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Molecular characterisation of Epstein-Barr virus-specific B cell response Archana Panikkar Master of Science A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Medicine

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Page 1: Molecular characterisation of Epstein-Barr virus-specific ...357566/s... · Primary Epstein-Barr virus (EBV) infection causes infectious mononucleosis (IM) in 50% of infected adolescents

Molecular characterisation of Epstein-Barr virus-specific B cell response

Archana Panikkar

Master of Science

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015

School of Medicine

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ABSTRACT

Primary Epstein-Barr virus (EBV) infection causes infectious mononucleosis (IM) in 50% of

infected adolescents and young adults. It has long been known that IM is associated with

the aberrant expansion of EBV-specific CD8+ T cells and can cause rare but serious

complications, including airway obstruction, splenic rupture, and haemolytic anaemia. IM is

also associated with an increased incidence of EBV-associated Hodgkin’s Lymphoma and

autoimmune diseases like multiple sclerosis. Despite significant research, it is not clear

why some individuals are more likely than others to develop clinical symptoms. In seeking

an explanation, we studied immune control during the early stages of EBV infection, before

cell-mediated immune responses come into play. We hypothesized that there is a defect in

immune control during the early stages of EBV infection. EBV infection induces a strong

humoral immune response and the antibodies produced against EBV are used as

diagnostic markers in the detection of EBV infection. While considerable knowledge has

been accumulated on T cell response during acute IM, we have little understanding of

virus-specific B cell immunity during primary EBV infection. Cross-sectional and

longitudinal analyses of humoral immune responses in this project revealed that patients

with acute IM have a severe deficiency of anti-EBV neutralizing antibodies, which was

associated with a lack of gp350-specific B cell responses and a significant reduction in

total memory B cells in peripheral blood. Furthermore, these patients also showed

dysregulation of tumour necrosis factor (TNF)-family members BAFF and APRIL and

increased expression of FAS on circulating B cells. This impairment of anti-viral B cell

responses was coincident with a dramatic loss of circulating myeloid and plasmacytoid DC

numbers during acute IM, which recovered to baseline following convalescence. The loss

of plasmacytoid dendritic cells was found to be much greater than that of myeloid DCs,

with slower recovery upon disease resolution. This is consistent with earlier published

findings of an absence of IFN-αin the plasma of IM patients, since plasmacytoid dendritic

cells are the main producers of IFN-α A number of possible causes for the loss of blood

DCs were also explored in this project. Finally, using in vitro studies, we demonstrated that

the loss of circulating DCs is intrinsically linked to the impaired activation and maturation of

antibody-secreting B cells. These observations delineate new mechanistic insight into

immune dysregulation during acute IM and its impact on anti-viral B cell responses that will

help us to design better therapeutic/prophylactic strategies to prevent acute IM symptoms.

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Declaration by Author

This thesis is composed of my original work, and contains no material previously published

or written by another person except where due reference has been made in the text. I

have clearly stated the contribution by others to jointly-authored works that I have included

in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical

assistance, survey design, data analysis, significant technical procedures, professional

editorial advice, and any other original research work used or reported in my thesis. The

content of my thesis is the result of work I have carried out since the commencement of

my research higher degree candidature and does not include a substantial part of work

that has been submitted to qualify for the award of any other degree or diploma in any

university or other tertiary institution. I have clearly stated which parts of my thesis, if any,

have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University

Library and, subject to the policy and procedures of The University of Queensland, the

thesis be made available for research and study in accordance with the Copyright Act

1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the

copyright holder(s) of that material. Where appropriate, I have obtained copyright

permission from the copyright holder to reproduce material in this thesis.

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Publications During Candidature

No publications

Publications Included in This Thesis

No publications included

Contributions of Others to the Thesis

Professor Rajiv Khanna from QIMR Berghofer Medical Research Institute was principal

supervisor of the candidate and provided substantial input in the conception and design of

this project. Dr. Corey Smith of the QIMR Berghofer Medical Research Institute was

associate supervisor and had substantial input in project design and interpretation of

research data.

Statement of parts of the thesis submitted to qualify for the award of another degree

None

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ACKNOWLEDGEMENTS

I am grateful to the many individuals whose help, encouragement and support made

it possible for me to complete this Ph.D.

First and foremost, my thanks go to Professor Rajiv Khanna, who encouraged me to

apply to the program and supported me generously during my tenure here. Under his

guidance, I have learned about immunology research, a field that was unfamiliar to me.

Dr. Corey Smith, my immediate supervisor, has been infinitely patient with me,

always willing to take time to teach me, clear my doubts and help me learn new skills. I

thank him for his support.

I thank Professor Denise Doolan and Professor Scott Burrows for serving on my

Ph.D. review panel. I thank Dr. Judy Tellam and Dr. Michelle Neller for helping me improve

my thesis. Dr. Nick Tellam, Dr. Kimberly Jones, Ms. Melissa Rist, Dr. Siok Tey, Dr.

Stephen Byrn, and Dr. Hamish Alexander were of invaluable help in getting samples from

study subjects.

Linda Jones, Vijayendra Dasari, Emily Edwards and Andrea Schuessler, my lab

colleagues past and present, have been good friends as well as sources of inspiration and

support through these three years. They and the rest of the Tumour Immunology Lab

made it a great work environment. I will always have fond memories of them.

I am grateful to Professor Jaap Middeldorp and MedImmune for providing gp350

protein and to CSL for providing Tetanus toxoid.

My gratitude goes to Dr.Henry Balfour and Dr.Andrew Hislop, our collaborators, as

well as to all the donors whose generosity made this study possible.

I thank University of Queensland Graduate School, Australia Centre for Vaccine

Development and QIMR Higher Degree Committee for supporting me with scholarships

and top-ups during my time here.

My husband and my two sons have been there for me always. As has my mother,

whose prayers and words of encouragement gave me the fortitude to continue during

difficult times.

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KEYWORDS

epstein-barr virus, infectious mononucleosis, innate immunity, humoral immunity, memory

b cells, antibody-secreting cells, dendritic cells , gp350, t follicular helper cells

AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS

(ANZSRC)

ANZSRC code: 110705, Humoral Immunity and Immunochemistry, 60%

ANZSRC code: 110707, Innate Immunity, 20%

ANZSRC code: 110309, Infectious Diseases, 20%

FIELDS OF RESEARCH (FOR) CLASSIFICATION

FoR code: 1107, Immunology, 70%

FoR code: 1103, Clinical Sciences, 30%

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TABLE OF CONTENTS

ABSTRACT ........................................................................................................................... I Declaration by Author .................................................................................................... ii Publications During Candidature .................................................................................. iii Publications Included in This Thesis ............................................................................ iii Contributions of Others to the Thesis ........................................................................... iii Statement of parts of the thesis submitted to qualify for the award of another degree . iii

ACKNOWLEDGEMENTS ........................................................................................................ IV KEYWORDS ........................................................................................................................ V AUSTRALIAN AND NEW ZEALAND STANDARD RESEARCH CLASSIFICATIONS (ANZSRC) ............ V FIELDS OF RESEARCH (FOR) CLASSIFICATION ....................................................................... V TABLE OF CONTENTS ................................................................................................... VI

TABLE OF FIGURES ....................................................................................................... IX

LIST OF ABBREVIATIONS............................................................................................... X

CHAPTER 1. INTRODUCTION ......................................................................................... 1

1.1. EPSTEIN-BARR VIRUS - GENERAL INTRODUCTION ....................................................... 1 1.2. GENOME STRUCTURE .............................................................................................. 2 1.3. LIFE CYCLE .............................................................................................................. 2

1.3.1. Latent Cycle ..................................................................................................... 3

1.3.2. Latent Gene Expression ................................................................................... 4 1.3.3. Lytic Cycle ........................................................................................................ 6

1.4. EBV-ASSOCIATED DISEASES .................................................................................... 7 1.4.1. Infectious Mononucleosis (IM) .......................................................................... 7 1.4.2. X-Linked Lymphoproliferative Disorder (XLP) .................................................. 8

1.4.3. Oral Hairy Leukoplakia (OHL) .......................................................................... 9

1.4.4. Burkitt’s Lymphoma (BL) .................................................................................. 9

1.4.5. Nasopharyngeal Carcinoma (NPC) .................................................................. 9 1.4.6. Hodgkin’s Lymphoma (HL) ............................................................................... 9

1.4.7. Post-Transplant Lymphoproliferative Disorder (PTLD) ................................... 10 1.5. EBV INFECTION OF B CELLS ..................................................................................... 10 1.6. IMMUNE RESPONSE TO EBV .................................................................................... 12

1.6.1. Innate Immune Response .............................................................................. 12

1.6.2. Cell-Mediated Immune Response .................................................................. 13 1.6.3. Humoral Response to EBV Infection .............................................................. 16

1.7. VACCINE DEVELOPMENT .......................................................................................... 17 1.8. IMMUNE EVASION .................................................................................................... 18 1.9. MOLECULAR CHARACTERIZATION OF EBV-SPECIFIC B CELL RESPONSE ...................... 20

1.9.1. Project Background ........................................................................................ 20

1.9.2. Aims and Scope of Thesis.............................................................................. 26

1.9.3. Hypothesis ..................................................................................................... 27 1.9.4. Project Aims ................................................................................................... 27

CHAPTER 2. MATERIALS AND METHODS .................................................................. 28

2.1. LIST OF CHEMICALS ................................................................................................. 28 2.2. EQUIPMENT ............................................................................................................ 29

2.3. MEDIA AND BUFFERS ............................................................................................... 29 2.4. STUDY COHORT ...................................................................................................... 30 2.5. CELL CULTURE TECHNIQUES ................................................................................... 30

2.5.1. Ficoll-Paque Separation of PBMC From Whole Blood ................................... 30

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2.5.2. Cryopreservation and Thawing of PBMC ....................................................... 31 2.5.3. Serum Separation and Storage ...................................................................... 31

2.5.4. Plasma Separation and Storage .................................................................... 31

2.6. PREPARATION OF RECOMBINANT PROTEIN ................................................................ 31 2.6.1. Protein Purification ......................................................................................... 31 2.6.2. Dot Blot .......................................................................................................... 32

2.6.3. Protein Concentration Estimation – Bradford Assay ...................................... 32

2.7. IMMUNOLOGICAL ANALYSIS ...................................................................................... 33 2.7.1. List of Antibodies Used in This Study ............................................................. 33 2.7.2. Flow Cytometric Analysis ............................................................................... 33 2.7.3. ELISpot .......................................................................................................... 33

2.7.4. Enzyme Linked Immunosorbent Assay (ELISA) ............................................. 34 2.7.5. Cytometric Bead Array (CBA) analysis .......................................................... 35 2.7.6. Apoptosis Assay ............................................................................................. 35 2.7.7. Intracellular Cytokine Assay ........................................................................... 36

2.7.8. Magnetic Bead Separation ............................................................................. 36

2.8. STATISTICAL ANALYSIS ............................................................................................ 36

CHAPTER 3. KINETICS OF B CELL SUBSETS DURING PRIMARY AND PERSISTENT EBV INFECTION ...................................................................................... 37

3.1. ABSTRACT .............................................................................................................. 37 3.2. INTRODUCTION ........................................................................................................ 38 3.3. MATERIALS AND METHODS ....................................................................................... 40

3.3.1. Flow Cytometry Analysis of B Cells ................................................................ 40 3.3.2. Conversion of Memory B Cells to Antibody-Secreting Cells ........................... 40

3.3.3. B Cell ELISpot ................................................................................................ 41 3.3.4. Enzyme Linked Immunosorbent Assay (ELISA) ............................................. 41 3.3.5. Cytometric Bead Array (CBA) Analysis .......................................................... 42

3.3.6. Annexin V Staining ......................................................................................... 42 3.3.7. Cytokine Influence and FAS/FAS L Killing ..................................................... 43

3.3.8. Statistical Analysis ......................................................................................... 43

3.4. RESULTS ................................................................................................................ 44 3.4.1. Phenotype and Frequency of B Cell Subsets ................................................. 44 3.4.2. The Mechanism of Reduction of B Cell Frequency ........................................ 51 3.4.3. Influence of Cytokines and FAS-Mediated Killing ........................................... 55

3.5. DISCUSSION ........................................................................................................... 56

CHAPTER 4. GP350-SPECIFIC B CELL RESPONSE DURING ACUTE IM ............... 59

4.1. ABSTRACT .............................................................................................................. 59 4.2. INTRODUCTION ........................................................................................................ 60 4.3. MATERIALS AND METHODS ....................................................................................... 61

4.3.1. EBV MA-gp350 Protein Purification ............................................................... 61 4.3.2. Dot Blot .......................................................................................................... 62 4.3.3. SDS–PAGE and Western Blotting .................................................................. 62

4.3.4. Protein Concentration Estimation-Bradford Assay ......................................... 63 4.3.5. Endotoxin Test ............................................................................................... 63 4.3.6. B Cell ELISpot ................................................................................................ 64 4.3.7. Fluorolink Cy5 Labelling ................................................................................. 64 4.3.8. Biotin Labelling of gp350 ................................................................................ 65

4.3.9. gp350 Tetramer Preparation .......................................................................... 66 4.3.10. B Cell Labelling Using gp350 Tetramers ..................................................... 66 4.3.11. Enzyme Linked Immunosorbent Assay (ELISA) of gp350 .......................... 66

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4.3.12. Titration of EBV Virus Pool ......................................................................... 67 4.3.13. Neutralisation Assay ................................................................................... 67

4.3.14. Pepmix EBV ................................................................................................ 68 4.3.15. Flow Cytometry of CD4+ T Helper Cells ...................................................... 68 4.3.16. Antigen-Specific CD4+ T Cell Expansion and Functional Analysis .............. 68 4.3.17. Intracellular Cytokine Staining (ICS) and Cell Surface Marker Staining ...... 68

4.3.18. Cell Surface Marker Staining of IM Samples .............................................. 69

4.4. RESULTS ................................................................................................................ 69 4.4.1. EBV MA-gp350 Purification and Quantification .............................................. 69 4.4.2. Frequency of EBV-Specific Memory B Cells .................................................. 70 4.4.3. gp350-Antibody Concentration in Plasma ...................................................... 75

4.4.4. Quantity and Quality of Antibody Produced During Primary EBV Infection .... 77 4.4.5. Circulating Memory CD4 Helper T Cells......................................................... 80 4.4.6. Presence of gp350-Specific CD4+ T Cells ...................................................... 82

4.5. DISCUSSION ........................................................................................................... 85

CHAPTER 5. PERIPHERAL BLOOD DENDRITIC CELL (DC) DYNAMICS DURING ACUTE EBV INFECTION ................................................................................................ 87

5.1. ABSTRACT .............................................................................................................. 87

5.2. INTRODUCTION ........................................................................................................ 88 5.3. MATERIALS AND METHODS ....................................................................................... 90

5.3.1. Flow Cytometry .............................................................................................. 90 5.3.2. In Vitro DC Culture ......................................................................................... 90

5.3.3. In Vitro Infection ............................................................................................. 90 5.3.4. Apoptosis (Annexin V Binding Assay) ............................................................ 90

5.3.5. Cytokine Influence .......................................................................................... 91 5.3.6. Migration to Lymphoid Organs ....................................................................... 91 5.3.7. DC-B Cell Interaction ..................................................................................... 91

5.4. RESULTS ................................................................................................................ 92 5.4.1. Frequency & Phenotype of Blood Dendritic Cells during EBV Primary Infection 92 5.4.2. Migration to Lymphoid Organs ....................................................................... 96

5.4.3. Effect of Cytokines on the Survival of DCs ..................................................... 97 5.4.4. Requirement of Dendritic Cells for Plasmablast (ASC) Formation ................. 99

5.5. DISCUSSION ......................................................................................................... 103

CHAPTER 6. DISCUSSION AND FUTURE DIRECTION ............................................ 106

REFERENCES................................................................................................................ 112

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TABLE OF FIGURES Figure 1. 1. Germinal Center (GC) ................................................................................................ 23 Figure 1. 2. DC-B cell-T cell interaction ......................................................................................... 24

Figure 3. 1. Detection of circulating B cell subsets in ex vivo PBMC ............................................. 44 Figure 3. 2. Ex vivo characterization of total B cells in PBMC and whole blood ............................. 45 Figure 3. 3. Ex vivo characterization of B cell subsets in PBMC during EBV infection ......... 46 Figure 3. 4. Analysis of in vitro-activated PBMC ...................................................................... 47 Figure 3. 5. Frequency of ASCs, total IgG-secreting cells and correlation with severity of infection ....................................................................................................................................... 48 Figure 3. 6. Frequency of B regulatory cells during EBV infection ......................................... 50 Figure 3. 7. Effect of EBV infection on conversion of MBC to ASC ......................................... 51 Figure 3. 8. Expression of TNF receptor superfamily. BAFF/APRIL receptors, during EBV infection ....................................................................................................................................... 52 Figure 3. 9. Concentration of TNF-family members BAFF and its homolog APRIL ................ 53 Figure 3. 10. FAS expression on B cells and FAS L concentration in plasma ........................ 54 Figure 3. 11. Cytokine influence in the reduction of B cells .................................................... 55

Figure 4. 1. EBV-membrane antigen gp350 purification ................................................................ 70 Figure 4. 2. B cell ELISpot analysis to EBV-MA gp350 and total IgG ............................................ 71 Figure 4. 3. Frequency of EBV-specific memory B cells per 106 PBMC .................................. 72 Figure 4. 4. Frequency of EBV-specific memory B cells as a percentage of total IgG-specific memory B cells ........................................................................................................................... 73 Figure 4. 5. Absolute count of EBV-specific memory B cells per litre of blood ............................... 74 Figure 4. 6. Measurement of gp350-antibody titre ......................................................................... 75 Figure 4. 7. Correlation of frequency of gp350-specific memory B cells and antibody titre with severity of infection ....................................................................................................................... 76 Figure 4. 8. Conventional EBV virus-neutralisation assay ............................................................. 77 Figure 4. 9. Enrichment of gp350-specific memory B cells for generation of monoclonal antibodies. Structure of gp350 (Szakonyi, Klein et al. 2006) ........................................................................... 79 Figure 4. 10. CD4/CD8 ratio .......................................................................................................... 80 Figure 4. 11. Frequency and phenotype of CD4+ T cell subsets during acute IM........................... 81 Figure 4. 12. gp350-specific CD4+ T cell response during acute IM .............................................. 82 Figure 4. 13. gp350-specific CD8+ T cell response during acute IM .............................................. 83 Figure 4. 14. Characterisation of gp350-specific CD4/CD8 T cells during persistent EBV infection ....................................................................................................................................... 84

Figure 5. 1. Peripheral blood dendritic cells during EBV infection .................................................. 93 Figure 5. 2. Frequency of DC subsets at different stages of EBV infection .................................... 94 Figure 5. 3. Frequency of DC subsets in whole blood and correlation to severity of infection ........ 95 Figure 5. 4. Migration to lymphoid organs ..................................................................................... 96 Figure 5. 5. Effect of IM plasma on DC survival ............................................................................ 97 Figure 5. 6. Effect of cytokines in the survival of blood DCs ................................................... 98 Figure 5. 7. Dendritic cell depletion and its effect on B cells ........................................................ 100 Figure 5. 8. Phenotypic analysis of in vitro-activated PBMC with and without the presence of DCs ............................................................................................................................................ 101 Figure 5. 9. Requirement of dendritic cells for antibody-secreting cell formation ......................... 102

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LIST OF ABBREVIATIONS

AF 700 alexa fluor 700 ICS intracellular cytokine staining

AIM acute infectious mononucleosis IFN interferon

APC allophycocyanin IgG immunoglobulin G

APC antigen presenting cell IL interleukin

BAFF B cell activating factor IM infectious mononucleosis

BCMA B cell maturation antigen LCL lymphoblastoid cell line

BCR B cell receptor LMP latent membrane antigen (EBV)

BL Burkitt’s lymphoma mDC myeloid dendritic cell

BSA bovine serum albumin MFI mean fluorescence intensity

BV brilliant violet MHC major histocompatibility complex

CBA cytometric bead array NK natural killer

CD cluster of differentiation NPC nasopharyngeal carcinoma

CTL cytotoxic T lymphocyte PBS phosphate buffered saline

Cy7 cyanin 7 PBMC peripheral blood mononuclear cells

DC dendritic cell PD-1 programmed cell death-1

DMEM Dulbecco’s modified Eagle’s medium

pDC plasmacytoid dendritic cell

DMSO dimethyl sulphoxide PE phycoerythrin

EBNA EBV nuclear antigen PE-Cy7 phycoerythrin-cyanin 7

EBV Epstein-Barr virus PerCp peridinin chlorophyll protein

EDTA ethylene diamine tetra-acetic acid

PerCp Cy5.5

peridinin chlorophyll protein cyanin 5.5

ELISA enzyme-linked immunosorbent assay

PWA Pokeweed mitogen

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ELISpot enzyme-linked immunospot assay

RCF/g relative centrifugal force

FACS fluorescence-activated cell sorting

RPMI-1640

Roswell Park Memorial Institute medium

FCS foetal calf serum RPM revolutions per minute

FITC fluorescein isothiocyanate RT room temperature

gp350 Epstein-Barr virus membrane antigen glycoprotein 350

SEM standard error of mean

GC Germinal centre TACI transmembrane activator and calcium-modulator and cyclophilin ligand

HIV human immunodeficiency virus TFH cell T follicular helper cell

HL Hodgkin’s lymphoma TNF tumour necrosis factor

HLA human leukocyte antigen VCA viral capsid antigen (EBV)

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CHAPTER 1. INTRODUCTION

1.1. EPSTEIN-BARR VIRUS – GENERAL INTRODUCTION

Herpesviruses are large DNA viruses with 100—200 kb genome size. There are at

least 25 viruses in the herpesviridae family, of which eight are known to infect humans and

establish life-long persistence in their hosts (Murphy 1995). However, while an estimated

90% of humans carry multiple types of herpesvirus from childhood, not everyone shows

signs of infection. Herpesviruses are notorious for the many mechanisms they employ to

evade the immune system (Hill, Jugovic et al. 1995, Ahn, Angulo et al. 1996, Abendroth,

Slobedman et al. 2000). The virus may remain latent for long periods of time, causing

infection only occasionally. Infection is usually mild in immunocompetent individuals, but

can be severe in individuals whose immune systems are compromised (Babcock, Decker

et al. 1999).

Herpesviridae are divided into three families—alpha, beta and gamma (Roizman,

Carmichael et al. 1981). Alpha herpesviruses are neurotropic viruses. They include herpes

simplex virus types I and II (HSV-I and HSV-II) and varicella-zoster virus. Beta

herpesviruses include human herpesvirus types 6 and 7 (HHV-6 and HHV-7) and

cytomegalovirus, which has the largest genome among herpesviruses. The gamma

herpesvirus family is lymphotropic and includes Epstein-Barr virus (EBV), also called

human herpes virus 4 (HHV4), and Kaposi’s sarcoma-associated herpesvirus (KSHV or

HHV8). These, in turn, are divided into lymphocryptovirus and rhadinoviruses. EBV is a

member of the lymphocryptovirus family.

Denis Burkitt, a surgeon working in Africa in the 1950s, was instrumental in the

discovery of EBV. He studied a cancer of the jaw that affects children in East Africa

(Burkitt 1958), which subsequently came to be named Burkitt’s lymphoma (BL). EBV was

isolated and documented in 1964 by Anthony Epstein and colleagues using cell lines

derived from BL (Epstein, Achong et al. 1964). EBV was the first identified case of a

human tumour virus (de-Thé, Geser et al. 1978). Subsequently, EBV has been associated

with a number of human non-malignant and malignant diseases of both lymphoid and

epithelial origin (Epstein, Achong et al. 1964, Wolf, zur Hausen et al. 1973, Rickinson,

Jarvis et al. 1975, Weiss, Movahed et al. 1989).

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1.2. GENOME STRUCTURE

Like all herpesviruses, EBV is an enveloped icosahedral virus. It has a protein core

with double stranded DNA surrounded by a nucleocapsid with 162 capsomers, the protein

tegument, and an outer envelope with glycoprotein spikes (Epstein, Barr et al. 1965). EBV

was the first herpesvirus to be sequenced. The complete DNA sequence of the EBV B95-8

strain was first published in 1984 (Baer, Bankier et al. 1984). A reference sequence for the

wild type EBV genome was created using the sequence from two viral genomes, the B95-

8 and Raji strains of EBV (de Jesus, Smith et al. 2003). The genome is composed of linear

double-stranded DNA of about 172kb. It includes highly repetitive terminal repeats, as well

as internal repeats that divide the genome into short and long sequence domains. The

genes are known according to their position on a BamH1 restriction endonuclease map of

the genome (Cheung and Kieff 1982).

Different strains of EBV have been identified, with variations at the DNA level

(Bornkamm, Delius et al. 1980). The main strains of EBV are broadly classified under two

distinct types, type I and type II, also known as type A and type B. They show sequence

homology, except for changes in DNA sequence of mostly latent genes (Sample, Young et

al. 1990). EBV type I is more common in Western societies and both types are found

equally in the African population (Apolloni and Sculley 1994). A clinical study based on

university students in the UK found that infection with type I strain of EBV is more likely to

cause infectious mononucleosis (IM), a clinical manifestation of EBV primary infection

(Crawford, Macsween et al. 2006). Multiple strains of EBV have also been detected in IM

patients (Tierney, Edwards et al. 2006).

1.3. LIFE CYCLE

EBV is usually transmitted orally, through saliva, and establishes infection within the

oropharynx. Although early studies indicated that primary infection occurs in epithelial

cells, more recent studies have suggested that circulating B cells in the oropharynx, and

not epithelial cells, are the primary site of infection (Allday and Crawford 1988, Niedobitek,

Agathanggelou et al. 1997). Absence of EBV infection in B cell-deficient patients, as well

as the requirement that B cells be in co-culture with epithelial cells in order for efficient

infection of epithelial cells in vitro, proves that EBV has a clear preference for B-

lymphocytes (Gratama, Oosterveer et al. 1988, Faulkner, Burrows et al. 1999, Faulkner,

Krajewski et al. 2000). Preferential binding to B cells is mediated through the interaction of

the major viral envelope glycoprotein gp350 with complement receptor CD21 on the B cell

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surface (Fingeroth, Weis et al. 1984). Janz and colleagues showed that even though

gp350 is not absolutely necessary for EBV infection, it is required for efficient EBV

infection of B cells (Janz, Oezel et al. 2000).

Although B cells remain the preferred site of EBV infection, the presence of

replicating virus in epithelial cells suggests that epithelial cells, too, play an important role

in EBV infection and transmission. Shannon-Lowe and colleagues showed that EBV is

transferred from the surface of infected B cells to epithelial cells (Shannon-Lowe, Neuhierl

et al. 2006, Shannon-Lowe and Rowe 2011). In this way, EBV can infect both the lymphoid

and epithelial compartments simultaneously.

There are two distinct stages in the EBV life cycle, the latent and lytic cycles. In B

cells, infection is predominantly latent, whereas in oropharyngeal epithelial cells the

infection is mostly lytic (Greenspan, Greenspan et al. 1985). Latency in epithelial cells is

found in EBV-positive epithelial tumour cells (Raab-Traub, Hood et al. 1983, Brooks, Yao

et al. 1992). In latent infection, only a limited number of genes are expressed that are

required for maintenance, proliferation and survival. In the lytic cycle, viral DNA is

replicated and mature virions are produced, which are then released to infect new cells

(Wang and Hutt-Fletcher 1998, Laichalk and Thorley-Lawson 2005).

1.3.1. Latent Cycle

The EBV viral genome resides as an episome in EBV-infected B cells (Decker,

Klaman et al. 1996). The episome, which is circularized EBV DNA, does not integrate into

the host genome. In vitro generated EBV-transformed lymphoblastoid cell lines (LCLs) are

similar to EBV-transformed B cells in vivo and proliferate indefinitely in cell culture (Pope,

Horne et al. 1968). These LCLs are used to study EBV-encoded genes expressed during

latent infection of B lymphocytes. EBV-immortalized B-lymphoblasts express nine virus-

encoded latent antigens, including six nuclear antigens (EBNA 1–6), three latent

membrane proteins (LMP1, 2A and 2B), two non-coding RNAs (EBERs) (Lerner, Andrews

et al. 1981, Hennessy, Fennewald et al. 1985, Dillner, Kallin et al. 1986, Longnecker and

Kieff 1990) and non-translated viral miRNAs (Pfeffer, Sewer et al. 2005). Primary infection

is followed by the expression of EBNA2 and EBNA5 (ENBA-LP). The other nuclear

antigens, membrane proteins and RNAs are expressed soon afterwards (Alfieri,

Birkenbach et al. 1991).

Three types of latency are found in B cells in vivo (Qu and Rowe 1992, Tierney,

Steven et al. 1994, Joseph, Babcock et al. 2000). Depending on the type of infected cells,

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the virus uses different expression patterns for each latency programmes (Babcock,

Hochberg et al. 2000):

Latency programme: This refers to either Latency 0, where the virus persists in

circulating memory B cells with no viral gene expression, or Latency I, where transient

expression of LMP2A, alone or together with the expression of EBNA1, is seen. The

expression of these viral proteins is necessary for maintaining persistent infection of

EBV in memory B cells. The latency programme in which EBNA1 is transcribed from

the Qp promoter (Nonkwelo, Skinner et al. 1996) is found in EBV-positive BL (Rowe,

Rowe et al. 1987, Chen, Zou et al. 1995, Bell, Groves et al. 2006).

Default or Latency II programme: EBNA1, along with latent membrane proteins LMP1

and 2, is expressed from the Qp promoter. Latency II gene expression can be found in

nasopharyngeal carcinoma (NPC), an epithelial carcinoma, and Hodgkin’s lymphoma

(HL) (Fåhraeus, Fu et al. 1988, Brooks, Yao et al. 1992, Deacon, Pallesen et al. 1993).

Growth programme: Also known as the latency III and this expression pattern is seen

in LCLs in vitro, latency III expresses all of the latent EBV genes. Expression is

initiated from the Wp promoter following infection and later from the Cp promoter

(Woisetschlaeger, Yandava et al. 1990, Shannon-Lowe, Adland et al. 2009). Latency

III infection can be found in IM and post-transplant lymphoproliferative disorder

(PTLD).

1.3.2. Latent Gene Expression

EBNA1 is a multifunctional DNA-binding viral protein, which binds to the latent origin

of replication (oriP) on the viral episome (Yates, Warren et al. 1985). This allows EBNA1 to

control replication of the viral DNA during cell division (Sample, Brooks et al. 1991,

Nonkwelo, Skinner et al. 1996). EBNA1 is the only viral protein that is expressed in all

three latency stages and continues to express when cells undergo lytic infection (Hsiao,

Lin et al. 2006, Malik-Soni and Frappier 2012, Sivachandran, Wang et al. 2012, Pinzon-

Charry, Woodberry et al. 2013). It is required for the efficient growth transformation of

primary B cells and for transactivation from the Cp viral promoter (Speck, Pfitzner et al.

1986). EBNA1 ensures the maintenance of the EBV episome in order to establish latency

(Rawlins, Milman et al. 1985). The absence of EBNA1 in an EBV mutant has been shown

to result in integration of the complete viral genome into the host genome (Humme,

Reisbach et al. 2003).

EBNA2 is one of the first nuclear antigens to be expressed after B cells are infected,

and it is essential for cellular transformation (Rooney, Howe et al. 1989, Alfieri, Birkenbach

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et al. 1991). EBNA2 is a transcriptional activator of several viral genes, including LMP1

and LMP2, as well as cellular genes CD23 and CD21 that are B cell antigens (Abbot,

Rowe et al. 1990). EBNA2 differs more extensively between type1 and type 2 strains of

EBV (Aitken, Sengupta et al. 1994, Cancian, Bosshard et al. 2011). EBNA5 is one of the

first nuclear antigens expressed, along with EBNA2, following infection (Alfieri, Birkenbach

et al. 1991). It enhances EBNA2-dependent transcription (Harada and Kieff 1997, Peng,

Zhao et al. 2004). EBNA5 and EBNA2 transcripts are encoded from the same primary

transcript, differing only in their second exon. The transcript also includes other EBNA

proteins (Dillner, Kallin et al. 1986, Rogers, Woisetschlaeger et al. 1990).

EBNA3A, 3B and 3C or 3, 4 and 6 are encoded by three adjacent genes and have

similar genomic organization (Sample, Young et al. 1990). EBNA3A and 3C are found to

be essential for virus-mediated growth transformation (Tomkinson, Robertson et al. 1993).

They may also be involved in transcriptional regulation, checkpoint control, LCL

proliferation, protein folding and cell cycle regulation (Robertson, Grossman et al. 1995,

Krauer, Kienzle et al. 1996, Parker, Touitou et al. 2000, Anderton, Yee et al. 2008).

LMP1 is essential for B cell transformation and it is involved in EBV-associated

malignancies (Kaye, Izumi et al. 1993, Cader, Vockerodt et al. 2013). LMP1 prevents

apoptosis by activating the NF-B pathway, which contributes to high-level expression of

anti-apoptotic proteins like Bcl-2, Bcl-XL, Bfl-1 and Mcl-1 (Finke, Fritzen et al. 1992, Wang,

Rowe et al. 1996, D'Souza, Edelstein et al. 2004). LMP1 is a homolog of CD40, which is a

B cell-activating surface molecule (Gires, Zimber-Strobl et al. 1997, Rastelli, Hömig-Hölzel

et al. 2008). Cells infected with EBV express LMP1 along with LMP2A, which can mimic B

cell receptor (BCR) signalling, and together they provide the necessary survival signal for

EBV-infected B cells to progress into the long-lived memory B cell population (Caldwell,

Wilson et al. 1998, Uchida, Yasui et al. 1999).

LMP2A and 2B are transcribed from the same strand by a bidirectional promoter

(Laux, Perricaudet et al. 1988). LMP2A blocks BCR signalling by latently-infected B cells

and induction of lytic EBV infection, thus promoting the survival of EBV-infected B cells. It

also mimics the presence of BCR and provides signals for B cell survival (Caldwell, Wilson

et al. 1998, Kilger, Kieser et al. 1998). LMP2A expression is consistently detected in EBV-

associated malignancies (Brooks, Yao et al. 1992, Busson, McCoy et al. 1992, Stewart,

Dawson et al. 2004). The specific function of LMP2B is not known, but recent studies have

reported that LMP2B can modulate the effect of both LMP1 and LMP2A (Rivailler, Quink et

al. 1999, Rovedo and Longnecker 2007, Rechsteiner, Berger et al. 2008).

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EBV encodes two non-coding RNAs—EBER1 and EBER2—which are abundantly

expressed in latently-infected cells and constitute a reliable marker for the detection of

EBV infection (Swaminathan, Tomkinson et al. 1991, Chang, Chen et al. 1992). EBERs

are not expressed during lytic infection (Greifenegger, Jager et al. 1998).They have been

shown to induce the production of IL-10, which stimulates B cell growth and suppresses

cytotoxic T lymphocytes (CTLs) (Kitagawa, Goto et al. 2000). EBERs provide resistance to

interferon-(IFN-)-induced apoptosis and are involved in the maintenance of

oncogenesis in BL cells (Komano, Maruo et al. 1999, Ruf, Lackey et al. 2005). Conversely,

EBER released from EBV-infected B cells also has been found to contribute to the

immunopathology during EBV infection by activating immune response and inducing pro-

inflammatory cytokines (Iwakiri, Zhou et al. 2009).

EBV encodes 44 mature microRNAs. They belong to two gene clusters, BHRF1

(miR-BHRF1) and BART (miR-BART) (Dölken, Malterer et al. 2010). BHRF1 miRNA is

expressed during latency III, and BART miRNA is expressed at low levels during latent

and lytic infections (Pfeffer, Sewer et al. 2005, Amoroso, Fitzsimmons et al. 2011). Recent

studies have shown that miRNAs are expressed at a higher level during the initial stages

of infection, which suggests that miRNA is involved during initial infection by EBV (Seto,

Moosmann et al. 2010). miRNAs inhibit apoptosis in newly-infected B cells and help

maintain BL cells in the absence of other viral gene expression (Vereide, Seto et al. 2014).

1.3.3. Lytic Cycle

All types of latency can be activated into a productive lytic cycle by the expression

of the BZLF1 gene, which acts as a switch between the lytic and latent cycles (Sinclair

2006). During the lytic cycle, about 80 genes are expressed that support replication of viral

DNA and synthesis of viral proteins (Pearson 1986). Lytic proteins are divided into

immediate early (IE), early (E) and late antigens (L). The IE genes, encoding the BZLF1

and BRLF1 proteins, are transcribed following infection, even when inhibitors of protein

synthesis are present (Biggin, Bodescot et al. 1987, Amon, Binne et al. 2004). IE proteins

are involved in transcriptional regulation, but they are not found in the mature virion.

BZLF1 acts as a switch to activate the lytic cycle, while BRLF1 is involved in the

expression of viral lytic genes (Feederle, Kost et al. 2000, Sinclair 2006). IE proteins also

are involved in the control of early protein synthesis. IE proteins regulate the expression of

E proteins BMLF1 and BMRF1, which may transactivate other E genes (Quinlivan, Holley-

Guthrie et al. 1993). The E proteins are involved in DNA replication and are divided into

EA-R (restricted) and EA-D (diffused) (Henle, Henle et al. 1970). Late proteins are the

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virus’s structural components and required for the packaging of viral DNA. They are the

viral capsid antigen (BNRF1) and the membrane antigen complex, the BLLF1(gp350),

BALF4 (gp110), BXLF2 (gH), BKRF2 (gL), BZLF2 (gp42) and BDLF3 (gp150) (Hummel,

Thorley-Lawson et al. 1984, Gong, Ooka et al. 1987, Heineman, Gong et al. 1988,

Mackett, Conway et al. 1990, Nolan and Morgan 1995, Johannsen, Luftig et al. 2004).

During the late stage, EBV episomes are replicated into linear concatemers which are then

cleaved and packaged into infectious virions (Gong and Kieff 1990, Papworth, Van Dijk et

al. 1997).

1.4. EBV-ASSOCIATED DISEASES

EBV is the causative agent of BL in Africa, NPC in China, and IM and HL in

Western societies. It has also been linked to PTLD, oral hairy leukoplakia, X-linked

lymphoproliferative syndrome, gastric carcinoma, NK/T cell lymphoma, some T cell

lymphomas, and carcinoma of the salivary glands.

1.4.1. Infectious Mononucleosis (IM)

IM was given the name glandular fever in the late 1800s. It was renamed Infectious

Mononucleosis in 1920 by Sprunt and Evans after they discovered the atypical

lymphocytes that are associated with this disease (Sprunt and Evans 1920). The causative

agent, EBV, was discovered in 1964 and the association between EBV and IM was first

made in1968 by Gertrude and Werner Henle (Henle, Henle et al. 1968).

Primary EBV infection often occurs within the first few years of life and stays

asymptomatic. IM is the result of delayed infection by EBV. In adolescents, about 50% of

those who contract EBV develop symptoms of IM (Pereira, Blake et al. 1969, Fleisher,

Henle et al. 1979). The characteristic symptoms are fever, lymphadenopathy and

pharyngitis. Additional symptoms include sore throat, headache, nausea, fatigue and

abdominal pain (Straus, Cohen et al. 1993). Although seen most commonly in young

adults, IM may sometimes manifest in children or older adults. In children, the symptoms

are not distinguished from many other childhood diseases, while in older adults the

symptoms are different from the typical disease (Horwitz, Henle et al. 1976, Kanegane,

Kanegane et al. 1997, Chan, Chiang et al. 2003, Cheng, Chang et al. 2007). Older adults

have prolonged fever and liver abnormalities without pharyngitis or lymphadenopathy

(Auwaerter 1999).

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The atypical lymphocytes commonly seen with IM are CD8+ T cells that are mostly

directed against lytic EBV antigens. The vigorous immune response by T cells contributes

to the symptoms of IM but at the same time is thought to be responsible for eliminating

most EBV-infected cells (Hislop, Annels et al. 2002). Confirmation of the clinical symptoms

of IM is carried out with serological testing, either for heterophile antibodies or specific

EBV antibodies (Sumaya and Ench 1985). IM patients show high titres of IgM and IgG

antibodies for viral capsid antigen (VCA) and EA. Antibodies for EBNA1 develop late after

EBV infection and remain at a constant detectable titre for life. IgG for VCA also persists

for life (Henle, Henle et al. 1974) . EBV is shed in oral secretions continually and at a high

concentration for months after acute infection (Fafi-Kremer, Morand et al. 2005). However,

the amount of virus shed is found to be unrelated to the severity of the illness (Balfour,

Holman et al. 2005). A study of IM during convalescence and the establishment of

persistent infection revealed that the majority of patients carry multiple strains of EBV

(Tierney, Edwards et al. 2006) that were found in three distinct compartments – the oral

cavity, the peripheral blood lymphocytes, and the cell-free fraction of blood plasma (Sitki-

Green, Edwards et al. 2004).

Clinical Complications during IM

Although complications during IM are not common among healthy individuals, some

have been reported. Splenic hemorrhage or splenic rupture, commonly related to trauma,

has been reported during the second week of infection (Smith and Custer 1946, Rutkow

1978, Maki and Reich 1982). Tonsillar swelling, if substantial, may cause upper airway

obstruction (Chan and Dawes 2001). Fulminant or fatal IM, hemolytic anemia and some

neurologic conditions also have been reported (Jenson 2000, Cohen 2003).

1.4.2. X-Linked Lymphoproliferative Disorder (XLP)

XLP is a rare, inherited, immunodeficiency disease that affects young males

(Sullivan, Byron et al. 1983, Grierson and Purtilo 1987). It is a fatal form of IM, in which the

immune system is unable to control primary EBV infection, even while it is able to resolve

other common viral infections like influenza. The defective gene is SH2D1A, which is an

important regulator of T and NK cells (Sayos, Wu et al. 1998). This defect results in

uncontrolled T cell activation and cytokine release in response to EBV infection, which

leads to extensive organ damage throughout the body. More than 50% of XLP patients

die, while the longevity of survivors is not known (Seemayer, Gross et al. 1995).

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1.4.3. Oral Hairy Leukoplakia (OHL)

OHL is a nonmalignant lesion of the tongue epithelium. It is an opportunistic

infection found in immunocompromised AIDS patients (Lau, Middeldorp et al. 1993). This

disease is an example in which the lytic cycle of EBV can be seen in the epithelial cells

localized to the sides of the tongue (Greenspan, Greenspan et al. 1985).

1.4.4. Burkitt’s Lymphoma (BL)

Endemic BL is a cancer that affects children in equatorial Africa and New Guinea

where there is high prevalence of malaria. This is usually a tumour in the jaw and shows

evidence of EBV DNA. Patients have a high titre of EBV-specific antibodies (Geser, Lenoir

et al. 1983). Sporadic BL is common in South America and North Africa and is mostly an

abdominal tumour. The tumour cells show a very characteristic translocation between

chromosomes 8 and 14 in Ig heavy chain locus and in some cases on chromosomes 2 or

22 on Ig light chain locus. This brings the c-myc gene from its usual regulatory position to

the constitutively expressed immunoglobulin (Ig) gene in B cells, which results in elevated

expression of the oncogene c-myc (Bernheim, Berger et al. 1983, Li, Van Calcar et al.

2003).

1.4.5. Nasopharyngeal Carcinoma (NPC)

NPC is a tumour of the epithelium and is found mostly in southern China and

Southeast Asia and less commonly in Northern Africa (Pagano, Huang et al. 1975,

Shanmugaratnam 1978). High titres of EBV-specific antibodies are found in the serum of

NPC patients (Henle and Henle 1976). NPC tumour cells express latency II antigens

EBNA1, LMP1 and LMP2 (Fåhraeus, Fu et al. 1988). It has been found that, besides EBV,

genetic and environmental factors contribute to the development of NPC (Ho, Ng et al.

1978, Armstrong, Armstrong et al. 1983).

1.4.6. Hodgkin’s Lymphoma (HL)

HL is most common in Western societies, where 40—50% of cases are positive for

EBV (Jarrett, Gallagher et al. 1991). The tumour shows prevalence in the age group of 25

to 30 (Gutensohn and Cole 1980). Studies have shown a three-fold to four-fold increase in

likelihood for individuals with a history of IM to also develop HL (Hjalgrim, Askling et al.

2000). HL and IM are characterized by the presence of mononuclear Hodgkin and

multinuclear Reed-Sternberg (HRS) cells. In HL the latency II program is observed

(Pallesen, Hamilton-Dutoit et al. 1991).

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1.4.7. Post-Transplant Lymphoproliferative Disorder (PTLD)

PTLD is a potentially fatal complication due to immunosuppression in transplant

patients (Crawford, Thomas et al. 1980). Polyclonal B cell lymphomas arise in individuals

with impaired T cell responses, resulting in uncontrolled proliferation of EBV-infected cells

(Babcock, Decker et al. 1999). EBV seronegative transplant recipients, especially pediatric

recipients, are more at risk of developing PTLD. Different manifestations of PTLD exist,

depending on factors including type of transplant and age. Latency III or growth program is

expressed during PTLD (Thomas, Hotchin et al. 1990).

1.5. EBV INFECTION OF B CELLS

Three patterns of latency are established after EBV infects B cells, based on the

type of cells infected in vivo. It also results in the transformation of B cells into

lymphoblasts in vitro (Babcock, Hochberg et al. 2000). EBV preferentially targets resting

memory B cells for latency and persists as an episome, circular virus DNA in the nuclei of

infected cells, thereby establishing lifelong persistence (Decker, Klaman et al. 1996,

Thorley-Lawson and Gross 2004).

Research on EBV infection and the mechanisms by which it activates B cells is

essential for understanding the virus and its associated diseases. Most EBV infections are

considered non-pathologic threats and they stay undetected by the host immune system

(Babcock, Hochberg et al. 2000). After infection, EBV-infected cells follow the normal B

cell development pathway. The virus activates the proliferation of newly-infected B cells by

expressing the latency III program, enters the germinal centre (GC) using the more

restricted latency II program, and persists in long-lived memory B cells by expressing the

latency I program (Thorley-Lawson and Babcock 1999, Souza, Stollar et al. 2005). In

some cases, EBV-infected GC B cells are found in the B cell lymphomas of HL, PTLD and

BL (Küppers, Klein et al. 1999).

GC B cells accumulate mutations during the process of somatic hypermutation

(SHM) and Ig class switching (CSR), which are required for the formation of a B cell with

high affinity BCR. The mutations are caused by genomic instability during double-stranded

DNA break required for SHM and CSR (Pasqualucci, Migliazza et al. 1998). These non-

functional B cells are usually eliminated in the GC by apoptosis (Lam, Kühn et al. 1997).

However, B cells carrying defective BCR in the malignant cells of HL, PTLD and BL are

not eliminated (Kanzler, Hansmann et al. 1996). Lymphoma cells in most of these

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malignancies are believed to be derived from these defective GC B cells that are rescued

from apoptosis (Brauninger, Spieker et al. 2003, Timms, Bell et al. 2003). EBV infection

has been found to be a likely event that rescues these pro-apoptotic B cells, since EBV-

positive cases of lymphoma show the accumulation of these GC B cells with harmful

mutations (Bechtel, Kurth et al. 2005). Studies from different groups have shown that, in

vitro, BCR-deficient GC B cells may be saved from apoptosis by EBV infection (Chaganti,

Bell et al. 2005, Mancao, Altmann et al. 2005).

EBV latent membrane proteins LMP1 and LMP2A, which can mimic CD40 and BCR

respectively, are thought to be responsible for the rescue of defective B cells both in vivo

and in vitro (Lalmanach-Girard, Chiles et al. 1993, Gires, Zimber-Strobl et al. 1997). Since

these two proteins are expressed in the later stages of EBV infection, two additional viral

proteins (BHRF1 and BALF1)—homologs of anti-apoptotic BCL-2—were found to be

transiently expressed in newly-infected B cells (Altmann and Hammerschmidt 2005). Both

LMP1 and LMP2A are expressed in EBV-positive HL and PTLD and could play a role in

lymphomagenesis. Conversely, a characteristic feature of BL is the translocation of the

proto-oncogene myc from its regulated position to the constitutively active immunoglobulin

position in B cells. The translocation event is suspected to occur during the Ig class switch

recombination, which is a destabilizing cellular event that occurs in GC B cells (Goossens,

Klein et al. 1998).

Analysis of EBV-infected cells from the tonsils of IM patients showed that EBV uses

two different strategies to spread in the B cell compartment, namely direct infection of

different subsets of B cells and clonal expansion of GC or memory B cells (Kurth, Spieker

et al. 2000). During IM, around 1% of peripheral B cells can be infected by EBV (Klein,

Svedmyr et al. 1976, Hochberg, Souza et al. 2004). The normal architecture of tonsillar

tissue is disrupted by proliferating EBV-infected B cells and cytotoxic T cells (CTLs) that

are responding to the virus (Anagnostopoulos, Hummel et al. 1995, Niedobitek,

Agathanggelou et al. 1997). It has also been reported that during IM EBV-positive B cells

undergo clonal expansion, without somatic hypermutation as in a normal GC (Kurth,

Spieker et al. 2000). Thus, EBV-infected B cells located in GC do not follow a normal GC

reaction to generate memory B cells during IM (Kurth, Hansmann et al. 2003). A study led

by Siemer found that EBV infection of B cells causes dysregulation of surface marker

expression required for the identity of different B cell subsets. Thus, EBV infection seems

to influence various factors and process of B cell differentiation (Siemer, Kurth et al. 2008).

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1.6. IMMUNE RESPONSE TO EBV

The immune system’s response to EBV is usually successful in limiting the primary

infection and controlling the lifelong latency state of the virus, which is why in most

instances the infection is asymptomatic. That is, immune control of EBV results in a

balance where host and virus coexist without disease in most carriers. Different branches

of immune response coordinate to achieve control at different points in the life cycle of

EBV. Host immune response is initiated by the innate immune system when it comes in

contact with the virus. Innate immunity is primarily mediated by dendritic cells (DCs) and

natural killer (NK) cells (Biron, Nguyen et al. 1999, Mocikat, Braumüller et al. 2003), with

macrophages, neutrophils, NKT cells and γδ T cells also playing a role (De Paoli, Gennari

et al. 1990, Martorelli, Muraro et al. 2012, Chung, Tsai et al. 2013).

1.6.1. Innate Immune Response

Dendritic cells are professional, antigen-presenting cells that are involved in the

innate activation of NK cells and in the induction of the adaptive immune response

(Lanzavecchia and Sallusto 2001, Ferlazzo, Pack et al. 2004). They accomplish this by

cross-presenting EBV antigens, which results in the priming of CD8+ and CD4+ T cells

(Bickham, Goodman et al. 2003). DCs are involved, directly and indirectly, in the

development of the humoral immune response by presenting antigens, providing

cytokines, and activating CD4+ T follicular helper cells (Wykes, Pombo et al. 1998,

Litinskiy, Nardelli et al. 2002, Cucak, Yrlid et al. 2009). There are two main subsets of

blood DCs, namely myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) (Romani, Gruner

et al. 1994, Grouard, Rissoan et al. 1997). EBV can infect subsets of DCs and monocytes,

and EBV infection prevents the conversion of monocytes to DCs (Li, Liu et al. 2002). It

also has been reported that EBV infection induces IFN- production by pDCs in in vitro

culture and in the humanized NOD-SCID mouse model (Lim, Kireta et al. 2007, Quan,

Roman et al. 2010). Furthermore, depletion of pDCs promotes EBV infection and

associated diseases in the humanized NOD-SCID mouse model, while enrichment of

pDCs results in better control of EBV infection (Lim, Kireta et al. 2007).

NK cells are able to reduce EBV transformation of B cells both in vitro and in vivo

(Thorley-Lawson, Chess et al. 1977, Baiocchi, Ward et al. 2001). Studies have also

reported a correlation between NK cell numbers, EBV viral load, and severity of symptoms

during IM (Williams, McAulay et al. 2005, Balfour, Odumade et al. 2013). A defect in NK

cell response during early EBV infection may contribute to the inability of individuals with

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X-linked lymphoproliferative disease (XLP) to control EBV infection (Parolini, Bottino et al.

2000). XLP patients also lack invariant NKT cells, which suggest that this subset of innate

immune cells could have a role in the early control of EBV infection (Chung, Tsai et al.

2013).

Among other immune cells, while macrophages play a role in clearing the virus

EBV-infected macrophages have reduced phagocytic ability (Lin and Li 2009). Neutrophils

also provide innate immunity to EBV infection as there is an increase in blood neutrophils

during initial infection and a transient neutropenia is observed in IM patients. The

interaction between EBV and neutrophils produces an opposing influence, with EBV

infection causing increased apoptosis of neutrophils while also causing enhanced antiviral

activity like respiratory burst and production of cytokines (Savard and Gosselin 2006).The

γδ T cell population expands during acute IM and remains elevated during convalescence,

which suggests a function in the control of primary EBV infection (De Paoli, Gennari et al.

1990).

1.6.2. Cell-Mediated Immune Response

The more common occurrence of EBV-associated diseases in T cell-

immunocompromised patients suggests that cell-mediated immune response is important

for the control of EBV infection (Khanna, Bell et al. 1999). The first evidence of virus-

specific T cell memory was provided by in vitro assays, where B cell proliferation from

seropositive individuals was found to regress after infection with the virus (Moss, Rickinson

et al. 1978, Rickinson, Moss et al. 1979). This response was primarily mediated by EBV-

specific MHC class I restricted CD8+ T cells (Moss et al., 1979), although MHC class II

restricted CD4+ T cells also are involved (Misko, Sculley et al. 1991).

The cellular events following EBV infection can either be asymptomatic, with no

evidence of major expansion of virus-specific T cells, or, in the case of IM, with massive T

cell activation and expansion with cytotoxic effector functions. Interestingly, comparable

high viral loads can be found in both types of infection (Silins, Sherritt et al. 2001). Both

asymptomatic and acute primary infections are followed by persistent latent infection of

memory B cells, which lasts for the lifetime of the host. If EBV-infected B cells receive the

signal to differentiate into plasma cells, or experience another similar event, it may lead to

reactivation of lytic EBV infection (Babcock, Decker et al. 1998, Laichalk and Thorley-

Lawson 2005).

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Immune Responses during IM

Clinical evidence from the study of IM patients, from the time of acute infection

through recovery and carrier state, suggests a strong cell-mediated immune response,

mainly involving CD8+ T cells (Callan, Tan et al. 1998). Most of what is known about CD8+

T cell response to EBV infection has been obtained from the study of IM patients. The

atypical lymphocytes found in large numbers in the blood of IM patients consist mostly of

EBV-specific CD8+ T cells (Tomkinson, Maziarz et al. 1989). This expanded population of

CD8+ T lymphocytes has been found to be antigen-driven and mostly specific for EBV lytic

antigens and to a lesser extent for EBV latent antigens (Steven, Annels et al. 1997). The

acute phase of IM is followed by a sharp reduction in CD8+ T cell numbers during recovery

(Callan, Fazou et al. 2000).

Despite having a large number of lytic epitope-specific CD8+ T cells in the blood of

IM patients, virus shedding from the oropharynx remains high for several months (Fafi-

Kremer, Morand et al. 2005). Comparative studies of blood and tonsil samples during

acute IM, recovery and long-term carrier states have shown that there is poor recruitment

of EBV-specific effectors in tonsils during acute IM (Hislop, Kuo et al. 2005). This was

found to be due to the lack of homing markers on the effector cells during IM. Latent

infection of the B cell pool is quickly controlled as latent epitope-specific CD8+ T cells

acquire homing markers more rapidly than the lytic epitope-specific memory T cells. In the

long-term carrier state, both latent and lytic antigen specific cells are enriched within the

tonsillar CD8+ T cells and both forms of infection are tightly controlled.

Immune Response by CD8+ T cells

Memory CTL responses in long-term virus carriers are highly focused on select

epitopes from EBV latent and lytic cycle antigens. During convalescence from acute

infection, the number of CD8+ T cells contracts, and a fraction are selected as long-term

memory T cells and are maintained thereafter for life (Dunne, Faint et al. 2002). These

memory T cells show marked immunodominance, in which epitopes from a particular

subset of latent or lytic antigens preferentially induce a strong response from CD8+ T cells,

while other antigens are subdominant targets and recognized less efficiently (Hislop,

Gudgeon et al. 2001).

Among EBV-latent antigens, the EBNA3 subset of nuclear antigens displays a

marked degree of immunodominance over other latent antigens (Khanna, Burrows et al.

1992, Murray, Kurilla et al. 1992). This preference for EBNA3 proteins and their derived

peptide epitopes was observed even during primary infection. CTL responses to other

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latent antigens were less frequently detected (Khanna, Burrows et al. 1992). Among the

epitopes that are presented during primary infection by CTLs, only a fraction is recognized

by memory CTL. So the makeup of long-term memory CTLs differs greatly from that

observed for CTLs following primary infection (Steven, Leese et al. 1996). The majority of

the CTL population—that is produced during a primary response—is heavily culled during

the establishment of memory (Catalina, Sullivan et al. 2001). This is particularly evident for

CTL-recognizing lytic cycle epitopes that are more heavily culled, relative to CTL-specific,

for latent cycle antigens (Hoshino, Morishima et al. 1999, Callan, Fazou et al. 2000). This

likely reflects the critical role of CD8+ T cells specific for latent epitopes in limiting the

expanding pool of latently infected B cells (Hislop, Annels et al. 2002).

CD8+ T cells specific for epitopes from EBV IE and E lytic antigens have been

detected in the blood of IM patients, and these responses, were shown to restricted

through a variety of HLA-A, B and C (Steven, Annels et al. 1997). Using tetrameric HLA-

peptide complexes, it has been found that a majority of the peripheral blood CD8+ T cells

targeting EBV can be specific for epitopes encoded by BZLF1 (Callan, Tan et al. 1998).

While T cells specific for lytic cycle antigens contract during convalescence, they can be

retained as long-lived memory T cells. Although T cells targeting lytic cycle antigens are

less in number for most individuals, they can still dominate over responses to EBV latent

antigens in some individuals (Tan, Gudgeon et al. 1999). CTLs from patients with acute IM

also show reactivity to late EBV antigens, such as the viral structural antigens gp350 and

gp85. These gp350 and gp85 specific memory CTLs are maintained at low levels following

recovery (Khanna, Moss et al. 1999). In vitro assays of CD8+ T cell response to EBV lytic

antigens show different levels of immunodominance, with IE the most abundant, followed

by E and then late proteins, indicating that the efficiency of epitope presentation decreases

with the advance of the lytic cycle (Pudney, Leese et al. 2005). These observations are

indicative of the strong immunodominance of IE-derived epitopes and E proteins whose

expression is directly activated by the IE proteins.

Immune Response by CD4+ T cells

Unlike CD8+ T cells, the CD4+ T cell pool remains relatively constant in the blood of

IM patients (Maini, Gudgeon et al. 2000). The response to EBV by CD4+ T cells has been

studied less than the response by CD8+ T cells. Even though the control of EBV infection

is mainly managed by MHC class I restricted CD8+ T cells, MHC class II restricted CD4+ T

cells have also been reported to play a role (Tomkinson, Maziarz et al. 1989, Precopio,

Sullivan et al. 2003). A number of CD4+ T cell epitopes from the viral glycoprotein gp350

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have been identified (Wallace, Wright et al. 1991). In addition, ex vivo studies have

identified multiple EBNA and LMP derived MHC class II epitopes, which have much lower

cognate T cell frequencies than MHC class I epitopes from the same antigens (Leen, Meij

et al. 2001, Long, Haigh et al. 2005). CD4+ T cells specific for epitopes from IE, E and also

several L lytic cycle proteins have been found in healthy carriers (Amyes, Hatton et al.

2003, Woodberry, Suscovich et al. 2005). Recent studies by Long et al. found that in

contrast to CD8+ T cells, CD4+ T cell responses to EBV lytic antigens are broadly

distributed across IE, E and L protein targets (Long, Leese et al. 2011). Adhikary et al.

have shown, using in vitro studies, that recognition of lytic structural protein by CD4+ T

cells could result in the detection of newly infected B cells before viral latency is

established (Adhikary, Behrends et al. 2006). Participation of CD4+ T cells during the early

stages of EBV infection has also been reported by other studies (Nikiforow, Bottomly et al.

2001). This suggests that such responses could probably control a recent B cell infection

event in vivo.

Using a dox-regulated expression system, in which antigen expression can be

controlled, Mackay and colleagues looked at antigen presentation to CD4+ and CD8+ T

cells. They found that newly synthesized antigens are readily presented by MHC class I

and recognized by CD8+ T cells, while MHC class II presentation and recognition by CD4+

T cells was delayed (Mackay, Long et al. 2009). This suggests that CD8+ T cells are more

effective during primary infection, while CD4+ T cells are important in controlling persistent

infection. In this scenario, CD8+ and CD4+ T cells would work together to control EBV by

targeting different stages of the EBV life cycle.

1.6.3. Humoral Response to EBV Infection

EBV infection causes a humoral immune response to a large number of virus-

encoded antigens. This response is prominent during the acute phase of IM, where the

initial humoral response is directed towards lytic antigens EA-R, EA-D, and the VCA. At a

later stage the response targets latent antigens such as EBNA1 (Henle, Henle et al. 1968).

Primary infection is characterized by the presence of IgM and IgG antibodies specific for

VCA and EA, detectable IgM and weak IgG against MA (Hewetson, Rocchi et al. 1973),

and an absence of antibodies against EBNA1. EBNA1-specific antibodies are not detected

until convalescence. The long-term virus carrier state leads to the persistence of IgG

against VCA, MA and EBNA1 for life (Henle, Henle et al. 1970).

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1.7. VACCINE DEVELOPMENT

The idea for a vaccine against EBV, to reduce clinical symptoms of acute IM and

control the more severe EBV-associated diseases, was first proposed by M.A. Epstein in

1976 (Epstein 1976, North, Morgan et al. 1982). The many diseases and malignancies

associated with EBV make the virus an important candidate for vaccine development. The

EBV structural antigen gp350 was considered from the beginning as a potential target,

based on the observation that this glycoprotein is the principal target for the virus-

neutralizing antibody response in vivo (Pearson, Dewey et al. 1970). Epitopes from gp350

has also been shown to induce a CTL response (Khanna, Bell et al. 1999). Peptide

epitopes from other lytic cycle antigens and latent antigens have also been investigated as

candidates for an epitope-based vaccine. An HLA-B8 restricted epitope from EBNA3 has

recently been used in a Phase I clinical trial. It was found to be well-tolerated and safe,

and the vaccine recipients seroconverted asymptomatically without IM (Elliott, Suhrbier et

al. 2008). One hurdle for peptide-based vaccines is the wide range of HLA types in

different individuals. The construction of recombinant or synthetic polyepitopes linking

minimum CTL epitopes in a continuous string has been shown to be highly effective in

inducing a protective CTL response (Thomson, Khanna et al. 1995, Thomson, Elliott et al.

1996) and offers one strategy for an epitope-based vaccine that provides broad HLA

coverage.

The EBV membrane protein gp350 is the best characterized vaccine target antigen

from EBV. This protein has been used in pre-clinical vaccine trials in animal models and in

clinical trials in humans. Vaccination with gp350 protected cottontop tamarins against

lymphoma and in common marmoset, vaccination reduced the viral load when compared

to the control (Morgan, Finerty et al. 1988, Mackett, Cox et al. 1996). The gp350 protein

was found to be highly immunogenic when complexed with immune stimulating complexes

(ISCOMS) or Adjuvant System 04 (AS04) from GlaxoSmithKline. Recombinant vaccinia

virus-expressing gp350 has also been tested in common marmosets and in a human trial

in China (Gu, Huang et al. 1995). Recent Phase I/II and Phase II studies using

recombinant gp350 complexed with AS04 demonstrated optimal immunogenicity after

three doses. All forms of gp350 were found to be safe and well tolerated. While no

protection from primary EBV infection was detected, recombinant gp350 in AS04 was

effective in reducing the symptoms of IM (Moutschen, Léonard et al. 2007, Sokal,

Hoppenbrouwers et al. 2007).

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1.8. IMMUNE EVASION

Viruses have developed various strategies to avoid detection and elimination by the

host’s immune system (Jackson, Nilsson et al. 1990, Gilbert, Riddell et al. 1993, York,

Roop et al. 1994, Lorenzo, Jung et al. 2002). Herpesviruses use their ability to establish

latency as a means to avoid detection. EBV achieves latency by regulating the expression

of latent genes in B cells and activating virus replication in lymphoid and epithelial cells

only occasionally. In order to accomplish this, the virus has developed mechanisms to

interfere with different levels of immune clearance. Down-regulation of viral genes,

inhibition of antigen processing and presentation and resistance to apoptosis are some of

the mechanisms employed by EBV for immune evasion (Vossen, Westerhout et al. 2002,

Ressing, van Leeuwen et al. 2003, Desbien, Kappler et al. 2009, Wycisk, Lin et al. 2011).

The most obvious strategy used by EBV to escape detection is to limit the number

and levels of viral proteins it expresses. EBV only expresses a few genes during latency.

The EBV nuclear antigen, EBNA1, is present in all latent and lytic cycles and in EBV-

associated malignancies. EBNA1 can limit its own synthesis to prevent proteasomal

degradation and antigen presentation. This is achieved by the glycine-alanine (GAr) repeat

domain in its N-terminus (Levitskaya, Coram et al. 1995, Tellam, Sherritt et al. 2001, Yin,

Manoury et al. 2003, Tellam, Smith et al. 2008). Further studies have shown that GAr

mRNA forms a G-quadruplex structure that prevents EBNA1 synthesis and presentation of

EBNA1 epitopes to CD8+ T cells (Murat, Zhong et al. 2014, Tellam, Zhong et al. 2014).

EBV also interferes with antigen presentation by down-regulating peptide

transporters (TAP) that are required for MHC class I presentation with BNLF2a protein

(Croft, Shannon-Lowe et al. 2009, Horst, Favaloro et al. 2011). EBV-encoded BILF1

protein functions by down-regulating cell surface expression of the MHC class I molecule.

It does so by binding to the molecule, internalizing and degrading it using lysosomes (Zuo,

Quinn et al. 2011). EBV-encoded BGLF5 is an exonuclease enzyme which inhibits cellular

protein synthesis (Knopf and Weisshart 1990, Rowe, Glaunsinger et al. 2007), causing

reduced cell surface expression of both MHC class I and class II molecules, which lowers

recognition of antigens by EBV-specific CD8+ and CD4+ T cells (Rowe, Glaunsinger et al.

2007, Zuo, Thomas et al. 2008, Croft, Shannon-Lowe et al. 2009, Horst, Favaloro et al.

2011).

Bim (Bcl-2 interacting mediator of cell death) regulates apoptosis during lymphocyte

development and the level of Bim is a critical factor in B cell survival. EBV is found to

repress the expression of Bim in EBV-infected B cells by down-regulation of Bim mRNA

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levels (Paschos, Smith et al. 2009). EBV also encodes two viral Bcl-2 proteins, BHRF1

and BALF1, which are anti-apoptotic proteins. These are expressed during the initial

stages of infection and also during the lytic phase (Henderson, Huen et al. 1993, Boya,

Pauleau et al. 2004).

BZLF2 encodes a glycoprotein, gp42, which is required for initial penetration and

infection of B cells (Nemerow, Houghten et al. 1989). The gp42 additionally functions as

an immune-modulator by binding directly to the chain of MHC class II molecules and

preventing T cell receptors from interacting with MHC class II/peptide complexes (Ressing,

Keating et al. 2005). EBV BARF1 blocks colony-stimulating factor CSF-1, which is required

for the proliferation of monocytes. Furthermore, BARF1 inhibits mononuclear cells from

producing IFN-, which is a key cytokine in the initiation of immune responses (Cohen and

Lekstrom 1999).

EBV subverts immune response by stimulating regulatory T cells through LMP1 to

secrete IL-10, which inhibits T cell proliferation and IFN-γ secretion (Marshall, Vickers et al.

2003). The viral homolog of cellular IL-10 is expressed by the BCRF1 gene and

contributes to immune evasion during the early phase of EBV infection (Miyazaki, Cheung

et al. 1993). LMP1 also functions as a constitutively active CD40 and aids in the survival of

EBV-infected B cells. Expression of LMP1 promotes cell growth and confers apoptosis

resistance (Henderson, Rowe et al. 1991, Middeldorp, Brink et al. 2003, Geiger and Martin

2006).

EBV-encoded miRNAs act as immune modulators. These miRNAs interfere in the

expression of host miRNAs and viral genes to protect the infected cell from the immune

response (Barth, Meister et al. 2011). EBV also uses miRNAs to regulate the expression of

LMP1, which is toxic to the cell (Lo, To et al. 2007). EBV-encoded miRNAs protect the

newly-infected B cells from apoptosis by down-regulating the pro-apoptotic Bim protein

(Seto, Moosmann et al. 2010, Marquitz, Mathur et al. 2011). During latent EBV infection,

the non-coding EBV-encoded RNAs, EBERs, confer resistance to apoptosis by IFN-,

thereby facilitating B cell transformation (Nanbo, Inoue et al. 2002, Ruf, Lackey et al.

2005). Thus, various strategies employed by the virus ensure the survival of EBV within

the host immune cell by allowing it to remain undetected for the lifetime of the host.

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1.9. MOLECULAR CHARACTERISATION OF EBV-SPECIFIC B CELL RESPONSE

1.9.1. Project Background

The B cell-dependent humoral immune response and the T cell-dependent cell-

mediated response have to work in concert to provide immunity against most pathogens.

The role of CD4+ T cells in humoral immunity has long been acknowledged (Miller, De

Burgh et al. 1965, Goodnow, Vinuesa et al. 2010). Recent research has shown that among

the CD4+ T subsets, T follicular helper cells are required for the development of T cell

dependent B cell responses (Breitfeld, Ohl et al. 2000, Schaerli, Willimann et al. 2000). It is

not widely accepted that B cells are involved in cell-mediated immune responses.

However, recent studies and clinical data from mice and humans have shown that B cells

are not just recipients of T cell help but are active contributors to different aspects of

cellular immunity (Cohen, Emery et al. 2006, Hauser, Waubant et al. 2008, Pescovitz,

Greenbaum et al. 2009). B cells are found to influence T cell responses by presenting

antigens, providing co-stimulatory molecules, and producing cytokines (Constant,

Schweitzer et al. 1995, Crawford, Macleod et al. 2006, Lund and Randall 2010).

Unlike T cells, which originate in the bone marrow and mature in the thymus, B cells

originate in the foetal liver and migrate to bone marrow after birth. Once bone marrow

develops B cells also originate in the bone marrow. B cells undergo selection and

maturation in the bone marrow (Raff, Megson et al. 1976). They produce antibodies and

activate the humoral immune response. The majority of B cells express MHC class II

molecules on the cell surface and present antigens to T cells. After interaction with a T

cell, the B cell divides and matures into antibody-producing plasma cells and memory B

cells. Antibodies produced by plasma cells provide the main defence against extracellular

pathogens by functioning in three ways: 1) Neutralization, in which antibodies bind to

pathogens or their products and block access to host cells; 2) Opsonization, in which

antibodies coat and tag pathogens, thereby promoting ingestion by phagocytes; and 3)

Complement activation, where a group of about 20 serum proteins are sequentially

activated and can directly lyse antibody-coated cells or enhance phagocytosis. Thus,

antibodies also act to enhance the function of the innate immune system (Murphy 2008).

Immunoglobulins (Ig) are glycoproteins that are either expressed on the surface of

B cells or secreted by plasma cells in response to an immunogen. They are grouped under

five classes (Berek, Berger et al. 1991): IgM, IgG, IgA, IgE and IgD, based on their heavy

chain structure (Brack, Hirama et al. 1978). Any of these heavy chain structures can

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combine with any of the two light chain structures ( or ) to form Ig or antibodies. Each Ig

contains two antigen-binding sites encoded by two identical light chains and heavy chains

linked by disulphide bonds. The heavy and light chains are coded by three different

families of genes, each one on a different chromosome. A single constant (C) region can

associate with many different variable (V) regions (Alt, Yancopoulos et al. 1984). The N

terminal region, consisting of a heavy and light chain, binds the antigen and is known as

the fragment antigen-binding (Fab). The C terminal region, consisting of a heavy chain

pair, does not bind the antigen but interacts with cells of the immune system and is called

fragment crystallizable (Fc). Therefore, the Fab contains the antigen-binding sites and Fc

region mediates effector functions (Gathings, Lawton et al. 1977). The maturation of a B

cell starts with the expression of the recombination activation genes RAG1 and RAG2,

which activate the gene recombination for the production of Ig (Schatz, Oettinger et al.

1989). The expression of IgM on the surface of a B cell leads to the progression of the

maturation process, which results in the production of an antibody-secreting plasma cell

(Fröland, Natvig et al. 1971).

B cells are activated by one of two mechanisms: a T cell independent response, or

a T cell dependent response. A T cell independent response is induced by antigens that

are usually non-protein and have many repeating epitopes that interact with multiple BCRs

and stimulate the production of IgM antibodies (Mond, Lees et al. 1995). This maturation is

mainly caused by polysaccharides and does not involve GC formation or B cells

undergoing affinity maturation and class switch recombination and generation of memory.

An example of this response is how certain plant lectins, like pokeweed mitogen, induce

proliferation and antibody production from mature B cells (Hammarstrom, Bird et al. 1979,

Richards, Watanabe et al. 2008). The majority of antibody responses to proteins are T cell-

dependent, requiring the help of T cells. B cell activation mainly occurs in the GC and

results in affinity maturation, class switching, and memory (Muramatsu, Kinoshita et al.

2000).

B cells play a crucial role in the host’s defence against infection, and this role is

characterized by a series of coordinated processes that include antigen recognition and

presentation, antibody secretion, and cytokine release. B cells mature and attain the ability

to recognize antigens in the bone marrow. They undergo several distinct development

stages in the bone marrow, including the elimination of auto-reactive cells from the

population (Hartley, Crosbie et al. 1991, Brink, Goodnow et al. 1992). The maturation of B

cells starts with somatic recombination, necessary for the production of Ig (Oettinger,

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Schatz et al. 1990, Bassing, Swat et al. 2002). Through this somatic event, a finite set of

genes produces a diverse array of gene recombination capable of recognizing a vast

number of molecular structures. Each B cell has a unique Ig, the BCR, which is specific for

a single antigen (Brack, Hirama et al. 1978, Rajewsky 1996, Murre 2007). Gene

rearrangement in the bone marrow occurs at the beginning of B cell maturation and is

completely independent of antigens (Tonegawa, Sakano et al. 1981, Lam, Kühn et al.

1997). After developing in the bone marrow, B cells migrate to the secondary lymphoid

organs, including the lymph nodes and the spleen, where circulating antigens from the

blood and lymph are drained. Here, the B cells interact with foreign antigens with the help

of T helper cells and DCs (Karrer, Althage et al. 1997, Junt, Moseman et al. 2007).

Antigen-activated B cells migrate to the follicles of peripheral lymphoid tissue—

including the spleen, lymph nodes, Peyer’s patch, and the tonsils—where activated B cells

start to divide and differentiate, forming GCs (Figure 1.1) (Hauser, Junt et al. 2007). B cells

divide, mature and become centroblasts in these GCs (Küppers, Zhao et al. 1993). For GC

response to be initiated it needs the interaction of BCR and CD40, two key receptors on B

cells that provide growth and differentiation signals to those cells (Uchida, Yasui et al.

1999). The initial interaction occurs at the margin between the primary follicles and T cell

areas in secondary lymphoid tissues. Antigen-activated B cells undergo clonal expansion

and somatic hypermutation in the dark zone of the GC. Somatic hypermutation modifies

the immunoglobulin variable region, thereby generating antibodies with higher affinity for

antigens (affinity maturation) (Li, Woo et al. 2004, Peled, Kuang et al. 2008). Centroblasts

differentiate into centrocytes and move into the light zone of the GC, where the B cells with

a functional BCR are selected by T helper cells and follicular dendritic cells (FDC). The

survival of a B cell depends on a functional BCR (Kraus, Alimzhanov et al. 2004). The

signals given by the T helper cells and FDCs result in the escape of the B cell from the

apoptotic pathway of the GC and their release from GCs. GC B cells that do not receive

signals through their BCR, or acquire mutations, are eliminated within the GC by apoptosis

(Liu, Joshua et al. 1989). Ig class switching also occurs in the GCs in which the original

IgM/ IgD heavy chain constant region is replaced by Ig isotype of IgG, IgA and IgE. The

differentiated memory B cell or plasma cell is then released from the germinal centers

(Kuppers 2003, Soulas-Sprauel, Le Guyader et al. 2007, Stavnezer, Guikema et al. 2008).

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Figure 1. 1. Germinal Center (GC)

Germinal centers provide the micro-environment inside the secondary lymphoid organs for the antigen activated B cells to develop into long lived plasma cells and memory B cells (Kuppers 2003).

Interaction between B cells, CD4+ T helper cells and dendritic cells are required for

the development of humoral immunity (Figure 1.2). CD4+ T follicular helper (TFH) cells are

specialized T cells that help B cells differentiate into antibody producing plasma cells and

memory cells. TFH cells also support germinal center formation and maintenance (Allen,

Okada et al. 2007).TFH cells have recently been found to be a distinct effector subset from

the other CD4+ T helper cells, the Th1, Th2, Th17 and regulatory T cells (Tregs). Bcl-6 is

the key regulator of TFH differentiation, and the expression of the chemokine receptor

CXCR5 guides TFH cells to the follicles (Haynes, Allen et al. 2007, Johnston, Poholek et al.

2009, Yu, Rao et al. 2009, Baumjohann, Okada et al. 2011). Various studies have found

that CD4+ T cells are important in the control of persistent viral infection (Battegay,

Moskophidis et al. 1994, Zajac, Blattman et al. 1998). Fahey and colleagues have shown

that viral persistence results in the redirection of CD4+ T cells from the Th1 response

induced during acute infection to TFH cells (Fahey, Wilson et al. 2011). In addition acute

viral infection has been found to generate both Th1 and TFH memory CD4+ T cells (Hale,

Youngblood et al. 2013).

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Figure 1. 2. DC-B cell-T cell interaction

Interaction of DC with T cells and B cells is important for humoral immune responses. DCs promote B cell differentiation into plasma cells by presenting antigens to B cells, providing cytokines and also by activating TFH cells. Signals from TFH cells are required for the differentiation of B cells into plasma cells and memory B cells. Adapted from Ueno et al (Ueno, Schmitt et al. 2010).

Dendritic cells (DCs) were discovered by Ralph Steinman in 1973 and are derived

from hematopoietic bone marrow stem cells (Steinman and Cohn 1973). Immature DCs

exist in the circulation or as tissue resident cells monitoring the surroundings for foreign

pathogens. They can sense pathogens through their pattern-recognition receptors (PRRs)

and once activated, mature DCs migrate to lymphoid organs to initiate the immune

response (Kadowaki, Antonenko et al. 2000, Jarrossay, Napolitani et al. 2001). DCs are

required to mediate the initiation of innate and adaptive immune responses and establish

immunity against pathogens. The two major blood DC subsets are pDCs and mDCs, and

both subsets are efficient antigen presenting cells (Tel, Schreibelt et al. 2013). In addition,

pDCs play a unique role in antiviral immunity by producing large amounts of type I

interferons, especially IFN-, when activated by the presence of viral and bacterial

components (Cella, Facchetti et al. 2000, Bauer, Redecke et al. 2001).

Type I interferons not only interfere with viral replication but also initiate events that

activate both innate and adaptive antiviral immune responses (Le Bon, Schiavoni et al.

2001). It has also been reported that pDCs are required for the conversion of memory cells

to plasma cells in a two-step process, with Type I interferons converting memory cells to

plasmablasts followed by IL-6 converting plasmablasts to antibody-secreting plasma cells

(Jego, Palucka et al. 2003). Type I interferons produced by pDCs also induce mDCs and

monocytes to produce two membrane proteins, B cell activating factor (BAFF) and a

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proliferation-inducing ligand (APRIL) which are required for B cell survival and proliferation

(Litinskiy, Nardelli et al. 2002). The fate of DCs in primary and persistent EBV infections is

not known. Decreased frequency and function of blood DC subsets have been reported

during infections, including HIV, Hepatitis B and malaria (Pacanowski, Kahi et al. 2001,

Duan, Wang et al. 2004, Pinzon-Charry, Woodberry et al. 2013). The reduced frequency of

pDCs and impaired production of IFN- also are thought to be crucial factors responsible

for viral infection in transplant patients (Lim, Kireta et al. 2006).

While T cell response to EBV infection has been studied extensively, the B cell

response during EBV infection has received less attention. It is known that EBV infection

results in the production of antibodies to a variety of virus-encoded antigens. Antibodies to

VCA, EA, as well as EBNA1, are used as diagnostic markers in the detection of primary

and persistent EBV infection (Henle, Henle et al. 1970). Most of the information regarding

EBV infection in vivo is from studies of IM patients. It is known that IM is caused by the

uncontrolled expansion of CD8+ T cells in response to the virus and the cytokines it

produces rather than by the virus itself. The reason for this aberrant expansion of CD8+ T

cells in only some individuals, leading to symptoms of IM, is not known. So the aims for

this project were to explore possible reasons for symptomatic EBV primary infection by

investigating the role of humoral immunity during acute IM. Considering the important role

of DCs in anti-viral immunity and in the development of humoral immune responses, the

fate of DCs during EBV primary infection also was explored.

Since B cells and antibody-secreting plasma cells are critical in providing protective

immunity, this study was designed to investigate the B cell-specific immune response

during EBV-associated infectious mononucleosis, and use this information towards

developing a vaccine for IM. Long-lived plasma cells, memory B cells and memory CD8+

and CD4+ T cells make up the different arms of the immune system, that provide

protective immunity. Quantitating memory B cell response is important for the successful

design of a vaccine. Recent studies on B cells specific for influenza, malaria and HIV used

B cell ELISpot for the quantification of antigen-specific memory B cells (Wrammert, Smith

et al. 2008, Doria-Rose, Klein et al. 2009, Weiss, Traore et al. 2010). Detailed phenotypic

analysis of the classification of B cells is possible by techniques including flow cytometry

and the B cell ELISpot. While techniques like ELISA and flow cytometry measure antibody

reactivity and specificity, ELISpot focuses on antibody-secreting cells and B cell responses

at a cellular level. The method was described in 1983 and the assay was developed for

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human memory B cells in 2003 by Crotty and colleagues (Czerkinsky, Nilsson et al. 1983,

Sedgwick and Holt 1983, Crotty, Aubert et al. 2004).

Using these techniques, this project studied B cell-mediated immune regulation

during and post IM. EBV membrane protein gp350 is the best-characterized candidate

protein for an anti-EBV vaccine. This is based on the observation that this glycoprotein

produces a virus-neutralizing antibody response after natural infection (Thorley-Lawson

and Poodry 1982). This protein was used to investigate the quality and quantity of virus-

neutralizing antibodies produced during primary and persistent EBV infection. The

frequency and phenotype of B cell subsets were studied to see if there is a deregulation in

the total B cell compartment as a result of IM.

Acute IM causes the up-regulation of various inflammatory and immunosuppressive

cytokines, as proven by various studies (Hornef, Wagner et al. 1995, Biglino, Sinicco et al.

1996, Corsi, Ruscica et al. 2004). Recently, Balfour et al. assessed the plasma of IM

patients for various cytokines that might contribute to disease symptoms, and found that

most cytokines tested were elevated during acute IM (Balfour, Odumade et al. 2013).

Consistently, IFN- was not detected in the plasma of IM patients. Since pDCs are the

main producers of IFN-, the study aimed to determine the role of peripheral blood DC

subsets during and post IM, to see whether lack of IFN- in the plasma of IM patients is

due to a defect in the DC population during acute IM.

1.9.2. Aims and Scope of Thesis

The objective of this research project was to find out why primary EBV infection

becomes symptomatic only in some individuals, resulting in Infectious Mononucleosis (IM).

IM is seen in about 50% of infected adolescents and young adults who acquire EBV

infection. IM is an immunopathological disease—symptoms arise because of an

uncontrolled expansion of CD8+ T cells in response to the virus and the cytokines it

produces. The reason behind this massive expansion of CD8+ T cells is not known.

Therefore, the focus of this project was to study the immune control of EBV during the

early stages of infection, before cell-mediated immune responses came into play. The

main focus was on the study of the humoral immune response, which is not well

understood. Various studies have described the involvement of DCs in the initiation of both

the innate and adaptive immune responses and their importance in the development of the

humoral immune response. Since DC function during EBV infection has received little

attention, this project also studied peripheral blood DCs during acute IM. The study of the

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humoral immune response focused on the virus-neutralizing antibody response, as well as

global B cell function, during acute IM.

1.9.3. Hypothesis

Symptomatic EBV primary infection (IM) occurs due to defective control of innate and/or

humoral immune responses during the early stages of EBV infection.

1.9.4. Project Aims

o To study the kinetics of different B cell subsets during primary and persistent

EBV infection.

o To investigate the B cell response against the EBV-membrane antigen gp350

during IM.

o To understand the role of peripheral blood DCs during EBV infection.

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CHAPTER 2. MATERIALS AND METHODS

2.1. LIST OF CHEMICALS

Acetic acid glacial BDH Chemicals, Poole, UK

Acrylamide and bis-acrylamide Bio-Rad Laboratories, CA, USA

Ammonium persulphate Bio-Rad Laboratories, CA, USA

-D methyl glucoside ACROS Organics, NJ, USA

Bromophenol blue Sigma-Aldrich, MO, USA

2-Mercaptoethanol Sigma-Aldrich, MO, USA

5-Bromo-4 chloro-3-indolyl phosphate

/nitroblue tetrazolium (BCIP/NBT)

Sigma-Aldrich, MO, USA

Concanavalin A sepharose GE Healthcare, Uppsala, Sweden

Coomassie brilliant blue Bio-Rad Laboratories, CA, USA

Carboxyfluoroescein diacetate succinimidyl ester (CFSE)

Sigma-Aldrich, MO, USA

Dimethyl sulphoxide (DMSO) Sigma-Aldrich, MO, USA

Ethanol Sigma-Aldrich, MO, USA

Ethylene diamine tetraacetic acid (EDTA) APS Chemicals Ltd, NSW, Australia

Ficoll-PaqueTM Plus GE Healthcare, Uppsala, Sweden

Glycerol APS Chemicals Ltd, NSW, Australia

Glycine APS Chemicals Ltd, NSW, Australia

Methanol Fronine Pty Ltd, NSW, Australia

Non-fat dry milk Bio-Rad Laboratories, CA, USA

Paraformaldehyde Sigma-Aldrich, MO, USA

Sodium carbonate BDH Chemicals, Poole, UK

Sodium bicarbonate BDH Chemicals, Poole, UK

Sodium dodecyl sulphate Bio-Rad Laboratories, CA, USA

Streptavidin-alkaline phosphatase Sigma-Aldrich, MO, USA

Tris Life Technologies, Vic, Australia

N,N,N’,N’-Tetramethylethylenediamine (TEMED) Sigma-Aldrich, MO, USA

Trypan blue Sigma-Aldrich, MO, USA

Trypsin CSL Ltd., Vic, Australia

Tween 20 Sigma-Aldrich, MO, USA

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2.2. EQUIPMENT

Analytical balance Sartorius AG, Gottingen, Germany

Autoclave, Tomy ES-315 Tomy, Tokyo, Japan

Bench top centrifuge, Sigma D-37520 Sigma, Osterode am Harz, Germany

CO2 incubator-Steri-Cult 200 Forma Scientific, GA, USA

Counting Microscope Olympus, CX41 Olympus, Tokyo, Japan

Cryo 1°C freezing container Nalgene, NalgeNunc, NY, USA

ELISpot Reader System AutoimmunDiagnostica (AID), Strassburg, Germany

Flow cytometry, FACS LSRII Becton Dickinson, CA, USA

Freezer -70°C Thermo Forma, GA, USA

Haemocytometer Sigma-Aldrich, Stenheim, Germany

Haemocytometer coverslips Menzel-Glaser, Menzel, Germany

Laminar Air Flow, Clyde Apac BH2000 Clyde-Apac, SA, Australia

MACS Separator Miltenyi Biotec, NSW, Australia

Microcentrifuge, Eppendorf 5415D Eppendorf, Hamburg, Germany

Milli-Q water supply Millipore Corporation, MA, USA

pH meter, Ionode Denver Instruments, CO, USA

Spectrophotometer Thermo Fisher Scientific, NY, USA

Vortex mixer Ratek, Vic, Australia

Water bath Grant Instruments Inc., Shepreth, UK

2.3. MEDIA AND BUFFERS

Foetal bovine serum (FBS) supplied by JRH Biosciences. FBS was heat-inactivated at

56°C for 60 min, aliquoted and then stored at -20°C for use.

RPMI-1640 (R0) (Gibco, Life Technologies) prepared by the QIMR Berghofer media

department, supplemented with L-glutamine and 100 U/mL of penicillin and 100 g/ml

streptomycin (Gibco).

R10 medium containing R0 medium with 10% FBS.

Freezing medium containing R10 with 10% DMSO from Sigma-Aldrich.

Dulbecco’s Modified Eagle Medium (DMEM) from Gibco, supplemented with 10%

FBS, 100 U/mL penicillin and 100 g/ml streptomycin sulphate.

F12 nutrient mixture from Gibco.

CTSTM OpTmizerTM serum free medium from Gibco

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Phosphate-buffered saline (PBS), prepared by the QIMR Berghofer Media

Department. PBS contained 2.85 g of Na2HPO4.2H2O, 0.625g of NaH2PO4.2H2O and

8.7 g of NaCl in 1L of Milli-Q water, with pH adjusted to 7.4.

Trypsin/versene with 0.5mM versene (EDTA) and 0.1% trypsin in Dulbecco’s PBS (80

g NaCl, 2g KCl, 11.5 g NA2HPO4, 2 g KH2PO4, 40 ml 0.5% v/v phenol red and 10 L

Milli-Q water).

Carbonate Buffer: 0.4 g of Na2CO3 and 0.73 g of NaHCO3 dissolved in 250 mL Milli-Q

water and pH adjusted to 9.4. The buffer was autoclaved and stored at 4°C.

BCIP/NBT Sigma FASTTM tablets were used for spot development in ELISpot assays.

One tablet per ELISpot plate was dissolved in 10 mL Milli-Q water just before use. The

solution was filtered through a 0.22 m membrane prior to use.

Resolving buffer for autoMACS separator contains PBS with 0.5% FBS and 2mM

EDTA.

2.4. STUDY COHORT

Blood was collected from volunteers from Brisbane (Australia) with informed consent

and project approval by the Human Research Ethics Committee of QIMR Berghofer.

Samples were obtained from 18 age-matched asymptomatic EBV carriers and 15 age-

matched EBV carriers with a history of IM. A cohort of 25 acute IM samples, which

contained 16 longitudinal samples from acute IM (AIM) through recovery and long-term

carrier state (post-IM), was obtained from collaborators in the USA and UK. The age range

for acute IM patients was 16.1-32.1 years (median: 19.7; mean: 20.7) and for

asymptomatic healthy carriers was 18-28 years (median: 24.0; mean: 24.7 years).

2.5. CELL CULTURE TECHNIQUES

2.5.1. Ficoll-Paque Separation of PBMC From Whole Blood

Blood samples were donated by healthy EBV-positive and EBV-negative donors.

EBV-positive donors included asymptomatic virus carriers, individuals with a history of IM,

and individuals during and after acute IM. Peripheral blood mononuclear cells (PBMC)

were isolated from fresh blood by density gradient centrifugation with Ficoll-Paque Plus.

Two 9 mL Vacuette collection tubes of blood were combined in 50 mL Falcon tubes and

diluted with R0. Diluted blood was then carefully overlaid onto 10 mL Ficoll-Paque, and

centrifuged at 450 g, with low acceleration (level 2/10) and low brake (level 1/10), at room

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temperature. The tubes were centrifuged for 20–30 min and the buffy coat at the interface

was carefully collected using a Pasteur pipette. The PBMC collected were washed by

centrifuging at 400 g for 10 min without brake, followed by washes with R0 and R10 at 200

g for 10 min. Cells were counted using a hemocytometer and stored in liquid nitrogen in

aliquots of 10 × 106 cells/vial.

2.5.2. Cryopreservation and Thawing of PBMC

Cells were pelleted by centrifugation at 200 g for 5 min. The supernatant was

aspirated and the cells were resuspended at 10 × 106 cells per 1 mL in freeze mix (R10

with10% DMSO). The cells were aliquoted into cryogenic storage vials and placed in a

Cryo 1°C freezing container in a -80°C freezer overnight. Vials of cells were then moved

into a liquid nitrogen dewar for long-term storage. Cells from liquid nitrogen were thawed in

a 37°C water bath, washed once in 10 mL of R10 and centrifuged at 200 g for 10 min. The

cells were then rested for a minimum of 1 h at 37°C in R10 and viable cells were counted

using trypan blue.

2.5.3. Serum Separation and Storage

A total volume of 8 mL of blood was collected in a Vacuette serum tube. The tube

was centrifuged at 1800 g with acceleration at level 7/10 and brake at level 2/10 for 10

min. The separated serum was stored as small aliquots at -80°C.

2.5.4. Plasma Separation and Storage

A 9 mL collection tube (Vacuette) of blood per donor was centrifuged at 200 g for 10

min at room temperature with acceleration of 5/10 and brake 3/10. The clear liquid,

plasma, was collected and centrifuged again at 1800 g for 10 min at room temperature

with low brake, and the plasma aliquoted and stored at -80°C.

2.6. PREPARATION OF RECOMBINANT PROTEIN

2.6.1. Protein Purification

Cells expressing the protein of interest were grown to a high density in medium

DMEM/F12 containing 10% heat-inactivated FBS, 100 U/mL of penicillin and 100 µg/mL of

streptomycin. The cells were maintained in serum-free medium and the secreted protein

was collected and concentrated using an Amicon Ultracel (Millipore) centrifugal filter. The

concentrated protein was then purified using a concanavalin A sepharose 4B column. It

was dialyzed using snake-pleated dialysis tubing in binding buffer (20 mM Tris–HCl pH

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7.4, 0.5 M NaCl in water). The column was equilibrated overnight and washed with three

times the column volume of binding buffer. The dialyzed protein in binding buffer was

loaded onto the column and the flow-through was collected. The protein was reloaded onto

the column three more times to facilitate maximum protein retention. The column was then

washed with six times the column volume of wash buffer (binding buffer with 25 mM –D

methyl glucoside) and eluted with increasing concentrations of –D–methyl glucoside. The

concentrated protein was then visualised by dot-blot and/or western blot and the

concentration estimated using the Bradford protein assay. The protein was then stored in

PBS as aliquots at -70°C until use.

2.6.2. Dot Blot

The dot blot was used to determine the presence of protein after its expression and

purification. On a strip of nitrocellulose membrane, the position of each different sample

was marked with a circle using a pencil. Then, 10 l of different samples from the protein

purification procedure (flow through, wash and elution samples), as well as positive and

negative controls, were blotted on the nitrocellulose membrane. Using a narrow-mouth

pipette tip, 1 l of sample was spotted in the centre of each circle. The membrane was

allowed to dry for 1 h at room temperature and blocked using 5% dry milk in PBST (PBS

with 0.05% Tween-20). After 1 h of blocking, the membrane was incubated with primary

antibody against the protein (or GFP, if fused with GFP), at a 1:3000 dilution in PBST at

room temperature on a rocker. The membrane was incubated with a secondary antibody

after washing with PBST. Goat anti-mouse HRP (SouthernBiotech, USA) was added to the

membrane at 1:3000 dilution and incubated for 1 h at room temperature. The protein was

detected using western lightning plus-ECL (PerkinElmer Inc. USA) and exposed to X-ray

film in the dark.

2.6.3. Protein Concentration Estimation – Bradford Assay

Protein concentration was estimated using Quickstart Bradford dye reagent (Bio-

Rad laboratories). Protein standards were made from 2 mg/mL BSA and PBS was used as

a blank. In a 96-well flat-bottom plate, seven serial dilutions of protein standards were

made in duplicate. The protein of interest and blank were also measured in duplicate,

using a spectrophotometer.

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2.7. IMMUNOLOGICAL ANALYSIS

2.7.1. List of Antibodies Used in This Study

Antibody Fluorochrome Suppliera Antibody Fluorochrome Supplier

CD3 PE-Cy7 / Alexa Fluor 700 1 / 2 CXCR3 APC 1

CD4 Pacific Blue 1 CXCR5 FITC 1 / 2

CD8 PerCP-Cy5.5 2 Fas PE-Cy7 2

CD11c APC 1 FasL PE 2

CD19 eFluor 450 3 HLA-DR PerCP 1

CD20 FITC 3 ICOS BV421 1

CD24 PE 1 IFN-γ Alexa Fluor 700 1

CD27 APC / APC-H7 1 IL-21 PE 3

CD38 PerCP-Cy5.5 1 Lineageb FITC 1

CD123 BV421 / PE 1 PD1 APC 3

BAFF-R FITC 1 TACI

APC 1

BCMA PE 2 TNF APC 1

CCR7 Alexa Fluor 700 1 a Supplier codes: 1: Becton Dickinson, 2: BioLegend, 3: eBioscience.

b Lineage antibody cocktail includes antibodies specific for CD3, CD14, CD19, CD20 and CD56.

2.7.2. Flow Cytometric Analysis

Phenotypic analyses of B cells and DCs were done using PBMC and flow

cytometry. Frozen PBMC stored in liquid nitrogen were thawed at 37°C, washed once in

R10 and rested for at least 1 h in a 37°C incubator. The cells were then counted and

500,000 cells/well were used for surface antigen staining in a 96-well V-bottom plate. The

cells were washed twice in PBS/2% FBS and incubated for 30 min at 4°C with

fluorochrome-conjugated antibodies diluted in PBS/2% FBS. The cells were washed twice

with PBS/2% FBS and fixed in 4% paraformaldehyde. Data were acquired on a

FACSCanto II or LSRFortessa flow cytometer and analysed using FlowJo software

(Treestar, USA).

2.7.3. ELISpot

Memory B cells in PBMC were quantified using the method developed by Crotty et

al., in which the memory B cells are converted to antibody-secreting cells in vitro. PBMC

were thawed and transferred to 24-well plates at 5 × 105 cells/well. The cells were

incubated at 37°C for six days in R10 containing 50 µM β-mercaptoethanol and an

optimized mix of polyclonal mitogens. This mix of polyclonal mitogens included pokeweed

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mitogen extract (PWA) at 1/100,000 dilution, Protein A from Staphylococcus aureus,

Cowan strain (SAC) at 1/10,000 dilution, CpG ODN 2006 at 2.5 g/mL (Sigma Aldrich)

(Crotty, Aubert et al. 2004) and IL-10 at 25 ng/mL (BD Biosciences) (Weiss, Traore et al.

2010).

The antigen of interest was immobilized on a 96-well ELISpot plate (Millipore

Multiscreen HTS IP) and incubated at 4°C overnight. Wells of the plate were coated with

either 25 g/mL of gp350, recombinant EBV membrane antigen expressed from CHO cells

(Hessing, van Schijndel et al. 1992), or control EBV non-neutralizing structural antigen

VCA (GenWay Biotech). Positive control wells were coated with 10 g/mL of tetanus

toxoid (CSL, Australia). To detect total IgG secreting cells, 15 g/mL capture anti-IgG

(Mabtech) was used. The next day the plates were washed with PBS and blocked with

R10 for 2–4 h at room temperature. For the detection of antigen-specific antibody-

secreting cells (ASC), the in vitro activated cells were washed and plated in triplicate in

antigen coated wells at 3 × 105 cells/ well. For the detection of total IgG secreting cells

3000 cells/well were plated in triplicate in wells containing capture anti-IgG. The plates

were then incubated at 37°C overnight.

The following day, excess cells were removed by washing wells with PBST. The

plates were then incubated with 1:500 biotinylated anti-IgG (Mabtech) for 2 h at room

temperature, followed by incubation with 1:1000 Streptavidin-AP (Sigma-Aldrich) for 1 h at

room temperature. Sigma Fast BCIP/NBT was then added to the wells and incubated at

room temperature until spots developed, at which time the reaction was stopped by

washing with water. Spot quantification was done using an ELISpot reader system.

2.7.4. Enzyme Linked Immunosorbent Assay (ELISA)

Protein in carbonate buffer pH9.4 was added to ELISA plates (EIA/RIA plate) and

incubated overnight at 4°C. The coated plates were washed with PBST and blocked with

PBS containing 5% non-fat dry milk (blotting grade blocker) for 2 h at room temperature.

The plates were washed and 25 l of serially diluted samples were added in duplicate and

incubated for 2 h at room temperature on a plate shaker. After further washing with

PBSTs, 25 l of HRP-conjugated goat anti-human IgG (1:1000 dilution; Chemicon), or goat

anti-human Ig (1:4000 dilution; Southern Biotech) was added and incubated for 1 h. After

washing with PBSTs, 25 l of TMB ELISA substrate solution was added to each well and

plates were incubated for 15 min at room temperature in the dark. Once the colour

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developed, the reaction was stopped by the addition of 25 l of 1 M HCL. Absorbance was

measured using a spectrophotometer at a wavelength of 450 nm.

2.7.5. Cytometric Bead Array (CBA) analysis

The supernatant from cell culture or plasma from donors was used to quantify

cytokine levels present in the samples. A cytometric bead-based multiplex assay kit (BD

Biosciences) was used to measure the different cytokines.The assay was done in

accordance with the manufacturer’s instructions, with minor modifications. A lyophilised

standard vial was reconstituted with 4 mL assay diluent and allowed to equilibrate for 15

min before aliquoting and storing at -80°C. An aliquot of standard was used to set up eight

2-fold dilutions of standard in assay diluent. The capture bead mix was made by adding

0.1 l of each bead per test. Each capture bead stock vial was vortexed for 15 sec to

resuspend beads thoroughly and resuspended using capture bead diluent for

serum/plasma. Each assay was set up in a 96-well V-bottom plate by mixing 12 l of

mixed capture beads and 10 l of standard or sample. The plate was then incubated at

room temperature on a shaker for 1 h. During this time, a pool of PE-conjugated detection

antibodies was made by combining 0.1 µl of each antibody with detection diluent. After the

1 h incubation, 12 l of detection antibody pool was added to the plate and incubated for

another 2 h at room temperature on a shaker. The beads were then washed with 175 l

wash buffer per well and centrifuged at 200 g for 5 min, before being resuspended in 300

l wash buffer for analysis. Data were acquired using a BD LSRFortessa flow cytometer

and analysed using FCAP Array software (BD Biosciences)).

2.7.6. Apoptosis Assay

Annexin V binds to phophotidylserine, which is exposed on the plasma membrane

off a cell when membrane integrity is lost during apoptosis. Annexin V detection kit from

BD Biosciences was used in combination with a dead cell stain (Live/Dead Fixable Aqua

Dead Cell Stain; Life technologies) to distinguish between dead and apoptotic cells. After

labelling PBMC with the dead cell stain and antibodies specific for cell surface markers,

the cells were washed with annexin binding buffer. Annexin V diluted in annexin binding

buffer was then added and cells were incubated for 15 min at room temperature in the

dark. The cells were then analysed by flow cytometry within 1 h of staining.

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2.7.7. Intracellular Cytokine Assay

PBMC were thawed and rested for 1 h in R10 at 37°C before being counted and

distributed in a 96-well V-bottom plate. The cells were stimulated by incubation with

mitogen for 4-6 h at 37°C, in the presence of 1 µl/mL GolgiPlug (Brefeldin A; BD

Biosciences). The cells were washed and stained with antibodies against cell surface

markers and incubated at 4°C for 30 min. The intracellular staining was performed after

fixing and permeabilizing the cells using Cytofix/Cytoperm solution. Permeabilizing

solution, a 10× solution diluted to 1× with deionized water was used for subsequent

washes and the cells were then incubated with antibodies specific for cytokines of interest

at 4°C for 30 min. Data were acquired by flow cytometry and post-acquisition analysis was

done using FlowJo software.

2.7.8. Magnetic Bead Separation

PBMC were labelled with MACS microbeads (Miltenyi Biotech) for the magnetic

separation of B cells and dendritic cells. Cells were counted and 1x106 PBMC were

resuspended in resolving buffer and incubated with microbeads for 15 minutes at 4°C. The

cells were washed and resuspended in 500l of resolving buffer and was used for

magnetic separation. The autoMACS separator was prepared and primed according to the

manufacturer’s instructions. Sample tubes were placed at the uptake port and the labelled

and unlabelled fractions were collected from port pos1 for positive (labelled) fraction and

port neg1 for the negative (unlabelled) fraction. Depending on the experiment, enrichment

for B cells and deletion for dendritic cells, appropriate separation program was chosen on

the separator.

2.8. STATISTICAL ANALYSIS

Statistical analysis was performed using Prism 6, GraphPad software. The data were

presented as mean ± standard error of the mean (SEM) or as percentages. Statistical

significance was determined using one way analysis of variance (ANOVA) for comparison

of more than two groups. The Wilcoxon matched pairs test was used to compare

measurements at two time-points for the same individual, and measurements for different

groups were compared by non-parametric Mann-Whitney test.

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CHAPTER 3. KINETICS OF B CELL SUBSETS DURING

PRIMARY AND PERSISTENT EBV INFECTION

3.1. ABSTRACT

The humoral immune response to EBV infection is a less explored area of research

that could provide insights into immune control during the early stages of EBV infection.

Therefore, this study was designed to understand the humoral immune response during

EBV infection, in particular during acute infectious mononucleosis (IM). Studies outlined

under Aim 1 addressed global B cell function, specifically the kinetics of two main subsets

of B cells - circulating memory B cells and long-lived plasma cells that provide protective

immunity. Analysis of the phenotype and frequency of B cell subsets from individuals at

different stages of EBV infection showed a significant reduction in the total B cell

population and in the memory B cell subset during acute IM. The frequency of circulating

memory B cells remained low even after recovery when compared to samples from

asymptomatic virus carriers. Moreover, the conversion efficiency of memory B cells to

antibody-secreting plasma cells following in vitro activation was significantly compromised

in individuals during and post-IM. Furthermore, IM patients also showed dysregulated

expression of tumour necrosis factor family members BAFF and APRIL and increased

FAS and FAS-L expression on circulating B cells and plasma respectively. Finally, in vitro

studies showed that the upregulation of FAS on B cells was intrinsically linked to IL-12

which is significantly elevated in the plasma of acute IM patients. These studies provide an

important mechanistic insight on the global B cell immunity following primary EBV infection

and may help us to design better prophylactic or therapeutic strategies to reduce the

disease burden due to primary EBV infection.

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3.2. INTRODUCTION

Humoral immune response plays a critical role in providing protection against

infection with viral pathogens. Two subsets of B cell memory—long-lived plasma cells in

bone marrow and memory B cells in the peripheral lymphoid systems—are essential for

protective immunity. Along with CD8+ and CD4+ memory T cells, they constitute

immunological memory. Following antigen encounter in secondary lymphoid organs, naïve

B cells mature into both plasma cells and memory B cells. Plasma cells transit through the

peripheral blood as plasmablasts and antibody-secreting cells (ASCs) then establish in the

bone marrow. Memory B cells circulate in the peripheral lymphoid system. While long-lived

plasma cells maintain high-affinity antibody levels in serum, memory B cells are capable of

rapid response to a secondary infection, converting to plasma cells that secrete antibodies

with high affinity (Slifka, Antia et al. 1998, Manz and Radbruch 2002, Purtha, Tedder et al.

2011). Both plasma cells and memory cells are, therefore, critical mediators in the

establishment of effective humoral immunity against viral challenge.

Recent observations have also revealed that B cell survival and maturation are

mediated by the B cell activation factor (BAFF) and its homolog APRIL (Schneider,

MacKay et al. 1999). Both BAFF and APRIL are TNF-family ligands and are produced

mostly by myeloid cells (Nardelli, Belvedere et al. 2001), including monocytes,

macrophages and dendritic cells, as well as by some non-lymphoid cells (Mackay and

Browning 2002). BAFF has three receptors on B cells—BAFF-R, TACI and BCMA—and it

binds to them with varying degrees of affinity (Day, Cachero et al. 2005). APRIL, however,

binds only to TACI and BCMA. BAFF-R is expressed on most B cell subsets and is found

to be the central receptor involved in B cell development, maturation and survival, while

TACI and BCMA are expressed on mature B cells (Laabi, Gras et al. 1994, Ng, Sutherland

et al. 2004, Darce, Arendt et al. 2007). Differential expression of BAFF-R has been shown

to be associated with B cell dysfunction, increased susceptibility to apoptosis (FAS

receptor expression) and reduced B cell numbers in a number of clinically important

settings, including HIV-viremic patients and patients with acute malaria infection (Moir,

Malaspina et al. 2004, Nduati, Gwela et al. 2011). Dysregulation in BAFF/BAFF-R

expression has also been associated with some B cell-related autoimmune diseases,

including Sjogren’s syndrome and systemic lupus erythematosus, both of which have been

associated with EBV infection (Sellam, Miceli-Richard et al. 2007).

Despite our thorough understanding of the role of B cell immunity in protection

against most viral pathogens, research into the impact of EBV infection on the B cell

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compartment has centred on B cells as the primary source of EBV infection, while little

attention has been paid to the induction of B cell immunity during primary EBV infection, or

the impact of EBV infection on the B cell compartment. The aim of this chapter is,

therefore, to investigate the impact of acute symptomatic EBV infection on the peripheral B

cell compartment. To explore the impact of acute EBV infection on B cells, longitudinal

analysis of the phenotype and frequency of B cells in peripheral blood was investigated in

a cohort of patients with acute IM, and in healthy latent virus carriers. This aim also studied

the impact of acute EBV infection on the expression of the BAFF receptors and their

ligands, and on the expression of FAS. Several cytokines are reported to be elevated

during EBV primary infection, IM and contribute to the symptoms of IM (Balfour, Odumade

et al. 2013). The influence of various cytokines on the survival of B cells was studied by in

vitro assay.

There was very little evidence of the activation of a strong B cell response in the

peripheral blood of IM patients, as the frequency of ASC remained low during IM, unlike

other acute viral infections (Doria-Rose, Klein et al. 2009, Lee, Falsey et al. 2010,

Wrammert, Koutsonanos et al. 2011, Wrammert, Onlamoon et al. 2012). There was a

reduction in the absolute number of peripheral B cells and in the proportion of memory B

cells in the blood. This loss of memory B cells in peripheral blood was associated with

poor in vitro conversion of memory B cells into ASCs, and with apparent dysfunction in the

expression of both BAFF-R and FAS on peripheral B cells.

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3.3. MATERIALS AND METHODS

3.3.1. Flow Cytometry Analysis of B Cells

The phenotypic analysis of B cells in PBMC was performed ex vivo using Flow

Cytometry. Specific human B cell markers for antibody-secreting cells (ASC) and memory

cells conjugated to flurophores were used as previously reported (Crotty, Aubert et al.

2004, Wrammert, Smith et al. 2008). The frequency and phenotype of ASCs and memory

cells were analysed using the following surface markers: CD3-PE Cy7, CD27-APC and

CD38-PerCp-Cy5.5, CD19–eFluor450 and CD20–FITC. The cells were washed with PBS

2% FCS and incubated with surface markers for 30 min at 4oC. The cells were fixed with

2% paraformaldehyde (Sigma Aldrich) in PBS. Data were acquired on a FACS Canto Flow

cytometer and analysed using FlowJo software.

To determine the differences in frequency of B regulatory cells in donors at various

stages of EBV infection, phenotypic analysis of B regulatory cells was done using flow

cytometry. The phenotype of Breg cells have been reported to be CD3-/CD19+ B cells that

express high CD24 and CD38 surface markers (Blair, Norena et al. 2010). The staining

was done using surface markers CD3–PECy7, CD19–eFluor450, CD24- PE and CD38

PerCpCy5.5 and data acquired and analysed by FACS Canto Flow cytometer and FlowJo

software.

Flow cytometric analysis was also used to study the expression of receptors of BAFF

(B cell activation factor) on B cells, as well as the expression of death receptor FAS on B

cells. BAFF binds to three receptors on B cells: BAFF-R, TACI and BCMA. PBMC were

stained with CD3-Alexa Fluor 700, CD19-eFluor450, CD27-APC-H7, BAFF-R-FITC, TACI-

APC, BCMA-PE and Fas-PECy7.

3.3.2. Conversion of Memory B Cells to Antibody-Secreting Cells

PBMC (5x105 cells/well on a 24-well plate) were cultured in R10 with 50 uM –

mercaptoethanol for six days. The medium was supplemented with an optimized mix of

polyclonal mitogens, which included pokeweed mitogen extract (PWA) at 1/100,000

dilution, Protein A from Staphylococcus aureus, Cowan strain (SAC) at 1/10,000 dilution,

CpG ODN 2006 at 2.5 ug/ml (Sigma Aldrich) (Crotty, Aubert et al. 2004) and IL-10 at 25

ng/ml (BD Biosciences) (Weiss, Traore et al. 2010).

The frequency and phenotype of antibody-secreting cells (ASCs) converted from

memory B cells were analysed using the following surface markers: CD3-PE Cy7, CD27-

APC, CD38-PerCp-Cy5.5, CD19-eFluor450 and CD20-FITC. Cells were washed with PBS

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2% FCS and incubated with surface markers for 30 min at 4oC. The cells were fixed with

2% paraformaldehyde (Sigma Aldrich) in PBS before analysis. Data were acquired on

FACS Canto Flow cytometer and analysed by FlowJo software.

3.3.3. B Cell ELISpot

Memory B cells in PBMC were quantified using the method developed by Crotty et al

(2004), in which memory B cells are converted to antibody-secreting cells in vitro. PBMC

were thawed and incubated at 37oC in a 24-well plate at 5x105 cells/ well for six days. The

cells were suspended in R10 with 50 uM -mercaptoethanol).The medium was

supplemented with an optimized mix of polyclonal mitogens, which included pokeweed

mitogen extract (PWA) at 1/100,000 dilution, Protein A from Staphylococcus aureus,

Cowan strain (SAC) at 1/10,000 dilution, CpG ODN 2006 at 2.5 µg/ml (Sigma Aldrich)

(Crotty, Aubert et al. 2004) and IL-10 at 25 ng/ml (BD Biosciences) (Weiss, Traore et al.

2010).

The antigen was immobilized on a 96-well ELISpot plate (Millipore Multiscreen HTS

IP) and incubated at 4oC overnight. The plate was coated with 5 µg/ml of capture anti-IgG

(Mabtech) for measuring the frequency of total IgG-secreting B cells. On the next day, the

plates were washed with PBS and blocked with R10 for 2–4 h at room temperature. The in

vitro-activated cells were washed and 3000 cells/well were plated in triplicate in antigen-

coated wells for the detection of total IgG secreting cells. The plates were then incubated

at 37oC overnight. The following day cells were washed with PBS containing 0.05%

Tween–20 (PBST) and incubated with 1:500 biotinylated anti-IgG (Mabtech) for 2 h at

room temperature followed by incubation with 1:1000 Streptavidin–AP (Sigma Aldrich) for

1 h at room temperature. The plates were then incubated with Sigma Fast BCIP/NBT at

room temperature until spots developed, following which the reaction was stopped by

washing with water. Spot quantification was done using an ELISpot reader (AID

Diagnostika).

3.3.4. Enzyme Linked Immunosorbent Assay (ELISA)

ELISA for BAFF/APRIL was done using ELISA kits (Human BAFF Quantikine ELISA

kit from R&D Systems and LegendMax Human APRIL ELISA kit from Biolegend)

according to manufacturer’s instructions. All reagents were brought to room temperature

before the start of the assay. Standards were prepared as per manufacturer’s instructions.

The plates were washed with wash buffer four times. Assay buffer, standards and samples

were added to their respective wells and sealed with adhesive strips and incubated at

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room temperature on a microplate shaker for 2–3 h. The plates were washed and BAFF

conjugate or APRIL detection antibody was added and the plates incubated at room

temperature for 1 h with shaking. BAFF ELISA was continued by adding substrate solution

and incubating for 30 min. APRIL ELISA was continued by incubating with Avidin–HRP

solution for 30 min followed by substrate solution for 15 min, with washing between each

step. The substrate reaction was stopped by the addition of stop buffer and the

absorbance read at 450 nm immediately using a spectrophotometer.

3.3.5. Cytometric Bead Array (CBA) Analysis

The plasma samples from 10 longitudinal donors during and post-IM were used to

quantify the cytokine levels present during EBV infection and compared with asymptomatic

latent virus carriers. BD Cytometric bead-based multiplex assay kit (BD Biosciences) was

used to measure FAS L in the plasma. The assay was done according to the

manufacturer’s instructions, with minor adjustments. A lyophilised standard vial from the

flex set was reconstituted with 4 ml assay diluents and allowed to equilibrate for 15 min

before aliquoting and storing at –80oC. An aliquot of standard was used to set up 8x2–fold

dilutions of standards in assay diluents. The capture bead mix was made by adding 0.1 µl

of each bead per test. Each capture bead stock vial was vortexed for 15 s to resuspend

the beads thoroughly and then resuspended using capture bead diluents for

serum/plasma. The assay was set up in a 96–well V-bottom plate by mixing 12 µl of mixed

capture beads and 10 µl of standard or sample. The plate was then incubated at room

temperature on a shaker for 1 h. PE detection mix was also made similar to capture bead

mix using detection diluents. After 1 h incubation, 12 µl of mixed PE detection reagents

were added and incubated for another 2 h at room temperature on a shaker. The plate

was washed with 175 µl wash buffer supplied in the kit and centrifuged at 200 g for 5 min.

The beads were then resuspended with 300 µl wash buffer for analysis. The data were

acquired on BD FACS LSRII flow cytometer and analysed on FCAP Array software.

3.3.6. Annexin V Staining

To determine the percentage of B cell apoptosis, Annexin V binding assays were

performed using Annexin V kit (BD Biosciences). Annexin V was used in combination with

a dead cell stain (Live/Dead Fixable Aqua Dead Cell Stain from Life Technologies) to

distinguish between dead and apoptotic cells. PBMC were washed with 1X Annexin

binding buffer (working concentration made by diluting one part of 10X Annexin V binding

buffer provided in the kit with nine parts distilled water) following staining with B cell

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markers. The B cell staining panel included CD3-Alexa Fluor 700, CD19- eFluor450, CD27

APC-H7, BAFF-R–FITC, TACI–APC and Fas–PECy7. After staining with the dead cell

stain and surface markers, the cells were incubated with 1 µl Annexin V (PE) in 100 µl

Annexin binding buffer for 15 min at room temperature in the dark. The cells were then

analysed using flow cytometry within 1 h of staining.

3.3.7. Cytokine Influence and FAS/FAS L Killing

To determine the mechanism responsible for reduced B cell numbers during acute

IM, the possibility of FAS-mediated apoptosis was explored in vitro. The cells were

cultured in serum free OpTmizer medium and various cytokines (R&D Systems) that were

upregulated in infectious mononucleosis samples were added individually at

concentrations of IL-4 (10 ng/ml), IL-6 (10 ng/ml), IL-10 (500 pg/ml), IL-12 (10 ng/ml), IFN-

(100 u/ml), TNF-(50 ng/ml) and TGF- (10 ng/ml). Culturing of cells with the addition of

IL-12 resulted in an increased expression of FAS on B cells. To induce cell death in vitro,

FAS-upregulated B cells were incubated with soluble FAS L (R&D Systems), FAS-L

aggregate made by incubating 100ng/ml of sFAS-L with N-terminal His tag with1g/ml of

anti-His antibody (R&D Systems), or anti-CD3 stimulated T cells that express high

membrane bound FAS-L. Cell viability was determined by flow cytometry. The cells were

stained with CD3/CD19/ Live/Dead marker and Annexin V and the percentage of CD3-

/CD19+ B cells that were Annexin V positive were determined as apoptotic cells.

Purified NA/LE mouse anti-human CD3 (BD Biosciences) at 2.5g/ml was coated on

48-well plate overnight at 4oC. PBMC (1x106) were added the next day and incubated for

three days for the activation of T cells. The activated T cells expressing high FAS-L was

then mixed with an equal number of PBMC cultured in IL-12 for three days. The PBMC

cultured with IL-12 were labelled with CFSE dye prior to culturing with IL-12 for three days

to label cells expressing high FAS. The cells were then incubated for 6 h and CFSE

labelled CD3-/CD19+ B cells were analysed for apoptotic cells by flow cytometry.

3.3.8. Statistical Analysis

Statistical analysis was performed using Prism 6, GraphPad Software. The data are

presented as mean +/- SEM or as percentages. Statistical significance was determined

using one way analysis (ANOVA) for comparison of more than two groups. The Wilcoxon

signed-rank test was used to compare measurements at two time points for the same

individual and comparison of different groups was done by the Mann Whitney test.

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3.4. RESULTS

3.4.1. Phenotype and Frequency of B Cell Subsets

It has recently been observed in a number of clinical settings that there is

perturbation in the peripheral blood B cell compartment during acute viral infections, and

that this is often associated with an expansion in the number of ASCs that are mostly

specific to the invading pathogen (Doria-Rose, Klein et al. 2009, Wrammert, Koutsonanos

et al. 2011). Therefore, in order to explore the impact of acute EBV infection on the

peripheral blood B cell compartment, the phenotype and frequency of B cell subsets were

analysed at different stages of EBV infection.

Figure 3. 1. Detection of circulating B cell subsets in ex vivo PBMC

Representative dot blot showing the gating strategy for ex vivo B cell subsets using PBMC from a

healthy virus carrier. Total B cells are gated as CD3-/CD19

+ cells, memory B cells as CD3

-

/CD19+/CD20

+/ CD27

+ cells, and antibody-secreting cells (ASC) as CD3

-/CD19

+/CD20

lo/CD27

hi and

CD38hi

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Peripheral blood mononuclear cells from a panel of 22 patients with acute EBV

infection, 12 patients during the early recovery phase (20-50 days or > six months from the

onset of symptoms), 30 long-term recovery patients (six months or more from the onset of

symptoms) and 17 age-matched long term virus carriers were assessed for the frequency

of total B cells, memory B cells (MBC) and antibody-secreting cells (ASCs).

Representative analysis of the gating strategy for an individual is shown in Figure 3.1.

Total B cells were defined as CD3-CD19+ cells, while MBC were identified by the co-

expression of CD20 and CD27 in CD19+ B cells. ASCs were identified, as previously

reported, based upon CD27 and CD38 expression in CD20lo B cells (Doria-Rose, Klein et

al. 2009). Somewhat unexpectedly, this analysis revealed that although B cells are the

primary source of EBV infection—and it has been reported that EBV-infected B cells

constitute up to 1% of peripheral B cells during IM—there was a significant reduction in the

proportion of CD3-CD19+ B cells (Figure 3.2A) during acute primary EBV infection.

Although not significant, there was also a reduction in the absolute number of B cells in

peripheral blood of the majority of IM patients during acute IM (Figure 3.2B).

Figure 3. 2. Ex vivo characterization of total B cells in PBMC and whole blood

Panel A: Analysis of 16 longitudinal samples from donors during and post IM to study the frequency of total B cells as CD3-

/CD19+ cells. Statistical analysis was done by Wilcoxon matched pair test for comparison within group with significant p value as *** p<0.001.

Panel B: Absolute count for total B cells is expressed as total PBMC count per litre of blood at time of harvest, 106cells/litre. 13 longitudinal samples from donors during and post IM were analysed.

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Following long-term convalescence, the proportion of peripheral B cells returned to

normal (Figure 3.3A). Analysis of the proportion of MBC in the total B cell population also

revealed a significant reduction during acute IM (Figure 3.3B). Interestingly, while MBC

numbers gradually recovered during convalescence, they still remained significantly lower

than the numbers for latent virus carriers, even after long-term recovery. Although

significant changes were evident in both total B cells and MBC populations during acute

infection, unlike other primary infections there was no evidence of a change in frequency

of ASCs in the peripheral blood of acute IM patients (Figure 3.3C).

Figure 3. 3. Ex vivo characterization of B cell subsets in PBMC during EBV infection

Panels A, B, C: Compilation of all data analysed from donors at different stages of EBV infection, with PBMC from 22 acute IM donors, 12 recovering post IM donors (20–50 days after onset of symptoms, post IM < 6months), 30 post-IM donors more than 6 months since start of symptoms, and 18 healthy virus carriers. PBMC were incubated with fluorescently labelled antibodies specific for CD3, CD19, CD20, CD27 and CD38 then assessed for the frequency of total B cells, MBCs and ASCs by flow cytometry

Panel A: ex vivo analysis of CD3-/CD19+ total B cells

Panel B: CD3-/CD19+/CD20+/CD27+ Memory B cells

Panel C: CD3-/CD19+/CD20lo/CD27hi and CD38hi antibody-secreting cells (ASCs).

Data shows mean, range and data points; statistical analysis was done by one-way ANOVA, Tukey’s multiple comparison with **** p<0.0001, *** p<0.001, ** p<0.01 and * p<0.05 and ns not significant.

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To characterise the memory B cells present in the blood of acute IM patients, PBMC

from donors at different stages of EBV infection were cultured for six days in the presence

of polyclonal mitogens that induce the differentiation of circulating memory B cells into

ASCs. Figure 3.4 shows the gating strategy from a representative sample where the

forward and side scatter plots for the lymphocyte gate were extended to include activated

B cells (Doria-Rose, Klein et al. 2009).

Figure 3. 4. Analysis of in vitro-activated PBMC

Representative dots blot showing the gating strategy of in vitro-activated PBMC. The PBMC were cultured with an optimized mix of polyclonal mitogens — pokeweed mitogen extract (PWA) at 1/100,000 dilution, Protein A from Staphylococcus aureus, Cowan strain (SAC) at 1/10,000 dilution, and CpG ODN 2006 at 2.5 µg/ml and IL-10 at 25 ng/ml — for six days. Using data gathered by flow cytometry, forward and side scatter plots for the lymphocyte gate are extended to include activated lymphocytes. Frequency of total B cells CD3-/CD19+ and antibody-secreting cells

as CD3-/CD19

+/CD20

lo/CD27

hi/CD38

hi are calculated.

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The efficiency of conversion of MBC to ASCs was studied by comparing the PBMC of

donors at different stages of EBV infection. Consistent with the ex vivo observations

showing a lack of ASCs in the peripheral blood of IM patients, conversion of MBC to ASCs

during acute EBV infection was significantly impaired (Figure 3.5A). To confirm these

observations, in vitro activated B cells from IM patients and healthy virus carriers were

assessed for the production of IgG using a B cell ELISpot assay. Consistent with the poor

in vitro conversion into ASCs, B cells cultured from patients acutely infected with EBV,

displayed a significant reduction in IgG production that recovered to normal levels

following disease resolution (Figure 3.5B). These observations provide further support for

significant perturbations in the B cell compartment during acute IM that result in low B cell

numbers and reduced conversion efficiency of memory B cells to antibody-secreting cells.

Figure 3. 5. Frequency of ASCs, total IgG-secreting cells and correlation with severity of infection

Panel A: Accumulated data from the conversion of circulating memory B cells in blood to antibody-secreting cells after six days’ culture in the presence of polyclonal mitogens. Data from donors at different stages of EBV infection are compared. Data are represented as percentages and statistical analysis was done by one-way ANOVA, Tukey’s multiple comparison with **** p<0.0001, *** p<0.001, ** p<0.01 and * p<0.05.

Panel B: Using B cell ELISpot analysis, the frequencies of IgG-secreting cells per million PBMC are compared for donors during and post IM, and healthy virus carriers. The p value was obtained by one-way ANOVA, Tukey’s multiple comparison with **** p<0.0001.

Panel C: Correlation of frequency of IgG-secreting cells with severity of infection. Severity of infection (SOI) scores assigned to acute IM patient donors ranged from zero to six, with asymptomatic infection being assigned a score of zero and severe symptoms with confinement to bed a score of six. The samples were grouped according to the severity of infection, with scores 0–3 being classified as mild infection and 4–6 as severe infection.

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The severity of EBV-infection has been recently shown to correlate with a number of

factors, including the viral load in peripheral blood and the peripheral CD8+ T cell numbers

(Balfour, Odumade et al. 2013). To determine if the abovementioned B cell defects are

associated with disease severity, a correlation between the severity of infection and

frequency of total IgG-secreting B cells was explored. Acute IM samples were scored from

mild to severe with scores ranging from 0 for asymptomatic infection, to 6 for severe illness

requiring confinement to bed or hospitalization. These samples were grouped, with scores

0–3 classified as mild symptoms and 4–6 as severe symptoms, and the results were

compared against the frequency of IgG-secreting B cells. Although not significant, likely

due to the low cohort size in this analysis, a trend was observed where increasing severity

of symptoms corresponded inversely with the incidence of IgG-secreting cells (Figure

3.5C).

In addition to assessing the frequency of memory B cells and ASC populations during

IM, the frequency of a recently-discovered B cell subset—B regulatory cells—were also

assessed for IM patients at different stages of acute EBV infection. B regulatory cells

(Bregs: CD3-CD19+CD24hiCD38hi) are known to contribute to maintaining immune

tolerance by balancing the Treg and Th1/Th17 populations (Flores-Borja, Bosma et al.

2013). They also are known to play an important role in autoimmunity, allergy, cancer, and

post transplantation (Blair, Norena et al. 2010, Newell, Asare et al. 2010, Schioppa, Moore

et al. 2011, Carter, Rosser et al. 2012). Since EBV infection—and a history of IM in

particular—have been implicated in various autoimmune diseases, a comparison was

done of the frequencies of Bregs during different stages of EBV infection to understand

their function during IM. Figure 3.6A shows the gating strategy for Breg cells as CD3-

/CD19+ B cells that express high levels of CD24 and CD38. A comparison of the frequency

of these cells in patients during IM and after recovery, or in healthy virus carriers, showed

no statistically significant differences (Figure 3.6B). These observations suggest that Bregs

are unlikely to play a significant role during symptomatic primary EBV infection.

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Figure 3. 6. Frequency of B regulatory cells during EBV infection

Panel A: The difference in frequencies of B regulatory cells (Bregs) at different stages of EBV infection was measured by flow cytometry. Representative dot plot shows the gating strategy for Breg cells as CD3-/CD19+ B cells that express high CD24 and CD38. PBMC were incubated with fluorescently labelled antibodies specific for CD3, CD19, CD24 and CD38 and analysed.

Panel B: Compilation of all data from donors with 13 acute IM samples, 12 recovering post IM samples (20–50 days after onset of symptoms), 11 post-IM samples more than six months since start of symptoms, and 10 healthy virus carriers are compared. Data are represented as percentage of CD3-/CD19+ B cells and statistical analysis was done by one-way Anova, Tukey’s multiple comparisons, with ns meaning no significant difference between donor groups.

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3.4.2. The Mechanism of Reduction of B Cell Frequency

Data presented above suggested that acute EBV infection is associated with global B

cell dysfunction. To explore the possibility whether direct infection of B cells with EBV

impacts upon the conversion of memory cells to ASCs, PBMC from seven healthy

volunteers were infected with EBV or mock infected for 6 h. These cells were washed and

incubated with mitogens to convert memory B cells to antibody-secreting cells. ELISpot

analysis of these donors was then performed to measure the IgG production and

compared between infected and uninfected samples. ELISpot analyses showed

comparable production of IgG between the two sets of samples, infected and mock treated

(Figure 3.7).

Figure 3. 7. Effect of EBV infection on conversion of MBC to ASC

PBMC from healthy donors were infected with EBV B95-8 and used for conversion of memory B cells to antibody-secreting cells. The frequency of total IgG secreting cells was quantified and compared with mock treated PBMC by B cell ELISpot assay.

To investigate other potential mechanism that results in reduced frequency of MBC

and total B cells during IM, the concentration of BAFF and its homolog APRIL and

expression of its receptors (BAFF-R, TACI and BCMA) on B cells were analysed. PBMC

were stained with antibodies specific for BAFF-R, BCMA and TACI and its expression on

CD3-/CD19+ B cells was assessed using flow cytometry. BAFF receptor expression on B

cells was analysed using PBMC from donors undergoing acute IM and follow-up samples

after six months of recovery. A longitudinal analysis of B cells from 12 IM patients showed

that the expression of BAFF-R on B cells is significantly decreased during acute IM, with

improved expression on disease resolution (Figure 3.8A). The percentage of B cells

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expressing TACI on CD19+CD27+ memory B cells was found to be similar between

different cohorts of EBV-infected individuals (Figure 3.8B). However, a significant increase

in BCMA expression was observed on B cells from acute IM patients when compared to

age-matched asymptomatic controls. Furthermore, the mean fluorescence intensity (MFI)

of BCMA expression was found to be significantly higher during acute EBV infection when

compared to latent infection (Figure 3.8C). This reversal in expression pattern of BAFF-R

and BCMA during acute IM could also suggest defective BAFF signalling that could have

an effect on the survival of B cells as no increase in ASCs was noticed during acute IM.

Figure 3. 8. Expression of TNF receptor superfamily. BAFF/APRIL receptors, during EBV infection

Panel A: Expression of the main B cell activation factor receptor (BAFF-R) on B cells measured as the mean

fluorescence intensity MFI by flow cytometry. A total of 15 longitudinal samples from donors during and post IM, and 12 healthy virus carriers, were analysed and compared.

Panel B: The expression of TACI, which is a common receptor for both BAFF and APRIL were measured on memory B cells (CD19+/CD27+ cells). PBMC from 6 longitudinal IM samples and 12 health carriers were analysed for TACI expression.

Panel C: The expression of APRIL- specific receptor BCMA was measured as the mean fluorescence intensity

MFI by flow cytometry. 9 longitudinal samples and 12 healthy virus carriers were analysed for BCMA expression.

The data are presented as mean+/- SEM and statistical analysis for the same sample at different time points was done by Wilcoxon analysis, while comparison between different groups was done using Mann-Whitney analysis. The significant p values were ** p<0.01 and * p<0.05.

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In the next set of experiments, the plasma concentration of BAFF and its homolog

APRIL were determined by ELISA using plasma samples from acute IM and age-matched

individuals. Interestingly, plasma BAFF levels appeared to be inversely proportional in

acute IM samples to the expression of BAFF-R. BAFF levels showed a significant increase

during acute IM, with levels decreasing as the disease resolved (Figure 3.9A). Samples

from post-IM and asymptomatic virus carriers showed similar concentration of BAFF in

plasma. Conversely, plasma levels of APRIL were found to be significantly decreased

during acute IM, with levels increasing after disease resolution (Figure 3.9B). Since APRIL

is reported to be critical for the survival of plasma cells in bone marrow (Belnoue, Pihlgren

et al. 2008), the reduced secretion of APRIL during acute IM could also impact upon the

survival of blood ASCs during acute IM. These data provide further support for the

dysregulation of BAFF signalling during acute IM.

Figure 3. 9. Concentration of TNF-family members BAFF and its homolog APRIL

Panel A: Concentration of B cell activation factor (BAFF) as measured by ELISA. Nine longitudinal plasma samples from donors during and post IM were compared to plasma from 10 healthy virus carriers.

Panel B: Concentrations of APRIL were measured by ELISA for 10 longitudinal plasma samples from donors during and post IM and compared to plasma from 11 healthy virus carriers.

The data are presented as mean +/- SEM and statistical analysis for the same sample at different time points was done by Wilcoxon analysis, while comparison between different groups was done using Mann-Whitney analysis. The significant p values were *** p<0.001, ** p<0.01,* p<0.05 and ns as not significant.

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To study whether the reduction in B cell numbers could be associated with an

increased susceptibility to apoptosis, the expression of FAS on B cells was compared in

IM samples and age-matched controls. A longitudinal analysis showed a significant

increase in the expression of FAS on circulating B cells during acute IM with the level

decreasing to reach level similar to age-match control upon disease resolution (Figure

3.10A). Furthermore, the levels of FAS-L were found to be significantly higher in the

plasma of acute IM patients when compared to asymptomatic latent virus carriers.

Following resolution of acute IM symptoms, FAS-L levels reduced to the level observed in

the plasma of asymptomatic latent virus carriers (Figure 3.10B). The increased level of

soluble FAS-L in the plasma of IM patients could be an indication of increased expression

of membrane-bound FAS-L on immune cells that could impact upon B cell survival.

Figure 3. 10. FAS expression on B cells and FAS L concentration in plasma

Panel A: FAS expression on CD19+ B cells from PBMC of 12 longitudinal IM donors and healthy

virus carriers were analysed by measuring the mean fluorescence intensity MFI by flow cytometry.

Panel B: FAS L concentration in the plasma of 10 longitudinal IM donors and 12 healthy virus carriers were measured by cytometric bead array from BD.

The data are presented as mean +/_ SEM and statistical analyses for the same sample at different time points was done using Wilcoxon analysis and comparison between different groups using Mann-Whitney analysis. The significant p values correspond to * p<0.05, **p<0.01, *** p<0.001.

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3.4.3. Influence of Cytokines and FAS-Mediated Killing

One mechanism by which FAS expression might be up-regulated in B cells during IM

is through cytokine mediated signalling. To assess this, PBMC from healthy donors were

incubated with different cytokines shown to be elevated in the plasma of IM patients.

These cytokines included IL-4, IL-6, IL-10, IL-12, IFN- , TNF- and TGF-Following

incubation, FAS expression was assessed on B cells by flow cytometry. Of all the

cytokines tested, exposure to IL-12 for 1-3 days resulted in the increased expression of

FAS on B cells (Figure 3.11A, 3.11B). None of the other cytokines showed an increase in

FAS expression. Next, the possibility of increased apoptosis by these B cells was tested

by the addition of FAS-L. Three different forms of FAS-L was used in the experiments as

monomeric soluble FAS-L was found to be less effective in triggering apoptosis when

compared to FAS-L aggregate or membrane bound FAS-L. FAS upregulated B cells were

incubated separately with soluble FAS-L (monomer or aggregated soluble FAS-L) or

membrane-bound FAS-L and evaluated for apoptosis by annexin V staining. There was no

noticeable increase in apoptotic cells when FAS upregulated cells were compared to

mock-treated cells (data not shown). This suggests that increased expression of FAS

alone is not enough to cause low B cell number, but that in combination with other factors

like low BAFF-R expression, could also impact the survival of B cells during acute IM.

Figure 3. 11. Cytokine influence in the reduction of B cells

Various cytokines reported to be elevated during acute IM were incubated with PBMC from healthy virus carriers in an in vitro setting. The cells were grown in serum-free medium and incubated with individual cytokines at concentrations of IL-4 (10 ng/ml), IL-6 (10 ng/ml), IL-10 (500 pg/ml), IL-12

(10 ng/ml), IFN- (100 u/ml), TNF- (50

ng/ml) and TGF- (10 ng/ml). The cells were cultured for 1–3 days and the expression of FAS on B cells was calculated as mean fluorescence

intensity MFI by flow cytometry.

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3.5. DISCUSSION

Global B cell function, and the frequency and phenotype of B cell subsets during EBV

infection have not been well studied. Understanding the kinetics of B cell subsets, MBCs

and ASCs could provide significant insights into immune control in the early stages of EBV

infection. The focus of studies outlined in this chapter was to quantify the two major B cell

subsets that provide protective immunity. Besides studying the frequency and phenotype

of B cell subsets, these studies also explored the various factors that could impact upon

the survival and function of B cells during acute EBV primary infection.

Symptomatic primary EBV infection is associated with a dramatic expansion of

predominantly effector CD8+ T cells and to a lesser extent NK cells. The research reported

here show that acute EBV infection is associated with a significant decrease in the number

of B cells in the peripheral blood. In this chapter, further evidence is provided

demonstrating that this reduction in B cell numbers in the peripheral blood is associated

with a reduction in the proportion of MBCs during acute IM, and is likely associated with

dysregulation in BAFF/APRIL signalling. These changes are also associated with poor in

vitro conversion of memory B cell populations into ASCs. This observation is

complimented by the report by Svedmyr et al where PBMC from an acute IM patient

showed severe reduction of Ig synthesis when activated by mitogen whilst response of B

cells before the symptomatic infection and after recovery were normal (Svedmyr, Ernberg

et al. 1984). Although it could be argued that the reduction in peripheral B cells is due to

direct infection of B cells by EBV and loss of these B cells in the peripheral blood as a

consequence of either EBV-induced lysis of infected B cells or clearing by EBV-specific T

cells, it has been shown that EBV typically infects around 1% of peripheral B cells during

acute IM (Klein, Svedmyr et al. 1976, Hochberg, Souza et al. 2004), suggesting that the

significant changes in the B cell compartment during symptomatic infection are not likely

due to EBV-induced specific recognition of EBV-infected B cells by EBV-specific CD8+ T

cells. It is also unlikely that direct EBV infection of B cells contributed towards the

inefficient in vitro conversion of MBCs into ASCs as in vitro infection of PBMC from healthy

donors with EBV did not prevent IgG production and was comparable to mock infected B

cells.

In addition to a reduction in the number of B cells in the peripheral blood during acute

EBV infection, there was also no evidence of an increase in the frequency of ASCs during

IM. This is in contrast to elevated ASCs that are evident in the blood during infection with

other viruses like influenza, dengue and respiratory syncytial virus (Lee, Falsey et al. 2010,

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Wrammert, Koutsonanos et al. 2011, Wrammert, Onlamoon et al. 2012). While detection

of antibody responses to EBV- antigens, particularly the viral capsid antigens, during IM

suggests that early ASC formation during the 4-6 week incubation period does occur,

dysfunction in the B cell compartment that is evident during IM suggests that continual

plasmablast formation or their survival may be disrupted during symptomatic infection.

This could potentially have a profound impact on the efficiency of EBV-specific antibody

production following infection, resulting in the poor control of virus.

While the direct mechanism that is leading to this global B cell dysfunction during IM

is not absolutely evident, it is clear that the cytokine milieu may play a role, as was evident

following in vitro observations showing that IL-12 can lead to the induction of FAS-

expression in B cells. Since IL-12 also plays a role in the promotion of B cell differentiation

and plasmablast formation, other mechanisms, such as those inducing dysregulation in

BAFF receptor signalling are also likely important mediators of B cell dysfunction during

acute IM. BAFF-BAFF-R signalling was reported to provide survival signals to B cells in

mice by inducing anti-apoptotic factor Bcl2 and together with B cell receptor (BCR)

regulates B cell survival (Rauch, Tussiwand et al. 2009). Since dendritic cells and CD4 T

cells play a direct role in the development of humoral immunity, these cells could influence

the B cell development and function. The function of these cells will be discussed in the

following chapters so as to present an overall picture of the immune response during acute

IM.

Dysregulation in BAFF signalling have been associated with other diseases,

including autoimmune disorders and primary antibody deficiencies that show a B cell

dysfunction (Sellam, Miceli-Richard et al. 2007, Kreuzaler, Rauch et al. 2012). Some of

these diseases show a strong correlation with EBV infection. Although highly speculative,

it is possible that this B cell dysfunction during acute EBV infection could contribute to its

potential role in B cell-associated autoimmune disorders. However, the role of EBV

infection in these disorders is not absolutely definitive and requires significant further

investigation to determine if the B cell dysfunction seen in both settings is related. It should

also be noted that changes in BAFF signalling are also evident during acute malaria

infection and in HIV infected individuals (Moir, Malaspina et al. 2004, Sellam, Miceli-

Richard et al. 2007, Nduati, Gwela et al. 2011). The reason for the inverse correlation in

the expression of BAFF and its main receptor BAFF-R on B cells during acute IM is not

clear. It could be that high levels of BAFF in the serum of IM patients cause receptor

endocytosis, or that reduced BAFF-R expression on B cells results in an increased BAFF

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level. A decrease in BAFF-R expression and expression of BCMA on peripheral blood B

cells is typically associated with the conversion of memory B cells into plasma cells (Avery,

Kalled et al. 2003); so the reversal in expression pattern of BAFF-R and BCMA during

acute IM could suggest premature maturation of B cells. Decreased expression of BAFF-R

and increased expression of FAS found during acute IM has also been reported in HIV-

viremic patients and has been associated with reduced B cell number during HIV infection

(Moir, Malaspina et al. 2004).

The study of global B cell function during acute IM revealed a previously

unappreciated defect in the peripheral B cell compartment with a reduced frequency of

memory B cells, the absence of antibody-secreting cells and impaired conversion of

memory cells to antibody-secreting cells during acute IM. It also showed a dysregulation

in BAFF signalling which is crucial for B cell survival and increased expression of death

receptor FAS. This change in phenotype of B cells during acute IM could result in

increased susceptibility to apoptosis and explain the reduction in B cell subsets. Therefore

the next aim in this project is to study the impact these apparent B cell defects could be

having upon the induction of an EBV-specific humoral immune response.

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CHAPTER 4. GP350-SPECIFIC B CELL

RESPONSE DURING ACUTE IM

4.1. ABSTRACT

Immunity to most pathogens requires the combined effort of both innate and adaptive

immune responses. During EBV infection, cell-mediated immunity, the CD8 T cell

response in particular, is primarily responsible for controlling infection. While the T cell

response to EBV has been well characterized, B cell and humoral immune responses

have largely been ignored. Antibodies produced against EBV—non-neutralising viral

capsid antigen (VCA) and EBV nuclear antigen (EBNA1)—are used as diagnostic markers

for the detection of EBV infection. It is known that the EBV membrane antigen EBV MA-

gp350 is the major target of the virus-neutralising antibody response after natural infection.

EBV MA-gp350 is the best-characterized vaccine antigen and it has been used in various

preclinical and clinical vaccine trials. While no protection from primary EBV infection has

been detected, neutralisation against gp350 has been found to be effective in reducing the

symptoms of IM. Studies in this chapter showed a defect in global B cell response during

acute infectious mononucleosis (IM). Sequential analysis of quantity and quality of virus-

specific antibody response were measured using B cell ELISpot and ELISA revealed a

reduction in memory B cells and reduced gp350 antibody titres. More importantly, IM

patients also showed a total lack of anti-EBV neutralising antibody response. Taken

together, these studies suggest that a dysregulation of both global and anti-viral B cell

responses is a critical factor in high levels of circulating viral load in acute IM patients.

.

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4.2. INTRODUCTION

The immune response to EBV is, by and large, successful in controlling infection and

ensuring lifelong latency of the virus. However, primary EBV infection can be associated

with severe disease, and is associated with an increased risk of a number of complications

during the latent stage of infection. The role of CD8+ T cells in the control of viral infection

and their contribution to the severity of clinical symptoms during IM are well understood.

The amplified response of CD8+ T cells to EBV causes IM in some, but could also lead to

the clearance of EBV-infected cells in others (Hislop, Annels et al. 2002). Although the

presence of the humoral or antibody response is used in the diagnosis of EBV infection,

the mechanism of B cell response to EBV infection is not well understood. However, it is

known that EBV membrane antigen EBV MA-gp350 is the major focus of virus-neutralising

antibodies after natural infection (Thorley-Lawson and Poodry 1982).

EBV MA-gp350 is required for efficient infection of B cells by EBV. The virus binds to

CD21, the complement receptor 2 (CR2) on B cells (Fingeroth, Weis et al. 1984). Until

recently, CD21 was the only known receptor for gp350, but a recent report stated that

complement receptor 1 (CR1 or CD35) on B cells also binds gp350 (Ogembo, Kannan et

al. 2013). This protein is found in abundance on the envelope of viral particles and on the

surface of EBV-infected cells (Beisel, Tanner et al. 1985). EBV MA-gp350 induces a virus-

neutralising antibody response against EBV after primary infection (Thorley-Lawson and

Poodry 1982). For this reason, the various preclinical vaccine trials in animal models and

clinical trials in humans have used gp350 as the immunogen, and it is the best-

characterized vaccine antigen against EBV (Epstein, Morgan et al. 1985, Morgan, Finerty

et al. 1988, Gu, Huang et al. 1995, Sokal, Hoppenbrouwers et al. 2007). Despite this focus

on gp350 as a vaccine candidate, very little research has been done to study the role of

the gp350-specific response during primary infection.

Interaction between CD4 T cells and B cells in the secondary lymphoid organs is

required for the development of humoral immunity (Garside, Ingulli et al. 1998). TFH cells

are specialized CD4+ T cells that reside in secondary lymphoid organs and support the

development and maturation of B cells (Junt, Fink et al. 2005). Interaction between B cells,

TFH cells and DCs in response to foreign antigens facilitates the formation of germinal

centres in secondary lymphoid organs and differentiation of B cells into plasma cells and

memory B cells (Hardtke, Ohl et al. 2005). Since TFH cells reside mostly in secondary

lymphoid organs like the lymph nodes, alternative approaches in human subjects has been

developed to study circulating memory TFH cells in blood, which have been shown to share

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functional properties with lymphoid TFH cells and correlate with antibody responses

(Breitfeld, Ohl et al. 2000, Morita, Schmitt et al. 2011, He, Tsai et al. 2013, Locci, Havenar-

Daughton et al. 2013).

The primary objective of the studies outlined in this chapter was to investigate the

kinetics of the EBV-specific B cell response in patients undergoing acute IM. To quantitate

B cell response to EBV, resting memory B cells were induced to convert to antibody-

secreting cells by in vitro stimulation using a mix of polyclonal mitogens. EBV membrane

antigen gp350 was used as the immunogen (Crotty, Aubert et al. 2004). This assay was

complemented by ELISA, which measured serum antibody titre during and post infection.

Together, they showed the response of the two arms of humoral immunity which provide

protection—circulating memory B cells in blood and long-lived plasma cells in bone

marrow. In addition, the capacity of antibodies in the plasma of IM patients to neutralise

the virus was also tested and compared by conventional method (Moss and Pope 1972).

The data showed reduced gp350-specific memory B cells, low gp350 antibody titre in

the plasma and no detectable level of neutralising antibody in patients undergoing IM. The

presence of CXCR5+ TFH cells in peripheral blood was found to be very low while CXCR3

expressing CD4+ T helper cell response was found to be the predominant CD4+ T cell

response during IM. EBV membrane antigen gp350 was found to be a very poor T cell

antigen.

4.3. MATERIALS AND METHODS

4.3.1. EBV MA-gp350 Protein Purification

CHO cells secreting truncated EBV MA-gp350 were kindly provided by Prof. Jaap

Middeldorp (Hessing, van Schijndel et al. 1992). The cells were grown to high density in

DMEM/Ham’s F12 (Life Technologies) at 1:1 ratio containing 10% heat-inactivated FCS,

100 units/ml of penicillin and 100 µg/ml of streptomycin. The cells were maintained in

serum-free DMEM/F12 medium and the secreted protein was collected and concentrated

using Amicon Ultracel-50K (Millipore).

The concentrated protein was then purified using con A sepharose 4B column (GE

Healthcare). It was dialyzed using snake-pleated dialysis tubing (Thermo Fisher) in binding

buffer (20 mM Tris–HCl pH 7.4, 0.5 M NaCl in water). The con A sepharose column was

equilibrated overnight and washed with three times the column volume of binding buffer.

The dialyzed protein in binding buffer was loaded onto the column and the flow-through

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was collected. The protein was reloaded onto the column three more times to facilitate

maximum protein retention. The column was then washed with six times the column

volume of wash buffer (binding buffer with 25 mM –D methyl glucoside) and eluted with

increasing concentration of –D–methyl glucoside (ACROS Organics, Thermo Fisher).

4.3.2. Dot Blot

This protocol was used to determine the presence of EBV MA-gp350 protein after its

expression and purification. On a strip of nitrocellulose membrane, the position of each

sample was marked as a circle with pencil. 10 µl of different samples from protein

purification (flow-through, wash and elution samples with different concentration of –D–

methyl glucoside), as well as positive and negative controls, were blotted on the

nitrocellulose membrane. Using a narrow-mouth pipet tip, 1 µl of the sample was spotted

each time to the centre of the circle. The membrane was allowed to dry for 1 h at room

temperature and blocked using 5% dry milk in PBST (PBS with 0.05% Tween–20). After 1

h of blocking, the membrane was incubated with OT6 mAb to gp350, a gift from Prof.

Middledorp, at 1:3000 dilutions in PBST at room temperature on a rocker. The membrane

was incubated with a secondary antibody after washing with PBST. Goat anti-mouse HRP

(Southern Biotech) was used as the secondary antibody at 1:3000 dilution and incubated

for 1 h at room temperature. The protein was detected using western lightning plus-ECL

(Perkin-Elmer), exposed on an x-ray film and developed in the dark.

4.3.3. SDS–PAGE and Western Blotting

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed to

confirm the presence of the protein EBV MA-gp350 after its purification by lectin

chromatography. The purified protein was analysed using a 6% SDS–PAGE based on the

molecular weight of the protein. A 6% gel (prepared from 30% acrylamide:bisacrylamide

stock solution, 0.4 M Tris–HCl pH 8.8 ,0.1% SDS, TEMED and ammonium persulphate

(APS) was loaded with molecular weight marker (Precision Plus Protein Standards from

Bio-Rad) and different concentration of the protein in reducing buffer (50 mM Tris0HCl pH

6.8, 50% Glycerol, 5% SDS, 0.05% BromoPhenolBlue and 0.25 M DTT). The samples

were prepared in reducing buffer and incubated at 95°C for 5–10 min before loading on the

gel. Electrophoresis was done on a Mini-Protein II cell gel apparatus (Bio-Rad) at room

temperature at 24 Amp for I h and stained with Coomassie Brilliant Blue (0.25%

Coomassie Blue, dissolved in 10% acetic acid and 50% methanol). Tris-Glycine buffer (25

mM Tris, 0.25 M Glycine and 0.1% SDS) was used as running buffer for electrophoresis.

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After electrophoresis, the protein was transferred to a nitrocellulose membrane,

Hybond-C (Amersham). The transfer was conducted in the cold room at 100 V for 1 h. The

membrane was blocked with 5% non-fat dry milk (Bio-Rad) and developed with gp350

mAb EBV OT6 provided by Prof. Middledorp (Hessing, van Schijndel et al. 1992) with a

1:3000 dilution for 1 h at room temperature, followed by incubation with secondary

antibody goat anti-mouse HRP (Southern Biotech) at 1:3000 dilution. The protein was

visualized by Western Lightning Plus-ECL (PerkinElmer) using an X-ray film.

4.3.4. Protein Concentration Estimation-Bradford Assay

The protein concentration was estimated using Quickstart Bradford dye reagent (Bio-

Rad). Protein standards were made from 2 mg/ml BSA (Sigma) and PBS was used as

blank. In a 96-well flat-bottom plate, seven serial dilutions of protein standards were made

in duplicate. The measurement was taken in a Spectrophotometer and each sample was

analysed in duplicate.

4.3.5. Endotoxin Test

The protein gp350, both homemade and commercial, were tested for Endotoxin

using ToxinSensor Endotoxin Detection System from GenScript. The sample pH was

between 6 and 8. Reagents were prepared in accordance with the manufacturer’s

instructions. Limulus Amebocyte Lysate (LAL) was reconstituted by adding 1.7 ml LAL

reagent. The chromogenic substrate was reconstituted similarly. Colour stabilizers 1, 2 and

3 were reconstituted in 10 ml of Stop solution for 1 and LAL water for 2 and 3. Standard

endotoxin solution was dissolved in 1 ml LAL water to get 6 EU, from which 1 EU/ml was

used to make dilutions for making the standard curve. The standard concentrations were

0.1, 0.04, 0.02, 0.01 and 0.005.

100 µl of standards, samples and blank were dispensed into endotoxin vials. 100 µl

of LAL was added to each vial and mixed by vortexing. The tubes were incubated at 37oC

for 45 min. After incubation, 100 µl of chromogenic substrate was added to each vial,

mixed gently and incubated for another 6 min at 37°C. This was followed by the addition of

500 µl of colour-stabiliser #1, followed by 500 µl each of colour-stabilizer #2 and #3, with

mixing between each addition. The absorbance of each reaction at 545 nm was read on a

spectrophotometer.

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4.3.6. B Cell ELISpot

Memory B cells in PBMC were quantified using the method developed by Crotty et al

(2004), in which memory B cells are converted to antibody-secreting cells in vitro. PBMC

were thawed and incubated at 37°C in a 24-well plate at 5x105 cells/ well for six days. The

cells were suspended in R10 (RPMI1640 with penicillin/streptomycin 100 IU/ml, 10% heat-

inactivated FCS, 50 µM -mercaptoethanol).The medium was supplemented with an

optimized mix of polyclonal mitogens, which included pokeweed mitogen extract (PWA) at

1/100,000 dilution, Protein A from Staphylococcus aureus, Cowan strain (SAC) at 1/10,000

dilution, CpG ODN 2006 at 2.5 µg/ml (Sigma Aldrich) (Crotty, Aubert et al. 2004) and IL-10

(BD Biosciences) at 25 ng/ml (Weiss, Traore et al. 2010).

The antigen was immobilized on a 96-well ELISpot plate (Millipore Multiscreen HTS

IP) and incubated at 4°C overnight. The plate was coated with 25 g/ml of gp350,

recombinant EBV membrane antigen expressed from CHO cells (Hessing, van Schijndel

et al. 1992), a control EBV non-neutralising structural antigen VCA (Gen Way Biotech),

and 10 µg/ml of tetanus toxoid (CSL) as a general antigen control. To estimate total IgG

secreting cells, 5 µg/ml of capture anti-IgG (Mabtech, Resolving Images) was used. On the

next day, the plates were washed with PBS and blocked with R10 for 2–4 h at room

temperature. The in vitro activated cells were washed and plated in triplicate to antigen-

coated wells at 3x105 cells/ well in order to detect antigen-specific antibody-secreting cells

(ASC). For the detection of total IgG-secreting cells, 3000 cells/well were plated in

triplicate to the wells containing capture anti-IgG. The plates were then incubated at 37°C

overnight. The following day, the excess cells were washed with PBS containing 0.05%

Tween-20 (PBST) and incubated with 1:500 biotinylated anti-IgG (Mabtech) for 2 h at room

temperature followed by incubation with 1:1000 Streptavidin-AP (Sigma Aldrich) for 1 h at

room temperature. The plates were then incubated with Sigma Fast BCIP/NBT (Sigma

Aldrich) at room temperature until spots developed, following which the reaction was

stopped by washing with water. Spot quantification was done using an ELISpot reader

(AID Diagnostika).

4.3.7. Fluorolink Cy5 Labelling

1 mg/ml gp350 protein was used for labelling with the Fluorolink Cy5 labelling kit from

GE Healthcare. The protein was mixed with sodium carbonate buffer pH 9.3, followed by

incubation with the dye, pre-weighed ready to label and supplied in the kit. The reaction

was incubated for 30 min at room temperature, with additional mixing every 10 min. The

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unconjugated dye was separated from the labelled protein using a gel filtration system that

was provided by the manufacturer.

CD19+ B cells were enriched from the PBMC of EBV sero-positive and sero-negative

donors by positive selection with an autoMACS separator. 20 µl of CD19 microbead was

incubated per 107 PBMC for 15 min in the dark in 100 µl buffer (PBS with 2 mM EDTA and

0.5% FCS) at 4°C. The cells were washed with 2 ml buffer and resuspended in 500 µl

buffer for magnetic separation using autoMACS separator (instrument and microbeads

from Miltenyi Biotec). The instrument was prepared and primed according to the

manufacturer’s instructions. Sample tubes were placed at the uptake port and the fraction

collection tubes at port pos1 to collect the positive (labelled) fraction and port neg1 for the

negative (unlabelled) fraction using the positive selection program on the separator.

Positive fraction which contains the CD19+ B cells were used to incubate with and without

Cy5 labelled gp350 protein (1 µg/sample well) and with and without unconjugated anti-

CD21 to block gp350 receptor on B cells. Enriched CD19+ B cells were stained with

Live/Dead fixable aqua, a live dead stain from Invitrogen, followed by anti-CD21 antibody

and then finally with Cy5 labelled gp350. B cells are then analysed by flow cytometry.

4.3.8. Biotin Labelling of gp350

Recombinant gp350 from MedImmune was biotinylated using the EZ-Link

Pentylamine-Biotin from Thermo Fisher. EZ-Link Pentylamine-Biotin has a terminal

primary amine which can bind to terminal carboxyl group on a protein. The reaction was

mediated by EDC, which is a carbodiimide crosslinker that activates the carboxyl group to

bind to primary amine, forming an amide linkage.

The protein (5 mg/ml) was dialysed into 25 mM MES buffer, pH 5.5 (Amresco) using

a Slide-A-Lyzer dialysis cassette (Thermo Fisher) overnight in the cold room. The protein

in MES buffer was concentrated using a 10 K Ultracel (Millipore) to 500 µl. Pentylamine-

Biotin was dissolved in water at 50 mM in 500 µl. Equal volumes of biotin solution and

protein were mixed. Just before use, an EDC solution was made in MES buffer at 20

mg/ml. 50 µl of the EDC solution was added to the protein-biotin mix and incubated at

room temperature for 2 h with shaking. Non-reacted Pentylamine-Biotin and EDC by-

products were removed by dialysis using Slide-A-Lyzer dialysis cassette. The biotinylated

protein was stored at 4°C, as recommended by the manufacturer.

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4.3.9. gp350 Tetramer Preparation

Biotinylated gp350 was used in the preparation of tetramers using Streptavidin–PE

(SAPE) premium grade (Molecular Probes, Invitrogen) and Streptavidin-APC (Prozyme-

Phycolink Strep-APC from Prozyme). The amount of protein and streptavidin conjugate

required for a 4:1 molar ratio was calculated as published in Current Protocols in

Immunology- Basic Protocol 4. The protocol for preparing MHC tetramer was adapted for

this purpose.

The streptavidin conjugate was added to the biotinylated protein in 5 steps of small

aliquots with 10 min interval between each addition. The tetramerisation was done on ice

and kept in dark between additions. Before cell staining, the tetramer preparation was

centrifuged for 5 min at maximum speed to remove aggregates.

4.3.10. B Cell Labelling Using gp350 Tetramers

PBMC were thawed and rested for 1 h in RPMI1640 with 10% FCS at 37°C. The

cells were counted and 200,000 cells were distributed per well in a 96-well V bottom plate.

The gp350 receptor CD21 on B cells was blocked using anti-CD21 antibody (R&D

Systems) to avoid the selection of all B cells. The blocking kept non-gp350-specific B cells

from being labelled.

The cells were washed twice in PBS with 2% FCS and incubated with and without

anti-CD21 antibody at a concentration of 10µg/ml. The cells were incubated at 4°C for 30

min. Then without washing the protein, gp350 (monomer or tetramer) was added to the

wells at 5 µg/well and incubated for another 30 min at 4°C. The cells were washed once

and incubated with CD19-eFluor450 to label B cells. The gp350-specific B cells were

sorted as CD19+, PE or APC positive cells by flow cytometry. The cells were also stained

with Live/Dead aqua (Life Technologies) to eliminate dead cells, followed by CD3/CD14

FITC (Beckman Coulter/BD Biosciences) to eliminate T cells and monocytes. The B cells

were then stained with both Strep-PE and Strep-APC. The gp350-specific B cells were

then visualized as live cells/CD3-/CD14-/CD19+ cells, which are double positive for PE and

APC.

4.3.11. Enzyme Linked Immunosorbent Assay (ELISA) of gp350

ELISA was done using ELISA plates (EIA/RIA plate from Costar and gp350 antigen

from MedImmune). 5 g/ml of protein was plated in carbonate buffer, pH 9.4, on ELISA

plates and incubated overnight at 4°C. The coated plates were washed in PBS containing

0.05% Tween–20, PBST and blocked with PBS containing 5% non-fat dry milk (blotting

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grade blocker from Bio-Rad) for 2 h at room temperature. The plates were washed and 25

µl of serially diluted plasma samples were added in duplicates and incubated for 2 h at

room temperature on a plate shaker. After further washing in PBST, 25 µl of 1:1000

dilution of HRP-conjugated goat anti-human IgG (Chemicon, Millipore) or 1:4000 dilution of

goat anti-human Ig (Southern Biotech) was added and incubated for 1 h. After washing in

PBST, 25 µl of 1xTMB ELISA substrate solution (eBiosciences) was added for colour

development and incubated for 15 min at room temperature in the dark, after which the

reaction was stopped by the addition of 25 µl of 1 M HCL. The absorbance was measured

using a spectrophotometer at the wavelength of 450 nm.

4.3.12. Titration of EBV Virus Pool

To titrate EBV virus pool B95-8, PBMC of an EBV seronegative donor were used. 50

µl of 1x105 cells/ well was plated in a 96-well plate and 50 µl of 10-fold serial dilution of

virus was added and incubated at 37°C for 1 h. Each dilution was done in six replicates

and the virus dilutions started with undiluted and 5 x 10-fold dilutions were used. The

plates were centrifuged at 300 x g/5 min, the medium removed and resuspended with

fresh R10. For the next four weeks, 100 µl of the medium was removed and replaced with

100 µl of fresh R10 and read after one month for the presence of transformation.

Transformation was indicated by the visible growth of cells in clumps and the TCID50 was

calculated as the effective dilution of plasma that inhibit 50% EBV transformation of B cells

and also using the Reed and Muench calculator (Reed and Muench 1938).

4.3.13. Neutralisation Assay

EBV seronegative PBMC were used for the assay. Plasma samples from IM patients

during infection and after recovery, along with age-matched healthy controls, were used

for the assay. The plasma was heat-inactivated at 56°C for 30 min before the assay. Equal

volume of 3-fold serially diluted plasma and 10 TCID50 of EBV B95-8 were mixed and

incubated in 96-well round bottom plates at 37°C for 2 h. Four replicates of 50 µl, each of

virus-plasma mixture, were then incubated with 50 µl of 1x105 cells/well for 1 h and the

plates were centrifuged at 300 x g/5 min. The medium was removed and resuspended with

200 µl of fresh medium (RPMI with 10% FCS) and plated in 96-well flat bottom plates. The

medium was changed once every week, and after four weeks the effective dilution of

plasma that inhibits virus transformation by 50% (EDI50) was calculated (Reed and

Muench 1938, Moss and Pope 1972, Sashihara, Burbelo et al. 2009).

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4.3.14. Pepmix EBV

gp350 Pepmix (JPT Technologies GmbH) was dissolved in 40 µl DMSO and diluted

in PBS as 1 µg/ml final concentration of each peptide, total of 224 peptides, per test for

stimulating PBMC. The aliquots were made as one single aliquot per test and stored at –

20°C.

4.3.15. Flow Cytometry of CD4+ T Helper Cells

The phenotypic analysis of a population of circulating TFH cells in peripheral blood

has been described by various recent studies. To evaluate the presence of these cells

during EBV infection, PBMC from different stages of EBV infection were stained with

surface markers, CD3–PE Cy7; CD4–Pacific Blue; CD45RA–AF700; CXCR5–FITC;

CXCR3–APC and ICOS–BV421 and PD–1 PE, and the frequency of these cells was

analysed by flow cytometry.

4.3.16. Antigen-Specific CD4+ T Cell Expansion and Functional Analysis

PBMC from healthy asymptomatic virus carriers and donors with a history of IM were

used to prepare EBV MA-gp350 specific CD4+ T cells. This was done by pulsing 1–2x106

PBMC with gp350 pepmix (JPT). The cells were incubated for 2 h at 37°C in a CO2

incubator. The cells were washed once in R10 and mixed with another 2–4x106 cells and

plated in a 48 well plate (2x106 cells/ well). On days 3, 5 and 7, interleukin (IL) -2 was

added at a concentration of 20 IU/ml.

On day 10, functional assay intracellular cytokine staining (ICS) was performed. ICS

measured the ability of cells to express cytokines IFN-, TNF-and IL-21. The cells were

stained with CD4-Pacific Blue, CD8- PerCPCy5.5 and CXCR5–FITC surface markers. The

intracellular staining was performed after re-stimulation with gp350 pepmix in the presence

of BrefeldinA/monensin (BD Biosciences) for 6 h. The cells were washed, fixed and

permeabilised with cytofix/cytoperm (BD Biosciences) and stained with IFN--AF700, TNF-

APC, and IL-21 PE. The CD4+ T cell analysis was performed by flow cytometry and

FlowJo software.

4.3.17. Intracellular Cytokine Staining (ICS) and Cell Surface Marker Staining

PBMC from IM patients were thawed and rested for 1 h in RPMI1640 with 10% FCS

at 37oC. The cells were counted and distributed in a 96-well V bottom plate. The cells were

stimulated by the addition of gp350 pepmix (JPT Technologies) overnight in the presence

of golgiplug, Brefeldin A (BD Biosciences) to the final concentration of 1 µl/ml for 4–6 h at

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37°C. The cells were washed and stained with surface markers, CD8- PerCPCy5.5, CD4-

Pacific Blue and incubated at 4°C for 30 min. The intracellular staining was performed after

fixing and permeabilizing the cells using cytofix/cytoperm solution. Permeabilizing solution,

10X solution diluted to 1X with deionized water, from BD Biosciences was used for

subsequent washes and cells were stained for cytokines using IFN--AF700, TNF-APC

and IL-21 PE and incubated at 4°C for 30 min. The cells were analysed by flow cytometry

and FlowJo software.

4.3.18. Cell Surface Marker Staining of IM Samples

Phenotypic analysis of B-cells was done on PBMC by Flow Cytometry. Frozen PBMC

stored in liquid nitrogen were thawed at 37°C, washed once in R10 and rested for at least

one hour in a 37°C incubator. The cells were then counted and 500,000 cells/ well were

used for surface antigen staining in a 96-well V bottom plate. The cells were washed twice

in PBS/2% FCS and incubated with fluorochrome conjugated antibodies diluted in PBS/2

% FCS for 30 minutes at 4°C. The cell surface markers were CD3-PE Cy7, CD4-Pacific

Blue, ICOS-BV421, CXCR5-FITC and CXCR3-APC, CD8-PerCpCy5.5 and PD1-PE. The

cells were washed twice in PBS/2 % FCS and fixed in 4 % paraformaldehyde. Data was

acquired on FACS LSRII Fortessa and analysed using FlowJo software.

4.4. RESULTS

4.4.1. EBV MA-gp350 Purification and Quantification

The recombinant gp350 used to measure B cell responses was expressed from

Chinese hamster ovary cells grown in serum-free medium. The cells were grown to 80%

confluence in serum containing DMEM/F12 medium, then moved to serum-free

DMEM/F12 and cultured for several weeks. Truncated gp350 protein secreted into the

medium was collected and concentrated using Amicon Ultracel-50K each week. The

protein was then purified using conA sepharose column and eluted by increasing

concentration of -D methyl glucoside. The presence of the protein was visualized using

dot blot analysis, SDS-PAGE and western blotting. The presence of purified protein in

different eluents after purification was checked first by dot blot analysis, and samples with

the protein were pooled together and dialysed into PBS, followed by SDS-PAGE and

western blot (Figure 4.1).

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Figure 4. 1. EBV-membrane antigen gp350 purification

SDS-PAGE and Western Blot analysis of the purification of EBV-MA gp350 expressed by CHO cells. The gp350 protein was purified using Con A Sepharose 4B column and visualized through coomassie blue staining and mAb EBV OT6.

Panel A: Truncated gp350 expressed by CHO cells was collected from the supernatant and concentrated using Amicon Ultracel-50K. Different volume of the supernatant was run on a 6% SDS-PAGE gel to confirm the presence of protein. Precision Plus protein standards were used as markers and 5 µl (lane 2), 10 µl (lane 3) and 20 µl (lane42) protein were loaded on the gel. After electrophoresis, the gel was stained with coomassie blue and the presence and size of protein were confirmed.

Panel B: Purified and concentrated EBV-MA gp350. Both gp350 and splice product gp220 were visible.

Panel C: Western Blot analysis of purified gp350 protein run on a 6% SDS-PAGE with 5 µl (lane 1

and 4), 10 µl (lane 3) and 20 l (lane 2) of protein loaded on the gel and transferred to a nitrocellulose membrane. The blot was then incubated with EBV-MA specific monoclonal antibody EBV OT6 and visualized with goat anti-mouse HRP and western lighting ECL.

Protein concentration was estimated using Bradford assay, with an average yield of 1

mg/ml per purification. The presence of endotoxin was ruled out by using a Toxin Sensor

LAL detection kit. The level of endotoxin was found to be less than 0.1 EU/g of protein.

4.4.2. Frequency of EBV-Specific Memory B Cells

Observations in the previous chapter suggested that during acute symptomatic EBV

infection, the B cell compartment is perturbed, with a reduction in total B cells and MBCs in

PBMC, which was associated with the poor in vitro conversion of MBCs into ASCs. To

explore the impact this B cell dysfunction has upon EBV-specific B cell immunity, the

kinetics of the gp350-specific memory B cell response was assessed. Longitudinal PBMC

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samples from 15 patients with IM were stimulated for 6 days with the B cell mitogen

cocktail, and then assessed for gp350-specific memory B cells by ELISpot.

Figure 4. 2. B cell ELISpot analysis to EBV-MA gp350 and total IgG

Representative ELISpot assay using in vitro-activated PBMC from donors at different stages of EBV infection. PBMC were cultured for six days in the presence of an optimized mix of polyclonal mitogens. 300,000 cells/well and 3,000 cells/well were incubated overnight on ELISpot plates coated in triplicate with gp350 antigen and total IgG capture antigen, respectively. Mitogens induce the conversion of memory B cells to antibody-secreting cells, and each individual spot represents a single antibody-secreting B cell (gp350/IgG). The frequency of antigen-specific memory B cells was calculated as a percentage of total IgG secreting B cells.

Panel A: PBMC from EBV-seropositive asymptomatic carrier and frequency of gp350 and total IgG-specific memory B cells.

Panel B: ELISpot from EBV seronegative donor and frequency of gp350 and total IgG-specific memory B cells.

Panels C, D, and E: ELISpot assay using PBMC from longitudinal samples from a donor during recovery (20-50 days post infection) and post-IM (six months after infection).

Figure 4.2 shows a representative ELISpot assay of gp350-specific and total IgG-

specific memory B cells using PBMC from asymptomatic EBV carriers, EBV-seronegative

donors as controls, and a longitudinal sample from a donor during illness, recovery, and

post IM. As expected, EBV-seronegative donors did not have EBV-specific memory B

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cells. As shown in the previous chapter, MBCs from acute IM displayed poor in vitro

conversion into ASCs. Similarly, there was a significantly lower frequency of gp350-

specific B cells during acute IM (Figure 4.3A).

Figure 4. 3. Frequency of EBV-specific memory B cells per 106 PBMC

Compilation of B cell ELISpot data using EBV membrane antigen (EBV-MA) gp350 which induces virus neutralising antibody response, EBV viral capsid antigen VCA, which is non-neutralising viral antigen, and general control antigen Tetanus Toxoid TT.

Panel A: Frequency of gp350-specific memory B cells per 106 PBMC with 11 acute IM samples, 11 post-IM samples and 18 age-matched asymptomatic virus carriers.

Panel B: Frequency of VCA-specific memory B cells with 8 longitudinal samples during and post-IM and 15 age-matched healthy virus carriers.

Panel C: Frequency of general antigen TT-specific memory B cells using 8 longitudinal samples and 15 age-matched healthy virus carriers.

The data are presented as mean+/_ SEM and statistical analysis for the longitudinal samples were calculated by Wilcoxon matched-pair analysis and the comparison between different groups by non-parametric Mann-Whitney analysis. The significant p values were **** p<0.0001, ** p<0.01 and *p<0.05.

To control for potential changes in the total memory B cell compartment,

commercially-available EBV viral capsid antigen (VCA), another structural antigen with no

neutralising properties, was used as a control EBV antigen and tetanus toxoid as a general

control antigen. Although not as significant, VCA and TT-specific ASCs were also reduced

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during acute IM (Figure 4.3A, 4.3B), suggesting that the reduction in the size of the

memory B cell compartment and poor in vitro conversion to ASCs was influencing the in

vitro conversion of gp350, VCA and TT-specific MBCs into ASCs.

Figure 4. 4. Frequency of EBV-specific memory B cells as a percentage of total IgG-specific memory B cells

Compilation of B cell ELISpot data using EBV antigens, gp350 and VCA, and control antigen Tetanus Toxoid TT normalised as the percentage of total IgG.

Panel A: Frequency of gp350-specific memory B cells per total IgG secreting memory B cells with 15 acute IM samples, 15 post-IM samples and 18 age-matched asymptomatic virus carriers.

Panel B: Frequency of VCA-specific memory B cells with 10 longitudinal samples during and post-IM and 15 age-matched healthy virus carriers.

Panel C: Frequency of general antigen TT-specific memory B cells using 12 longitudinal samples and 15 age-matched healthy virus carriers.

The data are presented as mean+/_ SEM and statistical analysis for the longitudinal samples were calculated by Wilcoxon matched-pair analysis and the comparison between different groups by non-parametric Mann-Whitney analysis. The significant p values were **** p<0.0001 and *** p<0.001.

The quantification of antigen-specific memory B cells using ELISpot assay is usually

performed by normalising the antigen-specific memory B cells as the percentage of total

IgG-secreting cells to account for B cell proliferation during the six day culture (Crotty,

Aubert et al. 2004). Therefore to further explore changes in the B cell response during IM,

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the frequency of gp350, VCA and TT-specific T cells was normalized as a proportion of

total IgG-secreting cells. These observations demonstrated that patients with acute IM had

a very low relative frequency of gp350-specific memory B cells when compared to age-

matched asymptomatic virus carriers (Figure 4.4A). The frequency of gp350-specific

memory B cells only reached numbers comparable to asymptomatic virus carriers during

convalescence. Conversely, the reduced absolute response to VCA and TT did not appear

attributable to a lower frequency of antigen-specific cells as a proportion of total IgG

secreting cells (Figure. 4.4B, 4.4C), and were more likely associated with the overall poor

in vitro conversion of memory B cells.

Figure 4. 5. Absolute count of EBV-specific memory B cells per litre of blood

Compilation of B cell ELISpot data using EBV antigens, gp350 and VCA, and control antigen Tetanus Toxoid TT using absolute count per litre of blood at the time of harvest.

Panel A: Frequency of gp350-specific memory B cells per litre of blood with 9 acute IM samples and 9 post-IM samples.

Panel B: Frequency of VCA-specific memory B cells with 8 longitudinal samples during and post-IM.

Panel C: Frequency of general antigen TT-specific memory B cells using 8 longitudinal samples during and post-IM.

Statistical analysis for the longitudinal samples was calculated by Wilcoxon matched-pair analysis. The significant p value ** p<0.01.

To confirm the lower frequency of gp350-specific B cells during acute IM, the

absolute number of antigen-specific MBCs per litre of blood at the time of harvest was

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calculated for samples where the whole blood count was available (Figure 4.5). This

analysis confirmed the low frequency of gp350-memory B cells during acute IM when

compared to post IM samples (Figure 4.5A), and demonstrated no significant difference in

the frequency of VCA or TT-specific memory B cells during and after acute IM (Figure

4.5B, 4.5C).

4.4.3. gp350-Antibody Concentration in Plasma

While ELISpot allows the quantification of EBV-specific memory B cells in peripheral

blood, ELISA determines the presence and measures the amount of EBV-specific antibody

titre in plasma produced by long-lived plasma cells. To see whether the frequency of

gp350-specific memory B cells correlate with antibody levels in the plasma, the plasma

samples from the same cohort of IM patients, during and after recovery, were used to

measure gp350 antibody titre with ELISA (Figure 4.6).

Figure 4. 6. Measurement of gp350-antibody titre

gp350 ELISA was performed to see whether gp350 antibody titre in the plasma of donors at different stages of EBV infection correlated with frequency of memory B cells in their blood as measured by B cell ELISpot.

Panel A: Data from 10 longitudinal IM samples and 10 age-matched healthy virus carriers measuring the gp350 total Ig antibody titre, which included IgA, IgM and IgG.

Panel B: Data from 10 longitudinal samples from IM donors and 10 age-matched healthy virus carriers measuring the gp350 total IgG antibody titre which complements the ELISpot analysis.

The data are presented as mean+/_ SEM. Statistical analysis for the longitudinal samples was done using Wilcoxon matched -pair analysis and comparison between different groups by non-parametric Mann-Whitney analysis. The significant p values were **** p<0.0001 and ** p<0.01.

Measures of total IgG and total Ig—aggregate of IgM, IgA and IgG—in longitudinal

plasma samples from 10 patients during and post-IM were compared against those from

age-matched asymptomatic latent virus carriers. Similar to the results obtained in the

ELISpot analysis, a significantly lower gp350 response was evident during acute IM in both

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total Ig titre (Figure 4.6A) and IgG titre (Figure 4.6B) when compared to the antibody titre

post-IM or in age-matched asymptomatic virus carriers.

To determine if reduced memory B cell response and plasma antibody levels

correlated with severity of infection, the cohort of patients was divided based upon a

clinical score of 4—6 or 0—3, then assessed for the number of gp350-specific memory B

cells and gp350-specific antibody titre during and following resolution of acute IM. These

observations revealed that there was no significant difference in the gp350-specific

memory B cell response during acute IM, in the resolving phase during the first six months

after IM, or in patients in long term recovery of more than 6 months after IM (Figure 4.7A).

Similarly, there was no difference in gp350-specific IgG titre in patients with, mild or severe

symptoms (Figure 4.7B).

Figure 4. 7. Correlation of frequency of gp350-specific memory B cells and antibody titre with severity of infection

SOI scores were assigned from 0–6 with a score between 0–3 classified as mild infection and 4–6 as severe. The percentage of gp350-specific memory B cells to total IgG+ memory B cells by ELISpot (Panel A) and gp350-IgG titre by ELISA (Panel B) was found to be similar in both groups with low frequency during acute IM and higher frequency with disease resolution Post IM-Recovery, 20-50 days after symptom starts (Post IM-R) as well as Post IM long term carrier state, more than six months from start of symptom (Post IM-LT).The data are presented as mean +/-SEM.

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4.4.4. Quantity and Quality of Antibody Produced During Primary EBV Infection

The conventional method to study the quality and quantity of neutralising antibody

produced looks at the ability of antibodies to inhibit transformation of B cells by EBV. This

assay was performed to confirm the ELISpot and ELISA data. Equal volume of 10 TCID50

of EBV B95-8 and heat inactivated plasma samples from donors were mixed and

incubated to let the antibodies in the plasma to neutralise the virus. This mixture was then

incubated with susceptible PBMC from EBV seronegative donors for 3-5 weeks to

determine the effective dilution of plasma that neutralise the virus.

Figure 4. 8. Conventional EBV virus-neutralisation assay

Efficiency of antibodies produced during and post IM to neutralise virus was compared between longitudinal plasma samples from IM patients and plasma from age-matched healthy virus carriers. The assay measures the ability of antibodies in plasma to inhibit EBV transformation of B cells.

Panel A: A representative well from neutralisation assay where antibodies in the plasma were not successful in neutralising the virus, as evidenced by EBV transformed B cells.

Panel B: representative well where antibodies in plasma efficiently neutralised virus, resulting in absence of EBV-transformed B cells.

Panel C: Compilation of data from all assays using 10 longitudinal IM plasma samples and samples from 10 age-matched healthy virus carriers.

Statistical analysis of the longitudinal samples was done by Wilcoxon matched-pair test and comparison between different groups by non-parametric Mann-Whitney test. The significant p values were *** p<0.001 and ** p<0.01.

Figures 4.8A and 4.8B shows representative wells from the neutralisation assay where

the antibodies in the plasma failed (Figure 4.8A) or succeeded (Figure 4.8B) in inhibiting

the virus transformation of B cells, as evidenced by the presence or absence of EBV

transformed B cells. The neutralisation assay used the plasma samples from 14 acute IM

samples and 10 post-IM samples of which 10 samples followed patients during IM to their

recovery > six months post-IM. Plasma samples from age-matched asymptomatic latent

virus carriers and EBV seronegative donors were used as positive and negative controls in

the assay. The neutralisation of the virus was below the detection level when using the

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plasma from acute IM patients while the plasma from both the post-IM samples as well as

asymptomatic virus carriers neutralised virus efficiently. This shows that the quality of virus

neutralising antibody made from recovered IM patients and asymptomatic virus carriers

are equally effective in neutralising the virus while the quality and/or quantity of

neutralising antibody produced remained poor during acute IM (Figure 4.8C).

Observations from the acute IM cohort suggest that gp350-specific B cell response in

patients is poor during acute symptomatic EBV infection. These observations support

findings from over forty years ago showing the delayed induction of neutralising antibodies

to EBV and suggest that primary EBV infection is associated with the inefficient priming of

gp350-specific memory B cells (Hewetson, Rocchi et al. 1973). Recent observations

during other human viral infections have shown that assessment of the B cell repertoire

during and post-infection can provide significant insight into the maturation of the antigen-

specific B cell response. These studies typically sorted either total ASCs, shown to be

enriched for antigen-specific cells during acute infection, or cells stained with fluorescently

labelled antigen, and then performed downstream B cell receptor and functional analysis

on recombinant monoclonal antibodies. As outlined in the previous chapter, there was no

evidence of an increase in ASCs during acute EBV infection, which would have provided a

potential source of gp350-specific B cells during acute IM. Additionally, as determined by

ELISpot assay, the frequency of gp350-specific B cells in both acute IM patients and

healthy virus carriers was found to be very low, constituting typically < 0.1% of the total B

cells. Since the frequency of gp350-specific B cells was so low, attempts were made to

develop a protocol for the enrichment of the gp350-specific B cell population in order to

isolate gp350-specific memory B cells to assess BCR usage and to generate monoclonal

antibodies. From the literature, two methods to enrich rare memory B cells were obtained

and tried (Figure 4.9). These methods involve labelling and sorting antigen-specific B cells

by flow cytometry using fluorescently labelled or biotinylated antigen. The method to

fluorescently label the antigen using Fluorolink Cy5 labelling Kit from GE (Potzsch,

Spindler et al. 2011) was tried first.

The CD19+ B cell population was sorted from the PBMC of healthy EBV seropositive

and seronegative donors using CD19 microbeads and autoMACS separator, both from

Miltenyi Biotec. To avoid selection of all B cells by labelled gp350 protein, the gp350

receptor CD21 on the B cells was blocked by unconjugated anti-CD21 antibody before

labelling with Cy5 labelled gp350 protein (Figure 4.9A). Panel 9A shows representative dot

plot of control where no protein or no anti-CD21 antibody was added, B cells labelled with

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Cy5 labelled gp350 without CD21 receptor block and B cell labelled with Cy5 labelled

gp350 with CD21 receptor blocked with anti CD21 antibody respectively. The frequency of

live cells that are Cy5+/CD19+ B cells was then quantified. While blocking with anti-CD21

antibody reduced the gp350 binding to CD21 on B cells a significant amount of non-

specific binding was still evident. To improve on this protocol, a technique was developed

using biotinylation of gp350 using the EZ-Link Pentylamine-Biotin from Thermo Scientific

and using dual fluorescently labelled Streptavidin for flow cytometry.

Figure 4. 9. Enrichment of gp350-specific memory B cells for generation of monoclonal antibodies. Structure of gp350 (Szakonyi, Klein et al. 2006)

Two methods for labelling rare memory B cells are shown. Panel A: First, fluorescently-labelled gp350 protein was used to enrich gp350-specific memory B cells using fluorolink Cy 5 labelling kit. Representative FACS plot of PBMC with 5A as control without the addition of gp350 protein, with Cy 5 labelled gp350 added, which resulted in all B cells being labelled since B cells express gp350 receptor CD21, and where unconjugated anti-CD21 was used to block CD21 receptor so that only gp350-specific memory B cells will be labelled by Cy5 labelled gp350 respectively.

Panel B: Next, biotinylated gp350 protein was tetramerised using Streptavidin-PE, as well as Streptavidin-APC. Frequency of gp350-specific memory B cells were gated as CD3

-/CD14

-/CD19

+ B cells which are

double positive for PE and APC. PBMC from both EBV seronegative and seropositive donors were used to evaluate the success of labelling.

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Previous comparison of tetrameric and monomeric antigens showed tetramers to be

more sensitive than monomers as the frequency obtained by tetramer labelling was larger

than from monomers (Franz, May et al. 2011). Biotinylated gp350 was used to make

tetramers using Streptavidin-PE and Streptavidin-APC. Tetramerisation of biotinylated

gp350 was done on the same day as labelling to avoid degradation of the tetramer. The

PBMC were stained by Live/Dead aqua followed by anti-CD21 antibody to block gp350

receptor and stained with gp350 tetramers and surface markers, CD3, CD14 and CD19 for

flow cytometry. The gp350-specific B cells were then analysed as live cells/CD3-/CD14-

/CD19+ B cells that are double positive for PE and APC. The aim of using dual colour for

tetramers was to enrich the gp350-specific B cells and avoid non-specific binding. Analysis

of EBV seropositive and seronegative donors showed that even though the blocking with

anti-CD21 antibody worked it is not complete and significant non-specific binding can still

be noticed with both fluorescent labelling and tetramer staining of B cells. This was made

clear by the presence of double positive B cells in both seropositive as well as

seronegative donors (Figure 4.9B). The low frequency of gp350-specific memory B cells

and the added complication of gp350 receptor CD21 being expressed on all B cells made

it difficult to specifically isolate gp350-specific memory B cells and this protocol was not

pursued further.

4.4.5. Circulating Memory CD4 Helper T Cells

One of the classic features of IM is a temporary reversal of the CD4/CD8 ratio during

acute IM (Hornef, Wagner et al. 1995, Maini, Gudgeon et al. 2000). This change is evident

in the cohort of patients investigated in this study as shown in Figure 4.10.

Figure 4. 10. CD4/CD8 ratio

Characteristic temporary reversal

of CD4/CD8 ratio during acute

IM and the restoration of T cell

homeostasis after disease

resolution. Statistical analysis

was done by one-way ANOVA,

Tukey’s multiple comparison

with *** p<0.001, ** p<0.01 and

* p<0.05.

These observations have suggested that the inflammatory response during IM is

primarily mediated by CD8+ T cells, with a reduced contribution by CD4+ T cells and NK

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cells (Tomkinson, Wagner et al. 1987, Balfour, Odumade et al. 2013). However, CD4+ T

cells play an important role in regulating the induction and maturation of both the cellular

and humoral immune response during viral infection (Hale, Youngblood et al. 2013). While

T helper (Th1) cells are critical for the induction of cellular immunity, the humoral immune

response is regulated by T follicular helper cells (TFH), a subset of CD4 T cells that help B

cells (Nakayamada, Kanno et al. 2011, Whitmire 2011). Therefore to explore their role

during EBV-infection, the frequency of TFH in the cohort of IM patients was assessed

during and after IM. Although TFH are classically found in germinal centres in secondary

lymphoid tissue, recent observations in a number of infectious settings have demonstrated

populations of circulating memory TFH cells in peripheral blood, characterised by the

expression of CXCR5, ICOS and PD1 (Haynes, Allen et al. 2007).

Figure 4. 11. Frequency and phenotype of CD4+ T cell subsets during acute IM

The frequency of CD4+ T cell subsets during and post IM were analysed. PBMC from donors at different stages of EBV infection was analysed by staining with surface markers CD4, CXCR5, CXCR3, ICOS and PD-1.

Panel A: The data from donors during and post IM showing the percentage of CD4+/CXCR5+ T cells that express ICOS and/or PD1.

Panel B: The data from donors during and post IM evaluating the frequency of CD4+/CXCR3+ T cells that express ICOS and /or PD1.

Statistical analysis was done by non-parametric Mann-Whitney test and the p value correspond to *** p<0.001.

To assess the presence of circulating TFH during EBV infection (both primary and

persistent), PBMC from donors during and post-IM were stained with CXCR5, ICOS and

PD1. PBMC were also stained with CXCR3 to characterise Th1 cells (Groom, Richmond

et al. 2012). These observations revealed that during acute EBV infection there was no

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increase in the frequency of circulating CD4+/CXCR5+/ICOS+/PD-1+ TFH cells (Figure

4.11A), although there was a dramatic increase in the proportion of CXCR3+ICOS+PD1+

cells, which likely correspond to a population of activated Th1 cells (Figure 4.11B). These

observations support the findings in the previous chapter showing no obvious induction of

circulating ASCs during acute IM, and suggest that the induction of TFH cells may also be

dysregulated during acute IM.

4.4.6. Presence of gp350-Specific CD4+ T Cells

To more specifically investigate the induction of CD4+ T cells responses during IM,

next, the presence of gp350-specific CD4+ T helper cells in blood was investigated.

Preliminary observations suggested that whole gp350 induced the non-specific

proliferation of CD4+ T cells in both EBV-seropositive and EBV-seronegative donors, which

supports previous studies by Hsiao, Lin et al that the binding of gp350 to CD21 receptor on

the B cell activates the human endogenous retrovirus HERV–K18 env, which encodes a

super antigen that can strongly stimulate T cells (Hsiao, Lin et al. 2006). Therefore, the

study of CD4+ T helper cells during primary and persistent infection was performed using a

gp350 pepmix of 15 mer peptides with 11 amino acid overlap. Intracellular cytokine

analysis was performed to investigate the induction of IFN-, TNF- and IL-21 producing

gp350-specific T cells.

Figure 4. 12. gp350-specific CD4+ T cell response during acute IM

Data from intracellular cytokine (ICS) staining of gp350-specific CD4 T cells. The cytokines produced after stimulation with gp350 pepmix (Panel A) and after stimulation with PMA and ionomycin (Panel B) for 12–16 h was studied in donors undergoing acute IM. The

frequency of CD4 T cells that produce IFN-,

TNF- and IL-21 are shown.

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Acute IM samples showed very low expression of IFN- and TNF- from CD4+ T cells

following stimulation with the gp350 pepmix (Figure 4.12A), suggesting poor induction of

gp350-specific Th1 cells despite evidence of a dramatic expansion of Th1 cells during IM,

which was also reflected in the strong IFN- and TNF- response following stimulation with

PMA and Ionomycin (Figure 4.12B). Similarly, stimulation with gp350 showed very little

activation of IL-21 production. To determine if the poor CD4+ T cell response to gp350

was also reflected in the gp350-specific CD8+ T cell response, CD8+ T cells from acute IM

patients were also assessed for IFN-, TNF- and IL-21 production following stimulation

with the gp350 pepmix. Similar to CD4+ T cells, the response of CD8+ T cells to gp350

was very low when compared to PMA and Ionomycin activated cells (Figure 4.13A &B).

Figure 4. 13. gp350-specific CD8+ T cell response during acute IM

Data from intracellular cytokine (ICS) staining of gp350-specific CD8+ T cells. The cytokines produced after stimulation with gp350 pepmix (Panel A) and after stimulation with PMA and ionomycin (Panel B) for 12–16 h was studied in donors undergoing acute IM. The frequency of CD8+ T cells that

produce IFN-, TNF-α and IL-21 are shown.

To determine if the poor response to gp350 was preferentially associated with IM, the

response in latent virus carriers with and without a history of IM was assessed. Due to the

inability to detect ex vivo responses in PBMC, cells from donors with and without the

history of IM were stimulated with the gp350 pepmix, cultured for 10 days in the presence

of IL-2, and then analysed on day 10 after re-stimulation with and without gp350 pepmix

for the production of IFN-, TNF- and IL-21. While there was evidence of a CD4+ T cell

response to gp350, latent virus carriers with and without a history of IM showed very little

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expansion of a gp350-specific T cell response, with a low frequency of IFN- TNF- and

IL-21 producing CD4+ T cells detected after 10 days in culture (Figure 4.14A).

Figure 4. 14. Characterisation of gp350-specific CD4/CD8 T cells during persistent EBV infection

Data from intracellular cytokine (ICS) staining of gp350-specific T cells. The cytokines produced after stimulation with gp350 pepmix was studied in healthy virus carriers, with (Post IM) or without (healthy carriers) symptomatic EBV primary infection. PBMC from donors with and without a history of IM were cultured for 10 days in presence of gp350 to expand gp350-specific T cell lines. These cells are used to study the cytokine profile of gp350-specific CD4+/CD8+ T cells by flow cytometry. The frequency of CD4+/CD8+ T cells that produce

IFN-, TNF- and IL-21 are shown.

Panel A: shows the response to gp350 by CD4+ T cells using PBMC from donors post IM and asymptomatic virus carriers.

Panel B: shows the response to gp350 by CD8+ T cells using PBMC from donors post IM and asymptomatic virus carriers.

Additionally, no significant difference in the response from volunteers with and

without a history of IM was evident. A similar response was also seen in CD8+ T cells;

where the response to gp350 was even lower (Figure 4.14B). These observations suggest

that gp350 is a poor immunogen for both CD4+ and CD8+ T cells, inducing very low

frequency CD4+ and CD8+ T cell responses during acute EBV infection, which did not

appear to improve in latent virus carriers.

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4.5. DISCUSSION

Study of humoral immune response during EBV infection has received very little

attention. Neutralising antibodies provide protective immunity after most infections and

vaccinations. They help control viral load during an active viral infection. In this chapter,

our study of humoral immunity—the virus-neutralising antibody response during acute

IM—showed a reduction in frequency of gp350-specific memory B cells, low gp350

antibody titre in the plasma and a complete lack of virus-neutralising capabilities for

antibodies produced during acute IM. Our findings also revealed a lack of circulating CD4+

TFH cells during acute EBV infection, and demonstrated that gp350 appears to be a poor

immunogenic target for CD4+ and CD8+ T cells. Lack of circulating TFH cells and poor

immunogenicity of gp350 potentially contribute to the ineffective induction of a gp350-

specific neutralising antibody response during IM, and thus poor control of the virus during

primary infection.

EBV membrane antigen gp350 is found on the outer coating of the virus, as well as

on the surface of EBV-infected cells (Pearson and Qualtiere 1978). gp350 is found in

abundance during EBV infection and antibodies against it have the capacity to neutralise

the virus (Hoffman, Lazarowitz et al. 1980, Moutschen, Léonard et al. 2007). Observations

as far back as 40 years ago showed that EBV-neutralising antibodies are slow to appear

during acute IM, with levels increasing and stabilising during convalescence, in contrast to

non-neutralising responses to the viral capsid antigen that peak during the first few weeks

of IM (Hewetson, Rocchi et al. 1973, Svedmyr, Ernberg et al. 1984). We found evidence

of very low gp350 response during acute IM, global dysfunction in the B cell

compartment—as outlined in the previous chapter—and likely poor immunogenicity of

gp350 contributing to inefficient virus neutralisation. Although it remains to be fully

explained, the poor immunogenicity of gp350 may explain the differential kinetics of

gp350-specific and VCA-specific B cell responses. While some early efficient processing

of the VCA antigen could promote an early antibody response to the EBV VCA, the poor

processing of gp350 may restrict the induction of a virus neutralising response. This

delayed neutralising antibody response could be an immune evasion mechanism of EBV

that facilitates the establishment of EBV persistence in the early stages of infection. This

lack of virus-neutralising antibody response also likely contributes to the increased viral

load that ultimately leads to symptomatic IM and a global dysfunction in B cell response

that may further complicate the induction of a neutralising antibody response. This is

supported by our findings and others that the establishment of an efficient neutralising

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response does not occur well into convalescence, often many months after exposure.

Unlike the massive expansion of CD8+ T cells during acute IM, CD4+ T cells do not

increase in number but still play an important role in the control of EBV infection. CD4+ T

helper cell subsets are reported to play a role in the control of early EBV infection and also

during persistent infection (Nikiforow, Bottomly et al. 2001, Mackay, Long et al. 2009).

CD4+ T cell subset (TFH cells) are also directly involved in humoral immune responses

(Breitfeld, Ohl et al. 2000). These cells influence the formation of germinal centres, signal

antigen-activated B cells via CD40L and provide cytokines IL-4, IL-10 and IL-21 to facilitate

the growth and differentiation of B cells (Bryant, Ma et al. 2007, King and Mohrs 2009).

Since TFH cells are mostly found in the secondary lymphoid organs, circulating memory

TFH cells in blood were studied instead. TFH cells (CXCR5+ CD4+ T cells, (Haynes, Allen et

al. 2007) were found to occur with low frequency, with resting phenotype and no

detectable level of activated TFH cells as measured by the expression of ICOS and PD1 in

the blood of IM patients. This is in agreement with the results from Aim 1 studies that

showed no increase in ASCs and decreased number of memory B cells during acute IM.

This suggests that despite the presence of high EBV viral loads, TFH cells do not persist, at

least in the blood during acute IM, unlike during other human viral infections (Breitfeld, Ohl

et al. 2000, Feng, Lu et al. 2011, Locci, Havenar-Daughton et al. 2013). This could also

impact the efficiency of B cell differentiation into memory cells and ASCs.

The majority of CD4+ T cell subsets found during acute IM were activated

CXCR3+CD4+ cells, previously reported to be Th1 cells (Groom, Richmond et al. 2012).

The frequency of activated Th1 cells decreased significantly with disease resolution, while

a low frequency of resting memory Th1 cells remained during and post IM. Despite the

obvious activation of a Th1 response, our data also showed that gp350 is a very poor T

cell immunogen. There was very little cytokine production by CD4+ T cells when stimulated

by gp350 as compared to control PMA and Ionomycin stimulation, and even in latent virus

carriers there was very little activation of a gp350-specific response. Although the

presence of gp350-specific Th1 cells has been reported (Adhikary, Behrends et al. 2006),

this analysis required repeated in vitro stimulation with recombinant protein to generate a

gp350-specific response. Our data suggest that while gp350-specific responses are

detectable they are sparse, even during the priming phase. This could also be part of the

immune evasion mechanism employed by EBV that makes gp350, abundant during EBV

infection, a poorly-recognised antigen to host immune cells. This data further support the

hypothesis that there is a defect in gp350-specific immune control during early infection.

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CHAPTER 5. PERIPHERAL BLOOD DENDRITIC CELL (DC)

DYNAMICS DURING ACUTE EBV INFECTION

5.1. ABSTRACT

Dendritic cells (DCs) are essential for the proper functioning of antiviral immunity and

the development of the humoral immune response. They promote B cell differentiation

directly through DC–B cell interaction and indirectly by providing cytokines and promoting

the development of TFH cells, which are specialized CD4+ T cells that help B cells.

Dendritic cells in blood are divided into two subsets: myeloid DCs or mDCs, and

plasmacytoid DCs or pDCs. These DCs are activated by a different set of Pattern

Recognition Receptor (PRR) ligands. Activated pDCs have the capacity to produce large

quantities of type I interferons, especially IFN-, which is a key cytokine in the initiation of

both innate and adaptive immune responses. Since dendritic cells and type 1 interferons

are both directly involved in humoral immune response, studies in this aim sought to

understand whether the impaired humoral immune response during acute EBV infection is

due to a dysregulation of peripheral blood DC subsets. Ex vivo analysis revealed that the

impairment of B-cell immunity during acute IM was coincident with a dramatic loss of

circulating myeloid and plasmacytoid DCs, which recovered to baseline following

convalescence. Furthermore, in vitro studies showed that the loss of circulating DCs may

be mediated by IL-10 which is consistently elevated in the plasma of acute IM patients.

Most importantly, we demonstrated that the loss of circulating DCs is intrinsically linked to

the impaired activation and maturation of antibody-secreting B-cells. Our findings show

defective innate immune control during acute infectious mononucleosis, which could have

an effect on humoral immune response.

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5.2. INTRODUCTION

Dendritic cells (DCs) belong to a group of antigen presenting cells (APCs) that

include B cells and monocytes. They provide a link between the innate and adaptive

immune systems. DCs reside in blood and peripheral tissues, constantly monitoring

incoming pathogens. They are activated upon directly sensing pathogens, or by other

innate lymphocytes (Kadowaki, Ho et al. 2001, Leslie, Vincent et al. 2002, Mocikat,

Braumüller et al. 2003). The activation of DCs is known to both initiate and stimulate innate

and adaptive immune responses (Lanzavecchia and Sallusto 2001, Ferlazzo, Pack et al.

2004). DCs are unique in their ability to induce a primary immune response in naïve T cells

and activate memory T cells by presenting processed antigens (Liu and MacPherson

1993, Klechevsky, Morita et al. 2008, Banchereau, Thompson-Snipes et al. 2012). They

present unprocessed antigens to naïve B cells and in so doing trigger humoral immune

response (Wykes, Pombo et al. 1998, Huang, Han et al. 2005). They produce B cell

activation factor BAFF, which is critical for B cell survival and growth (Schneider, MacKay

et al. 1999, Liu, Stacey et al. 2012). DCs also promote immune tolerance and

immunological memory (Brocker, Riedinger et al. 1997, Ludewig, Oehen et al. 1999).

Blood dendritic cells are divided into two subsets: myeloid DCs (mDCs), also known

as conventional DCs, and plasmacytoid DCs (pDCs). These two subsets have different

origins, phenotypes and functions. Myeloid DCs develop from a myeloid progenitor,

express CD11c and other myeloid markers, and secrete IL-12 when activated (Olweus,

BitMansour et al. 1997). More recent studies have shown that mDCs and pDCs have both

lymphoid and myeloid origins, and show flexibility in their development (Shigematsu,

Reizis et al. 2004). Plasmacytoid DCs express CD123 and CD303; when activated they

show a high capacity to produce Type I IFNs, especially IFN- (Siegal, Kadowaki et al.

1999). Both subsets express different toll-like receptors (TLRs) and vary in their responses

to different TLR ligands (Jarrossay, Napolitani et al. 2001, Kadowaki, Ho et al. 2001).

While mDCs express many TLRs and can respond to a variety of ligands from pathogens,

pDCs only express TLR 7 and 9 and specialize in recognizing bacterial and viral ligands

(Kadowaki, Ho et al. 2001).

The mDC and pDC populations are reduced during HIV, Hepatitis B and malaria

infections (Feldman, Stein et al. 2001, Pacanowski, Kahi et al. 2001, Duan, Wang et al.

2004, Pinzon-Charry, Woodberry et al. 2013). Chronic infection by HIV, hepatitis B and C,

and human papilloma viruses also result in lowered production of IFN-by pDCs because

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of impaired TLR signalling (Hasan, Caux et al. 2007, Gondois-Rey, Dental et al. 2009, Xie,

Shen et al. 2009). HIV and malaria infection also cause an accumulation of immature DCs,

which in HIV stimulates T cell tolerance rather than immunity (Krathwohl, Schacker et al.

2006, Pinzon-Charry, Woodberry et al. 2013). Infection of DCs with influenza A virus can

induce apoptosis. However, while mDCs are susceptible to influenza infection, pDCs

exhibit resistance and may secrete large amounts of IFN- in response to infection

(Thitithanyanont, Engering et al. 2007). It has been reported that in malaria infection a

decrease in DCs expressing BAFF results in decreased memory cell differentiation to

antibody-secreting cells (Liu, Stacey et al. 2012).

Although the impact of EBV on DC numbers and function in vivo has not been

thoroughly investigated, recent in vitro observations do suggest an interaction between

DCs and EBV. DCs are known to cross-present EBV antigens from infected B cells to

naïve T cells (Bickham, Goodman et al. 2003). EBV infection has been shown to result in

increased apoptosis of monocytes and inhibit development of dendritic cells (Li, Liu et al.

2002); and direct EBV infection of pDCs through MHC class II molecules is reported to

impair cell maturation (Severa, Giacomini et al. 2013). Other observations reveal that the

interaction of EBV with dendritic cells causes the production of large quantities of IFN-

from pDCs in in vitro culture and also in the humanized NOD/SCID mouse model (Lim,

Kireta et al. 2007, Quan, Roman et al. 2010). However, in contrast to these in vitro

observations, studies of cytokines in the plasma of infectious mononucleosis patients have

consistently found type I interferon IFN- to be absent, but other cytokines—including the

other type I interferon IFN-—to be elevated (Linde, Andersson et al. 1992, Hornef,

Wagner et al. 1995, Prabhu, Michalowicz et al. 1996, Balfour, Odumade et al. 2013). This

led us to hypothesize that blood dendritic cells, pDCs in particular, are involved in

symptomatic EBV primary infection IM since pDCs are the main producers of IFN- We

concluded that studying dendritic cells in acute IM patients would provide insights into

immune response during the early stages of EBV infection.

Studies in this chapter investigated the role of dendritic cells during EBV infection,

specifically during symptomatic primary EBV infection. The phenotype and frequency of

peripheral blood dendritic cell subsets during and post IM were compared to the DC

population in age-matched asymptomatic virus carriers. The data show a severe reduction

in dendritic cell subsets during acute IM and their gradual recovery upon disease

resolution. Since pDCs and the IFN- they produce are critical for the generation of

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plasma cells and Ig production, this aim also provided insights into factors that could affect

humoral immunity during EBV infection.

5.3. MATERIALS AND METHODS

5.3.1. Flow Cytometry

The frequencies of DC subsets during EBV primary and persistent infections were

measured using the four-colour dendritic value bundle from BD Biosciences. The DC

subsets were stained in accordance with the manufacturer’s instructions. Lineage cocktail

(lin1) in FITC included antibodies to surface markers CD3, CD14, CD16, CD19, CD20 and

CD56. The two different subsets of peripheral blood dendritic cells (mDCs and pDCs) were

identified. mDCs were characterized as populations expressing high levels of HLA-DR

(PerCp) and CD11c (APC), while pDCs were characterized as HLA-DR high cells which

express CD123 (PE) or CD123 (BV421). The data were acquired and analysed using flow

cytometry and FlowJo software.

5.3.2. In Vitro DC Culture

For the in vitro culture of DCs, 1x106 PBMC were cultured in FACS tubes (BD Falcon,

BD) to avoid cells adhering to the tissue culture plates. The cells were incubated in serum-

free OpTmizer medium from Invitrogen in the presence of IL-3 (20 ng/ml) and LPS (10

ng/ml), both from R&D systems. The cells were grown for 24 h and used in various in vitro

assays.

5.3.3. In Vitro Infection

PBMC (1x106) were cultured in FACS tubes (BD Falcon) to avoid cells adhering to

the tissue culture plates. The cells were incubated in serum-free OpTmizer medium from

Invitrogen in the presence of IL-3 (20 ng/ml) from R&D systems and EBV B 95-8. The cells

were grown for 24 h and stained for DC subsets and apoptosis.

5.3.4. Apoptosis (Annexin V Binding Assay)

Annexin V binding assays were performed using the Annexin V kit from BD in order

to determine the percentage of apoptotic dendritic cells. A live/dead marker—Live/Dead

Fixable Aqua Dead Cell Stain (Life Technologies)—was used to distinguish between dead

and apoptotic cells. PBMC were washed with 1X Annexin binding buffer (working

concentration made by diluting one part of 10X Annexin V binding buffer provided in the kit

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with nine parts distilled water), following staining with dendritic cell-specific surface

markers. The cells were incubated with 1 µl Annexin V (PE) in 100 µl Annexin binding

buffer for 15 min at room temperature in the dark. After incubation, 200 µl of 1X binding

buffer was added to each tube and analysed by flow cytometry within 1 h of staining.

5.3.5. Cytokine Influence

To test the role of cytokines in the reduction of dendritic cells during infectious

mononucleosis, PBMC from healthy donors were cultured in serum-free OpTmizer

medium in the presence of IL-3 and LPS. First, cells were incubated with 50% vol/vol

plasma from acute IM patients and age-matched asymptomatic virus carriers for 24 h, after

which the frequency of DCs and percentage of apoptotic DCs were measured by flow

cytometry. Next, various cytokines that are upregulated in infectious mononucleosis

samples were added individually at concentrations of IL-4 (10 ng/ml), IL-6 (10 ng/ml), IL-10

(500 pg/ml), IL-12 (10 ng/ml), IFN- (100 u/ml), TNF-(50 ng/ml) and TGF- (10 ng/ml).

Cytokines (R&D Systems) were added and the tubes incubated for 24 h in a 37oC

incubator, then analysed by flow cytometry with dendritic cell-specific surface markers and

Annexin V staining, both from BD.

5.3.6. Migration to Lymphoid Organs

The possibility of increased migration of blood DCs to lymphoid organs was tested by

studying the expression of lymphoid homing marker CCR7. The cells were incubated with

CCR7 (AF 700) for 10 min at 4oC before incubating with DC-specific surface markers for

another 20 min. DCs were then analysed for increased expression of CCR7 by flow

cytometry.

5.3.7. DC-B Cell Interaction

PBMC (1x107) from healthy donors were used to confirm the requirement of dendritic

cells for the conversion of memory B cells to antibody-secreting cells. Dendritic cells were

depleted using DC-specific microbeads from Miltenyi Biotec following manufacturer’s

instructions. The microbeads included CD304 (BDCA–4/Neuropilin–1) to deplete pDC and

CD1c (BDCA–1), and CD141 (BDCA–3) to deplete mDC subsets. 10 µl of each antibody

conjugated to biotin was incubated per 107 PBMC for 10 min in the dark in 100 µl buffer

(PBS with 2 mM EDTA and 0.5% FCS). The cells were washed with 2 ml of buffer, after

which 30 µl of anti-biotin microbeads were added before incubation for another 10 min.

The cells were washed with 2 ml buffer and resuspended in 500 µl buffer for magnetic

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separation using an autoMACS separator (the instrument and antibodies from Miltenyi

Biotec).

The instrument was prepared and primed in accordance with the manufacturer’s

instructions. The sample tubes were placed at the uptake port, while fraction collection

tubes were placed at port pos1 for the positive fraction (labelled) and port neg1 for the

negative fraction (unlabelled) using the deletes program on the separator. The cells were

then washed and resuspended in R10 containing an optimized mix of polyclonal mitogens

for the conversion of memory B cells to antibody-secreting cells using whole PBMC, DC-

depleted PBMC, and DC-depleted PBMC with DCs reconstituted. The conversion of

memory B cells to antibody-secreting cells, with and without dendritic cells, was analysed

by B cell ELISpot assay and flow cytometry after in vitro culture for six days.

5.4. RESULTS

5.4.1. Frequency & Phenotype of Blood Dendritic Cells during EBV Primary Infection

In order to study the impact of EBV infection on circulating DC populations,

phenotypic characterization was performed using the human DC four-colour assay kit as

described in the Materials and Methods section. The blood DC population was identified in

primary and persistent EBV infection as lineage-HLA-DR+ cells, which can be further

divided into the two major DC subsets—the CD11c+ myeloid (mDC) and the CD123+

plasmacytoid (pDC) dendritic cells. The lineage cocktail included CD3, CD14, CD16,

CD19, CD20 and CD56 antibodies conjugated to FITC for the elimination of T cells,

monocytes, B cells and NK cells. PBMC from asymptomatically-infected latent virus

carriers and individuals during and post-IM were analysed. Figure 5.1 shows the

longitudinal analysis of a donor undergoing acute IM (top panel) and after recovery, post-

IM (> six months later, middle panel). Figure 5.1 also shows the gating strategy for human

peripheral blood DCs (bottom panels) from a latent EBV carrier.

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Figure 5. 1. Peripheral blood dendritic cells during EBV infection

Seen here are representative dot plots obtained by flow cytometry of the gating strategy of peripheral blood dendritic cells from donors at different stages of EBV infection. The lineage-HLA-DR+ DCs are further divided as its major subsets as defined by the expression of CD11c+ myeloid DCs (mDCs) and CD123+ plasmacytoid DCs (pDCs). Lineage markers eliminate T cells, B cells, NK cells and monocytes. Top panels show the frequency of dendritic cells in a longitudinal sample from a donor during and post IM, while the bottom panel shows the frequency of DCs from an asymptomatic virus carrier. DCs and their subsets are shown as the percentage of PBMC.

A compilation of the data analysed from all volunteers at different stages of EBV

infection is shown in Figure 5.2. A significant reduction was noticed in the frequencies of

both blood DC populations during acute IM. Gradual recovery in both pDC (Figure 5.2A)

and mDC (Figure 5.2B) numbers was evident during convalescence, with numbers in long-

term patients similar to those seen in asymptomatically-infected individuals. Interestingly, a

more profound loss was evident for pDCs, which was associated with a slower recovery in

numbers during resolution of the infection.

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Figure 5. 2. Frequency of DC subsets at different stages of EBV infection

Panels A and B show the compilation of all data analysed from donors at different stages of EBV infection — 20 acute IM patients, 12 donors recovering from IM (20–50 days from the start of symptoms), 16 donors post IM (> 6 months from the start of symptoms), and 10 age-matched asymptomatic virus carriers. Panel A shows the frequency of pDCs, and Panel B shows the frequency of mDCs. The box plot shows mean, range and data points; statistical analysis was done by one-way ANOVA, Tukey’s multiple comparison with p values represent **** p <0.0001, ** p<0.01 and * p<0.05.

Analysis of the absolute number of pDCs and mDCs also confirmed a significant

reduction in peripheral DC populations during acute EBV infection (Figure 5.3A, 5.3B). The

number of pDCs (A) and mDCs (B) was estimated based on the total PBMC count per litre

of blood at the time of harvest.

Next, the severity of illness (SOI) was correlated with reduction in dendritic cell

frequency. Acute IM samples were scored for severity of illness. As mentioned in Chapter

3, the SOI scores assigned to acute IM patient donors ranged from zero to six, with

asymptomatic infection receiving a score of zero and severe symptoms requiring

hospitalization or confinement to bed a score of six. The samples were grouped according

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Figure 5. 3. Frequency of DC subsets in whole blood and correlation to severity of infection

Acute EBV infection is associated with an absolute loss in the number of peripheral dendritic cell populations that is associated with the severity of disease. The number of pDCs (A) and mDCs (B) was estimated based upon the total PBMC count per litre of blood at the time of harvest. Panel C shows correlation between frequency of DCs and severity of infection. Acute IM patient samples from collaborators at the University of Minnesota were grouped according to severity of infection, with 0–3 classified as mild and 4–6 as severe, where a score of 0 was assigned to donors with asymptomatic infection and a score of 6 to completely bed-ridden patients. Statistical analysis was done by non-parametric Mann Whitney test and the p values correspond to ** p<0.01 and * p<0.05.

to severity of infection, with scores 0–3 characterized as mild and 4–6 as severe. Samples

from six donors with mild symptoms and nine donors with severe IM symptoms were

analysed. The frequency of dendritic cell subsets in the samples was plotted against

severity of illness of the respective patients. A direct correlation was observed, with loss of

DCs, especially pDCs (Figure 5.3C), increasing as SOI scores increased. The number of

circulating pDCs was significantly lower in patients with an SOI of 4-6. Although not at

statistically significant levels, there was a trend towards a more severe reduction in mDC

populations in patients with an SOI of 4-6.

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5.4.2. Migration to Lymphoid Organs

Circulating blood DCs are either immature DCs migrating from the bone marrow to

peripheral tissue, or mature antigen-activated DCs migrating to lymphoid tissue to initiate

an immune response (de la Rosa, Longo et al. 2003). One possible explanation for the

loss of blood DC populations during acute IM is increased migration of DCs from blood to

lymphoid tissues where they are required to initiate adaptive immune response during an

acute infection. The expression of lymphoid homing marker CCR7 on blood DC subsets

was used to determine whether these cells showed evidence of enhanced migration.

Longitudinal samples from six donors during and post IM were analysed, along with six

age-matched asymptomatic virus carriers. No significant difference in the level of CCR7

expression on both pDCs (Figure 5.4A) and mDCs (Figure 5.4B) was evident during acute

IM when compared with levels in the latent carrier state. This does not rule out the

possibility of increased migration of blood DCs into lymphoid organs as the DCs

expressing high CCR7 could have already migrated to lymphoid organs when symptoms

first appeared.

Figure 5. 4. Migration to lymphoid organs

Differential expression of lymphoid

homing marker CCR7 is measured as

MFI in six longitudinal IM samples and

six asymptomatic virus carriers in DC

subsets. Panel A compares the MFI of

CCR7 expression in pDCs. Panel B

compares the MFI of CCR7 expression

in mDCs.

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5.4.3. Effect of Cytokines on the Survival of DCs

As with many acute infections, IM is associated with increased plasma levels of many

inflammatory and suppressive cytokines. Previous observations have suggested that some

of these cytokines have a detrimental effect on the survival of DC populations during acute

infection (Pinzon-Charry, Woodberry et al. 2013). Therefore, to assess the impact of

circulating plasma cytokines in IM patients on their DC subsets, PBMC from healthy

donors were incubated with plasma from acute IM patients and with plasma from age-

matched asymptomatic virus carriers for 24 h. The cells were cultured in serum-free

medium in the presence of IL-3, which is required for pDC survival, and the DCs were then

induced to mature using LPS. Incubation with plasma from acute IM patients caused a

marked loss of DCs in PBMC as compared to plasma from asymptomatic carriers (Figure

5.5A). Furthermore, these cells also showed down-regulation of surface HLA-DR

expression following incubation with acute IM plasma, which was more pronounced for lin-

CD123+ pDCs (Figure 5.5B) than in lin-CD11c+ mDCs (data not shown). A summary of

data based on 10 acute IM patients and five latent virus carriers is shown in Figure 5.5C.

Figure 5. 5. Effect of IM plasma on DC survival

Blood DCs in the PBMC of healthy donors were cultured in serum-free medium and induced to mature with the addition of LPS and IL-3, which are required for pDC survival. The cells were incubated with 50% v/v of medium alone, plasma samples from asymptomatic virus carriers, or plasma from acute IM patients. The percentages of lineage-HLA-DR+ cells were then compared. HLA-DR expression and percentage of apoptotic cells were analysed by flow cytometry.

Panel A shows the frequency of blood dendritic cells when incubated with medium alone, with plasma from asymptomatic virus carriers, and with plasma from acute IM patients.

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Panel B shows comparison of HLA-DR expression between medium alone with acute IM plasma and medium alone with asymptomatic virus carrier plasma.

Panel C is a summation of all data analysed from 10 acute plasma samples and 5 asymptomatic

plasma samples comparing the MFI of HLA-DR expression on lin-CD123+ pDCs.

Analysis by non-parametric Mann Whitney test; p values correspond to ** p<0.01 and * p<0.05.

Figure 5. 6. Effect of cytokines in the survival of blood DCs

Various cytokines upregulated in acute IM plasma were tested for their effect on the survival of DCs. Representative dot plots of a few cytokines tested are shown in Panel A. The cells were cultured in serum-free medium with LPS and IL-3, and various cytokines tested were added individually and incubated for 24 h, after which the frequency, HLA-DR expression, and percentage of apoptotic cells were analysed by flow cytometry. The frequency of DCs is represented as percentage of lymphocytes.

Panels B and C show the percentage of apoptotic cells in mock (medium-alone) and hIL-10 added blood DCs, with Panel B showing percentage of annexin-positive pDCs and Panel 6C showing

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annexin-positive mDCs from 10 donors. Statistical analysis was done by non-parametric Mann Whitney test and the p values correspond to ** p<0.01 and * p<0.05.

Panel 6D shows the representative histogram of HLA-DR expression when pDCs are incubated with hIL-10 and a representative histogram of HLA-DR expression when incubated with vIL-10, as a representative for all other cytokines that were tested, both compared to HLA-DR expression when cultured in medium-alone.

Next, we tested the influence of individual cytokines elevated during acute IM to see

whether any one of these cytokines, or a combination of them, could have caused

dendritic cell loss. Antiviral, inflammatory and suppressive cytokines found elevated during

acute IM were chosen for in vitro testing based on previously published findings by Balfour

and colleagues (Balfour, Odumade et al. 2013). PBMC from healthy volunteers were

cultured in serum-free medium in the presence of IL-3 and LPS, followed by incubation

with IL-4, IL-6, IL-10 (human and viral), IL-12, IFN-, TNF-, and TGF-. Figure 5.6A

shows a representative dot plot of the effect of a select cytokine on blood DCs. Of all the

cytokines tested, the addition of hIL-10, but not viral IL-10, was found to result in the loss

of dendritic cells (Figure 5.6A). An Annexin V binding assay was performed to determine

whether the loss was due to apoptosis. While there was increased apoptosis with the

addition of hIL-10 in both mDCs and pDCs, the loss of pDCs was greater than that of

mDCs (Figure 5.6B, Figure 5.6C). A reduction in HLA-DR expression was also evident on

pDCs with the addition of hIL-10. Figure 5.6D shows representative histograms of HLA-DR

expression comparing hIL-10 treated PBMC versus mock-treated PBMC.

5.4.4. Requirement of Dendritic Cells for Plasmablast (ASC) Formation

Previous studies have shown the importance of both pDCs and mDCs in the efficient

generation of ASCs (Jego, Palucka et al. 2003). Given the importance of dendritic cells in

promoting humoral immunity, and the findings that global and EBV-specific B cell immunity

are impaired during acute IM, the impact of DC depletion on the generation of ASCs was

tested in vitro. PBMC were depleted of dendritic cells using microbeads specific for

dendritic cell subsets: CD141, CD1c and CD303 (while CD141 and CD1c deplete mDCs,

CD303 depletes pDCs). Figure 5.7A shows the efficiency of DC depletion using PBMC

from a healthy donor. Then 1x106 untouched PBMC, PBMC depleted of DCs, and DC-

depleted PBMC with reconstituted DCs were cultured for six days using polyclonal

stimulators. On the sixth day, total IgG-secreting cells were quantitated using ELISpot

assay. Representative data from the ELISpot assay is shown in Figure 5.7B. The in vitro

cultured cells were also assessed for the presence of CD3-/CD19+/CD20lo/CD27hi/CD38hi

ASCs.

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Figure 5. 7. Dendritic cell depletion and its effect on B cells

Blood dendritic cells from the PBMC of healthy donors were depleted using DC-specific microbeads from Miltenyi Biotec. Panel A shows the DC population in PBMC from a healthy donor, top half, and effective depletion of DCs in the bottom half.

In Panel B, a representative ELISpot analysis shows the conversion of memory cells to antibody-

secreting cells. PBMC, DC-depleted PBMC, and PBMC with DC reconstituted from a healthy donor

were cultured with an optimised mix of polyclonal mitogens that convert memory B cells to

antibody-secreting cells after six days (B-stim). The frequencies of total IgG-secreting cells were

analysed in these three PBMC.

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A representative flow cytometric analysis is shown in figure 5.8. These observations

showed that the conversion of memory B cells to antibody-secreting cells and production

of IgG were severely reduced in DC-depleted PBMC.

Figure 5. 8. Phenotypic analysis of in vitro-activated PBMC with and without the presence of DCs

In vitro activated PBMC, in the presence and absence of dendritic cells, were subjected to phenotypic analysis by flow cytometry. The frequencies of total B cells (CD3-/CD19+) and antibody-secreting cells (CD3-/CD19+/CD20lo/CD27hi and CD38hi) were calculated as a percentage of lymphocytes. The lymphocyte gate was extended to include the activated B cell blasts.

The percentages of lymphocyte activation and total B cell numbers were significantly

reduced in DC-depleted PBMC when compared to untouched PBMC and DC-depleted

PBMC in which DCs were reconstituted (Figure 5.9A). The percentage of ASCs was also

reduced significantly in DC-depleted cultures (Figure 5.9B), which was associated with a

significant reduction in the number of IgG-secreting cells in DC-depleted populations

(Figure 5.9C). DC reconstitution restored IgG-secreting cells following in vitro stimulation.

To determine if B cells stimulated in the absence of DC showed evidence of increased

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apoptosis, an Annexin V binding assay was performed. The percentage of apoptotic B

cells in DC-depleted PBMC in culture was significantly higher when compared to

untouched PBMC and PBMC in which DCs were reconstituted (Figure 5.9D). This data

shows that DCs are required not only for the generation of antibody-secreting cells but

also for B cell survival in culture.

Figure 5. 9. Requirement of dendritic cells for antibody-secreting cell formation

Compilation of total B cells (Panel A), antibody-secreting cells (Panel B) by flow cytometry, and total IgG-specific memory B cells as measured by ELISpot assay (Panel C) were quantified using PBMC, with and without dendritic cells. PBMC were cultured in the presence of polyclonal mitogens for 6 days (B-stim). The percentages of annexin positive B cells were compared between the different PBMC to quantify apoptotic cells by flow cytometry in Panel D. Statistical analysis was done by one-way ANOVA, Tukey’s multiple comparison and the p values correspond to ****p<0.0001, ***p<0.001, ** p<0.01 and * p<0.05.

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5.5. DISCUSSION

The role of dendritic cells during the early stages of EBV infection and during acute

IM, is not well understood. The aim of this chapter, therefore, was to investigate the

potential role of DC in the immune dysfunction that is evident during acute IM. The

observations seen in this chapter demonstrated that in addition to dysfunction in the B cell

compartment during acute EBV infection, patients show a dramatic loss in circulating DC

populations. There was no evidence that this loss in circulating DCs was due to an

increase in the expression of lymphoid homing signals, though it did appear to be

associated with increased inhibitory cytokine levels, particularly of IL-10, which is known to

affect DC function and survival. Importantly these observations also suggest a potential

mechanism of B cell dysfunction in IM patients, by demonstrating a critical role for DCs in

promoting B cell maturation into ASCs.

The interaction of antigen-presenting cells with pathogens results in the initiation of

both innate and adaptive immune responses (Banchereau and Steinman 1998). In vitro

experiments and in vivo humanized mouse models show that pDCs and mDCs, the two

blood dendritic cell subsets, are activated when dendritic cells interact with EBV (Lim,

Kireta et al. 2006, Iwakiri, Zhou et al. 2009). This then leads to the production of Type I

interferons, mainly IFN- from pDCs. IFN- is a key cytokine in the initiation of both innate

and adaptive immune responses. Various studies measuring cytokines upregulated during

acute IM consistently show an absence of IFN- in the plasma of IM patients, although the

levels of other antiviral, inflammatory and immune-suppressive cytokines appear elevated

(Balfour, Odumade et al. 2013). Since pDCs are the main producers of IFN-, and

considering the importance of IFN- in the development of humoral immunity, the

frequency and phenotype of peripheral blood dendritic cells during primary EBV infection

was studied.

This is the first study to explore the fate of dendritic cells during EBV primary

infection. Longitudinal comparisons during acute infection and after recovery with age-

matched controls showed a severe reduction in dendritic cell population, both pDCs and

mDCs, during acute IM, with gradual recovery as the disease resolved. This loss of DCs,

especially pDCs, likely accounts for the lack of IFN-found in the plasma of IM patients.

Interestingly, the loss of pDCs was significantly greater and took longer to recover from

than the loss of mDCs. This could suggest some prolonged dysfunction in the DC

compartment following IM, although this possibility was not explored. However it is evident

that disease severity is associated with the loss of circulating pDC populations, suggesting

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that either the level of pDC loss is critical in the development of disease or that the

increased viral load and other inflammatory changes that occur during IM impact upon

pDC survival.

Various studies have reported a decrease in the frequency of dendritic cells during

viral infection. Viruses like HIV, HSV, measles and dengue can directly infect DCs and

affect their frequency and function (Feldman, Stein et al. 2001, Marovich, Grouard-Vogel

et al. 2001, Bosnjak, Miranda-Saksena et al. 2005, de Witte, Abt et al. 2006). A direct

cytopathic influence of EBV on the survival of dendritic cells has not been found, even

though in vitro experiments have shown that EBV can infect and impair pDC maturation

(Severa, Giacomini et al. 2013). It has also been shown that EBV infection of monocytes

results in their increased apoptosis and inhibits the development of monocyte precursors

to dendritic cells (Li, Liu et al. 2002). While the mechanism responsible for the loss of DCs

during acute IM has not been fully explained, it is evident that cytokines do play a role, as

shown by in vitro observations that incubation with IM plasma and hIL-10 results in the

loss of DCs. The effect of IL-10 on the survival of DCs has been reported in malaria as

well, where prolonged exposure to IL-10 resulted in the loss of DCs by apoptosis (Pinzon-

Charry, Woodberry et al. 2013). In vitro experiments in this study, too, showed a reduced

expression of HLA-DR when incubated with the plasma of IM patients or when incubated

with hIL-10. However, this phenomenon of low HLA-DR expression was not seen in ex

vivo experiments where the phenotype of blood DCs was studied in the PBMC of IM

patients.

Finally, to connect all three aims of this project, the requirement of dendritic cells in B

cell activation and generation of antibody-secreting cells was assessed. The requirement

of IFN- secreted by pDCs was reported to be essential for the production of Ig (Le Bon,

Schiavoni et al. 2001, Jego, Palucka et al. 2003) while mDCs provide BAFF and APRIL as

well as the IL-12 required for B cell survival and plasma cell differentiation respectively

(Dubois, Massacrier et al. 1998, Litinskiy, Nardelli et al. 2002). Since both DC subsets

were affected during acute IM and both are required for B cell survival and function, the

DC-depletion study was done by depleting total blood DCs. DC-depleted PBMC showed

less B cell activation and a reduction in ASC when compared to untouched PBMC, or

when DC-depleted PBMC were reconstituted with DC. These findings indicate that

dendritic cells are required for humoral immune response and that a defect in the DC

population results in a reduction in the generation of antibody-secreting cells. Contrary to

other published findings that depletion of pDCs led to the cessation of Ig production but

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not the death of B cells (Jego, Palucka et al. 2003), our results indicate that depletion of

both blood DC subsets affect Ig synthesis and the survival of B cells. Thus, defects in both

innate and humoral immune responses could contribute to the immune dysregulation

noticed in acute infectious mononucleosis.

The findings of this aim suggest that a loss of DC during the early stages of EBV

infection contributes to symptomatic IM. Dendritic cells are important in the control of viral

infection, as they are found in the periphery where pathogens enter the host. They can

sense pathogens and alert the immune system. Because of this, many pathogens target

dendritic cells as part of their immune evasion mechanism, but none has been found to be

as profound as during symptomatic EBV primary infection IM. Since dendritic cells play

both direct and indirect roles in humoral immune response, this defect could alter the

humoral immune response to EBV. Further studies on the mechanism of the loss of

dendritic cells could give insights into the dysregulation observed during acute IM and the

role played by peripheral blood dendritic cells in this disease.

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CHAPTER 6. DISCUSSION AND FUTURE DIRECTION

Delayed EBV infection causes infectious mononucleosis (IM) in more than 50% of

infected adolescents and young adults. IM is an immunopathological disease, in which the

symptoms result from an uncontrolled expansion of EBV-specific CD8+ T cells in some

individuals. It is not known why some individuals are more likely than others to develop IM.

The risk of developing IM increases with age, as does the severity of symptoms. The

reasons for this age-specific severity of IM are not known. One possible explanation for

this phenomenon is a concept termed heterologous immunity. This refers to T cell cross-

reactivity, where memory T cells specific to one virus get activated by an unrelated virus

and in doing so alters the host’s immune response (Selin, Varga et al. 1998). CD8+ T cells

specific to EBV lytic proteins have been found to cross-react with influenza-specific

proteins (Clute, Watkin et al. 2005). Another possible explanation is the difference in the

amount of virus received during primary infection. It is possible that a larger dose of virus

is transferred between young adults during kissing compared to less invasive methods of

infection during childhood. There is also a recent report that shows an imbalance in the

CD8+Tcell differentiation during early life of mice. CD8+T cells predominantly differentiate

into short lived effector cells with impairment in memory CD8+T cell formation (Smith,

Wissink et al. 2014). If this is true in humans, that could also explain the lack of

immunopathology in young children during primary EBV infection.

Although the disease is typically self-limiting, a history of IM is associated with an

increased risk of developing EBV-associated Hodgkin’s Lymphoma and certain

autoimmune disorders, including Multiple Sclerosis. Due to improved standards of living

the incidence of IM is increasing, especially in developed countries (Tattevin, Le Tulzo et

al. 2006). Certain similarities have been reported between IM and EBV-associated

diseases. The presence of multinucleated cells, Reed-Sternberg cells, and increased

susceptibility of individuals with HLA class 1 polymorphism was found in both IM and HL.

Similar B cell profiles with loss of naive and memory B cells and an overexpression of V4-

34 encoded Ig, which is associated with autospecificity, were found in IM and autoimmune

disease systemic lupus erythematosus (Mockridge, Rahman et al. 2004). The risk of MS

was reported to last for 30 years after IM, and persistent immunological changes caused

during IM were thought to be among the reasons for this increased risk (Nielsen,

Rostgaard et al. 2007). Thus, primary EBV infection could be a factor that determines the

risk of developing more serious EBV-associated diseases.

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Protection against most pathogens requires the induction of long-lived plasma cells

and memory B cells, the two arms of humoral immunity. The study of B cells during EBV

infection so far focused on the establishment and maintenance of persistent latent EBV

infection. So the objective was to understand humoral immune response to EBV,

especially during IM. We first examined global B cell response to EBV infection. We found

that unlike other acute infections—including malaria, dengue, hepatitis B and influenza,

which are accompanied by an increase in memory B cells and ASCs (Bocher, Herzog-

Hauff et al. 1999, Weiss, Traore et al. 2010, Wrammert, Koutsonanos et al. 2011,

Wrammert, Onlamoon et al. 2012)—acute IM causes a significant loss of MBCs and no

increase in the frequency of ASCs. Similar to the published findings on one IM patient

during incubation, acute IM and recovery (Svedmyr, Ernberg et al. 1984), our data show

that the ability of memory B cells to respond to mitogen activation and Ig production are

severely diminished during acute IM. The ability of memory B cells to convert to antibody

secreting cells and/or the survival of plasmablast seem to be compromised during acute

IM. This defect was not found to be a direct consequence of EBV infection, for in vitro

infection with EBV followed by mitogen activation showed normal conversion efficiency

and IgG synthesis. This suggests that other factors, such as virus-induced differential

expression of cytokines, contribute to the survival and functioning of B cells. Increased

expression of IL-6 and IFN- during acute IM has been reported to correlate positively with

severity of infection (Balfour, Odumade et al. 2013, Dunmire, Odumade et al. 2014).

Dunmire and colleagues also reported the lack of induction of nine interferon response

genes during IM that are upregulated during various other viral infections. Other studies,

too, have found a lack of IFN- production during acute IM (Hornef, Wagner et al. 1995,

Prabhu, Warwick et al. 1996), which is important for the generation of ASCs (Jego,

Palucka et al. 2003).

We next explored factors that could impact upon the survival of B cell subsets

during acute IM. BAFF, B cell activation factor, and its receptor expression on B cells are

crucial for the survival of B cells. Studies conducted in mice have shown that BAFF-BAFF-

R signalling is crucial to B cell homeostasis because it prevents apoptosis through various

signalling pathways (Rauch, Tussiwand et al. 2009). The reduced expression of BAFF-R

on IM B cells could have an effect on the survival of B cells. Similar to IM, other diseases

that result from B cell dysfunction—like autoimmunity—show a significant increase in

BAFF concentration in the plasma of patients (Sellam, Miceli-Richard et al. 2007,

Kreuzaler, Rauch et al. 2012). The presence of excess BAFF in the plasma of patients

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could be a marker for B cell dysfunction. BAFF homolog APRIL, which is reported to be

important for the survival of long-lived plasma cells in bone marrow (Belnoue, Pihlgren et

al. 2008), was reduced during acute IM; which could be a factor that affects plasma cell

survival. During acute infection, B cells also were found to express increased levels of

death receptor FAS and, in combination with BAFF dysregulation, this could compromise

B cell survival.

After studying global B cell function, our investigations turned to the quantity and

quality of virus-neutralising antibody response during acute IM. Humoral immune response

during acute IM showed a complete lack of virus-neutralizing antibodies, as measured by

the ability of antibodies in the plasma of IM patients to neutralise the virus. The frequency

of EBV-MA gp350-specific memory B cells, which could induce virus-neutralizing antibody

response and gp350-antibody titre, was extremely low in patients with IM. The poor

neutralising antibody response during acute IM could result in defective humoral immune

control and increased viral load, as seen in IM patients. In addition, there was no

detectable level of circulating memory TFH cells or gp350-specific TFH cells in the blood of

IM patients. EBV-MA gp350 was also found to be a very poor immunogen for both CD4+

and CD8+ T cells. While studies of peripheral blood have provided compelling evidence of

dysfunction in the humoral immune response during acute EBV infection, the tonsils are

the primary site of EBV infection and also one of the secondary lymphoid organs where

the initiation of immune response occurs. Therefore, analysis of the humoral immune

response, including the induction of gp350 immunity and TFH cells in tonsils, will likely

provide further insight into the dysregulated responses during IM. Despite its abundance

during acute infection, the lack of both a virus-neutralising antibody response and poor

induction of gp350-specific T cell responses suggest that inefficient handling of the gp350

antigen for presentation to both T and B cells may be an immune evasion mechanism of

EBV that promotes the successful establishment of a persistent infection.

The last aim of the project was to describe the function of dendritic cells during IM.

DCs have long been known to play a critical role in the induction of both cellular and

humoral immunity. Our data revealed a severe reduction in dendritic cell populations in

acute IM patients compared to DCs in latent virus carriers. Among the two DC subsets,

pDC loss was found to be far more severe than mDC loss, and pDC levels recovered at a

much slower rate than mDCs. The direct correlation between frequency of DC subsets and

severity of infection suggests a role for DC subsets in the development of IM. One

mechanism by which DC loss may be contributing to immune dysfunction during IM is by

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suppressing IFN- production, which is primarily produced by pDCs. Previous

observations have shown that IFN-produced by pDCs is critical for the efficient

differentiation of B cells into antibody-secreting plasmablasts (Le Bon, Schiavoni et al.

2001, Jego, Palucka et al. 2003). Indeed, the observations in this study support this

hypothesis, whereby it was demonstrated that DC depletion prevents the efficient

generation of antibody-secreting cells and Ig production. It has been reported that IFN-

secretion is required for the survival of pDCs in an autocrine manner (Kadowaki,

Antonenko et al. 2000). Various proteins encoded by EBV have been shown to target the

production of IFN-. EBV tegument protein LF2 and BZLF1 are two such proteins that

inhibits the function of IRF7 which is required for the production of type I interferons (Hahn,

Huye et al. 2005, Wu, Fossum et al. 2009). It is not clear whether the activities of these

EBV proteins results in the reduced secretion of IFN- that contributes to the loss of pDCs

or the reduced pDC number results in the reduced secretion of IFN-. Although

speculative, a plausible explanation is that the profound loss of DCs during acute IM may

be contributing to the lack of efficient maturation of B cells into ASCs during acute IM in

vivo, as evidenced by an absence of notable change in ASCs numbers in the peripheral

blood of IM patients.

Even though DCs influence the development of cellular and humoral immune

response, the loss of DCs does not seem to diminish antibody response to VCA during

acute IM, and the massive expansion of EBV-specific CD8+ T cells. It could be that the DC

loss observed during acute IM is confined to the blood DC population and that tissue-

resident DCs are not affected to the same extent. Our data does not address the impact of

the loss of blood DCs on the tissue specific DC pool during acute IM. It could also mean

that the blood DC loss occurs after the initiation of both humoral and cellular immune

response and is attributable to virus-induced environmental changes like prolonged

exposure to elevated cytokine levels as found during malaria infection (Pinzon-Charry,

Woodberry et al. 2013).

This study explored various reasons for the loss of pDCs. There was no evidence of

increased migration of blood DCs to lymphoid organs, or cytopathic effects of EBV, either

of which could have explained reduced blood DCs. We also tested, in vitro, the effect of IM

plasma and various cytokines that are elevated during acute IM. The results showed DC

loss when PBMC were incubated with IM plasma and a similar effect when they were

incubated with hIL-10. There was also a decrease in HLA-DR expression on pDCs, which

may not be relevant to IM as it was not found during ex vivo analysis of PBMC from IM

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patients. Further studies are required to elucidate the mechanism responsible for pDC loss

during acute IM. EBV infection could also result in other factors that affect the survival of

DCs. EBV-encoded protein BARF1 could be one such factor that could play a role in the

loss of DCs during IM (Cohen and Lekstrom 1999). The protein is produced during the

early stages of EBV infection and was found to inhibit IFN- secretion by blocking colony-

stimulating factor 1 (CSF-1). Whether this protein could also block type I interferon

regulatory genes and affect IFN- production remains to be explored. Studies into the role

of BARF1 protein during EBV infection could shed light into the lack of IFN- secretion and

DC loss.

Different models have been proposed over the years to explain symptomatic EBV

infection in adolescence and adults. Size of the viral load acquired during the initial

infection was suggested as one factor that could cause IM. This was found to be validated

in later studies where high viral load was found to correlate with severity of symptoms

(Williams, Macsween et al. 2004). Genetic differences with polymorphism in cytokine

genes such as IL-10 and IL-1 complex and variations in T cell response with HLA-A1 and

HLA-DR polymorphism were also suggested as possible factors contributing to developing

IM (McAulay, Higgins et al. 2007, Ramagopalan, Meier et al. 2011). Defective NK cell

control in the early stages of EBV infection has been suggested as another factor for

susceptibility to symptomatic EBV infection. A humanized mouse model depleted of NK

cells was found to have severe IM symptoms and was prone to EBV-associated

tumorigenesis (Chijioke, Muller et al. 2013, Azzi, Lunemann et al. 2014).

The above models provide alternate explanations for the cause of IM. Based on the

data from this study, we propose another possible model for immune dysregulation during

acute IM. IM causes a severe loss of blood DCs especially pDCs which, in turn, results in

the lack of IFN-, a key cytokine in the initiation of immune response. The absence of IFN-

, along with dysregulation in BAFF signalling, results in a poor virus neutralizing antibody

response, reduces memory B cell formation, and no increase in antibody-secreting cells

during acute IM. The outcome is an increase in viral load and uncontrolled expansion of

CD8+ T cells, which contribute to the clinical symptoms of IM. A few steps in this model

need clarification. The requirement of DC subsets for the survival and differentiation of B

cells was confirmed by our findings during the DC depletion study and also in research

undertaken by Jego and colleagues (Jego, Palucka et al. 2003). But the environmental

changes taking place during incubation and in the early days of IM—which influence

phenotypic changes in blood DCs and B cell subsets, and so affecting their survival—need

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further study. Also requiring study are the differential expression of neutralising and non-

neutralising antibody responses, which cause the high viral load, and the massive CD8+ T

cell expansion, which leads to symptomatic infection.

This study was able to bring forth the defects in the innate and humoral immune

responses that could contribute to symptomatic primary EBV infection, but the factors that

bring about this change need to be identified. Future work arising from this study should

include elucidating the mechanism of loss of dendritic cells, especially pDCs, during IM.

The factors that affect the phenotypic changes in B cells, which result in very poor

antibody production and reduced survival during acute IM, also need to be explained.

These insights will give a complete picture of the immune dysregulation that causes IM.

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