early immune responses to sars coronavirus in ferrets · early immune responses to sars coronavirus...

Early immune responses to SARS Coronavirus in ferrets

For the Degree of Philosophy of Doctorate

Abdolali Danesh

Graduate Department of Immunology

University of Toronto

© Copyright by Abdolali Danesh, 2012

ii

Early immune responses to SARS Coronavirus in ferrets

Abdolali Danesh

Doctor of Philosophy

Graduate Department of Immunology

University of Toronto

2012

Abstract

Severe acute respiratory syndrome (SARS) was defined as an invasive respiratory

disease in 2002, which originally came from China and rapidly spread all over the globe.

Acute pneumonia and lower respiratory tract involvement most affected the middle aged

individuals and elderly with a mortality rate of 11%. While SARS Corona virus (SARS-

CoV) has maintained its potential capacity to reemerge, clinical study of the immune

system of SARS patients, as well as controlled studies may lead to application of new

treatment strategies in future.

Throughout this work, I have focused on early immune responses to SARS-CoV

in humans and in ferrets. CXCL0 has been associated with alterations in the clinical

course of several infectious diseases, including SARS and influenza. Here I have cloned

ferret CXCL10 gene and have expressed its recombinant protein. I demonstrate that the

CXCL10 plasma level in SARS patients is associated with the severity of disease. I also

show that endogenous ferret CXCL10 exhibits similar mRNA expression patterns in the

lungs of deceased SARS patients and ferrets experimentally infected with SARS-CoV.

Type I interferons (IFNs) are indispensable parts of the innate immunity during early

iii

stages of infection. A clear distinction between genes upregulated by direct virus-cell

interactions and genes upregulated by secondary IFN production has not been made yet.

Here, I have investigated differential gene regulation in ferrets upon subcutaneous

administration of IFN-2b and during SARS-CoV infection. In vivo experiments

revealed that IFN-2b causes upregulation of abundant IFN response genes (IRGs),

chemokine receptors, and other genes that participate in phagocytosis and leukocyte

migration. SARS-CoV infection of ferrets leads to upregulation of varieties of IRGs and a

broad range of genes involved in cell migration and inflammation. This work allowed

dissection of several molecular signatures present during SARS-CoV infection, which are

part of a robust IFN antiviral response. Since localization of CD8+ Tcells may contribute

to tissue injury, I have characterized ferret CD8 gene and have generated reagents that

can be used in future studies with the aim of evaluating CD8+ T cells localization in the

ferret lung during infection with SARS-CoV.

iv

Key words:

Ferret, Animal model, SARS, Chemokines, CXCL10, Interferon, Gene expression, CD8

v

Co-authorships

The following thesis contains material from published papers and one written manuscript

co-authored by Charit Seneviratne, Cheryl M. Cameron, Mark E. Devries, Alyson A.

Kelvin, David Banner, Luoling Xu, Longsi Ran, Steven E. Bosinger, Thomas Rowe,

Marcus Czub, Colleen B. Jonsson, , Alberto J. León, Yuan Fang, Honglin Chen, Yi

Guan, Atsuo Ochi, Mark J. Cameron and David J. Kelvin.

All data analysis, experimental design and procedures were performed by Ali Danesh

within this thesis with the following exceptions. Charit Seneviratne designed and helped

with cloning and expression of ferret CXCL10 and CD8. Cheryl M. Cameron and

Thomas Rowe performed ferret experiments at BSL3 animal facility in Alabama, USA.

Colleen B. Jonsson supervised the BSL3 animal facility and helped with experimental

design and animal ethics protocols for the work performed in Alabama, USA. Mark E.

Devries helped with designing the degenerate primers and submission of ferret sequences

to the GeneBank. Alyson A. Kelvin ran real-time PCR on biopsies of lung tissues from

deceased SARS patients and critically reviewed the third chapter. David Banner helped

with immunoblots in chapter 2 and 4, and immunohsitochemistry in chapter 4. Luoling

Xu ran microarray experiments and helped with chemotactic assay. Longsi Ran and

Alberto Leon helped with microarray data analysis. Steven E. Bosinger reviewed chapter

2 and 4. Marcus Czub provided us with ferret total RNA for the first round of gene

sequencings. Yuan Fang, Honglin Chen and Yi Guan helped with sequencing ferret

genes. Atsuo Ochi helped with immunoblots in Chapter 4. He also designed experiments

and wrote the manuscript for Apendix I. Mark J. Cameron designed cytokine assay on

plasma of SARS patients, helped with microarray analysis and critically reviewed

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chapters 2 and 3. All Chapters (introduction, two published papers that a version of which

appears in this thesis, one manuscript and general discussion) were written by Ali Danesh

and David J. Kelvin. Appendix I (published paper) was written by Atsou Ochi, Ali

Danesh and David J. Kelvin.

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Acknowledgements

I would like to start with my special thanks to my supervisor Dr. David Kelvin. You

taught me how to work efficiently at UHN and University of Toronto. You let me work

on different areas of immunity of infectious diseases, which led to several publications.

Under your supervision, I learned how to deal with new scientific challenges in a world-

class research environment. I would like to thank Dr. Li Zhang and Dr. Rupert Kaul for

their extensive support, critical reviews of my research progress and for the enthusiasm

they brought to me.

Thanks to all members of Kelvin lab for their enormous support. Thanks to Dr. Mark

Cameron for all his support. His excellent scientific points of views were very important

to me. Thanks to Dr. Luoling Xu for all her helps and for providing us with necessary

reagents. I would like to thank Dr. Longsi Ran for all his help with microarray data

analysis.

I am in debt to my wife, Shideh, for all her dedication, support and inspiring hope and

courage to me. Thanks to my sister, my brothers and my parents for encouraging and

helping me in other aspects of my life.

I would like to thank the Canadian SARS Research Network, Toronto and Area nurses

and clinicians and clinical laboratory members for their support and contribution.

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Contents

Title page………………………………………………………………………………......i

Abstract……………………………………………………………………………….…...ii

Key words………………………………………………………………………………...iv

Co-authorships………………………………………………………………………….....v

Acknowledgments………………………………………………………………….……vii

Table of contents………………………………………………………………………….ix

List of figures……………………………………………………………………………xiv

List of tables……………………………………………………………………………..xvi

List of appendices……………………………………………………………………....xvii

List of abbreviations……………..…………………………………………………......xvii

ix

Table of contents

Chapter 1: introduction……………………………………………………………….....1

Overview…………………...…………………………………………………………..1

1 Coronaviruses………………...………………………………………………………..2

1.1 SARS-CoV………………………………………………………………………….....3

1.2 SARS-CoV Genomic Structure…………………………………………………….....4

1.3 SARS-CoV Receptors…………………………………………………………………5

1.4 SARS-CoV Life Cycle……………………………………………………………..….6

1.5 Characteristics of SARS-CoV…………………………………………………………7

1.6 Determination of TCID50……………………………………………………………..7

1.7 SARS Definition……………………………………..………………………………10

1.8 SARS diagnosis…………………………………………………………………...…10

1.9 SARS Treatment…………………………………………………………..…………11

1.10 SARS Vaccines..........................................................................................................11

2 Immune System...........................................................................................................12

2.1 Innate immune system……………………………………………………………….13

2.2 Adaptive immune system………………………………………………………….…16

2.3 Inflammation and lymphocyte activation....................................................................23

2.4 Interferons…………………………………………………………………………....27

2.5 Interferon regulated genes………………………………………………………...….30

2.6 CXCL10 (IP-10)………………………………………………………………..……31

2.7 Immune System and SARS……………………………………………………..……33

2.8 Immunopathology of SARS………………………………………………………….34

x

2.9 Cytokines and SARS……………………………………………………………...…35

2.10 IRGs and SARS…………………………………………………………………….36

2.11 SARS and leukopenia……………………………………………………………....37

3 SARS animal models……….………..………………………………………….……38

4 Hypothesis………………...………………………………………………………..…38

5 Objectives……….………..…………………………………………………………..39

Chapter 2: Upregulation of CXCL10 is associated with disease progression in

SARS-CoV- infected humans and ferrets……………………………………………..51

1 Introduction……….………….………………………………………………………51

2 Materials and methods………..……………………………………………………...54

2.1 SARS patients……………………………………………………………………..…54

2.2 Animals………………………………………………………………………………54

2.3 Titration of SARS-CoV……………………………………………………………...55

2.4 Viral load and clinical characteristics of infected ferrets.............................................55

2.5 Cytometric Bead Array (CBA)………………………………………………………56

2.6 Human lung biopsies and total RNA purification……………………………………57

2.7 Ferret total RNA purification and cDNA synthesis……………………………….…57

2.8 Real-time RT-PCR…………………………………………………………………...57

2.9 Amplification, cloning and sequencing of ferret-specific genes…………………….58

2.10 Generation of an expression vector for ferret CXCL10.............................................58

2.11 Cell culture, plasmid transfections, and protein purification.....................................59

2.12 Western Blot analysis………………………………………………………………59

2.13 Isolation of Peripheral Blood Mononuclear cells (PBMC) for

chemotaxis Assays…………………………………………………………………60

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2.14 Chemotaxis Assay……………………………………………………………….….60

2.15 Gene accession numbers……………………………………………………………61

2.16 Statistical analysis………………………………………………………………..…62

3 Results……….…………………………………………………………………...….62

3.1 CXCL9 and CXCL10 plasma level of patients with SARS…………………………62

3.2 Cloning and sequence analysis of ferret CXCL9, CXCL10, CXCL11

, and CXCR3………………………………………………………………………....67

3.3 Expression of recombinant ferret CXCL10 protein and detection by

western blot…………………………………………………………………………..73

3.4 Chemotactic activity of recombinant ferret CXCL10 protein…………………….…73

3.5 Stimulation of ferret PBMCs with LPS induces mRNA expression of

CXCL9, CXCL10 and CXCL11…………………………………….……………….77

3.6 Increased CXCL10 and CXCR3 mRNA expression in lung biopsies

from fatal outcome SARS patients at autopsy……………………….………………80

3.7 Expression of CXCL10 in lung biopsies of ferrets infected with

SARS-CoV…………………………………………………………………………...80

4 Discussion……………….……………..………………………………………..……83

Chapter 3: Early gene expression events in ferrets in response to SARS

coronavirus infection versus direct interferon-alpha2b

stimulation…………………………………………...……………………..91

1 Introduction……………………..……………….…………………………………....91

2 Materials and methods……………. ….……………………………………………...94

2.1 Ferrets……………………………………………………………………………......94

2.2 In vitro blood stimulation with IFN-2b…………………………………………….94

2.3 In vivo injection of ferrets with IFN-2b……………………………………………95

2.4 In vivo infection of ferrets with SARS-CoV………………………………………...95

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2.5 Intracellular staining…………………………………………………………………96

2.6 Cloning and sequencing of ferret-specific genes…………………………………….96

2.7 Gene accession numbers……………………………………………………………..97

2.8 Quantitative Real-Time PCR (qRT-PCR)………………………………………...…97

2.9 Microarray analysis…………………………………………………………………..98

2.10 Statistical analysis…………………………………………………………………..99

3 Results………………..……………………………………………..………………100

3.1 IFN-2b stimulation and SARS-CoV infection induced STAT1

phosphorylation and increased the expression levels of IRGs………………..…….100

3.2 Microarray analysis of IFN-2b injection or SARS-CoV infection

in ferret peripheral blood…………………………………………………...…..…..105

3.3 Microarray analysis of IFN-2b injection or SARS-CoV infection

in ferret lungs…………………………………………………………………….…115

3.4 Pathway and functional group differential gene expression patterns………………118

4 Discussion………………...…………………………………………………………127

Chapter 4: Cloning, expression and characterization of ferret CD8……………..137

1 Introduction……….…………..……………………………………………………..137

2 Materials and methods………………...………………………………………….…139

2.1 Antibodies…………………………………………………………………………..139

2.2 Animals……………………………………………………………………….…….139

2.3 Total RNA purification and cDNA synthesis………………………………………139

2.4 Amplification, cloning and sequencing of ferret CD8…………………...……….

2.5 Generation of ferret CD8 expression vector………………………………………140

2.6 Cell culture, plasmid transfection, and protein purification………………….…….140

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2.7 Western Blot analysis………………………………………………………………141

2.8 Gene accession numbers……………………………………………………………142

2.9 Isolation of Peripheral Blood Mononuclear cells (PBMC)…………………………142

2.10 Cell surface staining of ferret PBMCs…………………………………………….142

2.11 Immunofluorescent staining of ferret CD8-transfected COS-7 cells

with rabbit polyclonal antibody………………………………………………...…143

2.12 Generation of monoclonal ferret CD8 supernatant………………………….…..143

3 Results…….……….……………………………………………………………..…145

3.1 Cloning and sequence analysis of ferret CD8…………………………………….145

3.2 Expression of recombinant ferret CD8……………………………………………

3.3 Anti-human CD8 polyclonal antibody reacts with ferret PBMCs and

with CD8transfected COS-7 cells……………………………………………..…153

3.4 Generation of mouse anti-ferret CD8 monoclonal supernatant…………………...157

4 Discussion………….………….………………………………………………...….160

Chapter 5: General discussion…………………………………………………..........165

5.1 SARS correlation with CXCL10 in humans and ferrets …………………………...166

5.2 Early gene regulation events in SARS-CoV infection versus IFN-2b injection

in ferrets ……………………………………………………………………………168

5.3 Characterization of ferret CD8……………………………………………………...172

5.4 Future studies……………………………………………………………………….173

Appendix I: Cloning, Expression and Immunoassay Detection of

Ferret IFN-……………………………………………………………..181

1 Introduction……………………...………………………..…………………………181

2 Materials and methods…………….………..………………………………….……184

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2.1 Experimental animals…………………………………………………...…………..184

2.2 Total RNA purification and cDNA cloning of ferret IFN-………………………..1

2.3 Gene Transfection, Expression in COS-7 cells, and Protein

Purification……………………………………………………………………….…185

2.4 Western Blot analysis…………………………………………………………...….186

2.5 Isolation of Peripheral Blood Mononuclear Cells (PBMC)……………………...…186

2.6 Mouse B cell hybridoma preparation……………………………………………….187

2.7 Ferret IFN--specific ELISA………………………………………………….……187

2.8 Ferret IFN--specific ELISPOT assay………………………………..…………….188

2.9 Infection of ferrets with Influenza A virus…………………………………..……..188

3 Results……….…………..……………………………………………………..……189

3.1 Cloning of ferret IFN-cDNA and the expression of His6-,V5-tagged

recombinant IFN- in COS-7 transfectant cells………………………………….…189

3.2 Generation of monoclonal Abs specific for ferret IFN-γ…………...........................196

3.3 Utilization of monoclonal antibodies for the detection of ferret

IFN-by ELISA and ELISPOT immunoassays………………………………..…..199

4 Discussion……….…………………………….……………………………..……..207

Appendix II copyright wavers…………………………………………………..……214

List of figures:

Figure 1-1 Determination of TCID50 in a 96 well plate by cytopathic effect….…...…….9

Figure 1-2 Location, domains and ligands of human TLRs……….…………….………15

Figure 1-3 Antigen uptake and development of humoral and cell-mediated

adaptive immune responses……….………………………………………....18

Figure 1-4 Differentiation of CD4+ Th1 cells under the influence of

cytokines……………………………………………………………………....19

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cytokines………………………………………..…………………………....20


cytokines………………………………………………..…………………....21

Figure 1-7 Differentiation of CD4+ regulatory T cells under the influence of

cytokines…………………………………………………..………………....22

Figure 1-8 Development of adaptive immune response……….………………..……….26

Figure 1-9 Type I interferon signaling and upregulation of IRGs….……….………..….29

Figure 2-1 Mean CXCL10 level changes in plasma of different SARS groups.………...63

Figure 2-2 Mean CXCL9 level changes in plasma of different SARS groups….……….65

Figure 2-3 Multiple alignments of the primary amino acid sequences

of CXCL9, CXCL10, CXCL11, and CXCR3……………………..…..…….68

Figure 2-4 Phylogenetic analysis of ferret CXCL9, CXCL10, CXCL11

, and CXCR3………………………..………………………………..……... 70

Figure 2-5 The recombinant ferret CXCL10 protein migrates to

18.5kDa and is active in cell migration assays…………..…………...……...75

Figure 2-6 Induction of chemokine gene expression by lipopolysacharide………....…..78

Figure 2-7 CXCL10 and CXCR3 mRNA expression in lung tissues of

humans and ferrets infected with SARS-CoV…………………………...….81

Figure 3-1 In vitro phosphorylation of STAT1 in peripheral blood of ferrets

following stimulation with IFN-2b…………………………...………..…101

Figure 3-2 In vivo phosphorylation of STAT1 in ferret peripheral blood

mononuclear cells………….……………………………………...……..…103

Figure 3-3 Gene expression profile measured by qRT-PCR in peripheral blood

of ferrets stimulated with IFN-2b in vitro……………………..………….106

Figure 3-4 Microarray and qRT-PCR analysis of IRG expression in peripheral

blood and lung necropsies of ferrets injected with IFN-2b or

infected with SARS-CoV in vivo during the time course………… …..…...111

Figure 3-5 Intersect analysis of IRGs expression in blood and lung tissue

xvi

from IFN-2b injected and SARS–CoV infected ferrets….…………….…116

Figure 3-6 IRG pathway analyses of microarray datasets in lung necropsies

of IFN-2b injected and SARS-CoV infected ferrets……………………...120

Figure 3-7 Microarray analysis of immune response pathways in peripheral

blood and lung necropsies of ferrets injected with IFN-2b or

infected with SARS-CoV in vivo during the time course……….……….…124

Figure 4-1 Multiple alignment of the predicted full-length ferret CD8

amino acid sequence with CD8 sequences from other species

using Clustal-X software………………….…………………………...……146

Figure 4-2 Phylogenetic tree representing homology between ferret and

other known mammalian CD8 sequences……………………………..….149

Figure 4-3 COS-7 transfected cells express ferret CD8 recombinant protein………...151

Figure 4-4 Reactivity of ferret PBMCs and CD8-transfected COS-7 cells

with a polyclonal anti-human CD8α antibody…….…………………….….154

Figure 4-5 Flow cytometry staining and western blot analysis of cell culture

media from a hybridoma cell line and ferret CD8-transfected

COS-7 cells…………….……………………………………………….…..158

Appendix-Figure 1 Ferret IFN- cDNA……………...…………………………………190

Appendix-Figure 2 Secretion of ferret IFN- by COS-7 cell transfectants……....…….194

Appendix-Figure 3 Detection of ferret IFN- by a monoclonal anti-ferret

IFN- Ab………….………………………………………….……197

Appendix-Figure 4 Measurement of IFN- in the mitogen-stimulated ferret

PBMC culture supernatants by ELISA……………………………201

Appendix-Figure 5 Detection of increased levels of IFN- in ferret serum after

infection with H3N2 influenza A virus……………………………203

Appendix-Figure 6 ELISPOT assay for the IFN- producing cells in

mitogen-stimulated ferret PBMCs…………………………...……205

List of tables:

Table 2-1 Amino acid identity of ferret CXCL9, CXCL10, CXCL11 and

CXCR3 with known species sequences………………………………………72

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Table 3-1 Amino acid percentage identity of ferret immune genes

compared to known species………………………………………..…..……109

Table 3-2 Summary of differentially regulated genes in top functional groups

during the time course in IFN-2b-injected or SARS-CoV infected

ferrets………………………………………………………..………………110

Table 3-3 Interferon-regulated genes fold change during the time course

in IFN-2b-injected or SARS-CoV infected ferrets….………..……………114

Table 3-4 Comparison of canonical pathways between ferrets injected with

IFN-2b and those infected with SARS-CoV during the time

course...….…122

Table 3-5 Natural history of ferrets, infected with 103

TCID of

SARS-CoV……………..........................................................................……128

Table 4-1 The percentage amino acid identity between the ferret CD8α

amino acid sequence and CD8α protein sequences from other

mammals………………………………………………………………….…148

List of Appendices:

Appendix I Cloning, Expression and Immunoassay Detection of Ferret IFN-

List of abbreviations:

3CLpro

Chymotrypsin-Like Protease

ABSL3 Animal Biohazard Safety Level 3

ACE Angiotensin Converting Enzyme

ACE2 Angiotensin Converting Enzyme 2

APC Antigen Presenting Cells

ARDS Acute Respiratory Distress Syndrome

BLAST Basic Local Alignment search Tool

C3 Complement Component 3

CBA Cytometric Bead Array

CFB Complement Factor 3

CNS Central Nervous System

crKL V-crk sarcoma virus CT10 oncogene homolog

DAD Diffuse Alveolar Damage

DEMEM Dulbecco’s Modified eagle’s Medium

DNM1L Dynamin 1-Like

xviii

E Ag Small Envelope Antigen

EBI European Bioinformatics institute

ECL Enhanced Chemiluminiscent

EIF2AK2 Eukaryotic Translation Factor 2-alpha Kinase 2

ER Endoplasmic Reticulum

ERK Extracellular Signal-Regulated Kinase

FCGR Fragment C Gamma Receptor

FDR False Discovery Rate

GAS Gamma Activation Site

GTPase Guanosine Triphosphatase

HA Hemagglutinin-Acetylesterase

HAT Hypoxanthine Aminoprotein Thymidine

HCoV-229-E Human Coronavirus 229-E

HCoV-OC43 Human Coronavirus OC43

HCV Hepatitis C Virus

IFI Interefron Induced genes

IFI44L IFI44 Ligand

IFIT Interferon-Induced protein with Tetratricopeptide Repeat

IFN Interferon

IFNR Interferon Receptor

IL-8 Intracellular Cell Adhesion Molecule

iNOS Nitric Oxide Synthase

IP-10 Interefron-gamma inducible Protein 10

IRF Interferon Regulatory Factor

IRG Interferon Response Gene

ISG Interferon Stimulated Gene

ISGF Interferon Stimulatory Factor

ISRE Interferon-Stimulated Response Element

I-TAC Interferon-inducible T-cell Alpha Chemoattractant

ITGB Integrin Beta

JAK Janus Kinase

KLH Keyhole Limpet Hemocyanine

LCMV Lymphocytic Choriomeningitis Virus

LPS Lipopolysaccharide

M Matrix

M Ag Membrane Antigen

CDC Centre for Disease Control

M-CSF Monocyte-Colony Stimulating Factor

MFI Mean Fluorescent Intensity

MHC Major Histocompatibility Antigen

MHV Mouse Hepatitis Virus

MIG Monokine induced by gamma interferon

MT1H MethalloThionein 1 H

MVA Vaccinia Ankara

MX Myxovirus Resistance

N Ag Nucleocapsid Antigen

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NA Neuraminidase

NCBI National centre for Biotechnology Information

NK cell Natural Killer Cell

NOD Non-Obese Diabetic

NP Nucleocapsid

NS Not Significant

OAS Oligoadenylate Synthase

OASL OAS ligand

ORF Open Reading Frame

p38 Protein Kinase 38

PAMP Pattern Associated molecular Pattern

PBMC Peripheral Blood Mononuclear Cells

PBS Phosphate Buffered Saline

PIAS Protein Inhibitor of Activated STAT

PLIER Probe Logarithmic Error Intensity Estimate

PLP Papain-Like Protease

PMA Phorbol Myristate Acetate

Pol RNA-Dependent RNA Polymerase

Poly I:C Polyinosinic: Polycytidylic Acid

PSMB8 Proteasome Subunit Multifunctional beta 8

qRT-PCR Quantitative Real-time PCR

RLM-RACE RNA Ligase Mediated Rapid Amplification of cDNA

RSAD2 Radial S-adenocyl Methionine Domain Containing 2

S Ag Spike Antigen

SARS Acute Respiratory Syndrome

SARS-CoV SARS Coronavirus

SDC1 Syndecan 1

SDS Polyacrylamide Gel Electrophoresis

SOCS Cytokine Signaling

SRI Southern Research Institute

STAT Signal Transducer and Activator of Transcription

TCR T cell Receptor

TLR Toll-Like Receptor

TMB Tetramethylbenzidine

TOR2 SARS Coronavirus Toronto Strain 2

TPBS Phosphate Buffered Saline

TyK Tyrosine Kinase

USP18 Ubiquitin Specific Protein 18

UTR Untranslated region

WHO World Health Organization

1

Introduction

Overview

Emerging viral respiratory diseases that cause acute respiratory distress syndrome

(ARDS-like) diseases have been an important recent focus of the medical community,

virologists and immunologists. Severe acute respiratory syndrome (SARS) and pandemic

H1N1 influenza are two examples of respiratory diseases that have caused outbreaks in

different countries within the same decade, imposing social and economic crises1,2

.

SARS emerged as a new viral infectious disease in 2002. The etiological agent of

SARS was a novel virus, called SARS corona virus (SARS-CoV)3. The influenza-like

disease first was reported in China4. The viral infection mostly resulted in an acute

atypical form of pneumonia and lower respiratory tract involvement affecting middle-

aged individuals and the elderly5,6

. Prior to the outbreak of SARS, coronaviruses were

believed to be the causative agents of mild upper respiratory infections2,7

. Conversely, the

SARS corona virus was the first identified coronavirus to be the etiological agent of a

severe respiratory illness. SARS was spread to other Asian and European countries and to

North America by travelers6. It appeared as two separate outbreaks in spring of 2003 in

Toronto, infecting approximately 800 people and resulting in 43 deaths according to

World Health Organization (WHO) records (http://www.who.int/csr/sars/en/). SARS is

described as a disease with multi-organ involvement in severe cases. With a mortality

rate of 11%, SARS would be a potential disaster if it remerged into the human

population8.

Pandemic H1N1 influenza is a viral respiratory infection that was first identified

in 20079. Several strains of the virus have been identified, including pH1N1 A/mex/4108

2

and pH1N1 A/Cal079,10

. While the symptoms of pandemic influenza are similar to

seasonal influenza, in some individuals the infection is more virulent and can lead to

rapid development of ARDS and death11,12

. Individuals with pre-existing medical

conditions were more vulnerable to infection than patients with no medical history. In

high-risk patients, antiviral therapy with oseltamivir or zanamivir was thought to be

beneficial13

. Although the best strategy was the production and use of pandemic H1N1

vaccine, the unavailability of a large vaccine stock in poor or developing countries

reduced the efficacy of this method. Another issue is that a huge percentage of population

did not receive the vaccine in 2009 in developed countries such as the United States14-16

.

In both the case of SARS and pandemic H1N1 influenza infection, a balance

between different arms of immune system has been suggested to be essential in the

clearance of the disease with minimal side effects, while an imbalance may lead to tissue

injury17-19

. Clinical study of the immune system of infected patients, as well as research

conducted on appropriate animal models, may be of great value in the development of

new treatment strategies.

Throughout this work, with the aim of building ferrets as an immune model for

viral respiratory diseases, I have focused on early immune responses to SARS-CoV in

humans and in ferrets.

1. Coronaviruses

Coronaviruses are spherical, enveloped, RNA positive stranded viruses. They are

the largest RNA viruses with an approximate genome size of 27500-31500 bases20

.

Coronaviruses are classified as a genus in the family Coronaviradae and order

3

Nidovirales20

. Human Coronavirus 229E (HCoV-229-E) and Human Coronavirus OC43

(HCoV-OC43) are the main cause of the common cold after rhinoviruses7.

1.1 SARS-CoV

In early 2003, a new Coronavirus associated with a severe acute respiratory

syndrome was identified6. It was the first observation that a Coronavirus could be the

etiological agent of a deadly respiratory disease. At first it was hypothesized that SARS-

CoV may be a mutant human Coronavirus with newly acquired virulence, or the result of

recombination between separate human and animal Coronaviruses21

. It is now believed

that SARS-CoV is a previously unknown Coronavirus that through evolution obtained

the ability to infect humans20

. The bat, palm civet, raccoon dog, domestic cat, red fox and

Chinese ferret badger are all likely to have been reservoirs of SARS-CoV20,21

.

SARS-CoV has four structural proteins: Spike (S), nucleocapsid (N), small

envelope (E) and membrane (M)22

. Coronaviruses are divided into three groups based on

genome, natural host, antigenic and serologic similarities and differences20,22

.

Alphacoronaviruses (group I) usually infect mammals, different members of

betacoronaviruses (group II) can either infect mammals or birds, and

gammacoronaviruses (group III) infect birds. Replicase and structural gene analysis has

revealed that SARS-CoV is an early split from betacoronaviruses. Unlike other members

of this group, SARS-CoV does not have the gene for hemagglutinin esterase20

. In this

context some investigators have suggested designating a new group for SARS-CoV20,23

.

Two strains of SARS-CoV were isolated and sequenced in Canada and the United

States independently at the same time. Tor2 strain of the SARS-CoV was isolated from a

4

sample taken from a patient infected in Toronto and was sequenced at the Center for

Disease Control (BCCDC) in Vancouver24

. UKH-1, also known as the Urbani strain, was

sequenced in Atlanta, USA. After a short period of time, another strain was sequenced in

Taiwan and named SARS TW. The nucleotide sequences of these strains were different

at only 24 positions25

.

1.2 SARS-CoV Genomic Structure

A single stranded RNA of 29751 nucleotides with a 5’ cap and a polyadenylated

tail, comprises the SARS-CoV genome23

. The capped leader sequence is 60 nucleotide

long followed by an untranslated region of 204 nucleotides. Replicase, a non-structural

polyprotein is coded by two overlapping open reading frames (ORFs). ORF1a and

ORF1b are at positions 265-13398 and 13399-21485, respectively23

. Once ORF1a is

translated by host ribosome, a –1 frame shift facilitates the translation of ORF1b and the

synthesis of a single polyprotein. Cleavage of the polyprotein by proteases produces the

necessary proteins for viral replication. Structural proteins are located downstream of

ORF1b. The Spike gene is at position 21492-25259 and its product is a type I

glycoprotein made of S1 and S2 subunits23

. The second structural gene is Envelope (E) at

position 26117-26347, which its product is a 76 amino acid type II membrane protein26

.

The third structural gene is Membrane (M) at position 26398-2706324

. This gene

produces a transmembrane glycoprotein which its association with spike glycoprotein is

necessary for envelope formation and viral assembly. Nonstructural X1 and X2 genes are

positioned between S and E genes. X3, X4 and X5 are three other nonstructural genes,

which are located between M and N genes23,24

. The potential products of these

5

nonstructural proteins are not similar to any other known viral proteins. Nucleocapsid, a

glycoprotein with 422 amino acids in length is the product of the fourth structural gene

positioned at 28120-2933824,25

. At the end of the SARS-CoV genome there is a short

untranslated region23-26

.

1.3 SARS-CoV Receptors

Two types of receptors have been identified as SARS-CoV receptors. Angiotensin

converting enzyme 2 (ACE2) of the renin-angiotensin network is the main receptor for

SARS-CoV and binds to spike antigen27

. CD209 (L-SIGN), an integral type II

transmembrane protein is known as the second receptor for spike glycoprotein28,29

.

Angiotensin II causes vasoconstriction and controls sodium balance that results in blood

pressure homeostasis29

. The enzyme that catalyzes the conversion of angiotensin II from

angiotensin I is called angiotensin converting enzyme (ACE). ACE2 is an inhibitor of

angiotensin I and II; it converts angiotensin II to angiotensin 1-7. ACE2 is essential for

SARS infection in vivo. ACE2 knockout mice are resistant to lung infection with SARS-

CoV. Wild type mice infected with SARS-CoV showed decreased levels of ACE2.

Treatment of SARS-CoV-infected mice with recombinant spike antigen reduced the

pathology of the disease compared to untreated infected controls29

.

Gastric acid aspiration results in lung injury and sometimes leads to acute

respiratory distress syndrome (ARDS)30

. ACE2 plays a protective role in experimentally

induced acid injury in mice. It is shown that ACE2 knockout mice had increased lung

injury compared to the wild types. Rescue therapy with recombinant ACE2 was

6

associated with lesser degrees of lung injury in knockout mice30

. These findings

emphasize that ACE2 is important for SARS pathogenesis, not only as a viral receptor

but also in ARDS development29,30

.

1.4 SARS-CoV Life Cycle

Exposure of cells to SARS-CoV results in the interaction of Spike glycoprotein

with ACE2, which leads to fusion of virus envelope and cell membrane30

. The single

stranded positive RNA can then be translated directly by host ribosomes. Open reading

frames (ORF1a and ORF1b) translate the replicase protein, a single polypeptide of >7000

amino acids. Virally coded proteases such as chymotrypsin-like protease (3CLpro

) and

papain-like protease (PLP) cleave the polypeptide and produce RNA-dependent RNA

polymerase (Pol) and other proteins involved in viral replication30,31

. A negative stranded

RNA is copied from the genomic RNA by RNA-dependent RNA polymerase to produce

positive subgenomic mRNAs and is also as a template for the production of new positive

genomic RNA. Subgenomic mRNAs translate to structural proteins31

. Assembly of

nucleocapsid glycoprotein synthesized from mRNA and newly synthesized genomic

RNA occurs in the cytoplasm to form helical nucleocapsids. E and M proteins are

synthesized on membrane-bound polysomes and transported to the endoplasmic

reticulum (ER) and then to the Golgi apparatus. E and M interaction triggers the

formation of envelope and enclosure of the nucleocapsid31,32

. After synthesis on

membrane polysomes, Spike glycoprotein is added to the envelope and finally the new

virions are released by exocytosis. Glycosylation of all structural proteins occurs in the

7

Golgi apparatus. The Spike protein undergoes trimerization and cleavage to S1 and S2 in

the ER30-33

.

1.5 Characteristics of SARS-CoV:

SARS-CoV can be grown or titrated in FrhK-4 (fetal rhesus monkey kidney) or

Vero E6 (African green monkey epithelial) cells34

. SARS-CoV induces apoptosis in

epithelial cells of the lung35

. It also induces apoptosis leading to DNA fragmentation,

apoptotic body formation and mitochondrial damage in Vero E6 cells36

. Infection of

pneumocyte II cells in the lung of infected humans leads to syncytia formation36

. The

infectivity of SARS-CoV is lost after incubation at 37 °C for 15 minutes. Dried droplets

of the virus on plastic surfaces are stable for 5 days at room temperature and a relative

humidity of 40-50%. The infectivity of dried droplets was lost at high and humid

temperatures (38 °C, 80-90% relative humidity)37

. Common fixatives such as 70%

methanol, 10% bleach, 10% formaldehyde, and high (>12) or low (<3) PH inactivates the

virus)38

. In respiratory samples of infected patients, SARS-CoV survived for 5 days at 24

°C. The virus survived for 3 days in loose stool39

. Long term storage of the virus stock

can be performed in liquid nitrogen and the titrated working aliquots can be kept in -80

°C for months. These research specimens should be kept in locked tanks or freezers in a

BSL3 facility39

.

1.6 Determination of TCID50

8

TCID50 is an endpoint dilution technique that is used to quantify the amount of a

virus required to kill or generate a cytopathic effect in 50% of infected host cells. This

method was invented by Reed and Muench. TCID50 is more commonly used for titration

when the viruses do not grow in cell cultures or cannot form plaques. To determine

TCID50, serial dilutions of viral stock are prepared. Target cells infected with different

dilutions of the virus are added into different rows of a 96 well plate and incubated at 37

°C in a CO2 incubator. After approximately 3-7 days, depending on the virus and the

target cell, the percentage of cell death is counted for each dilution. Proportionate

distance (PD) is calculated as (% next above 50%)-50% / (% next above 50%) – (% next

below 50%). Then 50% end point is calculated and added to PD, and finally log TCID50

is calculated. To calculate TCID50/ml, TCID50 is divided by the volume of initial viral

inoculum added to each well40

(Figure 1-1).

9

Lysis

No lysis

Control

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

Dilution

70% endpoint

Figure 1-1: Determination of TCID50 in a 96 well plate by cytopathic effect

10

1.7 SARS Definition

Severe acute respiratory syndrome (SARS) was defined as the sudden onset of

severe respiratory symptoms in association with confirmation of infection by

(subsequently identified) SARS-CoV virus. Many SARS symptoms were non-specific

and similar to that of other types of pneumonia at onset41

. Fever, chills, myalgia, malaise,

headache, cough, dyspnea, diarrhea and nausea were common symptoms of SARS. A

confirmed case of SARS should demonstrate a fever of 38oC or higher, at least one of the

symptoms of lower respiratory tract involvement, radiographic evidence of lung

infiltrates and no alternative diagnosis of other pathogens, and at least one laboratory

finding of SARS, according to the World Health Organization (WHO) definition41

.

1.8 SARS diagnosis

Lung radiography of SARS patients was of great importance during the

outbreak42

. Airspace opacity in the lower lung lobes was found in many infected

individuals infected with SARS-CoV at the onset of the disease. Roughly 90% of patients

showed pulmonary opacities 7 days post-infection43

. Multifocal or bilateral lung

involvement occurred in 70% of patients44

. One of the unusual laboratory findings in

SARS patients was leuckopenia and lymphopenia accompanied by neutrophilia. Many

viral diseases, including Influenza A and Cytomegalovirus, are accompanied with

leukocytosis and lymphocytosis45

. However, the majority of SARS patients showed

leuckopenia, lymphopenia and neutrophilia with platelet counts of less than 150,000/l

(thrombocytopenia). Lactate dehydrogenase and creatine kinase were the only highly

11

elevated biochemistry markers46,47

. Electron microscopy evaluation of lung tissue

preparations and real-time PCR are thought to be the most accurate methods of laboratory

diagnosis48

. Anti-spike seroconversion from IgM to IgG and viral culture techniques are

accurate but time consuming42

.

1.9 SARS Treatment

At the initial outbreak of SARS, the therapeutic choice for treatment of SARS was

a combinational therapy of ribavirin and corticosteroids. Subsequently, laboratory

findings showed the inefficacy of ribavirin in the inhibition of SARS-CoV grown in Vero

E6 cells. Published clinical data was indicative of ineffectiveness of ribavirin as well49-51

.

Corticosteroids were the elective therapy during the second outbreak of SARS in

Toronto43-46

. Delayed lung injury accompanied with reduced viral load was observed in

treated SARS patients with corticosteroids46

. A small cohort of patients was treated with

interferon- and corticosteroids in Toronto47

. In an experimental model of SARS,

treatment of SARS-CoV-infected Cynomolgus macaques with pegylated IFN-

significantly reduced viral replication and lung injury52

. SARS-CoV neutralizing anti-

human IgG1 antibody against whole inactivated virus has also been effective in ferret

model53

.

1.10 SARS Vaccines

12

One of the first attempts to develop a SARS vaccine was the generation of

adenovirus vaccines that contained the SARS S1, M and N proteins. Rhesus monkeys

were immunized intramuscularly with this trivalent vaccine followed by a booster on day

28. Strong antibody responses against S1, and T cell responses against N were shown54

.

Yang et al. demonstrated the efficacy of a DNA vaccine targeting the spike glycoprotein

in mice55

. This DNA vaccine induced CD4+ and CD8

+ T cell responses and significant

antibody production. The SARS viral load was reduced significantly in immunized mice

challenged with SARS-CoV compared to unvaccinated mice56

. In one study, cellular and

humoral immune responses were detected in BALB/c mice after immunization with

plasmids containing M, N, Sa and Sb SARS-CoV antigens. M antigen stimulated the

highest cellular immune response, while Sa and Sb antigens stimulated the highest

humoral immune response57

.

However, some candidate SARS vaccines have also been shown to exacerbate

pathogenesis. In another study, the use of recombinant vaccinia Ankara (rMVA)

expressing SARS-CoV S or N antigens did not prevent SARS-CoV infection in ferrets,

and immunized ferrets exhibited strong inflammatory responses and focal liver necrosis

after SARS-CoV challenge58

.

2. Immune System

The complex and dispersed network of the immune system protects us against

pathogens and foreign substances. The immune system is the second line of defense after

13

natural barriers such as skin, mucus membrane and mucosal secretions. The immune

system of humans and other vertebrates consists of two arms; innate and adaptive59-61

.

2.1 Innate immune system

The first defense to dangers after breakdown of natural barriers is the mechanisms

of the innate immune system. The innate immune system reacts against pathogen and

non-pathogen foreign substances by mobilization of innate immune cells and various

innate macromolecules in a rapid and nonspecific manner. Importantly, the innate

immune response to a pathogen does not generate immunological memory for future

encounters with the same pathogen59,60

.

Main functions of the innate immune system are vast and range from cell

recruitment to removal of debris62

. Importantly, cytokine and chemokine secretion leads

to cell maturation and the recruitment of the immune cells to the site of infection62

.

Hallmarks of innate immune activation include the release of acute phase proteins such as

serum amyloid A and C-reactive protein and activation of complement62,63

. Finally,

elimination of foreign substances and dead cells is accomplished by phagocytic cells

through ingestion by the process of phagocytosis63,64

. The phagocytosed matter can then

be presented to adaptive immune cells for the downstream activation of the adaptive

immune system64,65

.

Proinflammatory cytokines and acute phase proteins are important mediators for

recruitment of the immune cells to the site of infection65

. Recruited cells along with other

14

resident immune cells such as macrophages and dendritic cells initiate inflammation at

the site of infection65,66

. Immune cells are capable of sensing pathogen-associated

molecular patterns (PAMPs) of the pathogens by their pattern recognition receptors

(PRRS)62,63

. PAMPS are conserved structures of pathogens that are sensed by the innate

immune cells63

. Much attention has been paid to the role of a famous group of PAMPS

called toll-like receptors (TLRs). A total of nine mammalian TLRs are known, each one

capable of detecting different or overlapping viral, bacterial or microbial

structures(Figure 1-2)60,62

. TLRs are specific for viral PAMPS for example the ligand for

TLR3 is double-stranded RNA, while TLR7 ligand is single-stransed RNA. TLR4 is an

example of a TLR, capable of recognizing bacterial lipopolysaccharide (LPS)63,64

.

Ligation on the cell surface of the TLRs with its ligand activates cell signaling pathways

that often lead to the production of cytokines and antimicrobial molecules60,62

. TLR

ligation leads to the recruitment of adaptor proteins such as MyD88 and TIRAP to the

intracellular portion of the receptor. These molecules activate downstream protein

kinases in order to amplify the signal and subsequent transcription factor initiation.

Transcription factors such as NF-kB and IRF3 translocate to the nucleus to influence

gene regulation and generation or suppression of inflammatory response63-65

.

15

TLR1TLR2,lipopeptide

TLR2TLR6,lipopeptides

TLR5,flagellin

TLR4,LPSTLR2,

peptidoglycan

TLR3, dsRNA

TLR7, ssRNA

TLR8, ssRNA

TLR9, CPG DNA

Endosome

Nucleus

Leucine –rich motif

Cysteine-rich motifTIR domain

Figure 1-2: Location, domains and ligands of human TLRs (Adapted from cellular

and molecular immunology, Abbas et. al, 2012)

16

2.2 Adaptive immune system

The second part of the immune system is the adaptive immune system. Its

function is to specifically eliminate the foreign invasion and support the non-specific

innate immune system. Two unique aspects of the adaptive immune system are

specificity and the increased magnitude of response during a second encounter with an

antigen (memory). The adaptive immune response is fully developed approximately two

weeks following antigen encounter60,61

. Adaptive immune system is centered by B and T

lymphocytes and works by specific recognition of individual antigens. Both B cells and T

cells are dependent on their antigen receptors for recognition of antigens61,65

(Figure 1-3).

The somatic hypermutaion and recombination of antigen recognition gene segments

(V(D)J) on B and T cells allows the generation of a vast pool of antigen receptors. Each

specific antigen receptor can only be expressed on one B or T lymphocyte clone67

. The B

cell receptor complex is composed of two parts, a surface innmuoglobulin that is specific

for a given antigen on each clone of B cells and a signal transduction part called ig- / ig-

(CD79) dimmer that expands to the cytoplasm and each chain contains an

immunoreceptor tyrosine-based motif (ITAM)68

. A certain clone of B lymphocytes that is

specific for unique antigen, expands and produces one type of immunoglobulin that is

specific for one epitope67,69

.

The T cell receptor (TCR) on the surface of T lymphocytes is responsible for

recognizing peptide antigen fragments that are presented by antigen presenting cells

(APC) in the context of the MHC66

. Cytotoxic T cells, the effector arm of cell-mediated

immune response, are CD8+ T cells that react with peptide-MHC I complex on the

surface of APCs. Another subset of the T cells is T helper cells (CD4+) that bind peptide-

17

MHC II complexes with their TCR. T helper cells are sub classified into Th1, Th2 and

Th17 cells based on their cytokine profiles65

. The role of Th1 cells is to boost the cell-

mediated immune response, mainly by secretion of IFN- and IL-12(Figure 1-4)66

. Th2

cells promote the humoral immune response and mainly by secreting IL-4, IL-5 and IL-

13 that act to activate and maturate B cells(Figure 1-5)66

. Th17 cells are involved in

inflammatory responses and release cytokines such as IL-17, IL-17a, IL-21 and IL-

22(Figure 1-6)65,70

.

Another subset of CD4+ T cells are regulatory T cells that primarily secrete TGF-

and/or IL-10, and are pivotal for the maintenance of self tolerance as well as regulation

of the homeostasis of T cells pool 70

(Figure 1-7).

18

Pathogen

IL-4

APC

Th1

cell

IFN-

Tcytotoxic

Cell lysis andphagocytosis

B cell

Humoralimmunity

Cell-mediatedimmunity

Infected cell

Plasma cell

Figure 1-3: Antigen uptake and development of humoral and cell-mediated adaptive

immune responses

19

DCIntracellularPathogen

Th0

NaïveT

Macrophage

DC

NKcellIFN-

IL-12

STAT1

STAT4

T-bet

IFN-

Th1 cell

IFN- Macrophage activationIgG class switch

Figure 1-4: Differentiation of CD4+ Th1 cells under the influence of cytokines

20

IgE class switchEosinophil activationMucosal secretion

DCParasite

Th0

NaïveT

DC

Mastcell

IL-4

STAT6GATA-3

NFAT

IL-4

IL-4IL-5IL-13

Th2 cell


21

DCBacteria, fungi

Th0

NaïveT

DC

Eosinphils?

IL-1IL-6

STAT3

IL-21 gene

InflammationBarrier function

IL-21

IL-17IL-22

Th17 cell

TGF-

RORtDC

IL-23


22

DC

Th0

NaïveT

DC

IL-10

FOXP3

IL-21 gene

Immune suppressionInducible regulatory T cell(CD3+CD4+CD25+FOXP3+

TGF-

CD25

Self peptide

Figure 1-7: Differentiation of CD4+ regulatory T cells under the influence of

cytokines

23

2.3 Inflammation and lymphocyte activation

T and B lymphocytes with their specific receptors for approximately 107 different

antigens are essential parts of the adaptive immune response. In their encounter with

antigen, each single clone of lymphocytes undergoes clonal expansion and thus, from a

small pool of lymphocytes a large group of effector cells is generated, which is highly

specific for a given pathogen60,71

. T cells play a central role in adaptive immunity. CD8+

cytotoxic T cells are involved in direct killing of virally infected cells via recognition of

processed antigen in the context of MHC I. CD4+ T cells provide help to B cells and

cytotoxic T cells after recognition of antigen-MHC II complex and expansion into Th1,

Th2 or Th17 subsets that was discussed earlier65,72

.

Activation of naïve T cells occurs in the secondary lymphoid organs, including

regional lymph nodes; mucosa associated lymphoid tissues and spleen. Naïve T cells

circulate in the body and enter these organs; if do not encounter antigens presented by

dendritic cells, they leave the organ and recirculate73

. Average time that a lymphocyte

stays in a secondary organ in the absence of its specific antigen is one day73

. Immature

dendritic cells at the site of viral infection take up antigens and after maturation migrate

to secondary lymphoid organs and present antigens to T cells. In steady estate, DCs

continuously migrate from tissues to the regional lymph nodes at a very low level but

their migration will be increased upon increased CCR7 expression during inflammatory

conditions74

. Migration of DCs starts a few hours after infection and maximizes in the

first or second day post infection75

. After antigen stimulation and clonal expansion, which

usually takes 4-8 days, effector T cells leave the secondary lymphoid organs and migrate

24

to the site of infection. Generation of effector cells will be faster if the number of specific

clones for a given pathogen is higher76,77

.

Activation of Naïve T cells and their entrance to lymph nodes is regulated by a

multistep process in which adhesion molecules (e.g. L-selectin and LFA-1) and

chemokines (CCL19 and CD21) play a key role. Naïve T cells through L-selectin weakly

adhere to L-selectin ligand on high endothelial venules (HEV) of the lymph nodes. Stable

adhesion of naïve T cells occurs through the interaction of LFA-1 on T cells and ICAM-1

on HEV. The avidity of ICAM-1 is increased by bound CCL19 and CCL21 on the

surface of HEV77

. Furthermore, interaction of these chemokines with their receptor

CCR7 leads to complete arrest of naïve T cells. Now naïve T cells are ready for

extravascular migration to the T cell zone where the CCL19 and CCL21 concentration is

the highest. Naïve T cells approximately stay for one day if antigen encounter does not

happen and then exit lymph nodes and recirculate and enter to other lymph nodes or

secondary lymphoid organs77,78

. A chemoattractant called sphingosine phosphate one

(S1P) and its receptor sphingosine phosphate one receptor (S1PR) is responsible for the

exit of naïve T cells. The blood level of S1P is high and the expression of S1PR on naïve

T cells is low because at high S1P concentrations, S1PR gets internalized. S1P

concentration in secondary lymphoid tissues such as lymph nodes is low. If T cell

activation does not happen, higher levels of S1PR on naïve T cells are expressed which

leads to the exit of T cells under the influence of higher concentration of S1P in

circulation79-81

.

Upon antigen encounter, activated T cells express CD69, which blocks S1PR

expression on T cells and causes their retention in lymph node. This provides T cells

25

enough time to undergo clonal expansion. At the end of expansion, the decrease of CD69

results in re-expression of S1PR on effector T cells and their exit from lymph node. Other

important events are the reduction of CCR7 and L-selectin expression, which is in the

favor of effector T cell exit81,82

.

Effector T cells migrate to the site of infection under the influence of other sets of

adhesion molecules and chemokine receptors. At the site of infection and in response to

pathogens, proinflammatory cytokines such as IL-1 are secreted, which increases the

expression of P-selectin, E-selectin and ICAM-1 on the surface of endothelial cells of

post capillary venules. Expression of CXCR3 on cytotoxic T cells attracts them toward

the high gradient of CXCL9, CXCL10 and CXCL1177,78

. Interaction of adhesion

molecules on endothelial cells with their ligands on T cells causes their rolling and arrest

and extravascular migration to the site of infection. Migration of other effector cells such

as Th1 cells, Th2 cells and Th17 cells is under the control of CXCR3 and CCR5; CCR4;

and CCR6 respectively72

. B cells use the same basic rules for their migration and

activation. Immature B cells leave the bone marrow and enter the red pulp of the spleen

and move into the white pulp in response to CXCL13 (CXCR5 ligand). B cells stay for

one day and then reenter the circulation after maturation and home to bone marrow,

regional lymph nodes or mucosa by similar mechanisms that explained for T cells74,78

(Figure 1-8).

26

APC

Pathogen

T cell

Plasma cell

Pathogen

Tcytotoxic

Infected cell

Apoptosis

B cellT cell

Memory

Day 0Antigenrecognition

Day 21Memoryformation

Day 14Adaptiveimmunity

Day 4-8Clonalexpansion

Magnitude of response

Effectorcell

B cell

Figure 1-8: Development of adaptive immune response (Adapted from cellular and

molecular immunology, Abbas et. al., 2012).

27

2.4 Interferons

The name interferons originate from their ability to “interfere” with viral

replication in host cells. Interferons are a family of cytokines involved in host immune

responses against viruses, bacteria and parasites. Type I interferons (IFN-,IFN-IFN-

IFN-and IFN- and type II interferon (IFN-) are involved in innate and adaptive

immune responses, respectively83

. Separate genes encode 13 members of IFN- family.

Type I IFNs are mainly secreted by monocytes/macrophages. There is only one IFN-

which is mostly secreted by fibroblasts. IFN- and IFN- are highly inducible by viruses

and synthetic double-stranded RNA83

. All type I interferons bind to the same

heterodimeric cell surface receptor made up from IFN receptor chain 1 (IFNAR1) and

IFN receptor chain 2 (IFNAR2)84

. Incubation of peripheral blood mononuclear cells

(PBMCs) of healthy individuals with SARS-CoV resulted in the production of IFN- as

well as IFN- in a dose-dependent manner85

. Inhibition of viruses by IFN- occurs

through interferon regulated genes (IRGs) such as 2’-5’ oligoadenylate synthase (OAS),

Myxovirus resistant 1 (MX1), interferon stimulated gene 15 (ISG15) and also through the

increase of NK cell activity, downregulation of MHC II expression and upregulation of

MHC I expression47

. Treatment of SARS patients with IFN- in conjunction with other

therapies such as corticosteroids may have provided clinical benefit during the SARS

outbreaks in Toronto86

.

IFN- is the 17 KD product of a single gene87

. Th1 CD4+ cells, CD8

+ cells and

NK cells are IFN- producing cells. IL-12 and IL-18 are capable of inducing the

production of IFN- and their synergistic function leads to production of higher levels of

28

IFN-88

. T cells produce IFN- upon stimulation by IFN- and IFN-. Similar to IFN-,

IFN- prevents viral replication by upregulation of many IRGs. The receptor for IFN- is

a heterodimer of two chains (IFNGR1 and IFNGR2)89

. IFN- activates

monocytes/macrophages and neutrophils and induces respiratory burst for killing the

phagocytosed pathogens and therefore boosts the innate immune system. Both MHC I

and MHC II are upregulated following the stimulation of cells with IFN-. Differentiation

of CD4+ cells to Th1 cells and inhibition of Th2 cells is under the influence of IFN-.

IFN- is a more potent activator of NK cells than type I interferons88,89

.

Signal transduction of IFN- and IFN- is distinct but partially overlaps.

Phosphorylation of Janus Kinase 1 (JAK1) and Tyrosine Kinase 2 (Tyk2) occurs after

ligation of IFN- to its receptor89

. This leads to phosphorylation of Signal Transducer

and Activator of Transcription 1 and 2 (STAT1 and STAT2). The STAT1-STAT2

heterodimer binds to Interferon Regulatory Factor 9 (IRF-9) and generates a complex

called Interferon Stimulatory Factor 3 (ISGF3). ISGF3 binds to Interferon-Stimulated

Response Element (ISRE) followed by activation and transcription of IRGs (Figure 1-

9)89,90

.

Stimulation of IFN- receptor by it’s ligand leads to phosphorylation of JAK1 and

JAK290

. This in turn leads to the phosphorylation and dimerization of STAT1. STAT1

dimers interact with Gamma Activation Site (GAS) in the nucleus that is located in the

promoter of many target genes such as IRF-1, IRF-9, Inducible Nitric Oxide Synthase-2

(iNOS-2) and Suppressor of Cytokine Signaling-1 (SOCS-1)90-92

.

29

IFNAR-2

IFN- /

IFNAR-1

Nucleus

JAK-1Tyk2

STAT1

STAT2

GAS

GAF

IRF9

ISGF3

ISIRE

ISG15CXCL10IRF5IRF7PKROAS

IRF1IRF2IRF8IRF9

Figure 1-9: Type I interferon signaling and upregulation of IRGs

30

2.5 Interferon regulated genes

Regulation of hundreds of genes downstream of the IFN signaling pathway

triggers the production of antiviral molecules in virally infected cells. In turn, viruses

have evolved strategies to avoid the IFN system. Understanding of virus-host interaction

is an exciting field of IFN research86

.

The downstream signaling pathways of type I and type II IFNs share similarities

but also have differences. While the STAT1-STAT2 heterodimer is present downstream

of type I interferons signaling pathway, the STAT1 homodimer is the dominant complex

downstream of the type II IFN pathway, accompanied with a lower engagement of

STAT1-STAT2 heterodimer93

. ISRE-mediated gene activation downstream of type I

interferons mainly leads to upregulation of ISG15, CXCL10, IRF-5, IRF-7, OAS, PKR,

while GAS-mediated gene activation downstream of type II interferon mainly leads to

upregulation of IRF-1, IRF-2, IRF-8 and IRF-994

. There are many other genes that are

shared between the two pathways (e.g. MX1, MHC I, etc). For a detailed list of IRGs,

please refer to IRGs database at http:www.lerner.ccf.org/labs/williams/.

Cross-talk also exists between type I and type II IFN pathways. Upon stimulation

of the type II IFN receptor and upregulation of IRF-1 and IRF-2, these two transcriptional

factors bind to the ISRE element, boosting the upregulation of downstream genes of type

I IFNs. Following the stimulation of type I IFNs, STAT5 and CrKL (V-crk sarcoma

virus CT10 oncogene homolog (avian)-like) are phosphorylated and heterodimerized, and

are then capable of binding to the GAS element and boosting the upregulation of the

Type II interferon signaling pathway93,94

.

31

IRGs exert their effect through different mechanisms, such as the direct targeting

of viral entry, the inhibition of protein synthesis or the degradation of viral RNA. Some

well-known IRGs and their functions are as follows; MX1 is a dynamin-like large

guanosine triphosphatase (GTPase), which has antiviral activity against a wide range of

RNA viruses. The antiviral activity of MX1 is effective at the early stages of the viral

cycle in the nucleus or cytoplasm95

. OAS is an adenylate synthetase that uses adenosine

triphosphate to synthesize 2’,5’-oligoadenylates. The latter activate latent RNase L that

is involved in the degradation of viral RNA96

. ISG15 is an ubiquitin-like enzyme that

covalently conjugates to a large number of cellular proteins; however this does not

usually lead to protein degradation. In the case of HIV-1, ISG15 inhibits the release of

virions97

.

2.6 CXCL10 (IP-10)

One of the IRGs downstream of IFN signaling pathway is a chemokine, CXCL10,

also known as IFN- inducible protein 10 (IP-10)98

. CXCL10 has been associated with

Th1-like diseases99

. Chemokines are a family of cytokines with chemoatractic, mitogenic

and immunomodulatory activities98

. The molecular weight of CXCL10 is 10 KD.

CXCL9, CXCL10 and CXCL11 are CXC chemokines, sharing the same seven

transmembrane G protein coupled receptor called CXCR3100

.

CXCL10 expression in thymic, splenic, and lymph node stromal cells is

constitutive at a low level101

. CXCL10 is highly inducible in endothelial cells,

monocytes, fibroblasts, keratinocytes, neutrophils, astrocytes, activated T cells99

. It has

32

been demonstrated that IFN-, IFN-, IFN-ß, TNF-, IL-1 ß, monocyte-colony

stimulating factor (M-CSF) and also exogenous factors including viruses and LPS can

induce CXCL10 production102

. The type of stimulant and stimulated cell may be

important in the magnitude of CXCL10 expression. For example, many cell types show

stronger response to IFN-, but skin fibroblasts are better responders to TNF-102

.

CXCR3 is a seven transmembrane receptor that is expressed on activated Th1

cells, activated CD8+ T cells, memory CD4

+ cells, memory CD8

+ T cells, NK cells,

dendritic cells, leukemic B cells, eosinophils and dividing microvascular endothelial

cells99

.

CXCL9, CXCL10 and CXCL11 function as chemoattractants for T lymphocytes,

B lymphocytes and natural killer cells103

, although their induction patterns are not the

same. In experimentally induced hepatitis of mice with adenovirus, accumulation of T

lymphocytes in liver is dependent on simultaneous upregulation of CXCL9 and

CXCL10104

. In an allograft rejection model, CXCL10 was expressed in the heart tissue

within 1 day, while CXCL9 was detected after 3 days post-transplantation105

. Infection of

mice with Lymphocytic Choriomeningitis Virus (LCMV) results in high levels of

CXCL10 expression in the pancreas 1 day post-infection, but there was no significant

change of CXCL9 and CXCL11106

.

CXCL10 level of expression is increased in psoriasis, multiple sclerosis,

rheumatoid arthritis, transplant rejection, inflammatory bowel diseases, viral meningitis,

pulmonary sarcoidosis, and chronic hepatitis, suggesting its correlation with Th1-type

diseases107

. CXCL10 expression could be beneficial or detrimental depending on the

33

disease type. Interestingly, non-obese diabetic (NOD) mice, vaccinated with CXCL10

DNA are protected from spontaneous diabetes108

. Neutralization of CXCL10 in mice

prevents the disease progression in Th1-dominant type I diabetes108

. As well,

neutralization of CXCL10 or blocking of its receptor (CXCR3) results in reduced HIV-1

replication109

. Furthermore, CXCL10 expression is necessary for the clearance of mouse

hepatitis virus (MHV)110

. Neutralization of CXCL10 led to increased mortality and

delayed viral clearance of MHV110

.

2.7 Immune System and SARS

The mortality rate in SARS patients older than 65 years was greater than 50%,

while children presented with mild symptoms111,112

. SARS patients treated with

corticosteroids showed less lung injuries and IFN- therapy was effective in SARS

treatment47,52

. These findings are suggestive of the immune system as a key player in

SARS containment and progress. Pathological studies also suggested an important role

for the immune system in SARS pathogenesis113

. It has been demonstrated that the

number of alveolar macrophages were significantly increased in the lung after infection

with SARS-CoV113

. Infiltration of B and T lymphocytes has been reported in lung

biopsies of deceased SARS patients. Patients that were not treated with corticosteroids

demonstrated greater infiltration of immune cells113

.

Measurement of blood cytokine levels in SARS patients at disease onset would be

valuable in determining the magnitude, specificity and quality of immune response to

SARS-CoV99-100-101

.

34

2.8 Immunopathology of SARS

The two types of alveolar cells are type I and type II pneumocytes also known as

“squamous alveolar cells” and “great alveolar cells”, respectively. Pneumocyte II is the

primary target of SARS-CoV114

. The interaction occurs between spike glycoprotein of the

virus and ACE2 on the surface of these cells. Pneumocyte II cells are located at the

angles between alveolar septa and make up 12% of the lung cells115

. They differentiate

into pneumocyte I cells and produce pulmonary surfactant proteins. Diffuse alveolar

damage (DAD) in SARS is suggested to be the result of penumocyte II destruction during

the immune inflammatory response against SARS-CoV116

. The pathology of SARS is

defined as acute phase, proliferative phase and fibrotic phase. DAD usually develops in

the acute phase of the infection. Pneumocyte II cells undergo regenerative hyperplasia in

the proliferative phase117

. Metaplasia of pneumocyte I cells, formation of fibrin in

pulmonary vessels and focal honeycomb fibrosis are other outcomes of infection with

SARS-CoV. Infiltration of CD3+ T cells, CD20

+ B cells, CD4

+ and CD8

+ cells to the lung

has been shown, which may lead to more injury in the lungs of SARS patients117

. CD8+

cells were the most common infiltrating cells, whereas CD56+ NK cells were absent.

Infiltration of lymphoid cells was lower in patients who received higher doses of

corticosteroids117,118

. It is believed that SARS is a systemic disease and systemic cytokine

activation might have a great impact on the multi organ involvement118,119

. Spleen

lymphoid depletion was observed in many SARS patients with complicated outcomes.

Lymphopenia and depletion of spleen from lymphoid cells may be correlated to cytokine

involvement120

.

35

Other pathological features of SARS that were reported in some patients might be

due to hypoxemia (O2 saturations less than 95% in blood). Pathological features of SARS

in non-respiratory organs, including skeletal muscle fiber necrosis, heart muscle fiber

necrosis, necrotic lesions in the adrenal gland, acute tubular necrosis of kidney and

hepatitis is suggested as hypoxemia effects115,120

. There was no evidence of encephalitis

or any specific damage to the brain in SARS patients117

.

2.9 Cytokines and SARS

The main role of cytokines is to orchestrate the innate and adaptive immune

responses against pathogens. TLRs are involved in the recognition of pathogens and the

activation of downstream transcription factors such as NFB and production of

proinflammatory cytokines121

. TNF-, IL-1 and IL-6 are three cytokines known as early

response cytokines122

. Chemokine and adhesion molecule upregulation by TNF-, lead to

the recruitment of leukocytes to the site of infection123

. Chemokine receptors are

expressed on many immune and non-immune cell types. They play an important role in

polarized immune responses. CXCL10 is an example of a chemokine that boosts Th1

response124

.

In vitro and in vivo studies have shown a strong association between blood

cytokines and the pathologic outcomes of SARS infection. The existence of a cytokine

storm in SARS patients with non-favorable outcomes has been suggested by many

investigators125

. In one study, the serum level of IFN-, IL-1, IL-6, IL-12, IL-8, MCP-1

and CXCL10 were significantly increased one week post-infection with SARS-CoV non-

36

favorable outcomes. While IFN- and CXCL10 (Th1 cytokines) were elevated in this

study, the level of IL-10 (anti-inflammatory) and IL-4 (Th2 cytokine) remained

unchanged125

. In another study IL-6 upregulation in the peripheral blood of SARS

patients was correlated to the severity of the disease. Furthermore, the level of IL-1 and

TNF- was not significantly changed in this study126

. While CXCL10 was elevated in

early and progressive stages of SARS, the level of IL-1, IL-2, IL-4, IL-10, IL-12, TNF-

, IFN- and RANTES was normal in another cohort of patients127

. Tang et al. has also

shown the adverse association of the CXCL10, CXCL9 and IL-8 levels with the severity

of the disease128

. Huang et al. found increased levels of IFN-, IL-18, TGF-, IL-6,

CXCL10, CCL2, CXCL9 and IL-8 in SARS patients129

. Elevated levels of IL-1 and

normal levels of IL-6, TNF-, IL-12 and IL-8 were observed in children infected with

SARS-CoV111

. A significant increase in inflammatory cytokines in the peripheral blood

could be an indication of high levels of these soluble factors at the site of infection and

leak to the blood stream. This may lead to infiltration of leukocyte subpopulations and

generation of exaggerated immune response at the site of infection. As the measurement

of cytokine levels in the lungs of SARS patients is difficult, the use of an appropriate

animal model would be of great importance.

2.10 IRGs and SARS

In most SARS patients the illness resolved with no need for hospitalization3,6

. As

stated above, activation of interferon regulated genes (IRGs) and modulation of innate

and adaptive immunity is partly controlled by interferons. Some scientists have linked the

37

severity of SARS with poor interferon responses1,6,18

. Other studies have shown a direct

correlation between high levels of expression of some IRGs such as CCL2 and CXCL10

with the severity of the disease129

. It was shown that single-nucleotide mutations in the

sOAS1 and MX1 gene were associated with poor outcome in SARS patients130,131

. The

peak expression level of IRGs was during the acute phase of the disease and their

resolution was associated with the recovery in patients with good outcome. IFNAR1 and

IFNGR1 were upregulated in patients with poor outcome, while majority of the

remaining IRGs were downregulated in these patients86

. Evaluation of IRG expression in

a controlled study in an animal model would be beneficial, as poor upregulation of IRGs

in SARS patients is associated with poor outcome. Also it would be important to find out

how the IRG expression can be improved, for example by using therapeutic agents.

2.11 SARS and leukopenia

Pulmonary infiltration and air-space consolidation at the acute phase of SARS

was shown by chest radiography115

. Leukopenia and significant reduction of lymphocyte

subpopulations in peripheral blood of SARS patients was also shown. Absolute count of

T lymphocytes (CD3+), T helper cells (CD4

+) and T cytotoxic cells (CD8

+) was

significantly decreased during the first week in one study132

. Similar observations were

reported by other investigators, showing the existence of leukopenia in SARS

patients120,133

.

38

3. SARS animal models

Several animal models have been used for the in vivo study of SARS-CoV

infection. SARS-CoV replicates in the lung of Cynomolgus macaques with minimal

clinical symptoms52,120,134

. The virus replicates in mice as well, but rapid clearance and

absence of the disease symptoms have limited the use of this animal model109

.

Ferrets are susceptible to infection with SARS-CoV and the virus replicates well

in their lungs134

. They are capable of transmitting the virus to other uninfected ferrets

housed with them. Ferrets shed the virus from pharynx two days post-infection. Infected

animals present a number of symptoms similar to those seen in humans, such as lethargy

and elevated body temperature134

. Histological changes and pathologic changes in lung

necropsies of infected ferrets have been shown135

. It has been suggested that the ferret is a

more appropriate animal model for SARS compared to murine and primate models.

Martina et al. have shown that from 6 ferrets infected with SARS-CoV, 4 showed SARS

symptoms such as pharyngeal shedding, lethargy and lung lesions and one died at day

7134-136

.

4. Hypothesis

My general hypothesis is that unbalanced expression of interferon regulated genes

at early stages of infection with SARS-CoV is correlated with the severity of illness.

Specifically, the high and persistent expression of CXCL10 is associated with

severity of SARS in humans. Moreover, SARS-CoV infection in ferrets is associated with

39

upregulation of CXCL10 and poor upregulation of protective IRGs (MX1, OAS1, OAS2,

IG15 and IFI44).

5. Objectives

The objective of this study was to evaluate early immune responses in ferrets after

their infection with SARS-CoV. Specifically, I sought to investigate the role of interferon

regulated genes such as CXCL10 during severe SARS infection in humans. With the aim

of utilizing ferrets as an immune animal model of SARS, SARS-CoV infected ferrets

were examined to determine if the IRG profiles were similar to those of SARS infected

human patients.

Here, I propose that high levels of CXCL10, an interferon regulated gene, is

associated with poor outcome and disease progression in SARS-CoV-infected humans

and ferrets.

Subsequently, I investigated the interferon regulated genes role in the generation

of an antiviral state in SARS-CoV-infected ferrets. Furthermore, I went on to determine

if the profile of ferret interferon regulated genes was similar to that of injected ferrets

with IFN-2b in the hopes of delineating the interferon signaling post SARS infection. I

propose that there are similarities and differences between SARS-CoV-infected ferrets

and IFN-2b-injected ferrets in terms of expression of interferon regulated genes, which

may suggest the benefits of IFN therapy in possible future outbreaks of SARS.

Furthermore, localization of CD8+ cells may contribute to tissue injury in the

ferret model of SARS. The characterized ferret CD8 gene and generated reagents for

ferret CD8 molecule in this thesis can be used in future studies to examine the

40

localization of CD8+ T lymphocytes in the lung of SARS-CoV infected ferrets and its

role in SARS pathology.

41

Reference List

1. Cowling,B.J. et al. Community psychological and behavioral responses through

the first wave of the 2009 influenza A(H1N1) pandemic in Hong Kong. J. Infect.

Dis. 202, 867-876 (2010).

2. Jin,Y. et al. Prevalence and clinical characteristics of human CoV-HKU1 in

children with acute respiratory tract infections in China. J. Clin. Virol. 49, 126-

130 (2010).

3. Lee,N. et al. A major outbreak of severe acute respiratory syndrome in Hong

Kong. N. Engl. J. Med. 348, 1986-1994 (2003).

4. Li,W. et al. Animal origins of the severe acute respiratory syndrome coronavirus:

insight from ACE2-S-protein interactions. J. Virol. 80, 4211-4219 (2006).

5. Lau,E.H. et al. A comparative epidemiologic analysis of SARS in Hong Kong,

Beijing and Taiwan. BMC. Infect. Dis. 10, 50 (2010).

6. Poutanen,S.M. et al. Identification of severe acute respiratory syndrome in

Canada. N. Engl. J. Med. 348, 1995-2005 (2003).

7. Breslin,J.J. et al. Human coronavirus 229E: receptor binding domain and

neutralization by soluble receptor at 37 degrees C. J. Virol. 77, 4435-4438 (2003).

8. Manocha,S., Walley,K.R. & Russell,J.A. Severe acute respiratory distress

syndrome (SARS): a critical care perspective. Crit Care Med. 31, 2684-2692

(2003).

9. Vincent,A.L. et al. Characterization of an influenza A virus isolated from pigs

during an outbreak of respiratory disease in swine and people during a county fair

in the United States. Vet. Microbiol. 137, 51-59 (2009).

10. Mahony,J.B. et al. Multiplex PCR tests sentinel the appearance of pandemic

influenza viruses including H1N1 swine influenza. J. Clin. Virol. 45, 200-202

(2009).

11. Dawood,F.S. et al. Emergence of a novel swine-origin influenza A (H1N1) virus

in humans. N. Engl. J. Med. 360, 2605-2615 (2009).

12. Perez-Padilla,R. et al. Pneumonia and respiratory failure from swine-origin

influenza A (H1N1) in Mexico. N. Engl. J. Med. 361, 680-689 (2009).

13. Deyde,V.M. et al. Detection of molecular markers of drug resistance in 2009

pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob. Agents

Chemother. 54, 1102-1110 (2010).

42

14. Nougairede,A. et al. Likely correlation between sources of information and

acceptability of A/H1N1 swine-origin influenza virus vaccine in Marseille,

France. PLoS. One. 5, e11292 (2010).

15. Horney,J.A., Moore,Z., Davis,M. & MacDonald,P.D. Intent to receive pandemic

influenza A (H1N1) vaccine, compliance with social distancing and sources of

information in NC, 2009. PLoS. One. 5, e11226 (2010).

16. Maurer,J., Uscher-Pines,L. & Harris,K.M. Perceived seriousness of seasonal and

A(H1N1) influenzas, attitudes toward vaccination, and vaccine uptake among

U.S. adults: does the source of information matter? Prev. Med. 51, 185-187

(2010).

17. Lau,Y.F., Tang,L.H., Ooi,E.E. & Subbarao,K. Activation of the innate immune

system provides broad-spectrum protection against influenza A viruses with

pandemic potential in mice. Virology 406, 80-87 (2010).

18. Giamarellos-Bourboulis,E.J. et al. Effect of the novel influenza A (H1N1) virus in

the human immune system. PLoS. One. 4, e8393 (2009).

19. Chen,J. et al. Cellular immune responses to severe acute respiratory syndrome

coronavirus (SARS-CoV) infection in senescent BALB/c mice: CD4+ T cells are

important in control of SARS-CoV infection. J. Virol. 84, 1289-1301 (2010).

20. Lau,S.K. et al. Ecoepidemiology and complete genome comparison of different

strains of severe acute respiratory syndrome-related Rhinolophus bat coronavirus

in China reveal bats as a reservoir for acute, self-limiting infection that allows

recombination events. J. Virol. 84, 2808-2819 (2010).

21. Li,F. Structural analysis of major species barriers between humans and palm

civets for severe acute respiratory syndrome coronavirus infections. J. Virol. 82,

6984-6991 (2008).

22. Yu,I.M. et al. Recombinant severe acute respiratory syndrome (SARS)

coronavirus nucleocapsid protein forms a dimer through its C-terminal domain. J.

Biol. Chem. 280, 23280-23286 (2005).

23. Woo,P.C. et al. Characterization and complete genome sequence of a novel

coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79, 884-

895 (2005).

24. Rota,P.A. et al. Characterization of a novel coronavirus associated with severe

acute respiratory syndrome. Science 300, 1394-1399 (2003).

25. Marra,M.A. et al. The Genome sequence of the SARS-associated coronavirus.

Science 300, 1399-1404 (2003).

43

26. Ortego,J., Escors,D., Laude,H. & Enjuanes,L. Generation of a replication-

competent, propagation-deficient virus vector based on the transmissible

gastroenteritis coronavirus genome. J. Virol. 76, 11518-11529 (2002).

27. He,Y., Li,J. & Jiang,S. A single amino acid substitution (R441A) in the receptor-

binding domain of SARS coronavirus spike protein disrupts the antigenic

structure and binding activity. Biochem. Biophys. Res. Commun. 344, 106-113

(2006).

28. Jeffers,S.A. et al. CD209L (L-SIGN) is a receptor for severe acute respiratory

syndrome coronavirus. Proc. Natl. Acad. Sci. U. S. A 101, 15748-15753 (2004).

29. Kuba,K. et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in

SARS coronavirus-induced lung injury. Nat. Med. 11, 875-879 (2005).

30. Imai,Y. et al. Angiotensin-converting enzyme 2 protects from severe acute lung

failure. Nature 436, 112-116 (2005).

31. Grigoriev,A. Mutational patterns correlate with genome organization in SARS

and other coronaviruses. Trends Genet. 20, 131-135 (2004).

32. Lai,M.M. SARS virus: the beginning of the unraveling of a new coronavirus. J.

Biomed. Sci. 10, 664-675 (2003).

33. Qinfen,Z. et al. The life cycle of SARS coronavirus in Vero E6 cells. J. Med.

Virol. 73, 332-337 (2004).

34. Stadler,K. et al. SARS--beginning to understand a new virus. Nat. Rev. Microbiol.

1, 209-218 (2003).

35. Lang,Z. et al. Pathological study on severe acute respiratory syndrome. Chin Med.

J. (Engl. ) 116, 976-980 (2003).

36. Yan,H. et al. SARS coronavirus induces apoptosis in Vero E6 cells. J. Med. Virol.

73, 323-331 (2004).

37. Chan,K.H. et al. The Effects of Temperature and Relative Humidity on the

Viability of the SARS Coronavirus. Adv. Virol. 2011, 734690 (2011).

38. Rabenau,H.F., Kampf,G., Cinatl,J. & Doerr,H.W. Efficacy of various

disinfectants against SARS coronavirus. J. Hosp. Infect. 61, 107-111 (2005).

39. Lim,W., Ng,K.C. & Tsang,D.N. Laboratory containment of SARS virus. Ann.

Acad. Med. Singapore 35, 354-360 (2006).

40. LaBarre,D.D. & Lowy,R.J. Improvements in methods for calculating virus titer

estimates from TCID50 and plaque assays. J. Virol. Methods 96, 107-126 (2001).

44

41. Leong,H.N. et al. Clinical and laboratory findings of SARS in Singapore. Ann.


42. Liang,G. et al. Laboratory diagnosis of four recent sporadic cases of community-

acquired SARS, Guangdong Province, China. Emerg. Infect. Dis. 10, 1774-1781

(2004).

43. Muller,M.P. et al. Early diagnosis of SARS: lessons from the Toronto SARS

outbreak. Eur. J. Clin. Microbiol. Infect. Dis. 25, 230-237 (2006).

44. Hui,D.S. et al. Severe acute respiratory syndrome: correlation between clinical

outcome and radiologic features. Radiology 233, 579-585 (2004).

45. Muller,M.P. et al. Can routine laboratory tests discriminate between severe acute

respiratory syndrome and other causes of community-acquired pneumonia? Clin.

Infect. Dis. 40, 1079-1086 (2005).

46. Fan,C.K. et al. Clinical and laboratory features in the early stage of severe acute

respiratory syndrome. J. Microbiol. Immunol. Infect. 39, 45-53 (2006).

47. Loutfy,M.R. et al. Interferon alfacon-1 plus corticosteroids in severe acute

respiratory syndrome: a preliminary study. JAMA 290, 3222-3228 (2003).

48. Schroeder,J.A. et al. Microwave-assisted tissue processing for same-day EM-

diagnosis of potential bioterrorism and clinical samples. Micron. 37, 577-590

(2006).

49. Leong,H.N. et al. Investigational use of ribavirin in the treatment of severe acute

respiratory syndrome, Singapore, 2003. Trop. Med. Int. Health 9, 923-927 (2004).

50. Wang,W.K. et al. Temporal relationship of viral load, ribavirin, interleukin (IL)-

6, IL-8, and clinical progression in patients with severe acute respiratory

syndrome. Clin. Infect. Dis. 39, 1071-1075 (2004).

51. Barnard,D.L. et al. Enhancement of the infectivity of SARS-CoV in BALB/c

mice by IMP dehydrogenase inhibitors, including ribavirin. Antiviral Res. 71, 53-

63 (2006).

52. Haagmans,B.L. et al. Pegylated interferon-alpha protects type 1 pneumocytes

against SARS coronavirus infection in macaques. Nat. Med. 10, 290-293 (2004).

53. ter Meulen,J. et al. Human monoclonal antibody as prophylaxis for SARS

coronavirus infection in ferrets. Lancet 363, 2139-2141 (2004).

54. Gao,W. et al. Effects of a SARS-associated coronavirus vaccine in monkeys.

Lancet 362, 1895-1896 (2003).

45

55. Yang,Z.Y. et al. A DNA vaccine induces SARS coronavirus neutralization and

protective immunity in mice. Nature 428, 561-564 (2004).

56. Huang,J., Ma,R. & Wu,C.Y. Immunization with SARS-CoV S DNA vaccine

generates memory CD4+ and CD8+ T cell immune responses. Vaccine 24, 4905-

4913 (2006).

57. Wang,Z., Yuan,Z., Matsumoto,M., Hengge,U.R. & Chang,Y.F. Immune

responses with DNA vaccines encoded different gene fragments of severe acute

respiratory syndrome coronavirus in BALB/c mice. Biochem. Biophys. Res.

Commun. 327, 130-135 (2005).

58. Czub,M., Weingartl,H., Czub,S., He,R. & Cao,J. Evaluation of modified vaccinia

virus Ankara based recombinant SARS vaccine in ferrets. Vaccine 23, 2273-2279

(2005).

59. Tan,A.T. et al. A longitudinal analysis of innate and adaptive immune profile

during hepatic flares in chronic hepatitis B. J. Hepatol. 52, 330-339 (2010).

60. Liu,G., Zhang,L. & Zhao,Y. Modulation of immune responses through direct

activation of Toll-like receptors to T cells. Clin. Exp. Immunol. 160, 168-175

(2010).

61. Ricklin,D., Hajishengallis,G., Yang,K. & Lambris,J.D. Complement: a key

system for immune surveillance and homeostasis. Nat. Immunol. 11, 785-797

(2010).

62. Kawai,T. & Akira,S. Innate immune recognition of viral infection. Nat. Immunol.

7, 131-137 (2006).

63. Akira,S., Uematsu,S. & Takeuchi,O. Pathogen recognition and innate immunity.

Cell 124, 783-801 (2006).

64. Kawai,T. & Akira,S. The role of pattern-recognition receptors in innate immunity:

update on Toll-like receptors. Nat. Immunol. 11, 373-384 (2010).

65. Iwasaki,A. & Medzhitov,R. Regulation of adaptive immunity by the innate

immune system. Science 327, 291-295 (2010).

66. Vari,F. & Hart,D.N. Loading DCs with Ag. Cytotherapy. 6, 111-121 (2004).

67. Dudley,D., Chaudhuri,J., Bassing,C. & Alt,F. Mechanism and control of V(D)J

recombination versus class switch recombination: similarities and differences.

Adv. Immunol. 86, 43-112 (2005).

68. Chu,P. & Arber,D. CD79: a Review. Appl. immunohistochem. Mol. Morphol. 9,

97-106 (2001).

46

69. Eason,D. et al. Mechanisms of antigen receptor evolution. Semin. Immunol. 16,

215-226 (2004).

70. Moss,R.B. et al. Th1/Th2 cells in inflammatory disease states: therapeutic

implications. Expert. Opin. Biol. Ther. 4, 1887-1896 (2004).

71. Kim,T.S., Sun,J. & Braciale,T.J. T cell responses during influenza infection:

getting and keeping control. Trends Immunol. 32, 225-231 (2011).

72. Locksley,R.M. Nine lives: plasticity among T helper cell subsets. J. Exp. Med.

206, 1643-1646 (2009).

73. Boyman,O., Letourneau,S., Krieg,C. & Sprent,J. Homeostatic proliferation and

survival of naive and memory T cells. Eur. J. Immunol. 39, 2088-2094 (2009).

74. Halin,C., Mora,J.R., Sumen,C. & von Andrian,U.H. In vivo imaging of

lymphocyte trafficking. Annu. Rev. Cell Dev. Biol. 21, 581-603 (2005).

75. Randolph,G.J., Ochando,J. & Partida-Sanchez,S. Migration of dendritic cell

subsets and their precursors. Annu. Rev. Immunol. 26, 293-316 (2008).

76. Moon,J.J. et al. Naive CD4(+) T cell frequency varies for different epitopes and

predicts repertoire diversity and response magnitude. Immunity. 27, 203-213

(2007).

77. Jenkins,M.K., Chu,H.H., McLachlan,J.B. & Moon,J.J. On the composition of the

preimmune repertoire of T cells specific for Peptide-major histocompatibility

complex ligands. Annu. Rev. Immunol. 28, 275-294 (2010).

78. von Andrian,U.H. & Mackay,C.R. T-cell function and migration. Two sides of the

same coin. N. Engl. J. Med. 343, 1020-1034 (2000).

79. Mora,J.R. & von Andrian,U.H. T-cell homing specificity and plasticity: new

concepts and future challenges. Trends Immunol. 27, 235-243 (2006).

80. Marzio,R., Mauel,J. & Betz-Corradin,S. CD69 and regulation of the immune

function. Immunopharmacol. Immunotoxicol. 21, 565-582 (1999).

81. Maceyka,M., Harikumar,K.B., Milstien,S. & Spiegel,S. Sphingosine-1-phosphate

signaling and its role in disease. Trends Cell Biol. 22, 50-60 (2012).

82. Spiegel,S. & Milstien,S. The outs and the ins of sphingosine-1-phosphate in

immunity. Nat. Rev. Immunol. 11, 403-415 (2011).

83. Hall,J.C. & Rosen,A. Type I interferons: crucial participants in disease

amplification in autoimmunity. Nat. Rev. Rheumatol. 6, 40-49 (2010).

47

84. Galligan,C.L. et al. Interferons and viruses: signaling for supremacy. Immunol.

Res. 35, 27-40 (2006).

85. Castilletti,C. et al. Coordinate induction of IFN-alpha and -gamma by SARS-CoV

also in the absence of virus replication. Virology 341, 163-169 (2005).

86. Cameron,M.J. et al. Interferon-mediated immunopathological events are

associated with atypical innate and adaptive immune responses in patients with

severe acute respiratory syndrome. J. Virol. 81, 8692-8706 (2007).

87. Yun,P.L., DeCarlo,A.A. & Hunter,N. Modulation of major histocompatibility

complex protein expression by human gamma interferon mediated by cysteine

proteinase-adhesin polyproteins of Porphyromonas gingivalis. Infect. Immun. 67,

2986-2995 (1999).

88. Smeltz,R.B. Profound enhancement of the IL-12/IL-18 pathway of IFN-gamma

secretion in human CD8+ memory T cell subsets via IL-15. J. Immunol. 178,

4786-4792 (2007).

89. Shtrichman,R. & Samuel,C.E. The role of gamma interferon in antimicrobial

immunity. Curr. Opin. Microbiol. 4, 251-259 (2001).

90. Uddin,S. & Platanias,L.C. Mechanisms of type-I interferon signal transduction. J.

Biochem. Mol. Biol. 37, 635-641 (2004).

91. Ramana,C.V., Gil,M.P., Schreiber,R.D. & Stark,G.R. Stat1-dependent and -

independent pathways in IFN-gamma-dependent signaling. Trends Immunol. 23,

96-101 (2002).

92. Haque,S.J. & Williams,B.R. Signal transduction in the interferon system. Semin.

Oncol. 25, 14-22 (1998).

93. Garcia-Sastre,A. & Biron,C.A. Type 1 interferons and the virus-host relationship:

a lesson in detente. Science 312, 879-882 (2006).

94. Takaoka,A. & Yanai,H. Interferon signalling network in innate defence. Cell

Microbiol. 8, 907-922 (2006).

95. Haller,O., Staeheli,P. & Kochs,G. Interferon-induced Mx proteins in antiviral host

defense. Biochimie 89, 812-818 (2007).

96. Bonnevie-Nielsen,V. et al. Variation in antiviral 2',5'-oligoadenylate synthetase

(2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a

splice-acceptor site in the OAS1 gene. Am. J. Hum. Genet. 76, 623-633 (2005).

97. Okumura,A., Lu,G., Pitha-Rowe,I. & Pitha,P.M. Innate antiviral response targets

HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc. Natl. Acad.

Sci. U. S. A 103, 1440-1445 (2006).

48

98. Luster,A.D. & Ravetch,J.V. Genomic characterization of a gamma-interferon-

inducible gene (IP-10) and identification of an interferon-inducible hypersensitive

site. Mol. Cell Biol. 7, 3723-3731 (1987).

99. Dufour,J.H. et al. IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient

mice reveal a role for IP-10 in effector T cell generation and trafficking. J.

Immunol. 168, 3195-3204 (2002).

100. Murphy,P.M. et al. International union of pharmacology. XXII. Nomenclature for

chemokine receptors. Pharmacol. Rev. 52, 145-176 (2000).

101. Villagomez,M.T., Bae,S.J., Ogawa,I., Takenaka,M. & Katayama,I. Tumour

necrosis factor-alpha but not interferon-gamma is the main inducer of inducible

protein-10 in skin fibroblasts from patients with atopic dermatitis. Br. J.

Dermatol. 150, 910-916 (2004).

102. Frangogiannis,N.G., Mendoza,L.H., Smith,C.W., Michael,L.H. & Entman,M.L.

Induction of the synthesis of the C-X-C chemokine interferon-gamma-inducible

protein-10 in experimental canine endotoxemia. Cell Tissue Res. 302, 365-376

(2000).

103. Belperio,J.A. et al. Role of CXCL9/CXCR3 chemokine biology during

pathogenesis of acute lung allograft rejection. J. Immunol. 171, 4844-4852

(2003).

104. Arai,K., Liu,Z.X., Lane,T. & Dennert,G. IP-10 and Mig facilitate accumulation of

T cells in the virus-infected liver. Cell Immunol. 219, 48-56 (2002).

105. Hancock,W.W. et al. Donor-derived IP-10 initiates development of acute allograft

rejection. J. Exp. Med. 193, 975-980 (2001).

106. Christen,U., McGavern,D.B., Luster,A.D., von Herrath,M.G. & Oldstone,M.B.

Among CXCR3 chemokines, IFN-gamma-inducible protein of 10 kDa (CXC

chemokine ligand (CXCL) 10) but not monokine induced by IFN-gamma

(CXCL9) imprints a pattern for the subsequent development of autoimmune

disease. J. Immunol. 171, 6838-6845 (2003).

107. Agostini,C. et al. Cxcr3 and its ligand CXCL10 are expressed by inflammatory

cells infiltrating lung allografts and mediate chemotaxis of T cells at sites of

rejection. Am. J. Pathol. 158, 1703-1711 (2001).

108. Shigihara,T. et al. CXCL10 DNA vaccination prevents spontaneous diabetes

through enhanced beta cell proliferation in NOD mice. J. Immunol. 175, 8401-

8408 (2005).

109. Glass,W.G., Subbarao,K., Murphy,B. & Murphy,P.M. Mechanisms of host

defense following severe acute respiratory syndrome-coronavirus (SARS-CoV)

pulmonary infection of mice. J. Immunol. 173, 4030-4039 (2004).

49

110. Liu,M.T. et al. The T cell chemoattractant IFN-inducible protein 10 is essential in

host defense against viral-induced neurologic disease. J. Immunol. 165, 2327-

2330 (2000).

111. Ng,P.C. et al. Inflammatory cytokine profile in children with severe acute

respiratory syndrome. Pediatrics 113, e7-14 (2004).

112. Xie,X., Chen,J., Wang,X., Zhang,F. & Liu,Y. Age- and gender-related difference

of ACE2 expression in rat lung. Life Sci. 78, 2166-2171 (2006).

113. Theron,M., Huang,K.J., Chen,Y.W., Liu,C.C. & Lei,H.Y. A probable role for

IFN-gamma in the development of a lung immunopathology in SARS. Cytokine

32, 30-38 (2005).

114. Chan,W.S. et al. Coronaviral hypothetical and structural proteins were found in

the intestinal surface enterocytes and pneumocytes of severe acute respiratory

syndrome (SARS). Mod. Pathol. 18, 1432-1439 (2005).

115. Franks,T.J. et al. Lung pathology of severe acute respiratory syndrome (SARS): a

study of 8 autopsy cases from Singapore. Hum. Pathol. 34, 743-748 (2003).

116. Chow,K.C., Hsiao,C.H., Lin,T.Y., Chen,C.L. & Chiou,S.H. Detection of severe

acute respiratory syndrome-associated coronavirus in pneumocytes of the lung.

Am. J. Clin. Pathol. 121, 574-580 (2004).

117. Ng,W.F., To,K.F., Lam,W.W., Ng,T.K. & Lee,K.C. The comparative pathology

of severe acute respiratory syndrome and avian influenza A subtype H5N1--a

review. Hum. Pathol. 37, 381-390 (2006).

118. Ding,Y. et al. The clinical pathology of severe acute respiratory syndrome

(SARS): a report from China. J. Pathol. 200, 282-289 (2003).

119. Sheng,W.H. et al. Clinical manifestations and inflammatory cytokine responses in

patients with severe acute respiratory syndrome. J. Formos. Med. Assoc. 104,

715-723 (2005).

120. Cui,W. et al. Expression of lymphocytes and lymphocyte subsets in patients with

severe acute respiratory syndrome. Clin. Infect. Dis. 37, 857-859 (2003).

121. Strieter,R.M., Belperio,J.A. & Keane,M.P. Host innate defenses in the lung: the

role of cytokines. Curr. Opin. Infect. Dis. 16, 193-198 (2003).

122. Neri,M. et al. Myocardial expression of TNF-alpha, IL-1beta, IL-6, IL-8, IL-10

and MCP-1 after a single MDMA dose administered in a rat model. Curr. Pharm.

Biotechnol. 11, 413-420 (2010).

50

123. Norris,D.A. Cytokine modulation of adhesion molecules in the regulation of

immunologic cytotoxicity of epidermal targets. J. Invest Dermatol. 95, 111S-120S

(1990).

124. Culley,F.J., Pennycook,A.M., Tregoning,J.S., Hussell,T. & Openshaw,P.J.

Differential chemokine expression following respiratory virus infection reflects

Th1- or Th2-biased immunopathology. J. Virol. 80, 4521-4527 (2006).

125. Wong,C.K. et al. Plasma inflammatory cytokines and chemokines in severe acute

respiratory syndrome. Clin. Exp. Immunol. 136, 95-103 (2004).

126. Zhang,Y. et al. Analysis of serum cytokines in patients with severe acute

respiratory syndrome. Infect. Immun. 72, 4410-4415 (2004).

127. Jiang,Y. et al. Characterization of cytokine/chemokine profiles of severe acute

respiratory syndrome. Am. J. Respir. Crit Care Med. 171, 850-857 (2005).

128. Tang,N.L. et al. Early enhanced expression of interferon-inducible protein-10

(CXCL-10) and other chemokines predicts adverse outcome in severe acute

respiratory syndrome. Clin. Chem. 51, 2333-2340 (2005).

129. Huang,K.J. et al. An interferon-gamma-related cytokine storm in SARS patients.

J. Med. Virol. 75, 185-194 (2005).

130. Hamano,E. et al. Polymorphisms of interferon-inducible genes OAS-1 and MxA

associated with SARS in the Vietnamese population. Biochem. Biophys. Res.

Commun. 329, 1234-1239 (2005).

131. He,J. et al. Association of SARS susceptibility with single nucleic acid

polymorphisms of OAS1 and MxA genes: a case-control study. BMC. Infect. Dis.

6, 106 (2006).

132. Li,T. et al. Significant changes of peripheral T lymphocyte subsets in patients

with severe acute respiratory syndrome. J. Infect. Dis. 189, 648-651 (2004).

133. Tang,X. et al. Measurement of subgroups of peripheral blood T lymphocytes in

patients with severe acute respiratory syndrome and its clinical significance. Chin

Med. J. (Engl. ) 116, 827-830 (2003).

134. Martina,B.E. et al. Virology: SARS virus infection of cats and ferrets. Nature

425, 915 (2003).

135. Marshall,E. & Enserink,M. Medicine. Caution urged on SARS vaccines. Science

303, 944-946 (2004).

136. See,R.H. et al. Severe acute respiratory syndrome vaccine efficacy in ferrets:

whole killed virus and adenovirus-vectored vaccines. J. Gen. Virol. 89, 2136-

2146 (2008).

51

Chapter 2

Upregulation of CXCL10 is associated with disease progression in

SARS-CoV- infected humans and ferrets.

(A version of this chapter is published in Molecular Immunology, Volume 45 (2008),

1288–1297 as “Cloning, expression and characterization of ferret CXCL10”.

Ali Danesh, Charit Seneviratne, Cheryl M. Cameron, David Banner, Mark E. Devries,

Alyson A. Kelvin, Luoling Xu, Longsi Ran, Steven E. Bosinger, Thomas Rowe, Marcus

Czub, Colleen B. Jonsson, Mark J. Cameron and David J. Kelvin, with permission from

Elsevier)

1. Introduction

The CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) chemokines, along

with their cognate receptor CXCR3 participate in the development and regulation of Th1

responses. The expression of CXCL9, 10, and 11 can be induced in virtually all cell

types by a variety of stimulants including IFN-γ, TLR activation and viral infection1-3

.

There is evidence in the literature that strongly suggests that CXCL10 is an important

regulator of Th1 polarized immune responses4, and that, in some instances, the CXCL9,

10 and 11:CXCR3 axis can generate pathological inflammatory conditions5-8

. While

several reports have identified an increased expression of CXCL9 or CXCL10 following

viral infection in animal models or humans5,7,9-11

, the role of these chemokines and their

receptor CXCR3 in the development of ARDS (acute respiratory distress syndrome)-like

diseases associated with pathogenic pulmonary viral infections is unknown. Support for

a role of this subfamily of chemokines in SARS comes from observations by ourselves

and other groups showing a correlation between increased systemic levels of CXCL10 in

SARS coronavirus (SARS-CoV)12-14

and H5N1-infected individuals15

, as well as

52

chronically elevated levels of systemic expression of CXCL10 in patients with poor

outcome post-infection12,14

.

It has been suggested that the ferret is a more appropriate animal model for SARS

compared to murine and primate models16

. Cynomolgus macaques (Macaca fascicularis)

fail to show clinical symptoms following infection with SARS-CoV, despite viral

replication in the species16,17

. In mice, the SARS-CoV replicates well, but the value of

the model as representative of the human condition has been debated due to rapid

clearance of the virus and the absence of clinical symptoms18

. In contrast, SARS-CoV

replicates well in ferrets with infected animals demonstrating a number of clinical

manifestations similar to those seen in humans, such as lethargy and elevated body

temperature16

. Furthermore, SARS-CoV infected ferrets display histological evidence of

pneumonitis, and a certain degree of mortality has been reported, which is also consistent

with the human disease16,17

. Replication of SARS-CoV occurs both in the upper and

lower respiratory tract, and development of ARDS-like disease also occurs in some

patients19,20

. Diarrhea has been reported in a portion of cases of human H5N1 infection,

and was also noted in many infected ferrets21-23

.

The ferret is the preferred animal model for influenza research, including that

with avian strains such as the H5N1 sub-type, as it closely mimics the human disease in

terms of susceptibility to infection, transmission, pathology and spectrum of symptoms

observed upon infection. For example, ferrets infected with highly pathogenic human

isolates of H5N1 virus develop rhinorrea and ocular discharge, anorexia, otologic

manifestations, fever, weight loss, dyspnea, pulmonary infiltrates and lymphophenia23-25

.

53

The major drawback to the use of the ferret model for either SARS-CoV or

influenza virus research is the general lack of immunological reagents. To investigate the

roles of CXCL9, 10, 11 and CXCR3 in SARS and avian influenza pathogenesis in the

ferret model, there is a need to develop ferret-specific antibodies to accurately assess

chemokine expression levels in infected animals, and to neutralize any effects these

chemokines might have in pathogenesis progression and clearance of infection.

As a first step towards this goal, we have cloned and sequenced the genes

encoding ferret CXCL9, CXCL10, CXCL11 and CXCR3 orthologues, and then

expressed and purified recombinant ferret CXCL10 protein. Next we showed that

expression of CXCL9, 10, and 11 were induced by LPS stimulation of ferret PBMC

cultures. We have also demonstrated that CXCL10 plasma level in different groups of

SARS patients is elevated. In addition, we have shown that levels of the mRNA

expression of CXCR3 and CXCL10 are increased in the lungs of deceased patients

infected with SARS-CoV during the SARS outbreak in Toronto. Furthermore, we have

confirmed that the increased CXCL10 expression observed in these patients is paralleled

in ferrets infected with SARS-CoV.

54

2. Materials and methods

2.1 SARS patients

Blood samples of suspected SARS patients were received every 7 days until day

28 or longer from University Health network hospitals in Toronto. Written consent was

obtained by hospital officials from patients or from their legal designates. Plasma was

harvested from collected blood for measurement of cytokines levels. Clinical data was

obtained from collaborating hospitals and entered in a database. Only confirmed cases

of SARS based on WHO definition of SARS (described in introduction) and a positive

RT-PCR test for SARS-CoV were included in this study.

2.2 Animals

Six-month-old male Fitch ferrets (Mustela putorius furo) were purchased from

Triple F Farms Inc. (Sayre, Pa. USA). Animals used for non-infectious experimentation

were housed at Toronto General Research Institute animal facility. The animal use

protocol was approved by Animal Care Committee of the University Health Network,

Toronto, Ontario. All experiments were conducted in accordance with committee

recommendations. Animals were quarantined and monitored for one week prior to tissue

and blood collection. The animals’ diet was based on a low fat/high protein regimen

recommended by Triple F Farms for small carnivores. Animal experiments involving

virus work were performed in the Animal Biohazard Safety Level 3 (ABSL3) facility at

Southern Research Institute (Birmingham, Alabama, USA), in accordance with the

approved protocols. Briefly, three male ferrets (castrated, descented), weighing

approximately 800-1000g, were infected intranasally with 103 TCID50 SARS-CoV virus

55

(TOR2 strain) in 1 mL PBS. An additional three animals were mock-infected using 1 mL

PBS alone. Infected animals were deeply anesthetized and euthanized via exsanguination

between days 5 and 7 post-infection, and lung tissue was obtained at necropsy and

immediately homogenized in Tripure reagent (Roche Diagnostics, Indianapolis, USA).

2.3 Titration of SARS-CoV:

A SARS-CoV Tor-2 strain provided by Dr. Feldman, University of Manitoba, was

propagated and titrated in Vero E6 cells at BSL3 animal facility of southern Research

Institute, Alabama U.S.A. 20,000 Vero E6 cells in 100 l cell culture media were added

to each well of a 96-well plate. Plate was incubated overnight at 37 °C in CO2 incubator.

The viral stock was serially diluted at 10-fold intervals from 10-1

to 10-8

. 100 l of each

viral dilution was added to wells of one row. Plate was incubated in the CO2 incubator for

another 3 days. The cytopathic effect in Vero E6 cells, which is the structural changes of

virally infected cells and leads to their lysis, was considered for determining the endpoint

and calculation of TCID50/ml. The endpoint was the lowest viral titer that caused the

lysis of infected cells in some but not all wells of a row in a 96 well plate. Each row of

the plate was representative of a titer as is described in chapter one.

2.4 Viral load and clinical characteristics of infected ferrets

The dose of SARS-CoV in my study was chosen based on smaller studies

performed in the Southern Research Institute by our collaborators, indicating that a lower

dose of the virus (103 TCID50/ml) generated more reproducible data. With this dose, less

56

variation of lung infectivity was observed and nasal turbinate and lung infectivity were at

its highest level. High doses of SARS-CoV (107 TCID50/ml) resulted in minimal nasal

turbinate infectivity. Ferrets were inspected daily for signs, symptoms, onset, severity and

duration of disease. The activity was scored at a scale of 1 to 3, with 3 as the worst.

Infected ferrets showed increased temperature and loss of appetite, which last for 7 days.

Sneezing started at day 3 and last until day 10 post-infection. Subcutaneous temperatures

showed increased levels in SARS-CoV infected ferrets from day 2 to 7 post-infection

with its peak at day 2 (38 °C), while the temperature of mock-infected ferrets was

between (36.5-37 °C). Viral levels in the nasal turbinates of infected ferrets peaked on

day 3 (107 TCID50), and dropped to undetectable level at day 7. Virus levels in the lungs

peaked at day 5 (105 TCID50) and dropped by day 7.

2.5 Cytometric Bead Array (CBA)

The level of CXCL9 and CXCL10 in the plasma of SARS patients were assayed

by chemokine cytometric bead array (CBA) kit (BD Biosciences, San Jose, CA).

Detection limits were 2 to 10000 pg/ml. Briefly, standards were reconstituted in assay

diluent and serially diluted to achieve a range of 0 to 10000 pg /ml. Fifty ul of mixed

capture beads, 50 ul standard / test samples and 50 ul PE detection reagent were added to

each tube. Tubes were incubated 3 hours in dark at room temperature. Beads were

washed with 1 ml wash buffer and re-suspended in 300 µl of 2% paraformaldehyde in

PBS. Data were acquired, using BD FACSCalibure flow cytometer. Results were

generated using BD CBA analysis software.

57

2.6 Human lung biopsies and total RNA purification

Total RNA was purified using Tripure mRNA kits (Roche Diagnostics,

Indianapolis, USA). Briefly, lung lower bilateral lobe biopsies collected from deceased

SARS patients at autopsy were homogenized in 2 mL Tripure reagent in a 50 mL

polypropylene tube at room temperature and chloroform extraction of RNA was

performed and then cleaned by DNase. Informed consent was obtained from subjects or

their substitute decision makers under the approval of the Research Ethics Boards of the

University Health Network and participating Toronto-area hospitals.

2.7 Ferret total RNA purification and cDNA synthesis

Ferret whole blood was diluted 1:1 with RPMI (Invitrogen, USA) and stimulated

with mitogens, lipopolysaccharide (LPS) (1 ug/mL), phorbol myristate acetate (PMA) (50

ng/mL) and ionomycin (0.1 mM), or polyinosinic: polycytidylic acid (poly I:C) (25

g/mL) by incubating at 37ºC in 5% CO2 for 1, 2, 4, 8, and 16 hours prior to RNA

purification. Paxgene RNA isolation method (Qiagen, Missisauga, Canada) was used for

RNA purification and then RNA was cleaned by DNase. Purified human or ferret total

RNA was reverse transcribed to cDNA using reverse transcriptase II (Invitrogen,

Carlsbad, USA).

2.8 Real-time RT-PCR

The following components were added to the reaction mixture plus cDNA to a

total volume of 10 µL in distilled water: 0.25 l cDNA, 250 nM forward gene-specific

primer, 250 nM reverse gene-specific primer, 5 L Cyber Green (Applied Biosystems,

58

Foster City, California, USA). In a single experiment, each reaction was performed in

triplicate. An ABI 7900 Sequence Detection System (Applied Biosystems, Foster City,

California, USA) was used for amplification. Initial denaturation was 15 minutes at

95oC, followed by 40 cycles of amplification. Each cycle consisted of a denaturation step

(15 seconds at 95oC) and an annealing/extension step (1 minute at 60

oC). Gene

expression levels were normalized to β-actin and data was analyzed by SDS 2.1 software

(Applied Biosystems, California).

2.9 Amplification, cloning and sequencing of ferret-specific genes

Purified RNA was reverse transcribed to cDNA using reverse transcriptase II

(Invitrogen, Carlsbad, USA). Gene specific degenerate primers were designed based on

multiple gene sequence alignment analysis of several species using the ClustalW (1.83)

web-based program from European Bioinformatics Institute (EBI) and then used to clone

the cDNAs for each gene. Primers were tested in silico using Primer Express (Applied

Biosystems, CA, USA). Standard PCR was performed using degenerate primers and

template cDNA. Specific bands were gel purified (Qiagen, Mississauga, Canada) and

cloned into the pCR 2.1-TOPO vector (Invitrogen, Calsbad, USA). Sequences of positive

clones were confirmed using ABI 3730XL DNA analyzers (Center for Applied

Genomics, Toronto, Ontario). Identification of genes was carried out by Basic Local

Alignment Search Tool (BLAST) analyses against National Centre for Biotechnology

Information (NCBI) data (http://www.ncbi.nlm.nih.gov/BLAST/).

2.10 Generation of an expression vector for ferret CXCL10

59

Kozak sequences were engineered at the 5’ end of the ferret CXCL10 cDNA and

the 3’ end termination codon was removed prior to sub-cloning into the pcDNA3.1/

His6.V5/ TOPO expression vector (Invitrogen, Mississauga, Canada). The removal of

the termination codon enabled the cloned gene to be expressed as a fusion protein that is

tagged at the C-terminus with the His6 and V5 epitopes. Sequencing was repeated to

ensure that no errors had been introduced and to verify that the gene had been inserted in

the correct open reading frame.

2.11 Cell culture, plasmid transfections, and protein purification

COS-7 cells were maintained in Dulbecco’s modified eagle’s medium (DMEM),

substituted with 10% fetal bovine serum (Invitrogen, Mississauga, Canada) at 37ºC, 5%

CO2. COS-7 cells (1x107) were transiently transfected with Effectene (Qiagen, Canada).

After 24-48 hours of incubation, conditioned media was run through Ni-NTA metal

immobilized affinity columns (0.5-1 mL). Bound protein was washed and eluted. Eluted

fractions were screened via Western blot using antibodies that specifically recognize the

epitope tags. Positive fractions were pooled and dialyzed against phosphate buffered

saline (PBS) at 4ºC and concentrated by lyophilization.

2.12 Western Blot analysis

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) was performed with pre-

cast gels (10-15%, Bio-Rad, USA). Protein blots were blocked with 5% milk

protein/Phosphate buffered saline-Tween 0.02% (TPBS) buffer for 1 hour at room

temperature followed by 16 hours incubation at 4°C with mouse-anti-His6 primary

60

antibody (Invitrogen, Canada) at 508 ng/mL. The blots were washed with TPBS and

incubated with goat-anti-mouse-HRP secondary antibody (Santa Cruz, USA) for 1 hour

at room temperature. Bands were visualized using enhanced chemiluminiscent (ECL)

reagents (GE healthcare, Canada). Goat polyclonal anti-human CXCL10 antibody (R&D

systems, USA) was used to demonstrate cross-reactivity with ferret recombinant

CXCL10.

2.13 Isolation of Peripheral Blood Mononuclear cells (PBMC) for Chemotaxis

Assays

PBMCs were isolated from ferret whole blood by centrifuging over Histopaque

density gradient (Sigma Chemicals, St. Louise, MO). Briefly, whole blood was diluted

1:1 with PBS (10 mL), layered over 5 mL Histopaque solution, and centrifuged at 400Xg

for 20 minutes. The enriched mononuclear cell layer at the Histopaque-plasma interface

was harvested, washed twice in PBS, and resuspended in chemotaxis binding buffer

(RPMI 1640, 1% bovine serum albumin, 25 mM HEPES).

2.14 Chemotaxis Assay

The Boyden chamber procedure was performed as described previously26

. Briefly,

a 25 L aliquot of recombinant ferret CXCL10 was diluted in chemotaxis medium was

placed in the lower compartment and 50 L of PBMC suspension (1x106

cells/mL) was

placed in the upper compartment. The two compartments were separated by a

polycarbonate filter (5 μm pore size; Neuroprobe, Cabin John, MD). The chamber was

incubated for 3 hours at 37ºC in humidified air with 5% CO2. At the end of the

61

incubation period, filters were removed fixed and stained with Diff-Quik (Harlew, Gibbs-

town, NJ). The number of migrating cells was counted in three high power fields (400X)

by light microscopy. Results are expressed as mean ± SD for at least three independent

experiments.

2.15 Gene accession numbers

Nucleotide sequences of the complete coding regions of CXCL9, 10 and 11 were

submitted to the GeneBank. Partial nucleotide sequence of CXCR3 coding sequence was

also submitted and the following accession numbers were obtained:

Ferret CXCL9 (EF492057), Ferret CXCL10 (EF492058), Ferret CXCL11

(EF492059), Ferret CXCR3 (EF492060).

2.16 Statistical analysis

The student’s t test or the Mann-Whitney rank sum test for two independent

populations was used for statistical analysis via SPSS for Windows V13.0 software

(SPSS Inc., Chicago, IL). P≤0.05 was considered significant.

62

3. Results

3.1 CXCL9 and CXCL10 plasma level of patients with SARS

Cross-sectional analysis of CXCL9 and CXCL10 levels was performed on plasma

of Toronto SARS patients. The mean CXCL10 levels in mild SARS and ICU non-fatal

groups at the onset of disease and 7 days post-onset was measured. The mean level of

CXCL10 in plasma was significantly higher in SARS patients compared to healthy

controls (Figure 2-1). At day 14, the CXCL10 plasma level was high only in ICU

patients. At this day, ICU fatal patients showed higher levels of CXCL10 compared to

ICU non-fatal patients. At day 28, CXCL10 only remained significantly increased in the

ICU-fatal group, while other groups had decreased levels of this chemokine (Figure 2-1).

CXCL9 is closely related to CXCL10 with overlapping functions and both of

these chemokines share the same receptor (CXCR3). However CXCL9 had a different

pattern of expression. Only the ICU non-fatal group presented a significant increase in

CXCL9 mean level at day 14 compared to healthy control group (Figure 2-2).

63

Figure 2-1. Mean CXCL10 level changes in plasma of different SARS groups.

Comparison of CXCL10 plasma concentration of mild SARS group (day 0, 7, 14, 28),

ICU non-fatal group (day 0, 7, 14, 28), and ICU fatal group (day 14, 28) with healthy

controls (day 0). Y-axis is representative of CXCL10 mean concentration as pg/ml.

Statistical analysis was performed using the student t test. P values show the degree of

significance for each condition compared to healthy controls (*P< 0.01)

64

Days post-infection

CX

CL

10

pg

/ml

0 7 14 280

2000

4000

6000

8000

10000Healthy control

SARS mild

ICU non-fatal

ICU fatal* *

*

*

* * *

*

305746

Line

65

Figure 2-2. Mean CXCL9 level changes in plasma of different SARS groups.

Comparison of CXCL9 plasma concentration of mild SARS group (day 0, 7, 14, 28), ICU

non-fatal group (day 0, 7, 14, 28), and ICU fatal group (day 14, 28) with healthy controls

(day 0). Y-axis is representative of CXCL9 mean concentration as pg/ml. Statistical

analysis was performed using the student t test. P values show the degree of significance

for each condition compared to healthy controls (*P< 0.01)

66

Days post-infection

CX

CL

9

pg

/ml

0 7 14 280

2000

4000

6000

8000

10000Healthy control

SARS mild

ICU non-fatal

ICU fatal

*

305746

Line

67

3.2 Cloning and sequence analysis of Ferret CXCL9, CXCL10, CXCL11, and

CXCR3

The cloning of ferret genes encoding CXCL9, CXCL10, CXCL11 and CXCR3

was performed using ferret cDNA derived from PBMC cultures stimulated with various

mitogens to induce cytokines and chemokines expression (see Methods). Each gene was

amplified by primers designed from consensus sequences derived from multiple

nucleotide sequence alignments among various species. In the case of CXCL10, the full-

length coding region was not fully sequenced due to the lack of sequence conservation at

the 5’ and 3’ end of the coding region. To address this issue, sequences upstream and

downstream of the open reading frame were sequenced by RNA ligase mediated rapid

amplification of cDNA ends (RLM–RACE). For CXCR3, we amplified and sequenced a

partial cDNA within the coding region. As expected, the predicted full-length amino acid

sequences for ferret CXCL9, CXCL10, CXCL11 (126, 98 and 94 amino acids

respectively) and the predicted partial CXCR3 sequence (229 out of 368 amino acids)

show significant homology to corresponding sequences in various other species (Figure

2-3A-D). Phylogenetic analysis was performed using ClustalW and phylogenetic trees

constructed using protein parsimony with a bootstrap value of 100. The ferret nucleotide

sequences were translated to amino acid sequences using “Translate” (ExPASy,

proteomics). Amino acid sequences of other species were obtained from Genbank

(http://www.ncbi.nlm.nih.gov/Genbank). The phylogenetic tree shown for genes

indicated a higher similarity existed between ferret gene and those of humans and order

carnivora than between the ferret and order rodentia (Figure 2-4A,B, Table 2-1).

68

Figure 2-3. Multiple alignments of the primary amino acid sequences of CXCL9,

CXCL10, CXCL11, and CXCR3. The putative translated full length protein sequences

of ferret CXCL9 (A), CXCL10 (B), CXCL11 (C) and the putative translated partial

sequence of ferret CXCR3 (D) were compared with orthologous protein sequences from

human, dog and or cow, where available.

CXCL9: EF492057, XM_591770, BC063122

CXCL10: EF492058, AB183191, NM_001565

CXCL11: EF492059, XM_594243, NM_005409

CXCR3: EF492060, AB185149, NM_001504

69

A

)

B

)

C

)

D

)

70

Figure 2-4. Phylogenetic analysis of ferret CXCL9, CXCL10, CXCL11, and

CXCR3. The full length putative protein sequences obtained for A) ferret CXCL9,

CXCL10, CXCL11 and B) partial putative protein sequence of ferret CXCR3 were

aligned with all available orthologues from cow, dog, human, mouse, rat, pig and frog

using ClustalW 1.83, and phylogenetic trees constructed using protein parsimony with a

bootstrap value of 100. All chemokines cluster specifically within their orthologous

groups.

Accession numbers:

CXCL9: NP_663705, NP_032625, NP_002407, XP_591770

CXCL10: NP_001008691, NP_620789, NP_067249, NP_001556, NP_001010949,

NP_001040016

CXCL11: NP_891997, NP_062367, NP_005400

CXCR3: NP_001011067, CAH64842, NP_445867, NP_034040, NP_001011673,

NP_0011495, NP_001011887

71

A)

B)

Human CXCL10

Rat CXCL11

Mouse CXCL11

Human CXCL11

Ferret CXCL11

Rat CXCL9

Mouse CXCL9

Human CXCL9

Cow CXCL9

Ferret CXCL9

Cow CXCL10

Pig CXCL10

Ferret CXCL10

Dog CXCL10

Rat CXCL10

Mouse CXCL10

96.049.1

49.9

84.7

98.8

84.8

100

99.6

97.979.5

100

92.575.4

Rat

Mouse

Pig

Cow

Human

Dog

Ferret

Frog

99.4

56.3

41.6

27.6

85.7

72

Species CXCL9 CXCL10 CXCL11 CXCR3

Human 68 80 86 91

Dog __ 81 __ 92

Cow 72 80 82 89

Mouse 58 67 63 86

Values shown represent % amino acid identity, with highest % for each gene in bold .

Table 2-1. Amino acid identity of ferret CXCL9, CXCL10, CXCL11 and CXCR3

with known species sequences

73

3.3 Expression of recombinant ferret CXCL10 protein and detection by western blot

A C-terminal His6/V5-tagged expression construct was generated in order to

produce recombinant ferret CXCL10 protein (predicted molecular weight of 18.5kDa,

including epitope tags). We transiently transfected COS-7 cells with this expression

construct and purified recombinant ferret CXCL10 by affinity chromatography. Eluted

fractions were subjected to western blot analysis using anti-HIS antibody. A single band

was noted at the expected molecular weight of 18.5 kDa. COS-7 cells transfected with

empty vector did not show reactivity with anti-His antibody, while recombinant ferret

IFN- reacted with same antibody at 34 KDa as a positive control (Figure 2-5A). As

recombinant ferret CXCL10 shows a high degree of homology with human CXCL10, we

tested whether our ferret recombinant CXCL10 would be recognized by an anti-human

CXCL10 polyclonal antibody. As shown in Figure 2-5B, ferret recombinant CXCL10

and human recombinant CXCL10 are reactive with a polyclonal goat anti-human

CXCL10 antibody.

3.4 Chemotactic activity of recombinant ferret CXCL10 protein

The purified recombinant ferret CXCL10 protein was tested for chemotactic

activity using ferret PBMC cultures in a Boyden chamber. Chemotactic migration of

ferret PBMCs increased upon treatment with ferret CXCL10 protein in a dose-dependent

manner. Maximal migration occurred at 200 ng/mL ferret CXCL10 protein, above which

concentration a declining response was observed. Therefore our CXCL10 induced

chemotaxis data conformed to a classical bell-shaped chemotaxis dose-response curve,

74

and was consistent with previous reports for recombinant human CXCL927

and

recombinant human IL-826

. Recombinant ferret CXCL10 attracted a statistically

75

Figure 2-5. The recombinant ferret CXCL10 protein migrates to 18.5kDa and is

active in cell migration assays. A) Anti-His6 western blot of purified recombinant

ferret CXCL10 (fCXCL10) protein (from elution 2) shows a single band at 18.5kDa

compared with ferret IFN- (fIFN-with a band at 34 KDa. B) Goat anti-human

CXCL10 (hCXCL10) polyclonal antibody is cross-reactive with ferret CXCL10

(fCXCL10) recombinant protein. C) Migration of ferret PBMCs following the exposure

to increasing doses of recombinant ferret CXCL10 protein. The results are expressed as

mean cell

count of three high-powered fields obtained in 3 experiments.* P< 0.05 compared to

migration induced by medium alone using student’s t test.

76

C)

* P<0.05

0

5

10

15

20

25

0 200 400 600 800 1000

Ferret CXCL10 concentration (ng/mL)

Mean

nu

mb

er

of

mig

rate

d c

ells / 3

fie

lds

*

*

*

*

*

Empty vector fCXCL10

fIFN-

A)

fCXCL10

B)

hCXCL10 Empty vector

77

significant greater number of PMBCs when compared to the negative control wells

(media alone) at all concentrations used in the assay (P<0.05) (Figure 2-5C). These

results confirmed that our recombinant ferret CXCL10 protein demonstrated biological

activity.

3.5 Stimulation of ferret PBMCs with LPS induces mRNA expression of CXCL9,

CXCL10 and CXCL11

We predict that stimulation of ferret PBMC cells with lipopolysaccharide (LPS)

should promote the secretion of pro-inflammatory cytokines and chemokines in

accordance to data from other species28-30

. Sequence analysis of ferret CXCL9, 10 and

11 genes enabled us to design primers for use in real-time PCR experiments in order to

detect gene expression of these chemokines. Initially, we set out to validate the

specificity of the primers and then aimed to test whether CXCL9, 10 and 11 transcription

is induced in ferret blood cells upon LPS treatment. Quantitative real-time PCR on

cDNA derived from ferret whole blood stimulated with LPS was performed and

demonstrated that CXCL9, CXCL10 and CXCL11 mRNA expression was induced at

various times following LPS treatment relative to unstimulated control cDNA. The peak

increase for CXCL9 (3-fold above unstimulated control, P<0.05) and CXCL11 (greater

than 8-fold above unstimulated control, P<0.01) occurred at 4 hours post-stimulation,

while for CXCL10 the peak increase (60-fold above unstimulated control, P<0.01)

occurred at 2 hours following LPS treatment (Figure 2-6).

78

Figure 2-6. Induction of chemokine gene expression by lipopolysacharide. Ferret

peripheral blood was stimulated with 0.1 mg/ml LPS for the time points indicated.

Utilizing primers specific for the ferret CXCL9, CXCL10, and CXCL11 genes, mRNA

expression was quantified. The Y-axis indicates the abundance of mRNA after

stimulation with LPS compared to untreated samples. Treatment time is indicated by X-

axis. Error bars indicate the SD of the relative quantities of the triplicates. Quantity

readings of triplicates at each time point are compared with quantity readings of

unstimulated triplicates, using student’s t test. The peak increases for CXCL9 and

CXCL11 occurred at 4 hours post-stimulation (P<0.05 and P<0.01 respectively), while

for CXCL10 was at 2 hours following LPS treatment (P<0.01).

79

mRNA expression

Time 0 1 h 2 h 4 h 8 h 16 h

Fold

in

cre

ase

0

10

20

30

40

50

60

70

CXCL10

mRNA expression

Time 0 1 h 2 h 4 h 8 h 16 h

Fold

incre

ase

0

2

4

6

8

10

CXCL11

mRNA expression

Time 0 1 h 2 h 4 h 8 h 16 h

Fold

incra

ese

0

1

2

3

4

5

CXCL9

* *

* * *

**

**

**

*

**

*P<0.05

**P<0.01

80

3.6 Increased CXCL10 and CXCR3 mRNA expression in lung biopsies from fatal

outcome SARS patients at autopsy

We and others have demonstrated increased levels of CXCL10 in the plasma of

SARS patients during acute and progressive illness12,14,31,32

. In order to compare the

expression of CXCL10 in lung biopsies of ferrets infected with SARS-CoV, we evaluated

the expression of CXCL10 and its receptor CXCR3 in the lungs of fatal outcome SARS

patients as well. Real-time PCR analysis was performed on cDNA purified from the lung

biopsies of 3 deceased SARS patients and 3 control patients who died from non-

respiratory illnesses. The results show high expression of CXCL10 and CXCR3 mRNA

in SARS patients (P<0.05) and no expression in control cadaveric lung biopsies (Figure

2-7A,B).

3.7 Expression of CXCL10 in lung biopsies of ferrets infected with SARS-CoV

In order to further dissect the ferret model of SARS-CoV infection and determine

if it is representative of the disease in humans on a molecular basis, we endeavored to

determine whether if the mRNA expression of the chemokine CXCL10 is increased in

SARS-infected ferrets. Real-time PCR analysis of CXCL10 gene expression in lung

tissue from both SARS-infected and uninfected ferrets was carried out. Consistent with

our findings in human SARS patients, ferrets experimentally infected with SARS-CoV

demonstrated a significant increase in CXCL10 mRNA expression in the lower

respiratory tract compared to uninfected control animals (P<0.05) (Figure 2-7C).

81

Figure 2-7. CXCL10 and CXCR3 mRNA expression in lung tissues of humans and

ferrets infected with SARS-CoV. A, CXCL10 and B, CXCR3 are significantly

increased in the lungs of fatal outcome SARS (P<0.05). RNA was isolated from bilateral

lower lung biopsies from 3 deceased SARS patients and from lower lung biopsies from 3

control cadaveric lungs. C, CXCL10 mRNA levels in the lungs of mock vs. SARS-CoV

infected ferrets. Data represent averages from 3 animals per group, performed in

triplicate wells. Asterisks indicate statistical significance using Mann-Whitney rank sum

test (P<0.05).

83

4. Discussion

The CXCL9, 10 and 11–CXCR3 axis has been reported to play a role in the

immune response following viral infection14,33-36

. The ferret represents an important

animal model for a number of human viral infectious diseases16,37,38

.

CXCL10 mainly recruits Th1 cells and NK cells to the site of infection1. The level

of this chemokine was increased in all patient groups at the onset of disease and remained

elevated during the disease course. The mean level of CXCL10 remained in the same

level as in mild SARS patients and ICU non-fatal groups at day 7 post-onset. While

CXCL10 was decreased at day 14 in the mild SARS group, it remained significantly

elevated in ICU groups implying that high level of this chemokine is associated with the

persistence of the disease. CXCL10 plasma level decreased to baseline in recovered

patients. Only ICU fatal patients had high levels of CXCL10 compared to other patients

groups or healthy controls.

CXCL10 is inducible in many cells of the respiratory system, including

endothelial cells, airway epithelial cells, monocytes and fibroblasts2,3

. It is likely that in

the course of SARS one or more of these cell types produce high levels of this

chemokine. High levels of CXCL0 in patients with a poor outcome may be associated

with increased damage to the lung. Production of CXCL10 in the lung may attract

lymphocytes to the site of infection and the development of ARDS13,18,31

. Neutralization

of CXCL10 in ferrets in future studies may reveal the precise role of this chemokine in

the pathogenecity SARS.

CXCL9, another chemokine with many overlapping functions with CXCL10 was

not significantly increased during the SARS-CoV infection.

84

As our findings in humans are a part of an uncontrolled retrospective study, we

went on to further study the role of CXCL10 in our ferret model. We report here for the

first time cloning and sequencing of the ferret genes for CXCL9, CXCL10, CXCL11 and

CXCR3. The ferret sequences of these chemokines and their cognate receptor were

highly conserved with human, dog and cow sequences.

Purified recombinant ferret CXCL10 was detected as a single band on anti-His6

western blots that ran close to the predicted molecular weight of 18.5kDa. Since

recombinant CXCL10 was expressed in a mammalian cell line (COS-7), it is reasonable

to assume that the recombinant protein is properly processed at the post-translational

level. Furthermore, we have demonstrated that recombinant ferret CXCL10 protein

exhibited biological activity in terms of stimulating chemotaxis in peripheral blood

mononuclear cells.

CXCL9, CXCL10 and CXCL11 are non-ELR chemokines found at increased

levels in a variety of viral diseases34

. Previous work has shown that CXCL10 mRNA

expression is strongly up-regulated in conjunction with CXCR3 in mice infected with

Lymphocytic Choriomeningitis Virus (LCMV), particularly in the pancreas and at early

stages of infection10

. More recently, it has demonstrated that LCMV infection of the

murine central nervous system (CNS) is associated with CXCL9 and CXCL11 up-

regulation33

. Several animal models have shown that inflammation-based pathology

induced by viral infections can be reduced by either neutralization of chemokines or

blockade of their receptor36,39,40

. Treatment of spontaneously diabetic NOD mice with

CXCL10 neutralizing antibodies suppressed the incidence of diabetes41

. In the MHV

85

mouse model of encephalitis, neutralization of CXCL9 or CXCL10 (or in CXCL10-null

mice) resulted in reduced neuropathology despite increased viral load5,11

.

The absence of immunological reagents for the ferret model of SARS and H5N1

has hindered a direct investigation into the role played by the CXCL9, 10 and 11-CXCR3

axis in the immmunopathogenesis of viral disease. The availability of the cloned genes

for ferret CXCL9, CXCL11 and CXCR3, will enable the expression and purification of

recombinant proteins. Biologically active recombinant purified CXCL10 protein has

been prepared and will be utilized immediately in a number of in vitro and in vivo assays.

Using these proteins we intend to generate specific monoclonal and polyclonal

neutralizing antibodies to investigate the involvement of these chemokines in the

pathogenesis of SARS and H5N1 diseases.

We and others have previously reported the elevation of CXCL10 in the serum of

SARS patients12,14,31

. Furthermore, the depletion of lymphocyte subsets from peripheral

blood might be partly due to the influence of CXCL10 in that up-regulation of CXCL10

in the lung might be influential in the recruitment and sequestration of lymphocyte

subsets to the site of infection14

. This in turn may lead to severe pathological

consequences as described in a number of viral infections including SARS and bird flu32

.

In conclusion, we have demonstrated increased gene expression of CXCL10 and

its receptor CXCR3 in lung biopsies of deceased SARS patients. We observed a similar

pattern of CXCL10 mRNA expression in the lungs of ferrets infected with SARS-CoV,

lending further support to the argument that ferrets are a highly representative model of

the human disease. We have identified a subfamily of chemokines and their cognate

receptor in ferrets and carried out an initial characterization of one of these chemokines.

86

This study is a fundamental step towards the development of a more readily available

array of immunological tools for use in investigating the mechanism of disease in this

natural model of viral infection.

87

Reference List

1. Cameron,M.J. & Kelvin,D.J. Cytokines and chemokines--their receptors and their

genes: an overview. Adv. Exp. Med. Biol. 520, 8-32 (2003).

2. Farber,J.M. Mig and IP-10: CXC chemokines that target lymphocytes. J. Leukoc.

Biol. 61, 246-257 (1997).

3. Rossi,D. & Zlotnik,A. The biology of chemokines and their receptors. Annu. Rev.

Immunol. 18, 217-242 (2000).

4. Krathwohl,M.D. & Anderson,J.L. Chemokine CXCL10 (IP-10) is sufficient to

trigger an immune response to injected antigens in a mouse model. Vaccine 24,

2987-2993 (2006).

5. Liu,M.T., Keirstead,H.S. & Lane,T.E. Neutralization of the chemokine CXCL10

reduces inflammatory cell invasion and demyelination and improves neurological

function in a viral model of multiple sclerosis. J. Immunol. 167, 4091-4097 (2001).

6. Qin,S. et al. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells

associated with certain inflammatory reactions. J. Clin. Invest 101, 746-754 (1998).

7. Ruth,J.H. et al. Selective lymphocyte chemokine receptor expression in the

rheumatoid joint. Arthritis Rheum. 44, 2750-2760 (2001).

8. Sorensen,T.L. & Ransohoff,R.M. Etiology and pathogenesis of multiple sclerosis.

Semin. Neurol. 18, 287-294 (1998).

9. Cameron,M.J., Bermejo-Martin,J.F., Danesh,A., Muller,M.P. & Kelvin,D.J. Human

immunopathogenesis of severe acute respiratory syndrome (SARS). Virus Res.

(2007).

10. Christen,U. et al. Cure of prediabetic mice by viral infections involves lymphocyte

recruitment along an IP-10 gradient. J. Clin. Invest 113, 74-84 (2004).



Immunol. 168, 3195-3204 (2002).

12. Cameron,M.J. et al. Interferon-mediated immunopathological events are associated

with atypical innate and adaptive immune responses in patients with severe acute

respiratory syndrome. J. Virol. 81, 8692-8706 (2007).

13. Cheung,C.Y. et al. Cytokine responses in severe acute respiratory syndrome

coronavirus-infected macrophages in vitro: possible relevance to pathogenesis. J.

Virol. 79, 7819-7826 (2005).

88




15. Peiris,J.S. et al. Re-emergence of fatal human influenza A subtype H5N1 disease.

Lancet 363, 617-619 (2004).

16. Martina,B.E. et al. Virology: SARS virus infection of cats and ferrets. Nature 425,

915 (2003).


303, 944-946 (2004).

18. Glass,W.G., Subbarao,K., Murphy,B. & Murphy,P.M. Mechanisms of host defense

following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary

infection of mice. J. Immunol. 173, 4030-4039 (2004).

19. Ng,W.F., To,K.F., Lam,W.W., Ng,T.K. & Lee,K.C. The comparative pathology of

severe acute respiratory syndrome and avian influenza A subtype H5N1--a review.

Hum. Pathol. 37, 381-390 (2006).

20. Ye,J. et al. Molecular pathology in the lungs of severe acute respiratory syndrome

patients. Am. J. Pathol. 170, 538-545 (2007).

21. Sidwell,R.W. & Smee,D.F. In vitro and in vivo assay systems for study of influenza

virus inhibitors. Antiviral Res. 48, 1-16 (2000).

22. Sweet C. Pathogencity of influenza viruses. Smith H. Microbiology Reviews 44,

303-330. 1980.

Ref Type: Generic

23. Zitzow,L.A. et al. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J.

Virol. 76, 4420-4429 (2002).

24. Govorkova,E.A. et al. Lethality to ferrets of H5N1 influenza viruses isolated from

humans and poultry in 2004. J. Virol. 79, 2191-2198 (2005).

25. Maines,T.R. et al. Avian influenza (H5N1) viruses isolated from humans in Asia in

2004 exhibit increased virulence in mammals. J. Virol. 79, 11788-11800 (2005).

26. Xu,L. et al. Modulation of IL-8 receptor expression on purified human T

lymphocytes is associated with changed chemotactic responses to IL-8. J. Leukoc.

Biol. 57, 335-342 (1995).

27. Liao,F. et al. Human Mig chemokine: biochemical and functional characterization.

J. Exp. Med. 182, 1301-1314 (1995).

89

28. Loos,T. et al. TLR ligands and cytokines induce CXCR3 ligands in endothelial

cells: enhanced CXCL9 in autoimmune arthritis. Lab Invest 86, 902-916 (2006).

29. Nau,G.J., Schlesinger,A., Richmond,J.F. & Young,R.A. Cumulative Toll-like

receptor activation in human macrophages treated with whole bacteria. J. Immunol.

170, 5203-5209 (2003).

30. Tassiulas,I. et al. Apoptotic cells inhibit LPS-induced cytokine and chemokine

production and IFN responses in macrophages. Hum. Immunol. 68, 156-164 (2007).

31. Chien,J.Y., Hsueh,P.R., Cheng,W.C., Yu,C.J. & Yang,P.C. Temporal changes in

cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory

syndrome. Respirology. 11, 715-722 (2006).

32. Ng,P.C. et al. Chemokine response in children with SARS. Arch. Dis. Child 90,

422-423 (2005).

33. Christensen,J.E., de Lemos,C., Moos,T., Christensen,J.P. & Thomsen,A.R.

CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in

immune surveillance of the lymphocytic choriomeningitis virus-infected central

nervous system. J. Immunol. 176, 4235-4243 (2006).

34. Luster,A.D. Chemokines--chemotactic cytokines that mediate inflammation. N.

Engl. J. Med. 338, 436-445 (1998).

35. Tschen,S.I. et al. CNS viral infection diverts homing of antibody-secreting cells

from lymphoid organs to the CNS. Eur. J. Immunol. 36, 603-612 (2006).

36. Xie,J.H. et al. Antibody-mediated blockade of the CXCR3 chemokine receptor

results in diminished recruitment of T helper 1 cells into sites of inflammation. J.

Leukoc. Biol. 73, 771-780 (2003).

37. Suguitan,A.L., Jr. et al. Live, attenuated influenza A H5N1 candidate vaccines

provide broad cross-protection in mice and ferrets. PLoS. Med. 3, e360 (2006).

38. Vos,A., Muller,T., Cox,J., Neubert,L. & Fooks,A.R. Susceptibility of ferrets

(Mustela putorius furo) to experimentally induced rabies with European Bat

Lyssaviruses (EBLV). J. Vet. Med. B Infect. Dis. Vet. Public Health 51, 55-60

(2004).

39. Carr,D.J., Chodosh,J., Ash,J. & Lane,T.E. Effect of anti-CXCL10 monoclonal

antibody on herpes simplex virus type 1 keratitis and retinal infection. J. Virol. 77,

10037-10046 (2003).

40. Dawson,T.C., Beck,M.A., Kuziel,W.A., Henderson,F. & Maeda,N. Contrasting

effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to

influenza A virus. Am. J. Pathol. 156, 1951-1959 (2000).

90

41. Shigihara,T. et al. CXCL10 DNA vaccination prevents spontaneous diabetes

through enhanced beta cell proliferation in NOD mice. J. Immunol. 175, 8401-8408

(2005).

91

Chapter 3

Early gene expression events in ferrets in response to SARS coronavirus

infection versus direct interferon-alpha2b stimulation

(A version of this chapter is published in Virology, volume 409, 2011, p102-112 as

“Early gene expression events in ferrets in response to SARS coronavirus infection

versus direct interferon-alpha2b stimulation”

Ali Danesh, Cheryl M. Cameron, Alberto J. León, Longsi Ran, Luoling Xu, Yuan Fang,

Alyson A. Kelvin, Thomas Rowe, Honglin Chen, Yi Guan, Colleen B. Jonsson, Mark J.

Cameron and David J. Kelvin, with permission from Elsevier.)

1. Introduction

Viral respiratory infections are a major worldwide cause of morbidity and

mortality1,2

. Emerging viral threats, such as the severe acute respiratory syndrome

coronavirus (SARS-CoV), avian influenza H5N1 and pandemic influenza H1N1 virus are

well-poised to cause epidemics or pandemics that could be socially and economically

disastrous3-5

.

For decades, ferrets have been used for the investigation of influenza infection

since they are susceptible to influenza viruses6. More recently, ferrets have also been

shown to be a good model of human SARS-CoV infection7. We have previously

characterized ferret cytokine and chemokine genes as well as have developed

immunological assays for evaluating the ferret immune system following SARS and

influenza infection8-10

.

Ligation of the interferon (IFN) alpha receptors 1 and 2 (IFNAR1 and IFNAR2)

with IFN- induces IFN signaling pathways and promotes IFN gene induction.

Formation of the signal transducer and activator of transcription 1 and 2 (STAT1-

STAT2) heterodimer occurs following the phosphorylation of Janus kinase 1 (JAK1) and

92

tyrosine kinase 2 (Tyk2) that are associated with IFNAR2 and IFNAR1, respectively11

.

These two kinases phosphorylate STAT1 and STAT2, which together form a complex

with interferon regulatory factor 9 (IRF9)12

. The interferon stimulatory factor 3 complex

(ISGF3) binds to interferon-stimulated response element (ISRE) and activates

transcription of IFN- inducible genes, including 2’-5’ oligoadenylate synthase 1

(OAS1), myxovirus resistance 1 (MX1), interferon stimulated gene 15 (ISG15) and many

other IFN-response genes (IRGs)13

.

IFN- stimulation ultimately promotes a cellular antiviral state which is

hallmarked by the upregulation of IRGs14

. Although IFN signaling gene upregulation

during viral infection has been the subject of previous reports, there is little information

regarding the host immune responses directly induced by viruses versus those that are

upregulated due to secondary IFN stimulation15-18

. Here we used our previously-

described ferret model19

to identify genes that were regulated by SARS-CoV infection

compared to IFN-2b stimulation in the ferret model to elucidate immune responses

during viral infection. We examined the phosphorylation status of signaling molecules in

IFN-2b stimulated peripheral blood mononuclear cells (PBMCs). We also analyzed the

in vivo gene expression profiles of ferret PBMCs and lung necropsies following IFN-2b

injection during the time course. Evaluation of gene expression patterns in PBMCs and

lung necropsies of SARS-CoV infected ferrets led us to the identification of 7

upregulated IRGs that also were upregulated in response to IFN-2b injection. Our

findings in ferrets suggested IFN-2b injection and SARS-CoV infection led to similar as

well as unique gene expression signatures in a global point of view. Increased knowledge

of the interaction of these gene expression signatures may improve our understanding of

93

the immune system of ferrets as a preferred animal model of severe respiratory viral

illnesses.

94


2.1 Ferrets

Male, 1 kg, six-month-old ferrets (Mustela putorius furo) were purchased from

Marshall Farms Inc. (Oak Park, IL) and housed at the Toronto General Research Institute

animal facility (Toronto, Canada) or at Southern Research Institute (Birmingham, AL,

USA). Ferrets were quarantined and monitored five days prior to tissue and blood

collection. The ferrets’ diet was based on a low fat/high protein regimen as recommended

by Marshal Farms. Animal protocols were approved and monitored by the Animal Care

Committee of the University Health Network or of the Southern research Institute.

2.2 In vitro blood stimulation with IFN-2b

Whole blood was drawn from 4 ferrets and diluted ¼ with cell culture media

(Invitrogen, CA). Two ml of diluted blood from 4 animals was stimulated with 0.001

g/ml IFN-2b (pegylated IFN-2b, Schering-Plough, Pointe-Claire, Canada) in separate

wells and incubated at 37oC (5% CO2) for 1 and 3 hours. PBS was added to 3 control

wells. The cultured blood was then harvested and injected to PaxGene tubes and RNA

was purified (Invitrogen, CA). One ml of blood stimulated with IFN-2b and or PBS was

also added to 10 ml Lyse/Fix buffer (BD Biosciences, USA) for evaluation of

phosphorylation status of signaling molecules at 0, 15, 30, 45, 60, 75 and 90 minutes,

using PhosFlow antibodies (BD Biosciences, USA).

95

2.3 In vivo injection of ferrets with IFN-2b

Subcutaneous injections of 1 ml of PBS (control) or 1 g/ml of IFN-2b were

performed on the back of each ferret. Two ml of blood was collected directly into

PaxGene tubes. One gram of the lung necropsy was added to TRIzol® reagent

(Invitrogen CA). Collected blood and lung tissues were used for RNA isolation, which

then used for microarray, and quantitative real-time PCR analysis. One ml blood was

added to Lyse/Fix buffer (BD Biosciences, San Jose, CA) for analysis of the signaling

molecule phosphorylation status.

2.4 In vivo infection of ferrets with SARS-CoV

Ferrets were infected with SARS-CoV in the Animal Biohazard Safety Level 3

(ABSL3) facility at Southern Research Institute (Birmingham, AL, USA), in accordance

with the approved protocols. Three male ferrets, weighing approximately 800–1000 g,

were infected intranasally with 103 TCID50 SARS-CoV TOR2 strain (isolated from a

patient in Toronto and sequenced at CDC, Vancouver, BC) in 1 mL PBS. An additional 3

animals were mock-infected with 1 mL PBS. Animals were anesthetized and blood and

lung necropsies were collected for RNA purification. Infection of ferrets with the above

mentioned dose results in weight loss, decreased activity temperature increase and

histology lesions with no mortality during the disease course19

. A summary of natural

history of ferrets infected with SARS-CoV has been provided as Table 3-5.

96

2.5 Intracellular staining

One ml of in vitro-stimulated blood with IFN-2b or PBS and/or 1 ml of blood

drawn from the IFN-2b or PBS injected ferrets and/or 1ml blood from infected ferrets

with SARS-CoV or mock controls (in vivo) was added to 10 ml Lyse/Fix buffer (BD

Biosciences, USA) and incubated in a 37oC water bath for 10 minutes. Tubes were then

centrifuged at 300g for 5 minutes and the cells were washed twice with cold PBS. One ml

Perm III (BD Biosciences, USA) was added to each tube and the tube was incubated on

ice for 20 minutes to permeabilize cells for intracellular staining. Cells were washed with

Perm/Wash (BD Biosciences, USA) and 106 cells were added to each tube for flow

cytometry. Twenty l of phosphorylated P-STAT1, P-STAT3, P-STAT4, P-STAT5, P-

p38 and P-ERK antibodies conjugated with Alexa-Fluor 488 was added to separate tubes

(BD Biosciences, USA). Matched isotype control was added to one tube as a negative

control. Tubes were incubated at room temperature in the dark for 30 minutes. Cells were

washed with cold Perm wash (BD Biosciences, USA) and fixed with 2%

paraformaldehyde in PBS. Twenty-thousand events were acquired with a BD

FACSCalibur (BD Biosciences, USA) and data were analyzed, using FlowJo software

(Tree Star Inc., USA).

2.6 Cloning and sequencing of ferret-specific genes

Cloning and sequencing was performed as described previously9. Briefly, purified

RNA was reverse transcribed to cDNA using Invitrogen RT-kits (Invitrogen, Carlsbad,

CA). Gene-specific degenerate primers were designed based on multiple gene sequence

alignment analysis of several species using ClustalW (1.83) and then used to clone the

97

cDNAs for each gene. Standard PCRs were performed and specific bands were gel-

purified (Qiagen, Mississauga, Canada) and cloned into the pCR 2.1-TOPO vector

(Invitrogen, Calsbad, USA). Sequences of positive clones were confirmed using an ABI

3730XL DNA analyzer (Applied Biosystems, Foster City, CA). The identification of

genes was performed using Basic Local Alignment Search Tool (BLAST) analyses

against National Centre for Biotechnology Information (NCBI) database.


STAT1 (EU835493), STAT2 (EU835988), MX1 (EU835483), OAS1

(EU835484), ISG15 (EU835986), ISG20 (EU835990), IRF7 (EU835985), IFI35

(EU835487), PKR (EU835989), P52RIPK (EU835488), CXCL8 (EU835489), SOCS3

(EU835987)

2.8 Quantitative Real-Time PCR (qRT-PCR)

The following components were added to the reaction mixture plus cDNA to a

total volume of 10 µL in distilled water: 0.2 l cDNA, 250 nmol forward gene-specific

primer, 250 nmol reverse gene-specific primer and 5 L Cyber Green (Applied

Biosystems, Foster City, CA). For every experiment, each reaction was performed in

triplicate. An ABI 7900 Sequence Detection System (Applied Biosystems, Foster City,

CA) was used for amplification. Initial denaturation was 15 minutes at 95oC, followed by

40 cycles of amplification. Each cycle consisted of a denaturation step (15 seconds at

95oC) and an annealing/extension step (1 minute at 60

oC). Expression levels were

98

normalized to β-actin and data was analyzed by SDS 2.1 software (Applied Biosystems,

Foster City, CA).

2.9 Microarray analysis

Briefly, 0.5 g of total RNA was isolated using Paxgene whole blood purification

kits or TRIzol® reagent. Oligonucleotide microarray analysis was performed using

Affymetrix two-cycle cRNA synthesis and IVT kits (Affymetrix, Santa Clara, CA).

cRNA samples (20 g) were labelled and hybridized to Affymetrix GeneChip Canine

Genome 2.0 Arrays to monitor the gene expression of over 18,000 Canis familiaris

mRNA/EST-based transcripts and over 20,000 non-redundant predicted genes. As

described earlier, canine arrays were used following the observation of high levels of

homology between canine and ferret nucleotide sequences (average of 89% identity)8,20

.

Table 3-1 demonstrates the amino acid identity of genes in this study compared to

available orthologues of human and mouse. The arrays were scanned using an Affymetrix

GCS3000 7G system according to standard Affymetrix protocols. Probe-level analysis

was performed using Probe Logarithmic Intensity Error (PLIER). The raw intensity

values for each individual target on the Affymetrix chips were pre-processed with

variance stabilization, log2-transformation and were then normalized against the time

zero datasets with ArrayAssist V 5.5.1 (Stratagene, USA). Student’s t-tests or EDGE time

course differential expression analysis21

were performed with Benjamini-Hochberg false

discovery rate (FDR) correction. Genes with a significant difference were selected for

agglomerative hierarchical clustering with Pearson distance metrics and average linkage

distance measurements between clusters using GeneLinker Platinum V 4.6.1 (Improved

99

Outcomes Software, Kingston, Canada). Ingenuity Pathway Analysis 5.0 software

(Ingenuity Systems Inc., Redwood City, CA) was used to annotate and organize the gene

expression data into networks and pathways. Pathways and functional categories were

considered as over-represented when Fisher’s exact test P value was ≤0.05. Datasets are

publicly available at the NCBI’s Gene Expression

Omnibus (http://www.ncbi.nlm.nih.gov/geo) accession number GSE22581.

2.10 Statistical analysis

T tests or EDGE analyses were used for statistical analysis considering a biological

filter of 1.5 fold change compared to controls and a P value of ≤0.05 as significant.

http://www.ncbi.nlm.nih.gov/geo

100

3. Results

3.1 IFN-2b stimulation and SARS-CoV infection induced STAT1 phosphorylation

and increased the expression levels of IRGs

We first investigated the phosphorylation of the IFN- receptor downstream

signaling molecule STAT1 to determine the signaling potential of IFN-2b in ferrets.

The phosphorylation status of STAT1 was evaluated using phosphorylated amino acid

specific monoclonal antibody for flow cytometry analysis that cross-reacted with the

phosphorylated ferret protein. Peripheral blood mononuclear cells (PBMCs)

demonstrated a significant STAT1 phosphorylation response 15 minutes post-stimulation

with IFN-2b in vitro compared to the control stimulated with PBS alone (Figue 3-1).

Since STAT1 phosphorylation was observed in vitro, we then determined whether

IFN-2b could activate STAT1 in vivo. Four ferrets were subcutaneously injected with 1

g/kg IFN-2b and peripheral blood samples were taken at 0, 3 and 6 hours post-

stimulation for flow cytometry examination. By 3 hours, samples extracted from all

ferrets demonstrated significant STAT1 phosphorylation in the PBMCs. Control ferrets

injected with PBS did not demonstrate marked STAT1 phosphorylation at any time point.

The STAT1 average mean fluorescent intensity (MFI) of the IFN-2b injected group was

significantly increased compared to the average of its control group (P<0.01) (Figure 3-

2A). These results indicated that STAT1 was also inducible by IFN-2b in vivo.

101

Figure 3-1. In vitro phosphorylation of STAT1 in peripheral blood of ferrets

following stimulation with IFN-2b. Average mean fluorescent intensities ( MFI) of

IFN-2b stimulated ferret whole blood in 3 wells from 3 ferrets and PBS-stimulated

blood in 3 wells from 3 ferrets were measured by flow cytometry (Y axis) in PBMCs

gates. The X axis indicates the MFI changes during the time course. Lines with black

diamonds: phosphorylated STAT1 in control ferret cells, lines with black squares:

phosphorylated STAT1 in ferret cells cultured with IFN-2b. *P<0.01 using student t

test.

102

* P<0.01

0

10

20

30

40

50

60

70

0' 15' 30' 45' 60' 75' 90'

Time course

MF

I

Mock

IFN-a2b injected

* *

* * *

IFN-2b

stimulated Moc

k

103

Figure 3-2. In vivo phosphorylation of STAT1 in ferret peripheral blood

mononuclear cells. A) In vivo stimulation with IFN-2b; the STAT1 average mean

fluorescent intensities (MFI) of IFN-2b-injected (n=4) and control (n=4) groups were

measured by flow cytometry during the time course (X axis) in the PBMC gate. The Y

axis indicates the MFI between STAT1 phosphorylation and isotype control. White

bars: Control group, black bars IFN-2b injected group. P<0.01, using student t test. B)

In vivo infection with SARS-CoV; the STAT1 average MFI in PBMCs of 3 SARS-CoV

infected ferrets (black bars) versus 3 mock controls (white bars) during the time course

(P<0.01). Bars are representative averages of MFI between STAT1 phosphorylation

and isotype control.

104

A)

B)

Day 0 Day 1 Day 2 Day 3 Day 4

PM

Cs M

FI

0.0

0.5

1.0

1.5

2.0

2.5

__ * __ *

0 h 3 h 6 h

PB

MC

s M

FI

0

1

2

3

4

5

__ *

__ *

IFN-2b injected Mock control

SARS-CoV infected Mock control

* P<0.01

105

IFN signaling is critical to successful antiviral responses during infection22

.

Therefore, we next investigated the phosphorylation status of STAT1 following SARS-

CoV infection. We infected ferrets with SARS-CoV or PBS control and measured the

phosphorylation of STAT1 by flow cytometry. Three ferrets infected with SARS-CoV

demonstrated significant STAT1 phosphorylation in PBMCs post-infection with a

maximum peak at day 3 (P<0.01). Control ferrets mock-infected with PBS did not

demonstrate significant STAT1 phosphorylation at any time point (Figure 3-2B).

Since STAT1 was phosphorylated following SARS-CoV infection and IFN-2b

injection, we investigated select IRG expression by qRT-PCR following in vitro

stimulation of ferret peripheral whole blood with IFN-2b. In vitro stimulation with IFN-

2b led to significant upregulation of STAT1 and several IRGs such as MX1, OAS1,

ISG15, ISG20, IRF7 and interferon-induced protein 35 (IFI35). As expected, activation

of IFN- receptor signaling initiated transcriptional activation of interferon response

genes (Figure 3-3).

3.2 Microarray analysis of IFN-2b injection or SARS-CoV infection in ferret

peripheral blood

We then assessed genome-wide gene expression following in vivo IFN-2b

administration in ferrets. Ferrets were subcutaneously injected with PBS (control) or IFN-

2b and blood samples were drawn for RNA isolation 1 and 2 days after injection.

Without a commercially available ferret microarray, the RNA was then used for

microarray analysis on the Affymetrix GeneChip Canine Genome 2.0 Array (see

106

Figure 3-3. Gene expression profile measured by qRT-PCR in peripheral blood of

ferrets stimulated with IFN-2b in vitro. Whole blood drawn from 8 ferrets was

stimulated with either IFN-2b or with PBS as the control group. qRT-PCR was

performed to measure the gene expression at the mRNA level. Values are shown as fold

increases after normalization against -actin. White and black bars represent the mean

value of control group and IFN-2b injected group respectively. STAT1 and downstream

IRGs were significantly upregulated during the time course (P<0.001***, P<0.01**,

using student t test).

107

0 h 1 h 3 h

0

2

4

6

8

10 STAT1

***

***

0 h 1 h 3 h

0

5

10

15

20

25

30 MX1

***

***

0 h 1 h 3 h

0

10

20

30

40

50

60 ISG15

**

**

0 h 1 h 3 h

0

20

40

60

80

100 OAS1

**

**

0 h 1 h 3 h

0

2

4

6

8

10

12

14ISG20

**

**

0 h 1 h 3 h

0

20

40

60

80 IRF7

**

**

0 h 1 h 3 h

0.0

0.5

1.0

1.5

2.0

2.5

3.0 IFI35

**

**

*** P<0.001 ** P<0.01

IFN-2b stimulated

Control

108

Materials and Methods), because ferret genes show a high degree of homology with

canine genes as we have previously established8,20,23

(Table 3-1). The peripheral blood

gene expression data from IFN-2b-injected group was normalized to the control group.

The t-test analyses showed the highest number of significant changes occurred at day 2,

with a total of 2717 upregulated and 1230 downregulated genes in peripheral blood of the

IFN-2b-injected ferrets (Table 3-2). A threshold of at least 1.5 fold-change and a p

value for the t-tests of less than 0.05 were chosen. The peak upregulation of a cluster of

IRGs, including MX1, OAS1, OAS2, ISG15, IFI44 and ubiquitin specific protein 18

(USP18), occurred at day 1, while peak upregulation of IRGs such as JAK1, JAK2,

protein inhibitor of activated STAT1 (PIAS1) IRF1, interferon- receptor 1 (IFNGR1),

and eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) occurred at day 2

post-injection (Figure 3-4A and Table 3-3).

After assessing the large scale gene expression profile following ferret in vivo

IFN-2b stimulation, we validated the expression of selected IRGs by qRT-PCR

according to the availability of the ferret specific primers. We found STAT1 and IRGs

such as MX1, OAS1 and ISG15 were significantly upregulated in ferrets injected with

IFN-2b compared to the controls (Figure 3-4B). The lack of ferret sequences for other

IRGs prevented us from confirming the upregulation of these genes.

To determine if STAT1 phosphorylation was correlated with IRG activation in

our ferret animal model of SARS-CoV infection19

, we went on to analyze host gene

expression following SARS-CoV infection. The gene expression data at day 2 post-

infection with SARS-CoV was normalized to the mock control dataset. Unfortunately,

109

Table 3-1. Amino acid percentage identity of ferret immune genes compared to

known species. Gene symbol Dog Human Mouse

STAT1 90 89 87

STAT2 79 86 75

STAT3 97 98 98

MX1 89 78 72

OAS1 79 70 65

ISG15 87 73 64

ISG20 81 75 72

IRF1 93 89 79

IRF2 100 94 93

IRF7 80 73 65

IFI35 82 74 71

PKR 100 100 98

P52RIPK 88 86 81

CXCL8 90 76 52

CXCL9 NA* 68 58

CXCL10 81 80 67

CXCL11 NA 86 63

NFKB 98 97 93

NFKB inhibitor 100 94 87

CXCR3 92 91 86

CCL5 96 83 76

SOCS3 100 100 97

SOCS5 98 95 88

IFN-α 71 54 48

IFN-β 75 57 44

IFN-γ 86 63 48

TNF-α 95 88 77

IL-1β 71 61 58

IL-4 84 47 36

IL-6 75 58 38

IL-16 78 65 67

CCL13 80 NA NA

TLR3 95 89 87

TLR7 100 96 92

*NA = not available

110

Table 3-2. Summary of differentially regulated genes in top functional groups during

the time course in IFN-2b-injected or SARS-CoV infected ferrets.

IFN-2b SARS-CoV

Day 1 Day 2 Day 1 Day 2

BL LG BL LG LG BL LG

Total upregulated genes 716 82 2717 512 4222 138 1014

Total downregulated genes 51 147 1230 550 1248 414 894

Cellular process ↑ 440 44 1581 266 2180 69 542

Cellular process ↓ 15 83 577 318 570 229 427

Metabolic process ↑ 328 30 1212 175 1573 52 369

Metabolic process ↓ 8 58 383 247 392 188 297

Intracellular signaling cascade

↑ 50 3 205 40 271 12 63

Intracellular signaling cascade

↓ 2 11 81 0 80 29 60

Cell cycle ↑ 48 6 160 23 178 6 45

Cell cycle ↓ 0 11 36 44 38 30 29

Immune response ↑ 32 6 82 18 125 9 44

Immune response ↓ 0 2 32 0 39 7 29

Number of regulated genes in different functional categories with at least 1.5-fold change

and a significant t-test of p<0.05 (↑ upregulated, ↓ downregulated).

111

Figure 3-4. Microarray and qRT-PCR analysis of IRG expression in peripheral

blood and lung necropsies of ferrets injected with IFN-2b or infected with SARS-

CoV in vivo during the time course. A) Ferrets were injected with IFN-2b or with

PBS. Whole blood or lung necropsies were collected at day 1 and day 2. mRNA was

purified, converted to cRNA and ran on the Affymetrix Canine gene chip II. Hierarchal

microarray EDGE analysis demonstrated a cluster of IRGs, which were significantly

upregulated (red color). B) Upregulation of IRGs was confirmed at peak time-points with

qRT-PCR, where ferret specific primers were available. White and black bars are the

mean gene expression level of control and injected ferrets respectively, normalized to

actin. C) Differential upregulation of IRGs observed following the microarray analysis of

blood and lung necropcies of ferrets infected with SARS-CoV. D) Upregulation of 4

IRGs was confirmed by qRT-PCR.

A) IFN-a2b

IRGs

Ctrl D1 D2 Ctrl D1 D2Blood Lung

Blood Lung

24 h

ST

AT

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* *

* *

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B) IFN-a2b

IFN-a2b injected

Mock control

112

305746

Rectangle

305746

Rectangle

305746

Typewritten Text

305746

Typewritten Text

24h

305746

Typewritten Text

24h

C) SARS-CoV

IRGs

Ctrl D2 Ctrl D1 D2

Blood Lung

Blood Lung

48 h

ST

AT

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0

1

2

3

4

5

6 *

48 h

OA

S1

/

ac

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0

2

4

6

8

10

12

14 *

48 h

MX

1 /

ac

tin

0

1

2

3

4

5

6 *

48 h

ISG

15 /

acti

n

0

10

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*

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OA

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8

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12

14 *

D) SARS-CoV

SARS-CoV infected

Mock control

113

114

Table 3-3. Interferon-regulated genes fold change during the time course in IFN-

2b-injected or SARS-CoV infected ferrets.

Symbol Gene Name

IFN-α2b SARS-CoV

Day 1 Day 2 Day 1 Day 2

Blood Lung Blood Lung Lung Blood Lung

Interferon-regulated genes (IRGs)

EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 1.9 1.7 2.4 - 1.8 - -

IFI35 interferon-induced protein 35 - - - - 2.1 - 1.5

IFI44 interferon-induced protein 44 5.8 4.1 4.3 1.6 5.2 5.5 7.0

IFI44L interferon-induced protein 44-like 3.7 2.7 3.2 - 2.7 3.1 3.8

IFIT1

interferon-induced protein with tetratricopeptide repeats

1 - - - - 1.7 - -

IFNGR1 interferon gamma receptor 1 - - 3.8 - 1.7 - -

IRF1 interferon regulatory factor 1 - - 3.0 - 1.7 - 1.6

ISG15 ISG15 ubiquitin-like modifier 11.0 14.3 2.4 1.8 35.2 14.0 10.8

JAK1 Janus kinase 1 (a protein tyrosine kinase) - - 2.2 - - - -

JAK2 Janus kinase 2 (a protein tyrosine kinase) - - 2.1 - - - -

MX1 myxovirus (influenza virus) resistance 1, interferon-

inducible protein p78 (mouse) 3.8 1.6 2.5 - 5.7 2.4 4.4

OAS1 2.,5.-oligoadenylate synthetase 1, 40/46kDa 4.0 2.5 1.9 - 11.4 4.6 2.1

OAS2 2.-5.-oligoadenylate synthetase 2, 69/71kDa 2.1 2.2 - - 5.2 4.1 2.9

OASL 2.-5.-oligoadenylate synthetase-like - - - - - 2.2 -

PIAS1 protein inhibitor of activated STAT, 1 - - 2.4 - - - -

PSMB8

proteasome (prosome, macropain) subunit, beta type, 8

(large multifunctional peptidase 7) - - - - 1.7 - 1.7

RSAD2 radical S-adenosyl methionine domain containing 2 2.9 - 2.7 - - 9.3 2.1

SOCS1 suppressor of cytokine signaling 1 - - - - - 10.2 -

STAT1 signal transducer and activator of transcription 1, 91kDa 1.5 2.6 1.9 - 3.0 2.7 2.1

USP18 ubiquitin specific peptidase 18 4.9 - 3.7 - - 6.0 -

Upregulated genes relative to controls, showing ≥1.5-fold increase and t-test’s p<0.05.

115

blood samples from day 1 post-infection did not meet minimal RNA quality for

microarray analysis, preventing us from performing a time-course study on the peripheral

blood. There were 138 upregulated and 414 downregulated genes (p < 0.05, > 1.5 fold

change) as ascertained by t-test analysis at day 2 post-infection (Table 3-2). IRGs,

including STAT1, MX1, OAS1, OAS2, ISG15, IFI44, suppressor of cytokine signaling 1

(SOCS1), radical S-adenosyl methionine domain containing 2 (RSAD2), USP18 and

OAS ligand (OASL) were significantly upregulated (Figure 3-4C and Table 3-3). The

upregulation of STAT1, MX1, OAS1 and ISG15 were validated with qRT-PCR (Figure

3-4D). These gene expression and STAT1 phosphorylation findings suggested that

robust IFN responses were activated following SARS-CoV infection 2 days post-

infection.

Interferon canonical pathway analysis confirmed the similarities between the

expression patterns of IRGs at day 2. STAT1, MX1, OAS1, USP18, RSAD2, ISG15 and

IFI44 were upregulated in the peripheral blood of IFN-2b-injected and SARS-CoV-

infected ferrets. In contrast, OASL, OAS2 and SOCS1 were upregulated during SARS-

CoV infection alone (Figure 3-5A).

3.3 Microarray analysis of IFN-2b injection or SARS-CoV infection in ferret lungs

Since SARS-CoV infection causes severe lung pathology we went on to compare

and contrast the genes upregulated by IFN-2b stimulation and SARS-CoV infection in

the lungs of ferrets. Microarray analysis was performed on lung necropsies of IFN-2b-

injected ferrets compared to controls. The peak gene expression occurred at day 2 with a

116

Figure 3-5. Intersect analysis of IRGs expression in blood and lung tissue from IFN-

2b injected and SARS–CoV infected ferrets. Venn diagrams are representative of

IRGs upregulation and indicate the total number of regulated genes. The time points were

chosen according to the highest expression levels of IRGs. For more information, refer to

Table 3-3.

117

A) Blood, day 2

SARS-CoV IFN-2b

IFI44 ISG15 MX1 OAS1 RSAD2 STAT1 UPS18

OAS2 OASL SOCS1

EIF2AK2 IFI44L IFNGR1 IRF1 JAK1 JAK2 PIAS1

3699 304 248

SARS-CoV IFN-2b

130 5371 99

B) Lung, day 1

EIF2AK2 IFI44 IFI44L ISG15 OAS1 OAS2 MX1 STAT1

IFI35 IFIT1 IFNGR1 IRF1 PSMB8

Total regulated genes

Total regulated genes

Upregulated IRGs

Upregulated IRGs

118

total of 512 upregulated and 550 downregulated (p < 0.05) genes (Table 3-2).

Interestingly, the strongest upregulation of several IRGs, such as STAT1, MX1, OAS1,

OAS2, ISG15, IFI44, IFI44 ligand (IFI44L) and EIF2AK2, occurred on day 1 (Figure 3-

4A and Table 3-3). There was a marked increase in the total number of regulated genes

from lung necropsies of SARS-CoV-infected ferrets compared to lungs from IFN-2b-

stimulated ferrets. The SARS-CoV infected ferrets had a peak in gene expression at day 1

with 4222 upregulated versus 1248 downregulated genes (p < 0.05) (Table 3-2). Both the

number of upregulated IRGs and the expression levels peaked at day 1, including

STAT1, MX1, OAS1, OAS2, ISG15, IRF1, interferon-induced protein with

tetratricopeptide repeats 1 (IFIT1), IFI35, IFI44, IFI44L, proteasome subunit

multifunctional beta 8 (PSMB8), EIF2AK2 and IFNRG1. JAK1 was the only IRG that

was downregulated at day 1 (Figure 3-4C). The upregulation of STAT1, MX1, OAS1 and

ISG15 was validated with qRT-PCR on lung necropsies of ferrets injected with IFN-2b

or infected with SARS-CoV (Figure 3-4D). The comparison of microarray results

between the lung tissue of IFN-2b-injected and SARS-CoV-infected ferrets at day 1

revealed commonalities in the expression patterns of most IRGs. STAT1, MX1, OAS1,

OAS2, ISG15, IFI44, IFI44L and EIF2AK2 were among the overlapping genes (Figure

3-5B).

3.4 Pathway and functional group differential gene expression patterns

To further model the pathways involved in the host response to SARS-CoV and

the direct effects of IFN-2b administration, functional analysis of the regulated genes

119

was performed using Ingenuity Pathway Analysis software. For each experimental group,

genes showing changes in their expression levels were mapped into high-level Gene

Ontology categories: cellular process, metabolic process, intracellular signaling cascade,

cell cycle and immune response (Table 3-2). The number of genes present in each

functional category is representative of the level of biological activity in each

experimental group with respect to the controls. Analysis of the IFN signaling canonical

pathway showed the upregulation of STAT1, MX1, OAS1, OAS2, ISG15 and IFI44 in

lung necropsies of IFN-2b injected and SARS-CoV infected ferrets (Figure 3-6).

Functional classification of upregulated genes showed that IFN-2b induces

increased expression of phagocytosis-related genes, such as Fc fragment of IgG, high

affinity Ia, receptor CD64 (FCGR1A) and dynamin 1-like (DNM1L), leukocyte

transendothelial migration genes, such as integrin beta 1 and 2 (ITGB1 and ITGB2), and

upregulation of chemokine receptors, chemokine C-C motif receptor 3, 7 and 9 (CCR3,

CCR7, CCR9) and chemokine C-X-C motif receptor 4 (CXCR4) (Table 3-4). These

results suggest that IFN-2b is able to activate specific functions of the leukocyte

responses in blood samples after exposure.

The lungs of ferrets infected with SARS-CoV showed broader immune responses

than IFN-2b-injected ferrets, as demonstrated by the higher number of regulated genes

in several functional categories related to the activation of the immune responses,

including: complement and coagulation, cell adhesion molecules and leukocyte activation

(Figure 3-7).

120

Figure 3-6. IRG pathway analyses of microarray datasets in lung necropsies of IFN-

2b injected and SARS-CoV infected ferrets. Ingenuity pathway analyses indicated

similar patterns of IRGs upregulation, downstream of STAT1 signaling pathway in lung

tissue from ferrets A) injected with IFN-2b and B) infected with SARS-CoV.

IFN-a2b injected ferrets

SARS CoV infected ferrets

B)

-4 0 4

A)

121

122

Table 3-4. Comparison of canonical pathways between ferrets injected with IFN-2b

and those infected with SARS-CoV during the time course.

Symbol Gene Name

IFN-α2b SARS-CoV

Day 1 Day 2 Day

1 Day 2


Apoptosis

BAX BCL2-associated X protein 1.8 - 2.4 - 4.4 - -

CASP3 caspase 3, apoptosis-related cysteine peptidase - - 1.6 - 1.5 - -

CASP6 caspase 6, apoptosis-related cysteine peptidase 1.9 - 2.5 - - - -

CASP7 caspase 7, apoptosis-related cysteine peptidase - - 1.9 - - - -

CASP8 caspase 8, apoptosis-related cysteine peptidase - - 1.6 - - - -

ENDOD1 endonuclease domain containing 1 - - 1.7 - - - -

TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 1.8 - 2.0 1.5 2.3 2.5 2.6

Cell cycle

CCNA1 cyclin A1 - - 1.6 - - - -

CCND3 cyclin D3 - - 2.1 - - - -

CCNE1 cyclin E1 - - 1.6 - 1.6 - 1.5

CDK7 cyclin-dependent kinase 7 - - 1.9 - - - -

SMAD2 SMAD family member 2 - - 2.0 - - - -

SMAD4 SMAD family member 4 1.6 - 3.3 - - - -

Chemokines/Chemokine receptors

CCL14 chemokine (C-C motif) ligand 14 - - - - 2.6 1.7 -

CCL17 chemokine (C-C motif) ligand 17 - - - - 2.2 - -




CCL4 chemokine (C-C motif) ligand 4 - - - - 1.5 - 1.6

CCR3 chemokine (C-C motif) receptor 3 - - 1.6 - - - -



CXCL12

chemokine (C-X-C motif) ligand 12 (stromal cell-derived

factor 1) - - 1.8 - 3.5 - -

CXCL14 chemokine (C-X-C motif) ligand 14 - - 1.8 - - - -

CXCR3 chemokine (C-X-C motif) receptor 3 - - - - - - 1.7

CXCR4 chemokine (C-X-C motif) receptor 4 1.7 - 2.2 - - - -

123

Table 3-4. (continued)

Symbol Gene Name

IFN-α2b SARS-CoV

Day 1 Day 2 Day

1 Day 2


Fc gamma R-mediated phagocytosis

ARPC1A actin related protein 2/3 complex, subunit 1A, 41kDa - - 2.0 - 3.0 - -

ARPC1B actin related protein 2/3 complex, subunit 1B, 41kDa 1.6 - 1.9 - 3.6 - -

ARPC5 actin related protein 2/3 complex, subunit 5, 16kDa 2.1 - 4.0 - - - -

CFL2 cofilin 2 (muscle) - - 1.8 - - - -

DNM1L dynamin 1-like - - 1.8 - - - -

FCGR1A Fc fragment of IgG, high affinity Ia, receptor (CD64) 1.6 - 1.5 - - - -

LYN v-yes-1 Yamaguchi sarcoma viral related oncogene homolog - - 1.9 - - - -

PTPRC protein tyrosine phosphatase, receptor type, C 2.2 - 4.9 - 1.7 - -

SYK spleen tyrosine kinase - - 1.6 - - - -

Glycolysis-gluconeogenesis

ACSS1 acyl-CoA synthetase short-chain family member 1 1.5 - 1.6 - - - 1.5

ACSS2 acyl-CoA synthetase short-chain family member 2 - - 2.4 - 1.9 - -

LDHA lactate dehydrogenase A 2.1 - 2.1 - - - -

LDHB lactate dehydrogenase B - - 1.9 - 3.1 - -

LDHC lactate dehydrogenase C 1.9 - 2.0 - - 1.7 -

PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 - - 1.5 - 1.8 - -

PDHB pyruvate dehydrogenase (lipoamide) beta 1.5 - 1.7 - - - -

PGM1 phosphoglucomutase 1 - - 1.8 - - 2.0 -

Leukocyte transendotelial migration

CXCR4 chemokine (C-X-C motif) receptor 4 1.7 - 2.2 - - - -

ITGA4

integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4

receptor) 2.1 - 2.6 - - - -

ITGB1

integrin, beta 1 (fibronectin receptor, beta polypeptide,

antigen CD29 includes MDF2, MSK12 - - 1.9 - - - -

ITGB2

integrin, beta 2 (complement component 3 receptor 3 and 4

subunit) - - 1.6 - 3.0 - -

Wnt signaling pathway

APC adenomatous polyposis coli - 1.5 1.8 1.7 1.7 - -

AXIN2 axin 2 - - 2.1 - - - 1.6

CSNK1A1 casein kinase 1, alpha 1 2.1 - 3.3 - 1.8 - -

CSNK1E casein kinase 1, epsilon - - 1.6 - 2.0 - -

CTNNB1 catenin (cadherin-associated protein), beta 1, 88kDa 1.8 - 3.5 - 2.0 - -

LEF1 lymphoid enhancer-binding factor 1 - - 2.3 - 1.6 - -

SMAD2 SMAD family member 2 - - 2.0 - - - -

SMAD4 SMAD family member 4 1.6 - 3.3 - - - -

TCF7 transcription factor 7 (T-cell specific, HMG-box) - - 1.5 - 1.6 - -

Upregulated genes relative to controls, showing ≥1.5-fold increase and t-test’s p<0.05.

124

Figure 3-7. Microarray analysis of immune response pathways in peripheral blood

and lung necropsies of ferrets injected with IFN-2b or infected with SARS-CoV in

vivo during the time course. Ferrets were injected with IFN-2b or with PBS. Whole

blood or lung necropsies were collected at different time-points. mRNA was purified and

used for microarray EDGE analysis. Three immune pathways that play key roles in early

immune responses (“Leukocyte activation”, “cell adhesion molecules” and “complement

and coagulation”) were used to identify similarities and differences. Red and blue colors

are representative of upregulation and downregulation, respectively. A) IFN-2b

injected ferrets. B) SARS-CoV infected group.

Leukocyteactivation

A) IFN-a2b

Ctrl D1 D2 Ctrl D1 D2

Blood Lung

Cell adhesionmolecules

Complementand coagulation

125

Leukocyte activation

B) SARS-CoV

Ctrl D2 Ctrl D1 D2

Blood Lung

Cell adhesion molecules

Ctrl D2 Ctrl D1 D2

Blood Lung

Complement and coagulation

Ctrl D2 Ctrl D1 D2

126

127

4. Discussion

Type I IFNs are a critical component of the innate immune response during viral

infections. The function of many downstream genes has been studied in-depth, however,

it is likely that the presence of the virus and subsequent TLR-mediated signaling are

required to deploy full IRG-mediated antiviral activity24

. In this study we investigated the

gene signatures induced following subcutaneous administration of IFN-2b in ferrets.

We also analyzed the signaling pathways during an infection with SARS-CoV, and by

means of comparative analysis we profiled IFN gene responses in the context of a

respiratory infection. We used an experimental model of infection with SARS-CoV in

ferrets, which causes mild symptoms without mortality. The pathological features of this

model were previously published19

and a summary of the clinical information can be

found in Table 3-5.

We assessed the capacity of subcutaneous administration of IFN-2b to activate

antiviral responses in ferrets. The activation levels of several intracellular signaling

proteins were studied by using phospho-specific antibodies and subsequent FACS

analysis. STAT1 plays a key role downstream of IFN signaling while STAT3 and STAT5

are thought to be involved at a lesser extent, and/or weak participation of STAT4,

mitogen activated protein kinase 38 (p38) and Extracellular Signal-Regulated Kinase

(ERK)25

. In vitro incubation of ferret PBMCs with IFN-2b led to strong

phosphorylation of STAT1, weak phosphorylation of STAT3 and STAT5 and no

phosphorylation of STAT4, p38 and ERK. Furthermore, the activation of the STAT1

signaling pathway in vitro was confirmed at the mRNA level with the presence of many

128

Table 3-5. Natural history of ferrets, infected with 103

TCID of SARS-CoV (adapted

from Chu et al, Virology, Vol. 374, 2008).

SARS-CoV-infected Mock controls

Total number of ferrets 42 18

Peak temperature day 2 (38oC) day 2 (36.9oC)

Sneezing 17/42 0/18

Diarrhea 5/42 1/18

Mortality 0/42 0/18

Lowest WBC count 4.0 x 103 /l (day 2) 8.0 x 103 /l (day 2)

Lowest lymphocyte count 1.8 x 103 /l (day 2) 5.0 x 103 /l (day 2)

Peak viral titer in nasal wash day 2

Peak viral titer in lung day 5

129

downstream IRGs, including MX1, OAS1, OAS2, ISG15, and IFI44. The in vivo effects

of IFN-2b were also investigated. STAT1 showed increased phosphorylation levels in

the peripheral blood at early hours post-injection, while STAT3 and STAT5 remained

unchanged. Moreover, we did not observe mRNA gene expression of interleukin 8 (IL-8)

and Suppressor of Cytokine Signaling 3 (SOCS3) at the mRNA level, suggesting that

STAT326

and STAT527

, respectively, do not participate in vivo in response to IFN-2b.

The global numbers of regulated genes found in the microarray results constitute

good estimators of the intensity of the host response at different time-points. In vivo

effects of IFN-2b can be observed 24 hours after the injection and their peak is reached

48 hours post-injection. IRGs are markedly increased in both blood and lung tissue,

however the responses in the blood show greater breadth and magnitude as compared

with the responses observed in lung tissue (Table 3-3). This suggests that the

administration protocol of IFN-2b used in this study is only capable of inducing a

limited activation in lung tissue. Therefore, alternative protocols including direct

administration of IFN-2b into the respiratory tract or subcutaneous administration at

higher doses should be explored in order to achieve stronger antiviral responses at the

infection sites. Gene expression during SARS-CoV infection, on the other hand, shows

the presence of strong antiviral and inflammatory responses in the lungs 24 hours post-

infection, fading on day 2 post-infection in both blood and lung tissue.

As expected, IFN-2b stimulates the increased expression of a variety of IRGs

that play a central role in the clearance of viral infections, including MX1, OAS1, OAS2

and ISG15. They exert their effects through different mechanisms of action, such as

direct targeting of viral entry, inhibition of protein synthesis or degradation of viral RNA.

130

MX1 is a dynamin-like large guanosine triphosphatase (GTPase), which has antiviral

activity against a wide range of RNA viruses. The antiviral activity of MX1 is effective at

the early stages of the viral cycle in the nucleus or cytoplasm22

. OAS is an adenylate

synthetase, which uses adenosine triphosphate to synthesize 2’,5’-oligoadenylates. The

latter activates latent RNase L that is involved in the degradation of viral RNA28

. ISG15

is an ubiquitin-like enzyme that covalently conjugates to a large number of cellular

proteins; however this does not usually lead to protein degradation. In the case of HIV-1,

ISG15 inhibits the release of virions29

.

Upregulation of similar sets of IRGs by SARS-CoV and IFN-2b were observed,

including 7 IRGs (STAT1, ISG15, MX1, OAS1, OAS2, IFI44 and IFI44L) in the

peripheral blood and lung tissue of both groups. In contrast, several IRGs, including

IFI35, IFIT1 and PSMB8, were only upregulated in the lungs during SARS-CoV

infection. These results suggest that the expression of certain IRGs lie beyond the direct

control of IFN-α2b, and additional signals such as activation of TLR-signaling by viral

components are probably required to assemble a fully functional antiviral response.

Although the induction of IRGs by IFN-α2b is the hallmark feature of IFN

responses, a full understanding of the biological effects of antiviral IFNs requires a

comprehensive study of the additional functional responses triggered by IFN-α2b. In the

blood of ferrets injected with IFN-α2b, the upregulation of genes that participate in

glycolysis-gluconeogenesis (e.g. acyl-CoA synthetases and lactate dehydrogenases)

(Table 3-4) are indicators of higher levels of metabolic activity. Moreover, IFN also

induces the expression of genes related with apoptosis (e.g. caspases and TNFSF10) and

cell cycle (e.g. cyclins and SMAD family members). It is unclear whether IFN-α2b alone

131

is capable of inducing apoptosis and/or cell replication in vivo, however, the upregulation

of these genes may indicate that PBMCs are now more responsive to signals capable of

triggering cell cycle events. Upregulation of chemokine receptors, such as CCR3, CCR7,

CCR9 and CXCR4 may indicate that IFN-α2b can increase the responsiveness of PBMCs

to locally-produced chemokines. Likewise, increased levels of genes that are involved in

leukocyte transendothelial migration and Fc-gamma receptor-mediated phagocytosis

(Table 3-4) suggest that IFN-α2b enhances leukocyte responses30

. Interestingly, a number

of genes that are part to the Wnt signaling pathway were found to be upregulated (Table

3-4). This indicates that in vivo administration of IFN-α2b also has effects over

lymphocyte maturation and differentiation31

.

The lungs of ferrets infected with SARS-CoV show the upregulation of a broader

variety of genes, as compared with IFN-α2b administration, and depicts a more complex

biological environment dominated by the antiviral responses, leukocyte infiltration and

other inflammatory responses (Figure 3-7B). A number of chemokine ligands, such as

chemokine C-C motif ligand 2 (CCL2), CCL4, CCL14, CCL19 and CCL25, and cell

adhesion molecules, such as activated leukocyte cell adhesion molecule (ALCAM) and

intercellular adhesion molecule 1 (ICAM1) are upregulated during SARS-CoV infection,

but these were not induced by the administration of IFN-α2b (Figure 3-7A and Table 3-

4). SARS-CoV also induced the upregulation of genes of the complement system such as

complement component 3 (C3) and complement factor B (CFB) (Figure 3-7B). Taken

together, these results depict how IRGs and other arms of the innate immune responses

are capable of resolving the respiratory infection caused by SARS-CoV infection.

132

Previously, gene regulation has been investigated using microarray analysis with

the intent on revealing molecular pathways imperative to H1N1 infection32

. Here we

investigated gene regulation of SARS-CoV infected and IFN-2b injected ferrets.

Microarray analysis was conducted on RNA from lungs and blood on day 1 and day 2.

The number of upregulated genes was quantified and compared to the number of

downregulated genes for each sample type. The number of downregulated genes was

greater than upregulated genes in the day 1 IFN-2b lungs and in the day 2 SARS-CoV

infected blood samples. To expose the molecular signature of this finding we then broke

down the genes from each group into their respective functional pathways: cellular

process, metabolic process, intracellular signaling cascade, cell cycle, and immune

response. Interestingly, we found that for every functional pathway the day 1 IFN-2b

injected lungs had more downregulated genes than upregulated genes (except the

Immune response) where blood samples from the same animals had the opposite trend of

more upregulated genes. Furthermore, the SARS-CoV infected animals had the opposite

trend where day 1 lungs had more upregulated genes and in the blood of day 2 there were

more downregulated genes. These findings may be indicative of the activity of the

stimulant IFN-2b compared to SARS-CoV. Moreover, the difference in the number of

genes regulated shows that IFN-2b and SARS-CoV have different spatial stimulation

which may be an important finding when determining the therapeutic efficacy of IFN-

2b. It is possible that the increase in gene expression in the blood samples following

IFN-2b injection is indicative of activation of systemic immunity where the SARS-CoV

infection had an increase of lung gene expression signifying possible local inflammation.

133

Type I IFNs play a critical role during antiviral responses, however their functions

in vivo have not yet been fully resolved. Additional research is required to define the

optimal IRGs profile that is present during successfully cleared viral infections.

Moreover, fine tuning of the IRGs responses may achieve more prolonged and wider

protection by therapeutic agents such as attenuated vaccines against respiratory viruses.

134

Reference List

1. Kolling,U.K. et al. Leucocyte response and anti-inflammatory cytokines in

community acquired pneumonia. Thorax 56, 121-125 (2001).

2. Thompson,W.W. et al. Mortality associated with influenza and respiratory syncytial

virus in the United States. JAMA 289, 179-186 (2003).

3. Dawood,F.S. et al. Emergence of a novel swine-origin influenza A (H1N1) virus in

humans. N. Engl. J. Med. 360, 2605-2615 (2009).

4. Dushoff,J., Plotkin,J.B., Viboud,C., Earn,D.J. & Simonsen,L. Mortality due to

influenza in the United States--an annualized regression approach using multiple-

cause mortality data. Am. J. Epidemiol. 163, 181-187 (2006).

5. Weiss,R.A. & McMichael,A.J. Social and environmental risk factors in the

emergence of infectious diseases. Nat. Med. 10, S70-S76 (2004).

6. HULL,R.B. & LOOSLI,C.G. Adrenocorticotrophic hormone (ACTH) in the

treatment of experimental air-borne influenza virus type A infection in the ferret. J.

Lab Clin. Med. 37, 603-614 (1951).


915 (2003).

8. Cameron,C.M. et al. Gene expression analysis of host innate immune responses

during Lethal H5N1 infection in ferrets. J. Virol. 82, 11308-11317 (2008).

9. Danesh,A. et al. Cloning, expression and characterization of ferret CXCL10. Mol.

Immunol. 45, 1288-1297 (2008).

10. Ochi,A. et al. Cloning, expression and immunoassay detection of ferret IFN-

gamma. Dev. Comp Immunol. 32, 890-897 (2008).

11. Marijanovic,Z., Ragimbeau,J., van der,H.J., Uze,G. & Pellegrini,S. Comparable

potency of IFNalpha2 and IFNbeta on immediate JAK/STAT activation but

differential down-regulation of IFNAR2. Biochem. J. 407, 141-151 (2007).

12. Takaoka,A. & Yanai,H. Interferon signalling network in innate defence. Cell

Microbiol. 8, 907-922 (2006).

13. Uddin,S. & Platanias,L.C. Mechanisms of type-I interferon signal transduction. J.

Biochem. Mol. Biol. 37, 635-641 (2004).

135

14. Chevaliez,S. & Pawlotsky,J.M. Interferon-based therapy of hepatitis C. Adv. Drug

Deliv. Rev. 59, 1222-1241 (2007).




16. Chelbi-Alix,M.K. & Wietzerbin,J. Interferon, a growing cytokine family: 50 years

of interferon research. Biochimie 89, 713-718 (2007).





19. Chu,Y.K. et al. The SARS-CoV ferret model in an infection-challenge study.

Virology 374, 151-163 (2008).

20. Rowe,T. et al. Modeling host responses in ferrets during A/California/07/2009

influenza infection. Virology 401, 257-265 (2010).

21. Storey,J.D., Xiao,W., Leek,J.T., Tompkins,R.G. & Davis,R.W. Significance

analysis of time course microarray experiments. Proc. Natl. Acad. Sci. U. S. A 102,

12837-12842 (2005).

22. Haller,O., Staeheli,P. & Kochs,G. Interferon-induced Mx proteins in antiviral host

defense. Biochimie 89, 812-818 (2007).

23. Fang,Y. et al. Molecular characterization of in vivo adjuvant activity in ferrets

vaccinated against influenza virus. J. Virol. 84, 8369-8388 (2010).

24. Bosinger,S.E. et al. Global genomic analysis reveals rapid control of a robust innate

response in SIV-infected sooty mangabeys. J. Clin. Invest 119, 3556-3572 (2009).

25. Li,H., Gade,P., Xiao,W. & Kalvakolanu,D.V. The interferon signaling network and

transcription factor C/EBP-beta. Cell Mol. Immunol. 4, 407-418 (2007).

26. Gharavi,N.M. et al. Role of the Jak/STAT pathway in the regulation of interleukin-

8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo. J.

Biol. Chem. 282, 31460-31468 (2007).

27. Barclay,J.L., Anderson,S.T., Waters,M.J. & Curlewis,J.D. Regulation of suppressor

of cytokine signaling 3 (SOC3) by growth hormone in pro-B cells. Mol. Endocrinol.

21, 2503-2515 (2007).

136

28. Bonnevie-Nielsen,V. et al. Variation in antiviral 2',5'-oligoadenylate synthetase

(2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a

splice-acceptor site in the OAS1 gene. Am. J. Hum. Genet. 76, 623-633 (2005).

29. Okumura,A., Lu,G., Pitha-Rowe,I. & Pitha,P.M. Innate antiviral response targets

HIV-1 release by the induction of ubiquitin-like protein ISG15. Proc. Natl. Acad.

Sci. U. S. A 103, 1440-1445 (2006).

30. Corssmit,E.P. et al. Effects of interferon-alpha (IFN-alpha) administration on

leucocytes in healthy humans. Clin. Exp. Immunol. 107, 359-363 (1997).

31. Staal,F.J., Luis,T.C. & Tiemessen,M.M. WNT signalling in the immune system:

WNT is spreading its wings. Nat. Rev. Immunol. 8, 581-593 (2008).

32. Shapira,S.D. et al. A physical and regulatory map of host-influenza interactions

reveals pathways in H1N1 infection. Cell 139, 1255-1267 (2009).

137

Chapter 4

Cloning, expression and characterization of ferret CD8 (A version of this chapter is submitted to journal of “Veterinary Immunology and

Immunopathology”, as Cloning and characterization of ferret CD8 and

development of a ferret CD8 hybridoma clone, Danesh et al.)

1. Introduction

Ferrets (Mustela putorius furo) are considered to be the preferred animal model

for studying viral respiratory diseases such as influenza and SARS1-3

. The ferret model

has been used in influenza research for several years and mimics the human season of

influenza4,5

. As a result, ferrets have been used for testing for the safety and efficacy of

live attenuated influenza vaccines. Currently, anti-sera hemagglutinin assays in ferrets are

used to measure antigenic distance in evaluation of new influenza vaccines6.

Specific immune responses have a significant impact on outcomes of infectious

respiratory diseases7,8

. Despite the prominent use of ferrets in medical research, the

immune system of these animals remains poorly characterized9. Recently we reported the

characterization of the ferret chemokines, CXCL9, CXCL10 and CXCL11, which are

important in migration of mononuclear cells to sites of infection10

. In order to evaluate

the immune response during the course of a viral infection, it is important to be able to

determine the activity of the immune cells, such as T cells11

. T lymphocytes are essential

components of immune responses that have yet to be characterized in the ferret

model12,13

. The lack of ferret specific antibodies capable of detecting T lymphocyte

surface molecules has hindered the evaluation of specific subsets of T cells, either in vivo

or in vitro, in ferret infectious disease models.

CD8 is a cell surface molecule that is used to distinguish cytotoxic T

lymphocytes14

. The CD8 molecule is expressed either as a heterodimer of and chains,

138

or as a homodimer of the chain15

. CD8+ cells comprise a subset of cell-mediated

immunity, cytotoxic T cells, that are essential in the eradication of virally infected cells or

tumors7,16

. CD8 is a co-stimulatory molecule for the T cell receptor and is necessary for

the recognition of the MHC I-peptide complex on antigen presenting cells15,17

. The

infiltration of CD8 positive cells to the site of infection has been demonstrated in several

studies18-22

.

In this study, we report for the first time the cloning of full-length ferret

CD8cDNA coding sequence. We expressed and characterized recombinant ferret

CD8 protein using polyclonal antibodies. We employed the expressed recombinant

protein to screen hybridoma supernatants derived from mice immunized with intact ferret

peripheral blood mononuclear cells (PBMCs). From these screenings, we identified a

parental hybridoma cell line whose secreted antibody specifically reacts with over-

expressed ferret CD8. We characterized this hybridoma cell line with both

immunoblotting and flow cytometry with recombinant CD8. The development of a

monoclonal antibody that recognizes ferret CD8 provides an important reagent for the

study of cytotoxic T cells in ferret models of viral infection.

139


2.1 Antibodies

Monoclonal anti-V5 and polyclonal anti-HIS antibodies were obtained from

Invitrogen (Mississauga, Canada). Polyclonal anti-human CD8 (H-160), FITC-

conjugated goat anti-rabbit IgG antibody, PE-conjugated donkey anti-mouse IgG, and

HRP-conjugated goat anti-mouse HRP, were purchased from Santa Cruz Biotechnology

(Santa Cruz, California).

2.2 Animals

Ferrets (Mustela putorius furo) were obtained from Triple F farms (Sayre,

Pennsylvania), and housed at the Toronto General Research Institute animal facility as

described previously10

. Animals were used in accordance to an animal use protocol for

non-infectious experimentation, which was approved by Animal Care Committee of the

University Health Network.

2.3 Total RNA purification and cDNA synthesis

Ferret whole blood was diluted 1:1 in RPMI (Invitrogen) and cultured at 37oC in

5% CO2 after stimulation with 1 g/mL LPS, 50 ng/mL PMA and 0.1 mM ionomycin, or

25 g/mL poly I:C. Cultures were harvested and collected in Paxgene RNA tubes and

total RNA extraction was performed (Qiagen, Mississauga, Canada). Total RNA was

transcribed to cDNA using Superscript II reverse transcriptase (Invitrogen).

140

2.4 Amplification, cloning and sequencing of ferret CD8

Degenerate primers were designed according to multiple nucleotide sequence

alignment of known CD8 sequences using ClustalW from European Bioinformatics

Institute (EBI). The primers were 5’-GGCCGAAGGGCGCAACTT-3’ (forward) and 5’-

GCCACACAGGATCCATCTCCA-3’ (reverse). A cDNA fragment of 729 base pairs

was amplified by standard PCR using these primers. The amplified cDNA fragment was

excised from an agarose gel and purified (Qiagen). The purified fragment was subcloned

into pCR 2.1 TOPO vector (Invitrogen). Plasmid DNA was isolated from positive clones

and sequenced using an ABI 3730XL DNA analyzer (Center for Applied Genomics,

Toronto, Ontario). The ferret CD8 cloning sequence was identified by a Basic Local

Alignment Search Tool (BLAST) search of the NCBI databases.

2.5 Generation of ferret CD8expression vector

PCR-based subcloning was used to generate an expression vector for ferret

CD8. The primers used to amplify ferret cDNA were 5’-GCCATGGCCTCTCGGGT

GACCCCCC-3’ (forward) and 5’-GACATATTTCTCTGAAGGGCT-3’ (reverse). These

primers engineered at 5’ Kozak sequence and the 3’ termination codon removed. The

cDNA was subcloned into the pcDNA-3.1/His6/V5-TOPO expression vector (Invitrogen),

which produces a fusion protein tagged at the C-terminus with His6 and V5 epitopes

when transfected into mammalian cell lines.

2.6 Cell culture, plasmid transfection, and protein purification

141

COS-7 cells were maintained in Dulbecco’s modified eagle’s medium (DMEM),

substituted with 10% fetal bovine serum (Invitrogen) at 37ºC, 5% CO2. COS-7 cells

were transiently transfected with Effectene (Qiagen). After 48 hours, the cells were

harvested and lysed in 1% NP40 buffer. Aliquots of the lysates were prepared for

western blots by boiling in Laemmli buffer. The soluble portion of the cell lysates was

run through Ni-NTA metal immobilized affinity column and bound protein was washed

and eluted into several fractions (Novogen, San Diego, California). Fractions from

washes and elutions were evaluated by western blot with anti-V5. Positive fractions were

pooled and dialyzed into PBS at 4ºC.


SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) was performed with pre-

cast gels (10-15%, Bio-Rad), and transferred to nitrocellulose membranes. The

membranes were blocked with 5% milk in phosphate buffered saline-Tween 0.02%

(TPBS) followed by 16 hours incubation with primary antibodies at the appropriate

dilutions. The blots were washed with TPBS and incubated with a goat-anti-mouse-HRP

secondary antibody. Following the final wash, bands were visualized using enhanced

chemiluminiscent (ECL) reagent (GE healthcare, UK). In some instances, ferret CD8

was immunoprecipitated with anti-His antibody (Invitrogen) or polyclonal anti-human

CD8 prior to western blot analysis.

142


Complete coding sequence of ferret CD8 was submitted to the GeneBank and an

accession number was obtained (EF492056). Accession numbers used for the multiple

sequence alignment and phylogenetic tree are: P01731 (mouse), P33706 (dog), P41688

(cat), P07725 (rat), P30433 (orangutan), AAH25715 (human), AAI51260 (cow) and

AAT52341 (pig).

2.9 Isolation of Peripheral Blood Mononuclear cells (PBMC)

Ferret PBMCs were isolated by centrifugation through Histopaque solution

(Sigma Chemicals). Briefly, 5 mL whole blood was diluted 1:1 with PBS, layered on 5

ml Histopaque solution and centrifuged at 400 g for 20 minutes. The enriched

mononuclear cell layer at the Histopaque-plasma interface was harvested, washed twice

and re-suspended in PBS.

2.10 Cell surface staining of ferret PBMCs

Ferret blood was collected in EDTA tubes and PBMCs were isolated as described

above. 1 x 106

cells/100 L PBS was incubated with 0.2 g primary antibody for 20

minutes at room temperature. Cells were washed and re-suspended in 100 L PBS. 0.2

g secondary antibody was added to each tube and incubated for 20 minutes while

protected from light. Cells were washed with PBS and fixed with 2% paraformaldehyde

in PBS. 10000 events were acquired using a FACS Calibure flow cytometer and Cell

Quest software (Becton Dickinson). The acquired data was analyzed using Flow Jo 7.2

software (Tree Star, Inc.).

143

2.11 Immunofluorescent staining of ferret CD8-transfected COS-7 cells with

rabbit polyclonal antibody

COS-7 cells were transiently transfected with the ferret CD8 expression vector.

Transfected cells were fixed with 4% formaldehyde in PBS and permeablized with

Triton-X100 (0.3%). Primary antibody solutions, diluted in 5% FCS in PBS, were added

to tubes and incubated at 37oC for 1 hour (anti-V5 antibody and polyclonal anti-human

antibody at a concentration of 0.02 g/l). Appropriate secondary antibodies (anti-mouse

IgG-PE or anti-rabbit IgG-FITC) were added to tubes at a concentration of 0.02 g/l

and incubated for 1 hour at 37oC, while protected from light. The cells were washed and

the coverslips were mounted onto slides with Gold antifade with DAPI (Invitrogen).

Images were captured from prepared slides using an LSM 510 META NLO microscope

(Zeiss).

2.12 Generation of monoclonal ferret CD8 supernatant

Mouse immunizations and hybridoma production were performed by Promab

Technologies Inc. Balb/c mice were immunized by three separate injections of ferret

PBMCs. Animals were sacrificed and splenocytes were harvested for fusion with the

Sp2/0 mouse myeloma cells. Single cell lines of hybridomas were cultured and cell

culture media from growing cells were used for screening by cell-based ELISA. Briefly,

96-well plates were coated with 200,000 ferret PBMCs/well. Plates were blocked with

5% BSA in PBS. 100 l hybridoma media was added to each well and plates were

incubated for 1 hour at room temperature. Plates were washed five times with PBS. 10 l

HRP-labeled anti-mouse IgG (0.02 g/l) was added to each well and incubated at room

144

temperature for 1 hour. Following three washes with PBS, tetramethylbenzidine (TMB)

was added to each well. Positive wells were visualized by color change and positive

clones were further grown and expanded. Later, supernatants from hybridoma cells, that

tested positive in ELISA assays, were screened with ferret CD8-transfected COS-7

cells, using western blots and flow cytometry.

145

3. Results

3.1 Cloning and sequence analysis of ferret CD8

Cloning of full-length ferret CD8 coding sequence was performed using ferret

cDNA derived from mitogen-stimulated PBMCs as described in the methods section.

Primers used for cloning were designed from inter-species consensus sequences after

multiple nucleotide sequence alignment of various species (Figure 4-1). The predicted

full-length amino acid sequence of ferret CD8 (242 amino acids) is homologous to

CD8 sequences of other species (Figure 4-1B, Table 4-1), with highest amino acid

identity to dog (78%) and cat (77%). Phylogenic analysis of amino acid sequences

confirms that ferret CD8 protein is most closely related to dog CD8(Figure 4-2).

3.2 Expression of recombinant ferret CD8

A C-terminal His6/V5-tagged expression construct was generated to produce

recombinant ferret CD8 protein by transfection in mammalian cells. COS-7 cells were

transiently transfected with the ferret CD8 expression construct and whole lysates were

subjected to western blot analysis using the anti-V5 antibody. A band was detected at the

expected molecular weight (38 kDa) in protein derived from CD8-transfected COS-7

cells, while no band was detected in the untransfected control lane (Figure 4-3A).

Affinity purified recombinant ferret CD8 also migrated to 38 kDa when subjected to

western blot analysis using anti-V5 antibody (Figure 4-3B).

146

Figure 4-1. Multiple alignment of the predicted full-length ferret CD8 amino acid

sequence with CD8 sequences from other species Clustal-X software. The predicted

amino acid sequence of ferret CD8 was aligned with CD8 protein sequences from

other mammalian species using Clustal-X software. The numbers to the right of the

sequences represent amino acid positions. Putative signal peptide, extracellular,

transmembrane, and cytoplasmic domains are indicated. Available sequences for CD8

used in the alignments were mouse (Mus musculus) (P01731), dog (Canis lupus

familiaris) (P33706), cat (Felis catus) (P41688), rat (Rattus norvegicus) (P07725), human

(Homo sapiens) (AAH25715), cow (Bos taurus) (AAI51260) and pig (Sus scrofa)

(AAT52341).

147

Signal peptide Extracellular domain

Transmembrane domain Cytoplasmic domain

148

Table 4-1. The percentage amino acid identity between the ferret CD8α amino acid

sequence and CD8α protein sequences from other mammals.

Species Percent Identity with Ferret (%)

Human 66

Dog 78

Cat 77

Cow 61

Pig 59

Mouse 45

Rat 50

149

Figure 4-2. Phylogenetic tree representing homology between ferret and other

known mammalian CD8 sequences. A phylogenetic tree was constructed using

CLUSTAL W and MEGA 3.1 packages and bootstrapped 100 times. Bootstrapping

confidence values are between 66 and 100. The numbers on the nodes of the dendrogram

represent bootstrap values. The full-length ferret nucleotide sequence was translated to

amino acid sequence using “Translate” (ExPASy, proteomics). The sequences used for

phylogenetic analysis were mouse (Mus musculus) (P01731), dog (Canis lupus

familiaris) (P33706), cat (Felis catus) (P41688), rat (Rattus norvegicus) (P07725), human

(Homo sapiens) (AAH25715), cow (Bos Taurus) (AAI51260), and orangutan (Pongo

pygmaeus) (P30433).

151

Figure 4-3. COS-7 transfected cells express ferret CD8 recombinant protein. COS-7

cells were either untransfected or transfected with pcDNA-3.1/His6/V5-TOPO expression

vector using Effectene. A) Western blot performed on lysate of COS-7 cells transfected

with ferret CD8, using anti-V5. B) Western blot performed on purified ferret CD8

recombinant protein, using anti-V5.

152

49

38

28

14

kDa

Anti-V5

62

A)

Anti-V5

B)

64

50

36

22

kDa CD8 a

Transfected COS-7

cell lysatesPurified Protein

153

3.3 Anti-human CD8 polyclonal antibody reacts with ferret PBMCs and with CD8

transfected COS-7 cells

Ferret PBMCs were stained with a commercially available polyclonal anti-human

CD8 antibody (H-160). Using flow cytometry, 32% of PBMCs were observed to cross-

react with this antibody (Figure 4-4A). The polyclonal antibody also detected a protein

band with a molecular weight corresponding closely with that of endogenous CD8 in

lysates derived from ferret splenocytes (Figure 4-4B). No protein band was detected in

lysates from the ferret brain-derived cell line, Mpf, which does not express CD8 (Figure

4-4B). H-160 was also tested for cross-reactivity with the expressed recombinant ferret

CD8. Lysates were prepared from transfected COS-7 cells and immunoprecipitated with

anti-His or with H-160. The immunoprecipitates were subjected to western blot analysis

with anti-His or H-160, and a 38 kDa band was detected consistently (Figure 4-3C). To

confirm the cross-reactivity of H-160 antibody with recombinant CD8 protein, we

performed immunofluorescence staining. COS-7 cells transfected with ferret CD8 were

co-stained with H-160 and anti-V5 antibody. Fluorescence microscopy revealed that the

H-160 signal (green) overlapped with the anti-V5 signal (red). Surprisingly, we observed

that the transiently expressed ferret CD8 protein was accumulated in the cytoplasm and

very little expression was seen on the cell surface. We further tested for CD8

expression on the cell surface by flow cytometry. Indeed, transfected cells labeled with

H-160 were reactive in flow cytometry as compared to untransfected cells (Figure 4-4E).

These results suggest that the recombinant protein shares antigenicity with human CD8.

Furthermore, although transfected cells exhibit predominantly cytoplasmic expression of

recombinant CD8, they do express some ferret CD8 protein on the cell surface. However,

154

Figure 4-4. Reactivity of ferret PBMCs and CD8-transfected COS-7 cells with a

polyclonal anti-human CD8α antibody. A) Ferret PBMCs were stained with a

polyclonal anti-human CD8 antibody and subjected to flow cytometry analysis. Results

were compared to an isotype (secondary antibody alone) control. B) Western blot

analysis of ferret splenocytes with rabbit anti-human antibody. Protein from lysates

derived from a ferret brain cell line (Mpf) was used as a negative control. C) Ferret CD8a

transfected COS-7 cells lysates was immunoprecipitated either with anti-His or with anti-

human CD8 antibody and western blots were probed with anti-His and or anti-human-

CD8 antibodies. D) Immunostaining of ferret CD8-transfected COS-7 cells with rabbit

polyclonal anti-human CD8 and the epitope tag antibody (anti-V5) on separate

channels. The cells were stained with the nuclear stain DAPI and results were compared

to untransfected COS-7 cells. E) Ferret CD8-transfected COS-7 cells were stained with

polyclonal anti-human CD8 subjected to flow cytometry. Results were compared to

untransfected COS-7 cells.

155

Secondary antibody aloneAnti-human CD8

(H-160)

A)

kDa

38

Anti-human CD8

(H-160)

28

49

B) C)

kDa

49

38

28

17

WB: Anti-His-tag

A B C A B C

A: COS-7 cell lysate,

B and C: His-tagged ferret CD8

transfected COS-7 extracts

HH

L L

IP:

Anti-human

CD8 (H-160)

Scattered plot

156

D)

Untransfected COS-7 cells and

polyclonal anti-human CD8 (H-160)

CD8α transfected COS-7 cells and

polyclonal anti-human CD8 (H-160)

E)

COS-7 cells transfected with ferret CD8

anti-human CD8

(H-160) Anti-V5

DAPI Merge

157

polyclonal antibodies are not appropriate for quantitative flow cytometric analysis of T

lymphocytes. We tested a panel of commercially available antibodies (clones SK1, SK2,

G42-8, RPA-T8, OX-8 and 341), however, none exhibited cross-reactivity with ferret

PBMCs.

3.4 Generation of mouse anti-ferret CD8 monoclonal supernatant

In parallel to the cloning and sequencing of ferret CD8, we began

immunizations of mice with ferret PBMCs to generate hybridoma clones expressing

antibodies that recognize cell surface markers. Following ELISA-based screening of

over 1000 clones, we tested the ELISA-positive hybridoma supernatants in flow

cytometry staining of ferret PBMCs. We identified several parental clones that exhibited

a positive profile in flow cytometry analysis of PBMCs, including a clone designated

8F9. Ferret CD8-transfected COS-7 cells and untransfected cells were stained with 8F9

antibody and subjected to flow cytometry. The results demonstrated that 8F9 was

reactive with transfected cells as compared to untransfected cells (Figure 4-5A). Using

western blots analysis on lysates from CD8transfected COS-7 cells, we detected a

band at 38 kDa with 8F9, which aligned with an identical western blot performed in

parallel with anti-V5 (Figure 4-5B). 8F9 did not detect a band in lysates from

untransfected cells (Figure 4-5B).

158

Figure 4-5. Flow cytometry staining and western blot analysis of cell culture media

from a hybridoma cell line and ferret CD8-transfected COS-7 cells. A) COS-7 cells,

transiently transfected with ferret CD8, were stained with hybridoma supernatant (8F9)

and subjected to flow cytometric analysis. Results were compared to an isotype

(secondary antibody alone) control. B) Protein derived from ferret CD8-transfected

COS-7 cell lysates was subjected to western blots using the hybridoma supernatant from

clone 8F9. Identical blots were probed with anti-V5. Protein derived from untransfected

COS-7 cells was used as a negative control.

159

Secondary antibody alone

A)

B)

8F9

A: Transfected COS-7 cells probed with 8F9 parental clone

B: untransfected COS-7 cells probed with 8F9 parental clone

C: Transfected COS-7 cells probed with anti-V5

38 kDa

A B C

38 kDa

Scattered plot

160

4. Discussion

We previously reported the importance of characterization of the ferret as an

animal model to study respiratory infectious diseases, and subsequently characterized a

subfamily of ferret chemokines10

. Others have used ferret as an animal model for

influenza and SARS-CoV infection and have emphasized specific advantages that the

ferret has over other existing animal models, including mice and monkeys1-3

. A major

disadvantage for the use of ferrets in influenza and SARS research is the general lack of

immunological reagents, such as antibodies that can distinguish subsets of T

lymphocytes. To address this problem, we have cloned and sequenced the ferret CD8

gene. The predicted amino acid sequence of ferret CD8 contains 242 amino acids and

includes a highly conserved putative signal peptide of 21 amino acids. We observed

lower homology in the extracellular domain between ferret orthologues, which may

explain the inability of commercially available CD8 monoclonal antibodies to recognize

ferret CD8.

To express recombinant ferret CD8, we used plasmid transfections in COS-7

cells, which are a cell line derived from the kidney of African green monkey that does not

express CD8. We assayed and detected ferret recombinant CD8using western blot

analysis of transfected CD8-COS-7 cell lysates with anti-V5. The molecular weight of

the recombinant protein correlated with both the predicted molecular weight of ferret

CD8, and what had been reported previously for other species23

. Next, we tested

commercially available polyclonal antibodies to validate the recombinant protein. A

polyclonal anti-human CD8 antibody, which we found to be reactive with ferret PBMCs,

cross-reacted in flow cytometry, western, and immunofluorescence analysis with COS-7

161

cells over-expressing ferret CD8. These results confirmed that recombinant ferret CD8

shares antigenicity with other CD8 orthologues.

The expressed ferret CD8protein was used to screen ferret-specific cell surface

antibodies. To develop these monoclonal antibodies, we screened hybridoma

supernatants derived from mice immunized with intact ferret PBMCs using cell-based

ELISA, flow cytometry staining of ferret PBMCs, and western blots of recombinant

CD8over-expressed in COS-7 cells. Up to this point, we have identified a single

mouse IgG secreting clone, 8F9, which detects recombinant ferret CD8 protein in

western blot and flow cytometry.

It has been demonstrated that SARS- and H5N1- infected patients had post-

mortem lymphocyte infiltration to the lung, consisting mainly of CD8 positive cells24

.

Depletion of lymphocyte subsets from peripheral blood is under the influence of

chemokines and their migration to the lung have been suggested25-27

. The infiltration of

lymphocytes into the lung may lead to pathological consequences and, in some cases, to

the development of acute respiratory distress syndrome (ARDS)24

. Large-scale

production and purification of this monoclonal antibody will enable us to demonstrate the

presence or absence of cytotoxic T lymphocytes in lung infiltrates of experimentally

infected ferrets with SARS-CoV and H5N1 virus.

In summary, we have demonstrated for the first time the cloning, characterization,

and expression of ferret CD8. We used the recombinant protein to identify a

monoclonal hybridoma clone that recognizes ferret CD8 This study represents an

important step towards the development of reagents to characterize distinct T lymphocyte

populations in ferret models of infectious disease.

162

Reference List

1. Darnell,M.E. et al. Severe acute respiratory syndrome coronavirus infection in

vaccinated ferrets. J. Infect. Dis. 196, 1329-1338 (2007).


915 (2003).

3. Peltola,V.T., Boyd,K.L., McAuley,J.L., Rehg,J.E. & McCullers,J.A. Bacterial

sinusitis and otitis media following influenza virus infection in ferrets. Infect.

Immun. 74, 2562-2567 (2006).

4. Lambkin,R. et al. Strong local and systemic protective immunity induced in the

ferret model by an intranasal virosome-formulated influenza subunit vaccine.

Vaccine 22, 4390-4396 (2004).

5. Small,P.A., Jr., Waldman,R.H., Bruno,J.C. & Gifford,G.E. Influenza infection in

ferrets: role of serum antibody in protection and recovery. Infect. Immun. 13, 417-

424 (1976).

6. Gupta,V., Earl,D.J. & Deem,M.W. Quantifying influenza vaccine efficacy and

antigenic distance. Vaccine 24, 3881-3888 (2006).

7. Belz,G.T., Bedoui,S., Kupresanin,F., Carbone,F.R. & Heath,W.R. Minimal

activation of memory CD8(+) T cell by tissue-derived dendritic cells favors the

stimulation of naive CD8(+) T cells. Nat. Immunol. 8, 1060-1066 (2007).

8. Zheng,B. et al. Rectification of age-associated deficiency in cytotoxic T cell

response to influenza a virus by immunization with immune complexes. J.

Immunol. 179, 6153-6159 (2007).

9. Svitek,N. & von,M., V. Early cytokine mRNA expression profiles predict

Morbillivirus disease outcome in ferrets. Virology 362, 404-410 (2007).

10. Danesh,A. et al. Cloning, expression and characterization of ferret CXCL10. Mol.

Immunol. 45, 1288-1297 (2008).

11. Hauge,S., Madhun,A.S., Cox,R.J., Brokstad,K.A. & Haaheim,L.R. A comparison of

the humoral and cellular immune responses at different immunological sites after

split influenza virus vaccination of mice. Scand. J. Immunol. 65, 14-21 (2007).

12. Boon,A.C., de Mutsert,G., Fouchier,R.A., Osterhaus,A.D. & Rimmelzwaan,G.F.

Functional profile of human influenza virus-specific cytotoxic T lymphocyte

activity is influenced by interleukin-2 concentration and epitope specificity. Clin.

Exp. Immunol. 142, 45-52 (2005).

163

13. Hoji,A. & Rinaldo,C.R., Jr. Human CD8+ T cells specific for influenza A virus M1

display broad expression of maturation-associated phenotypic markers and

chemokine receptors. Immunology 115, 239-245 (2005).

14. Willemsen,R.A. et al. CD8 alpha coreceptor to improve TCR gene transfer to treat

melanoma: down-regulation of tumor-specific production of IL-4, IL-5, and IL-10.

J. Immunol. 177, 991-998 (2006).

15. Pinto,R.D., Nascimento,D.S., Vale,A. & Santos,N.M. Molecular cloning and

characterization of sea bass (Dicentrarchus labrax L.) CD8alpha. Vet. Immunol.

Immunopathol. 110, 169-177 (2006).

16. Vikman,S. et al. CD8(+) T cells against multiple tumor-associated antigens in

peripheral blood of midgut carcinoid patients. Cancer Immunol. Immunother.

(2007).

17. Somamoto,T., Yoshiura,Y., Nakanishi,T. & Ototake,M. Molecular cloning and

characterization of two types of CD8alpha from ginbuna crucian carp, Carassius

auratus langsdorfii. Dev. Comp Immunol. 29, 693-702 (2005).

18. Anglen,C.S., Truckenmiller,M.E., Schell,T.D. & Bonneau,R.H. The dual role of

CD8+ T lymphocytes in the development of stress-induced herpes simplex

encephalitis. J. Neuroimmunol. 140, 13-27 (2003).

19. Brown,T.J. et al. CD8 T lymphocytes and macrophages infiltrate coronary artery

aneurysms in acute Kawasaki disease. J. Infect. Dis. 184, 940-943 (2001).

20. Chen,H.C. et al. Lymphocyte activation and hepatic cellular infiltration in

immunocompetent mice infected by dengue virus. J. Med. Virol. 73, 419-431

(2004).

21. Foxwell,A.R., Kyd,J.M., Karupiah,G. & Cripps,A.W. CD8+ T cells have an

essential role in pulmonary clearance of nontypeable Haemophilus influenzae

following mucosal immunization. Infect. Immun. 69, 2636-2642 (2001).

22. Wang,Y., Lobigs,M., Lee,E. & Mullbacher,A. CD8+ T cells mediate recovery and

immunopathology in West Nile virus encephalitis. J. Virol. 77, 13323-13334

(2003).

23. Shaw,A.S. et al. Short related sequences in the cytoplasmic domains of CD4 and

CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein

kinase. Mol. Cell Biol. 10, 1853-1862 (1990).

24. Ng,W.F., To,K.F., Lam,W.W., Ng,T.K. & Lee,K.C. The comparative pathology of

severe acute respiratory syndrome and avian influenza A subtype H5N1--a review.

Hum. Pathol. 37, 381-390 (2006).

164

25. Aggarwal,A., Agarwal,S. & Misra,R. Chemokine and chemokine receptor analysis

reveals elevated interferon-inducible protein-10 (IP)-10/CXCL10 levels and

increased number of CCR5+ and CXCR3+ CD4 T cells in synovial fluid of patients

with enthesitis-related arthritis (ERA). Clin. Exp. Immunol. 148, 515-519 (2007).




27. Hsieh,M.F. et al. Both CXCR3 and CXCL10/IFN-inducible protein 10 are required

for resistance to primary infection by dengue virus. J. Immunol. 177, 1855-1863

(2006).

165

General Discussion

SARS-CoV is an emerging infectious disease of the 21st century that claimed

more than 840 lives worldwide1. SARS is a severe respiratory disease in middle aged and

elderly humans and can be a potential threat that may cause outbreaks if it re-emerges in

future1-3

. The acute phase of SARS infection has been linked with the lack of a Th2

response and the existence of a Th1 response accompanied by increased proinflammatory

cytokines4,5

. While the role of Th1-mediated immune responses has been wildly studied

the role of innate immunity during the early stages of SARS is still not well understood.

Our human cytokine and gene expression data suggest that patients with poor outcome

show distinctive features in their innate immune responses6. Taken together, I believe that

interferon regulated genes (IRGs) play a key role during the early hours post-infection

with SARS-CoV and they might determine the degree of severity. This prompted the

investigation of the interferon responses in ferrets as are the preferred animal model of

SARS7. Ferrets have shown greater susceptibility to SARS-CoV compared to mice

8.

SARS-CoV replicates well in ferrets. SARS infection in ferrets mimics human symptoms

accompanied with histological evidence of pneumonitis and a percentage of mortality. In

addition, unlike mice, ferrets transmit the disease7,8

. The drawback to study the immune

system of ferrets was the lack of immunological reagents for this model9,10

. I started

building my new immune model of SARS infection in ferrets with the aim of transposing

our gene expression findings from human patients into a more controlled experimental

environment, and paying special attention to the interferon responses.

166

5.1 SARS correlation with CXCL10 in humans and ferrets:

The clinical presentation of SARS in humans had a diverse range from

asymptomatic infection in some cases to respiratory failure in others1-3,11

. In our studies,

IFN-was significantly increased in SARS patients with mild disease compared to the

patients who needed critical care6. Treatment of SARS patients with corticosteroids

delayed the lung injury in some patients and IFN-α therapy was reported to be effective

in SARS treatment12

; together, these findings point to the important role of the immune

response in the containment of SARS disease progress5,13,14

. We also found that IFN-

was increased in SARS patients with mild disease compared to patients who needed

critical care6.

I propose that an exaggerated immune response during the early stage of SARS

infection, featured by strong expression of some IRGs such as chemokines, may break

down the homeostasis of the immune system in those patients that were not able to clear

the virus at early stages of the infection. CXCL10 is one the IRGs that drew my attention

as an important player in SARS containment or spread. I have shown that the level of

CXCL10 was increased in mild SARS patients and ICU SARS patients compared to

healthy controls. ICU SARS patients had significantly higher levels of CXCL10

compared to the mild SARS patients, moreover, the highest levels of CXCL10 were

observed in those ICU patients who died. Some of the IRGs, such as CXCL10 and CCL2

are important regulators of cell migration to the site of infection and dysregulation of

these cheomokines may be associated to severity of the disease14,15

. Other investigators

have studied the level of proinflammatory and Th1/Th2 cytokines in SARS patients, and

although different profiles were defined, the levels of CXCL10 were consistently

167

upregulated in different groups of patients5,13,16,17

. Some investigators have suggested that

a “cytokine storm” is the main determinant of poor outcome in SARS patients5,13,15

. In

our previous gene expression study in SARS patients6

and also in this study, the levels of

proinflammatory and Th1/Th2 cytokines were examined. Patients with poor outcome did

not show an increased expression of proinflammatory cytokines and our data does not

support the “cytokine storm” hypothesis. Instead, a CXCL10 response seems to correlate

with the severity of the disease, which may enforce its effects by recruiting infiltrating

cells in the lung. Infiltration of cells to the lung under the influence of different

combinations of chemokines is an interesting field that could be addressed in future

studies in ferrets. Identification of the genes that display a strong correlation with SARS

pathology can be beneficial in different aspects. For example, modulation of CXCL10

activity might represent a novel therapeutic approach, either by direct neutralization of

CXCL10 or by blocking its receptor (CXCR3) and CXCL10 signalling. These

hypotheses can be tested further in a future ferret study. Moreover, strong upregulation of

CXCL10 but not other closely related chemokines, such as CXCL9 was a distinctive

feature of SARS that is not seen during influenza infection even though both diseases

may present some similar symptoms at onset. Therefore, CXCL10 can be considered as a

specific biomarker for early diagnosis of SARS. The comparison of SARS and influenza

infection in terms of disease course and CXCL10 expression pattern is an interesting

topic and needs to be the subject of further investigation.

CXCL10 is an important regulator of Th1 polarized immune responses18

and in

some instances, can promote inflammation and pathological events19-21

. However, the

role of this chemokine and its receptor CXCR3 in the development of ARDS-like

168

diseases associated with pathogenic pulmonary viral infections is not well understood. I

have sequenced for the first time the full mRNA sequence of ferret CXCL10, which is

highly conserved with its human homolog. Later, I expressed the recombinant ferret

CXCL10 and demonstrated its biological activity in a chemotaxis assay as well as its

cross-reactivity with a polyclonal antibody against human CXCL10. The purified

CXCL10 protein can be used for generating ferret specific monoclonal antibodies that

detect and or neutralize ferret CXCL10 for future studies.

I have shown the presence of increased expression of CXCL10 in the lung

biopsies of deceased SARS patients. Infected ferrets with SARS-CoV showed a similar

pattern of CXCL10 gene expression at mRNA level in their lungs. This similarity, further

supports that ferrets are the preferred animal model of SARS7. The role of CXCL10 in

lung pathological outcomes following SARS-CoV infection in ferrets can be further

tested by means of neutralization studies.

5.2 Early gene regulation events in SARS-CoV infection versus IFN-2b injection in

ferrets:

We previously demonstrated that imbalanced immune responses and deviated

expression of IRGs are associated to poor outcome in SARS patients6. Patients who

needed hospitalization or were transferred to ICU showed high levels of CXCL10 and

CCL2 in their serum. IRGs such as MX1, methallothionein 1H (MT1H), Syndecan 1

(SDC1) and IFIT3 were upregulated in mild SARS patients but not in ICU SARS

patients6. Other investigators have shown a similar correlation between SARS severity

and the expression pattern of chemokines4-6,13

. It has been shown that a poor IFN

response is associated with poor outcome in SARS patients12

. We observed increased

169

levels of IFN- in the plasma of our mild SARS patients compared to ICU SARS

patients. The level of IFN- in plasma was also increased in all mild SARS patients6. In

order to obtain a broader perspective of the role of IRGs and other immune mediators, we

went on to analyze the gene expression patterns in the peripheral blood and lung

necropsies of SARS-CoV-infected ferrets.

STAT1, a key molecule involved in IFN- and IFN- signaling, showed higher

levels of phosphorylation in the peripheral blood, and its mRNA levels were increased in

both the peripheral blood and the lungs of SARS-CoV-infected ferrets. Interestingly, the

lungs showed a higher number of total regulated genes and also more upregulated IRGs,

as compared with the blood. I defined a cluster of protective IRGs (STAT1, MX1, OAS1,

OAS2, ISG15 and IFI44), which were upregulated in both peripheral blood and lung

tissues of infected ferrets with SARS-CoV.

We used 6 month old ferrets, which are considered as young adults. SARS

infection in young ferrets produces mild symptoms and low mortality rate, which is

similar to the pattern found in young patients11

. The pattern of upregulated IRGs in the

young ferrets possessed many similarities with the findings in mild SARS patients, the

majority of which were young adults. Moderate expression of CXCL10 and formation of

an antiviral estate by protective IRGs seems to be a desired environment for the clearance

of the virus and the resolution of the disease, while strong upregulation of CXCL10 and

decreased expression of protective IRGs may lead to poor outcome. In this regard, I

suggest a future study in which senior ferrets (approximately 4 year old) would be

infected with SARS-CoV for investigating the pattern of IRG expression in their blood

170

and lung. For such an experiment, I hypothesize that increased CXCL10 expression and

decreased expression of protective IRGs in old versus young ferrets is associated with

severity of symptoms and higher mortality rates in old ferrets.

Interaction of IFNs with their receptors leads to activation of downstream

signaling, regulation of target genes and, generally, the establishment of an antiviral

estate22,23

. The IFN-family is a diverse group of cytokines, sharing 50% homology

amongst the group members24

. Although the gene expression patterns of IFN- have

been studied in human and mouse, the role of the different IFN- subtypes has not been

studied in detail in animal models. The gene expression patterns following IFN-2b and

ribavirin treatment in the peripheral blood of hepatitis C virus (HCV) patients was the

subject of a study by Taylor et al.25

. They used microarray analysis to evaluate the cyclic

changes in gene expression during the course of the treatment. However, the use of

combined therapy agents in that study and the lack of groups of patients and healthy

controls that received IFN-2b alone, prevented to clearly distinguish the direct effect of

IFN-2b on the peripheral blood. They have also studied the gene expression patterns of

human PBMCs after in vitro stimulation with IFN-2b26

. To my knowledge, there is no

published data on gene regulation in the lung necropsies of mouse or ferrets after

treatment with IFN-2b.

Here I hypothesize that IFN-2b can be used as a therapeutic agent and is able to

reduce the severity of SARS-CoV infection in ferrets. I have shown the responses

induced by IFN-2b injection and SARS-CoV infection in ferrets, both in vitro and in

vivo, in order to show the possible benefits of IFN-2b therapy. I have shown for the first

171

time that stimulation of ferret cells with IFN-2b, or subcutaneous injection of IFN-2b,

leads to phosphorylation of STAT1 and upregulation of protective IRGs in ferret PBMCs

and in their lungs. I found that genes associated with immune pathways such as leukocyte

activation pathway, cell adhesion molecules pathway and the complement pathway were

upregulated in the peripheral blood and lung necropsies from ferrets. Many genes of the

functional pathways, including cellular process, metabolic process, intracellular signaling

and cell cycle were also upregulated in blood and lung tissue. I have found that IFN-2b

regulates different arms of the immune response at early hours post administration in

ferrets. I have been able to identify a cluster of important protective IRGs that play a

critical role in providing an antiviral estate in the peripheral blood and lung necropsies of

injected ferrets with IFN-2b. Expression of an overlapping array of protective IRGs in

the lungs of the SARS-CoV-infected ferrets and IFN-2b injected ferrets may be an

indication of the potential benefit of using IFN-2b as a therapeutic agent. However as

mentioned earlier, more direct evidence for the efficacy of IFN-2b therapy needs to be

further investigated in future studies to conclude that such a therapy is effective. This

could be achieved by infecting young and senior ferrets with SARS-CoV and their

subsequent treatment with IFN-2b. Here, if SARS remerges in future, early treatment of

high risk diagnosed SARS patients, such as elderly patients or those with other medical

complications, may help to control the severity of the infection. This could reduce the

need of long-term hospitalization and its huge costs. Prophylaxis treatment of high risk

individuals such as professional health workers during an outbreak may also be valuable,

especially when current antiviral therapies have not yet proven their full efficacy in

SARS infection11,12

.

172

Microarray gene expression analysis revealed differences between SARS-CoV

infection and IFN-2b injection in my study as well. There were more upregulated IRGs

in the lung of infected ferrets with SARS-CoV compared to their blood. On the other

hand, ferrets injected with IFN-2b showed higher number of upregulated IRGs in the

peripheral blood than in the lung tissue. These differences can be the result of the

localized lung infection in SARS, versus the systemic effects caused by IFN-2b

injection. Immune responses and functional pathways followed the same trend as well. I

believe that, in a future study, it may be interesting to consider the use of higher doses of

subcutaneous IFN-2b, multiple doses, and/or to explore the intranasal administration of

IFN-2b to achieve stronger antiviral responses in the lungs. I speculate that faster and

stronger upregulation of protective IRGs such as MX1, OAS1, OAS2 and ISG15 limits

the viral growth and leads to a reduction in the lung damage. Viral confinement within

the infected tissue may limit the production of high levels of chemokines such as

CXCL10. It has been shown that SARS-CoV can directly upregulate this chemokine in

vitro4. As we and others have shown, high levels of CXCL10 are associated with poor

outcome during SARS-CoV infection5,6,13,15

, which might be the result of the recruitment

of an excessive number of immune cells into the lung and their contribution to self-

damage.

5.3 Characterization of ferret CD8

Many SARS patients at the onset of the disease presented unilateral or bilateral

air-space consolidation2. Pulmonary infiltration was increased in patients with poor

outcome, according to chest X-rays2,27

. Significant reduction of lymphocyte

173

subpopulations in peripheral blood of SARS patients have been reported by many

investigators28,29

. Absolute count of T lymphocytes (CD3+

), T helper cells (CD4+

), T

cytotoxic cells (CD8+

), B cells and NK cells were substantially reduced during the acute

phase of the disease, as compared to the healthy control group28,29

. Immunohistochemical

evaluation of sections prepared from the lung of deceased SARS patients has suggested

the infiltration of CD20+

B cells, CD8+

T cells and CD4+

T cells to the lung30

. The

number of CD8+ cells was notably higher than CD4

+ cells, whereas CD56

+ NK cells were

absent30

. Patients who had received higher doses of steroids had lower degrees of

lymphoid cells infiltration30,31

. In the last chapter of my thesis I cloned, expressed and

characterized the full-length ferret CD8cDNA coding sequence. The aim of this part of

my project was to be able to evaluate the infiltration of CD8+ cells in the lungs of

infected ferrets with SARS-CoV in future studies. CD8+ cells express CXCR3 on their

cell surface and could migrate to the site of infection under the influence of CXCL1032

. I

hypothesize that under the influence of CXCL10, CD8+ cells migrate to the lung of

infected ferrets with SARS-CoV. I believe that neutralization of CXCL10 with

monoclonal antibodies or the inhibition of its receptor CXCR3 by means of small-

molecule antagonists, will reduce the infiltration of CD8+ cells in the lung of ferrets

infected with SARS-CoV, and subsequently decrease symptoms severity and mortality

rates.

5.4 Future studies

The observation of lymphopenia in peripheral blood of SARS patients and the expression

of CXCL10 in lung necropsies of deceased SARS patients might be an indication of rapid

174

migration of lymphocytes to the lung under the influence of the CXCR3-CXCL10 axis.

However, monitoring host immune responses in an uncharacterized animal model

requires the generation of more reagents with modern technologies.

Sequencing the entire genome of ferrets would be a priority for developing desired

reagents at large scale. Here I suggest sequencing the ferret entire genome using a high-

throughput method which is fast and cost-effective. The history of nucleotide sequencing

backs to 1970s and the RNA genome of a bacteriophage was the first complete genome

made available33

. The chain-termination method was the first DNA sequencing method,

developed a few years later. In this method, single stranded DNA, DNA primers, DNA

polymerase and radioactive labeled dNTPS were used for sequencing the DNA

fragments. The limitation of this technology is the non-specific binding of the primer to

the DNA34

. Dye-terminator technology was the next method of choice, and is still used

today. It was improved for automated sequencing, which is fast and uses fluorescent

labeling for safety and ease of analysis. However unequal light emission peak heights are

a disadvantage of this method that may cause errors35

. Another disadvantage of all

methods so far discussed is their limitation in sequencing large DNA fragments. Only

short DNA fragments of up to 1000 base pairs can be sequenced by these methods. High-

throughput sequencing, which does not utilize standard methods such as dye reporters has

developed to produce hundreds of thousands or millions of sequences at once with a low

cost36

. The first next generation sequencing technology was a bead-based method called

Massively Parallel Signature Sequencing (MPSS)37

. This technology was complex and

expensive and finally was bought and modified by Illumina, using reversible dye-

terminator technology in which DNA molecules are attached to primers on a slide and

175

amplified to local colonies38

. DNA nanoball sequencing is one the next generation

sequencing methods, in which small fragments of genomic DNA can be amplified and

used for unchained sequencing by ligation to read the nucleotide sequences. Large

numbers of DNA nanoballs at low cost can be sequenced and assembled by this method

for sequencing the whole genome39

.

In situ hybridization would be another technique that could be used for evaluation of gene

expression40

. Making recombinant proteins is time-consuming, but using RNA In Situ

hybridization could be a fast approach for poorly characterized animal models. Having

the gene sequences, one can produce fluorescently labeled complementary RNA to utilize

it for determining the expression level of genes of interest. Antibody phage display

technology is a fast approach for producing large libraries of antibodies against desired

targets. This can be performed by fusion of the DNA coding sequences of the target

protein to one of the genes encoding the phage surface proteins. However affinity

selection of these antibodies needs the expression of recombinant protein in an expression

vector and exposing the phage antibodies to these recombinant antibodies to immobilized

recombinant target antigen41

.

The sequenced genes by high-throughput technology can be used to express immune

genes at the protein level as recombinant proteins. These recombinant proteins can be can

be used for the generation of hybridoma clones and the development of monoclonal

antibodies. Generated antibodies can be used for development of ELISA kits to measure

endogenous gene products at the protein level. Neutralizing antibodies also can be used in

neutralization assays.

176

Sequences obtained by one of these modern technologies can be further used to produce

ferret microarray chips. Sequences also can be used for designing ferret specific primers

for confirmation of microarray results. Expression of cytokine, chemokine, cell surface

markers and transcription factors at the mRNA level in the tissue biopsies can also be

assessed by In Situ hybridization.

In this thesis I have shown that the expression pattern of CXCL10 in the lung of infected

ferrets with SARS-CoV was similar to its expression in the lung of deceased SARS

patients. I suggest that CXCL10 may play an important role in the severity of the disease

and I propose future studies in order to neutralize CXCR3-mediated signaling by using

small-molecule inhibitors such as AMG479. I also suggest the neutralization of CXCL10

with a neutralizing antibody generated by phage or hybridoma technologies in future.

Ferrets may be infected with SARS-CoV and treated with CXCL10 neutralizing

antibodies. In this future study, the survival of treated ferrets compared to untreated

ferrets could be evaluated. Additionally, ferret specific monoclonal anti-CXCL10

antibodies could be used for measurement of CXCL10 at the protein level. Other factors

that should be assessed include the infiltration of leukocytes to the lung and gene

expression profiles in the lung or peripheral blood of ferrets. This could be achieved

using custom made ferret specific microarray chips. CXCL10 is an example of an IRG

that may play a key role in the pathogenesis of SARS.

There are other IRGs that may be protective against infection with SARS-CoV. I have

shown in the third chapter of my thesis that SARS-CoV infection and IFN-2b injection

in ferrets have their unique, as well as overlapping gene expression patterns. I suggest

that IFN-2b can be used as a therapeutic agent in a future study to investigate whether

177

this cytokine is able to reduce the severity of SARS-CoV infection in ferrets. It may be

valuable to explore the use of higher doses of IFN-2b in order to induce stronger IFN

responses in the lungs. I suggest the use of ferret specific microarray chips to evaluate the

gene expression patterns in treated versus untreated animals. Real-time PCR and in situ

hybridization could be used to confirm the microarray results. Finally, I propose the use

of alternative administration routes of IFN-2b, such as intranasal administration, for

future studies. It is important to mention that I used one single dose of IFN-2b in my

experiments. Considering treatments with multiple doses may also be beneficial for

creating a long-lasting IRGs response, as in day 2 post-injection of IFN-2b, I observed a

decline in the number and the expression levels of IRGs.

178

Reference List

1. Leong,H.N. et al. Clinical and laboratory findings of SARS in Singapore. Ann.


2. Hui,D.S. et al. Severe acute respiratory syndrome: correlation between clinical

outcome and radiologic features. Radiology 233, 579-585 (2004).

3. Lee,N. et al. A major outbreak of severe acute respiratory syndrome in Hong

Kong. N. Engl. J. Med. 348, 1986-1994 (2003).

4. Spiegel,M. & Weber,F. Inhibition of cytokine gene expression and induction of

chemokine genes in non-lymphatic cells infected with SARS coronavirus. Virol.

J. 3, 17 (2006).

5. Wong,C.K. et al. Plasma inflammatory cytokines and chemokines in severe acute

respiratory syndrome. Clin. Exp. Immunol. 136, 95-103 (2004).

6. Cameron,M.J. et al. Interferon-mediated immunopathological events are

associated with atypical innate and adaptive immune responses in patients with

severe acute respiratory syndrome. J. Virol. 81, 8692-8706 (2007).

7. Martina,B.E. et al. Virology: SARS virus infection of cats and ferrets. Nature

425, 915 (2003).

8. Chu,Y.K. et al. The SARS-CoV ferret model in an infection-challenge study.

Virology 374, 151-163 (2008).




303, 944-946 (2004).

11. Poutanen,S.M. et al. Identification of severe acute respiratory syndrome in

Canada. N. Engl. J. Med. 348, 1995-2005 (2003).



13. Huang,K.J. et al. An interferon-gamma-related cytokine storm in SARS patients.

J. Med. Virol. 75, 185-194 (2005).

14. Cheung,C.Y. et al. Cytokine responses in severe acute respiratory syndrome

coronavirus-infected macrophages in vitro: possible relevance to pathogenesis. J.

Virol. 79, 7819-7826 (2005).

179




16. Krathwohl,M.D. & Anderson,J.L. Chemokine CXCL10 (IP-10) is sufficient to

trigger an immune response to injected antigens in a mouse model. Vaccine 24,

2987-2993 (2006).

17. Glass,W.G., Subbarao,K., Murphy,B. & Murphy,P.M. Mechanisms of host

defense following severe acute respiratory syndrome-coronavirus (SARS-CoV)

pulmonary infection of mice. J. Immunol. 173, 4030-4039 (2004).



Immunol. 168, 3195-3204 (2002).

19. Liu,M.T., Keirstead,H.S. & Lane,T.E. Neutralization of the chemokine CXCL10

reduces inflammatory cell invasion and demyelination and improves neurological

function in a viral model of multiple sclerosis. J. Immunol. 167, 4091-4097

(2001).

20. Ruth,J.H. et al. Selective lymphocyte chemokine receptor expression in the

rheumatoid joint. Arthritis Rheum. 44, 2750-2760 (2001).

21. Christen,U. et al. Cure of prediabetic mice by viral infections involves

lymphocyte recruitment along an IP-10 gradient. J. Clin. Invest 113, 74-84

(2004).

22. Hall,J.C. & Rosen,A. Type I interferons: crucial participants in disease

amplification in autoimmunity. Nat. Rev. Rheumatol. 6, 40-49 (2010).

23. Galligan,C.L. et al. Interferons and viruses: signaling for supremacy. Immunol.

Res. 35, 27-40 (2006).

24. Chen,J., Baig,E. & Fish,E.N. Diversity and relatedness among the type I

interferons. J. Interferon Cytokine Res. 24, 687-698 (2004).

25. Taylor,M.W., Tsukahara,T., McClintick,J.N., Edenberg,H.J. & Kwo,P. Cyclic

changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in

peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10

weeks of treatment. J. Transl. Med. 6, 66 (2008).

26. Taylor,M.W. et al. Global effect of PEG-IFN-alpha and ribavirin on gene

expression in PBMC in vitro. J. Interferon Cytokine Res. 24, 107-118 (2004).

27. Ye,J. et al. Molecular pathology in the lungs of severe acute respiratory syndrome

patients. Am. J. Pathol. 170, 538-545 (2007).

180

28. Cui,W. et al. Expression of lymphocytes and lymphocyte subsets in patients with

severe acute respiratory syndrome. Clin. Infect. Dis. 37, 857-859 (2003).

29. Tang,X. et al. Measurement of subgroups of peripheral blood T lymphocytes in

patients with severe acute respiratory syndrome and its clinical significance. Chin

Med. J. (Engl. ) 116, 827-830 (2003).

30. Ng,W.F., To,K.F., Lam,W.W., Ng,T.K. & Lee,K.C. The comparative pathology

of severe acute respiratory syndrome and avian influenza A subtype H5N1--a

review. Hum. Pathol. 37, 381-390 (2006).

31. Franks,T.J. et al. Lung pathology of severe acute respiratory syndrome (SARS): a

study of 8 autopsy cases from Singapore. Hum. Pathol. 34, 743-748 (2003).

32. Lindell,D.M., Lane,T.E. & Lukacs,N.W. CXCL10/CXCR3-mediated responses

promote immunity to respiratory syncytial virus infection by augmenting

dendritic cell and CD8(+) T cell efficacy. Eur. J. Immunol. 38, 2168-2179 (2008).

33. Fiers,W. et al. Complete nucleotide sequence of bacteriophage MS2 RNA:

primary and secondary structure of the replicase gene. Nature 260, 500-507

(1976).

34. Sanger,F. & Coulson,A.R. A rapid method for determining sequences in DNA by

primed synthesis with DNA polymerase. J. Mol. Biol. 94, 441-448 (1975).

35. Rosenthal,A. & Charnock-Jones,D.S. New protocols for DNA sequencing with

dye terminators. DNA Seq. 3, 61-64 (1992).

36. Hall,N. Advanced sequencing technologies and their wider impact in

microbiology. J. Exp. Biol. 210, 1518-1525 (2007).

37. Brenner,S. et al. Gene expression analysis by massively parallel signature

sequencing (MPSS) on microbead arrays. Nat. Biotechnol. 18, 630-634 (2000).

38. Mardis,E.R. Next-generation DNA sequencing methods. Annu. Rev. Genomics

Hum. Genet. 9, 387-402 (2008).

39. Porreca,G.J. Genome sequencing on nanoballs. Nat. Biotechnol. 28, 43-44 (2010).

40. Jin,L. & Lloyd,R.V. In situ hybridization: methods and applications. J. Clin. Lab

Anal. 11, 2-9 (1997).

41. Hoogenboom,H.R. Overview of antibody phage-display technology and its

applications. Methods Mol. Biol. 178, 1-37 (2002).

181

Appendix I

Cloning, Expression and Immunoassay Detection of Ferret IFN-

(A version of this Appendix is published in Developmental and Comparative

Immunology, volume 32 (8), (2008), 890-7, as “Cloning, Expression and Immunoassay

Detection of Ferret IFN-”

Atsuo Ochi, Ali Danesh, Charit Seneviratne, David Banner, Mark E. Devries, Thomas

Rowe, Luoling Xu, Longsi Ran, Markus Czub, Steven E. Bosinger, Mark J. Cameron,

Cheryl M. Cameron, and David J. Kelvin, with permission from Elsevier.)

1. Introduction

Influenza A viruses infect millions of people world-wide resulting in high

morbidity and mortality rates, particularly in the very young and elderly 1-4. Certain

highly pathogenic avian influenza A viruses, such as the H5N1 strain, are a significant

threat to global health. H5N1 influenza has a high mortality rate, even in healthy young

adults, and while human to human transmission has been limited, mutation or genetic

reassortment of H5N1 influenza A virus may create a strain readily capable of human to

human transmission and with pandemic potential.

Cytokines are important mediators of the immune response and have been shown

to play a pivotal role in host immune defense to microbial infection 5. Interferon gamma

(IFN- has been recognized as a multipotent cytokine due to its broad range of

regulatory functions during virus-induced host immune responses 6,7

. IFN- can directly

influence the differentiation of T cells and promote Th1-type immune responses in CD4+

and CD8+ T cells

8,9. Virtually all immune cell types express receptors for IFN- and are

influenced by this cytokine. Signaling by IFN- leads to up-regulation of class II major

histocompatibility complex (MHC) expression, which in turn increases the antigen

presentation to CD4+ T cells by macrophages, dendritic cells, and B cells

10,11. IFN- also

182

increases the expression of class I MHC, resulting in enhanced stimulation of antigen-

specific CD8+ T cells

12,13. Upon stimulation of these cells by IFN-, CD8

+ T cells are

important in the eradication of intracellular virus during recovery from viral infections 14-

16. Moreover, high levels of IFN- bias the polarity of CD4

+ T helper cells towards a Th1

phenotype, characterized by production of IL-2 and IFN- 17,18

. A Th1-type immune

response is fundamental to the development of host immunity against many pathogens,

including influenza A virus 19-21

. IFN- is also necessary for T cell-mediated viral

clearance and limitation of latent viral infections 22

. Moreover, IFN- production is

considered to be critical in determining vaccine efficacy 23,24

. Therefore, measurement of

IFN- production in both natural and experimental influenza A virus infections will

improve our understanding of the antiviral immune response, which may assist the

development of more effective vaccines.

Several animal models have been developed for testing the efficacy of influenza vaccines

and antiviral therapies, including mice, rats, guinea pigs, nonhuman primates and

ferrets25-28. Ferrets (Mustela putorius furo) have been used by researchers to identify

novel viruses, determine strain toxicity and evaluate vaccines 29. Ferrets are naturally

susceptible to human strains of influenza virus and display many of the same symptoms

that appear in humans, including sneezing, nasal discharge, weight loss and an increased

body temperature 30

. Indeed, the Spanish flu virus (H1N1) was first identified in ferrets

31. Ferrets therefore represent a fundamentally important model for studying the

transmission and pathogenesis of influenza. A current limitation to the ferret influenza

model, however, is the lack of species-specific immunological reagents required to assess

host responses in vaccinated and infected animals. In the current study, we report the full

183

length cloning of the ferret IFN-cDNA, expression of the recombinant cytokine, and the

generation of monoclonal antibodies suitable for ELISA and ELISPOT detection of

natural ferret IFN-.

184


2.1 Experimental animals

Six-month old male ferrets (Mustela putorius furo) were purchased from Triple F

Farms Inc. (Sayre, PA. USA) and housed at the Southern Research Institute (SRI) BSL-2

animal facility (Birmingham, AB). Approval by animal care committee at SRI was

obtained for all procedures. After arrival, ferrets were quarantined and monitored for one

week prior to tissue and blood collection. Animal diets were based on a low fat, high

protein regimen.

Female Balb/c mice (4-6 wks old) were purchased from Jackson Laboratories

(Bar Harbor, ME, USA).

2.2 Total RNA purification and cDNA cloning of ferret IFN-

Ferret whole blood was diluted 1:1 with RPMI 1640 cell culture media

(Invitrogen, Carlsbad, USA) and stimulated with mitogens (LPS (1 g/ml, Sigma

Chemicals, St. Louise, MO, USA), PMA (50 ng/ml, Sigma), ionomycin (0.1 mM, Sigma)

and poly I:C (25 g/ml, Sigma) and incubated at 37ºC in 5% CO2 for 2, 4, 8, and 12

hours prior to RNA purification. Paxgene RNA isolation method (Qiagen, Mississauga,

Canada) was used. cDNA was synthesized from purified total RNA by reverse

transcriptase II (Invitrogen). A 907 bp cDNA fragment was amplified by PCR using

consensus sequence primers based on nucleotide sequence alignment of IFN- from

multiple species, including dog, cat, and pig. The PCR fragment was isolated, cloned and

sequenced to confirm that it encompassed the full-length coding sequence for ferret IFN-

. An expression vector for IFN- was generated by PCR-based subcloning using the

185

following primers: 5’-CGATGAATTATACAAGCTATATCTTA-3’ (forward) and 5’-

TTTCGATGCTCTG CGGCCGGGAAA-3’ (reverse). The resulting 501kb fragment

was ligated into pCR 3.1-TOPO vector (Invitrogen). Nucleotide sequences of positive

clones were confirmed by dideoxy sequencing using ABI 3730XL DNA analyzers

(Center for Applied Genomics, Toronto, Ontario). Gene identification was carried out by

Basic Local Alignment Search Tool (BLAST) analyses against National Centre for

Biotechnology Information (NCBI) databases at National Institutes of Health, USA.

Multiple sequence alignment was performed using Clustal X included in the Lasergene

Software package (DNAStar Inc., Madison, WI).

2.3 Gene Transfection, Expression in COS-7 cells, and Protein purification

PCR was used to generate a Kozak sequence at the 5’ end of the ferret cDNA, and

to remove 3’ end termination codons before sub-cloning into pcDNA3.1/His6.V5/TOPO

expression vector (Invitrogen) to produce a His6/V5 tagged form of the protein.

Expression constructs were sequenced to verify sequence and orientation.

COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)

(Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen) at 37ºC, 5% CO2.

COS-7 cells were transiently transfected with plasmid encoding the recombinant ferret

IFN- using Effectene (Qiagen). After 24-48 hours, the cell culture supernatant from the

IFN--transfected cells was run through Ni-NTA metal immobilized affinity column

(Novagen, EMS Chemicals, San Diego, CA, USA) to bind HIS-tagged recombinant ferret

IFN- protein. The purified protein was washed, eluted and fractions were collected.

Eluted fractions were screened by immunoblotting using an antibody recognizing the V5

186

epitope (Invitrogen). Fractions containing the IFN- protein were pooled and dialyzed

against phosphate buffered saline (PBS) at 4ºC and concentrated by spin column

(Nanosep 10k OMEGA, Pall Life Science, East Hills, NY, USA). The protein

concentration was determined by protein assay kit (Pierce, Rockford, IL, USA).


10-15% SDS-Poylacrylamide gel electrophoresis (SDS-PAGE) was performed

with pre-cast gels (Bio-Rad, USA), or 10% NuPAGE pre-cast gels (Invitrogen). Protein

was transferred to nitrocellulose and the membranes were blocked with 5% fat free milk,

0.01% Tween-20 in PBS (TPBS) for 1 hour at room temperature. Membranes were

incubated at 4C for 4-16 hours with monoclonal anti-V5 Ab (1:1000) (Invitrogen) or

hybridoma culture supernatant (1:50). Hybridized membranes were washed with TPBS

and incubated with goat-anti-mouse-HRP (1:5000) (Santa Cruz, CA, USA) for 1 hour at

room temperature. Protein blots were visualized using enhanced chemiluminescent (ECL)

reagents (GE Healthcare, Canada).

2.5 Isolation of Peripheral Blood Mononuclear Cells (PBMC)

PBMCs were isolated from ferret blood gradient centrifugation through

Histopaque solution (Sigma). Briefly, whole blood was diluted 1:1 with PBS (10 ml),

layered on to 5 ml Histopaque solution, and centrifuged 400 xg for 20 min. The enriched

mononuclear cell layer at the Histopaque-plasma interface was harvested and used as the

source of PBMCs.

187

2.6 Mouse B cell hybridoma preparation

Recombinant ferret IFN- (50 g) and 2 mg of keyhole limpet hemocyaine (KLH)

(Calbiochem, San Diego, CA, USA) were diluted in 0.5 ml PBS. Five l of

glutaraldehyde was added and the mixture was allowed to incubate at room temperature

for 1 hour. Due to the appearance of aggregates, the whole mixture was washed on a spin

column (Nanosep 10k OMEGA, Pall Life Science) and concentrated to 0.1 ml volume.

Following centrifugation, 0.5 ml PBS was added and the mixture was centrifuged again.

After two rounds of PBS addition and centrifugation, the mixture was made up to 0.5 ml

in PBS and used as the source for the priming antigen. Mice were immunized with 25 l

antigen suspension in emulsified Complete Freund's Adjuvant. Mice were injected an

additional two times at bi-weekly intervals with 5 g of recombinant ferret IFN-. Three

days after the third injection, spleen cells were removed and isolated for fusion with

Sp2/0-Ag14 using polyethylene glycol (Roche, Mannheim, Germany) and hypoxanthine

aminopterin thymidine (HAT) resistant hybridomas were selected. Hybridoma cells were

screened for the reactivity against IFN- by ELISA using Nunc MaxiSorp 96 well plates

coated with ferret IFN- (100 l, 0.1 g/ml).

2.7 Ferret IFN--specific ELISA

A 96-well ELISA plate (MaxiSorb, Nunc) was coated with 100 l/well

monoclonal anti-IFN- (2 g/ml) overnight at 4C. The wells were blocked with 150 l

1% BSA in PBS for 1 hour at 37C. Supernatants from mitogen-stimulated PBMC

cultures or serum from influenza A virus infected ferrets were loaded into each well at

dilutions described in the text and incubated for 1 hour at 37C. Wells were washed with

188

PBS/0.5% Tween-20 and then incubated for 1 hour at room temperature with biotin-

conjugated anti-IFN- antibody (1 g/ml in 0.5% Tween-20/1%BSA). The wells were

washed three times with PBS/0.5% Tween-20 before incubation with HRP-Avidin for 30

minutes. The substrate, (o-phenylenediamine, Sigma) was applied for 15 minutes at

room temperature. Colorimetric changes were quantitated using an automated ELISA

reader (Quant, BIO-TEK Instruments, Winooski, VT, USA).

2.8 Ferret IFN--specific ELISPOT assay

PVDF plates (Millipore, MAIPS4510) or MaxiSorp plates (Nunc) were coated

with a monoclonal anti-ferret IFN- antibody as the capture antibody, and blocked with

1% BSA-PBS. Ferret PBMCs were cultured in the presence of stimulating reagents as

described in the text for 18 hours. Wells were washed with water to remove cells and

then captured IFN- was detected by a biotin-conjugated detection antibody coupled to

HRP-avidin (Sigma). The ELISPOT was developed using DAB (Vector Laboratories,

Burlingame, CA, USA).

2.9 Infection of ferrets with Influenza A virus

Male ferrets (castrated, descented) weighing approximately 800-1000 g were

infected intranasally with 106 EID50 influenza virus (H3N2 strain: A/Panama/2007/99) in

1 ml PBS. An additional three animals were mock-infected using PBS diluent alone.

Animals were euthanized humanely six days post-infection, and serum was obtained

using 10 ml SST vacutainer tubes.

189

3. Results

3.1 Cloning of ferret IFN- cDNA and the expression of His6-,V5-tagged

recombinant IFN- in COS-7 transfectant cells

A cDNA encoding ferret IFN- was cloned from isolated total RNA derived from

mitogen-stimulated ferret PBMCs as described in the materials and methods. The cDNA

sequence and predicted amino acid sequence for ferret IFN- are depicted in Figure

Appen.-1A. Amino acid sequence homology to the predicted ferret IFN- was highest in

the Eurasian badger (Meles meles) (97%), followed by the canine (86%) and feline (83%)

sequences (Fig. Appen.-1B). The homology of ferret IFN- to human and mouse IFN-

was 63% and 48%, respectively. Phylogenetic analysis using BLAST (see Figure

Appen.-1C) indicates that closest orthologue of ferret IFN- is that of the badger. The

ferret IFN- cDNA was subcloned into the His6-,V5-fusion protein expression vector and

subsequently transfected into COS-7 cells to produce recombinant IFN- protein. Ferret

IFN- protein was secreted from the COS-7 transfectants and migrated as a 35-45 kDa

band when subjected to immunoblotting experiments using a V5-specific antibody

(Figure Appen.-2). Since the estimated molecular weight of ferret IFN- is ~17kDa as the

monomer, the band detected in Coomassie blue staining and immunoblotting is constant

with the dimerized form of IFN-.

190

Appendix-Figure 1: Ferret IFN- cDNA. (A) Full length ferret IFN- cDNA sequence

including 80 base pairs in the 5’ untranslated region (UTR), 501 base pairs of coding

sequence with predicted amino acid sequence, and 404 base pairs in the 3’UTR. (B)

Alignment of the amino acid sequences of ferret, Eurasian badger, rabbit, cat, dog, mouse,

and human IFN- precursor proteins (accession numbers Y11647, P30123, P46402,

P42161, P01580 and P01579, respectively) is shown. Asterisks indicate positions

displaying identical amino acid residues in all sequences in the alignment, and periods

indicate positions displaying semiconserved substitutions. Scores of amino acid

homology between ferret IFN- and IFN- from different species are shown in the lower

panel. (C) Phylogenetic tree showing the relationship between ferret and other known

vertebrate IFN-sequences. This tree was constructed using CLUSTAL W and MEGA

3.1 packages and bootstrapped 10,000 times. †Bootstrapping confidence values are

between 66 and 100. The Gene peptide accession numbers for IFN-γ are: badger,

CAA72346; dog, AAD314233; panda, ABE02189; cat, BAA06309; rhinoceros,

ABC18310; donkey, AAC42595; pig, ABG56234; dolphin, BAA82042; sheep,

ABD64367; buffalo, BAE75855; cow, NP_776511; armadillo, AAZ57195; woodchuck,

AAC31963; rabbit, BAA24439; human, P01579; monkey, AAM21477; mouse, P01580;

rat, NP_620235; chicken, CAA69227; zebrafish, BAD06253.

191

A)

192

B)

193

C)

194

Appendix-Figure 2: Secretion of ferret IFN- by COS-7 cell transfectants. Detection

of recombinant ferret IFN- by V5-tag-specific immunoblotting. Secreted recombinant

ferret IFN- was enriched using a His-affinity column. The first three eluate fractions,

shown in lanes 1, 2 and 3 respectively, were analysed by V5-specific immunoblotting.

The arrow indicates the dominant protein band observed in fraction 2 and 3.

196

3.2 Generation of monoclonal Abs specific for ferret IFN-

In the early attempts to monitor IFN- in ferret cell cultures, we tried

commercially available cytometric bead arrays, which employ human or mouse IFN--

specific antibodies. Neither human nor mouse-specific arrays resulted in the positive

detection as antibodies did not cross react with ferret IFN-. We are therefore prompted

to generate monoclonal antibodies specific to ferret IFN-. To produce ferret IFN-

specific antibodies, recombinant IFN-was conjugated to a carrier protein, KLH (keyhole

limpet hemocyanin) using glutaraldehyde. The resulting KLH-IFN- complex was

injected i.p. into Balb/c mice. Following fusion of splenocytes isolated from IFN--

immunized mice with hybridoma parent cells, IFN--reactive B cell hybridomas were

established. Hybridoma clones, selected by ELISA reactivity, were tested for specificity

by immunoblotting against recombinant ferret IFN- and supernatants from mitogen-

stimulated ferret PBMCs (Figure Appen.-3). Clone 3E7 recognized a 36 kDa band in

samples of recombinant ferret IFN-, demonstrating that the ferret IFN- reactive

monoclonal antibody can also detect the dimerized form of IFN- (Figure Appen.-3).

Additionally, a weak band at ~65 kDa was consistently observed indicating a possible

detection of another oligomeric form of ferret IFN- (Figure Appen.-3). Interestingly, the

study of culture supernatants derived from mitogen-stimulated PBMCs showed

prominent bands at 60-70 kDa and the band at 36 kDa was only weakly detectable

(Figure Appen.-3). The data indicates that the native ferret IFN- exists as an oligomeric

form (likely a dimer of the dimer), while the recombinant IFN- exists predominantly as a

single dimer.

197

Appendix-Figure 3: Detection of ferret IFN- by a monoclonal anti-ferret IFN- Ab.

Western blot analysis of recombinant ferret IFN- using monoclonal Abs (LEFT PANEL).

Lysates from COS-7 cells transfected with recombinant ferret IFN- samples were

analyzed by Western blot using a monoclonal antibody established from mouse

immunized by recombinant ferret IFN- and shown in the left panel (RIGHT PANEL).

Supernatants derived from ferret PBMC cultures stimulated with PMA plus ionomycin,

were analyzed by Western blot using anti-ferret IFN- monoclonal antibody. Arrows at

the right of each panel indicate the dimers for lower molecular weight protein bands and

putative tetramer as higher molecular weight species.

199

3.3 Utilization of monoclonal antibodies for the detection of ferret IFN-by ELISA

and ELISPOT immunoassays

An important application for monoclonal anti-ferret IFN- antibodies is the

detection and quantitation of IFN- protein in biological samples. To this end, we tested

our monoclonal antibodies for utility in a ferret IFN--specific ELISA assay. To select

the antibody pair for IFN- recognition in ELISA assays, we tested monoclonal

antibodies derived from different clones. The clone 3E7 was conjugated to biotin and

used as the detection antibody against Ferret IFN- and antibodies from five other clones

were used to coat the assay wells. Of the five monoclonal anti-Ferret IFN- antibodies

that we screened in this manner, the clone 1E3 consistently showed the presence of Ferret

IFN- in mitogen-stimulated ferret spleen cell supernatants. Therefore, we decided to

continue using 1E3 as the ELISA capture antibody paired with 3E7 as the detection

antibody. As shown in Figure Appen.-4A, an ELISA was carried out using the paired

capture and biotin-conjugated detection antibodies, and demonstrated an increase in

optical density that correlated directly with the concentration of purified recombinant

IFN-. The increase in optical density exhibited a smooth logarithmic correlation

between 10 pg/ml and 10 ng/ml of recombinant IFN- protein (R2 = 0.9826). Using this

standard curve, we measured the concentration of IFN- in in vitro stimulated PBMC

supernatants. IFN- was present only in samples that were treated with PMA in

conjunction with a Ca2+ ionophore (Figure Appen.-4B). The results indicate that the

monoclonal antibodies selected are applicable for ferret IFN--specific ELISA.

Our primary scope in developing the ferret IFN--specific monoclonal antibodies

was to develop a reagent with which to measure IFN- immune responses in tissues or

200

cells derived from influenza A virus-infected ferrets. To this end, an ELISA using the

capture-detection monoclonal antibody pair was used to assess the level of IFN- in sera

obtained from influenza A-infected ferrets. The assay showed substantial levels of

circulating IFN- on day 6 post-infection (Figyre Appen.-5). The level of IFN- in serum

from the non-infected control ferret was below the detection limit. These results show

that the ELISA assay using our anti-ferret IFN- monoclonal antibodies will be

invaluable in monitoring systemic IFN- responses during a host response against virus

infection.

ELISPOT IFN- assays are also a key tool in the determination and quantitation

of IFN--secreting cells. To develop a ferret-specific IFN- ELISPOT assay we

employed the same set of monoclonal ferret IFN- antibodies, clone 3E7 and 1E3.

ELISPOT was performed on ferret PBMCs that had been stimulated with PMA plus

ionomycin. As demonstrated in Figure Appen.-6, increasing numbers of IFN- secreting

cells were detected in direct proportion to the number of stimulated cells plated, while the

number of IFN- positive cells did not increase above background when increasing

numbers of unstimulated cells were plated. The results indicate that 3E7 and 1E3 anti-

ferret IFN- monoclonal antibodies will be invaluable for performing ELISPOT analysis

of IFN- levels in ferret tissue.

201

Appendix-Figure 4: Measurement of IFN- in the mitogen-stimulated ferret PBMC

culture supernatants by ELISA. (A) Standard curve for ferret IFN-ELISA. ELISA

plate well were coated with a monoclonal anti-ferret antibody generated in our laboratory.

Recombinant ferret IFN- was sequentially diluted and loaded to the antibody-coated

wells. Captured ferret IFN- was detected by a second monoclonal anti-ferret IFN-

antibody generated in our laboratory conjugated to biotin, using the avidin-HRP detection

method. Logarithmic dilution was used to derive a standard curve for downstream

applications of the ELISA. (B) IFN- in mitogen stimulated ferret PBMC supernatants.

ELISA utilizing the monoclonal ferret IFN- antibody as a capture antibody was

performed on COS-7 cells treated with PMA, ionomycin or both. Results represent the

mean values of triplicate samples.

202

A)

B)

203

Appendix-Figure 5: Detection of increased levels of IFN- in ferret serum after

infection with H3N2 influenza A virus. Serum from two ferrets (Animals A and B) was

taken 6 days post-infection. Sera from infected ferrets and one uninfected control were

loaded on to ELISA plates coated with the ferret IFN- specific capture antibody-coated

and the samples were analyzed as indicated in Figure Appen.-4. Results represent the

means of triplicate samples.

205

Appendix-Figure 6: ELISPOT assay for the IFN- producing cells in mitogen-

stimulated ferret PBMCs. ELISPOT assay was performed in the same manner outlined

for Figure Appen.-4 for capture and detection. COS-7 cells were plated in serial dilution

and stimulated with PMA plus ionomycin for 18 hours and IFN-γ secreting cells were

detected by biotinylated capture antibody. The y-axis depicts the number of IFN- spot

forming cells per well; total cells per well are indicated on the x-axis. Data shown are the

average of triplicate samples.

207

4. Discussion

Experimental models that would allow the testing of effective treatments or

vaccines for infection by novel influenza A viruses are currently in high demand. Ferrets

have been used as an animal model of infection with influenza A viruses to test the

severity of the disease and also to evaluate efficacy of potential vaccines32

. IFN-

synthesis in vivo is a central regulator in host immune responses against viral infection.

In this study we describe the cloning of a full length cDNA for IFN- and expression of

the recombinant IFN- protein. Furthermore, we describe the generation of two

monoclonal antibodies specific for ferret IFN- and the subsequent development of

immunoassays for the detection of native IFN-γ. We anticipate that the IFN-γ

immunoassays established in this study will be useful in gaining insight into ferret

antiviral responses and in other immune processes in general. Previous studies on IFN-γ

have shown that there is a strict species-specific activity of IFN-γ. IFN- genes isolated

from diverse species such as guinea pig, turkey, rhino, and catfish have been previously

described in the literature33-37

. Since ferrets are susceptible to human viral diseases,

including influenza A virus 38

and SARS-coronavirus 30

, we expect that the availability of

the ferret IFN-γ assay system described in this study will greatly expand the utility of the

ferret infection model.

Unexpectedly, the monomeric form of IFN-γ (17kDa) was detectable at only low

levels in recombinant ferret IFN- samples. Instead, a dominant band at ~36 kDa was

consistently observed during Western blotting whereas a weak band at 65-70 kDa was

also observed in the same sample. Conversely, when native IFN- in mitogen-stimulated

PBMC culture supernatants were investigated with monoclonal antibodies, dominant

208

bands were identified at 65-70 kDa and the ~36 kDa band was barely detectable.

Oligomeric forms of IFN-γ were very stable aggregates, as treatment with reducing

reagents, guanidine or urea did not disrupt the IFN-γ into its monomeric form. Since the

IFN-γ dimer was also observed in supernatants from mitogen-stimulated ferret PBMC

cultures, it is unlikely that the dimerization is caused by the epitope tags present on the

recombinant protein. The recombinant IFN-γ dimer was found to have biological

function consistent with known IFN-γ activity 39

. Treatment of ferret PBMC cultures

with the dimerized recombinant IFN-γ increased the surface presentation of class II MHC,

suggesting that the recombinant ferret IFN- is able to maintain biological function in the

dimerized form. We observed, however, a IFN- specific band in Western blotting of

mitogen-stimulated PBMC culture supernatants at a molecular mass at 65 kDa. This

suggests that the probable form of ferret IFN- in vivo exists in tetrameric form rather

than a dimeric form. Our observation of recombinant ferret IFN- seems to contradict the

recent report on guinea pig IFN-γ in which recombinant IFN-γ was shown to exist

primarily as a monomer 40

. Recombinant guinea pig IFN- was expressed in bacteria,

and the purified protein was capable of stimulating the class II MHC expression in guinea

pig cells. It has been reported previously that human IFN-γ shows a dominant band at

approximately 65 kDa in immunoblotting and human IFN-γ specific receptors are shown

to be reactive with IFN-γ dimers 41

. Other groups have demonstrated that carbohydrate

modification alters the molecular nature of human IFN- 42

. Taken together, these

observations suggest that it is not unreasonable to find that ferret IFN-γ may exist in

monomeric, dimeric and oligomeric forms. Subtle variation in amino acid sequences

between two species may result in differences in post-translational modification by

209

glycosylation, which could result in differential association of monomers into oligomers.

Alternatively, the origin of cells secreting IFN-, such as CD8+ T cells or NK cells, may

determine the differences observed in IFN-γ oligomerization. The interaction between

ferret IFN-γ and its receptor under physiological conditions may be mediated by IFN-γ

dimerization or oligomerization. Whether or not dimerization is the mechanism of IFN-γ

activation in other species obviously requires further investigation.

Cloning and expression of biologically active ferret IFN-γ is critical in the

development of immunoassays such as ELISPOT and ELISAs for detection of interferon

production and interferon producing cells. These reagents will be invaluable in the

assessment of vaccine efficacy against influenza A and other emerging infectious viruses.

210

Reference List

1. Morens,D.M. & Fauci,A.S. The 1918 influenza pandemic: insights for the 21st

century. J. Infect. Dis. 195, 1018-1028 (2007).

2. Poland,G.A., Jacobson,R.M. & Targonski,P.V. Avian and pandemic influenza: An

overview. Vaccine (2007).

3. Thomas,J.K. & Noppenberger,J. Avian influenza: A review. Am. J. Health Syst.

Pharm. 64, 149-165 (2007).

4. Mills,C.E., Robins,J.M., Bergstrom,C.T. & Lipsitch,M. Pandemic influenza: risk of

multiple introductions and the need to prepare for them. PLoS. Med. 3, e135 (2006).

5. Muller,U. et al. Functional role of type I and type II interferons in antiviral defense.

Science 264, 1918-1921 (1994).

6. Huang,S. et al. Immune response in mice that lack the interferon-gamma receptor.

Science 259, 1742-1745 (1993).

7. Bouley,D.M., Kanangat,S., Wire,W. & Rouse,B.T. Characterization of herpes

simplex virus type-1 infection and herpetic stromal keratitis development in IFN-

gamma knockout mice. J. Immunol. 155, 3964-3971 (1995).

8. Muller,U. et al. Functional role of type I and type II interferons in antiviral defense.

Science 264, 1918-1921 (1994).

9. Whitmire,J.K., Tan,J.T. & Whitton,J.L. Interferon-gamma acts directly on CD8+ T

cells to increase their abundance during virus infection. J. Exp. Med. 201, 1053-

1059 (2005).

10. Steeg,P.S., Moore,R.N., Johnson,H.M. & Oppenheim,J.J. Regulation of murine

macrophage Ia antigen expression by a lymphokine with immune interferon

activity. J. Exp. Med. 156, 1780-1793 (1982).

11. Wadee,A.A., Kuschke,R.H. & Dooms,T.G. The inhibitory effects of

Mycobacterium tuberculosis on MHC class II expression by monocytes activated

with riminophenazines and phagocyte stimulants. Clin. Exp. Immunol. 100, 434-439

(1995).

12. Maudsley,D.J. & Morris,A.G. Regulation of IFN-gamma-induced host cell MHC

antigen expression by Kirsten MSV and MLV. II. Effects on class II antigen

expression. Immunology 67, 26-31 (1989).

13. Thomas,H.E., Parker,J.L., Schreiber,R.D. & Kay,T.W. IFN-gamma action on

pancreatic beta cells causes class I MHC upregulation but not diabetes. J. Clin.

Invest 102, 1249-1257 (1998).

211

14. Refaeli,Y., Van Parijs,L., Alexander,S.I. & Abbas,A.K. Interferon gamma is

required for activation-induced death of T lymphocytes. J. Exp. Med. 196, 999-

1005 (2002).

15. Maraskovsky,E., Chen,W.F. & Shortman,K. IL-2 and IFN-gamma are two

necessary lymphokines in the development of cytolytic T cells. J. Immunol. 143,

1210-1214 (1989).

16. Smith,P.M., Wolcott,R.M., Chervenak,R. & Jennings,S.R. Control of acute

cutaneous herpes simplex virus infection: T cell-mediated viral clearance is

dependent upon interferon-gamma (IFN-gamma). Virology 202, 76-88 (1994).

17. Bradley,L.M., Dalton,D.K. & Croft,M. A direct role for IFN-gamma in regulation

of Th1 cell development. J. Immunol. 157, 1350-1358 (1996).

18. Constant,S.L. & Bottomly,K. Induction of Th1 and Th2 CD4+ T cell responses: the

alternative approaches. Annu. Rev. Immunol. 15, 297-322 (1997).

19. Halford,W.P., Halford,K.J. & Pierce,A.T. Mathematical analysis demonstrates that

interferons-beta and -gamma interact in a multiplicative manner to disrupt herpes

simplex virus replication. J. Theor. Biol. 234, 439-454 (2005).

20. Karupiah,G., Fredrickson,T.N., Holmes,K.L., Khairallah,L.H. & Buller,R.M.

Importance of interferons in recovery from mousepox. J. Virol. 67, 4214-4226

(1993).

21. Katze,M.G., He,Y. & Gale,M., Jr. Viruses and interferon: a fight for supremacy.

Nat. Rev. Immunol. 2, 675-687 (2002).

22. Steed,A.L. et al. Gamma interferon blocks gammaherpesvirus reactivation from

latency. J. Virol. 80, 192-200 (2006).

23. Lee,S., Gierynska,M., Eo,S.K., Kuklin,N. & Rouse,B.T. Influence of DNA

encoding cytokines on systemic and mucosal immunity following genetic

vaccination against herpes simplex virus. Microbes. Infect. 5, 571-578 (2003).

24. Asanuma,H., Sharp,M., Maecker,H.T., Maino,V.C. & Arvin,A.M. Frequencies of

memory T cells specific for varicella-zoster virus, herpes simplex virus, and

cytomegalovirus by intracellular detection of cytokine expression. J. Infect. Dis.

181, 859-866 (2000).

25. Alarcon,J.B., Hartley,A.W., Harvey,N.G. & Mikszta,J.A. Preclinical evaluation of

microneedle technology for intradermal delivery of influenza vaccines. Clin.

Vaccine Immunol. 14, 375-381 (2007).

26. Ben Yedidia,T. & Arnon,R. Towards an epitope-based human vaccine for

influenza. Hum. Vaccin. 1, 95-101 (2005).

212

27. Giebink,G.S. Otitis media: the chinchilla model. Microb. Drug Resist. 5, 57-72

(1999).



29. Reid,A.H., Taubenberger,J.K. & Fanning,T.G. The 1918 Spanish influenza:

integrating history and biology. Microbes. Infect. 3, 81-87 (2001).

30. Roberts,A. & Subbarao,K. Animal models for SARS. Adv. Exp. Med. Biol. 581,

463-471 (2006).

31. Reid,A.H., Taubenberger,J.K. & Fanning,T.G. The 1918 Spanish influenza:

integrating history and biology. Microbes. Infect. 3, 81-87 (2001).



33. Jeevan,A. et al. Production and characterization of guinea pig recombinant gamma

interferon and its effect on macrophage activation. Infect. Immun. 74, 213-224

(2006).

34. Loa,C.C., Hsieh,M.K., Wu,C.C. & Lin,T.L. Molecular identification and

characterization of turkey IFN-gamma gene. Comp Biochem. Physiol B Biochem.

Mol. Biol. 130, 579-584 (2001).

35. Milev-Milovanovic,I. et al. Identification and expression analysis of interferon

gamma genes in channel catfish. Immunogenetics 58, 70-80 (2006).

36. Morar,D. et al. Cloning, sequencing and expression of white rhinoceros

(Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of

rhinoceros IFN-gamma specific antibodies. Vet. Immunol. Immunopathol. 115, 146-

154 (2007).

37. Svitek,N. & von,M., V. Early cytokine mRNA expression profiles predict

Morbillivirus disease outcome in ferrets. Virology 362, 404-410 (2007).

38. Maher,J.A. & DeStefano,J. The ferret: an animal model to study influenza virus.

Lab Anim (NY) 33, 50-53 (2004).

39. Farrar,M.A. & Schreiber,R.D. The molecular cell biology of interferon-gamma and

its receptor. Annu. Rev. Immunol. 11, 571-611 (1993).

40. Jeevan,A. et al. Production and characterization of guinea pig recombinant gamma

interferon and its effect on macrophage activation. Infect. Immun. 74, 213-224

(2006).

213

41. Kudo,S. & Kawano,K. Association and dissociation properties of natural human

interferon gamma. J. Chromatogr. B Biomed. Sci. Appl. 723, 25-30 (1999).

42. Fukuta,K. et al. Remodeling of sugar chain structures of human interferon-gamma.

Glycobiology 10, 421-430 (2000).

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