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NATURAL KILLER AND T-CELL IMMUNITY PRE-TRANSPLANT AS A PREDICTOR FOR

CYTOMEGALOVIRUS POST-TRANSPLANT

by

Shanil S Keshwani

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Institute of Medical Science University of Toronto

© Copyright by Shanil S Keshwani (2015)

ii

Natural Killer and T-Cell Immunity Pre-Transplant as a Predictor

For Cytomegalovirus Post-Transplant

Shanil S Keshwani

Masters of Science

Institute of Medical Science University of Toronto

2015

Abstract

Pre-transplant CMV specific T-cell and NK-cell responses could predict the risk of CMV

reactivation following transplantation. We tested this hypothesis in a large cohort of

CMV seropositive transplant recipients enrolled in five centers. No patient received

antiviral prophylaxis and all had regular viral load monitoring. PBMCs were stimulated

with live virus followed by immunophenotyping of T and NK-cell subsets including CD4,

CD8, T-reg populations (CD4+CD25+ FOXP3+), CD16, CD56, NKG2C, and IFN-γ. The

primary outcome was viremia requiring antiviral therapy within the first 3-months post-

transplant. A total of 272 CMV R+ transplant patients were analyzed. Transplant types

included kidney (65.4%), liver (23.5%), and other (11.1%). CMV viremia occurred in

83/272(30.7%) and disease in 10/272(3.7%). This study provides novel insight into pre-

transplant evaluation of T and NK cell phenotypes and subsequent risk of CMV

reactivation.

iii

Acknowledgments

I wish to express my sincere gratitude to my supervising committee (Dr. Atul Humar, Dr.

Deepali Kumar, and Dr. Shahid Husain) who gave willingly of their time to continually

provide me with advice and support during the undertaking of this research. Collectively,

their efforts provided an excellent research atmosphere and a learning experience that

was second to none.

I would also like to thank my wonderful colleagues Luiz Lisboa, Adrian Egli, Daire

O’Shea, Jared Fairbanks, Mario Fernandez, Muhtashim Mian, Nikhil Patil, Andre Siegel,

Victor Ferriera, Sang Hoon Han, Justin Manuel, Peter Ashton, and Vanessa Rojas all of

whom it has been a pleasure to work alongside and who have each contributed greatly

to the achievement of my work. More broadly, I would like to acknowledge the many

research assistants, post-doctoral fellows, laboratory technicians, and graduate

students within the Li Ka Shing and Multi-Organ Transplant who together create a world

class learning and research environment.

I would also like to thank Dr. Marcelo Cypel, Dr. Chung-Wai Chow, and Dr. Adriana

Zeevi for accepting to be in my defense committee at a very short notice. I really

appreciate their time and commitment.

Lastly, but most importantly, I thank my family; my parents, my brother, and my fiancé.

Their love and support provides me a solid foundation from which I can successfully

pursue such clinical and research endeavors. I dedicate this work to them.

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Contributions

Shanil Keshwani (author) solely prepared this thesis. All aspects of this body of work,

including the planning, execution, analysis, and writing of all original research and

publications was performed in whole or in part by the author. The following contributions

by other individuals are formally and inclusively acknowledged:

Dr. Atul Humar (Primary Supervisor and Thesis Committee Member) – mentorship;

laboratory resources; guidance and assistance in planning, execution, and analysis of

experiments as well as manuscript/thesis preparation.

Dr. Deepali Kumar (Thesis Committee Member) – mentorship; laboratory resources;

guidance and assistance in planning, execution, and analysis of experiments as well as

manuscript/thesis preparation.

Dr. Shahid Husain (Thesis Committee Member) – mentorship; guidance in interpretation

of results.

Dr. Nicholas Mueller and the Swiss Cohort Group– providing clinical samples and

clinical data.

Peter Ashton– ordering reagents and helping me out in PBMC isolation.

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Table of Contents

Acknowledgments ........................................................................................................ iii

Contributions .............................................................................................................. iiiv

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

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

List of Figures ............................................................................................................... ix

List of Abbreviations .................................................................................................... xi

Chapter 1 CYTOMEGALOVIRUS INFECTION AFTER ORGAN

TRANSPLANTATION: AN INTRODUCTION ................................................ 1

1.1 Human Cytomegalovirus: A Herpes Virus……………………………………………2

1.2 Biology of Cytomegalovirus…………………………………………………………….3

1.3 Immune Responses Specific Against CMV…………………………………………..6

1.3.1 NK-Cell Responses Against CMV……………………………………………..6

1.3.2 T-Cell Responses Against CMV…………………………………………...…14

1.4 CMV Epidemiology………………………………………………………………….....17

1.5 CMV Infection…………………………………………………………………….…....18

1.5.1 Direct Effects of CMV Infection in Solid Organ Transplant Patients……..18

1.5.2 Indirect Effects of CMV Infection in Solid Organ Transplant Patients……21

1.5.3 Risk Factors for CMV Infection in Solid Organ Transplant Patients…...…23

1.5.4 Laboratory Diagnosis for CMV……………………………………………….24

vi

1.5.5 Definition of CMV Infection……………………………………………….…..29

1.5.6 Antiviral Drugs for CMV Prevention and Treatment…………………….….30

1.5.7 Strategies for CMV Prevention and Treatment……………………………..33

1.5 Study Rationale………………………………………………………………………...34

Chapter 2 HYPOTHESIS AND EXPERIMENTAL APPROACH ...................................35

2.1 Study Aims…………………………………………………………………………..….36

2.2 Study Hypothesis………………………………………………………………………37

Chapter 3 METHODOLOGY .........................................................................................38

3.1 Patient Population……………………………………………………………………..39

3.2 Clinical Definitions………………………………………………………………..……40

3.3 Peripheral Blood Mononuclear Samples……………………………………………40

3.4 Viral Strain……………………………………………………………………………...41

3.5 Stimulation and Staining by Flow Cytometry.……………………………………….42

3.6 Statistical Analysis……………………………………………………………………..43

Chapter 4 RESULTS .....................................................................................................44

4.1 Patient Population and Outcomes……………………………………………….…..45

4.2 CD4 and CD8 T-Cell Interferon-γ Response………………………………………..47

4.3 Predictive Value of Pre-Transplant Testing…………………………………………48

vii

4.4 CD4 and CD8 T-Cell Response in D+/R+ and D-/R+ Subgroups………………….50

4.5 Regulatory T-Cell Subset (CD4+CD25+FoxP3+) Response…………………..…51

4.6 Specific NK-Cell Phenotypes Are Associated With CMV Reactivation…………..55

4.7 Specific NK-Cell Phenotypes Are Associated With CMV Protection……………..56

4.8 NK-Cell Interferon- γ Responses Are Associated With CMV Protection……..….58

4.9 NKG2C Expression is Associated With CMV Protection in D+/R+ Patients……..59

Chapter 5 GENERAL DISCUSSION AND CONCLUSION ...........................................61

5.1 General Discussion and Conclusion for T-Cell………………………………….….62

5.2 General Discussion and Conclusion for NK-Cell……………………………….….65

Chapter 6 FUTURE DIRECTION ..................................................................................69

6.1 Future Directions……………………………………………………………………....70

REFERENCES ..............................................................................................................77

viii

List of Tables

Table 4-1. Clinical Characteristics of Patients and Outcome…………………….46

Table 4-2. CMV specific T-cell response in patients with and

without CMV Viremia………………………………………………………..47

Table 4-3. CMV specific T-cell response in patients with and

without CMV disease………………………………………………………..49

Table 6-1. Drugs licensed for prophylaxis, pre-emptive therapy

and treatment of CMV infection……………………………………………74

ix

List of Figures

Figure 1-1. A Cartoon Depicting the Structure of the HCMV Virion………………...4

Figure 1-2. Summary of NK Cell Biology …………………………………………….….8

Figure 1-3. Flow Analysis of NK-cell Subsets ………………………………………...10

Figure 1-4. NK Cell in Regulatory Network ……………………………………….......12

Figure 1-5. Flow Analysis of T-cell subsets ………………………………………......16

Figure 1-6. Overview of Cytomegalovirus (CMV) Infection: Direct

and Indirect effects…………………………………………………….……..20

Figure 1-7. A Model for CMV Pathogenesis After Solid Organ

Transplantation………………………………………………………….……22

Figure 1-8. CMV- Histology Owl's Eye Inclusions…………………………………....26

Figure 4-1. CD4 T-Cell Response in D+R+ and D-R+ Subgroups……………..…..50

Figure 4-2. CD8 T-Cell Response in D+R+ and D-R+ Subgroups…………….…...51

Figure 4-3. T-Regulatory Cell Baseline Response (CD4+CD25+FoxP3+)………...52

Figure 4-4. Absolute T-Regulatory Cell Response (CD4+CD25+FoxP3+)………..54

Figure 4-5. CD56bright, CD16 Neg NK-cell Response…………………………….……56

Figure 4-6. CD56dim, CD16neg NK-cell Frequency in CMV Viremia

Patients………………………………………………………………………...57

Figure 4-7. CD56dim, CD16+, IFN-γ+ NK-cell Response……………………..……….58

x

Figure 4-8. CD56bright, CD16Neg, NKG2C+ NK-cell Response in

D+R+ Patients with CMV Viremia………………………………………….59

Figure 4-9. CD56bright, CD16Neg, NKG2C+ NK-cell Response in

D+R+ Patients with CMV Disease…………………………………..……..60

Figure 6-1. CD56dim, CD16pos Showing Dose-Dependent Immunosuppressive

Drug Response ………………………………………………………..……..71

Figure 6-2. CD56dim, CD16pos, NKG2Cpos Showing Dose-Dependent

Immunosuppressive Drug Response…………………………………….72

xi

List of Abbreviations

CMV Cytomegalovirus

NK Natural Killer

PBMC Peripheral Blood Mononuclear Cell

IFN-γ Interferon gamma

T-Reg T-Regulatory

R+ Recipient Positive

R- Recipient Negative

D+ Donor Positive

D- Donor Negative

HSV Herpes Simplex Virus

VZV Varicella Zoster Virus

EBV Epstein Barr Virus

HHV Human Herpes Virus

kpb kilo-base pairs

HLA Human Leukocyte Antigen

OKT3 Orthoclone Muromonomab

ATG Anti-Thymocyte Globulin

TNF Tumor Necrosis Factor

NF-kB Nuclear Factor Kappa-light-chain-enhancer of activated B cells

DC Dendritic Cell

IE Immediate Early

gB Glycoprotein B

gH Glycoportein H

TLR Toll-Like Receptor

HSCT Hematopoietic Stem Cell Transplant

CTL Cytotoxic T Lymphocyte

HIV Human Immunodeficiency Virus

AIDS Acquired Immune Deficiency Syndrome

PBL Peripheral Blood Lymphocyte

xii

TBB Transbronchial Biopsy

IHC Immunohistochemistry

CNS Central Nervous Syndrome

BOS Bronchiolitis Obliterans Syndrome

CAV Cardiac Allograft Vasculopathy

mTOR Mammalian Target of Rapamycin

NAT Nucleic Acid Test

QNAT Quantitative Nucleic Acid Test

ELISA Enzyme-Linked Immunosorbent Assay

IgG Immunoglobulin G

IgM Immunoglobulin M

BAL Broncho alveolar Lavage

PCR Polymerase Chain Reaction

DNA Deoxyribonucleic Acid

G-CSF Granulocyte-Colony Stimulating Factor

IVIG Intravenous Immunoglobulin

HFF Human Foreskin Fibroblast

MMF Mycophenolate Mofetil

IQR Inter-quartile Range

TAP Transporter Associated with Antigen Porcessing

APC Antigen Presenting Cell

HCV Hepatitis C Virus

KIR Killer Immunoglobulin Receptor

CDV Cidofovir

1

Chapter 1

CYTOMEGALOVIRUS INFECTION AFTER ORGAN TRANSPLANTATION: AN INTRODUCTION

2

1.1 HUMAN CYTOMEGALOVIRUS: A HERPES VIRUS

Cytomegalovirus (CMV) is one of the most common opportunistic infection after organ

transplantation. It has the potential to cause significant morbidity, graft lost, and

adverse outcomes in this patient population. CMV replication is controlled by the innate

and adaptive immune system. Due to immunosuppression used to prevent organ

rejection, the host immune response is impaired after transplantation. Prediction of CMV

reactivation and progression to disease after transplantation using clinical risk

stratification and viral load testing remains difficult. In addition, pre-transplant donor and

recipient serostatus is used to stratify post-transplant risk with CMV donor seropositive /

recipient seronegative (D+/R-) patients considered at highest risk; R+ patients are

considered at intermediate risk of CMV reactivation (Razonable 2008; Kotton et al.

2013).

The herpesviridae are a large group of double-stranded enveloped DNA viruses.

Currently, over 150 members of the herpes family have been identified. There are eight

different herpes viruses known to infect humans. These include: Herpes Simplex Virus

type 1 and type 2 (HSV), Varicella Zoster Virus (VZV), Cytomegalovirus (CMV) and

Epstein Bar Virus (EBV) and Human Herpes Viruses (HHV) 6, 7 and 8 (Grinde 2013).

Cytomegalovirus (CMV) was identified in 1956 and was first isolated from the salivary

gland and kidneys of two dying toddlers. Cytomegalic intranuclear inclusion bodies were

discovered from them. Two other laboratories isolated CMV at approximately the same

time. Thus, CMV was initially called salivary gland virus or salivary gland inclusion

disease virus. In 1960, Weller et al. proposed the use of the term cytomegalovirus

(Craig et al. 1957). CMV was first isolated in a renal transplant recipient in 1965

(Brennan 2001).

3

1.2 BIOLOGY OF CYTOMEGALOVIRUS

CMV is the largest virus that infects humans, 150-200 nm in diameter. The genome

consists of over 200 kilo-base pairs (kbp) double-stranded DNA. Compared to other

herpes viruses, the genome of CMV encodes for a two to three times larger number of

gene products. CMV has four structural elements; core, capsid, tegument and envelope.

The core contains the linear double-stranded DNA and is surrounded by a

proteinaceous layer defined as the tegument or matrix, which, in turn, is enclosed by a

lipid layer containing a large number of viral glycoproteins (Mocarski 2002).

4

Figure 1-1. A Cartoon Depicting the Structure of the HCMV Virion. Adapted with

permission from Dr. Marko Reschke. (Tomtishen III 2012). The double-stranded DNA

virus is contained within a polyhedral viral capsid. The viral capsid is surrounded by the

tegument, which mainly consists of proteins that aid in reproduction and concealment

from the host’s immune system. The membranous envelope that surrounds the

tegument is made by the Golgi bodies in the host cell. The viral glycoproteins assist in

entrance of the virus, development of new viruses, and spreading of the virus from one

cell to the next.

In target cells, the entry of CMV has been examined in-vitro in fibroblasts, endothelial

cells and phagocytes (Compton 1995). The process begins when there is attachment

and fusion of the virion with the cell membrane. The key player in this process is the

Glycoproteins of the viral envelope (Adlish et al. 1990; Compton et al. 1992; Compton et

al. 1993; Kari & Gehrz 1992; Soderberg et al. 1993; Taylor & Cooper 1990; Compton

5

1995). Studies show that HLA class I (Human Leukocyte Antigen class I) molecules

have a role in viral entry (Grundy et al. 1987). Following viral entry, the capsid moves to

the nucleus and the process of transcription and translation of early and late antigens

starts following a very stringent timetable (Michelson-Fiske et al. 1977). Infected cells

will be damaged and further replication and dissemination of the virus will be stopped

with the cellular immune response. During a state of decreased immunity i.e. by the use

of immunosuppressive drugs, the virus comes in a stage of latency and may reactivate.

The potent monoclonal drug OKT3 (Orthoclone Muromonomab) and the polyclonal

antithymocyte globulin (ATG) are known as potent inducers of CMV reactivation. This is

probably mediated by TNF-alpha release during OKT3 treatment. TNF-alpha induces

the transcription factor NF-kB that causes enhancement of the major Immediate Early

enhancer/promoter gen of CMV (Prösch et al. 1995).

CMV has the potential to infect large number of human cell types which includes

fibroblasts, granulocytes, monocytes, macrophages, dendritic cells, epithelial and

endothelial cells (Sinzger et al. 1995; Sinzger et al. 1996). It can also cause disease in

most organs, such as pneumonitis, myocarditis, gastrointestinal disease, retinitis,

hepatitis, nephritis and pancreatitis. CMV establishes latent infection in the host after

primary infection like other herpes viruses and remains mainly in monocytes and

CD34+ bone marrow progenitor cells (Emery 2012; Noriega et al. 2014). Latent CMV is

defined by the carriage of the CMV genome devoid of active replication but with the

capacity of the CMV genome to reactivate under specific stimuli (Björkström et al.

2013). Only a few viral genes are expressed in the latent phase and few viral proteins

are produced. The infected cell is therefore not detected by the host immune system.

The exact mechanisms that control latency are unclear.

During the acute infection, the foremost cell-type in blood infected with CMV is

monocytes that may serve as means of transport, while differentiation into tissue

macrophages authorizes the full replication cycle (Smith et al. 2004). Monocytes are

acknowledged as the major infected cell type in peripheral blood and the foremost sites

of CMV latency in vivo (Taylor-Wiedeman et al. 1991; Taylor-Wiedeman et al. 1993;

Rice et al. 1984). The most probable theory on how CMV spread to different tissues is

6

either by exploiting cells as a means of transportation for virus shedding or by trafficking

of free virions in the blood (Haspot et al. 2012). The CMV genome is carried by CD34+

progenitors in bone marrow (Mendelson et al. 1996). Viral reactivation can occur after

inflammatory stimuli when monocytes differentiate to macrophages or dendritic cells

(DCs) (Reeves et al. 2005; Söderberg-Nauclér et al. 1997). The CMV genome does not

produce infectious virus during latency and there is a lack of IE transcription; samples

from tissues with CMV-disease show an elevated frequency of CMV-infected

macrophages expressing late viral genes (Sinzger et al. 1995; Gnann et al. 1988).

Furthermore, the virus has established strategies to prolong viral replication, evade

lysosomal degradation and avoid destruction of the infected cells as well as other

functions to remain persistent in macrophages (Söderberg-Nauclér & Nelson 1999).

1.3 IMMUNE RESPONSES SPECIFIC AGAINST CMV

The CMV-specific immunity includes both innate and adaptive immune responses.

Crucial players in immune control of CMV include NK-cells, CD8+ and CD4+ T-cells.

The primary infection, occurring in most cases during childhood, gives rise to a cell

mediated defense. CMV infected cells will present CMV antigens on their surface to

be recognized by T-cells specific against CMV (Catry 2013).

1.3.1 NK-Cell Responses Against CMV

The first line of the host defense in controlling CMV infection is activation of Natural

Killer (NK) cells, early production of pro-inflammatory cytokines, and the activation of

the innate immune cell effector function. NK cells are innate lymphoid cells constituting

the third most abundant lymphocyte population in the peripheral blood (Vivier et al.

2012a; Romo et al. 2011; Björkström et al. 2013; Sanos et al. 2011). The past fifteen

years witnessed unpredictable advances in our knowledge of NK cells, their surface

receptors, and the molecular mechanisms involved in their function.

7

NK cells are known as the foremost component of the innate immune system and they

have two major functional properties. The first one is cytotoxicity and they are usually

contributed by CD56dim cells. They can be subdivided further into natural cytotoxic

activity and antibody-dependent cell-mediated cytotoxicity (ADCC). Tumor cells and

virally infected cells in the absence of prior stimulation or immunization contributes to

the natural cytotoxicty while the Fc part of the antibody triggers ADCC, directed against

antibody-coated target cells, in that case the NK cell FcγR CD16. Resting NK cells

have cytotoxic activity, but cytotoxicity increases intensely when NK cells are stimulated

by cytokines like IL-2, IL-12, IL-15, IL-18, and many others (Smyth et al. 2002;

Carayannopoulos & Yokoyama 2004; Ralainirina et al. 2007a). The release of the

cytotoxic molecules perforin and granzymes mediates NK cell-mediated lysis of target

cells. The second important function of NK cells is cytokine production and they are

usually contributed by CD56bright cells. Activation of NK cell receptors and/or stimulation

by cytokines present in the microenvironment triggers the cytokine producing NK cells.

In particular, NK cells are an essential source of IFN-γ. Therefore, NK cells contribute to

innate immune responses by activation of cytotoxicity and cytokine production leading

to the first line of defense of the organism before adaptive immunity has developed

(Smyth et al. 2002; Carayannopoulos & Yokoyama 2004; Ralainirina et al. 2007a).

8

Figure 1-2. Summary of NK Cell Biology. Reproduced with permission of

FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY in the

format Republish in a thesis/dissertation via Copyright Clearance Center.

(Ralainirina et al. 2007b) (a) The three effector functions of NK cells: natural

cytototoxicity, antibody-dependent cellular cytotoxicity (ADCC), and cytokine production.

Although cytotoxic activity is already present in resting NK cells, it increases strongly

upon stimulation with activating cytokines, for example, IL-2 or IL-15. (b) Concept of the

balance between activating and inhibitory messages governing NK cell functions.

Healthy cells express normal amounts of MHC Class I molecules [ligands for NK cell

inhibitory receptors (IR)] but few ligands for activating NK cell receptors and are thus

spared by NK cells. In contrast, diseased cells frequently down-regulate expression of

MHC Class I molecules, and expression of activating ligands (AL) is increased. Thus,

NK cells receive an excess of activating messages and proceed to target cell lysis. AR,

Activating receptors.

9

NK cell can be divided into four subpopulation on the basis of the relative expression of

the markers CD16 (or FccRIIIA, low-affinity receptor for the Fc portion of

immunoglobulin G) and CD56 (adhesion molecule mediating homotypic adhesion): (1)

CD56bright CD16neg) (50–70% of CD56bright), (2) CD56bright CD16dim (30–50% of

CD56bright), (3) CD56dim CD16neg), and (4) CD56dim CD16pos (Poli et al. 2009; Cooper et

al. 2001). In healthy individuals, a population (3) is numerically in the minority and their

role is largely unknown. They are often intensely expanded in human immunodeficiency

virus infection but are hyporesponsive under these conditions. The CD56dim CD16pos NK

cells represent at least 90% of all peripheral blood NK cells and are therefore the major

circulating subset. A maximum of 10% are CD56bright NK cells (Poli et al. 2009; Cooper

et al. 2001).

10

Figure 1-3. Flow Analysis of NK-cell subsets. In a healthy volunteer, majority of the

NK cells are CD56dim and CD16pos. They contribute around 90% of NK cells. Viral and

chronic diseases can alter the CD56dim and CD56bright NK cells.

Natural killer (NK) cells are important innate effectors in immunity and also have a role

in the regulation of the adaptive immune response. They have been shown in different

contexts to stimulate or inhibit T cell responses. Most recent findings have expanded

our understanding of the mechanisms essential for this regulation. Regulation by NK

cells have revealed that it can result from both direct interactions between NK cells and

11

T cells, as well as indirect interactions with antigen presenting cells and the impact of

NK cells on infected cells and pathogen load (Crouse et al. 2015).

Some indirect enhancement of T-cell response by NK cells involves dendritic cell

maturation and antigen cross presentation. The outcome involves NK cell produced

IFN-γ induced maturation of dendritic cells in vitro, leading to IL-12 production and

increased co-stimulation on dendritic cells (Gerosa 2002), NK cell produced IFN-γ in

vivo promotes IL-12 production by dendritic cells leading to an enhanced CD8+ T cell

response and tumor clearance (Mocikat et al. 2003), NK cell produced IFN- γ following

dendritic cell vaccination in vivo promotes IL-12 production by dendritic cells leading to

CD4+ T cell help independent CD8+ T cell activation (Adam et al. 2005), and NK cell

killing of target cells releases antigen for cross presentation and activation of T cell

responses (Krebs et al. 2009).

Some direct enhancement of T-cell response by NK cells involves CD4+ T-cell

differentiation. The outcome involves NK cell produced IFN-γ signaling on CD4+ T cells

promotes differentiation into TH1 helper cell (Martín-Fontecha et al. 2004), human

tonsilar NK cell secreted IFN-γ assists in TH1 cell polarization (Morandi et al. 2006), and

NK cell produced IFN-γ signaling on CD4+ T cells promotes differentiation into TH1

helper cells and protection from L. major infection (Laouar et al. 2005).

Some indirect impairment of T-cell response by NK cells involves reduced antigen

presentation and reduced stimulatory capacity of antigen presenting cells. The

outcomes involves NK cell mediated elimination of dendritic cells or MCMV infected

dendritic cells leading to reduced dendritic cell vaccine efficacy and reduced CD4+ and

CD8+ T cell responses and increased viral persistence in Ly49H+ mice (Andrews et al.

2010; Hayakawa et al. 2003), and reduced CD8+ and CD4+ T cell response to LCMV

(Cook & Whitmire 2013).

Some direct impairment of T-cell response by NK cells involves reduced CD8+ T-cell

activation and T-cell elimination. The outcome involves NK cell secreted IL-10 following

MCMV infection leads to a reduced CD8+ T cell response and protection from

12

immunopathology (Lee et al. 2009), NK cell mediated killing of CD4+ and CD8+ T cells

(Rabinovich et al. 2003; Cerboni et al. 2007; Lu et al. 2007; Soderquest et al. 2011).

Figure 1-4. NK Cell in Regulatory Network. Adapted by permission from

Macmillan Publishers Ltd: Nature Immunology, ©2011. (Colonna et al. 2011) (a) NK

cells recognize MCMV-infected cells via Ly49H-m157 interaction, which triggers

cytotoxicity and proliferation. (b) When MCMV infection induces abnormal activation and

proliferation, Ly49H+ NK cells produce IL-10, which limits CD8+ T cell–mediated tissue

damage. (c) Ly49H+ NK cells can also lyse MCMV-infected dendritic cells (DCs),

thereby reducing T cell priming and viral control. (d) NK cells can eliminate LCMV-

specific CD4+ T cells, impairing viral control or preventing excessive T cell response. '?'

indicates that the receptors mediating this response are not known. (e) CD4+ T cell

responses to immunogens can stimulate NK cells via IL-2 and DC-derived IL-12. (f)

Neutrophils are required for the functional competence of NK cells via an unknown

mechanism.

13

Initial phases of CMV infection elicit the innate immune responses with induction of

interferons, inflammatory cytokines, recruitment and activation of NK-cells via binding of

Glycoprotein-B (gB) and Glycoprotein-H (gH) to Toll like receptor 2 (TLR2) and

subsequent TLR2 dependent activation of NFKP (Compton et al. 2003; Boehme et al.

2006). Despite the fact that CMV is immunomodulatory mechanisms to avoid NK-cell

mediated killing, NK-cells are thought to have an imperative role in the defense against

CMV. Recurrent herpesvirus infections and serious episodes of CMV-disease tends to

occur with individuals lacking NK-cells (Biron et al. 1989). Several studies have

supported the importance of innate immunity particularly the NK cells to defend against

CMV (Carroll et al. 2012; Loh et al. 2005; Min-Oo & Lanier 2014). NK cells play a crucial

role in early immune responses after HSCT because they are the first lymphoid

population recuperating after the allograft. In humans, Guma and colleagues showed

expansion of NKG2C+ NK cells associated with individuals who are CMV seropositive

(Mónica Gumá et al. 2006) and furthermore, studies suggests that CMV promotes NK-

cell development after HSCT (with cord blood graft) as a more rapid NK-cell maturation

together with expansion of NKG2C+ NK cells in patients undergoing CMV reactivation is

observed (Della Chiesa et al. 2012).

Expansion of NKG2C+ NK cells with CMV-associated cells have been reported in

various human disease settings including immunodeficiency (Kuijpers et al. 2008), HIV

infection (Monica Gumá et al. 2006), acute hantavirus infection (Björkström et al. 2011),

and after HSCT (Foley, Cooley, Verneris, Curtsinger, et al. 2012; Foley, Cooley,

Verneris, Pitt, et al. 2012). NKG2C+ NK-cells transplanted from seropositive donors

display amplified function in response to a secondary CMV event compared with

NKG2C+ NK-cells from seronegative donors. It would appear like NKG2C+ memory-like

NK-cells are transplantable and dependent on CMV antigens in the recipient for clonal

expansion of NK-cells formerly exposed to CMV in the donor (Foley, Cooley, Verneris,

Curtsinger, et al. 2012).

14

1.3.2 T-Cell Responses Against CMV

The predominant control mechanism continues to be the cellular immune response.

This is the main reason why severe forms of CMV- disease occur entirely, with

exception of congenital infection, in severely cellular immunosuppressed individuals.

CMV-specific INF-γ producing CD8+ Cytotoxic T-Lymphocytes (CTLs) protect against

CMV-retinitis in patients with AIDS (Jacobson et al. 2008) and in HSCT-patients the

reconstitution of CMV-specific CD8+ cells are shown to be correlated with protection

against disease (Li et al. 1994; Reusser et al. 1991). CMV-specific T-cells have also

been used in adaptive treatment with transfer of cellular immunity (Uhlin et al. 2012).

Through activation of B-cells, humoral immunity develops which leads to production of

CMV-specific antibodies. It is thought that these antibodies can deactivate viral particles

but they cannot help in the dismissal of the CMV infected cells (Heller et al. 2006;

Casazza et al. 2006).

The CMV-specific CD8+ CTLs will clonally expand and mature and kill infected cells.

Using the cytolytic effector proteins perforin and granzyme, or inducing apoptosis via

Fas Ligand, studies show the existence of virus-specific CD4+ T-cells that can kill

infected cells directly like CD8+ T-cells (Whitmire 2011).

Keeping CMV under constant control is "costly" for the immune system. In adults (but

not elderly) around 5-10% of the total peripheral blood CD4+ and CD8+ T- cells

recognize CMV elements and looking only at the memory cells, approximately 10% are

CMV specific. The specificity of the CMV-reactive T-cells is rather extensive, covering

about 70% of the total open reading frames of the CMV genome (Casazza et al. 2006).

However, the scenario tends to change when the individual gets older. In the Swedish

longitudinal OCTO-immune study, it was shown that a combination of high CD8+ and

low CD4+ percentages in addition to poor T-cell proliferation in peripheral blood

lymphocytes (PBL) was associated with a higher 2-year mortality in very old (86-92 year

old) Swedish individuals (Olsson et al. 2000). These alterations were associated with

evidence of CMV infection. The authors suggested that a combination of old age,

lymphocyte activation due to chronic infection, such as CMV, and a related imbalance in

15

unknown factors that regulate the homeostatic mechanisms in the immune system

might have played some role to an increased risk for the decreased survival observed in

the OCTO study. Khan et al. showed that the clonally expanded memory CD8+ T-cells

seen in CMV-seropositive healthy donors were characterized by the lack of CD28

expression and the increased expression of CD57 (CD28- CD57+ CCR7-) and he

indicated that such cells frequently are oligoclonal, anergic, and directly correlated with

poor immune response (Khan et al. 2002).

The CD8+ CMV-specific response is extremely diverse and is directed towards more

than 70% of the CMV open reading frames in a young adult (Elkington et al. 2003). The

CTLs recognize structural, early, and late antigens along with immunomodulators

including pp28, pp50, pp150, pp65, gH, gB, US2, and others.

Several studies have examined the impact of chronic CMV infection on memory T-cell

homeostasis and the differentiation phenotype of antigen-experienced CTL. The main

CD8+ effector T-cell population during acute CMV infection has the CD45RA- CCR7-

CD45RO+ CD27+ CD28+/- phenotype. However, in chronic CMV infections, the markers

CD27 and CD28 do not seem to appear on the cell surface and the population is either

CD45RO- as in the effector memory cell population or shows the CD45RO+ as in

terminally differentiated effector T-cells re-expressing CD45RA (Gamadia et al. 2003;

Appay et al. 2002).

Studies show that CD4+ depleted mice get an augmented incidence of recurrent CMV

infections (Polić et al. 1998). Studies also show that in humans, there is increasing

evidence that not only HLA class I / CD8+ T-cell responses but also HLA class II / CD4+

T-cells are also important for the control of CMV (Einsele et al. 2002; Sester et al.

2005). Low levels of CMV-specific CD4+ T- cells correlates with susceptibility to CMV-

infections in lung transplant recipients (Sester et al. 2005), and studies show that in

HSCT-patients detectable CD4+ T helper responses defends against CMV-disease and

the recovery of the CMV-specific T-cells is vital for both the persistence of adoptively

transferred CMV-specific CTL (Walter et al. 1995) and the endogenous reconstitution of

the same (Reusser et al. 1991).

16

Studies show that in CMV-exposed individuals, 9% of the circulating CD4+ memory T-

cell population is directed against CMV epitopes. Broad antigen recognition has been

revealed for CD4+ T-cells just as for CD8+ T-cells. In healthy individuals, greater than

30% of them are directed against glycoprotein B (gB) antigens. Most CD4+ T-cell

responses against gB and gH are directed towards highly conserved regions (Sylwester

et al. 2005).

Figure 1-5. Flow Analysis of T-cell subsets. T-cell responses were measured by flow

cytometry. This flow analysis is of a healthy volunteer having a normal range of CD4

and CD8 population.

17

1.4 CMV EPIDEMIOLOGY

Global human population shows approximately 70% seroprevalence for CMV (Emery

2012) and the rates fluctuate from 45-95%, depending on age, country and socio-

economic conditions (Cannon et al. 2010). CMV infection is mostly attained during

early childhood and there is a rise in adolescence. The virus is expelled in body fluid

(urine, saliva, tears, semen and breast milk and cervical secretion) for a long period

after infection (Landolfo et al. 2003). Virus transmission occurs with close contact like

that among family members and children in day care centers (via urine or saliva).

Sexual transmission is observed between partners via semen and cervical secretion.

CMV may be spread in various ways like via the placenta during maternal viremia,

through secretion in the birth canal or from breast milk. Transmission of CMV by

blood transfusion or blood products may occur, but it is less likely after the use of

filtered blood was introduced. CMV is transferred with solid organ and bone marrow

transplantation when the donor is CMV seropositive.

Based on the epidemiological studies, there is a relationship between CMV and

development of cancers and various inflammatory diseases like atherosclerosis

(Sorlie et al. 2000), inflammatory bowel diseases (Dimitroulia et al. 2006), rheumatoid

arthritis (Pierer et al. 2012), systemic lupus erythematosus (Pérez-Mercado & Vilá-

Pérez 2010), and Sjogren's syndrome (Shillitoe et al. 1982). There are a number of

studies where CMV proteins and nucleic acids are identified within the tissue of

different cancer types, e.g. colon cancer (Harkins et al. 2002), breast cancer (Harkins

et al. 2010), prostate cancer (Samanta et al. 2003), salivary gland tumors (Melnick et

al. 2012), glioblastoma (Cobbs et al. 2002; Stragliotto et al. 2013), neuroblastoma

(Wolmer-Solberg et al. 2013), and rhabdomyosarcoma (Price et al. 2012).

18

1.5 CMV INFECTION

In an immunocompetent host, the primary CMV infection is generally asymptomatic or

presents as a flu-like syndrome. Only a minor portion of infected individuals are affected

with acute CMV disease, mononucleosis syndrome. The clinical signs include fever,

pharyngitis, sometimes cervical lymphadenitis, and hepatitis. The spleen may be

enlarged. Atypical lymphocytes are observed in the blood and laboratory findings

usually vanish after six weeks. Fatigue commonly continues for quite a few weeks to

months. It is a rare to develop severe disease with organ-specific complications

(Baumgratz et al.; Eddleston et al. 1997).

1.5.1 Direct Effects of CMV Infection in Solid Organ

Transplant Patients

In organ transplant patients, CMV is one of the most clinically significant opportunistic

infections. The virus has the potential to cause severe CMV disease, ranging from CMV

syndrome to tissue invasive disease. CMV syndrome is a flu-like illness and the clinical

symptoms may be characterized by malaise, fever, thrombocytopenia, leucopenia, and

mild elevation of liver enzymes. Each type of organ transplantation has a different

occurrence of tissue-invasive disease. In patients without anti-viral prophylaxis, the

overall likelihood of developing CMV infection or disease in lung transplant recipients

ranges from 54 to 92 percent (Zamora et al. 2005; Duncan et al.; Ettinger et al. 1993).

The most significant risk factor for CMV infection and disease is organ donor (D) and

recipient (R) serostatus with CMV D+/R- recipients at the maximum risk (Zamora et al.

2005; Duncan et al.; Ettinger et al. 1993). CMV has remained the most frequent

opportunistic infection after organ transplantation despite an antiviral strategy, causing

pneumonitis, myocarditis, hepatitis, nephritis, gastrointestinal disease, pancreatitis and

retinitis (Snydman et al. 2011).

19

CMV has a tendency for invading the transplanted organ. For example, in lung

transplant patients the most common form of tissue invasive CMV disease is

pneumonitis. If not treated, CMV pneumonitis can be life threatening. In single lung

transplant recipients, the disease affects the transplanted lung almost exclusively

(Buffone et al. 1993). The clinical signs include fever, cough, tiredness, dyspnea and

hypoxia. The clinical symptoms of CMV pneumonitis and acute rejection are the same.

In order to differentiate between infection and acute rejection, a transbronchial lung

biopsy (TBB) may be needed. The treatments are opposite; acute rejection is treated

with increased immunosuppressive therapy while CMV disease is treated with antiviral

drugs and reduced immunosuppressive therapy. Following lung transplantation, CMV

pneumonitis has been presented to be a risk factor for invasive aspergillosis (Husni et

al. 1998). The second most common tissue-invasive disease in lung transplant

recipients is gastrointestinal CMV disease. During the early era of heart transplantation,

myocarditis and pneumonitis were severe complications of the CMV disease with

myocarditis being very unique to heart transplant recipients. To confirm the diagnosis,

an endomyocardial biopsy is required that shows viral inclusion bodies or by performing

immunohistochemistry (IHC). Myocarditis has the potential to cause severe arrhythmia,

cardiac dysfunction, and even sudden cardiac death (Kindermann et al. 2012; Kytö et

al. 2005; Partanen et al. 1991; Baumgratz et al.). In the heart transplant recipients, CMV

syndrome and gastrointestinal disease continue to remain the most common forms of

CMV disease.

Gastrointestinal CMV disease occurs in all types of solid organ transplants. Vomiting,

nausea, epigastric pain, dysphagia, diarrhea, abdominal cramps and severe

gastrointestinal bleeding are the symptoms related to the disease. CMV disease can

lead to ulceration or rupture in any part of the gastrointestinal tract with stomach,

proximal small bowel, and caecum being the most common location. Variable lesions

from erythema to deep ulcers are noticed in the endoscopy (Ljungman et al. 2002). For

the diagnosis, a biopsy and the detection of the virus with histological examination and

IHC are required.

20

Solid organ transplant patients rarely get diagnosed with retinitis. The symptoms include

blurring or loss of central vision, scotomata ("blind spots"), floaters, or photopia

("flashing lights"). Based on the characteristic retinal changes, ophthalmologist can

diagnose retinitis. Retinitis is infrequent and central nervous system (CNS) disease is

uncommon in organ transplant recipients (Kotton et al. 2013).

As the CMV virus has a predilection for invading the transplanted organ, hepatitis is

most frequent in liver transplant recipients, while nephtritis generally only in kidney

transplant recipients (Razonable & Humar 2013).

Figure 1-6. Overview of Cytomegalovirus (CMV) Infection: Direct and Indirect

effects. Adapted with permission from Fishman and Rubin (Fishman & Rubin

1998).

21

1.5.2 Indirect Effects of CMV Infection in Solid Organ

Transplant Patients

CMV has possible indirect effects in addition to the direct effects of invasive CMV

infection which are both general and transplant specific (Figures 1-6 and 1-7). These

conditions are called indirect effects of CMV infection as they are not directly related to

viral invasion of the tissue. Elevated risk of bacterial, fungal and viral infection (Fishman

et al. 2007; Husni et al. 1998; Munoz-Price et al. 2004), new-onset diabetes mellitus

after transplantation (Van Laecke et al. 2010) and acute rejection (Grattan et al.;

Monforte et al. 2009; Paraskeva et al. 2011) are the possible general indirect effects.

Possible transplant-specific indirect effects after lung transplantation include

bronchiolitis obliterans syndrome (BOS) (Chmiel et al. 2008; Duncan et al. 1994; Snyder

et al. 2010; Zamora 2002) and in heart transplant patients include cardiac allograft

vasculopathy (CAV) (Grattan et al.; Potena et al. 2009). Peribronchiolar infiltration of

lymphocytes which leads to fibrous scarring in the bronchioles and progressive airflow

obstruction is involved in the pathogenesis of BOS (Estenne et al. 2002). An initial

endothelial injury followed by intimae hyperplasia and the proliferation of vascular

smooth cells that lead to the diffuse luminal stenosis of the coronary arteries is involved

in the pathogenesis of CAV (Avery 2003). Other probable indirect effects after renal

transplantation are chronic allograft nephropathy (Arthurs et al. 2008; Freeman 2009;

Reischig et al. 2009), accelerated hepatitis C virus recurrence and vanishing bile duct

syndrome after liver transplantation (Bosch et al. 2012; Gao & Zheng 2004;

Lautenschlager et al. 1997).

A model of CMV pathogenesis after solid organ transplantation is described by Emery

(Emery 2012) (Figure 1-7). Latent infection is transferred with the donor organ (red

spots). The CMV virus becomes activated and thereafter the local spread of the virus

occurs in the transplant organ over the next several days. The virus may spread through

the blood to infect other target organs. The high levels of replication, DNAemia, are

associated with CMV disease. In addition, early graft infection may contribute to the

indirect effects shown in the figure 1-7.

22

Figure 1-7. A Model for CMV Pathogenesis After Solid Organ Transplantation.

Adapted by permission from Oxford University Press and V.C. Emery: QJM ©2011

(Emery 2012). A model for CMV pathogenesis after solid organ transplantation. The

donor organ harbours a small number of cells with latent infection (red dots), which

become activated through the effects of the proinflammatory environment on the major

immediate early promoter shortly after transplant. Subsequent local spread of virus in

the infected organ ensues over the next 7 days, which may then spread through the

blood to infect other target organs, which contributes to the overall level of CMV

DNAemia. If left untreated, these high levels of replication will be associated with the

direct effects of CMV infection. In addition, early graft infection may contribute to acute

organ malfunction, occurrence of other opportunistic infections and also long-term graft

and patient survival. GI: Gastrointestinal.

http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=3328974_hcr262f1.jpg

23

1.5.3 Risk Factors for CMV Infection in Solid Organ

Transplantations

Serostatus

The impact of CMV serostatus is important. Seronegative (R-) recipients who receive

organs from a CMV-positive donor (D+) run the highest risk of CMV disease (as a result

of the reactivation of latent virus from the transplanted organ). Seropositive (R+)

recipients who receive organs from a CMV- positive donor (D+) or CMV-negative donor

(D-) have an intermediate risk of CMV disease. Patients with D-/R- serostatus run the

lowest risk of CMV infection, but they may acquire infection through natural

transmission in the community settings, or by blood transfusion, if the blood is not

leukocyte depleted or CMV negative.

Type of organ

Depending on the type of organ transplanted, the incidence of CMV infection and

disease varies. McDevitt found an occurrence of CMV disease in kidney transplant

recipients of 8%, in liver 20%, in heart 25%, in lung or heart-lung 39% and in pancreas

50% (McDevitt 2006). The highest risk of developing CMV disease are lung and

intestinal transplant recipients and the hypothesis behind increased CMV disease may

be the larger amount of lymphoid tissue in lung and intestinal transplant organs, and

also the higher immunosuppression (Balthesen et al. 1993).

Immunosuppression

Various factors including the type of immunosuppressive drug, the dose, and duration of

the treatment can have an impact in the development of CMV infection or disease.

During the first three to six months after transplantation, the dosage is particularly high.

Studies show that anti-thymocyte globulin (ATG) has been associated with an increased

risk of CMV disease (Jamil et al. 2000). Recent studies suggest that mammalian target

of rapamycin (mTOR) inhibitors are associated with a lower risk of CMV infection

(Andreassen et al. 2014; Kobashigawa et al. 2013; Viganò et al. 2010).

24

Acute rejection

There is a bidirectional association between CMV and acute rejection as acute rejection

produces a proinflammatory environment that can reactivate CMV and the treatment for

acute rejection is augmented immunosuppression. On the contrary, CMV upregulates

antigens and the consequence is alloreactivity which increases the risk for acute

rejection (Zamora 2004; Tolkoff-Rubin et al.; Snydman et al. 2011).

Blood transfusion

The transfusion of blood products continues to be a risk factor if the blood contains

leukocytes. Leuko-depleted blood products have considerably reduced the risk of

transfusion-transmitted CMV (Kekre et al. 2013; Thiele et al. 2011).

1.5.4 Laboratory Diagnosis for CMV

Serology, histopathology, viral culture, pp65 antigememia, and nucleic acid tests (NAT)

are the laboratory tests that are available to diagnose CMV. Serological testing and viral

cultures from multiples sites was the cornerstone of diagnosis in the early period. In

today’s era, viral load (quantitative nucleic acid tests (QNAT)) or antigenemia are the

standards for the diagnosis and monitoring of CMV infection and disease. CMV infection

can be termed CMV viremia (culture, PCR or antiginemia), CMV antigenemia (viral

antigen testing), and CMV DNAemia (NAT) depending on the method that is applied.

Serology

CMV-IgM and IgG antibodies are detected by serology. Enzyme-linked immunosorbent

assay (ELISA) is one of the techniques most frequently used to detect CMV-specific

antibodies. The CMV IgM antibody response following primary infection slightly

precedes IgG antibody development. After the onset of infection, the CMV IgM antibody

touches a plateau in the first months and then gradually declines in the following three

25

to six months. The quickest assay to detect immunity is CMV IgG antibodies since it

persist for lifetime. Both the organ donor and the recipient should undergo CMV IgG

serology testing before transplantation (Kotton et al. 2013). After transplantation, CMV-

IgM and IgG antibodies have a restricted value for the diagnosis of CMV disease

(Humar et al. 2005). Due to the high level of immunosuppression after transplantation,

there is a delayed or impaired ability to produce antibodies. The transfusion of blood

products may produce a false positive test (via the passive transfer of antibodies).

Seroconversion (the appearance of IgM and IgG antibodies in a previously seronegative

individual) was used to diagnose CMV infection in the early periods. Another possibility

was to detect a fourfold rise in CMV IgG titres in paired specimens attained at least two

or four weeks apart.

Histopathology

Tissue-invasive CMV disease can be confirmed by performing histopathology. Large

cells (cytomegalia) with viral inclusion bodies ("owl's eye") are typical morphological

changes found in a biopsy from an affected organ (Figure 1-8).

26

Figure 1-8. CMV- Histology Owl's Eye Inclusions. CMV is a lytic virus and it leads to

a cytopathic effect in-vitro and in-vivo. The pathologic trademark of CMV infection is an

enlarged cell with viral inclusion bodies. The microscopic description given to these cells

is most commonly an "owl's eye". Although considered diagnostic, such histological

findings may be minimal or absent in infected organs.

(http://www.asm.org/division/c/photo/cmv1.jpg)

The method is used together with IHC and monoclonal antibodies to detect CMV

antigens. For detecting CMV organ involvement, the histological detection of owl's eye

inclusion bodies is an exceedingly specific method; however, its sensitivity is low. In

today’s era, this method has been still used albeit less frequently compared to the early

era of transplantation (Stewart & Cary 1991; den Bakker 2013). Due to its invasive

procedure, there is a major drawback and restriction. A biopsy could be required to

distinguish between acute rejection and CMV infection if the transplanted organ is

affected. A biopsy for histopathology is also necessary when the symptoms continue

http://www.asm.org/division/c/photo/cmv1.jpg

27

regardless of the treatment of CMV disease, but CMV testing in the blood is negative,

which may occur in some cases of gastrointestinal disease (Eid et al. 2010). Broncho-

alveolar lavage (BAL) fluids, particularly, alveolar macrophages appear to be the cell

containing CMV can also be used for the detection of large cells with viral inclusion

bodies and CMV antigen detection by IHC (Chemaly et al. 2004).

Viral culture

CMV can be isolated from numerous specimen types including blood, urine,

cerebrospinal fluid, BAL fluid, and from tissue biopsy where viral culture is highly

specific for the detection of CMV. However, one major shortcoming is time since the

culture of human fibroblasts usually takes two to four weeks and the sensitivity is poor. .

As a result, the test has restricted use in diagnosing infection or disease in transplant

recipients. A positive blood culture is specific and predictive of CMV disease. Since

seropositive recipients may shed CMV in their secretions, the detection of CMV in

cultures from other sites does not predict active disease. For example, a positive viral

culture from urine is generally not specific for active CMV disease (Pillay et al. 1993).

Viral culture is the method used when phenotypic antiviral drug resistance testing is

requested. However, phenotypic procedures are generally too time-consuming for the

clinical diagnosis of drug resistance.

The Antigenemia Assay

A semi-quantitative test that detects pp65 antigen in CMV-infected peripheral blood

leukocytes is the antigenemia assay (Razonable, Paya, et al. 2002). This test has been

performed at quite a few centers to diagnose acute CMV infection and to guide pre-

emptive therapy since it has higher sensitivity than cultures (Baldanti et al. 2008).

However, a major limitation is the need to process the clinical sample within a few hours

(6¬8 hours) as the test heavily relies on leukocytes. Leukopenia is thus a drawback; an

28

absolute neutrophil count of less than 1,000/mm3 weakens the performance of the

assay (Kotton 2013).

Quantitative Nucleic Acid Tests (QNAT)/Polymerase Chain Reaction (PCR)

In today’s age, Quantitative nucleic acid tests (QNAT)/polymerase chain reaction (PCR)

are the most frequently used molecular assay (Hirsch et al. 2013; Cardeñoso et al.

2013). The method is implemented for the diagnosis of active disease, monitoring the

response to the therapy, and monitoring when pre-emptive therapy is used as a

prophylactic approach. Most laboratories use real-time PCR. The progresses with real-

time PCR are a broader linear range, more rapid turnaround time and reduced risk of

carryover contamination compared with the previously used conventional PCR method

(Piiparinen et al. 2004). In order to detect CMV viral load, both whole-blood and serum

specimens are used. It is essential to use the same specimen when monitoring with

quantitative real-time PCR over time. Since blood measures both cell- free and

intracellular viruses, whole blood often gives a higher viral load compared with plasma

(Razonable, Brown, et al. 2002). There has been inconsistency in the test results (viral

load) from laboratories at different centers due to the lack of normalization (Pang et al.

2009). There are differences in commercial detection reagents, primers and probes

targeting different genes, methods for extracting nucleic acid and calibration, among

others and this could explain difference in the results. An international reference for the

quantification of CMV nucleic acid, which enables assay calibration and standardization

among laboratories, was released by the World Health Organization (WHO) in the year

2010. In a recent multinational study, five different laboratories were able to

demonstrate good reproducibility in viral load values when using a commercial test that

was calibrated to the WHO standard (Hirsch et al. 2013). In early era of CMV diagnosis,

only qualitative CMV PCR was available and it is also a sensitive test, but it is incapable

to differentiate low- level from high-level viral replication and is therefore not as useful a

test when monitoring the effect of CMV treatment. Diagnosis of drug resistance is now

routinely performed by genotypic resistance testing.

29

1.5.5 Definition of CMV infection

The following definitions are adapted from Ljungman et al.(Ljungman et al. 2002):

Primary infection is the detection of CMV infection in an individual previously found to

be CMV seronegative.

Reinfection or superinfection is the detection of a CMV strain that is distinct from the

strain that was the cause of the patient's original infection.

Reactivation is assumed if the CMV strain detected in the previous infection is found to

be indistinguishable from the strain causing the new episode.

The following definitions are in accordance with Kotton et al. (Kotton et al. 2013), and

Razonable et al. (Razonable & Humar 2013):

CMV infection: evidence of CMV replication regardless of symptoms (differs from latent

CMV)

Asymptomatic CMV infection: evidence of CMV infection without clinical symptoms

CMV disease: evidence of CMV infection with attributable symptoms classified as:

CMV syndrome: viral syndrome with fever and/or malaise, leukopenia and/or

thrombocytopenia.

Tissue-invasive CMV disease: symptoms and signs of disease and CMV detected by

immunohistochemistry (IHC) with CMV-specific antibodies in a biopsy from the affected

organ. The definitive diagnosis relies on the detection of CMV in the tissue specimen,

with the exception of central nervous system disease and retinitis.

30

1.5.6 Antiviral Drugs for CMV Prevention and Treatment

Some of the drugs that have been evaluated for CMV prophylaxis in various transplant

recipients are acyclovir, ganciclovir, valganciclovir, Foscarnet, Cidofovir, and immune

globulin preparations.

Acyclovir/valacyclovir

Acyclovir is a homologue of ganciclovir and a nucleoside analogue of guanosine. In

transplant patients, acyclovir was sometimes used as CMV prophylaxis during the early

1990’s. Valacyclovir is the prodrug of acyclovir. The suggested prophylaxis dose of

valacyclovir is 2000 mg p.o. four times daily (Razonable & Humar 2013), but studies

show that a lower dose of valacyclovir, 1000 mg three times daily to D+/R- may also be

effective (Sund et al. 2001; Sund et al. 2013). However, valacyclovir is not

recommended for the treatment of CMV disease.

Ganciclovir

Ganciclovir is a homologue of acyclovir and nucleoside analogue of guanosine. In order

for drug to be effective, phosphorylation of the drug is required. Its mechanism of action

is through the inhibition of virally encoded DNA polymerase (Crumpacker 1996). The

dose has to be adjusted for renal function since ganciclovir is excreted in urine.

Clearance is directly correlated to the glomerular filtration rate. The plasma half-life is

two to four hours and the intracellular half-life of ganciclovir triphosphate is about 16.5

hours. The drug causes toxicity to the bone marrow and neutropenia. For severe

neutropenia, granulocyte colony stimulating factor (G-CSF) can be used together with

ganciclovir, if needed, to increase the leukocyte count. Oral ganciclovir is not

recommended for treatment and the prophylaxis dose is 1,000 mg three times daily.

31

Valganciclovir

Valganciclovir is a valine ester of ganciclovir, a prodrug of ganciclovir. The mechanism

of this drug is activation via a viral protein kinase HCMV UL97 and subsequent

phosphorylation by cellular kinases. It is well absorbed after oral administration and

rapidly hydrolysed to ganciclovir in the liver and intestinal wall. The bioavailability of

ganciclovir from valganciclovir tablets is approximately 60%. A dose of 900 mg of

valganciclovir daily can achieve systemic exposure similar to 5mg/kg of i.v. ganciclovir

daily (Asberg et al. 2010). The adverse effects are similar to ganciclovir, therefore,

valganciclovir thus has to be adjusted for renal function and is associated with bone

marrow suppression, particularly leukopenia. Valganciclovir can be used as treatment in

mild or moderate CMV disease. The recommended treatment dose of valganciclovir is

900 mg twice daily and the prophylaxis dose is 900 mg once daily.

Foscarnet

Foscarnet directly inhibits the CMV DNA polymerase and is a pyrophosphate analogue

(Crumpacker 1992). The drug is principally used for the treatment of ganciclovir-

resistant CMV in transplant patients. The most common adverse effects are renal

impairment, anemia, electrolyte imbalance, and granulocytopenia. The recommended

treatment dose is 60 mg/kg i.v. every eight hours and it is not recommended for

prophylaxis.

Cidofovir

Cidofovir is a nucleotide analogue of cytosine and is converted by cellular enzymes to

cidofovir triphosphate, which is an active inhibitor of viral DNA polymerase (Andrei et al.

2015). Dose-dependent nephrotoxicity is the main adverse event. The treatment dose is

5 mg/kg once weekly x 2 and then every two weeks thereafter. In the cases of

32

ganciclovir resistance, cidofovir is the alternative and is not recommended for

prophylaxis. Very limited studies have been performed for this drug.

As per the guidelines from the American Society of Transplantation, the recommended

doses of prophylaxis and treatment are adapted (Razonable & Humar 2013).

Intravenous immunoglobulin (IVIG)

CMV-specific immunoglobulin (CMV-IVIG) in combination with antiviral agents has been

used as prophylaxis in some settings. When severe CMV pneumonitis occurs, CMV-

IVIG or IVIG is sometimes used in combination with i.v.ganciclovir. The effect is thought

to be immunomodulatory and limits acute inflammatory events (Zamora 2004).

However, the efficacy of this approach is unknown.

Ganciclovir resistance

Ganciclovir resistance is caused by several factors including prolonged exposure to the

drug, suboptimal ganciclovir levels and intensive immunosuppression. Lung transplant

recipients, CMV D+/R- patients, and patients with a high viral load of CMV DNA in blood

may develop ganciclovir resistance. Ganciclovir resistance is caused by mutations in

the viral UL97 (coding for viral protein kinase, which is responsible for the

phosphorylation of ganciclovir) or UL54 genes (coding for CMV DNA polymerase). UL97

mutations appear first in about 90% of cases, but UL54 mutations may follow later in

patients treated with ganciclovir. Mutation in UL54 is associated with a higher level of

resistance to ganciclovir or cross-resistance to foscarnet or cidofovir (Limaye 2002).

Mutations in UL97 do not affect foscarnet or cidofovir and the drugs can be used as

treatment.

33

1.5.7 Strategies for CMV Prevention and Treatment

Universal prophylaxis

In a universal prophylaxis strategy, all patients at risk of CMV infection undergo the

administration of antiviral prophylaxis for a fixed period of time. Antiviral medication

commence immediately or very early after transplantation and most often continue for

three to six months (Kotton et al. 2013) and could be prolonged for lung transplant

recipients (Palmer et al. 2010). The benefits of universal prophylaxis are that it is easy

to administer, a lesser amount of monitoring with QNAT is needed, and the drug also

protects from other herpes viruses like herpes simplex virus and varicella zoster virus

(Kotton et al. 2013). However, the drawbacks include increased drug toxicity, drug-

related cost, and the risk of becoming resistance to the drug. After the termination of the

antiviral drug, there is a high possibility of developing CMV disease (i.e. late-onset

disease) (Schoeppler et al. 2013).

Pre-emptive therapy

In a pre-emptive strategy, patients are monitored at regular intervals to detect early viral

replication. Antiviral treatment is introduced once viral replication reaches a certain

threshold. These laboratory methods include the QNAT for the detection of CMV DNA

from blood or serum and CMV pp65 antigenemia assay. Treatment is thus given to

prevent the progression of asymptomatic infection to disease. The downsides of pre-

emptive approach are that it involves frequent monitoring and can be challenging to run-

through if the patients live far away from the hospital or laboratory. The viral load may

increase very rapidly when CMV is reactivated and Emery et al. reported a doubling

time of approximately 24 hours (Atabani et al. 2012). In order to compare the difference

in viral load, only one assay and one specimen type, either whole blood or plasma,

should be used. Compared to plasma, whole blood often gives a higher viral load. The

benefits are reduced toxicity, reduced drug cost, and a lower rate of late-onset disease.

34

Treatment

Antiviral therapy is given to patients with symptomatic CMV disease, most often i.v.

ganciclovir with two to three weeks' duration. In mild or moderate CMV disease,

valganciclovir is an alternative. The treatment should be continued until CMV is

undetectable and in today’s era this is often monitored once a week with CMV DNA

from blood or serum.

1.6 STUDY RATIONALE

The rationale and clinical importance behind finding pre-transplant biomarkers that

predict CMV viremia post-transplant is to then allow tailoring treatments which includes

the immunosuppressive drug regimens and antiviral prophylaxis in the best possible

way to prevent CMV viremia or disease post-transplant. Our study used live viral

stimulation which is a novel approach whereas other studies have used peptides and

viral lysates to look at pre-transplant adaptive and innate responses to predict CMV

viremia and disease. The rationale behind using live virus in our experiments is to

reproduce as faithfully as possible what the immune system would deal with - for

instance the interaction with protein complexes that are found in the envelope that could

potentially be disrupted when making a lysate. Of course that may also allow for the

infection of susceptible PBMC types (e.g. monocytes, dendritic cells) and therefore for

immune evasion mechanisms to affect antigen presentation to T and NK cells.

Despite an increasing awareness of the importance of innate immunity, the roles of

natural killer (NK) cells in CMV infection have not been clearly defined and studies like

the one which I would be showing in my thesis would give a better perspective on the

roles of NK cells.

35

Chapter 2

HYPOTHESIS AND EXPERIMENTAL APPROACH

36

2.1 Study Aims

The aim of my studies was to find pre-transplant predictive biomarkers that would

predict CMV viremia and disease post-transplant. Specific aims include:

1) To determine whether CMV-specific T-cell responses which include the CD4+

and CD8+ response may be useful for predicting the risk of CMV reactivation and

disease following transplantation.

2) To determine whether baseline non-CMV-specific T-Regulatory cell response

which include the CD4+CD25+FoxP3+ response may be useful for predicting the

risk of CMV reactivation and disease following transplantation.

3) To determine whether NK cell responses which include CD56Br and CD56dim

response assessed pre-transplant would help predict the risk of CMV reactivation

and disease following transplantation.

4) To determine the role of the surface marker, NKG2C, on NK cells that would help

predict CMV viremia and disease.

The CMV-specific T-cell response may include both CD4+ and CD8+ T-cell responses.

Several studies have demonstrated that post-transplant development of T-cell immunity

is important for the control of CMV replication and that the longitudinal evaluation of

such response may be clinically useful for prediction of CMV reactivation. Another

aspect of the cell mediated immune response includes regulatory T cells (Tregs). We

have previously shown that post-transplant changes in Treg populations occur in

response to CMV replication and in conjunction with more standard measures of cell

mediated immunity may be predictive of viral control (Egli et al. 2012).

The first line of the host defense in controlling CMV infection is activation of Natural

Killer (NK) cells, early production of pro-inflammatory cytokines, and the activation of

the innate immune cell effector function (Vivier et al. 2012b; Caligiuri 2008; Romo et al.

2011; Sanos et al. 2011; Björkström et al. 2013). NK cells are innate lymphoid cells

constituting the third most abundant lymphocyte population in the peripheral blood

37

(Romo et al. 2011; Sanos et al. 2011; Björkström et al. 2013). The past fifteen years

have seen several advances in our knowledge of NK cells, their surface receptors, and

the molecular mechanisms involved in their function.

2.2 Study Hypothesis

We hypothesized that pre-transplant CD4+ and CD8+ T-cell responses in addition to T-

Regs response would be useful for predicting the risk of CMV reactivation and disease

following transplantation. Recent data show that pre-transplant CD4+ and CD8+ T-cell

responses may also be useful for predicting the risk of CMV reactivation following

transplantation (Bestard et al. 2013; Cantisán et al. 2013; López-Oliva et al. 2014). In

addition, the pre-transplant frequencies of regulatory T cells may also play a role in

CMV reactivation post-transplant. However, post-transplant factors such as type of

transplant, use of induction and immunosuppression may modify the risk.

We also hypothesized that pre-transplant CD56Br and CD56dim NK cell responses

would be useful for predicting the risk of CMV reactivation and disease following

transplantation. In addition, the expansion and higher expression of the NK cell memory

marker specific for CMV would help to protect against CMV viremia and disease. Cross-

sectional studies showed that NKG2C+ NK cells can represent more than 25% of

peripheral NK cells in individuals infected with CMV while accounting for less than 2% of

NK cells in CMV-seronegative individuals (Jost & Altfeld 2013; Mónica Gumá et al.

2006; Gumá et al. 2004). More recently, Lopez-Verges et al. characterized the changes

in NK cells longitudinally in a group of individuals with acute CMV infection and showed

a preferential expansion of NKG2C+ NK cells (Lopez-vergès et al. 2011).

To help answer this question, we evaluated a large cohort of CMV seropositive

transplant recipients, who did not receive antiviral prophylaxis and were managed using

a pre-emptive strategy. We characterized whether measurement of CMV specific pre-

transplant T-cell and NK cell responses could predict reactivation of CMV post-

transplant.

38

Chapter 3

METHODOLOGY

39

3.1 Patient Population

The patient population consisted of a subset of patients who were recruited as part of

the Swiss Transplant Cohort Study. This cohort study collects prospective samples and

clinical data from solid organ transplant patients enrolled from five centers in

Switzerland (Koller et al. 2013). All patients provided written informed consent for

inclusion in the cohort study and for testing of samples. In addition this study was

reviewed and approved by the Swiss Transplant Cohort Scientific Committee. The

inclusion criteria for this study were 1) organ transplant recipient (including islet), 2) pre-

transplant CMV seropositivity, and 3) no antiviral prophylaxis specific for CMV

prevention, and 3) regular viral load monitoring post-transplant as part of a pre-emptive

strategy.

Frozen PBMCs were shipped to Canada. We chose patients from Switzerland and not

Canada because our collaborator had already recruited patients from various regions of

Switzerland. We also chose this cohort because none of the patients received antiviral

prophylaxis and so this represented a natural history cohort. This would have been

difficult to find in Canadian transplant centers.

A total of 435 patients were eligible for the study. Of these, 272 patients had pre-

transplant peripheral blood mononuclear cell (PBMC) samples available. These

samples had been cryopreserved and after thawing, had cells with at least 70% viability

that could be used for assessment of T-cell responses. Weekly testing for CMV viremia

was performed by the local laboratory at each of the 5 transplant centers for the first 12-

weeks post-transplant. All patients were followed for 6-months post-transplant for the

development of CMV viremia or symptomatic CMV disease.

40

3.2 Clinical Definitions

CMV Viremia

CMV viremia was defined as the detection of CMV in the blood by the site specific CMV

PCR assay (Ljungman et al. 2002). The primary end-point in this study was the

incidence of viremia requiring pre-emptive antiviral therapy.

CMV Disease

Patients were followed for the development of active CMV disease for 12 months after

transplantation. CMV disease was defined according to standard clinical criteria and

included CMV syndrome and tissue invasive disease. These were defined based on the

American Society of Transplantation definitions for use in clinical studies (Humar &

Michaels 2006).

3.3 Peripheral Blood Mononuclear Samples

Between 8 and 16ml of blood was collected in an EDTA blood collection tube (Becton

Dickinson). PBMCs were prepared by density gradient centrifugation using Ficoll-

PaqueTM Plus (GE Healthcare). Cells accumulating at the interface were washed twice

in PBS and resuspended in culture medium consisting of RPMI1640 (Life Technologies)

+10% heat inactivated fetal bovine serum (Life Technologies) + 2mM glutamine (Life

Technologies) +1% penicillin/streptomycin. PBMCs were cryopreserved until use. The

same cryopreservation protocol was followed at each site.

The protocol for PBMC isolation used by the group results in high yields of leukocyte

recovery, including monocytic (monocytes, dendritic cells). It is very important not to lay

more than 20 ml of 1:1 blood-PBS mix over Ficoll in order to maximize the recovery; the

1:1 mix proportion is also very relevant. PBS must be at room temperature. The

41

intermediate spin step is meant to wash Ficoll away. The last spin step, at low speed, is

meant to deplete platelets from the sample. If cryopreserving the isolated cells, spin for

a last time at 300g for 10 min after cell count; drain the complete media and resuspend

in FBS 10% DMSO. All spin steps are performed at room temperature.

3.4 Viral Strain

Laboratory adapted Towne strain of CMV was used for all assays. To determine viral

titers we infected Human foreskin fibroblasts (HFF-1) cells from ATCC (SCRC1041).

HFF-1 cells were maintained in Dulbecco’s modified Eagle’s medium (ATCC)

supplemented with 2 mM glutamine, 1% penicillin/streptomycin and 10% fetal bovine

serum. HFF cells were infected with CMV Towne strain. Cells and supernatant were

harvested post-infection and a standard 14-day plaque assay was used to determine

viral titers (Boeckh & Boivin 1998; Chou & Scott 1988).

Plaque media was prepared by adding 10 ml FBS+22 ml L-Glutamine to DMEM 2X with

glutamine using 0.2 micron filter pore. In a jar make 2% Sea plaque agar using PBS and

warm it to 56° C. Mix it with 1:1 plaque media and add 4 ml in each well. Once it

solidifies, turn the well upside down and incubate at 37° C for around 2 weeks. After 2

weeks of incubation, add 4mL of 10% buffered formalin onto the top of the agar plugs in

each well at room temperature for 3-4hrs. Aspirate the formalin and discard it in a

separate container. Immediately remove the agar plugs using a curved spatula being

careful not to disturb the cell monolayers. Gently rinse the wells with tap water. Stain

with 1 part of 0.8% crystal violet in 50% ethanol with 3 parts of PBS (1:4). Add around

1ml in each well. Wash away the staining using tap water. Dry plates over night by

inverting it.

42

3.5 Stimulation and Staining by Flow Cytometry

PBMCs were resuspended in culture media at a concentration of 3 x 105 cells/ml per

condition for 6 hours. The negative control had the media alone (unstimulated). The

positive control was stimulated with phorbol 12-myristate 13-acetate (PMA) and

ionomycin as previously described by Egli et al. (Egli et al. 2012). PBMCs were

stimulated with live CMV Towne Strain virus at a MOI of 0.03. Brefeldin A at 10μg/ml

was added for the last 4h of culture. Markers for identifying T-cell and NK-cell subsets

were CD3, CD4, CD8, CD25, FoxP3, CD16, CD56, and NKG2C along with IFN-γ (all

antibodies from Biolegend, R&D Systems, BD Biosciences or eBioscience).

Cells were then surface labelled with anti-CD3-FITC (eBioscience), anti-CD4-PE Texas-

Red (BD Biosciences), anti-CD8-Brilliant Violet (Biolegend), anti-CD25-PE-Cy7

(eBioscience), anti-CD16-Pacific Blue (eBioscience), anti-CD56-APC-Cy7

(eBioscience), and anti-NKG2C-PE (R&D Systems) before fixing with intracellular

fixation buffer and intracellular permeabilization buffer (eBioscience). Next, intracellular

staining was performed with anti-FoxP3-APC (eBioscience) and anti-IFN-γ-PerCP-

Cy5.5 (eBioscience).

Ten-color flow cytometry was performed using a LSR Fortessa II flow cytometer (Becton

Dickinson, New Jersey, USA.). Two hundred microliters of the concentrated specimen

was added to each tube, which contained antibodies and fluorochromes. Data (50,000-

100,000 events per tube) were acquired and analyzed using FlowJo, LLC (Oregon,

USA) software. Nonviable cells were excluded by using Live/Dead Stain. Samples in

which cells showed a viability of 70% or more were considered for this study. Antigen

expression was depicted as fluorescence intensity on dual parameter scattergrams.

Using a pulse geometry gate (such as FSC-H x FSC-A), doublets were easily

eliminated. Cell clumps will take longer than single cells when they pass through the

laser intercept and this affects the area of the signal. Singlets were used to analyze the

data. Negative controls were gated based on fluorescence minus one (Tung et al.

2007). The inspection of the data was also done using the back gating tool that allows

to define what cells would fall in the final population. All assays were optimized and

43

validated in healthy volunteer samples (n=7) using fresh and cryopreserved samples,

prior to testing of patient samples.

3.6 Statistical Analysis

Statistical analyses were performed using SPSS (version 22.0, Chicago, Ill.) and

GraphPad Prism (version 5.0, La Jolla, CA). Patient data were compared using the chi-

square test for categorical variables and the Mann-Whitney U test and Wilcoxon test for

continuous variables, when appropriate. A two-tailed p-value of <0.05 was considered

significant.

44

Chapter 4

RESULTS

45

4.1 Patient Population and Outcomes

We evaluated a cohort of 272 pre-transplant CMV seropositive solid organ transplant

(SOT) recipients. Baseline demographics are outlined in Table 4-1. Transplant types

included kidney (178/272; 65.4%), liver (64/272; 23.5%), heart (12/272; 4.4%), islets

(7/272; 2.6%), and others (11/272; 4.1%). None of the patients received antiviral

prophylaxis and all patients underwent regular virologic monitoring for the first 12-weeks

post-transplant. Immunosuppression consisted primarily of tacrolimus, mycophenolate

mofetil (MMF), and glucocorticoids. Induction therapy was used in 200/272 (73.5%)

patients and consisted of either basiliximab (175/272; 64.3%) or anti-thymocyte globulin

(ATG) (25/272; 9.2%). All transplant recipients were CMV seropositive pre-transplant

and 170/272 (62.5%) had a seropositive donor. The incidence of CMV viremia requiring

pre-emptive antiviral therapy in the first 12-weeks post-transplant was 83/272 (30.5%).

In the CMV viremia group, 10/83 (12%) had symptomatic CMV disease (predominantly

CMV viral syndrome).

A comparison of baseline clinical variables in patient who developed viremia vs. those

who did not is shown in Table 4-1. In the CMV viremia group, the patients were slightly

older compared to the non-viremic group (mean age 54.4 ± 13.5 vs 50.2 ± 14.0;

p=0.028). The incidence of CMV viremia was significantly higher in D+/R+ patients

(60/170; 35.3%) vs. D-/R+ patients (23/102; 22.5%); p= 0.03) . No significant difference

in induction therapy and immunosuppressive regimens in the two groups were observed

(Table 4-1). No cases of CMV disease occurred in the absence of detectable viremia.

46

Table 4-1. Clinical Characteristics of Patients and Outcome

Variable

CMV viremia

(n=83)

No CMV viremia

(n=189)

p-value

Gender, n

Male/Female

50/33

122/67

0.590

Age, Mean ± SD 54.4 ± 13.5 50.2±14.0 0.028

Transplanted organ, n (%)

Kidney

Liver

Others

52 (62.7%)

20 (24.1%)

11 (13.2%)

126 (66.7%)

44 (23.3%)

19 (10.0%)

0.080

CMV Serostatus, n (%)

D+/R+

D-/R+

60 (72.3%)

23 (27.7%)

110 (58.2%)

79 (41.8%)

0.030

Immunosuppression, n (%)

MMF

Tacrolimus

CsA

Glucocorticoids

Others

15 (18.0%)

14 (16.9%)

16 (19.3%)

33 (39.8%)

5 (6.0%)

53 (28.1%)

40 (21.2%)

14 (7.4%)

68 (36.0%)

14 (7.3%)

0.11

Induction Therapy, n (%)

Basiliximab

Antilymphocyte globulin

47 (56.6%)

8 (9.6%)

128 (67.7%)

17 (9.0%)

0.160

CMV Disease 10 (12.0%) 0 (0.0) p<0.001

Categorical variables were compared using Fisher’s Exact Test and continuous variables using Mann-Whitney U Test. D=donor;

R=recipient

47

4.2 CD4 and CD8 T-Cell Interferon-γ Response

A pre-transplant CMV-specific CD4 T-cell response was observed in 63/272 (23.2%)

patients. A CD8 T-cell response was observed in 201/272 (73.9%) patients (Table 4-2).

Table 4-2: CMV specific T-cell response in patients with and without CMV Viremia

% of patients with positive response

Total

CMV viremia

n=83

No CMV viremia

n=189

P-value

Total Cohort CD4+IFNγ+

n=272

63/272

(23.2%)

21/83

(25.3%)

42/189

(22.2%)

0.64

Total Cohort CD8+IFNγ+

n=272

201/272 (73.9%)

60/83

(72.3%)

141/189

(74.6%)

0.77

D+/R+ CD4+IFNγ+

n=170

41/170

(24.1%)

15/60

(25.0%)

26/110

(23.6%)

0.85

D-/R+ CD4+IFNγ+

n=102

22/102

(21.6%)

6/23

(26.1%)

16/79

(20.3%)

0.57

D+/R+ CD8+IFNγ+

n=170

127/170

(74.7%)

44/60

(73.3%)

83/110

(75.5%)

0.85

D-/R+ CD8+IFNγ+

n=102

74/102

(72.5%)

16/23

(69.6%)

58/79

(73.4%)

0.79

48

The quantitative range (frequency distribution) of CD4+ and CD8+ T-cell responses is

shown in Figures 4-1 and 4-2. No specific demographic factor was associated with

likelihood of either CD4+ or CD8+ T-cell response. For example, no significant

difference in likelihood of a response was observed in the pre-liver transplant vs. the

pre-kidney transplant patients (p=NS for both CD4 and CD8 responses). Similarly, age

at transplantation was not significantly different in responders vs. non-responders.

Relative to CD4 responses, the likelihood of CD8 response was significantly greater

(p<0.001). However, the responses were significantly correlated with each other (i.e. a

patient with a CD4 response was more likely to also have a CD8 response; p<0.001).

4.3 Predictive Value of Pre-Transplant Testing

We then analyzed whether the pre-transplant CMV specific CD4 and CD8 T cell

response was able to predict the development of CMV viremia in the first 12-weeks

post-transplant. (Table 4-2). For CD4+ T-cells, the percentage of patients that produced

a positive response to CMV stimulation pre-transplant in the group that subsequently

developed viremia was 21/83 (25.3%) compared to 42/189 (22.2%) in the group that did

not develop viremia (p=0.64). For CD8+ T-cells, the percentage of patients that had

positive response to CMV stimulation pre-transplant was 60/83 (72.3%) in the group

that subsequently developed viremia compared to 141/189 (74.6%) in the non-viremic

group (p=0.77). Similarly there was no difference in the percentage of patients that had

CD4+ or CD8+ T-cell responses in the CMV disease vs. no disease subgroup (Table 4-

3).

49

Table 4-3: CMV specific T-cell response in patients with and without CMV disease

# of patients with positive response

Total

n=272

CMV disease

n=10

No CMV disease

n=262

P-value

Total Group CD4+IFN-γ+

n=272

63/272

(23.2%)

1/10

(10.0%)

62/262

(23.7%)

0.46

Total Group CD8+IFN-γ+

n=272

201/272 (73.9%)

6/10

(60.0%)

195/262

(74.4%)

0.29

D+/R+ CD4+IFNγ+

n=170

41/170

(24.1%)

0/8

(0.0%)

41/162

(25.3%)

0.20

D-/R+ CD4+IFNγ+

n=102

22/102

(21.6%)

1/2

(50.0%)

21/100

(21.0%)

0.39

D+/R+ CD8+IFNγ+

n=170

127/170

(74.7%)

4/8

(50.0%)

123/162

(75.9%)

0.11

D-/R+ CD8+IFNγ+

n=102

74/102

(72.5%)

2/2

(100.0%)

72/100

(72.0%)

0.52

50

4.4 CD4 and CD8 T-Cell Response in D+/R+ and

D-/R+ Subgroups

Next, we analyzed the predictive value of pre-transplant CD4 and CD8 responses in

donor seropositive and donor seronegative subgroups (Tables 4-1 and 4-2 and Figures

4-1 and 4-2). There was no significant difference in D+R+ and D-R+ subgroups in terms

of predictive value for pre-transplant measurement of CD4 or CD8 T-cells response

(p=NS for all comparisons). Similarly, no difference was observed in the incidence of

subsequent CMV disease (Table 4-3).

D+/R+ CD4+ T-Cell Response

D+R

+ No V

irem

ia

D+R

+ CM

V V

irem

ia

0.0

0.5

1.0

2.0

3.0

4.0

5.0p=0.85

IFN

- +

Fre

qu

en

cy (

%)

D-/R+ CD4+ T-Cell Response

D-R

+ No V

irem

ia

D-R

+ CM

V Virem

ia

0.0

0.5

1.0

2.0

3.0

4.0

5.0p=0.57

IFN

- +

Fre

qu

en

cy (

%)

Figure 4-1. CD4 T-Cell Response in D+R+ and D-R+ Subgroups. Each dot

represents individual patients. Solid lines represent median values. The %IFN-γ

frequency in the D+R+ and D-R+ showed no significance and was unable to predict CMV

viremia based on the CD4 T-Cell response.

51

D+/R+ CD8+ T-Cell Response

D+R

+ No V

irem

ia

D+R

+ CM

V V

irem

ia

0

5

10

15 p=0.85

IFN

- +

Fre

qu

en

cy (

%)

D-/R+ CD8+ T-Cell Response

D-R

+ No V

irem

ia

D-R

+ CM

V Virem

ia

0

5

10

15p=0.79

IFN

- +

Fre

qu

en

cy

Figure 4-2. CD8 T-Cell Response in D+R+ and D-R+ Subgroups. Each dot

represents individual patients. Solid lines represent median values. The %IFN-γ

frequency in the D+R+ and D-R+ showed no significance and was unable to predict CMV

viremia based on the CD8 T-Cell response.

4.5 Regulatory T-Cell Subset

(CD4+CD25+FoxP3+) Response

Next, we analyzed regulatory T-cell (T-reg) subsets. The frequency of (non-CMV

specific) T-regs in the whole cohort ranged from .04% to 11.50% (median frequency

2.35%) (Figure 4-3). Patients with CMV viremia had a higher baseline T-reg frequency

(median 2.61% (IQR 0.25%-8.89%)) compared to the group that did not develop

subsequent viremia (median 2.11% (IQR 0.04%-11.50%); p=0.035 (Figure 4-3).

52

No V

irem

ia

CM

V Virem

ia

0

1

2

3

4

5

6

7

8

9

10

11

12

p=0.035

% T-Reg at Baseline (Unstimulated)T

-Reg

Fre

qu

en

cy (

%)

Figure 4-3. T-Regulatory Cell Baseline Response (CD4+CD25+FoxP3+). Each dot

represents individual patients. Solid lines represent median values. The result shows

that patients who had a higher baseline of non-CMV specific T-Reg frequency are at a

higher risk to developing CMV viremia.

53

Next we assessed whether changes in T-Reg frequency were observed following 6 hour

stimulation with live virus. Both groups had a significant decline of measured T-regs

frequency when stimulated with virus for 6 hours (median 1.05% (IQR 0.0%-8.26%);

p<0.001) (Figure 4-4).

54

No V

irem

ia (U

nstim

ulate

d)

No V

irem

ia (V

iral

Stim

ulatio

n)

Vire

mia

(Unst

imula

ted)

Vire

mia

(Viral

Stim

ulatio

n)

0

1

2

3

4

5

6

7

8

9

10

11

12

p<0.001

p<0.001

Absolute T-Reg Frequency

T-R

eg

Fre

qu

en

cy (

%)

Figure 4-4. Absolute T-Regulatory Cell Response (CD4+CD25+FoxP3+). Each dot

represents individual patients. Solid lines represent median values. The result shows

that upon stimulation with live virus for 6 hours, both the viremic and non-viremic group

showed significant decline of non-CMV specific T-Reg frequency.

55

4.6 Specific NK-cell Phenotypes Are Associated

With CMV Reactivation

A subset of NK cells which was CD56bright and CD16 negative was more frequent in

patients with subsequent viremia requiring anti-viral therapy (median 10.1%) compared

to patients with no viremia (median 5.07%); p= 0.008 (Figure 4-5). In a healthy

volunteer, CD56bright cells which include CD56bright, CD16 dim and CD56bright, CD16neg

are around 10%. However, during chronic infections this subset of NK cells may be

elevated and expanded since CD56bright NK cells maybe the immature precursors to well

differentiated CD56dim cells162,163. These results correlate with our cohort of patients who

were diagnosed with end-stage renal and liver diseases.

56

Figure 4-5. CD56bright, CD16neg NK-cell Response. Each dot represents individual

patients. The %CD56bright CD16neg frequency showed statistical significance and was

able to show that it is elevated in CMV viremic patients.

4.7 Specific NK-Cell Phenotypes Are Associated

With CMV Protection

A subset of NK Cells which was CD56dim and CD16 negative was expressed highly in

patients with no viremia (median 16.3%) compared to patients with subsequent viremia

requiring anti-viral therapy (median 8.05%). It was statistically significant with a p-value

of 0.037 (Figure 4-6). In a healthy volunteer, the proportion of CD56dim cells is

approximately 90% and the majority is CD56dim and CD16pos. The ratio of CD56dim to

57

CD56bright is altered during chronic infection, hematopoietic stem cell transplantation,

transporter associated with antigen processing (TAP) deficiency, and other diseases.

Figure 4-6. CD56dim, CD16neg NK-cell Frequency in CMV Viremia Patients. Each dot

represents an individual patient. Higher expression of %CD56dim CD16neg frequency

showed statistical significance and was able to display protective effect against

developing CMV viremia.

58

4.8 NK-cell Interferon-γ Responses Are

Associated With CMV Protection

In a subset of NK cells which was CD56dim and CD16 positive, we saw that a robust

IFN-γ response was able to protect patients from CMV disease, but not CMV viremia.

The results were statistically significant with p-value of 0.017 (Figure 4-7). Probable

reasons for weak or no response IFN-γ from CMV disease patients may be due to cells

being anergic, factors influencing signal regulation, or the lack of professional antigen

presenting cells (APCs).

Figure 4-7. CD56dim, CD16+, IFN-γ+ NK-cell Response. Each dot represents an

individual patient. Higher expression of %IFN-γ on CD56dim CD16+ frequency showed

statistical significance and was able to display protective effect against developing CMV

disease.

59

4.9 NKG2C Expression is Associated With CMV

Protection in D+R+ Patients

In a subset of NK cells which was CD56bright and CD16 negative, an increased

expression of NKG2C was able to protect the D+R+ patients from CMV viremia (median

0.40 vs -1.69) (Figure 4-8) and CMV disease (median 0.21 vs -4.14) (Figure 4-9). The

results were statistically significant with a p-value of 0.036 and 0.045 respectively.

However, the results were not significant for D-R+ patients.

Figure 4-8. CD56bright, CD16neg, NKG2C+ NK-cell Response in D+R+ Patients with

CMV Viremia. Each dot represents an individual patient. Higher expression of

%NKG2C on CD56dim CD16neg NK cells showed statistical significance and was able to

display protective effect against developing CMV viremia upon stimulation with live

virus.

60

Figure 4-9. CD56bright, CD16neg, NKG2C+ NK-cell Response in D+R+ Patients with

CMV Disease. Each dot represents an individual patient. Higher expression of

%NKG2C on CD56dim CD16neg NK cells showed statistical significance and was able to

display protective effect against developing CMV disease upon stimulation with live

virus.

61

Chapter 5

GENERAL DISCUSSION AND CONCLUSION

62

5.1 General Discussion and Conclusion for T-cell

We analyzed pre-transplant CMV-specific cellular responses for their role to predict

CMV reactivation post-transplant in a large cohort of CMV-seropositive SOT recipients

not receiving antiviral prophylaxis. Our study showed that pre-transplant CD4+ and

CD8+ T-cell responses were not associated with CMV viremia in the first 12-weeks

post-transplant. No differences in predictive value were observed between the D+/R+

subgroup and the D-/R+ subgroups. Although the incidence of viremia was high

(30.5%), the overall incidence of CMV disease was low as would be expected in a pre-

emptive strategy. Nevertheless, pre-transplant T-cell responses were also not predictive

of development of subsequent CMV disease. We also assessed non-specific regulatory

T-cell frequencies in this cohort. A novel finding was that high pre-transplant T-reg

frequencies were associated with an increased risk of CMV viremia post-transplant

(median 2.61% (IQR 0.25%-8.89%) compared to the group that did not develop

subsequent viremia (median 2.11% (IQR 0.04%-11.50%); p=0.035). The reactivation of

CMV after transplantation likely involves a series of contributing factors that impact the

risk such as the type, and dosage of immunosuppression drugs, the occurrence of

acute rejection, cold-ischemia time and HLA compatibility and the concurrent

reactivation of other viruses (Mengelle et al. 2015; Razonable & Humar 2013). Since

many of these contributing factors are in the post-transplant period, it is not surprising

that a single time-point pre-transplant assessment of cell mediated immune response in

the transplant candidate may be a poor predictor of post-transplant outcomes.

Post-transplant CMV-specific cellular immunity may have greater relevance in predicting

CMV outcomes. Several studies evaluating repeated measures of post-transplant cell

mediated immune response have been very promising for allowing further refinement of

prevention and treatment strategies. For example, in a multi-center study evaluating the

utility of CD8+T cell response measurement at the end of prophylaxis in 127 D+/R-

patients, a CMI response (using the Quantiferon-CMV assay) was associated with

reduced likelihood of late-onset CMV disease (Manuel et al. 2013). We have also

shown that patients with CMV viremia who have detectable CMV specific CD4 T-cell

63

responses are more likely to spontaneously clear viremia without the need for antiviral

therapy (Lisboa et al. 2012).

Studies assessing the use of CMV-specific cell-mediated immunity testing in the pre-

transplant setting to predict post-transplant outcomes have yielded more conflicting

results, but have utilized differing assays, and study populations. Bestard et al.

evaluated pre-transplant testing in a cohort of 137 kidney transplant patients including

109 R+, and 28 D+/R- patients, including subsets that received either prophylaxis or

pre-emptive therapy (Bestard et al. 2013). They used an ELISPOT based IFN-γ assay

and stimulated PBMCs with either CMV lysate, or a pp65 and IE-1 peptide pool. The

study found that low pre-transplant IE-1-specific IFN-γ responses predicted post-

transplant CMV antigenemia and symptomatic disease, but that response to viral lysate

or pp65 was not predictive. Cantisán et al. evaluated a cohort of 23 lung and 32 kidney

transplant patients (44/55 R+, 8/55 D+/R-, 3/55 D-R-) (Cantisán et al. 2013). They

found that 30/44 (68%) R+ patients had postive CD8+ T-cell response detectable pre-

transplant as determined by the Quantiferon-CMV assay (a percentage of positives very

similar to what we obtained). The assay was able to successfully predict the

development of post-transplant CMV replication with a negative and postive predictive

value of 83.7% and 75% respectively. López-Oliva et al. assessed 15 CMV

seropositive kidney transplant patients using flow cytometry and intracellular cytokine

staining on pre-transplant stimulated with either IE-1 or pp65 antigens. CD8 but not CD4

T-cell responses to IE-1 were signifcantly higher in patients who did not develop CMV

infection during the first year post-transplant (López-Oliva et al. 2014). The type of ex-

vivo antigen stimulation for eliciting immune response likely plays an important role in

the assay. Our study used live viral stimulation; alternatives included overlapping

peptide pools and viral lysate. Each method of stimulation likely has advantages and

disadvantages. Although perhaps more technically demanding, we chose live virus

stimulation in order to potentially allow for the full repertoire of antigen presentation - for

instance, live virus allows the interaction with protein complexes that are found in the

envelope that could potentially be disrupted when making a lysate (Boehme et al.

2006). A potential downside of this approach may result from infection of susceptible

64

PBMC types (e.g. monocytes, dendritic cells) and activation of viral immune evasion

mechanisms affecting antigen presentation to T and NK cells (Sinzger 2006).

We also found the percentage of seropositive patients who had a positive CD4+ T-cell

response to be low while the CD8+ T-cell response was better preserved. This may be

due to a combination of factors including premature immunologic ageing in patients with

chronic disease such as end-stage renal or liver disease (Betjes & Litjens 2015).

A novel finding that merits further study was the higher baseline Treg frequency

observed in the patients who subsequently developed CMV viremia. The role of T-regs

has not been well studied in relation to CMV reactivation post-transplant. In a study of

30 patients with CMV viremia, a lower T-reg frequency was observed in patients who

had spontaneous clearance vs. those with progression (Egli et al. 2012). Similarly,

CMV-induced regulatory T cells can functionally suppress effector T cells during

recurrent CMV infections (Schwele et al. 2012).

Our study had some limitations. Some heterogeneity in the study population existed

since different transplant types were assessed. However, immunosuppressive

regimens were similar and sample size for the liver and kidney patients was large

enough to be analyzed separately with no differences observed compared to the overall

cohort. It would have been great if we had the doses of immunosuppressive drug

regimen since higher doses of immunosuppressive drugs contributes to CMV viremia

and disease. Our study also didn’t look into the HLA matching between donor and

recipient and several studies show it may some role in affecting the viral replication

parameters (Aldridge et al. 2015; Egli et al. 2007; Shabir et al. 2013). Also, the number

of patients with CMV disease was low (n=10) because a pre-emptive strategy was

employed. However, the primary end-point we evaluated was CMV viremia and since

no patient received antiviral prophylaxis, this study in effect represents as close to a

“natural history” study as is possible within the current standard of care. Other cell

types may play a role in post-transplant CMV replication including NK cells and these

were not assessed in the present study (Gonzalez et al. 2014). Finally, different viral

load assays were used in different centers. For this reason, and the fact that patients

65

were treated based on viremia, we did not analyze quantitative viral load as an

outcome. Strengths of our study include the very large sample size, the absence of

antiviral prophylaxis and the fact that all patients were CMV seropositive pre-transplant.

In summary, our study demonstrates that pre-transplant CD4+ and CD8+ T-cell

responses as determined by the methods we employed are unable to predict CMV

reactivation and disease occurring after transplant and would unlikely to be of significant

clinical utility. We did note some novel findings related to Treg frequencies and risk of

CMV reactivation that would be interesting to explore further.

5.2 General Discussion and Conclusion for NK-

cell

The purpose of this study was to determine if pre-transplant CMV-specific cellular

responses can predict post-transplant CMV in the early post-transplant period. We

were able to show that pre-transplant NK-Cell responses which include the CD56bright

and CD56dim NK cells may be of some importance in predicting CMV viremia or disease

for the first 12-weeks post-transplant. Similar to other studies, this study also showed

changes in NKG2C receptor and the importance of NKG2C phenotype as a potential

CMV-specific subset of NK cells.

Gumá et al had reported that the serological status for other herpesviruses (i.e. EBV

and HSV-1) showed no correlation with the numbers of NKG2C+ cells and CMV

coinfection accounts for increased proportions of NKG2C+ cells observed in HIV-1+

patients, a conclusion further supported by other reports in which this variable was

considered (Gumá et al. 2004; Mónica Gumá et al. 2006). Similarly, an expansion of

NKG2C+ NK cells was observed in HIV-1 infection and chikungunya virus infection, but

again only in those individuals also infected with HCMV, whereas such an expansion

was not observed in recurrent HSV-2 infection (Gumá et al. 2004; Mónica Gumá et al.

2006). Recently, NKG2C+ NK-cell expansions have been reported in several acute and

66

chronic viral infections (i.e. Hantavirus, Chikungunya, HCV and HBV), and are

systematically associated with CMV co-infection, thus suggesting that the pre-existing

CMV mediated redistribution of the NK-cell compartment is amplified (Gumá et al. 2004;

Mónica Gumá et al. 2006; Jost & Altfeld 2013; Mela & Goodier 2007; Muntasell et al.

2013; Brunetta et al. 2010; Béziat et al. 2012; Petitdemange et al. 2011). It remains to

be elucidated whether prior exposure to CMV primes NKG2C+ NK cells to expand more

rapidly in response to other viruses, or whether different viral infections can trigger the

expansion of these NKG2C+ NK cells (Jost & Altfeld 2013). From this standpoint, it is

possible that CMV coinfection becomes a potentially important confounding variable in

clinical studies focused on the cellular NKR distribution.

CD56bright NK cells are known for production of various cytokines and may be important

in early immune responses and in the shaping of the adaptive response (IFN-γ) as well

as playing the role of regulatory NK cells (IL-10) (Akuffo et al. 1999). Another important

and developing concept is the observation of increases or reductions in the percentages

of CD56bright NK cells in various diseases (Poli et al. 2009). It can be questioned why

these cells are expanded in several clinical settings. What are the potential mechanisms

leading to the expansion? One hypothesis may be that CD56dim NK cells have a high

turnover under these conditions and have to be replaced, and consequently their

precursor cells (CD56bright) are released in high numbers from the bone marrow and/or

the lymph nodes (Poli et al. 2009; Caligiuri 2008). On the other hand, CD56bright NK cells

and their cytokine production might be important on their own in certain diseases and

they would therefore selectively expand (Poli et al. 2009). Are these expansions a

consequence of or a predisposing factor of the disease? Are they beneficial or

deleterious for the host? The same questions of course also arise regarding the

reductions or the absence of CD56bright NK cells. Steady progress in this field can be

expected, and it is hoped that new discoveries will provide insight into the true

relevance of the CD56bright NK cell population in human well-being and disease.

Three recent papers have finally shown, quite convincingly, that CD56bright NK cells are

very likely precursor cells of the CD56dim subset (Strowig et al. 2008; Romagnani et al.

2007; Ouyang et al. 2007). Indeed, CD56dim NK cells display shorter telomeres than

67

CD56bright NK cells from peripheral blood and lymph nodes. This implies that the latter

are less mature than the former (Strowig et al. 2008; Romagnani et al. 2007; Ouyang et

al. 2007).

In healthy donors, CD56bright cells contribute to not more than 10% of all peripheral

blood NK cells (Poli et al. 2009; Cooper et al. 2001). However, during re-formation of

the immune system after hematopoietic stem cell transplantation, expansions above this

low proportion have been described where the first lymphocytes to appear in blood are

CD56bright NK cells. This population also escalates in patients who are treated daily with

a low dose of IL-2 (Chan et al. 2007). Interestingly, there are collective numbers of

studies describing expansions of CD56bright NK cells in different diseases, and the list of

diseases will likely be added to in the future. Zimmer et al. showed that in two cases of

transporter associated with antigen processing (TAP) deficiency CD56bright NK cells

represent all peripheral blood NK cells (Carson & Caligiuri 1996; Poli et al. 2009).

A converse situation is described by Saraste et al. who treated a group of 11 multiple

sclerosis patients with IFN-β and detected an expansion of CD56bright NK cells with a

simultaneous decrease of CD56dim cells after 12 months of treatment (14.7 ± 2.5% of all

NK cells were CD56bright) (Poli et al. 2009; Zimmer et al. 2007). Kubo et al. likewise

noticed an intense expansion of CD56bright NK cells during the convalescent phase in a

case report of a human herpes virus type 6-associated acute necrotizing

encephalopathy in a young child. Here, the authors speculate, without any

demonstration, that the CD56bright NK cells produce high levels of inflammatory

cytokines (as found in the serum of the patient) and that a high percentage of these

cells are a risk factor for the development of encephalitis during infection with human

herpes virus type 6 (Poli et al. 2009; Kubo et al. 2006). In a cohort of female patients

chronically infected with hepatitis C virus (HCV) compared with HCV resolvers and

normal uninfected controls, total NK cell percentages among lymphocytes were reduced

in the first group whereas the proportion of CD56bright cells among total NK cells was

increased. These cells produced more IFN-γ than CD56bright NK cells than the other two

groups, and overall NK cell cytotoxicity was not impaired (Poli et al. 2009; Golden-

Mason et al. 2008).

68

Interestingly, several recent observations show that in some diseases, the percentage

of CD56bright NK cells is reduced. In patients with coronary heart disease, overall NK cell

numbers are diminished as is the cytotoxic activity, and in addition, there is a tendency

towards lower percentages of CD56bright NK cells in the patients compared with normal

controls. The rationale of this study was the fact that infections are considered to be a

risk factor for coronary heart disease and that NK cells participate in immune responses

against viruses and bacteria (Poli et al. 2009; Hak et al. 2007). Similarly, studies in a

small number of patients with allergic rhinitis and/or asthma, Scordamaglia et al. found a

significantly decreased percentage of CD56bright NK cells compared with non-allergic

individuals. This reduced percentage has a consequence of weak IFN-γ production and

diminished interactions with DC, so that in allergic diseases, NK cells might be

incapable to satisfactorily shape adaptive immunity in the T helper type 1 direction (Poli

et al. 2009; Scordamaglia et al. 2008). All these data will have to be established by

larger series but they provide thought-provoking indications.

Our study used live viral stimulation whereas other studies used peptides and viral

lysates. The rationale behind using live virus in our experiments is to reproduce as

faithfully as possible the proteins that the immune system would encounter - for

instance the interaction with protein complexes that are found in the envelope which

could potentially be disrupted when making a lysate. Of course that may also allow for

the infection of susceptible PBMC types (e.g. monocytes, dendritic cells) and therefore

for immune evasion mechanisms to affect antigen presentation to T and NK cells.

In summary, our results suggest that pre-transplant CD56bright and CD56dim NK cell

responses play an important role in controlling CMV replication and may be a more

important predictor than T-cell responses for CMV in the early post-transplant period

when patients receive no universal prophylaxis. Assessment of specific NK cell

phenotypes should be further evaluated for prediction of CMV reactivation and the

importance of NKG2C+ phenotype as a potential CMV-specific subset of NK cells could

be further evaluated as a biomarker.

69

Chapter 6

FUTURE DIRECTIONS

70

6.1 Future Directions

Factors contributing to CMV replication and disease besides the innate and adaptive

immune response include the potency of immunosuppression given after

transplantation. Although immunosuppressive drugs suppress T-cell proliferation and

activation, the extent to which they affect NK cells is not well studied. Our labs have

been involved in assessing NK cell phenotypes and has been studying their phenotypic

change when treated with immunosuppressive drugs and stimulated with live virus.

In our pilot study, based on ten CMV seropositive healthy volunteers, PBMCs were

treated with immunosuppressive drugs including MMF, tacrolimus, and sirolimus. They

were also stimulated with live CMV Towne strain virus with a MOI of 0.03.

Preliminary results suggested that specific NK cell phenotypes show dose-dependent

response upon treatment with various drug regimens and live virus stimulation. MMF

and Tacrolimus showed dose-dependent decrease of CD56dim% cell frequency

[p<0.001] while Sirolimus was able to maintain CD56dim% cell frequency. Sirolimus has

been associated with a lower incidence of CMV infection in transplant recipients (Ozaki

et al.).

71

Figure 6-1. CD56dim, CD16pos Showing Dose-Dependent Immunosuppressive Drug

Response. PBMCs when stimulated with live virus and treated with varying doses of

immunosuppressive drugs showed changes in the % frequency of CD56dim and CD16pos

cells. US: unstimulated, VS: Viral Stimulated, D1: Dose 1 (For MMF it was 100ng/ml,

For Tacrolimus and Sirolimus it was 5ng/ml), D2: Dose 2 (For MMF it was 1000ng/ml,

For Tacrolimus and Sirolimus it was 10ng/ml), D3: Dose 3 (For MMF it was 10000ng/ml,

For Tacrolimus and Sirolimus it was 20ng/ml).

Preliminary results also suggested that NKG2C expression showed a dose-dependent

response upon treatment with various drug regimens and live virus stimulation.

%NKG2C cell frequency increases initially (from 30.5% to 68.0%) when treated with

MMF and stimulated with CMV, but decreases when the dose is 10000ng/ml [p<0.001].

There was no difference observed on Tacrolimus with increasing dose. However,

%NKG2C cell frequency decreases with an increasing dose of sirolimus and CMV

72

stimulation (from 44.5% to 28.0%) [p<0.001]. Our future research will focus on how

alterations in immunosuppression may further effect NK cell function and the control of

CMV.

Figure 6-2. CD56dim, CD16pos, NKG2Cpos Showing Dose-Dependent

Immunosuppressive Drug Response. PBMCs when stimulated with live virus and

treated with varying doses of immunosuppressive drugs showed changes in the %

frequency of NKG2C on CD56dim and CD16pos NK cells. US: unstimulated, VS: Viral

Stimulated, D1: Dose 1 (For MMF it was 100ng/ml, For Tacrolimus and Sirolimus it was

5ng/ml), D2: Dose 2 (For MMF it was 1000ng/ml, For Tacrolimus and Sirolimus it was

10ng/ml), D3: Dose 3 (For MMF it was 10000ng/ml, For Tacrolimus and Sirolimus it was

20ng/ml).

73

Anti-viral prophylaxis can sometimes be beneficial, however, it can cause severe side

effects such as injury to the bone marrow (ganciclovir, valganciclovir, and cidofovir) and

severe injury to the kidneys (cidofovir and foscarnet) (see table 5-1). Furthermore, all

four drugs target the same enzyme (DNA polymerase) required by CMV-infected cells

for further viral replication. Strains of CMV that are becoming resistant to ganciclovir,

foscarnet, and cidofovir are emerging and further research needs to be conducted to

develop new therapies especially for the management of cytomegalovirus (CMV)

disease in high-risk patients.

74

Table 6-1. Drugs licensed for prophylaxis, pre-emptive therapy and treatment of

CMV infection. Recent patents on anti-infective drug discovery by BENTHAM.

Reproduced with permission of BENTHAM in the format Thesis/Dissertation via

Copyright Clearance Center. (Steininger 2007).

One promising drug in development by Merck & Co. Inc is letermovir (formerly called

AIC246) or MK-8828. The drug shows promising results in terms of good oral

bioavailability, its lack of toxicity, and the apparent absence of drug-drug interactions.

Letermovir has a novel mechanism of action, applying its antiviral effect by interfering

with the viral pUL56 gene product and in the progression disrupting the viral terminase

complex. This agent exhibits substantial potential as a substitute to more toxic antivirals

75

in patients at high risk for CMV disease, principally in the transplantation setting

(Verghese & Schleiss 2013).

Another emerging anti-CMV therapy is an analogue of cidofovir (CDV) known as

brincidofovir (formerly called CMX001). The mechanism of action is that it remains intact

in plasma and transport drug directly to the target cell which leads to an enhanced

cellular uptake and high intracellular levels of cidofovir diphosphate which competitively

obstructs the integration of deoxycytidine triphosphate into viral DNA by viral DNA

polymerase and this disrupts further chain elongation (Quenelle et al. 2010). Unlike its

parent compound cidofovir, this drug can be absorbed when taken orally. Importantly,

levels of brincidofovir do not build up in the kidney and impair renal function.

Brincidofovir was initially developed as a protective drug against the likelihood of

biological warfare with smallpox virus. However, this drug has potent activity in

laboratory experiments against a range of DNA viruses, including common herpes

viruses (HSV-1 and HSV-2) (Quenelle et al. 2010). In a well-designed phase II clinical

trial, brincidofovir was capable of decreasing the risk of developing CMV-related

disease among participants who received hematopoietic stem cell transplants (Marty et

al. 2013). As this drug accrues inside CMV-infected cells resulting in extended anti-CMV

activity, brincidofovir needs to be taken only twice weekly. Brincidofovir is being

developed by Chimerix, Inc. and is currently undergoing phase III clinical trials.

The prospect of NK cells as an immunotherapy has been publicized in numerous clinical

trials, but barriers to their wider efficacy are due to both their tolerance to self-major

histocompatibility complex molecules and their susceptibility to immunosuppressive

elements that are present at the sites of many tumors. These elements include

inhibitory cytokines, myeloid-derived suppresser cells, and Tregs. Although the range of

immunosuppressive elements within tumors is overwhelming, most converge on and

inhibit signaling pathways that are also targets of activating ligands so that tipping the

balance in favor of NK-cell activation may require the elimination of only one or a few

inhibitory components (McGrath 2014). Clinical trials like these can be incorporated for

viral treatments like CMV.

76

Studies have shown that activated NK-cells can bypass T-regulatory cell modulation.

McGrath et al. showed the importance of studying endogenous IL-15 production. This

homeostatic cytokine accumulates after lymphodepletion and becomes available for the

expansion of adoptively transferred lymphocytes, including NK cells. Critically, it does

not promote Treg expansion and can render effector lymphocytes resistant to Tregs

(McGrath 2014). Therefore, further exploring the arena of IL-15 and CMV development

would be of great potential.

Enhancing the effect of NK cell immunotherapy by selectively blocking or activating NK

receptors is a booming area of research. Blocking CD94/NKG2A or inhibitory KIRs of

NK cells might reduce the NK inhibitory signal, leading to enhanced NK activity,

whereas upregulating the expression of activating receptors, such as NKG2D on NK

cells, or their ligands (ULBPs and MICA/B) on tumor cells, could increase the NK

activating signal (Yoon et al. 2015). Thus, manipulating NK-cells for better CMV defense

is also an avenue worth exploring.

77

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