UNIVERSITI PUTRA MALAYSIA

TRANSFECTION OF HEPG2 CELLS WITH BACTERIOPHAGES T7 AND M13 DISPLAYING REGIONS OF

HEPATITIS B SURFACE ANTIGEN

TANG KIE HIE

FBSB 2008 17

TRANSFECTION OF HEPG2 CELLS WITH BACTERIOPHAGES

T7 AND M13 DISPLAYING REGIONS OF

HEPATITIS B SURFACE ANTIGEN

By

TANG KIE HIE

Thesis Submitted to the School of Graduate Studies, Universiti Putra

Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

December 2008

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

TRANSFECTION OF HEPG2 CELLS WITH BACTERIOPHAGES T7

AND M13 DISPLAYING REGIONS OF HEPATITIS B SURFACE ANTIGENS

By

TANG KIE HIE

Dec 2008

Chairman: Associate Professor Dr Tan Wen Siang, PhD

Faculty: Biotechnology and Biomolecular Sciences

Hepatitis B is a major public health problem worldwide that may lead to chronic

liver diseases, cirrhosis and hepatocellular carcinoma. It is caused by hepatitis B

virus (HBV) which is responsible for 500,000 deaths annually. The preS domain

of HBV particularly amino acids 21-47, is believed to be involved in the initial

step of HBV infection through attachment to hepatocyte. In order to study virus-

cell interactions, both T7 and M13 bacteriophages have been genetically

modified to display regions of Hepatitis B surface antigens. In the present study,

the recombinant T7 and M13 phages were used to transfect human

hepatocarcinoma cell line HepG2. Interestingly, T7-PreS1/2 carrying the second

half of preS1 (residues 60-108), but not T7-PreS1/1 which carried the first half of

preS1 (residues 1-47), was demonstrated to be the most effective in transfecting

HepG2 cells in a dose- and time-dependant manner. The DNA of phage could be

recovered from cell lysate and confirmed by PCR whereas infectious form of

internalized phage was measured by plaque forming assay. Under fluorescence

microscope, internalized phage exhibited the appearance as fluorescent dots. On

the other hand, M13-PreS carrying the preS region showed low efficiency of

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transfection, thus suggesting that the interaction is also dependant on the valency

of targeting ligand. Surprisingly, unmodified phages (MOI ≈ 1.25 × 105) were

also capable of transfecting HepG2 at low efficiency and were thought to be

taken up by nonspecific uptake. Dynamic light scattering analysis has been

carried out to determine the thermostability of recombinant phages. The display

of HBV surface antigen did not affect the stability of phages since no significant

differences in thermostability were observed between control and recombinant

phages. The ability and stability of recombinant phages to transfect HepG2 cells

demonstrate the potential of phage displayed system as gene therapy for liver

cancer.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

TRANSFEKSI SEL HEPG2 DENGAN BAKTERIOFAJ T7 DAN M13 YANG MEMPAMERKAN KAWASAN ANTIGEN PERMUKAAN

HEPATITIS B

Oleh

TANG KIE HIE

Dis 2008

Pengerusi: Profesor Madya Dr Tan Wen Siang, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Hepatitis B merupakan masalah utama kesihatan awam sedunia yang

menyebabkan penyakit hati kronik, sirosis dan barah hati. Penyakit ini

disebabkan oleh virus hepatitis B (HBV) dan menjadi punca kematian sebanyak

500,000 setiap tahun. Kawasan liputan preS HBV terutamanya asid amino 21-47,

dipercayai terlibat dalam jangkitan awal HBV melalui pelekatan pada hepatosit.

Untuk mempelajari interaksi virus-sel, genetik bakteriofaj T7 and M13 telah

diubah-suai untuk mempamerkan kawasan-kawasan antigen permukaan Hepatitis

B. Dalam kajian ini, faj rekombinan T7 and M13 digunakan untuk menjangkiti

sel barah manusia HepG2. Yang menariknya, T7-PreS1/2 yang membawa

separuh kedua preS1 (residu 60-108), dan bukannya T7-PreS1/1 yang membawa

separuh pertama preS1 (residu 1-47), didapati paling berkesan dalam transfeksi

sel-sel HepG2 bergantung kepada dos faj dan masa. DNA faj boleh didapati di

dalam sel dan disahkan dengan PCR manakala faj berjangkit dalaman dapat

dikira melalui ujian pembentukan plak. Faj dalaman dapat dilihat sebagai bintik-

bintik pendarfluor dengan mikroskop pendarfluor. Sementara itu, M13-PreS

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yang membawa kawasan preS menunjukkan transfeksi dengan effisiensi yang

rendah, mencadangkan bahawa perhubungan tersebut juga bergantung kepada

valensi ligan penujuan. Yang menakjubkannya, faj (MOI ≈ 1.25 × 105) yang

tidak diubah-suai juga berupaya menjangkiti HepG2 dengan effisiensi transfeksi

yang rendah dan dijangka melalui mekanisma tidak spesifik. Analisis serakan

cahaya dinamik telah dijalankan untuk menentukan kestabilan haba faj

rekombinan. Pameran antigen permukaan HBV didapati tidak menjejaskan

kestabilan faj memandangkan tiada perbezaan ketara diperhatikan antara

kestabilan haba faj kawalan dan rekombinan. Oleh itu, kebolehan dan kestabilan

faj dalam menjangkiti sel HepG2 telah menunjukkan potensi sistem pameran faj

sebagai terapi gen untuk barah hati.

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ACKNOWLEDGEMENTS

I wish to thank a lot of wonderful people whose valuable support and

encouragement made possible this study and thesis completion. First, I wish to

express my deepest appreciation to my supervisor, Associate Professor Dr. Tan

Wen Siang for being very supportive and patient throughout the period of this

study. His guidance and ideas have definitely inspired me in doing science and

greatly motivated myself in overcoming problems. I am also very grateful to my

second supervisor, Professor Datin Paduka Dr. Khatijah Mohd. Yusoff, for her

helpful discussions and suggestions. Her invaluable advices and constructive

criticisms really enlightened me.

Many thanks go to the staff members from the Department of Microbiology and

Institute of Biosciences for helping in my experiments. I wish also to

acknowledge members of Laboratories 134 and 143, especially Ong Swee Tin,

Michelle Ng, Ho Chin Woi, Yap Wei Boon, Jason Lee, Loh Lip Nam, Senthil

Kumar, Chew Few Ne, Chong Fui Chin, Rattana Wongchupan, Tang Kah Fai,

Eddie Chia, Nicole Ngai and Erin Lim. Many thanks for their sharing of

scientific knowledge and inspirations. I will not forget the wonderful and joyful

time we spent in the laboratories.

A personal appreciation goes to my housemates: Cheng Ai, Hock Ngiik, Peng

Choo and Ley Fong. Thank you very much for accompanying and encouraging

me during the toughest time. I will always remember the care and love that you

all gave throughout the stay.

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Last but not least, I would like to extend my thanks to my parents and brothers

for their patience, sacrifices, understanding and never-ending love that brought

me to the completion of this study.

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I certify that a Thesis Examination Committee has met on 1 December 2008 to conduct the final examination of Tang Kie Hie on her thesis entitled “Transfection of HepG2 Cells with Bacteriophages T7 and M13 Displaying Regions of Hepatitis B Surface Antigens” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of the Examination Committee were as follows: Wan Zuhainis Saad, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Norazizah Shafee, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Syahida Ahmad, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences (Internal Examiner) Wan Kiew Lian, PhD Associate Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (External Examiner) ________________________ BUJANG KIM HUAT, PhD Professor and Deputy Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 19 March 2009

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: TAN WEN SIANG, PhD Associate Professor Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Supervisor) KHATIJAH YUSOFF, PhD Professor Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) ________________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 19 March 2009

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DECLARATION

I hereby declare that this thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degrees at UPM or other institutions. _______________ TANG KIE HIE Date: 16 January 2009

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TABLE OF CONTENTS Page ABSTRACT ii ABSTRAK iv ACKNOWLEDGEMENTS vi APPROVAL viii DECLARATION x TABLE OF CONTENTS xi LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF ABBREVIATIONS xv CHAPTER

1. INTRODUCTION Introduction 1

2. LITERATURE REVIEW 2.1 Hepatitis B Virus (HBV) 3 2.1.1 HBV Morphology 3 2.1.2 Genomic Organisation and Viral Transcripts 3 2.1.3 Hepadnavirus Replication 6 2.1.4 Surface Antigens 9 2.1.5 Transmission 12 2.1.6 Treatment of HBV 13 2.1.7 Prevention of HBV 14 2.1.8 Hepatocellular Carcinoma 15 2.2 Bacteriophage T7 16 2.2.1 Morphology and Structure 16 2.2.2 Life Cycle of T7 phage 17 2.3 Bacteriophage M13 19 2.3.1 Mophology and Structure 19 2.3.2 Life Cycle of Filamentous Phages 20 2.4 Phage Display and Applications 23 2.5 Biosafety of Phages for Applications in Human 25

3. MATERIALS AND METHODS

3.1 Materials 27 3.1.1 Media, Buffers and Solutions 27 3.1.2 Cell Lines 29 3.1.3 Bacteria Strains and Plasmids 29 3.1.4 Bacteriophages 29 3.1.5 Primers 30 3.2 Preparation of Bacteriophages 31 3.2.1 Bacteriophage T7 31 3.2.2 Bacteriophage M13 34

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3.2.3 Amplification of DNA 36 3.2.4 Agarose Gel Electrophoresis 37 3.2.5 Light Scattering Analysis 38 3.3 Maintenance of Cell Line 39 3.3.1 Thawing the Cells 39 3.3.2 Subculturing the Cells 39 3.3.3 Storing the Cells 40 3.3.4 Counting the Cells 40 3.4 Transfection of Cell Line with Bacteriophages 41 3.4.1 Seeding 41 3.4.2 Transfection 41 3.4.3 Harvesting the Cells 41 3.4.4 DNA Extraction 42 3.5 Immunofluorescence 43

4. RESULTS

Identification of Recombinant Phages and Phagemid 44 Verification of Recombinant Phage by PCR 45

Confirmation of Recombinant Phages by DNA 46 Sequencing

Analysis of Transfected HepG2 DNA by PCR 48 Phage Internalization 53

4.3.1 Plaque Assay 53 4.3.2 Immunofluorescence 55

Dynamic Light Scattering Measurement 59

5. DISCUSSION 5.1 Display of PreS1 and S Regions on T7 Phage 64 5.2 Display of PreS Region on M13 Phage 66 5.3 Transfection of HepG2 with Phages 67 5.4 Fluorescence Microscopy 68 5.5 Phage Internalization via Receptor-Mediated Endocytosis 69 5.6 Dynamic Light Scattering Analysis of Phages 74

6. CONCLUSION 76 REFERRENCES 80 APPENDICES 93 A1 T7Select 415-1b Vector Map 93 A2 pCANTAB 5E Vector Map 94 BIODATA OF THE STUDENT 96

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LIST OF TABLES

Table Page

2.1 Applications of phage display 24

3.1 Media 27

3.2 Buffers and solutions 28

3.3 Bacteria strains 29

3.4 Phagemid 29

3.5 Bacteriophages 30

3.6 Oligonucleotides for amplification of phage DNA 30

3.7 Ratio of CsCl:TE in centrifuge tube 32

3.8 PCR conditions for primers 37

4.1 DLS data for purified T7 phages measured at various temperatures 60

4.2 DLS data for M13 phages measured at various temperatures 62

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LIST OF FIGURES

Figure Page

2.1 Transcriptional and translational map of HBV 5

2.2 The hepadnaviral replication cycle 8

2.3 Schematic representation of L protein 9

2.4 A T7 phage particle 16

2.5 Replication cycles of lytic phages 18

2.6 A filamentous bacteriophage M13 particle 19

2.7 M13 life cycle 21

4.1 Diagrams of vector map and regions amplified by primers for confirmation of recombinant phages and phagemid

44

4.2 Verification of recombinant phages by PCR 46

4.3 Chromatogram showing the sequencing results of T7-S (a), T7-PreS1/1 (b), T7-PreS1/2 (c) and pCANTAB5E-PreS (d)

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4.4 PCR analysis of extrachromosomal DNA from T7-transfected (24 h) HepG2 cells

49

4.5 PCR analysis of extrachromosomal DNA from T7-transfected (72 h) HepG2 cells

50

4.6 PCR analysis of extrachromosomal DNA from M13-transfected HepG2 cells

52

4.7 In vitro internalization assay of transfected HepG2 cells 54

4.8 Detection of internalized T7 phages in HepG2 cells by fluorescent microscopy

56

4.9 Detection of internalized M13 phages in HepG2 cells by fluorescent microscopy

58

5.1 A schematic diagram of the wild type T7 and T7Select 415-1b vector

65

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LIST OF ABBREVIATIONS

α alfa

β beta

ε epsilon

λ lambda

°C degree centigrade

μ micron

AFB1 aflatoxin B1

AUG start codon

aa amino acid

BSA bovine serum albumin

bp base pair

CsCl cesium chloride

cccDNA covalently closed circular DNA

dsDNA double-stranded deoxyribonucleic

DHBV duck HBV

DLS dynamic light scattering

DNA deoxyribonucleic acid

DNase deoxyribonuclease

DR direct repeat

Dт translational diffusion coefficient

dNTP deoxynucleoside triphosphate

EDTA ethylenediamine tetraacetic acid

ELISA enzyme-linked immunosorbent assay

xv

EN enhancer

FBA fluorescent-bacteriophage assay

FBS fetal bovine serum

GFP green fluorescence protein

GSHV ground squirrel hepatitis virus

g gram

h hour

HBIG Hepatitis B Immune Globulin

HBV hepatitis B virus

HBcAg hepatitis B core antigen

HBeAg hepatitis B e antigen

HBsAg hepatitis B surface antigen

HCC hepatocellular carcinoma

HCl hydrochloride

HepG2 human hepatocellular liver carcinoma cell line

IgE Immunoglobulin E

kDa kilodalton

kb kilobase

L-HBsAg large surface antigen

M Molar

MHC major histocompatibility complex

M-HBsAg medium surface antigen

mW miliwatt

mRNA messenger ribonucleic acid

NTE sodium-tris-EDTA

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NaCl sodium chloride

NaOH sodium hydroxide

N-terminal amino-terminal

nm nanometer

OD optical density

ORF open reading frame

PBS phosphate-buffered saline

PC precore

PCR polymerase chain reaction

PEG polyethylene glycol

PreS1 N-terminal region of L-HBsAg comprising 108 or 119

amino acid

PreS2 region of M and L-HBsAg comprising 55 amino acid

pH Puissance hydrogene

pHSA polymerized human serum albumin

pfu plaque forming unit

pgRNA pregenomic ribonucleic acid

RF replicative form

RME receptor-mediated endocytosis

RNA ribonucleic acid

RNase ribonuclease

Rh hydrodynamic radius

rcDNA relax-circular deoxyribonucleic acid

SCCA1 squamous cell carcinoma antigen 1

SOC small outer capsid

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xviii

S-HBsAg small surface antigen

ssDNA single-stranded deoxyribonucleic acid

TLM translocation motif

TAE Tris-acetate-EDTA

TE Tris-EDTA

WHV woodchucks hepatitis virus

xg centrifugal force

V volt

v/v volume/volume

vol volume

w/v weight/volume

CHAPTER 1

INTRODUCTION

Hepatitis B virus (HBV) is the prototype of Hepadnaviridae, a family

which includes related hepatitis viruses which infect woodchucks (WHV),

ground squirrels (GSHV) and Pekin ducks (DHBV) (Summers and Mason,

1982). Worldwide, two billion people have been infected with HBV of which

400 million are chronically infected. Chronic infection of HBV causes

progressive liver diseases and hepatocellular carcinoma (HCC) that responsible

for around 500,000 deaths each year (Perz et al., 2006).

To date, alpha interferon therapy can be used for treatment of chronic

HBV infection. Nevertheless, it is ineffective in more than 50% of carriers

(Hoofnagle and Di Bisceglie, 1997) and it is associated with several side effects

(Torre and Tambini, 1996). Other therapy using nucleoside analogs and

inhibitors of virus replication have led to development of drug-resistant mutants

due to high rate of viral turn over and error-prone polymerase (Locarnini and

Omata, 2006).

A vaccine derived from inactivated hepatitis B surface antigen (HBsAg)

purified from human plasma was first licensed in the United States, followed by

yeast- and mammalian-derived hepatitis B vaccines which are now widely used

(Shouval et al., 1994; Mahoney, 1999). The ultimate goal of hepatitis B

vaccination is to control HBV-related chronic liver diseases and hepatocellular

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carcinoma. However, HBV with molecular variants in the ‘a’ determinant of

HBsAg make it unsusceptible for recognition and neutralisation by anti-HBsAg

antibodies.

Gene therapy has been recognized as one of the most promising

approaches in curing cancers (Verma and Sonia, 1997). The technology requires

a gene delivery vehicle capable of targeting a therapeutic gene to the selected cell

type with high efficiency without stimulating significant immune, inflammatory

and cytotoxic responses (Michael and Curiel, 1994). Hence, the use of phage

display systems for mammalian gene transfer is of great interest due to their

stability, direct linkage between genotype and phenotype, inexpensive mass

production or purification and excellent safety profile in mammalian cells

(Larocca and Baird, 2001). By displaying targeting sequences on the phage coat,

bacteriophage vectors which exhibit no natural tropism for mammalian cells may

confer mammalian cell-type specific tropism through receptor-mediated

endocytosis (RME) (Varga et al., 2000), thus offer an advantage in targeting

treatment of cancer.

In the present study, T7 and M13 phages displaying regions of HBsAg

which are believed to be involved in infectivity and attachment site for

hepatocytes, were used for targeting human hepatocellular carcinoma cell line

(HepG2). Therefore, this study was aimed to examine the ability of genetically-

targeted T7 and M13 phages in delivering their genes and internalise to HepG2

cells. The thermostability of T7 and M13 particles was also tested with dynamic

light scattering (DLS).

CHAPTER 2

LITERATURE REVIEW

2.1 Hepatitis B Virus (HBV)

2.1.1 HBV Morphology

Human HBV is an enveloped DNA virus grouped under the family

Hepadnaviridae and genus Orthohepadnavirus (Ganem and Varmus, 1987).

Internal to the envelope is the viral nucleocapsid that comprises of core proteins,

a partially double stranded DNA genome of 3.2 kb and a polymerase protein (P)

(Ganem, 1991). HBV has three forms of surface antigens (HBsAg): large L-

HBsAg, middle M-HBsAg and small S-HBsAg that are embedded in the

envelope of the virion. During HBV infection of an individual, HBsAg particles

containing only HBsAg are produced in great abundance. These noninfectious

particles exist as spherical and filamentous forms of 22 nm in diameter, which is

smaller than the 42 nm infectious Dane particles or HBV virion (Dane et al.,

1970).

2.1.2 Genomic Organisation and Viral Transcripts

A hepadnavirus virion contains two single-stranded linear DNA

molecules of different sizes which paired in an overlapping fashion with breaks

at both strands, producing a circular partially double stranded molecule. The

3

negative (−) strand is about 3200 bases long, which serves as the template for all

viral mRNA transcription, whereas the positive (+) strand varies in length, from

1700 to 2800 bases long. A protein, P is covalently attached to 5’ end of the (−)

strand (Gerlich and Robinson, 1980), while the (+) strand ends in a 19-base-long

RNA molecule. At the 5’ end of both strands, there is a pair of directly repeated

11-base-long sequences, designated as DR1 and DR2 that play crucial role in the

initiation of viral DNA synthesis (Lien et al., 1986; Will et al., 1987).

As shown in Figure 2.1, there are four open reading frames (ORF)

present in the DNA, which are C, P, S and X (Wei and Tiollais, 1999). ORF C

contains two in-frame start codons, AUGs, which encode the structural protein of

the HBV nucleocapsid (Pasek et al., 1979), and a hydrophobic polypeptide

bearing hepatitis B e antigen (HBeAg). Instead of incorporating into HBV

virions, HBeAg is secreted from cells and accumulating in the serum. The ORF P

sequence overlaps the end of ORF C region and continues nearly to the end of

the DNA molecule. It encodes the viral polymerase which is utilised in the viral

genomic replication as well as the protein primer molecule that is attached to the

5’ end of (−) strand DNA (Bosch et al., 1988). Within the ORF P is the ORF S

which occurs with three in-frame initiation codons, resulting in the synthesis of

three related HBsAg. These proteins of 39 (L-HBsAg), 33 (M-HBsAg) and 24

kDa (S-HBsAg) in size are found on the surface of the virion (Valenzuela et al.,

1979; Wong et al., 1985).

Although there are four ORFs in HBV genome, only three mRNAs are

synthesized. In addition, the product of ORF X is not well understood. It appears

4

to encode possibly three trans active regulatory proteins that facilitate the

transcription from cellular and viral promoter sequences (Seeger and Mason,

2000).

Figure 2.1: Transcriptional and translational map of HBV. The inner circles represent the rcDNA. It comprises of a complete (−) strand DNA and an incomplete (+) strand DNA with the reverse transcriptase and a capped RNA oligomer attached to its 5’ end respectively. The position of direct repeat sequences, DR1 and DR2, and the enhancers, EN1 and EN2 are indicated. Three major viral RNAs, the core (C) or pgRNA, preS (L) mRNA an S mRNA are depicted by the outer circle, are controlled by several internal promoters at different sites but are all terminated at a common 3’ end, indicated by the letters A. In between the outer and inner circles are the four protein-coding regions which include precore (PC) and core genes, polymerase gene and X gene. The envelope genes, L, M and S overlap with the polymerase ORF (adapted from Seeger and Mason, 2000).

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2.1.3 Hepadnavirus Replication

A hepadnavirus particle contains predominantly relaxed-circular DNA

molecules (rcDNA) with a complete minus (−) strand and a partially synthesized

plus (+) strand. A small amount of linear DNAs as a result of in situ priming is

also found. The (−) strand is covalently attached to a protein, the viral reverse

transcriptase at 5’ end while the (+) strand ends in a 19-base-long RNA (Voyles,

2002).

The life cycle of HBV (Figure 2.2) begins by attaching to the host cell

membrane via its envelope proteins and the virus release its genome into the

nucleoplasm (Lu et al., 1996; Kann et al., 2007). During initiation of infection,

the (+) strand is elongated until both strands are of essentially equal length. Both

the protein and RNA at the 5’ end are removed from the strands and a covalently

closed circular DNA (cccDNA) is formed when the viral genome reaches the

nucleus. The formation of cccDNA indicates successful initiation of infection as

the cccDNA serves as a template for mRNA transcription (Tuttleman et al.,

1986).

Replication begins with the synthesis of the pregenomic RNA (pgRNA)

molecule which is larger than its genome (Figure 2.2), and has terminal repeats

since same set of bases is copied twice. The pgRNA would serve as a template

for synthesis of core (C) protein (HBcAg) and preC, viral envelope (S), viral

polymerase (P) and X proteins (Summers and Mason, 1982). The P protein binds

to the epsilon (ε) stem-loop structure at the 5’ end of its own mRNA template

and initiates encapsidation into nucleocapsids that are assembled in the cytosol

6