© CO

PYRI

GHT U

PM

UNIVERSITI PUTRA MALAYSIA

HEPATITIS B CORE PARTICLE AS A DISPLAY PLATFORM FOR FOREIGN EPITOPES

YAP WEI BOON

FBSB 2011 18

© CO

PYRI

GHT U

PM

HEPATITIS B CORE PARTICLE AS A DISPLAY PLATFORM FOR FOREIGN EPITOPES

By

YAP WEI BOON

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement for the Degree of Doctor of Philosophy

December 2011

© CO

PYRI

GHT U

PM

ii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Doctor of Philosophy

HEPATITIS B CORE PARTICLE AS A DISPLAY PLATFORM FOR FOREIGN EPITOPES

By

YAP WEI BOON

December 2011

Chairman: Professor Tan Wen Siang, PhD

Institute: Faculty of Biotechnology and Biomolecular Sciences

Hepatitis B core (HBc) particle is a useful display platform for foreign epitopes due to

its high immunogenicity and stability. HBc small and large particles are made up of 180

and 240 subunits of hepatitis B core antigen (HBcAg) with triangulation numbers, T=3

and T=4, respectively. The foreign epitopes are presented preferentially at the amino

(N), carboxy (C) terminus or the immunodominant region of HBcAg without disturbing

the formation of HBc particles. The effects of N-terminally inserted peptides with

various lengths (14, 17, 22 and 25 amino acid residues) were first investigated in this

study. A polyhistidine tag was inserted at the N-terminus of HBcAg with or without

extraneous peptide linkers. The His-tagged HBc particles were purified with an IMAC-

based column. The yield improved with an increase in the length of N-terminal

extensions. His-�-L-HBcAg carrying 20 extraneous amino acids at the N-terminus of

© CO

PYRI

GHT U

PM

iii

HBcAg, was bound by anti-His monoclonal antibody and showed the highest colour

intensity in the ELISA. The antigenicity of HBc particles was barely affected by the N-

terminal insertion and they were reactive against an anti-HBc monoclonal antibody and

human anti-HBc antisera. In addition, they formed virus-like particles with an average

diameter of about 30 nm when observed under a transmission electron microscope

(TEM).

In order to authenticate the application of His-tagged HBc VLPs as an alternative

diagnostic reagent in HBV infections, His-�-L-HBcAg was purified in substantial

amount with immobilized metal affinity-expanded bed adsorption chromatography

(IMA-EBAC). IMA-EBAC allows a single-step purification of His-tagged HBc

particles directly from the unclarified Escherichia coli lysate. The duration needed in

the protein purification process using IMA-EBAC is shorter (from two to three hours)

than sucrose density gradient ultracentrifugation which requires two to three days. The

yield of purified His-tagged HBcAg was shown to be 100-fold higher than that obtained

with sucrose density gradient ultracentrifugation. An optimal adsorption of His-tagged

HBc VLPs to the Streamline Chelating adsorbent was achieved at pH 8.0. The majority

of the host cell proteins were eliminated by using wash buffers A (pH 8.0) and B (pH

6.0) supplemented with 10% (v/v) glycerol and 50 mM imidazole, respectively.

Approximately 56% of the His-tagged HBc particles were recovered from the bacterial

homogenate at pH 7.0 by using 500 mM imidazole and 1.5 M NaCl. The purified His-

tagged HBc particles remained antigenic and intact throughout the entire protein

purification and they also formed HBc particles (30 nm) as verified by TEM. This

© CO

PYRI

GHT U

PM

iv

implies that the purified His-tagged HBc VLPs can be a potential reagent in the

diagnosis of HBV infections.

The C-terminal region of the nucleocapsid (N) protein of Nipah virus, namely NP401-532,

is vulnerable to bacterial proteases. Since HBc particle is highly stable and antigenic,

NP401-532 was displayed at the N-terminus (NP401-532HBc) and the immunodominant loop

(HBc79-80NP401-532) of HBc protein. NP401-532HBc and HBc79-80NP401-532 showed

improved stability compared to that of NP401-532 when subjected to proteinase K

treatment. In responses to the anti-NiV antisera, the antigenicity of the N-terminally

inserted NP401-532 was enhanced whereas NP401-532 presented at the immunodominant

loop was only detectable in its denatured form. The N-terminus of HBc protein is

therefore preferable to its immunodominant loop with regard to the enhanced stability

of the C-terminal domain of NiV N protein.

© CO

PYRI

GHT U

PM

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PARTIKEL TERAS VIRUS HEPATITIS B SEBAGAI PLATFORM PEMAPARAN UNTUK EPITOP ASING

Oleh

YAP WEI BOON

Disember 2011

Pengerusi: Profesor Tan Wen Siang, PhD

Fakulti: Bioteknologi dan Sains Biomolekul

Partikel teras hepatitis B (HBc) merupakan sebuah platform pemaparan yang amat

berguna kepada epitop asing kerana partikel sedemikian berkebolehan tinggi untuk

merangsang penghasilan antibodi dalam perumahnya. Partikel teras HBV yang kecil

dan besar diperbuat daripada 180 dan 240 salinan protein teras (HBcAg), dengan

nombor triangulasi T = 3 dan T = 4. Kebanyakan epitop asing telah dipaparkan pada

bahagian amino (N), karboksi (C) atau imunodominan HBcAg tanpa memusnahkan

kebolehannya untuk membentuk partikel HBc. Pengaruh beberapa peptida dengan

kepanjangan yang berlainan (14, 17, 22 dan 25 residu asid amino) telah dikaji dalam

bahagian pertama kajian ini. Tag polihistidin telah dipamerkan tanpa atau bersama

dengan sambungan peptida di bahagian amino HBcAg. Partikel HBc yang bertagkan

polihistidin telah ditulenkan terus berlandaskan IMAC. Hasil penulenan partikel HBc

didapati bertambah dengan kepanjangan peptida yang disambungkan di bahagian

© CO

PYRI

GHT U

PM

vi

aminonya. His-�-LHBcAg yang mempunyai 20 asid amino berlebihan di bahagian

amino HBcAg dikesan oleh antibodi monoklonal anti-His dan menunjukkan intensiti

yang paling tinngi dalam ELISA. Keantigenan partikel HBc turut tidak terganggu oleh

paparan peptida ketika diuji dengan antibodi monoklonal anti-HBc dan sampel serum

pesakit hepatitis B. HBcAg yang ditagkan dengan polihistidin juga membentuk partikel-

partikel teras yang seiras dengan virus sebenar (VLPs) dengan diameter kira-kira 30 nm

semasa diperhatikan di bawah mikroskop elektron (TEM).

Dalam usaha untuk mengesahkan penggunaan VLPs HBc sebagai reagen diagnostik

yang berkesan, His-�-L-HBcAg telah ditulenkan dalam jumlah yang besar dengan

mengaplikasikan sistem kromatografi penjerapan molekul melalui logam statik pada

lapisan penjerap yang berkembang (IMA-EBAC). IMA-EBAC membolehkan

penulenan partikel HBc secara langsung daripada pecahan sel Escherichia coli tanpa

proses penjernihan. Jangka masa yang diperlukan dalam proses penulenan protein IMA-

EBAC adalah lebih singkat (dari dua hingga tiga jam) berbanding dengan ultra-

pengemparan ketumpatan sukrosa yang memerlukan dua hingga tiga hari. Hasil

penulenan HBcAg yang bertagkan His ditunjukkan 100 kali ganda lebih tinggi daripada

yang diperoleh melalui ultra-pengemparan ketumpatan sukrosa. Kadar penjerapan

optimum partikel HBc yang bertagkan His pada penjerap Streamline Chelating tercapai

pada pH 8.0. Hampir kesemua sisa protein bakteria E. coli telah diluruhkan dengan

menggunakan larutan penimbal A (pH 8.0) dan B (pH 6.0) yang mengandungi 10% (v/v)

gliserol serta 50 mM imidazol masing-masing. Kira-kira 56% partikel teras HBV telah

ditulenkan pada pH 7.0 dengan menggunakan 500 mM imidazol dan 1.5 M NaCl.

© CO

PYRI

GHT U

PM

vii

Partikel HBc bertag-His yang ditulenkan masih antigenik dan utuh selepas proses

penulenan IMA-EBAC. Hal ini dapat dibuktikan melalui data yang diperoleh dalam

ELISA dan pembentukan partikel HBc (30 nm) seperti yang ditunjukkan melalui TEM.

Oleh sebab demikian, partikel teras HBV yang ditagkan dengan polihistidin

sememangnya berpotensi tinggi untuk dijadikan reagen dalam diagnosis jangkitan HBV.

Bahagian C nukleokapsid (N) Nipah virus (NiV), yakni NP401-532, amatlah sensitif

terhadap tindakan protease bakteria. Justeru, NP401-532 telah dipamerkan di bahagian

amino (NP401-532HBc) dan imunodominan (HBc79-80NP401-532) HBcAg kerana kestabilan

dan keantigenannya yang tinggi. Pemaparan NP401-532 di atas partikel teras HBV telah

berjaya memperbaiki kestabilannya dalam tindak balasnya dengan proteinase K. Selain

itu, keantigenan NP401-532 terhadap sampel serum penyakit NiV telah banyak

dipertingkatkan melalui pemaparannnya di bahagian amino HBcAg. Walau

bagaimanapun, NP401-532 yang dipaparkan di bahagian imunodominan HBcAg hanya

dapat dikesan apabila dinyahaslikan. Sesungguhnya, NP401-532 lebih sesuai dipaparkan di

bahagian amino HBcAg berbanding dengan bahagian immunodominannya demi

meningkatkan kestabilan dan keantigenannya.

© CO

PYRI

GHT U

PM

viii

ACKNOWLEDGEMENTS

Many people have endeavoured together with me to make me whom I am today.

Although a brief acknowledgement may not suffice to remark the toils and hard work

that we went through, I duly want to address my deepest gratitude to them. First and

foremost, the supervisory committee members: Prof. Dr. Tan Wen Siang, Assoc. Prof.

Dr. Tey Beng Ti and Assoc. Prof. Dr. Noorjahan Banu Mohamed Alitheen. Their

willingness to leave every page of manuscripts had refined my work. Their seamless

advice and encouragement have strengthened my confidence and enthusiasm towards

research.

Warmest thanks to my family. I may always count on their shoulders whenever I am

tired and down. My parents, although barely understand my work, are always being

supportive and understanding. Nonetheless, members from Laboratories 134 and 143,

Faculty Biotechnology and Biomolecular Sciences, and Laboratory of Biochemical

Engineering, Faculty Engineering, UPM. Special thanks to seniors who were my guides

when I was in dilemmas and doubts: Michelle, Swee Tin, Kie Hie and Kah Fai.

Last but not least, Graduate Research Fellowship (GRF), Universiti Putra Malaysia that

has supported my three-years-study. Boundless thank you to all of them. Without them,

I am only a plain ‘Yap Wei Boon’.

© CO

PYRI

GHT U

PM

ix

I certify that a Thesis Examination Committee has met on 19 December 2011 to conduct the final examination of Yap Wei Boon on his Doctor of Philosophy thesis entitled “Hepatitis B Core Particle as a Display Platform For Foreign Epitopes” in accordance with the Universities and University College 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 Doctor of Philosophy. Members of the Examination Committee were as follows: Wan Zuhainis Saad, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairperson) Chong Pei Pei, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Cheah Yoke Kqueen, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) David Bhella, PhD Honorary Lecturer Centre for Virus Research University of Glasgow UK (External Examiner) _____________________________ SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 25 January 2012

© CO

PYRI

GHT U

PM

x

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee were as follows:

Tan Wen Siang, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)

Tey Beng Ti, PhD Associate Professor Faculty of Engineering Universiti Putra Malaysia (Member)

Noorjahan Banu Mohamed Alitheen, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)

________________________________ BUJANG BIN KIM HUAT, PhD

Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

© CO

PYRI

GHT U

PM

xi

DECLARATION

I declare that the thesis is my original work except for the quotations and citations, which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

__________________ YAP WEI BOON Date: 19 December 2011

© CO

PYRI

GHT U

PM

xii

TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xvii LIST OF FIGURES xviii LIST OF ABBREVIATIONS xxi CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 2.1 Hepatitis B virus (HBV) 7 2.1.1 Genome organization 9 2.1.2 Virion structures 10

2.1.3 Viral replication 15 2.1.4 Immunopathogenesis 18 2.2 Hepatitis B core antigen (HBcAg) 21 2.2.1 HBcAg as a carrier platform 26 2.3 Nipah virus (NiV) 30 2.3.1 Virion structures and their roles in viral 33 replication 2.3.2 Nucleocapsid (N) protein of NiV 37 2.4 Cell disruption 41 2.5 Protein purification 43 2.5.1 Conventional protein purification processes 43

2.5.2 Expanded bed adsorption chromatography 45 (EBAC) 2.5.2.1 Principles of EBAC 46 2.5.2.2 Immobilized metal affinity-expanded 52 bed adsorption chromatography (IMA-EBAC)

2.5.2.3 Applications of EBAC 55

© CO

PYRI

GHT U

PM

xiii

3 GENERAL MATERIALS AND METHODS 3.1 Construction of recombinant plasmids 58 3.1.1 Plasmid extraction 58 3.1.2 Polymerase chain reaction (PCR) 59 3.1.3 Agarose gel electrophoresis 60 3.1.4 DNA quantification 61 3.1.5 Restriction enzyme (RE) digestion 62 3.1.6 Gel extraction of RE digested pRSETB and 62 PCR products 3.1.7 Cloning of insert into pRSETB 63 3.2 Transformation of E. coli strain BL21 (DE3) with 63 recombinant plasmids 3.2.1 Preparation of E. coli strain BL21 (DE3) 63 competent cells 3.2.2 Introduction of recombinant plasmids into 64 E. coli strain BL21 (DE3) competent cells 3.3 Screening of positive transformants 64 3.3.1 Cycle sequencing of the inserted genes in 64 pRSETB 3.4 Production of recombinant protein 65 3.4.1 Cultivation of E. coli harbouring the 65 recombinant plasmids 3.4.2 Disruption of E. coli using ultrasonication 66 3.4.3 Protein purification with HisTrap HP column 66 3.4.4 Protein purification with sucrose density 67 gradient ultracentrifugation 3.5 Protein analysis 68 3.5.1 Sodium dodecyl-sulphate polyacrylamide gel 68 electrophoresis (SDS-PAGE) 3.5.2 Western blotting 69 3.5.3 Enzyme-linked immunosorbent assay (ELISA) 70 3.5.4 Transmission electron microscopy (TEM) 71 3.6 Protein quantification 72 3.6.4 The Bradford assay 72

© CO

PYRI

GHT U

PM

xiv

4 N-TERMINALLY HIS-TAGGED HEPATITIS B CORE ANTIGENS: CONSTRUCTION, EXPRESSION, PURIFICATION AND ANTIGENICITY

4.1 Introduction 73 4.2 Materials and Methods 76 4.2.1 Construction of recombinant plasmids encoding 76 the N-terminally His-tagged HBcAgs 4.2.2 Protein solubility analysis 79 4.2.3 Sodium dodecyl-sulphate polyacrylamide gel 79 electrophoresis (SDS-PAGE) 4.2.4 Western blotting 80 4.2.5 Purification of His-tagged HBcAgs 80 4.2.6 Enzyme-linked immunosorbent assay (ELISA) 81 4.2.7 Transmission electron microscopy (TEM) 82 4.3 Results 82 4.3.1 Construction of N-terminally His-tagged 82 HBcAgs 4.3.2 Expression of the recombinant protein in E. coli 83 strain BL21 (DE3) 4.3.3 Solubility analysis of His-tagged HBcAgs at 85 different temperatures 4.3.4 Purification of His-tagged HBcAgs with 86 immobilized metal affinity chromatography 4.3.5 Protrusion of His-tag on the surface of core 89 particles 4.3.6 Antigenicity of His-tagged HBcAgs 91 4.4 Discussion 93 4.5 Conclusion 97

5 PURIFICATION OF HIS-TAGGED HEPATITIS B CORE ANTIGEN FROM UNCLARIFIED BACTERIAL HOMOGENATE USING IMMOBILIZED METAL AFFINITY-EXPANDED BED ADSORPTION CHROMATOGRAPHY

5.1 Introduction 98 5.2 Materials and Methods 100 5.2.1 Materials 100 5.2.2 Cultivation of cells expressing His-tagged 100 HBcAg 5.2.3 Cell disruption 101 5.2.3.1 Ultrasonication 101

5.2.3.2 Bead milling 101 5.2.4 Measurement of viscosity 101 5.2.5 Optimization of adsorption and elution of 102 His-tagged HBcAg

© CO

PYRI

GHT U

PM

xv

5.2.5.1 Binding pH 102 5.2.5.2 Elution conditions 102 5.2.6 Equilibrium adsorption isotherm 103 5.2.7 Operation of EBA column 104 5.2.7.1 Bed expansion characteristics 104

5.2.7.2 Dynamic binding capacity 104 5.2.7.3 Direct purification of His-tagged 105 HBcAg from unclarified feedstock 5.2.8 Ultracentrifugation 106 5.2.9 Protein analysis 106 5.2.10 Enzyme-linked immunosorbent assay (ELISA) 107 5.2.11 Calculations 107 5.3 Results 108 5.3.1 Homogenization 108 5.3.2 Optimization of the binding pH 109 5.3.3 Elution conditions 110 5.3.4 Equilibrium adsorption isotherm 113 5.3.5 Bed expansion characteristics 114 5.3.6 Dynamic binding capacity 116 5.3.7 Direct purification of His-tagged HBcAg from 117 unclarified feedstock 5.3.8 Antigenicity of His-tagged HBcAg 120 5.4 Discussion 123 5.5 Conclusion 129

6 DISPLAY OF THE ANTIGENIC REGION OF NIPAH VIRUS NUCLEOCAPSID PROTEIN ON HEPATITIS B VIRUS CAPSID

6.1 Introduction 130 6.2 Materials and Methods 131 6.2.1 Construction of the recombinant plasmids 131 6.2.2 Expression of NP401-532, NP401-532HBc, 134 HBc79-80NP401-532 in E. coli

6.2.3 Purification of NP401-532, NP401-532HBc and 134 HBc79-80NP401-532 with a HisTrap HP column 6.2.4 Sucrose density gradient ultracentrifugation 134

6.2.5 Proteolytic assay 135 6.2.6 SDS-PAGE and Western blotting 135 6.2.7 Enzyme-linked immunosorbent assay (ELISA) 135 6.2.8 Transmission electron microscopy (TEM) 136 6.3 Results 136

6.3.1 Construction, expression and purification 136 of NP401-532, NP401-532HBc and HBc79-80NP401-532 6.3.2 The stability of NP401-532, NP401-532HBc, 139 HBc79-80NP401-532 6.3.3 Sucrose density gradient sedimentation and 140

© CO

PYRI

GHT U

PM

xvi

transmission electron microscopy (TEM) 6.3.4 Antigenicity of NP401-532, NP401-532HBc, 142 HBc79-80NP401-532

6.4 Discussion 145 6.5 Conclusion 148

7 � GENERAL CONCLUSION AND FUTURE RECOMMENDATIONS 7.1 General conclusion 149

7.2 Future recommendations 152

REFERENCES 155 APPENDICES 170 BIODATA OF STUDENT 178 LIST OF PUBLICATIONS 179

1.pdfHEPATITIS B CORE PARTICLE AS A DISPLAY PLATFORMFOR FOREIGN EPITOPESABSTRACTTABLE OF CONTENTSCHAPTERREFERENCES