. RePLICA’s change in sensitivity versus specificity as the assay duration

progresses for the elk cerebral cortex seed-homogenate dilution-sets in Figure 2. The

shaded region represents the time range where RePLICA’s sensitivity and specificity are

≥95%. The dashed vertical line represents where sensitivity equals specificity. Likelihood

ratio (LR) is shown when specificity is < 100%.

47

CWD+ cerebral cortex homogenates seeded rPrP-conversion as soon as 5 hours. No

CWD+ cerebral cortex seed-homogenates diluted at 1.00E-01 yielded a ThT positive

signal, and remained inhibitory over the course of the RePLICA’s duration (85 hours).

Further diluting the CWD+ cerebral cortex homogenate to 1/30 (3.33E-02), or further

resulted in consistent CWD+ Rxns (Figure 2).

Diluting CWD+ brain homogenate by 3.69E-03 resulted in the most efficient CWD+

Rxns. CWD+ replicates in this dilution-set had undergone conversion between 5 and 10

hours. The mean CWD+ Rxn time for all dilutions was 12.99 ±9.11 hours. Replicates at

this dilution also exhibited the least amount of variance, thus the most consistent, in

conversion time of all CWD+ dilution-sets. Therefore, diluting elk cerebral cortex to

3.69E-03 (~1/270) appears to provide the optimal seeding-conditions. This suggests in a

diagnostic setting, diluting cerebral cortex to ~1/270 may provide the best possible

sensitivity for detecting weak CWD+ elk tissue samples from preclinical elk, without

over diluting the homogenate.

Table 1 compares how the other CWD+ brain dilutions compared to the 3.69E-03

dilution. Dilutions between 1.11E-02 and 1.36E-04 did not significantly differ between

one another in terms of conversion time, with means fluctuating between 7.30 hours and

11.14 hours. Virtually no difference was seen between the 3.69E-03 and 1.23E-03

dilutions. This shows once inhibitory factors have been diluted away, a surprising amount

of flexibility exists in terms of optimally seeding the assay.

48

Established cut-off times were between ~31 hours and 45 hours, exhibiting 100%

sensitivity and 100% specificity (p < 0.001) (Figure 2). The 4.54E-05 dilution-set

achieved 100% sensitivity at 92% specificity with a likelihood ratio of 12 at 59.23 hours.

The likelihood ratio indicates that conversion reactions occurring between ~45 hours

(where the dilution last maintain 100% specificity) and ~59 hours are 12 times more

likely to have been seeded by a CWD+ associated event.

The ROC curve sensitivity and specificity coordinates for the amalgamated dilution-set

data is shown in Figure 3. More than 70 of 100 CWD-positive seeded wells can be

expected to generate signals between 5 hours and 15 hours. No reaction occurs before ~5

hours. A broad corridor exists between ~34 hours and ~59 hours where both sensitivity

and specificity are ≥ 95%, intersecting one another at ~47 hours (Figure 3). This is a

remarkable result, as some RT-QuIC publications note not much sensitivity is gained

beyond 45 hours [171].

In RePLICA, beyond ~47 hours the likelihood ratio drops significantly as specificity

declines, increasing the potential for false-positive results. In a diagnostic setting,

between 30 and 34 hours represents a conceivable time range where cut-off times are

likely to be established to maintain high specificity. An elk brain seed-homogenate (or

sample) generating a ThT signal around this time should be thoroughly scrutinized, and

repeated in higher replicate numbers.

The CWD status of the cerebral cortex (CWD+ vs. CWD-) was the variable with the most

profound effect on conversion time ( =0.94, p < 0.001). No statistical interactions

49

existed within the combined data from both FLUOstars suggesting either of the machine

significantly influenced rPrP-conversion times with respect to the CWD status of the

seeds ( 0.001, p=0.764). CWD- Rxns appeared to occur rather stochastically.

Seventy one of 84 CWD- seed-homogenates remained non-reactive. Of the 13 CWD-

Rxns that occurred, the earliest conversion time was after 46 hours, 10 hours past the

beginning of the cut-off time zone (30-34 hours).

50

3.3.3 Lymphoid Tissue Optimization

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

100

1000

10000

100000

CWD- (1.00E-01)

CWD- (3.33E-02)

CWD- (1.11E-02)

CWD- (3.69E-03)

CWD- (1.23E-03)

CWD- (1.36E-04)

CWD- (4.54E-05)

CWD+ (1.00E-01)

CWD+ (3.33E-02)

CWD+ (1.11E-02)

CWD+ (3.69E-03)

CWD+ (1.23E-03)

CWD+ (1.36E-04)

CWD+ (4.54E-05)

CWD+ (4.09E-04)

CWD- (4.09E-04)

Hours

Flu

ore

sc

en

ce

Un

its

Figure 4. RePLICA ThT fluorescence curves generated by CWD+ and CWD- elk

ileocecal lymph node seed-homogenates. Red-shaded data points (squares) are ThT

fluorescence curves of CWD+ Rxns. Blue-shaded data points (circles) are ThT

fluorescence curves of CWD- Rxns. The dashed-horizontal line indicates the ThT

fluorescence threshold (500 RFU) at which point the rPrP-conversion times were

recorded. Data points decrease in shading according to seed-dilution factors, listed to the

right (n = 12 per dilution).

51

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

4.54E-05

1.36E-04

4.09E-04

1.23E-03

3.69E-03

1.11E-02

3.33E-02

1.00E-01

< 38.91 hrs

< 25.20 hrs

< 21.68 hrs

< 20.57 hrs

< 45.58 hrs

< 49.29 hrs

< 38.54 hrs

< 39.46 hrs

RO

C C

uto

ff Tim

es

Hours (Fluorescence 500 RFU)

Tis

su

e H

om

og

en

ate

/Seed

Dilu

tio

n

Figure 5. The ReLICA CT-plot for CWD+ Rxns and CWD- Rxns shown in Figure 4 for

elk ileocecal lymph node seed-homogenates. Each data point represents the time (hours)

an rPrP-conversion event occurred (ThT signal), defined by exceeding the 500RFU

threshold. Data points are arranged in rows, respective to the dilution-set labeled on the

left axis. Red-shaded data points (squares) are represent CWD+ Rxns. Blue-shaded data

points (circles) represent CWD- Rxns. Data points at 85 hours were non-reactive.

Double-headed arrows illustrate the ROC curve cut-off time determined for each

dilution-set. Exact cut-off times are listed on the right axis. Posted cut-offs represent

100% sensitivity and 100% specificity. The grey-shaded region depicts the cut-off time

zone. The vertical line represents where the RePLICA was 100% specific (~31 hrs). Error

bars represent the mean and 1 standard deviation.

52

Table 2. Mean conversion times for RePLICA reactions seeded by dilutions of CWD+

elk ileocecal lymph node.

Mean conversion time (Fluorescence ≥500 RFU)

Optimum dilution†

( Sub-Optimum dilutions

‡

( Mean difference

*

1.11E-02 ( = 4.97)

1.00E-01 ( = 14.85) -9.88* (< 0.001)

3.33E-02 ( = 11.21) -6.24* (= 0.001)

3.69E-03 ( = 5.43) -0.46 (= 1.000)

1.23E-03 ( = 6.36) -1.39 (= 0.976)

4.09E-04 ( = 7.63) -2.66 (= 0.574)

1.36E-04 ( = 9.23) -4.27 (= 0.064)

4.54E-05 ( = 15.60) -10.62* (< 0.001)

†The “Optimum dilution” represents the dilution factor of a 10%(w/v) CWD+ seed-

homogenate (elk ileocecal lymph node) providing the shortest mean rPrP-conversion time

(ThT signal).

‡“Sub-optimum dilutions” are dilutions of a 10%(w/v) CWD+ seed-homogenate (elk

ileocecal lymph node) which had lengthier mean rPrP-conversion times. “Mean

difference” indicates how many hours, on average, the “Optimum dilution” of a

10%(w/v) CWD+ seed-homogenate (elk cerebral cortex) produced an rPrP-conversion

event sooner than the respective “Sub-optimum dilution”.

*Mean differences and significance (p-values) were determined using univariate ANOVA

with Tukey HSD post-hoc testing.

53

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850

10

20

30

40

50

60

70

80

90

100Specificity%

Sensitivity%

LR

Hours

%an

d L

ikelih

oo

d R

ati

o (

LR

)

Figure 6. RePLICA’s change in sensitivity versus specificity as the assay duration

progresses for the elk ileocecal lymph node dilution-sets in Figure 5. The shaded region

represents the time range where RePLICA’s sensitivity and specificity are ≥95%. The

dashed vertical line represents where sensitivity equals specificity. Likelihood ratio (LR)

is shown when specificity is < 100%.

54

0 20 40 60 80

0

5000

10000

15000

CWD+ (1.00E-01)

CWD- (1.00E-01)

A

Hours

Flu

ore

sce

nce

Un

its

0 20 40 60 80

0

5000

10000

15000

CWD+ (3.33E-02)

CWD- (3.33E-02)

B

Hours

Flu

ore

sce

nce

Un

its

0 20 40 60 80

0

5000

10000

15000

CWD+ (1.11E-02)

CWD- (1.11E-02)

C

Hours

Flu

ore

sce

nce

Un

its

0 20 40 60 80 100

0

5000

10000

15000

CWD- (3.69E-03)

CWD+ (3.69E-03)

D

log

10

RF

U

log10 Hours

Hours

Flu

ore

sce

nce

Un

its

Figure 7. The effect of dilution on CWD+ and CWD- Rxns’ kinetic profiles and

conversion time, seeded by ileocecal lymph node, in the RePLICA buffer system. Panels

are organized where seeding-homogenates become more dilute, panel A being the least

diluted. The grey fitted-curve in panel A is represents a Gompertz sigmoidal function

(Equation 1, R2 = 0.9525). The solid black curve in panel D (and inset) is a power-series

function reflecting the transition from the lag-phase to exponential phase (Equation 2, R2

= 0.9778). The dashed curve illustrates a second function of ThT signal fluorescence

decay (Equation 3, R2 = 0.2013).

55

Similar to the cerebral cortex seed-homogenates, diluting the lymph node seed-

homogenates significantly affected the time a CWD+ Rxn would occur ( = 0.596, p <

0.001). Neither FLUOstar influenced CWD+ Rxns more than the other ( = 0.008, p =

0.428), indicating that results from CWD+ tissues were highly reproducible between the

FLUOstars. The earliest CWD+ Rxn seeded by ileocecal lymph node occurred at 4 hours.

In contrast to brain, lymphoid tissues displayed far less conversion inhibition in less

diluted homogenates. Conversion reactions seeded by CWD+ lymph node homogenates

displayed shorter lag-phases than reactions seeded by brain tissues.

In addition, lymphoid tissues did not require as much dilution as brain tissues to

overcome inhibitory effects. Diluting lymph homogenate by 1.11E-02 in seed-diluent

prior to seeding the RePLICA will optimally seed CWD+ Rxns (Table 2). Dilutions

between 1.11E-02 and 4.09E-04 did not perform significantly better than the other,

although the 1.11E-02 dilution exhibited the least amount variance. Therefore, the 1.11E-

02 dilution was the most consistently performing dilution without having to further

sacrifice PrPCWD

content. This dilution (~1/90) was selected as the optimal seeding-

dilution for lymphoid tissues, in terms of diagnostic purposes.

Since lymphoid homogenates seeded conversion reactions sooner than brain

homogenates, the shaded ROC curve cut-off zone in Figure 5 also advanced by

approximately 10 hours. In addition, a 100% sensitive and 100% specific cut-off time

could be established for each tested dilution-set. Figure 6 is a graphical representation of

ROC curve sensitivity versus specificity coordinates over the duration of the RePLICA

assay for the dilutions of ileocecal lymphoid tissues. By 31 hours, all dilutions were

56

100% sensitive and specific for deciphering between CWD+ from CWD- seed-

homogenates reactions.

It can be expected that approximately 95 of 100 true CWD+ lymphoid seed-homogenates

will elicit CWD+ Rxns within 17 hours. The earliest CWD- Rxn occurred at 32 hours

(Figures 4 and 5), far beyond the expected conversion time for CWD+ Rxns. The cut-off

time zone where both sensitivity and specificity were ≥95% was quite broad, extending

between 18 and 52 hours. This is likely due to the more volatile seeding nature of

lymphoid tissues. However, ~31 hours seems to be a cut-off time shared by all dilutions-

sets where 100% sensitivity and specificity could be achieved (Figures 5 and 6).

Like brain tissues, rPrP-conversion times for lymphoid tissues (Figure 5) were dominated

by the CWD status of the seeds ( = 0.949, p < 0.001). In this particular instance, one of

FLUOstars had shown a higher tendency to produce more CWD- Rxns than the other (p

< 0.05), however the effect size ( = 0.032) was so small the differences were

irrelevant. This did not impact the ability to accurately decipher CWD+ from CWD-

lymph node.

In addition to characterizing the assay for diagnostic purposes, the diluted lymph node

seed-homogenates provide some unique insight pertaining to their seeding behaviour in

the assay (Figure 7). Diluting CWD+ lymph node homogenate from 1.00E-01 to 3.69E-

03 increases rPrP-conversion efficiency. This is indicated by reduced lag-phases and tight

conversion time replicates, showing little variance (Figure 7). The panels in Figure 7

show the mean lag-phase for CWD+ Rxns is reduced from ~15 to ~5 hours, by diluting

57

lymph node homogenate from 1.00E-01 to 1.11E-02, respectively (also seen in Table 2).

The precision in conversion time is remarkably consistent between replicates (n=12).

However, a more interesting result was the propensity for CWD- lymph node seeds to

unanimously seed CWD- Rxns conversions when diluted at 1.00E-01. Upon further

dilution of the CWD- lymph homogenates, the occurrence of conversion diminished. This

effect was not seen from brain tissues, indicative something inherent about lymph node

tissue is able to cause rPrPc aggregation. In addition, the fluorescence kinetic profiles of

the CWD- lymph node seeded CWD- Rxns curves (Figure 7A) are distinguishable from

profiles of CWD+ Rxns. All 12 CWD- Rxns show lower maximum RFU values than the

lowest CWD+ Rxn.

Moreover, close examination of the data points between CWD+ and CWD- Rxns show

CWD- Rxns have greater vertical distances between the data points, especially during the

beginning of the exponential phase of the curve. This perhaps indicates the nucleation

and fibrilizing dynamics of CWD- Rxns occur at a faster rate compared to the CWD+

Rxns. It is interesting to note that despite their apparently slower fibrilizing rate, CWD+

Rxns seed conversion between 3 to 10 times sooner than the CWD- Rxns. This suggests

CWD+ Rxns may occur via a different mechanism than CWD- Rxns conversions. The

CHAPS-NDSB detergent combination of the RePLICA system may provide

opportunities to structurally characterize PrP-fibril assembly, seeded by infectious elk

CWD.

58

The fluorescence curve profile of the 1.00E-01 CWD+ lymph node dilutions exhibit a

sigmoidal behaviour not seen in lower dilutions (Figure 7A). The amyloid formation

kinetics of CWD+ Rxns seeded by lymph node dilutions of 1.00E-01 could be fit with the

following Gompertz sigmoidal function:

Equation 1:

[ (

) ] ,

The value “ ” is the upper asymptote (max fluorescence) of the system; and are

constants, and is time and is the baseline fluorescence. The sigmoid function is an

accurate description of the model (R2 = 0.9525) and supported by a replicates fit test (p =

1.000). This function could not be applied to CWD+ Rxns seeded by more dilute lymph

node seed-homogenates.

CWD+ lymph node dilutions of 3.33E-02, and lower, show a sharp exponential increase

to a maximum RFU between 10,000 and 15,000. They are then followed by phase of ThT

signal decay. Figure 7D illustrates two separate events contributing to this fluorescence

profile. The transition from lag-phase to exponential-phase of the ThT signal can be

characterized by a power-series (Figure 7D) (GraphPad PRISM 5.0):

Equation 2:

,

59

When a maximum or peak RFU value is achieved by around 14 hours, a decay function

follows:

Equation 3:

( )

The value “ ” in equation 3 is the decay rate constant , is time, is the initial

fluorescence value beginning the decay phase (or the maximum fluorescence achieved by

the exponential phase), and is the bottom plateau (GraphPad PRISM 5.0). It might be

surmised that decay phase is due to ThT being intercalated into rPrP amyloid, quenching

the fluorescent properties [102]. The lack of this effect in Figure 7A, seeded by the

1.00E-01 dilutions, suggests the fibrils and/or amyloid are forming differently. Given

every one of the 12 CWD+ replicates and 12 CWD- replicates exhibit the same profile,

the process is repeatable and unlikely to be a coincidence.

The RePLICA conversion system provides a consistent platform, where timed seeded-

conversion of rPrPc is statistically able to decipher between CWD+ and CWD- elk brain

and lymphoid tissues. Both CWD+ and CWD- lymphoid seed-homogenates appeared

more volatile at seeding rPrP-conversion than brain tissues, though accuracy of the test

was unaffected. The assay possesses good precision regarding rPrP-conversion time by

CWD+ seeds. Optimal seeding-conditions were successfully determined for brain and

lymphoid tissues. A 10%(w/v) brain homogenate should be diluted by 3.69E-03 (~1/270)

in the CHAPS-NDSB seed-diluent. A 10%(w/v) lymph node homogenate should be

60

diluted to 1.11E-02 (~1/90) in the CHAPS-NDSB seed-diluent. Seeding RePLICA rPrP-

conversion reactions with these seed homogenates gives the best CWD+ Rxn efficiency.

In addition, the RePLICA buffer system showed CWD- lymphoid tissues possess an

ability to seed rPrP-conversion when under diluted. This was not seen by brain tissues. In

addition kinetics profiles of amyloid formation by CWD+ tissues can be distinguished

from CWD- seeded conversion. The precision and reproducibility of the RePLICA buffer

system may allow for more perspectives on PrPd seeded fibril assembly, perhaps even

structurally.

61

Performance of the RePLICA Conversion System on a Multianimal Chapter Four:

Elk Panels.

4.1 Introduction

The following experiment addresses the second project objective: to simulate a diagnostic

test scenario using the optimized in vitro rPrP-conversion seeding-conditions on panels of

multiple elk brain and lymphoid tissues. Elk tissues were supplied from a previous time-

course experiment conducted at the Lethbridge Laboratory. The elk had been orally

challenged with CWD+ material. Groups of 2 CWD+ (challenged) and 1 CWD- sham

inoculated had been euthanized at various time-points (days) post-inoculation (DPI). The

objective was to put-to-test the validity of timing rPrP-conversion as a plausible

diagnostic method to detect elk CWD, using the diagnostic methodology and criteria

outlined in Chapter 3.

4.2 Methods and Materials.

Cerebral cortex and tonsil tissues from 4 CWD- (control) and 10 CWD+ elk, euthanized

at different DPIs, were homogenized as previously described (Chapter 2). Brain (cerebral

cortex) and tonsil homogenates were diluted in the CHAPS-NDSB seed-diluent were

diluted accordingly to their optimal seeding-parameters as determined (Chapter 3).

62

Therefore, cerebral cortex was diluted to 3.69E-03, and tonsil (lymphoid tissue) was

diluted to 1.11E-02. RePLICA reactions were setup, seeded, and conducted as described

in Chapter 2. Elk animal/tissue IDs remain as originally randomly assigned.

63

4.3 Results and Discussion

4.3.1 Multianimal Brain Panel

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

Elk#169 LM 784DPI +/Clin+(ea

Elk#180 MM 760DPI -/Clin-

Elk#181 LM 760DPI -/Clin-

Elk#168 MM 748DPI +/Clin+

Elk#148 MM 685DPI +/Clin+(di

Elk#142 MM 650DPI +/Clin+

Elk#48 MM 525DPI +/Clin-

Elk#46 LM 525DPI -/Clin-

Elk#49 MM 525DPI +/Clin-

Elk#189 MM 400DPI -/Clin-

Elk#188 MM 400DPI +/Clin-

Elk#187 MM 400DPI +/Clin- < 44.5 Hrs100%Sens.100%Spec.

A

Hours (Fluorescence 500 RFU)

Elk

Sam

ple

(D

ilu

tio

n:

1.1

1E

-02)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

Elk#169 LM 784DPI +/Clin+(ea

Elk#180 MM 760DPI -/Clin-

Elk#181 LM 760DPI -/Clin-

Elk#168 MM 748DPI +/Clin+

Elk#148 MM 685DPI +/Clin+(di

Elk#142 MM 650DPI +/Clin+

Elk#48 MM 525DPI +/Clin-

Elk#46 LM 525DPI -/Clin-

Elk#49 MM 525DPI +/Clin-

Elk#189 MM 400DPI -/Clin-

Elk#188 MM 400DPI +/Clin-

Elk#187 MM 400DPI +/Clin-

B< 33.0 Hrs100%Sens.100%Spec.

Hours (Fluorescence 500 RFU)

Elk

Sam

ple

(D

ilu

tio

n:

3.6

9E

-03)

64

Figure 8. A RePLICA CT-plot of elk cerebral cortex seed-homogenates from multiple

animals. Sub-optimally diluted seeds (1.11E-02) are shown in Panel A. Optimally diluted

seeds (3.69E-03) are shown in Panel B. “DPI” indicates the disease incubation period for

the respective elk prior to euthanization. Solid squares represent CWD+

inoculated/clinically symptomatic elk. Half squares are CWD+/asymptomatic elk. Circles

are CWD- elk. Black data points are elk with PRNP 132MM, while grey are 132LM. The

shaded area represents the time zone where ROC curve cut-off times were typically

established for cerebral cortex seed-homogenates from Figure 2. The dashed vertical line

represents the cut-off time for the particular dilution of the homogenates in the trial (cut-

off data listed upper-right (p < 0.001)).

65

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

Elk#169 LM 784DPI +/Clin+

Elk#180 MM 760DPI -/Clin-

Elk#181 LM 760DPI -/Clin-

Elk#168 MM 748DPI +/Clin+

Elk#148 MM 685DPI +/Clin+

Elk#142 MM 650DPI +/Clin+

Elk#48 MM 525DPI +/Clin-

Elk#46 LM 525DPI -/Clin-

Elk#49 MM 525DPI +/Clin-

Elk#189 MM 400DPI -/Clin-

Elk#188 MM 400DPI +/Clin-

Elk#187 MM 400DPI +/Clin-

100%LR: n/a

99%LR:96

98%LR:48

96%LR:24

100% Sensitive

97%LR:32

Specificity

Hours (Fluorescence 500 RFU)

Elk

Sam

ple

(D

ilu

tio

n:

3.6

9E

-03)

Figure 9. A RePLICA CT-plot depicting how sensitivity and specificity can be

interpreted in the cut-off time zone as the assay’s duration progresses. The data account

for two amalgamated RePLICA runs (each seeded with 3.69E-03 dilutions of elk cerebral

cortex seed-homogenates), the use of two different batched of elk rPrPc substrate, and 2

FLUOstar plate-readers. Eight replicates of each individual seed-homogenate were run

per microplate, per FLUOstar, per trial/rPrPc batch (n = 24 per elk homogenate). ROC

curve cut-off times (vertical lines) were established using CWD+/clinical PRNP 132MM

elk (black squares), and all CWD- control elk (circles). Half-filled squares are

CWD+/asymptomatic elk. Grey data points are PRNP 132LM elk. By vertical line “1”

(~35 hours), the ROC curve cut-off is 100% sensitive and specific with respect to the

ROC curve controls. The following lines indicate 100% sensitivity, though decreasing

specificity (listed at right). The shaded area is the cut-off time zone identified for brain

homogenate seeds, defined by Figure 2. Error bars means and standard deviation.

66

RePLICA reactions seeded by cerebral cortex homogenates having been diluted to 1.11E-

02 (sub-optimal, Figure 8A) and 3.69E-03 (optimal, Figure 8B) in seed-diluent prior to

seeding. This is an important figure because it demonstrates the effect of inadequate

dilution of the seed-homogenate impairing the proper detection of a weak CWD+ sample.

The significant difference lies within the CWD+ Rxns seeded by Elk#169. This animal is

of PRNP 132LM genotype, and was allowed to incubate CWD for 784 days. Figure 8A

shows a 1.11E-02 dilution of the brain homogenate yielded only 3/8 conversion reactions

before the 45 hour cut-off, 6/8 CWD+ Rxns the end of the cut-off time zone (~31 to 59

hours), and 2/8 replicates were non-reactive. In contrast, when Elk#169 seed-homogenate

was optimally diluted to 3.69E-03, 7/8 CWD+ Rxns occurred before the 33 hour cut-off

time.

The assay was 100% sensitive for CWD+ PRNP 132MM elk, euthanized at 525DPI and

later. RePLICA could not accurately detect CWD in CWD+ elk euthanized at 400DPI. At

the 1.11E-02 dilution, one false-positive (CWD- Rxn) conversion was present by

Elk#189, at 25 hours (45 hours cut-off), confounding a positive result from the CWD+

Elk #188. In Figure 8B, the optimally diluted brain seed-homogenates (3.69E-03)

accelerated all CWD+ Rxns by 5 to 10 hours. Two positive CWD+ Rxns were generated

by the two CWD+ elk (Elk#187 and Elk#188) euthanized 400DPI. Seeding propensity

had increased with respect to the CWD- controls Elk#46, Elk#181, and Elk#180,

however no negative control seeded an CWD- Rxns conversion prior to the ascribed 33

hour cut-off.

67

The assay in Figure 8B was repeated for consistency purposes using a different elk rPrPc

substrate batch and different aliquots of elk cerebral cortex homogenates diluted to

3.69E-03. The repeated RePLICA results are overlaid with the results from Figure 8B

(see Figure 9). None of the CWD+ Rxns were affected by individual machine effects (

= 0.005, p = 0.385). Conversion times of all seed-homogenates did differ slightly

depending on elk rPrPc batch (p < 0.001), though the differences were negligible (

=

0.117), which support the assay’s consistency.

A ROC curve analysis was performed on the amalgamated data to see how the data might

be interpreted in a diagnostic setting (Figure 9). ROC curves were generated using

CWD+ Rxns from CWD+/clinical PRNP 132MM elk and all CWD- control elk. Cut-off

times for 100% sensitivity are drawn on Figure 9, with corresponding decreasing

specificity as the assay duration progresses. Both amalgamated RePLICA trials in Figure

9 generated 100% sensitivity and specificity at 34.5 hours, very close to the ~33 hours

cut-off for the 3.69E-03 dilution calculated in Figure 2. This further highlights the

reproducibility of the assay, and support for a 30 to 34 hour assay cut-off time. The third

cut-off time in Figure 9 at 48 hours shows 100% sensitivity and 98% specificity (LR:48).

This includes 1 false-positive from Elk#46, though more importantly, has identified

21/24 replicates from Elk #169, and 2 positive replicates from each of the preclinical elk,

#187 and #188.

Figure 9 depicts exemplifies how to interpret RePLICA data in a diagnostic setting,

allowing for a type of grading scale over a cut-off time-zone. The example should be the

CWD+ Rxns by Elk#169. The mean rPrP-conversion times seeded by elk #168, #148,

68

#142, #48, and #49 are between are between 9 and 14 hours, and not significantly

different from one another (p = 0.570). Elk#169 has a mean conversion time of 28.8

hours—distributed nearly exactly the established cut-off time—with a wide variance,

supporting the need to scrutinize rPrP-conversion times in this range. The distribution of

Elk#169 rPrP-conversion times are significantly different from both the CWD- controls

(p < 0.05) and CWD+ results from elk #168, #148, #142, #48, and #49 (p < 0.05).

69

4.3.2 Multianimal Tonsil Panel

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Elk#169 LM 784DPI +/Clin+

Elk#168 MM 748DPI +/Clin+

Elk#181 LM 760DPI -/Clin-

Elk#180 MM 760DPI -/Clin-

Elk#148 MM 685DPI +/Clin+

Elk#142 MM 650DPI +/Clin+

Elk#46 LM 525DPI -/Clin-

Elk#188 MM 400DPI +/Clin-

Elk#187 MM 400DPI +/Clin-

Elk#189 MM 400DPI -/Clin-

Elk#52 MM 300DPI+/Clin-

Elk#51 LM 300DPI +/Clin-

AR

eP

LIC

A T

rial #

1

Hours (Fluorescence 500 RFU)

Elk

Sam

ple

ID

(D

iluti

on

: 1.1

1E

-02)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

Elk#169 LM 784DPI +/Clin+

Elk#168 MM 748DPI +/Clin+

Elk#181 LM 760DPI -/Clin-

Elk#180 MM 760DPI -/Clin-

Elk#148 MM 685DPI +/Clin+

Elk#142 MM 650DPI +/Clin+

Elk#46 LM 525DPI -/Clin-

Elk#188 MM 400DPI +/Clin-

Elk#187 MM 400DPI +/Clin-

Elk#189 MM 400DPI -/Clin-

Elk#52 MM 300DPI+/Clin-

Elk#51 LM 300DPI +/Clin-

B

Re

PL

ICA

Tra

il #2

Hours (Fluorescence 500 RFU)

Elk

Sam

ple

ID

(D

iluti

on

: 1.1

1E

-02)

70

Figure 10. A RePLICA CT-plot of elk tonsil seed-homogenates from multiple animals.

Panels A and B show the performance of two different batches of elk rPrPc, seeded by

CWD+ and CWD- elk tonsil homogenate, repeated on two different RePLICA trials,

using two FLUOstar plate-readers. The shaded area is the cut-off zone established for

lymphoid tissues (Figure 5). The vertical dashed-lines represent a ROC curve cut-off of

100% sensitivity/specificity for each panel (specific to the ROC curve controls). ROC

curve cut-off times were established from CWD+ /clinical PRNP 132MM elk (black

squares), and all CWD- elk control (circles) data. Half-filled squares are CWD+

/asymptomatic elk. Grey data points are elk which are PRNP 132LM. Error bars means

and standard deviation. Sample number breakdown: n = 32/elk (total), 16/elk/panel,

8/elk/FLUOstar/panel.

71

The RePLICA correctly identified CWD+ tonsils from elk euthanized at 400DPI and later

(Figure 10), including Elk#169 which had varying CWD+ Rxns times in the brain tissue

panel. Elk#48 and #49 from the brain tissue panel were exchanged for Elk#51 and

Elk#52 which were euthanized at 300DPI. Elk#48 and #49 were already known to have

PrPCWD

in the tonsil, however it had yet to be detected in the tonsils in animals

euthanized at 300DPI. Since CWD infectivity is can sometimes be better found in lymph

tissues before brain [10, 51], it was anticipated the RePLICA would be able to detect

CWD in these tissues. Unfortunately, the RePLICA was not able to detect CWD in the

elk euthanized at 300DPI.

For some reason there was a large, significant difference in conversion times between the

two tested elk rPrPc batches used on the separate trials (

= 0.421, p < 0.001), seen in

Figure 10A versus 10B. It is unclear why the tonsils showed more variability than the

brain tissues. An equipment change had recently been employed around the time the

assay was conducted (new sonicating cuphorn), which could have been more damaging

to the CWD+ seeding body (CHAPS-DRM/microdomains, etc.). Other factors might

include an unknown reagent quality issue, or perhaps miscalculation of buffer

components. In any case, an interesting effect is seen where despite the large significant

difference, it did not change the capability of the assay in terms of specificity or

sensitivity.

The ROC cut-off time can move accordingly to a shared decrease (or increase) in rPrP-

conversion efficiency between CWD+ and CWD- seeded reactions. The first elk rPrPc

batch (Figure 10A) showed accelerated CWD+ Rxns, though volatility within CWD-

72

Rxns was also increased. For the second elk rPrPc batch (Figure 10B), though conversion

lag-phases for CWD+ Rxns were 10 hours longer, little to zero CWD- Rxns occurred.

In Figure 10, the cut-off times for each trial (A and B) were calculated using the

CWD+/clinical PRNP132MM elk as positive control values, and all CWD- control elk as

negative data. The cut-off time calculated for the first elk rPrPc batch (Figure 10A) was

23 hours, and 30 hours for the second batch (Figure 10B). To the diagnostician’s

advantage, the ROC curve cut-off time can be recalculated to account for different rPrP-

conversion efficiencies. When the conversion activity of the individual tonsil

homogenates are compared relative to themselves versus either elk rPrPc batch, the effect

is negligible ( = 0.065).

When the total variance contributed by all factors in Figures 10A and 10B is considered,

this interaction effect only accounted for 0.4% of the variance in the corrected model.

This also accounts for the aforementioned unknown variables affecting rPrP-conversion

efficiencies between Figures 10A and 10B. No significant three-way ANOVA

interactions between individual tonsil seed-homogenate, FLUOstar used, or elk rPrPc

batch was found to influence PrP-conversion time ( = 0.037, p = 0.239). Therefore,

CWD+ and CWD- seeded conversion efficiencies move relative to one-another.

The 100% sensitivity and specificity cut-off time for the optimal lymph tissue dilution

(1.11E-02) was ~25 hours (Figure 5). Despite that conversions in Figure 10B appear to be

sub-optimal in terms of the lengthened lag-phase, the calculated cut-off time of 30 hours

73

still matches the cut-off time of ~31 hours for all the lymph tissue dilutions previously

determined (see Figure 5).

Both panels of brain and tonsil tissues performed well within the anticipated parameters

as per the RePLICA optimization experiments (Chapter 3). Optimally seeded RePLICA

reactions with the CHAPS-NDSB buffer system perform with consistency on both brain

and tonsil tissues. RePLICA was successful in detecting CWD in brains of preclinical

PRNP 132MM elk which were incubating CWD for 525 days or more. The assay could

detect CWD in the tonsils of PRNP 132MM elk incubating CWD for 400 days or more.

It was shown that the conversion time of CWD+ and CWD- seeded reactions appear to

move relative to one another. The ROC curve determined cut-off time is mobile, and can

be adjusted to accommodate the system’s conversion efficiency.

For use as a diagnostic assay, it is important to scrutinize any rPrP-conversion reactions

occurring within the cut-off time-zone, which appears to begin around the 30 hour mark

(depending on tissue type). By example, brain tissues from Elk#169 displayed a mean

CWD+ Rxn time situated directly over established cut-off time. If this tissue was tested

using only a single reaction well at the optimally seeded dilution, there is a ~1:8 chance

the CWD+ Rxns time would occur after the established cut-off, though remain in the cut-

off zone. The validity of a conversion reaction can be verified through replicates and a

distribution analysis of the seed-homogenate’s mean conversion time. A model similar to

the one in Figure 9 would help interpretations.

74

Evaluating the Sensitivity of the RePLICA Conversion System versus Chapter Five:

Bioassay

5.1 Introduction

The following experiment addresses the third objective which is to evaluate the

RePLICA’s sensitivity for elk CWD infectivity using diagnostic criteria based on rPrP-

conversion time, determined in the first objective. Bioassay is used to determine the

infectious potential of a biological material to facilitate prion-disease, and is carried-out

with either tissue-culture or animal models [22, 32]. The assay is time-sensitive: Post-

inoculation, a latent period precedes the dichotomous outcome of whether or not the

animal becomes sick with a TSE. As the infectious material is diluted pre-inoculation, the

latent period is increased [22].

It has been reported that the RT-QuIC is at least as sensitive for detecting PrPd as

bioassay models [50, 171], however the analysis is focused a dichotomous outcome

whether or not a ThT signal is generated. Though recognized as being synonymous to the

latent-period of disease, rPrP-conversion time (or end of the lag-phase) is not formally

considered in the data analysis. Incorporating time as a scalar outcome creates another

dimension for analysis. RePLICA has set criteria to include rPrP-conversion time to

validate any given ThT signal. Therefore, rPrP-conversion events can also be transformed

into a continuous outcome (time), useful for generating a quantifiable response ratio in

conjunction to the dilution of CWD+ seed-homogenates.

75

The term seeding-potential could be used to describe the ability of all seed-homogenate

components to generate a ThT positive signal in the RePLICA conversion buffer system.

Seeding-potential (SD50) has been paralleled to LD50 for bioassay, where 50% of PrPd-

seeded rPrP-conversion replicates are deemed positive by an established set of criteria

[50, 171]. In the case of RT-QuIC, it is the dilution of seed-homogenate where half the

PrPd-seeded conversion reactions gave ThT positive signals.

In the following experiment, RePLICA was tested for its limit of detection for CWD

infectivity, using a CWD+ titred brain homogenate kindly supplied by the CFIA.

Bioassay results for this exact CWD+ titred brain homogenate have been published [22].

5.2 Methods and Materials

The CWD+ titred brain homogenate was a pool of CWD+ brain homogenates from 3 elk.

The homogenate had been stored at -80oC as 20%(w/v) brain tissue in PBS. The stock

20%(w/v) CWD+ titred homogenate in PBS was thawed and diluted to a 10%(w/v)

homogenate with 2%(w/v) CHAPS, 2M NDSB-201, PBSk [10mM Na2HPO4, 2mM

KH2PO4, 137mM NaCl, 2mM KCl, pH 7.4], 2mM EDTA, cOmplete™ Protease

Inhibitors (Roche). Therefore, the final buffer composition of the 10%(w/v) homogenate

was 1%(w/v) CHAPS, 1M NDSB-201, in PBSk. To ensure adequate solubilization, the

homogenate was sonicated at 37oC for 20 seconds. All dilution factors of the pooled

76

titred CWD+ homogenate in the published bioassay study [22] and the presented

RePLICA data are with respect to a 10%(w/v) concentration.

A CWD- brain homogenate control was made by pooling equal portions of CWD- brain

homogenates from Elk#180 and Elk#189, (see: Figure 8). These homogenates were

pooled for consistency purposes. These homogenates were selected to keep the PRNP

genotypes consistent with methionine at PrPc position 132 in both the brain seed-

homogenates and rPrPc substrate within the conversion system.

In the bioassay protocol, the homogenate had been serially diluted by factors of 10 in

PBS, down to 1.00E-11 [22]. The LD50 reported for the homogenate was reported as

1.00E-06, with respect to the Tg(CerPrP-M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay models [22]. The dilutions of the titred CWD+ homogenate and CWD-

homogenate used herein were more gradual, diluted by ~1/3 (33.3µL into 66.7µL) in

seed-diluent (1%(w/v) CHAPS, 1M NDSB-201, PBSk). The dilution series went from

1.00E-1 down to 6.87E-09. This dilution gradient should provide a clear, quantifiable

seeding-potential for each dilution-set. The titred brain homogenate was run on a total of

4 RePLICA trials, with an emphasis on the dilutions between 1.23E-03 and 6.87E-09.

Exact replicate numbers for each dilution-set are listed in Figure 11. The RePLICA trials

were conducted as described in Chapter 2.

The sensitivity for RePLICA was characterized in two ways: The first way was to

identify the penultimate sensitivity, defined as the last dilution of the CWD+ titred brain

homogenate where 50%, or more, of its replicates produced CWD+ Rxns before 31

77

hours. This is set according to the defined cut-off time-zone from the optimization of

cerebral homogenate (see Figure 2). One hundred percent specificity at 31 hours is also

supported in Figure 3. For further reference, individual ROC curve cut-offs were

calculated for each dilution-set (Figure 11).

The second way was to create a dose-response curve to determine the EC50 (half-effective

concentration) of CWD+ titred homogenate in the system. The intent was to characterize

a relationship between CWD+ Rxns time and CWD infectivity in bioassay. The log10 for

each CWD+ dilution was taken for the dose variable. The reciprocal of each replicate’s

CWD+ Rxns time was taken as the response variable. Thus, the shorter the CWD+ Rxn

time, the greater response value. The following function was fit to the mean responses

values from each CWD+ dilution-set, using PRISM v5.0 (Graphpad):

Equation 4.

(

) [

]

The value “ is the rPrP-conversion time in hours for the RFU threshold to be

met in the response term. The values and are the maximum and minimum

response values in the data, respectively. is the half effective dose achieving a

maximal response. The “ ” value is the of each dilution, while is the slope.

78

5.3 Results and Discussion

5.3.1 Sensitivity of RePLICA versus Bioassay

0 5 10 15 20 25 30 35 40 45 50 55 60 65

6.87E-09

2.06E-08

6.19E-08

1.86E-07

5.58E-07

1.68E-06

5.03E-06

1.51E-05

4.54E-05

1.36E-04

4.09E-04

1.23E-03

3.69E-03

1.11E-02

n = 12

n = 12

n = 24

n = 24

n = 24

n = 24

n = 48

n = 48

n = 36

n = 36

n = 24

n = 24

n = 24

n = 24

Hours (Fluorescence 500 RFU)

Dilu

tio

n (

Bio

as

sa

ye

d H

om

og

en

ate

)

79

Figure 11. The RePLICA CT-plot for elk CWD+ Rxns seeded by a Tg(CerPrP-

M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay titred CWD+ brain

homogenate. Each data point represents the time (hours) an rPrP-conversion event

occurred, defined by a ThT fluorescence signal exceeding the 500RFU threshold.

Conversion reactions seeded by the titred elk CWD+ brain homogenate are red-shaded

squares used in Bian et al (2010). CWD- Rxns are shaded-blue circles. Data point

shading is associated with more dilute seed-homogenate, decreasing in concentration

from top to bottom. Dilution factor and replicate numbers (n =) for each dilution-set are

listed on the left. Data was generated with a total of 4 independent RePLICA trials, two

different elk rPrPc batches, and using two FLUOstar plate-readers. Double-headed arrows

are individual ROC curve cut-offs determined for each CWD+/- dilution-set. The grey-

shaded area is the critical zone established for brain seed-homogenates dilutions (Figure

2). Error bars represent the median and interquartile range.

80

-8-7-6-5-4-3-21

2

4

8

16

32CWD+Bioassayed Pool

EC50 Response(14.5 hrs)

Cut-off (31 hours)

EC50 Dose(2.22E-06)

log10 Dilution

[Ho

urs

RF

U>

500]-1

x 1

00

Figure 12. A dose-response curve of the CWD+ Rxns seeded by dilutions of a

Tg(CerPrP-M132)1536+/-

and Tg(CerPrP-E226)5037+/-

titred CWD+ elk brain

homogenate. The red dose-response curve was generated from data in Figure 11 using

Equation 4. Error bars are 95% confidence intervals for each dilution-set’s mean response

value (rPrP-conversion time). Dashed lines represent the 99% prediction bands of where

any given CWD+ Rxn replicate will generate a response at the respective dilution. The

solid horizontal line represents the 31 hour cut-off time. The determined EC50 translates

to 2.22E-06 with a mean response value equivalent to 14.5 hours for an rPrP-conversion

time.

81

Serial dilutions of the titred CWD+ brain homogenate (Figure 11) performed

synonymously to the dilutions shown in Figure 2. The best performing CWD+ dilution

(shortest rPrP-conversion time with the least conversion time variance) was the brain

seed-homogenate diluted to 3.69E-03. Again, this proved to be the optimal seeding

dilution of the titred homogenate. Consistencies between RePLICA trials, FLUOstars,

and dilution-sets were evaluated using multifactorial ANOVA, respective to the

following factors: dilution factor, each of the 4 RePLICA trials, both FLUOstar plate-

readers. A three-way ANOVA between the factors showed no overall significant

differences or statistical interactions ( = 0.024, p = 0.921). Conversion times seeded by

the titred CWD+ homogenate was insignificantly influenced by either FLUOstar, in

conjunction with the 4 separate RePLICA trials ( = 0.015, p = 0.994).

In Figure 11, the 6.18E-08 dilution-set of the titred CWD+ homogenate was the last to

seed more than half of the CWD+ Rxn replicates before the critical time zone beginning

at ~31 hours (SD50) . This identical CWD+ titred homogenate caused disease in 2/6

Tg(CerPrP-M132)1536+/-

and 3/6 Tg(CerPrP-E226)5037+/-

mice, at a dilution of 1.00E-

06 [22]. This suggests the seeding-potential within this particular RePLICA system

statistically possesses ~16 fold greater sensitivity for detecting CWD-infectivity in this

brain material than the mouse bioassay model. This level of sensitivity coincides well

with reported RT-QuIC results, showing sensitivities are between 10 and 100 fold for

sensitive [50, 171].

A key observation was the 1.86E-07 and 6.19E-08 dilutions of the CWD+ titred

homogenate, displaying mean CWD+ Rxns times of 29.6 and 34.5 hours, respectively.

82

These means are situated directly over the beginning of the established cut-off time zone.

The 6.19E-08 dilution seeded rPrP-conversion in 14 of 24 replicates prior to 31 hours,

while each of the next lower dilutions (2.06E-08 and 6.87E-09) yielded only 1 of 24

replicates before this time. The seeding-potential of the RePLICA essentially vanished

after the 6.19E-08 dilution.

Excluding the optimal brain homogenate dilution (3.69E-03), all other CWD+ dilution-

sets between 1.11E-02 and 5.58E-7 had mean CWD+ Rxn times between 8 and 18 hours.

Although, given their CWD+ Rxn time variances, Tukey HSD post-hoc testing suggests

they are not significantly different from one another (α = 0.05). Conversely, the 1.86E-07

and 6.19E-08 dilutions belonged to their own subset of mean CWD+ Rxn times, not

shared with any other CWD+ or CWD- dilution-set (p < 0.05).

The EC50 for titred CWD+ brain homogenate was calculated to be 2.22E-06, shown in

Figure 12. The lower 99% prediction-band for the data becomes no longer viable at a

theoretical dilution of 4.26E-07 with a response value equivalent to ~19 hours.

Comparing to Figure 11, the CWD+ 5.58E-07 dilution-set was the last to have a mean

CWD+ Rxn time not significantly different from the other positive dilutions up to 1.23E-

03. It was also the last dilution-set to achieve 100% sensitivity and specificity.

The RePLICA’s EC50 value—2.22E-06—reported for the titred CWD+ homogenate may

have an interesting biological relevancy: it is only 2 fold greater than 1.00E-06 LD50 for

this homogenate, reported in Bian et al [22]. This EC50 value might even represent a

more precise LD50, because the dilution gradient used herein for seeding RePLICA

83

reactions is a factor of 3, not 10 fold as used in the cited bioassay [22]. When examining

the data in Bian et al in more detail, 3/6 Tg(CerPrP-E226)5037+/-

mice had succumb to

disease at the 1.00E-05 and 1.00E-06 dilutions, and only 2/6 Tg(CerPrP-M132)1536+/-

mice had been ill at the 1.00E-06 dilution. As such, the 1.00E-06 may actually be slightly

below the LD50 for the homogenate. This demonstrates that RePLICA may have the

capacity to estimate, or perhaps determine, a titre of TSE-infectivity by timing rPrP-

conversion using high replicate numbers. This should be repeated with a variety or rPrPc

substrate PrPd-seed combinations.

The 1.86E-07 and 6.19E-08 dilutions of the titred CWD+ homogenate identify a unique

concentration range of PrPd seed and/or associated conversion co-factors. This is because

they are at the limit of detection for RePLICA. Because these dilutions are lower than the

LD50 of 1.00E-06 for the mice reported by Bian et al [22], it would be important to test if

optimally-seeded CWD+ material producing a mean CWD+ Rxn time of 30 to 35 hours

in the RePLICA is infectious. Recalling the multianimal brain panel in Figure 9, the mean

rPrP-conversion time seeded by Elk#169 was within this critical time region after being

optimally diluted to 3.69E-03.

The question arises, if the Elk#169 brain homogenate diluted to 3.69E-03 seeds

RePLICA CWD+ Rxns reactions like the titred CWD+ brain homogenate at a dilution of

6.19E-08, might this suggest a 10%(w/v) brain homogenate from Elk#169 is as lethal to

the Tg(CerPrP-M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay models as a

~1.67E-05 dilution of the titred CWD-positive brain homogenate? At the moment, it can

84

only be hypothesized whether or not the maximum lethality of an Elk#169 brain

homogenate is near its LD50 to this mouse model.

CWD+ brain homogenates seeded into RePLICA as neat 10%(w/v) or at 1.00E-01

(1%(w/v)) are inhibitory (Figure 1). Transgenic mouse models (for cervid PrPc) have

been successfully challenged with 10%(w/v) and 1%(w/v) brain homogenates [50],

therefore do not have to dilute away inhibitory factors as in the RePLICA. Though

RePLICA could statistically detect less CWD infectivity by dilution through the CHAPS-

NDSB seed-diluent, it suffers a ~1/270 dilution penalty for optimal seeding by sacrificing

PrPd quantity in the diluting process. Accounting for these inhibitory dilutions, RePLICA

is likely exactly as sensitive as for CWD the cited bioassay model [22].

The RePLICA displays a comparable sensitivity for CWD infectivity as the Tg(CerPrP-

M132)1536+/-

and Tg(CerPrP-E226)5037+/-

mouse bioassay model for the identical

CWD+ elk brain homogenate. It is interesting to note that the incubation time for the

CWD+ challenged Tg(CerPrP-E226)5037+/-

mice of Bian et al is also reflected in

RePLICA CWD+ Rxn times. Mice challenged with lesser diluted inocula (1.00E-02

through 1.00E-04) show incubation times between 126 and 147 days, with comparable

variance of ±4 to ±12 days [22]. Mice challenged with inocula from 1.00E-05 through

1.00E-06 show incubation times between 263 and 248 days, with comparable variance of

±79 and ±51 days [22].

It appears as two subsets of CWD+ incubation means, with the latter dilutions showing

twice the incubation time and significantly more conversion-time variance. The same was

85

true concerning the 1.86E-07 and 6.19E-08 dilutions versus the other CWD+ dilutions in

the RePLICA (p < 0.05). The EC50 for RePLICA was determined at a dilution of 2.22E-

06, with a response value equal to ~14.5 hours. If this response time is doubled, it closely

reflects the beginning of the ~31 cut-off ROC curve cut-off time zone for brain

homogenates. The increased variance in CWD+ Rxn times is also seen.

This is additional supporting evidence that the consistency of RePLICA has strong

correlations to the cited bioassay model [22]. To have achieved the most ideal conversion

conditions to reflect solely this Tg(CerPrP-E226)5037+/-

mouse bioassay model is highly

unlikely. There are potential biological relevancies that should be explored further using

the RePLICA method of analysis.

86

General Conclusions Chapter Six:

In vitro rPrP-conversion methods are highly sensitive tools for prion detection. In this

study, an in vitro rPrP-conversion method, called RePLICA, was developed using aspects

from two existing methods: Amyloid Seeding Assay and RT-QuIC. RePLICA shows that

timing rPrP-conversion is a feasible approach to quantify elk CWD prion-activity in

biological tissues. The main objective of this study which was to differentiate between

CWD+ and CWD- elk tissues was achieved. ROC curve coordinates were used to

establish a cut-off time to accurately time rPrP-conversion as a reliable diagnostic

measure of prion-infection. RePLICA shows it can be used in a diagnostic setting to

detect elk CWD in brain and lymphoid tissues very consistently, and proves as sensitive

as bioassay.

In addition, another interesting use of RePLICA would be the construction of CT-plots.

The rPrP-conversion time (or duration of the lag-phase) is synonymous to the latent

period of a bioassay. Through high replicate numbers, a CT-plot of RePLICA appears

remarkably similar to an Incubation-Time assay for inoculated mice and hamsters

(reviewed in [1]).

Future research should include attempts to improve upon detecting PrPd in bodily fluids

which are relatively non-invasive to collect, such as blood and urine. In addition,

RePLICA may prove useful to titre prion-infectivity using other PrPd types and tissue

sources, and compare assay results to bioassay models. For instance, a homogenate of

87

classical or atypical forms of BSE could be “titred” via RePLICA, using an rPrPc for a

particular bioassay model such as Syrian hamster. In addition, it may also be predictive of

certain cross-species transmission barriers, which is always a concern with prions.

88

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