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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
i
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.
ii
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.
iii
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
iv
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.
v
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%
vi
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
vii
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
viii
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
ix
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
x
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
xi
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)
1
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).
2
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
3
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,
4
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
5
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).
6
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
7
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).
8
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).
9
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).
10
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
11
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).
12
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
13
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).
14
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
15
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
16
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).
17
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).
18
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
19
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.
20
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
21
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,
22
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).
23
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).
24
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
25
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
26
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
27
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.
28
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
29
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
30
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
31
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
32
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.
33
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
34
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
35
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.
36
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.
37
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.
38
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
39
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.
40
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
41
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
42
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
43
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.
44
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
45
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.
46
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.
47
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.
48
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.
49
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.
50
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.
51
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
52
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.
53
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.
54
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.
55
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.
56
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,
57
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
58
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.
59
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.
.
60
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
61
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
62
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.
63
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.
64
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
65
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.
66
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
67
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).
68
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
69
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).
70
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
71
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
72
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
73
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,
74
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
75
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
89
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
90
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
109
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
110
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
111
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.
112
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