imran riaz malik - higher education...

Studies on the use of HBV epitope Chimeras

By

Imran Riaz Malik

School of Biotechnology,

National Institute for Biotechnology and Genetic

Engineering (NIBGE), Faisalabad &

Quaid-i-Azam University, Islamabad, Pakistan

2014

Studies on the use of HBV epitope Chimeras

A dissertation submitted for partial fulfillment of the degree of

DOCTOR OF PHILOSOPHY IN

BIOTECHNOLOGY

By

Imran Riaz Malik

School of Biotechnology,

National Institute for Biotechnology and Genetic

Engineering (NIBGE), Faisalabad &

Quaid-i-Azam University, Islamabad, Pakistan

2014

Declaration

I hereby declare that the work presented in the following thesis is my own

effort, except where otherwise acknowledged, and that the thesis is my own

composition. No part of this thesis has been previously presented for any

other degree.

Imran Riaz Malik

Dedicated to,

My Affectionate

Parents

Mr. Muhammad Riaz Malik (Late)

&

Mrs. Razia Begum

Research is what I’m doing when I don’t know what I’m doing.

Wernher Von Braun (1912-1970)

“In the Name of Allah, the Most Gracious, the Most Compassionate!”

Table of Contents Acknowledgements………………………………………………………………. I

List of Figures…………………………………………………………………….. III

List of Tables……………………………………………………………………… V

List of Abbreviations………………………………………………….................... VI

Abstract…………………………………………………………………………… IX

1. Introduction and Review of Literature........…………………………... 1

1.1. Introduction to viral hepatitis………………………………………………… 1

1.2. Hepatitis B Virus…………………………………………………………….. 1

1.2.1. History……………………………………………………………... 2

1.2.2. Epidemiology……………………………………………................ 3

1.2.3. Genome……………………………………………………………. 4

1.2.4. Infection…………………………………………………………… 5

1.2.5. Treatment………………………………………………………….. 6

1.2.6. Viral Life cycle ………………………………………………….... 8

1.2.7. Model Systems……………………………………………………. 10

1.2.8. Immune responses…………………………………………………. 10

1.2. 8.1. Cellular Immune Response……………………………… 12

1.2.8.2. Humoral Immune Response……………………………... 15

1.2.9. Vaccine…………………………………………………............... 16

1.2.9.1. Plasma derived vaccines……………………………….... 16

1.2. 9.2. Recombinant vaccine……………………………………. 17

1.2.9.3. Vaccines under development……………………………. 19

1.2.9.3.1. DNA vaccines…………………………………. 19

1.2.9.3.2. Therapeutic Vaccines………………………….. 19

1.3. HBV epitopes ……………………………………………………………….. 20

1.3.1. Pre-S HBV epitopes……………………………………………….. 21

1.3.2. HBV core epitopes………………………………………………... 21

1.4. Studies on HBV antigens for New Vaccine Development………………… 23

1.5. Aims of study ……………………………………………………………… 26

2. Materials and Methods………………………………………………... 27

2.1. Chemicals and Reagents……………………………………………………. 27

2.2. Bacterial Strains, Plasmids and Constructed Vectors……………………… 27

2.3. Bacterial Growth Media/Conditions……………………………………….. 29

2.4. Experimental Animals……………………………………………………… 29

2.5. Collection of Samples……………………………………………………… 29

2.6. Primers / Oligonucleotides…………………………………………………. 29

2.7. Genetic Techniques………………………………………………………… 31

2.7.1. HBV DNA isolation……………………………………………... 31

2.7.2. Plasmid DNA isolation…………………………………………. 32

2.7.3. Polymerase Chain Reaction (PCR)……………………………… 32

2.8. Purification, Digestion and Cloning of Amplified DNA fragments……… 32

2.9. Construction of Recombinant Plasmids and Chimeric Plasmids…………. 33

2.9.1. pIJM.HBs-Ag............................................................................... 33

2.9.2. pIJM.HBc-Ag............................................................................... 34

2.9.3. Construction of Chimeric Expression Plasmids / Vectors………... 34

2.9.3.1. pIJMcsc-1……………………………………………... 35

2.9.3.2. pIJMcsc-2……………………………………………... 36

2.9.3.3. pIJMcsc-3……………………………………………… 37

2.9.3.4. pIJMcsc-4……………………………………………… 38

2.9.3.5. pIJMcsc-5……………………………………………… 39

2.10. Competent cells and transformation experiments………………………… 40

2.11. DNA Sequence analysis………………………………………………….. 40

2.12. Expression of Chimeric genes…………………………………………… 40

2.13. Gel electrophoresis……………………………………………………….. 41

2.14. Biochemical techniques………………………………………………….. 42

2.14.1. Purification of His-6 chimeric proteins by talon affinity

chromatography……………………………………………………….. 42

2.14.2. Determination of protein………………………………………. 43

2.14.3. Protein analysis by SDS-PAGE……………………………….. 43

2.14.4. Western blotting……………………………………………….. 45

2.14.4.1. Visualization of histidine tagged proteins for

anticore antibody…………………………………….. 45

2.14.2 Detection of histidine tagged proteins for

antiPreS antibody…………………………………… 45

2.15. Immunization……………………………………………………………... 46

2.15.1. Enzyme Linked Immunosorbent Assay………………………… 46

2.15.2. ELISpot Assay………………………………………………….. 46

2.15.2.1. Detection of pIJMcscs-5 specific CTLs………………. 47

3. Results…………………………………………………………………. 48

3. 1. Analysis of PCR products…………………………………………………. 48

3.2. Cloning of PCR products…………………………………………………… 49

3.3. DNA Sequence Analysis of cloned genes and translation to Amino acids…. 52

3.3.1. Hepatitis B surface gene, 681bp…………………………………... 52

3.3.2. Hepatitis B Core gene, 549bp........................................................... 52

3.3.3. Cloned Chimeric genes……………………………………………. 52

3.4. Expression and Purification of Chimeric Proteins…………………………… 58

3.4.1. SDS-PAGE analysis……………………………………………….. 58

3.4.2. Further characterization of chimeric csc-5 harboring PreS1………. 61

3.5. Determination of immunogenicity…………………………………………… 62

3.5.1. Immune response induced by subcutaneous immunization………… 62

3.5.1.1. csc-5 protein……………………………………………… 63

3.6. ELISPOT Assay……………………………………………………………… 64

3.6.1. Immunogenicity of Chimeric HBcAg expressing PreS1…………… 64

3.7. Other Chimeric Proteins evaluated for Immunogenicity…………………….. 65

3.7.1. csc-1 protein………………………………………………………... 65

3.7.2. csc-2 protein………………………………………………………... 67

3.7.3. csc-4 protein………………………………………………………... 67

4. Discussion……………………………………………………………… 69 5. References……………………………………………………………… 76 Appendix…………………………………………………………………………

I

Acknowledgements

All praises and thanks to Allah Almighty, the Lord of all creations, the beneficent,

the most merciful and the most compassionate, who created man and taught him manners

and is the source of entire knowledge and wisdom endowed to mankind. All respect and

reverence for the Holy Prophet (PBUH) whose teachings are complete guidance for

humanity.

This thesis is the results of years of work and learning that would not have been

possible, and definitely not as much fun, without all the people who helped, supported

and encouraged me in different ways, especially, I would like to thank the following

persons: First of all, I would like to thanks Higher Education Commission (HEC)

Pakistan for financial support to conduct the present research.

No words can be enough to express my indebtedness to my supervisor,

Dr. Javed Anver Qureshi, for his brilliant versatility, devoted guidance, being so very

supportive and dependable, unflinching support and immense patience at all stages of this

work.

I feel myself highly obliged to my Co-Supervisor, Dr. Moazur Rahman, for his

learned guidance useful training and dynamic supervision during whole PhD research

work. I am also thankful to Mr. Naveed Altaf Malik for his learned guidance and

sympathetic attitude through the progress of my thesis.

Special thanks are extended to Dr. Shahid M. Baig, Head, Health Biotechnology

Division (HBD), who is extremely supportive of all research activities going on in this

department. I express my sincere gratitude to Director NIBGE, for providing excellent

education and research facilities to accomplish this work.

I am pleased to acknowledge Dr. Matti Sallberg, Department of clinical

Microbiology, Karolinska Institutet, University Hospital Stockholm, Sweden for his

extensive cooperation and guidance and encouragement during my stay at his lab. I am

highly indebted to him and lab fellows there especially Lars Frelin, Gustaf Ahlen,

Anthony Chen, and Anett Brass who shared their knowledge and techniques open

heartedly.

II

My research group, Gene cloning; thank you all! Naeem Riaz, Fozia Nasreen,

Abdul Ghaffar, M. Salahuddin, Nida, Aqsa, saima , Rahim ullah, Majid and my sweet

friends Ismail, Rahat, Tariq, Abdur Rahman, and Abdul Jabbar for paving the way and

always helping me.

My acknowledgment will be incomplete if I would not express my sincere

feelings to my valuable and loving brothers, Waqar Hussain Tahir, Muhammad Sohail

Shahzad and sweet Sisters for always being very supportive.

I would like to give my heartfelt appreciation to my wife, Uzma Imran who has

accompanied me with her love, unlimited patience, understanding and encouragement.

Without her support, I would never be able to accomplish this work.

My sweet daughter, Wania Noor and my nephews and nieces, Tauseef, Talha,

Danial, Fasih, Sabih, Areeba, Eshaal,Tooba, Fahad and Nimra, the greatest gift and

meaning of life, for lighting up the cloudiest day and always making me smile.

I would like to express my gratitude to my parents Mr and Mrs Muhammad Riaz

Malik for they are the foundation rock upon which I strive to learn and whose love I can

never repay. I would like to give my heartfelt appreciation to my parents, who brought

me up with their love and encouraged me to pursue advanced degrees.

Imran Riaz Malik

III

List of Figures

Figure 1.1. Geographical Distribution of hepatitis B virus………………… 3

Figure 1.2. Hepatitis B Genome…………………………………………… 4

Figure 1.3. Life cycle of HBV replication………………………………… 9

Figure 1.4. Immune response to HBV infection………………………….. 11

Figure 1.5. Flow chart of CD4+ differentiation into Th1 and Th2 cells…... 13

Figure 1.6. CD8+ cells interacting with infected cell………………………. 14

Figure 1.7. The presence of antibodies over the course of infection………. 15

Figure 2.1. Cloning of pIJM.HBs-Ag............................................................ 33

Figure 2.2. Cloning of pIJM.HBc-Ag……………………………………… 34

Figure 2.3.Cloning of amplified DNA products into pET28a prokaryotic

expression plasmid and construction of pIMJcsc-1…………….. 35

Figure 2.4. Cloning of amplified DNA products into pET28a prokaryotic

expression plasmid and construction of pIMJcsc-2……………. 36


expression plasmid and construction of pIMJcsc-3……………. 37


expression plasmid and construction of pIMJcsc-4……………... 38


expression plasmid and construction of pIMJcsc-5…………….. 39

Figure 2.8. Outline of purification procedure for chimeric proteins………… 42

Figure 3.1. Amplified PCR products analysed on 1% agarose gel and viewed

under UV light…………………………………………………… 48

Figure 3.2. Analysis of PCR products and generated restriction digest

DNA fragments along with 50 base pair ladder “M”……………… 49

Figure 3.3. Analysis of PCR products and generated restriction digest DNA

fragments along with 1Kb ladder “M”………………………………………… 50

Figure 3.4. Analysis of PCR products and generated restriction digest DNA

fragments along with 1Kb and 100 base pair ladder “M”……………. 51

Figure 3.5. Nucleotide and amino acid sequence of csc-1gene………………… 53

IV

Figure 3.6. Nucleotide sequence and amino acid sequence of csc-2 gene……… 54

Figure 3.7. Nucleotide and amino acid sequence of csc-3 gene………………… 55



Figure 3.10. 10% polyacrylamide gel electrophoresis of proteins in the

presence of sodium dodecyl sulfate………………………………. 59

Figure 3.11. Purified expressed proteins by talon affinity chromatography……. 60

Figure 3.12. Western blot analysis of the csc-5 for antiHBc…………………… 61

Figure 3.13. Western blot analysis of the csc-5 for antiPreS……………………. 62

Figure 3.14. Mean antiHBcAg and HBsAg (1-42) IgG antibody titer after

one (w2, w4) or two (w6)s.c immunizations with 10ug of the

chimeric protein(csc-5) in C57BL/6J……………………………… 63

Figure 3.15. Evaluation of the immunogenicity of two doses (10ug and 15ug) of

Chimeric protein (csc-5) subcutaneously immunized C57BL/6J

mice by enzyme linked immunosorbent Assay (ELISpot) assay…… 65

Figure 3.16. Antigenicity of csc-1 protein synthesized and purified …………….. 66

Figure 3.17. Antigenicity of csc-2 protein synthesized and purified……………... 67

Figure 3.18. Antigenicity of csc-4 protein synthesized and purified……………... 68

V

List of Tables

Table 1.1. MHC restriction, co-receptor and function of each T cell type…… 12

Table 1.2. Vaccines produced by manufacturers and country………………… 18

Table 2.1. Bacterial strains, standard cloning vectors, recombinant and

Chimeric construct…………………………………………………. 28

Table 2.2. Oligonucleotides used for amplification of target sequences of HBV

Genome…………………………………………………………….. 30

Table 2.3. Oligonucleotides used for amplification of constructed chimeric

DNA fragments from cloned Chimeric Plasmids………………… 31

Table 2.4. Composition of separating and stacking SDS-PAGE gels………….. 44

Table 2.5. Composition of sample loading (4X) and running buffer (10X)……. 44

VI

List of Abbreviations Anti-HBc Antibodies to HBcAg

Anti-HBs Antibody to HBsAg

bp Base pair

CTL Cytotoxic T lymphocyte

DNA Deoxyribonucleic acid

EDTA Ethylene diamine tetraacetic acid

ELISpot Enzyme linked immunospot Assay

ELISA Enzyme Linked Immunosorbent Assay

ExPASy Expert Protein Analysis System

g Gram

GnCl Guanidine chloride

HBcAg Hepatitis B core antigen

HBsAg Hepatitis B surface antigen

HBV Hepatitis B virus

HCC Hepatocellular carcinoma

HIV Human immunodeficiency virus

ICs Immunogenic complexes

IFNs Interferon

IPTG Isopropyl β-D-thiogalactoside

Kanr Kanamycine resistance gene

kDa Kilodaltons

LB Luria Bertani

MHC Major-histocompatibility-complex

MIR Major Immunodominant region

mRNA Messenger RNA

NLS Nuclear localization signal

NC Nitrocellulose membrane

OD Optical density

PCR Polymerase chain reaction

VII

PEG-IFNα Pegylated interferon alpha 2a

SDS-PAGE Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis

SFC IFNɣ spot forming cells

TAE Tris-acetate-EDTA buffer

TE buffer Tris EDTA buffer

TEMED Tetramethyl ethylene diamine

Th Humoral T helper

TNF Tumor necrosis factor

TNFα Tumor necrosis alpha

Tris Tris( 2-hydroxymethyl)-aminomethane

ZVS Varicella zoster virus

VIII

List of amino acids and their abbreviations

Amino acid Three letter code Single letter code Glycine Gly G

Alanine Ala A

Valine Val V

Leucine Leu L

Isoleucine Ile I

Methionine Met M

Phenylalanine Phe D

Tryptophan Trp W

Proline Pro P

Serine Ser S

Threonine Thr T

Cystein Cys C

Tyrosine Tyr Y

Asparagine Asn N

Glutamine Gln Q

Aspartic acid Asp D

Glutamic acid Glu E

Lysine Lys K

Arginine Arg R

Histidine His H

IX

Abstract

Hepatitis B virus (HBV) is a common cause of viral hepatitis with significant health

complications including cirrhosis and hepatocellular carcinoma. Major advances have

been made in the treatment of Hepatitis B recent years. However, much remains to be

accomplished because current antiviral drugs do not eradicate infection. The currently

licensed hepatitis B vaccines, consisting of recombinant hepatitis B surface antigen

(HBsAg) and alum, are highly effective and induce protective antibody titers in 95% of

vaccinated individuals after 3 immunizations before onset of HBV infection.

Present studies were aimed to develop chimeric vaccine (proteins) that may stimulate B-

and T- cell responses and generate enhance immune response to treat the HBV patients

after infection. In this regard, five HBV chimeric vaccine constructs were designed and

developed using the HBcAg gene encoding amino acids 1-78 and amino acids 80-144.

Amino acid- position 79 where deleted and replaced with preS epitopes sequences. All

five chimeric constructs (pIJMcsc-1, pIJMcsc-2, pIJMcsc-3. pIJMcsc-4 and pIJMcsc-5)

contained HBcAg as a carrier molecule for the epitopes of preS regions primed HBcAg-

specific antibodies response. These constructs (chimeric plasmids) were transformed into

E.coli to see the protein product on SDS-PAGE. The expressed proteins (csc-1, csc-2,

csc-3. csc-4 and csc-5) were purified based on affinity chromatography using His tag at

the C terminal of expressed proteins.

The purified chimeric proteins were further subjected to Enzyme linked Immunosorbent

assay (ELISA) and western blotting assays using secondary anti- rabbit AP conjugated

antibody and secondary anti-mouse AP-conjugated antibody respectively. All five

chimeric proteins showed positive antibody response. In vivo study was performed for

chimeric protein (csc-5) only. This chimeric protein (csc-5) was evaluated in C57BL/6J

mice for activation of humoral and cellular immune responses, primed both HBcAg-

specific T cells and antibodies to preS1.

X

Result obtained indicated that the csc-5 protein can induce HBV-specific antibodies and

T cells, two functions that well complement the activity of antiviral compounds. Thus,

the csc-5 protein may be a future component in therapy for chronic HBV infections

where the host is gaining a sustained control of the HBV infection and also a viable

approach to develop an effective bi-functional therapeutic vaccine as an add-on for

treatment of chronic HBV.

Introduction and Review of Literature

Chapter 1


1


1.1. Introduction to viral hepatitis

The word hepatitis is the general term that refers to any inflammation (-itis) of the liver

(hepato). Hepatitis means inflammation of liver. Viruses that primarily attack the liver are

called hepatitis viruses. Currently, six hepatotropic viruses of clinical interest have been

discovered in humans, the hepatitis A, B, C, D, E and G. These viruses cause a wide

range of hepatic pathology, from transitory to chronic infections and from subclinical to

fulminant liver disease, liver cirrhosis and hepatocellular carcinoma. (Gillcrist, 1999).

Types A, B, and C hepatic viruses are the most common. Those at risk for viral hepatitis

include workers in the health care profession, people with multiple sexual partners,

intravenous drug abusers, and hemophiliacs. Blood transfusion is a rare cause of viral

hepatitis. All hepatitis viruses can cause acute hepatitis whereas viral hepatitis types B

and C can cause chronic hepatitis. When the liver is inflamed, it does not perform these

functions well, which brings about many of the symptoms, signs, and problems

associated with hepatitis (NIH, USA 2008).

Other viruses (NIH, USA 2008) have been isolated that cause hepatic inflammation but

are not specifically hepatotropic. These include Cytomegalovirus, Epstein-Barr virus,

Human immunodeficiency virus (HIV), Herpes simplex virus, Varicella zoster virus

(ZVS).

1.2. Hepatitis B Virus

The hepatitis B virus (HBV) is a small, enveloped DNA virus belongs to the

Hepadnaviridae family under the Orthohepadnavirus genus (Grimm et al., 2011). It has

global prevalence with 350 million people chronically infected, out of which 4.5 million

in Pakistan. (Liaqat et al., 2011) Pakistan lies in an endemic region with 3-5% HBV

carrier rate in the country (Awan et al., 2010; Liaqat et al., 2011). The virus is

transmitted through blood or other bodily fluids, with the most common mode of


2

transmission being sexual transmission. The major cause for chronic infections is mother-

to-child transmission at birth. Chronic HBV infection may lead to cirrhosis and

eventually develop into hepatocellular carcinoma. (Yang et al., 2009).

1.2.1. History

Viral hepatitis B is a disease that was first described in the fifth century BC, when

Hippocrates described epidemic jaundice referring to persons infected with acute

hepatitis B virus as well as other agents capable of infecting the liver. Epidemics of

jaundice have been described throughout history and were particularly common during

various wars in the 19th and 20th centuries.

MacCallum in 1947 was the first who introduced the terms, hepatitis A and hepatitis B in

order to categorize infections and serum hepatitis. Blumberg in 1963 discovered a

previously unknown protein in the blood of an Australian aborigine while searching for

polymorphic serum proteins (Blumberg et al., 1967). This protein was designated as the

Australian (Au) antigen. It became apparent that this protein was related to type B

hepatitis B. By 1968, other investigators, notably Prince, Okochi and Murakami had

established that the Au antigen (now known as the hepatitis B surface antigen) was only

found in the serum of type B hepatitis infected patients (Prince, 1968; Okochi and

Murakami, 1968).

Virus-like particles in the serum of patients suffering from type B hepatitis B was found

in 1970 by Dane (Dane et al., 1970). These particles were designated as the hepatitis B

virus. Non-related hepatitis viruses were discovered later, but the hepatitis B virus

retained its name. The viral nature of these particles by detecting an endogenous DNA-

dependent DNA polymerase within its core were confirmed by (Kaplan et al., 1973).

Discovery of this polymerase allowed Robinson to detect and characterize the HBV

genome (Robinson and Greenman, 1974a).


3

1.2.2. Epidemiology

The distribution of hepatitis B infection varies greatly all over the world (Figure 1.1)

High prevalence rate is found in Southeast Asia, China, and Africa. Areas with low doses

of endemicity include North America, Western Europe, and Australia, due to horizontal

transmission among young adults from lifestyle and occupational exposure (Kane, 1995;

Jinlin et al., 2005).

Figure 1.1: Geographical Distribution of hepatitis B virus (Jinlin et al., 2005).

Seven genotypes (A-G) and four serotypes (adw, ayw, adr, and ayr) of hepatitis B virus

have been reported (Kao and Chen, 2002) based upon the nucleotide sequence divergence

in the HBV strapped from face protein sequence. In Europe, Genotype A and D are

predominant (Zanetti et al., 1988); genotype B and C in Asia and genotype D are more

frequent in Middle-East. In Asia, Genotype B appears to be associated with a higher

HBeAg clearance and less development of cirrhosis and hepatocellular carcinoma (Fung

and Lok, 2004).Genotype D is present worldwide and more prevalent in Mediterranean

area, near and Middle East and South Asia. In Pakistan, Genotype C and D are the most

common HBV genotypes and are associated with increased severity and less response to

interferon therapy (Liaqat et al., 2011).


4

1.2.3. Genome

Hepatitis B virus is a member of the hepadnaviridae family and a 3.2Kb small circular

DNA containing four genes (core, surface, polymerase and X) which encodes seven

proteins (Seeger et al., 2007) (Figure 1.2).

Figure 1.2: Hepatitis B Genome (Seeger et al., 2007)

The surface gene of HBV encodes Large (L), Middle (M), and Small (S) proteins. These

proteins share a common C-terminal sequence (S protein; 226 amino acid, aa) that has

four transmembrane domains. The M protein has preS2 antigen (55aa) additionally, while

the L protein has preS1 antigen (108 or 119 amino acid, aa depending on the serotype)

and preS2 antigens additionally (Neurath and Kent, 1988).

The core gene of hepatitis B virus encodes two polypeptides. The first translation product

is of a 183 amino acid long, 21kDa protein which assembles intracytoplasmically into the

viral nucleocapsids HBcAg (Galibert et al., 1979).A second product is initiated at the Pre

C AUG preceding the core start site in frame which results in a 25kDa precore/core

fusion protein (Ou et al., 1986).Viral polymerase is encoded by the P gene of about 2500

base pairs that acts as a reverse transcriptase function.


5

Hepatitis B X gene has numerous activities that are relevant to HBV associated

pathogenesis, especially hepatocarcinogenesis. However, the role of HBx in the HBV life

cycle and HCC are not yet understood (Feitelson and Lee, 2007; Murakami, 1999;

2001).The function of HBx is related to its location and level of expression (Henkler et

al., 2001; Murakami, 2001; Shin et al., 2006).

1.2.4. Infection

It has been described that HBV infection may result in subclinical or asymptomatic

infection, acute self-limited hepatitis, or fulminant hepatitis requiring liver

transplantation. Person infected with HBV may also develop acute or chronic HBV

infections (Edwards and Bouchier, 1991)

Acute infection: The hepatitis B surface antigen (HBsAg) is a reliable marker of

hepatitis B virus infection, and a negative test for the HBsAg makes hepatitis B virus

infection very unlikely but not impossible. It appears in the blood late in the incubation

period and before the prodromal phase of acute type B hepatitis; it may be present for

only a few days, disappearing even before jaundice has developed, but it usually last for

3-4 weeks and may persist for up to 3 months. It should therefore be sought as soon as

possible in acute hepatitis. Antibody to HBsAg (anti-HBs) usually appears after about 3

months and persist for many years or perhaps permanently. Anti-HBs imply either that

infection has occurred at some time or that individual has been vaccinated (Edwards and

Bouchier, 1991)

The hepatitis B core antigen (HBcAg) is not found in the blood, but antibody to it (anti-

HBc) appears early in the illness and rapidly reaches a high titre which then subsides

gradually and persists. Anti-HBc is initially of IgM type and IgG antibody appear later.

Anti-HBc (IgM) can sometimes reveal an acute hepatitis B viral infection when the

HBsAg has disappeared and before anti-HBs has developed. The hepatitis B e antigen

(HBeAg) appears only transiently at the outset of the illness and is followed by the


6

production of antibody (anti-HBe). The HBeAg reflects active replication of the virus in

the liver (Edwards and Bouchier, 1991).

Chronic infection: Chronic hepatitis B virus infection is marked by the presence of the

HBsAg and anti-HBc (IgG) in the blood. Rarely, anti-HBc (IgG) alone is the sole

evidence of chronic infection. Usually, the HBeAg or anti-HBe is also present; the

HBeAg is thought to indicate continued active replication of the virus in the liver while

anti-HBe implies that the replication is occurring at a much lower level or that the viral

DNA has become integrated into the hepatocyte DNA (Edwards and Bouchier, 1991).

1.2.5. Treatment Approved drugs are advised for the treatment of HBV that includes interferon-α, PEG –

interferon and antiviral drugs like lamivudine, adefovir, dipivoxil, entecavir and

telbivudine (Gilroy and Mukherjee, 2008). Most of the present treatment such as

nucleoside analogues and/ or interferon has limited success (Nassal, 1997; Zoulim, 2001).

Alpha Interferon (IFN-α): Alpha interferon has been used since the early 1970s

(Greenberg et al., 1976). It is believed to act by both immunomodulatory effects and

antiviral actions (Zuckerman and Thomas, 1998).Its effectiveness is approximately 33%

in achieving HBeAg seroconversion (Lee, 1997). It has been proven that about 90% of

HBeAg seroconversion is sustained and 21% of patients with HBeAg seroconversion will

lose HBsAg over the following 5 years (Krogsgaard, 1998). The major shortcoming of

IFN-α is the high frequency of side effects such as flue like symptoms, bone marrow

suppression, psychiatric symptoms and thyroid dysfunction (Lee, 1997).

Lamivudine: A nucleoside analog, Lamivudine which is a potent inhibitor of HBV-

DNA replication both in vitro and vivo (Lai et al., 1998; Doong et al., 1991; Zoulim and

Trepo, 1998; Luscombe and Locarnini, 1996; Dienstag et al., 1999a; 1999b). However,

cessation of the drug leads to rapid relapse of viral replication unless HBeAg

seroconversion occurs (Lai et al., 1998; Zoulim and Trepo, 1998; Severini et al.,

1995).The major advantage of lamivudine over interferon-alpha is the lack of side effects.


7

However, prolonged medication leads to development of mutation within the tyrosine-

methionine-aspartate- aspartate (YMMD) motif of the polymerase gene (Perrillo et al.,

1996; Markowitz et al., 1996; Bartholomeusz et al., 1997; Honkoop et al., 1997; Ling et

al., 1996; chayama et al., 1998; Tipples et al., 1996) which usually associated with rise of

ALT and HBV-DNA.

Adefovir dipivoxil: Adefovir dipivoxil is a phosphonated acyclic purine nucleoside

analog with broad spectrum antiviral activity (Heathcote et al., 1998; Naesens et al.,

1997; De Clercq, 1991; Heijtink et al., 1993; 1994).

Entecavir: Entecavir is a carbocyclic deoxyguanosine analog and is an effective

inhibitor of HBV both in vitro and vivo (Genovesi et al., 1998; Innaimo et al., 1997;

Yamanaka et al., 1999). It has a much lower 50% effective concentration than lamivudine

indicating higher potency as an anti-HBV agent and non competitive inosine

monophosphate dehydrogenase inhibitor.

Combination antiviral therapy: Frequent use of a single nucleotide analog agent in

the treatment of chronic HBV infection leads to an emergence of drug resistance HBV

mutants (Markowitz et al., 1996; Bartholomeusz et al., 1997; Heijtink et al., 1994). It has

been reported that Famiciclovir treatment can produce changes in the domain B of the

polymerase gene, whereas lamivudine produces changes in the domain B and C of the

polymerase gene ((Markowitz et al., 1996; Bartholomew et al., 1997; Heijtink et al.,

1994).

Several studies demonstrate that the treatment with lamivudine alone, or in combination

with lamivudine alone, or in combination with interlukin-12 (IL-12), result in the

restoration of the HBV-specific CD4+ and CD8+ immune response in chronic infected

individuals. However, the therapeutic effect was sustained in those patients (Boni et al.,

2001, 2003; Rigopoulou et al., 2005).


8

Current treatment strategies: Currently, pegylated interferon alpha 2a (PEG-IFNα)

or nucleos (t) ide analogues, such as adefovir, entecavir (ETV), lamivudine, telvivudine,

and tenofovir (Conjeevaram and Lok, 2003; Janssen et al., 2005; Lau et al., 2005;

Dienstag, 2008) are being used for the treatment of HBV infection. However, the efficacy

of these therapies in preventing liver cirrhosis and HCC is still limited. Treatment with

PEG-IFNα leads to a sustained antiviral response in only one third of patients, regardless

of combining the therapy with polymerase inhibitors. The treatment with nucleos (t) ide

analogues significantly suppresses HBV replication that leads to a decrease of

necroinflammtion in the liver. Furthermore, the long-term treatment is subsequently

associated with the appearance of drug resistance HBV strains that is often the cause of

the therapy failure (Raney et al., 2003; Locarnini and Mason, 2006). Therefore, the new

approaches in treating chronic hepatitis B are urgently needed. Recommended treatment

of chronic hepatitis B with interferon-α and/ or nucleo (t) analogues does not lead to a

satisfactory result. Induction of HBV-specific T cells by therapeutic vaccination or

immunotherapies may be an innovative strategy to overcome HBV persistence.

Vaccination with commercially HBV vaccines did not result in effective control of HBV

infection, suggesting that new formulations of therapeutic vaccines are needed (Kosinka

et al., 2010).

1.2.6. Viral Life cycle

In vivo, the hepatocytes are the primary replication site for HBV and can infect up to

100% of the hepatocytes (Guidotti et al., 1999: Thimme et al., 2003). The mechanism of

viral entry in to the hepatocytes is at present unknown, but the large S protein is thought

to be involved but the receptor(s) on the hepatocytes are still unknown. The replication of

HBV is unique among the DNA viruses since it involves a RT step of RNA pregenome

(Nassal, 1997). After entering probably via endocytosis (Glebe and Urban et al., 2007)

and uncoating, the open circular viral genome is transported into the nucleus (Kann et al.,

2007) via nuclear localization signal (NLS). In the nucleus DNA repair enzymes process

the viral minus and plus strands to produced the covalently closed circular (cccDNA) that

serves as the template for transcription of the viral pre-genomic and messenger RNA(


9

mRNA). Two HBV enhancers, Enh I and Enh II positively regulate transcription of the

HBV promoters together with transcription factors that bind to these promoters (Seeger

and Mason et al., 2000; Doitsh and Shaul, 2004). Within the newly produced

nucleocapsid particles, new minus strand DNA is synthesized by RT of the pregenomic

RNA. The newly formed DNA minus strand serves as a template for the plus strand.

Some of the core particles transport the developing genome back to the nucleus a process

that effectively amplifies the HBV copy number in the cell. Other core particles associate

with viral envelope proteins in the ER and are secreted out of the hepatocytes as

infectious virions to initiate new rounds of infections in susceptible as shown in Figure

1.3.(Fields et al., 1996; Chisari, 1996; Nassal, 1997; Seeger and Mason, 2000; Cann,

2001). Direct after HBV infections the replication is not very efficient, and HBV DNA

and HBV antigens cannot be detected in serum or liver until four to seven weeks post

infection (Fong et al., 1994).

Figure 1.3: Life cycle of HBV replication.


10

1.2.7. Model Systems

Human and chimpanzees share a common ancestry and are 98.8% genetically identical.

Unlike recently, the chimpanzee was the only animal besides humans to propagate

hepatitis B. (Ebersberger et al., 2002). HBV infection develops into acute hepatitis as in

humans (Bertoni et al., 1998; Will et al., 1982) without establishing persistence. Many

successful studies have been done in chimpanzees; however, there are several ethical

issues in using chimpanzees.

Small animal models are easily to work with than chimpanzees. Transgenic mouse

models for HBV allows for the study of pathological effects of one or multiple viral

proteins (Lerat et al., 2002). Chisari (1985) generated a transgenic mouse model carrying

the preS-S gene and expressing HBsAg in tissue from birth. Immunizations with human

HBsAg could not elicit an anti-HBs response showing that these are immunologically

tolerant to HBsAg. The HBsAg-Tg mouse model provided a model for the stage in HBV

infection when viral DNA has integrated into host genome (Chisari, et al., 1985).

Another alternative are the use of surrogate animal models. The woodchuck and pekin

duck are good examples of naturally occurring HBV infections. As the virus have

adapted to each host, the natural course of infection can not be expected to be exactly the

same as human hepatitis B. The woodchuck model develops HCC in almost all

chronically infected animals (Cote at el., 2000) and has revealed much in HBV viral

replication, chronic infection and HCC development. The duck model is rarely associated

with liver disease and HCC despite viral replication, and has therefore been used for

antiviral studies in vivo (Mason et al., 1994; Luscome et al, 1996).

1.2.8. Immune responses

The immune responses to HBV and their role in the pathogenesis of hepatitis B are

incompletely understood. Correlative clinical studies (Chisari and Ferrari, 1995)

indicated that in acute, self-limited hepatitis B, Strong T-cell responses to many HBV

antigens are readily demonstrable in the peripheral blood. These responses involve both


11

major-histocompatibility-complex (MHC) class II-restricted, CD4+ helper T cells and

MHC class I-restricted, CD8+ cytotoxic T lymphocytes. The antiviral cytotoxic T

lymphocyte response is directed against multiple epitopes within the HBV core,

polymerase and envelope proteins; strong helper T-cell responses to C and P proteins

have also been demonstrated in acute infection (Chisari and Ferrari, 1995; Chisari, 1996).

By contrast, in chronic carriers of HBV, (Chisari and Ferrari, 1995; Chisari, 1996) such

virus-specific T-cell responses are greatly attenuated, at least as assayed in cells from the

peripheral blood. However, antibody responses are vigorous and sustained in both

situations (although free antibodies against HBsAg are not detectable in carriers because

of the excess of circulating HBsAg). This pattern strongly suggests that T-cell responses,

especially the responses of cytotoxic T lymphocytes, play a central role in viral clearance

(Figure 1.4).

Figure 1.4: Immune response to HBV infection (Chisari and Ferrari, 1995; 1996).

It is generally acknowledged that the humoral antibody response contributes to the

clearance of circulating virus particles and the prevention of viral spread within the host

while the cellular immune response eliminates infected cells. The T cell response to the

hepatitis B virus (HBV) is vigorous, polyclonal and multispecific in acutely infected

patients who successfully clear the virus and relatively weak and narrowly focused in

chronically infected patients, suggesting that clearance of HBV is T cell dependent.


12

Persistent HBV infection is characterized by a weak adaptive immune response, thought

to be due to inefficient CD4+ T cell priming early in the infection and subsequent

development of a quantitatively and qualitatively ineffective CD8+ T cell response. Other

factors that could contribute to viral persistence are immunological tolerance, mutational

epitope inactivation, T cell receptor antagonism, incomplete down-regulation of viral

replication and infection of immunologically privileged tissues. However, these pathways

become apparent only in the setting of an ineffective immune response, which is,

therefore, the fundamental cause. Persistent infection is characterized by chronic liver

cell injury, regeneration, inflammation, widespread DNA damage and insertional

deregulation of cellular growth control genes, which, collectively, lead to cirrhosis of the

liver and hepatocellular carcinoma.

1.2. 8.1. Cellular Immune Response

Regulation of HBV occurs through non-cytolytic down regulation of viral replication,

which means that viral clearance results without killing the infected cells. Generally, this

occurs through the release of cytokines by virus-inactivated lymphomononuclear cells

(Rehermann and Nascimbeni, 2005). As a result, virus-specific CD8+ and CD4+ T cells

play key effector and regulatory roles in hepatitis B antiviral immunity. Though CD8+ T

cells are the main effector cells that cause viral clearance, CD4+ T cells are necessary to

facilitate the induction and maintenance of CD8+ T cells (Guidotti and Chisari,

2006). The Table 1.1 demonstrates the co-receptor, MHC restriction, and function of each

T cell type.

Table. 1.1: MHC restriction, co- receptor and function of each T cell type.

T cell type Co-receptor MHC restriction Function Helper T cell CD4 Class II B cell help macrophage

activation

help for CD8 T cells cytokine secretion

Cytotoxic T cell CD8 Class I Killing virus infected cells, killing tumor cells?


13

CD4+ T cell Response: Activated HBV-specific CD4+ T cells are multispecific.

However, strong helper T cell responses are found against certain peptides in both the

HBcAg and HBeAg following resolution of acute HBV infection. While HBeAg has

been shown to induce a Th2 immune response, HBcAg stimulated a Th1 response.

Additionally, CD4+ T cell responses are detected against polymerase and X antigens

(Chang and Lewin, 2007). Because the Th2 response to HBeAg was dominant to the Th1

response to the HBcAg, the HBcAg-specific T cells were depleted in vivo (Huang et al.,

2006).

It is important that both Th1 and Th2 responses are generated because they induce

different responses. The Th1 cells stimulate macrophages, which can clear virus particles,

while the Th2 cells stimulate humoral immunity, in which B cells are stimulated to

Figure 1.5: Flow chart of CD4+ differentiation into Th1 and Th2 cells (Huang et al.,

2006)

generate immunoglobulins for opsonization. However, different doses of virus seem to

generate different responses. For example, low doses of virus produce a Th1-mediated

response while high doses of virus produce a Th2-mediated response (Huang et. al.,

2006). Flow chart of CD4+ differentiation into both Th1 and Th2 cells and their functions

is presented in Figure 1.5.


14

CD8+ T cell Response: Figure 1.6. Indiçâtes a CD8+ cell interacting with an infected

cell. CD8+ T cells are the main effector cells in the viral clearance of HBV. Experiments

that depleted CD8+ T cells in chimpanzees after infection with HBV resulted in viral

persistence of HBV, indicating the importance of these cells. CD8+ T cells produce IFN-

γ, which clear HBV through destabilization of the viral capsid, degradation of viral

proteins, and post-transcriptional degradation of HBV RNA. The combined effect of

cytokines, such as IFN- γ (which can also be produced by HBV specific Th1 CD4+ T

cells), and cytolytic activity leads to the destruction of the virus without excessive liver

damage (Chang and Lewin, 2007). Figure 1.6 shows a CD8+ cell interacting with an

infected cell.

Figure.1.6: CD8+ cells interacting with infected cell (Chang and Lewin, 2007).


15

1.2.8.2. Humoral Immune Response

The humoral immune response is critical to long-term HBV clearance (Chang and Lewin,

2007). Acting in conjunction with the cellular arm of the adaptive immune system, the

humoral immune response produces antibodies that help to control HBV infection.

Although both arms of the adaptive immune response aid in clearing infection, neither

arm can completely eradicate infection by itself (Bertoletti and Gehring, 2006).

The humoral immune response produces anti-envelope antibodies, which can be detected

in human sera following HBV infection. Antibodies to each of the HBV proteins have

been detected, although every antibody is not detected in each individual with exposure

to HBV (Vranckx, 1983). In fact, the presence of different antibodies indicates different

stages of infection. For example, individuals that display anti-HBs (surface antigen)

antibodies are thought to have cleared infection, as anti-HBs antibodies are known to

Figure1.7: The presence of these antibodies over the course of infection (Vranckx,

1983).

contain the spread of infection in the host, remove and destroy viral particles, and prevent

reinfection by blocking antigen binding sites on the host’s cells. These are known as


16

neutralizing antibodies and can be found in individuals that have recovered from

infection or those that have been vaccinated (Figure 1.7). Chronic HBV carriers,

however, do not have surface proteins. Instead, antibodies to HBV core proteins (anti-

HBc) can be detected. Unlike anti-HBs antibodies, anti-HBc antibodies are persistent

throughout the course of infection and do not appear to neutralize HBV infection.

Additionally, the presence of anti-HBc antibodies do not seem to stimulate the production

of anti-HBs antibodies (Huang et al., 2006).

Furthermore, different strains of the hepatitis B virus are composed of different protein

combinations, stimulating the generation of different antibody subclasses. More

specifically, differences in IgG subclasses in response to antigens correspond to different

stages of infection and different types of immune response. For example, individuals

with chronic HBV infection exhibit higher concentrations of total IgG and IgG1 than

individuals that have recovered from infection. Moreover, chronic carriers exhibit

different ratios of different IgG subtypes when compared with vaccinated or recovered

individuals. In the case of anti-HBc, chronic carriers demonstrated an anti-HBc IgG

subclass pattern of IgG1 > IgG3 > IgG4 whereas recovered individuals displayed a

different pattern: IgG3 > IgG1 > IgG4 (Huang et. al., 2006).

1.2.9. Vaccine

Drs. Blumberg and Millman developed the first hepatitis B vaccine, which was initially a

heat-treated form of the virus. Vaccines against HBV were first made from the serum of

people who had been HBV infected and subsequently cleared the infection. Because

these individuals naturally produced anti-HBsAg IgG and then cleared the infection, they

were immune to reinfection with HBV. The serum from these individuals was taken and

purified antibody was passively administered. Since, then much improvement has been

in the development of vaccine against Hepatitis B virus and is as below:

1.2.9.1. Plasma derived vaccines

In 1981, the FDA approved a more sophisticated plasma-derived hepatitis B vaccine for

human use (see Table 1.2). This early HBV vaccine was prepared from blood donors that


17

had high HBsAg and appeared to be in good health. The 22nm HBsAg particle was

separated from infectious 42 nm HBV virions by ultracentrifugation and was subjected

to multiple steps to inactive the viral particles that included formaldehyde and heat

treatment (or “pasteurization”). The inactivation process destroys HBV and most other

known animal viruses, including human retroviruses. This vaccine was administered at

20ug HBsAg with 0.5mg alum/ dose in 1ml and thismerosal as a preservative. This is

currently licensed in the United States as Hepatavax-B (Merck, Sharp and Dohn, West

Point, PA). However, the plasma-derived HBV vaccines are no longer in common use

(Hollinger, 1991).

1.2. 9.2. Recombinant vaccines In the later 1980s, the plasma-derived vaccines fell out of favor because of concerns

regarding contamination of the blood supply with the human immunodeficiency virus

(HIV), which causes AIDS. This pushed the development of recombinant vaccines that

could be produced in eukaryotic vectors and were free of any contaminating human

viruses (Poiesz et al., 1984), utilizing recombinant DNA technology. In an infected

individual, HBsAg is present in two forms: as a protein on the surface of the 42 nm virion

(also called the Dane particle) and as a secreted 22 nm particle that is a hollow sphere of

surface antigen. Using the yeast Saccharomyces cervisiae, researchers created a vector

which contained the coding sequences for this surface antigen of HBV, HBsAg. The key

to this success was that the yeast assembled this 22 nm protein in the same way that

excess surface antigen assembles and is secreted in humans. Therefore, the artificial

surface antigen resembled the naturally occurring particle was created (Valenzuela et al.,

1982).

Shortly after the yeast vector was created, researchers from France created a similar

particle that could induce the correlate of immunity. This time, the recombinant plasmid

was transfected into Chinese hamster ovary (CHO) cells. Also, this plasmid was different

than the recombinant yeast plasmid because it included an extra piece of the genome.

While the yeast plasmid included only the S gene of HBV, the CHO clones used the S

gene plus the pre-S2 portion of the genome, creating a vector that began one start codon


18

upstream in the genome. In this case as well, the 22-nm particle containing the

immunogenic HBsAg was produced by the artificial system. The researchers who created

this vaccine tested its efficacy against the efficacy of HBsAg molecules purified from

human systems, and found the efficacies to be identical. This lent support to the idea that

these artificial particles are as immunogenic as the naturally (Da Villa, G. et. al. 1998;

Mahoney and Kane 1999). Since then, Recombinant vaccines have been produced by

many manufactures (See Table 1.2).

Table 1.2: Vaccines produced by manufacturers and country.

NUMBER TYPE OF VACCINE

BRAND NAME

MANUFACTURER COUNTRY

1 Plasma derived Hepaccine-B Chiel Jedang South Korea

2 Plasma derived Hepavax B Korea Green Cross South Korea

3 Recombinant DNA Enivac-HB Centro de Ingeniera Genetica Y Biolotecnologia

Cuba

4 Recombinant DNA Hepavax-Gene Korea Green Cross South Korea

5 Recombinant DNA Euvax B LG Chemical South Korea

6 Recombinant DNA Recombivax H-B

Merck Sharpe and Dohme

United States

7 Recombinant DNA Engerix-B SmithKline Beecham Belgium

8 Combined Hib and recombinant DNA

Comvax Merck Sharpe and Dohme

United States

9 Recombinant DNA (mammalian cell)

Genhevac B Pasteur Merieux Connaught

France

10 Combined hepatitis A and B (recombinant)

Twinrix SmithKline Beecham Belgium

11 Combined DTP and recombinant

Tritanrix-HB SmithKline Beecham Belgium

12 Recombinant DNA (mammalian cell)

Heprecombe Swiss Serum and Vaccines Institute

Swizterland


19

1.2.9.3. Vaccines under development

1.2.9.3.1. DNA vaccines DNA vaccine concept was introduced by Tang and Johnston (1992) and described

intradermal delivery of plasmid DNA generated antibody response against expressed

protein, a possibility for genetic therapy. Many researchers worked on these lines and

demonstrated independent studies expressing transgenes through intramuscular or

intradermal immunization with plasmid DNA encoding a foreign protein, and expressed

in situ that elicited a protective immunity against the pathogen (Ulmer et al., 1993; Fynan

et al., 1993; Wang et al., 1993; Lagging et al., 1995). Although different formulations,

delivery techniques and plasmids were used, DNA vaccines were established as a future

vaccine platform. Today, several DNA vaccine trials in human have shown that it may

not be as simple as originally envisioned (Calarota et al., 1998). It has been demonstrated

that plasmid DNA encoding HBV surface antigen (HBsAg) and core antigen (HBcAg)

elicits vigorous humoral and cellular response in many species (Rahman et al., 2000;

Davis et al., 1996; Kwissa et al., 2000; 2003; Riedl et al., 2002).However, the vector

used for animal testing cannot be applied in human studies.

The earliest phase I clinical trial for a DNA vaccine was hepatitis B (Liu and Ulmer,

2005).This trial demonstrate that the DNA vaccine platform is well tolerated and safe, as

no adverse events were reported( Foiretti et al., 2010).

1.2.9.3.2. Therapeutic Vaccines

Over 20 years, continuous efforts have been undertaken to develop a therapeutic vaccine

for chronic hepatitis B to enhance the virus-specific immune responses and overcome

persistent HBV infection. Numerous clinical trials of therapeutic immunization as

combination of the HBsAg protein vaccines with antiviral treatment with lamivudine did

not lead to a satisfactory improvement of the therapies (Dahmen et al., 2002; Horiike et

al., 2005; Vandepapeliere et al., 2007). The strategies designed to specifically stimulate

HBV specific T cell responses were also not successful (Heathcote et al., 1999; Mancini-

Bourgine et al., 2004; Yang, 2006).


20

The lipopeptide-based vaccine containing a single cytotoxic T lymphocyte (CTL) epitope

derived from HBV nucleocapsid was able to induce a vigorous primary HBV-specific T

cell response in naïve subjects (Vitiello et al., 1995). However, in HBV chronic carriers,

the vaccine initiated only poor CTL activity and had no effect on HBeAg/anti-HBe

seroconversion (Heathcote et al., 1999). The DNA vaccine expressing small and middle

envelope proteins proved to elicit the HBV-specific cellular immune response in chronic

HBV carriers, however, this effect was only transient(Mancini-Bourgine et al., 2004).

The therapeutic vaccine-based HBsAg complexed with human anti-HBs was proposed by

the group of Wen et al., (Wen et al., 1995). It was demonstrated that this immunogenic

complexes (ICs) administered to HBeAg-positive patients led to decrease of HBV DNA

in serum, HBeAg seroconversion (Yao et al., 2007). Currently, the IC-based vaccine is

the only one that entered phase III of clinical trials in chronic hepatitis B patients (Xu et

al., 2008). Even through the IC-based vaccine led to antiviral effect, clearance of HBV

was not observed in treated patients. Therefore, some steps have been undertaken to

combine the IC-based vaccine with nuleos (t) ide analogues treatment. The ongoing

clinical trial will show whether IC are effective as a therapeutic vaccine in chronic

hepatitis B.

1.3. HBV epitopes In general, it is widely accepted now that the HBc carrier is capable of ensuring a high

level of B cell and T cell immunogenicity to foreign epitopes (Milich et al., 1995;

Pumpens et al., 1995; Schodel et al., 1996; Ulrich et al., 1998; Pumpens and Green,

1999; Murray and Shiau, 1999). In addition to the ability of the HBc carrier moiety to

provide T cell help to inserted sequences, the HBc capsid mediates the T-cell-

independent character of the humoral response to inserted epitopes, due to the high

degree of repetitiveness of the epitopes and the proper spacing between them (Fehr et al.,

1998).


21

1.3.1. Pre-S HBV epitopes

The envelope protein-S of HBV-PreS1 protein located on the envelope of the HBV play

an important role in virus assembly, entry into the host cell, induction of host

immunogenic reaction, and pathogenesis (Poisson et al., 1997). Recombinant preS

product could also be developed as a protein vaccine that elicits B and T cell immune

responses on a broader range of MHC haplotypes (Milich et al., 1990; Pride et al., 1998;

Hui et al., 1999a; Shouval, 2003).

B cell epitopes: B cell determinants were mapped to residues 12-32, 32-53 and 94-117

of preS1 region (Milich, 1988; Neurath et al., 1989a). Five distinct antibody-binding sites

within the preS1 region of HBsAg/P43 of the adw subtype: preS1 (16–27), preS1 (32–

53), preS1 (41–53), preS1 (94–105) and preS1 (106–117), were determined in mice

(Milich et al., 1987). A putative B-cell epitope biased to the C-terminal region, preS1

(57–119), was identified in a commercially available immunoglobulin prepared from

chronically HBV infected donors (Park et al., 2000). Additionally, many epitopes of

monoclonal antibodies directed against preS1 were also mapped such as preS1 (30–35)

(Kuttner et al., 1999), preS1 (19–26), preS1 (37–45) (Maeng et al., 2000) and preS1 (37–

43) (Pizarro et al., 2001).

T cell epitopes: A dominant T cell recognition site was identified in the N-terminal

residues 21-28 of preS1 sequence (Jin et al., 1988; Ferrari et al., 1989). Many T-cell

epitopes in preS1 region also were identified, such as preS1 (21–30), preS1 (29–48)

(Ferrari et al., 1992), and preS1 (94–117) (Milich et al., 1987).

1.3.2. HBV core epitopes

HBc particles induce the strongest B cell, Th cell and CTL responses, among other HBV

polypeptides, and function as both T-cell-dependent and T cell-independent antigen (

Milich et al., 1995). Recently, the enhanced immunogenicity of HBcAg was explained by

its ability to be presented by B cells as the primary antigen-presenting cell in mice

(Milich et al., 1997a).


22

B cell epitopes: B cell epitopes were mapped within the MIR on the tips of the spike

carrying so-called c and el epitopes (Salfeld et al., 1989; Sallberg et al., 1991). Only one

group (Colucci et al., 1988) positioned the main B cell epitope to the right of aa 80, at the

region 107-118. The other HBe epitope, e2, was assigned originally to the region 130-138

(Salfeld et al., 1989). Later, human and murine anit-HBe antibodies were found to

recognize a linear region around position 130 within the amino acid stretch 126-135

defined now as the e2 epitope and the most essential sequences there are 129-PPA-131

129-PP-Y-132 (Sallberg et al., 1991). Refinement of HBc regions exposed or internalized

at the surface of the particle with monoclonal antibodies led to the final conclusion that

sequence 127-133, along with region 78-83, occupies a superficial position on the native

HBc shell, whereas other regions, for example, 133-145 and 9-20, were hidden (Sallberg

et al., 1993).

Th epitopes: In acutely infected hepatitis B patients, the immunodominant Th epitope

50-69 is recognized irrespective of HLA background, two further important epitopes are

1-20 and 117-131 and a series of other sequences covering practically the whole HBc

polypeptide were described ( Chisari and Ferrari, 1995). In mice, examination of the fine

specificity of Th cell recognition revealed predominant epitopes specific for each murine

strain, dependent on the H-2 haplotype: 120-140 ( haplotype H-2s, b), 100-120 (

haplotype H-2f, q), and 85-100 ( H-2d mice ) ( Milich et al., 1987).

CTL epitopes: A single HLA-A2 restricted CTL epitope amino acid 18-27 has been

identified in man (Chisari and Ferrari, 1995). Additionally, an HBc epitope 141-151 has

been defined by CTL clones from patients with acute hepatitis B, which is restricted by

both HLA-Aw68 and HLA-A31 molecules. Recent studies allowed the direct isolation of

a naturally processed HBc peptide 88-96 recognized by HLA-A11-restricted CD8+ CTLs

(Tsai et al., 1996). In mice, the HBc peptides 93-100( Kuhrober et al., 1996) and 87-96 (

Kuhrober et al., 1997) were found as CTL epitopes in the context of Kb binding ( H-2b

mice ) and Kd binding ( H-2d mice ) respectively. In macaques, the long-lived CTL

response was directed against HBc peptide 63-71 (Townsend et al., 1997). Peptides 84-

95 and 88-95 were predicted as good candidates for CTL epitopes (Ehata et al., 1992).


23

1.4. Studies on HBV antigens for New Vaccine Development

The preS protein of hepatitis B surface antigen was cloned and expressed in eukaryotic

cells (Qin et al., 2003), Plants (Gao et al., 2003) Transgenic mice (Xiong et al., 2003),

and yeast (Lu et al., 2002). Expression vectors (pET, pT7-7, pGEX2T, and pTXBI

(NEB)), pS300) were used to express preS protein (Delos et al., 1991; Wei et al., 2002

Nunez et al., 2001; Chen et al., 2003).

A role of LHBsAg in protective immunity has been suggested by the presence of both B

and T cell epitope (Gerlich, 1990). It has been reported that MHsAg of 281 amino acid

residues appears to be dispensable for HBV virion assembly (Gerken et al., 1991).

Attempts have been made to develop Recombinant preS product as a protein vaccine that

elicits B and T cell immune responses on a broader range of MHC haplotypes (Milich et

al., 1990; Pride et al., 1998; Hui et al., 1999b; Shouval., 2003). The preS1 regions of

amino acids 12-20 and 89-90 amino acid bind specifically to a cell surface protein, p80

(Ryu et al., 2000), while the 13 residues of the carboy-terminus of the preS1 bind

efficiently to the core particles (Poisson et al., 1997).

The preS1 region has been shown to be a particularly efficient immunogen at the T and B

cell level. A dominant T cell recognition site was identified in the N-terminal residues

21-28 of preS1 sequence (Jin et al., 1988; Ferrai et al., 1989) while B cell determinants

were mapped to residues 12-32, 32-53 and 94-117 (Milich, 1988; Neurath et al., 1989a).

Fusion of the first 42 residues of the preS1 to either site N or C terminus of the preS1

protein allowed efficient secretion of the modified particles and rendered the linked

sequence accessible at the surface of the particle and in mice elicited high titers of preS1

specific antibodies which recognized the authentic L protein (Prange et al., 1995).

PreS1 and PreS2 domains in addition to humoral response harbor T-cell specific and

antigenic determinants that may play an important immunogenic role in terms of

augmenting antibody titres to HBsAg( antiHBs) and circumvention of genetic non-


24

responsiveness to the S-antigen in mice (Caselmann, 1995). To explore the structure-

function relationship of the preS1, large quantities of the pure preS1 are required.

However, purification of intact preS1 has not been successful due to the susceptibility of

the polypeptide to degradation during and after its expression in soluble form in bacteria

(Kim and Hong, 1995; Delos et al., 1991). The preS1 was expressed in insoluble form in

bacteria, which needed additional refolding step (Lin et al., 1991).

Zhao et al (2002), studied the therapeutic T cell vaccine for the treatment of chronic

hepatitis B by improving the cellular immunization of HBsAg vaccine with the

coexpression of the preS1(1-42) and the core (1-44) antigen of HBV in E.coli.

Unfortunately, there are no suitable measures to treat HBV infection so far. It is

necessary to develop new anti-virus methods, such as therapeutic vaccines that could be

more effective for viral remediation (Lau, 2000; Li and Tang, 2000; Malik and Lee,

2000; Torresi and Locarnini, 2000; Kubba et al., 2003).

Hepatitis B core antigen is a major viral nucleocapsid protein consisting of a 19-21.5kDa

polypeptide which shares the antigenic sites for both HBc and HBeAg antigens

(Takayuki et al., 1988). HBcAg is a very powerful immunogenic and induces strong

humoral T helper (Th) and cytotoxic T cell (CTL) responses and functioning as both T

cell dependent and T-cell independent antigen (Milich et al., 1997a, b) .It is involved in

capsid formation, packaging of the pregenome reverse transcriptase complex, trafficking

of the capsid in the cell and envelopment ( Koschel et al., 1999).

Zheng et al. (1992) demonstrated that disulfide bond formation is not essential for the

assembly of HBcAg capsid. It is a maturing process; the protein assembled initially in the

reducing environment of the E.coli cytoplasm is probably fully reduced, but during

isolation and storage, oxidation occurs, especially involving highly reactive C-terminal

cysteins. The results obtained with the E.coli expressed HBc are mainly in agreement

with these observations showing that HBc purified under denaturing and reducing

conditions could reform nucleocapsids by taking advantage of the self-assembly


25

properties of HBcAg. HBcAg has four cysteine residues that a large number of potential

disulfide bonding patterns are possible as Cys 183, Cys 161 and Cys 107 are involved in

disulfide bond resulting in dimer formation

Konig et al., considered that the interaction domain of HBcAg protein subunit aa78-117

conducted dimerization while aa113-aa143 did polymerization. To expose foreign

epitopes on the surface of chimeric HBcAg capsids, they can be added to the N-terminus

of HBcAg (Clarke et al., 1987; Moriarty et al., 1990; Schodel et al., 1992), or to the C

terminus of C terminally truncated HBcAg (Borisova et al., 1987; Borisova et al, 1998;

Schodel et al, 1992; Stahl et al, 1989) or inserted at position 144, upstream to the

arginine rich region of full length HBcAg. However, Schodel et al., 1992 showed that the

most promising location of inserted epitopes from the point of view of immunogenicity

seems to be the site of an outer loop predicted to be on the surface of HBcAg in the

vicinity of position 80 ( Argos and Fuller, 1988 ).

HBcAg has therefore been suggested as a carrier moiety of foreign epitopes for vaccines.

Insertion of a pres epitope at the internal site resulted in the most efficient antiPreS

antibody response. Furthermore, immunization with hybrid HBc/preS particles

exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These

results indicated that the position of the inserted B cell epitope within HBcAg is critical

to its immunogenicity (Schodel et al., 1992).

HBcAg aggregates upon expression in E. coli (Cohen and Richmond, 1982). As a

precursor of the capsid assembly the core dimers have the ability to spontaneously

aggregate to particles (Zhou and Standring, 1992). Core dimers are very stable structures

which can be dissociated to monomers only under strong denaturing and reducing

conditions during SDS-PAGE. Self assembly is mediated by protein-protein interactions

of the core protein domain from the N-terminus to amino acid 144. The following

arginine rich domain is responsible for nucleic acid binding but it is not necessary for

assembly (Birnbaum and Nassal, 1990).It has been described by several groups that

fusion of heterologous sequences to the C-terminus allows in some cases the detection


26

with specific antibodies indicating the outside localization of the foreign epitopes (von

Brunn et al., 1993; Borisova et al., 1998). Since the HBc capsids contains holes on their

speculated surfaces (Bottcher et al., 1997; Conway et al., 1997). It was speculated that

the foreign polypeptide chains emerged through the holes thus becoming localized on the

surface and accessible to antibodies. (Crowther et al., 1994).

1.5. Aims of study

A major problem in chronic hepatitis B virus (HBV) infections is that treatment with

specific antivirals is life-long since they rarely induce sustained responses. An attractive

option is therefore to combine antiviral therapy with some type of immune stimulator,

such as a therapeutic vaccine. Substantial body of evidence now supports arguments for

the use of HBcAg particles as carriers of immunological determinants of various types.

The opportunity to display multiple epitopes on single, highly immunogenic particles

with strong T cell epitopes emphasizes their value as candidates for vaccines or

components of vaccines. The envelope proteins of HBV antigens are known to elicit

virus-neutralizing and protective antibodies HBcAg is highly immunogenic and can

prime specific antibodies and can be utilize by using HBcAg as a carrier for foreign

epitopes. However, it may also be of advantage to simultaneously improve the

neutralizing antibody response to the surface (S) region of HBV.

Several lines of evidence suggest that a key target for the cellular immune response is the

HBV core antigen (HBcAg). However, it may also be of advantage to simultaneously

improve the neutralizing antibody response to the surface (S) region of HBV. Therefore,

a small laboratory study was designed to evaluate the combined effect of HBsAg with the

ability of HBcAg to act as a carrier for HBsAg-derived sequences since this may

constitute an appropriate platform to be used as a therapeutic vaccine.

Materials and Methods

Chapter 2


27


2.1. Chemicals and Reagents

All the general laboratory chemicals, and molecular biology reagents used in this study

were of a high purity ‘or its equivalent. General laboratory materials and biochemical

reagents were obtained from:

Sigma-Aldrich Chemical Company Ltd., http:/ www.sigmaaldrich.com

Bio-Rad Laboratories, http:/ www.bio-rad.com

Roth, http:// www.roth.com

Reagents for molecular biology and protein purification were purchased from:

Sigma-Aldrich Chemical Company Ltd., http:// www.sigmaaldrich.com

Bio-Rad Laboratories, http:/ www.bio-rad.com

Fermentas, http:/ www.fermentas.com

New England Biolab, http:/ www.neb.com

Promega, http:/ www.promega.com

Qiagen, http:/ www.qiagen.com

Roche Diagnostics, http:/ www.roch.com

Invitrogen Life Technologies, http:/ www.inbitrogen.com

Clontech, http:// www.clontech.com

2.2. Bacterial Strains, Plasmids and Constructed Vectors

Bacterial strains, standard plasmids and constructed plasmids/vectors used in this study

are listed in Table 2.1. Glycerol stocks were prepared by mixing bacterial cultures (liquid

growth, 800 µl) with glycerol (200 µl) and stored at -70°C.

http://www.sigmaaldrich.com/

http://www.bio-rad.com/

http://www.roth.com/

http://www.sigmaaldrich.com/

http://www.bio-rad.com/

http://www.fermentas.com/

http://www.neb.com/

http://www.promega.com/

http://www.qiagen.com/

http://www.roch.com/

http://www.inbitrogen.com/

http://www.clontech.com/


28

Table 2.1: Bacterial strains, standard cloning vectors, recombinant and chimeric constructs.

Strains Description Source/Reference

Top 10 E.coli

F-, mcrA, ∆(mrr-hsdRMS-mcrBC), ᵩ 80 lacZ ∆M15,∆lac X74, deoR, recA1, araD139∆(ara-leu) 7697, galU, galK, rpsL( StrR), endA1 nupG,

Invitrogen, USA

BL21 (DE3) E.coli

F-, ompT, hsdS(rB- mB-), gal, dcm, λ(DE3) Novagen, USA

Standard Cloning Vectors

pTZ57R/T AmpR, Lac Z gene, T7 Promoter, Multiple cloning sites,f1(1G), rep(pMB1)

Fermentas USA

pET28a KanR , Multiple cloning site, lac region, f1 origin, pBR322 origin, 6XHis tag, T7Promoter, T7 terminator

Invitrogen, USA

Recombinant Plasmids / Vectors Constructs

pIJM.HBs-Ag pTZ57R/T contains full length hepatitis B surface gene

This study

pIJM.HBc-Ag pTZ57R/T contains full length hepatitis B core gene This study

Chimeric Vectors/ Plasmids Constructs

pIJMcsc-1 Harbour’s core1-78, PreS12-32 and core 80-144 aa in pET28a expression vector under T7 promoter

This study

pIJMcsc-2 Harbour’s core1-78, PreS32-53 and core 80-144aa in pET28a expression vector under T7 promoter

This study

pIJMcsc-3 Harbour’s core 1-78, PreS124-147 and core 80-144 aa in pET28a expression vector under T7 promoter

This study


This study


This study


29

2.3. Bacterial Growth Media/Conditions

Luria Bertani (LB) medium as described by (Miller 1972 See Appendix 2.1) was used as

growth medium for the growth of bacterial strains. LB-agar plates were prepared by

autoclaving LB liquid medium containing 1.5% agar. Where necessary, ampicillin or

kanamycin was added in the LB medium at the concentration of 100µg/ml and 50µg/ml.

E.coli cells harbouring the relevant plasmid were grown overnight in 5ml LB liquid

medium containing appropriate antibiotic and then transferred into 50ml LB medium in

falcon tubes. The tubes were then further placed at 37°C in a Shaking incubator (New

Brunswick incubator shaker) for 16-18 hours. The bacterial cells (cell pellets) were

obtained by centrifugation (4000rpm x g, 4°C, 10 min) were either stored at -20°C or

used immediately for plasmid DNA isolation.

2.4. Experimental Animals

Inbred mice C57BL/6J were obtained from B& K Universal, Charles River Laboratories,

Sulzfeld, Germany. The local committee on Animal Ethics, at Karolinska Institutet,

Sweden accorded the approval of all experiments mentioned in this dissertation.

2.5. Collection of Samples

Blood samples were obtained from patients admitted at Liver centre, DHQ Hospital,

Faisalabad, Pakistan. All the patients had symptoms such as appetite loss, fatigue, nausea,

vomiting, and jaundice. Blood samples (5cc) from these patients were collected in

vaccutainers containing EDTA as anticoagulant. The blood was gently mixed with

anticoagulant and allowed to stand for 30 min at room temperature and centrifuge at

4,000 rpm for 10 min to separate plasma and serum. Plasma was stored at -20°C till

further use.

2.6. Primers / Oligonucleotides

Primers used in PCR amplification of epitopes and cloned chimeric DNA fragments are

presented in Table 2.2, and 2.3. Primers were either selected from the available literature

or designed using Primer 3 software supported by manual calculations and finally

verified for specificity using the National Center for Biotechnology Information BLAST


30

server. Oligoes synthesized from Sigma Aldrich Pty Ltd and were obtained in lyophilized

form. Stock solutions of primers (200 ul) were prepared in Tris. EDTA buffer pH 8.0

whereas working solutions were prepared in nuclease free sterile water (Astral scientific).

Solutions were stored at 20°C.

Table 2.2: Oligonucleotides used for amplification of target sequences of HBV genome.

Name Amplified region DNA Sequence + restriction site in primers Nucleotide

Position 5-

3(bp)

PCR

product

size(bp)

T/B cell

response

References

HBSF1 Full length HBs-Ag GCG AAG CTT ATG GAG AAC ATC ACA TCA GG HindIII

157

HBSR1 Full length HBs-Ag GTC CTC GAG TTA AAT GTA TAC CCA AAG XhoI

837

681

T cell

Nader, 2005

HBCF1 Full length HBc-Ag AGC GAA TTC ATG GAC ATT GAT CCT TA EcoRI

1903

HBCR1 Full length HBc-Ag CAC CTC GAG CTA ACA TTG AGA TTC CCG XhoI

2454

549

T helper cell

Azizi et al.,2007

HBC1-78F Core region ( 1-78 amino acid)

GCGGCCCATGGACATTGATCCTTATAAAG NcoI

1903

HBC 1-78R Core region ( 1-78 amino acid)

CGA GTC GAC ATC TTC CAA ATT ACC AC SalI

2136

234

B cell

This study

HBC 80-144F Core region ( 80-144amino acid)

AC AAG CTT ATA TCC AGG GAC CTA GTA GTC HindIII

2140

HBC 80-144 R Core region ( 80-144amino acid)

CA CTC GAG TCG GAA GTG TTG ATA AGA TAG XhoI

2334

195

B cell

This study

HBPreS124-147F

PreS epitope ( PreS 124-147)

TTAGTCGACTGCACGACTCCTGCT SalI

526

HBpreS124-147R

PreS epitope ( PreS 124-147)

TTGAAGCTTGCAATTTCCGTCCGA HindII

598

72

B cell

Peterson, 1987

HBPreS1-42F PreS epitope ( PreS 1-42)

TTAGTCGAC ATG GGG CAG AAT CTT TC SalI

2850

HBPreS1-42R PreS epitope ( PreS 1-42)

TTGAAGCTTGTCTGGCCAGGTGTCCT HindIII

2978

129

B cell

Prange et al., 1995

HBS112-32F PreS epitope ( PreS 12-32)

GA GTC GAC GGA TTC TTT CCC GAC SalI

2883

HBS112-32R PreS epitope ( PreS 12-32)

GG AAG CTT CCA ATC TGG ATT TGC GGT HindIII

2945

63

B cell

Xinchun et al., 2003

HBS132-53F PreS epitope ( PreS 32-53)

TA GTC GAC TGG GAC TTC AAT CCC A SalI

2943


TA AAG CTT CCC GAA TGC TCC AGC T HindIII

3008

66

B cell

Xinchun et al., 2003

HBPreS1-42F PreS epitope ( PreS 1-53)

TTAGTCGAC ATG GGG CAG AAT CTT TC SalI

2850


TA AAG CTT CCC GAA TGC TCC AGC T HindIII

3008

159

This study


31

Table 2.3: Oligonucleotides used for amplification of constructed chimeric DNA fragments from cloned Chimeric Plasmids.

Name Amplified region DNA Sequence + restriction site in primers PCR

product

size(bp)

References

Core region ( 1-78 amino acid)


csc-1

Core region ( 80-144amino acid)


510

This study



csc-2



513 This study



csc-3



519 This study



csc-4



606 This study



csc-5



576 This study

2.7. Genetic Techniques

General procedures for cloning and DNA manipulations were performed as described by

Sambrook et al., (1989). All DNA manipulations and vector construction experiments

were performed using E.coli top 10 strains

2.7.1. HBV DNA isolation

HBV-DNA was extracted and isolated from plasma according to Kao et al., (2000). 50 µl

lysis buffer (Appendix 2.2) was added to an eppendorf tube containing 100 µl plasma

sample and gently vortexed. Samples were incubated at 37°C for 30 min, room

temperature for 60 min, and then heated at 95°C for 15 min. The samples were cooled

and kept at -20°C for 15 min and finally centrifuged for 5 min at 10,000 rpm. The

resulting supernatant was transferred into an autoclaved eppendorf treated with phenol

: chloroform: isoamyl alcohol (25:24:1). The DNA was precipitated with 3M sodium


32

acetate (pH: 4.3) and washed with 70% ethanol; vacuum dried and then dissolved in 10

µl TE buffer (Appendix 2.3).

2.7.2. Plasmid DNA isolation

Plasmid DNA was extracted and isolated from the bacterial cells using the Qiagen mini-

prep system (Qiagen Ltd.) following the manufacturer’s instructions and then stored at -

20°C until needed for further use.

2.7. 3. Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) technique was developed in 1983. This technique

was used to amplify the target DNA sequences from isolated DNAs from blood and

bacterial plasmids containing HBV DNA sequences. In all amplification reactions

performed, the amplification of each desired target sequence was optimized by following

a series of amplification steps. A series of polymerase chain assays (reactions) were

performed to amplify DNA fragments from HBV genome (extracted from patient

samples) or recombinant plasmid constructs. The PCR assays performed to amplify target

sequences are presented in Appendix. 2.6.

2.8. Purification, Digestion and Cloning of Amplified DNA fragments

The amplified DNA products (681bp; 549bp, 234bp, 195bp; 159bp, 129bp; 72bp, 66bp

and 63bp) were purified, digested with respective restriction enzymes and cloned into

previously restricted TA cloning vector or pET28a expression vector dephosphorylated

with alkaline phosphatase. Restricted amplified DNA products were purified by a gel

extraction method using the QIAquick gel extraction kits (Qiagen, inc. USA). A typical

cloning (ligation) reaction contained 50ng of restricted vector DNA. Molar vector: insert

of 1:1 and 1:3 were used. Cloning (ligation) reactions was performed in a 20 µl reaction

volume using T4DNA ligase system (Fermentas) for overnight at 16°C. Orientation of

the inserts was checked by respective restriction enzyme digestions. The constructs were

named as pIJM.HBs-Ag; pIJM.HBc-Ag; pIJMcsc-1; pIJMcsc-2; pIJMcsc-3; pIJMcsc-4, and


33

pIJMcsc-5 (with PG linkers flanking the inserted sequence). Construction strategy of

recombinant / chimeric vectors/ plasmids is presented below:

2.9. Construction of Recombinant and Chimeric Plasmids

2.9.1. pIJM.HBs-Ag

The amplified DNA product Hepatitis B surface gene, 681bp bearing Xho1 and Hind III

restriction enzyme sites was cloned into pTZ57RT cloning vector between LacZ and T7

promoter using standard cloning procedures as described in Promega Inc. USA Cloning

guide. The generated construct is diagrammatically represented in Figure 2.1 and was

named as pIJM.HBs-Ag.

Figure 2.1: Cloning of pIJM.HBs-Ag.The amplified DNA fragment “681bp” coding 227

AAs” was cloned into pTZ57R/T at XhoI and HindIII restrictions sites to form

pIJM.HBs-Ag.


34

2.9.2. pIJM.HBc-Ag

The amplified DNA product hepatitis B core gene, 549bp bearing EcoRI and Xho1

restriction enzyme sites was cloned into pTZ57RT cloning vector between LacZ and T7

promoter using standard cloning procedures as described in Promega Inc. USA Cloning

guide. The construct generated is represented in Figure 2.2 and was named as pIJM.HBc-

Ag.

Figure 2.2: Cloning of pIJM.HBc-Ag. The amplified DNA fragment “549bp” coding 183

AAs” was cloned into pTZ57R/T at EcoRI and XhoI restrictions sites to form pIJM.HBc-

Ag.

2.9.3. Construction of Chimeric Expression Plasmids / Vectors

To develop chimeric expression vectors, a stepwise cloning strategy was adopted. These

chimeric constructs contained Pre-S DNA fragment and two epitopes from hepatitis core

regions. Each of the DNA fragments (234bp, 195bp, 159bp, 129bp, 72bp, 66bp and

63bp) was amplified using specific set of primers listed in Table 2.2 by PCR specific

reactions. In general, a cassette of three amplified DNA fragments was made by inserting

Pre-S DNA in the center of the two hepatitis core epitopes. This cassette was inserted in

pET28a expression vector at the C-terminal attachment of His 6 by respective restriction

enzymes and ligation procedures. Details of cloning prokaryotic expression vectors

“Chimeras” are presented below:


35

XhoI HindIII

2.9.3.1. pIJMcsc-1

Three DNA fragments from the genes coding preS12-32, (63bp); HBcAg amino acid

residues from 1 to 78 (234bp) and from 80 to 144 (195bp) were cloned in pET28a

expression vector using positional cloning approach. A cassette containing the three

amplified DNA fragments was made and inserted into of pET28a at NcoI and XhoI

restriction enzyme positions which formed a chimeric plasmid (Figure 2.3), and was

named as pIJMcsc-1.

A

B

C

D

Figure 2.3: Cloning strategy of amplified DNA products into pET28a prokaryotic expression plasmid and construction of pIJMcsc-1. The amplified DNA fragment “234bp”coding1-78AAs” was cloned into pET28a (A) at Sal1 and Nco1 restriction sites, generating as plasmid “B”. The amplified DNA fragment “ 63bp” coding for PreS 12-32 AAs” was ligated at Sal1 and HindIII sites of plasmid “B” to generate plasmid “C); Finally, the amplified DNA product “ 195bp” bearing HBc80-144AAs” was cloned into plasmid “C” at restriction sites HindIII and Xho1 to form pIJMcsc-1 designated as plasmid D.

PCR amplified DNA “63bp” “PreS12-32”AAs

Ligation

HindIII

Amplified DNA product: 195bp “80-144 AAs

Ligation

pHBc1-78/PreS12-32 5666bp

NcoI SalI


Ligation

SalI


36

NcoI SalI

2.9.3.2. pIJMcsc-2

Three DNA fragments from the genes coding preS132-53(66bp); HBcAg amino acid

residues from 1 to 78 (234bp) and from 80 to 144 (195bp) were cloned in pET28a



restriction enzyme positions which formed a chimeric plasmid (Figure 2.4), and was

named as pIJMcsc-2.

A

B

C

D Figure 2.4: Cloning strategy of amplified DNA products into pET28a prokaryotic expression plasmid and construction of pIJMcsc-2. The amplified DNA fragment “234bp”coding1-78AAs” was cloned into pET28a (A) at Sal1 and Nco1 restriction sites to form plasmid “B”. The amplified DNA fragment “ 66bp” coding for PreS 32-53 AAs” was then ligated at Sal1 and HindIII sites of plasmid “B” to generate plasmid “C); Finally, the amplified DNA product “ 195bp” bearing HBc80-144AAs” was cloned into plasmid “C” at restriction sites HindIII and Xho1 to form pIJMcsc-2 designated as plasmid D.

PCR amplified DNA “66 bp” “PreS32-53”AAs

SalI HindIII

I

XhoI HindIII

Ligation

Ligation

Ligation




37

NcoI SalI

PreS124-147

pHBc1-78/Pres124-147 5675bp

2.9.3.3. pIJMcsc-3

The amplified DNA fragments from the genes coding preS124-147(72bp); HBcAg amino

acid residues from 1 to 78 (234bp) and from 80 to 144 (195bp) were cloned in pET28a



restriction enzyme positions which formed a chimeric plasmid (Figure 2.5) and was

named as pIJMcsc-3.


A

B PCR amplified DNA “195bp” “HBc80-144 AAs” C

D

Figure 2.5: Cloning strategy of amplified DNA products into pET28a prokaryotic expression plasmid and construction of pIJMcsc-3. The amplified DNA fragment “234bp”coding1-78AAs” was cloned into pET28a (A) at Sal1 and Nco1 restriction sites, generating as plasmid “B”. The amplified DNA fragment “ 72bp” coding for PreS 124-147 AAs” was ligated at Sal1 and HindIII sites of plasmid “B” to generate plasmid “C); Finally, the amplified DNA product “ 195bp” bearing HBc80-144AAs” was cloned into plasmid “C” at restriction sites HindIII and Xho1 to form pIJMcsc-3 designated as plasmid D

Ligation


Ligation

SalI HindIII

XhoI HindIII

Ligation


38

XhoI HindIII

PreS1-53


2.9.3.4. pIJMcsc-4

The genes encoding genes preS1-53 (159bp), HBcAg amino acid residues from 1 to 78

(234bp), and from 80 to 144 (195bp) were cloned in pET28a expression vector using

positional cloning approach. A cassette containing the three amplified DNA fragments

was made and inserted into of pET28a at NcoI and XhoI restriction enzyme positions

which formed a chimeric plasmid (Figure 2.6) and was named as pIJMcsc-4.

A

B PCR amplified DNA “195bp” “HBc80-144 AAs” C D

Figure 2.6: Cloning strategy of amplified DNA products into pET28a prokaryotic expression plasmid and construction of pIJMcsc-4. The amplified DNA fragment “234bp”coding1-78AAs” was cloned into pET28a (A) at Sal1 and Nco1 restriction sites, generating as plasmid “B”. The amplified DNA fragment “ 159bp” coding for PreS 1-53 AAs” was ligated at Sal1 and HindIII sites of plasmid “B” to generate plasmid “C); Finally, the amplified DNA product “ 195bp” bearing HBc80-144AAs” was cloned into plasmid “C” at restriction sites HindIII and Xho1 to form pIJMcsc-4 designated as plasmid D.

NcoI SalI

Ligation


SalI HindIIII


Ligation

Ligation


39

PreS1-42


XhoI HindIII

2.9.3.5. pIJMcsc-5 The genes encoding preS1-42 (129bp), HBcAg amino acid residues from 1 to 78 (234bp)

and from 80 to 144 (195bp) were cloned in pET28a expression vector using positional

cloning approach. A cassette containing the three amplified DNA fragments was made

and inserted into of pET28a at NcoI and XhoI restriction enzyme positions which formed

a chimeric plasmid (Figure 2.7) and was named as pIJMcsc-5.


A

B

C PCR amplified DNA “195bp” “HBc80-144 AAs”

D Figure 2.7: Cloning strategy of amplified DNA products into pET28a prokaryotic expression plasmid and construction of pIJMcsc-5. The amplified DNA fragment “234bp”coding1-78AAs” was cloned into pET28a (A) at Sal1 and Nco1 restriction sites, generating as plasmid “B”. The amplified DNA fragment “ 129 bp” coding for PreS 1-42 AAs” was ligated at Sal1 and HindIII sites of plasmid “B” to generate plasmid “C); Finally, the amplified DNA product “ 195bp” bearing HBc80-144AAs” was cloned into plasmid “C” at restriction sites HindIII and Xho1 to form pIJMcsc-5 designated as plasmid D.

NcoI SalI

Ligation


Ligation

SalI HindIII

Ligation


40

2.10. Competent cells and transformation experiments

The cloned recombinant and chimeric DNA constructs (prepared previously) were

transformed into E.coli Top10 and/ BL21 (DE3) competent cells. The competent cells

were prepared as described (Promega p. no. 52). A 200µl aliquot of chemically-

competent cells (Appendix 2.7) was thawed on ice in 1.5ml microcentrifuge tubes.

Approximately 30 ng plasmid DNA was added to 200 µl of competent cells and mixed

gently. The cells were incubated on ice for 30 min and heat shocked at 42°C for 45

seconds and subsequently incubated on ice for 5 min. 1ml of LB medium was added to

the cells and incubated at 37°C for 1h at 200 rpm. A 200 µl aliquot of the transformation

mixture was plated on a pre-warmed LB agar containing the appropriate antibiotic. The

inverted plate was incubated overnight at 37°C. The transformants (colonies) were

selected on LB agar plates supplemented with ampicillin (100µg/ml), X.gal (40µg/ml)

and IPTG (0.5mM). The transformants were named as E.coli: IJM.HBs-Ag; IJM.HBc-Ag;

IJM.csc-1; IJM.csc-2; IJM.csc-3; IJM.csc-4; and IJM.csc-5 (Appendix 2.9).

2.11. DNA sequencing analysis

DNA sequencing analysis of the Recombinant and Chimeric plasmids was carried out on

an ABI 3100 capillary sequencer, using BigDye. The DNA sequence of purified cloned

genes (HB Surface; HB Core; csc-1; csc-2; csc-3; csc-4 and csc-5) was performed at the

DNA sequencing Facility NIBGE, Faisalabad. DNA sequence of the surface, core,

chimeric genes and deduced amino acids sequence was analyzed using programs in the

ExPASy (Expert Protein Analysis System).

2.12. Expression of Chimeric genes

For expression experiments, constructs were transformed into E.coli host BL21 (DE3) as

described in section 2.6.9. Strains harbouring relevant plasmids were recovered from

frozen glycerol stocks (-70°C) by streaking onto LB-agar plates containing kanamycin


41

(50 µg/ml) and incubated at 37°C for overnight. Subsequently, single colony was

transferred to LB liquid medium (Appendix 2.1) supplemented with 50µg/ml kanamycin

in 10 ml falcon tubes and were grown 2-3 h in shaker at 37°C, 200 rpm until OD reached

upto 0.5-0.6. Then took 0.5-0.8% of above freshly grown culture in 250 ml and induced

expression by the addition of auto-induction medium for overnight at 37°C as described

by (Studier, 2005). The cells were harvested by centrifugation at 4000 rpm at 4°C.

Expression solutions used for various constructs are described in Appendix 2.8.

2.13. Gel electrophoresis

Amplified PCR products were resolved on 1-2% agarose gel, prepared by melting 1-2

grams of agarose in 100 ml 1X TBE buffer (0.89M Tris-Borate, 0.025 M EDTA), in a

microwave oven for 2-5 min. Ethidium bromide (final concentration 0.5µg/ml) was

added to the gel to stain the DNA for visualization after electrophoresis. PCR products

were mixed with bromophenol blue dye (0.25 % bromophenol in 40% sucrose solution)

and electrophoresis was performed at 100 volts for 30-40 min in 1X TBE buffer.

Resolved PCR products were detected by placing the gel on UV transluminator (Life

Technology, USA).


42

2.14. Biochemical techniques

Standard biochemical techniques as described elsewhere (talon affinity chromatography,

western blotting and ELISA) were employed for the purification, characterization and

immunological assays.

2.14.1. Purification of His-6 chimeric proteins by talon affinity chromatography

The constructed chimeric proteins (csc-1, csc-2, csc-3, csc-4, and csc-5) expressed in E.coli were

purified using talon affinity chromatography (Clontech, USA). Purification strategy of chimeric

proteins is presented in Figure 2.8.

Cell pellet (50ml bacterial culture) + 5ml (50mM sodium Phosphate, 300mM NaCl, pH 7.00) + Lysozyme

Sonication and centrifugation, 4000rpm,

10min, 4°C

Pellet +5ml of resuspension buffer Supernatant (Soluble protein) centrifugation 5000 rpm,

10 min, 4°C

Supernatant incubate Cell debris

with talon resins for 1h, 4°C

Pour resin with protein sample into column

Wash twice with 10 ml of wash buffer (50 mM sodium phosphate, 300 mM NaCl and 6M GnCl) to remove non specific proteins

Eluted protein with 4 ml of elution buffer containing 250 mM Imidazole

Collect purified eluted protein in 10 fractions of 0.4-0.5ml volume

Figure 2.8: Outline of purification procedure for chimeric proteins.


43

2.14.2. Determination of protein

Bio-Rad Bradford Protein Assay ( Bradford, 1976) a dye binding assay, in which the

absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 dye shifts

from 465nm to 595nm when binding to protein occurs. First of all, pipet out 800 µl of

dH2O and 1µl of each sample solution diluted in into a tube. Add 200 µl of dye reagent

concentrate to each tube, vortex and incubate at room temperature for at least 5 min. The

samples should incubate at room temperature for no more than 1 h. Measure absorbance

at 595nm and use the standard curve to calculate the concentration of protein.

2.14.3. Protein analysis by SDS-PAGE

SDS-PAGE was performed based the method of Laemmli (1970) using a Bio-Rad Mini

PROTEAN II apparatus. A 10% or 12% separating gel (Table 2.4) was prepared by

mixing reagents 1, 2 and 3A in a flask and the solution was degassed for 15 min under

vacuum. Reagent 5-6 was added with gentle mixing. The gel was poured, overlaid with

distilled water and allowed to set for at least 30 min. A 3% stacking gel (Table 2.4) was

prepared in the same way using reagent 3B instead of 3A. The distilled water was

removed from the top of the separating gel, and then the stacking gel was poured with a

10-lane 0.75mm thick comb inserted and allowed to set for at least 90 min. The gel was

transferred to the electrophoresis tank, which was filled with running buffer (Table 2.5).

Protein samples (15µl) were mixed with 5µl sample loading buffer (Table 2.3) and β-

Mercaptoethanol were added with brief vortexing; the solutions were then heated for at

70°C for 10 min. The solution was then held at room temperature to cool down before

loading into the lanes of the gel. Electrophoresis was performed at constant voltage

(100V) using the running buffer (Table 2.5) until the dye front reached the bottom of the

gel (~90 min).

Protein bands were visualized by Coomassie Blue staining. The gel was soaked

sequentially on an orbital platform in fixing solution (65: 25: 10 water/2-propanol/glacial

acetic acid) for ~ 3h, staining solution (65: 25: 10 water/2-propanol/glacial acetic acid

containing 0.025% Commassie Brilliant Blue R-250) for ~20h, and then several changes


44

of destaining solution (90:10 water/glacial acetic acid) over~24h or until the background

was faded sufficiently.

Table 2.4: Composition of separating and stacking SDS-PAGE gels.

Separating gel Stacking gel No Component

12% 15% 3%

1 H2O 3.34ml 2.35ml 6.35ml

2 Acrylamide (30%) 4ml 5ml 1ml

3A Separating gel buffer

Tris-HCl (1.5M, pH 8.8)

2.5ml 2.5ml

3B Stacking gel buffer

Tris-HCl (0.5M, pH 6.8)

2.5ml

4 20%SDS 50µl 50µl 50µl

5 TEMED 5µl 5µl 5µl

6 Ammonium persulphate 100µl 100µl 100µl

Total Volume 10ml 10ml 10ml

Table 2.5: Composition of sample loading (4X) and running buffer (10X).

Sample loading buffer 4x(10ml)

Running buffer (10x)

Components Quantity;

Final concentration

Components Quantity;

Final

concentration

Glycerol 5.0g ( 5.4M) Glycine 144g(190mM)

SDS 1.0g (10%) Tris-HCl 30.0g(25mM)

EDTA 37.2mg(10mM) SDS 1g (0.1%)

Dissolve in Tris-HCl (0.5M, pH 6.8) and adjust pH to

6.8 with 1M NaOH. Commassie Brilliant Blue dye

was added to give a deep color. Stored at -20°C.

Adjust volume to 1 liter with

milliQ water


45

2.14.4. Western blotting

Following the separation of protein by SDS-PAGE, Western blotting as described by

(Ahlen et al., 2009; 2005) was used for target protein identification. For western blot

analysis, proteins were transferred electrophoretically to nitrocellulose membrane using

iBlot system as described by Invitrogen.

2.14.4.1. Visualization of histidine tagged proteins for anticore antibody

Detection of chimeric protein for anticore on western blots was performed using

Secondary anti-rabbit AP-conjugated antibody (Sigma). After iBlot transfer system, the

nitrocellulose membrane was subjected with blocking solution as described by

Invitrogen. Then Wash membrane twice (2x) in 10 ml dH2O for 5 min and incubate the

membrane with 10ml of Primary Antibody Solution (polyclonal rabbit anti-hepatitis B

virus core antigen (Ref: B0586, Lot: 10027138 Dako) for 1h. The nitrocellulose

membrane was then washed 4 times with antibody wash solution for 5 min. Membrane

incubated with Secondary anti-rabbit AP-conjugated antibody (Sigma) for 30 min.

Subsequently, the blot was again washed 4 times with antibody wash and 3 times with

dH2O and drained off excess liquid and place on the clean sheet of transparency plastic.

Apply 2.5ml of the Chemiluminescent Substrate to the membrane surface. Let the

reaction develop for 5 min. Develop the membrane in the Gene Gnome equipment for 1

to 4 min.

2.14.4.2. Detection of histidine tagged proteins for antiPreS antibody

Detection of chimeric protein for antiPreS on western blots was performed using

Secondary anti-mouse AP-conjugated antibody (Sigma). After iBlot transfer system, the

nitrocellulose membrane was subjected with blocking solution as described by

Invitrogen. Then Wash membrane twice (2x) in 10ml dH2O for 5 min and incubate the

membrane with 10ml of Primary Antibody Solution (polyclonal mouse anti-hepatitis B

virus PreS antigen (Kind gift of antiPreS from Paul Pumpen, university of Latvia) for 1h.

The nitrocellulose membrane was then washed 4 times with antibody wash solution for 5


46

min. Membrane incubated with Secondary anti-mouse AP-conjugated antibody (Sigma)

for 30 min. Subsequently, the blot was again washed 4 times with antibody wash and 3

times with dH2O and drained of excess liquid and place on the clean sheet of

transparency plastic. Apply 2.5ml of the Chemiluminescent Substrate to the membrane

surface. Let the reaction develop for 5 min. develop the membrane in the Gene Gnome

equipment for 1 to 4 min.

2.15. Immunization

Inbred mice C57BL/6J mice were obtained at 6 weeks of age from Charles River

Laboratory. Group of four female mice were immunized subcutaneously at the base of

tail. Chimeric (csc-5) purified protein was used to immunize mice with 10 µg of primary

dose and 15 µg of booster dose. Chimeric purified csc-5 protein was emulsified in

Incomplete Freund’s adjuvant (IFA). Mice were bled preimmunization and at various

times after primary and booster immunizations for anticore and antiPreS antibody

determinations.

2.15.1. Enzyme Linked Immunosorbent Assay

Enzyme linked immunosorbent assay was used to measured anticore and antiPreS

antibodies as described (Nystrom et al., 2010) by using chimeric protein (1µg/ml) and

anti-rabbit IgG and anti-mice IgG were used as the secondary antibody. The data were

expressed as the antibody titer representing the highest dilution yielding three times the

optical density of the preimmunization sera. Blood samples were collected through eye

puncture, and the spleen was isolated for the immunological assays.

2.15.2. ELISpot Assay

ELISpot assays were used to detect the presence of cytokine which are response for the T

cell proliferation as described by (Nystrom et al., 2010). In brief, splenocytes from group

of four mice were pooled and immediately tested for presence Hepatitis B core and PreS

specific T cells with a commercial anti-IFN-ɣ ELISpot assay (MabTech, Stockholm,

Sweden), using known positive control, Chimeric csc-5 protein to stimulate CTLs for 36

h before detection. Each group was performed in duplicate and spot counts were


47

calculated as mean number of IFNɣ spot forming cells (SFC)/106 cells. The number of

spots was scored using the Aid ELISpot reader system version, 2.6, Autoimmune

Diagnostika, Germany.

2.15.2.1. Detection of pIJMcsc-5 specific CTLs

ELISpots 96 wells plates were coated with 10µg/ml anti-IFN gamma (AN18) and about

200 µl per well and incubate overnight at 4°C.After washing and blocking with cell

medium ( 2h, 37°C). Prepared cells and dilute upto 2x106/ml and then plate out purified

immune cell 100µl/well (200.000cell/well). The plates were then washed extensively

after incubation of 36-40 h in 37°C in 5% CO2. The biotinylated anti-human IFN-ɣ was

added at 2µg/ml and incubates for 2 h at 37°C. Plates were then washed, and 1:1000

diluted streptavidin-peroxidase was added and incubated for 1 h at room temperature,

followed by addition of developing reagent (Bio-Rad). Spots were developed for 10 min,

and the reaction was stopped by washing plates with distilled water. The plates were

dried and the spots enumerated using the ELISpot automated reader.

Results

Chapter 3

Results

48

Results

3.1. Analysis of PCR products

The PCR-DNA products 681bp; 549bp, 234bp, 195bp; 159bp, 129bp; 72bp, 66bp and

63bp were obtained by the PCR procedures as described in Materials and Methods using

specific primers (Table 2.2). The results are presented in Figure 3.1.

(b)

(a)

(c)

Figure 3.1: Amplified PCR products analysed on 1% agarose gel and viewed under UV light. lane M: 50bp DNA marker. Fig.a) lanes 1, 3 and 4: PCR product 681 bp. HBs-Ag; Fig.b: PCR product 549 bp, HBc-Ag and Fig. c: lane 1.PCR product “PreS12-32” 63 base pair, lane 2: PCR product HBc1-78” 234 base pair, lane 3: PCR product “PreS1-42” 129base pair , lane 4: PCR product “PreS124-147” 72bp, lane 5: PCR product“HBc80-144” 195bp, lane 6: PCR product “PreS32-53”66 bp, lane 7: PCR product “PreS1-53” 159bp.

Results

49

3.2. Cloning of PCR products

The amplified PCR products obtained were ligated into cloning vectors or expression

vector. The map of recombinant (pIJM.HBs-Ag, pIJM.HBc-Ag), and chimeric constructs

(pIJMcsc-1, pIJMcsc-2, pIJMcsc-3, pIJMcsc-4, and pIJMcsc-5) are shown in Figures 2.1,

2.2, 2.3, 2.4, 2.5, 2.6, 2.7 (Materials and methods). The constructed recombinant plasmids

and chimeric constructs were transformed into E. coli BL21 (DE3) cells and the

corresponding transformants were selected (See table 2.9 in Annexure). Recombinant

(pIJM.HBs-Ag, pIJM.HBc-Ag) and chimeric plasmids ((pIJMcsc-1, pIJMcsc-2, pIJMcsc-

3, pIJMcsc-4, and pIJMcsc-5) were isolated from the transformants and finally the

constructs were confirmed by restriction enzyme digestion. The generated restricted

DNA fragments from recombinant plasmids “pIJM.HBs-Ag and pIJM.HBc-Ag were

analyzed on 1% agarose gel and results obtained are presented in Figure 3.2.

(a)

(b)

Figure 3.2: Analysis of PCR products and generated restriction digest DNA fragments along with 50 base pair ladder “M”. a) HindIII and XhoI restriction pattern of pIJM.HBs-Ag, b) EcoRI and XhoI restriction pattern of pIJM.HBc-Ag.

The respective cloned cassette of chimeric amplicon was amplified using primers listed in

Table 2.3 (see materials and methods) from the Plasmids obtained from the transformants

(E.coli: E.coli: IJMcsc-1, E.coli: IJMcsc-2, E.coli: IJMcsc-3, E.coli: IJMcsc-4 and E.coli:

IJMcsc-5). The PCR products and the restriction analysis of the cloned “chimeric

cassettes (csc-1, csc-2, csc-3, csc-4 and csc-5) obtained are presented in Figures 3.3. and

3.4.

Results

50

Figure 3.3: Analysis of PCR products and generated restriction digest DNA fragments along with 1Kb ladder “M” .a). PCR product “csc-1” 510 base pair , b) NcoI and XhoI restriction pattern of pIJMcsc-1; c) PCR product “csc-2” 513 base pair , d) NcoI and XhoI. restriction pattern of pIJMcsc-2; e) PCR product “csc-3” 519 base pair and f) NcoI and XhoI digest of pIJMcsc-3.

Results

51

Figure 3.4: Analysis of PCR products and generated restriction digest DNA fragments along with 1Kb and 100 base pair ladder “M” .a). PCR product “csc-4” 606 base pair , b) 606 bp and 5369 bp restriction pattern of pIJMcsc-4; c) PCR product “csc-5” 576 base pair , d) NcoI and XhoI restriction pattern of pIJMcsc-5.

Results

52

3.3. DNA Sequence analysis of cloned genes and translation to amino acids.

The DNA sequence of purified cloned genes (HBs-Ag; HBc-Ag; csc-1; csc-2; csc-3, csc-4

and csc-5) were analyzed on an ABI 3100 analyzer as described in Materials and

Methods. Chromatograms obtained were evaluated using computer program Sequencer

v.4.1.2 (Gene Codes Corporation). The DNA sequence of “a” determinant HBs-Ag (681

bp) and HBc-Ag (549bp) obtained from the chromatograms was compared with gene

sequences from Gene bank to identify any aberrant nucleotide base-pair change. The

nucleotide sequence of cloned chimeric genes was translated into amino acids using

computer program ExPasy server and Justbio.com.

3.3.1. Hepatitis B surface gene, 681bp

The nucleotide sequence of cloned full length hepatitis B surface gene (681bp) isolated

from HBV chronic patient had 100% homology with the nucleotide sequence having

accession number gb|HQ008866.1| available in Gene bank. The comparative data of the

nucleotide sequence of cloned Hepatitis B surface gene and Gene bank sequence

gb|HQ008866.1 is presented in Annexure Fig 2.6. The DNA sequence of cloned HBs-Ag

had submitted to Gene bank with accession number AM991999.

3.3.2. Hepatitis B Core gene, 549bp

The sequence data of cloned DNA core region “549” bp DNA fragment had 100%

homology with the DNA sequence listed in the Gene bank as “X02496.1”.

3.3.3. Cloned Chimeric genes

The DNA sequencing and corresponding amino acids data of the cloned chimera“csc-1,

csc-2, csc-3, csc-4 and csc-5 are presented in Figs.3.5, 3.6, 3.7, 3.8, 3.9. The DNA

sequence obtained for chimeric constructs confirmed the insertion DNA sequence of

Hepatitis B surface epitopes in the core gene. Green represents: Core 1-78amino acid and

80-144 amino acid, others represents: PreS epitopes: 12-32, 32-53, 124-147, 1-53 and 1-

42 amino acids.

http://www.ncbi.nlm.nih.gov/nucleotide/317016963?report=genbank&log$=nuclalign&blast_rank=2&RID=NWJVT0GY01N

Results

53

A NcoI ccatggacattgatccttataaagaatttggagctactgtggagttactctcgtttttgccttctgacttctttccttcagtacgagatcttctagataacgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaatactgtgctggggggaactaatgactctagctacctgggtgggtggtaatttggaagatgtcgacggattctttcccgaccaccagttggatccagccttcagagcaaacaccgcaaa tc SalI cagattggaagcttatatccagggacctagtagtcagttatgtcaacactaatatgggc HindIII ctaaaattcaggcaactattgtggtttcacatttcttgtctcacttttggaagagaaacagttatagagtatttggtgtcttttggagtgtggattcgcactcctccagcttatagaccaccaaatgcccctatcttatcaacacttccgctcgagcaccaccaccaccaccac

XhoI

B 5'3' Frame 2 Met D I D P Y K E F G A T V E L L S F L P S D F F P S V R D L L D N A S A L Y R E A L E S P E H C S P H H T A L R Q A I L C W G E L Met T L A T W V G G N L E D V D G F F P D H Q L D P A F R A N T A N P D W K L I S R D L V V S Y V N T N Met G L K F R Q L L W F H I S C L T F G R E T V I E Y L V S F G V W I R T P P A Y R P P N A P I L S T L P L E H H H H H H

C 1 -ATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCT - 60 1 - M D I D P Y K E F G A T V E L L S F L P - 20 61 - TCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAACGCCTCAGCTCTGTATCGGGAA - 120 21 - S D F F P S V R D L L D N A S A L Y R E - 40 121 - GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATACTG - 180 41 - A L E S P E H C S P H H T A L R Q A I L - 60 181 - TGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGGTAATTTGGAAGATGTCGAC - 240 61 - C W G E L M T L A T W V G G N L E D V D - 80 241 - GGATTCTTTCCCGACCACCAGTTGGATCCAGCCTTCAGAGCAAACACCGCAAATCCAGAT - 300 81 - G F F P D H Q L D P A F R A N T A N P D - 100 301 - TGGAAGCTTATATCCAGGGACCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTAAAA - 360 101 - W K L I S R D L V V S Y V N T N M G L K - 120 361 - TTCAGGCAACTATTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTTATA - 420 121 - F R Q L L W F H I S C L T F G R E T V I - 140 421 - GAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCAAAT - 480 141 - E Y L V S F G V W I R T P P A Y R P P N - 160 481 - GCCCCTATCTTATCAACACTTCCGCTCGAG - 510 161 - A P I L S T L P L E X - 180

Figure 3.5: Nucleotide and amino acid sequence of csc-1 gene; A represents, nucleotide sequence alongwith restriction enzyme sites; B: deduced amino acid translation of csc-1gene and C: alignment of DNA sequence to amino acids sequence.

http://expasy.ch/cgi-bin/dna_sequences?/work/expasy/tmp/http/seqdna.22038,2

Results

54

A NcoI

ccAtggacattgatccttataaagaatttggagctactgtggagttactctcgtttttgccttctgacttctttccttcagtacgagatcttctagataacgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaatactgtgctggggggaactaatgactctagctacctgggtgggtggtaatttggaagatgtcgactgggacttcaatcccaacaagga SalI cacctggccagacgccaacaaggtaggagctggagcattcgggaagcttatatccagggacctagtagtcagttatgtcaacac

HindIII Taatatgggcctaaaattcaggcaactattgtggtttcacatttcttgtctcacttttggaagagaaacagttatagagtatttggtgtcttttggagtgtggattcgcactcctccagcttatagaccaccaaatgcccctatcttatcaacacttccgctcgagcaccaccaccaccaccac XhoI

B

5'3' Frame 2

Met D I D P Y K E F G A T V E L L S F L P S D F F P S V R D L L D N A S A L Y R E A L E S P E H C S P H H T A L R Q A I L C W G E L Met T L A T W V G G N L E D V D W D F N P N K D T W P D A N K V G A G A F G K L I S R D L V V S Y V N T N Met G L K F R Q L L W F H I S C L T F G R E T V I E Y L V S F G V W I R T P P A Y R P P N A P I L S T L P L E H H H H H H

C

1 - ATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCT - 60 1 - M D I D P Y K E F G A T V E L L S F L P - 20 61 - TCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAACGCCTCAGCTCTGTATCGGGAA - 120 21 - S D F F P S V R D L L D N A S A L Y R E - 40 121 - GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATACTG - 180 41 - A L E S P E H C S P H H T A L R Q A I L - 60 181 - TGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGGTAATTTGGAAGATGTCGAC - 240 61 - C W G E L M T L A T W V G G N L E D V D - 80 241 - TGGGACTTCAATCCCAACAAGGACACCTGGCCAGACGCCAACAAGGTAGGAGCTGGAGCA - 300 81 - W D F N P N K D T W P D A N K V G A G A - 100 301 - TTCGGGAAGCTTATATCCAGGGACCTAGTAGTCAGTTATGTCAACACTAATATGGGCCTA - 360 101 - F G K L I S R D L V V S Y V N T N M G L - 120 361 - AAATTCAGGCAACTATTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACAGTT - 420 121 - K F R Q L L W F H I S C L T F G R E T V - 140 421 - ATAGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCACCA - 480 141 - I E Y L V S F G V W I R T P P A Y R P P - 160 481 - AATGCCCCTATCTTATCAACACTTCCGCTCGAG - 513 161 - N A P I L S T L P L E X - 180

Figure 3.6: Nucleotide and amino acid sequence of csc-2 gene; A represents, nucleotide sequence alongwith restriction enzyme sites; B: deduced amino acid translation of csc-2 gene and C: alignment of DNA sequence to amino acids sequence.

http://expasy.ch/cgi-bin/dna_sequences?/work/expasy/tmp/http/seqdna.24931,2

Results

55

A

NcoI ccAtggacattgatccttataaagaatttggagctactgtggagttactctcgtttttgccttctgacttctttccttcagtacgagatcttctagataacgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaatactgtgctggggggaactaatgactctagctacctgggtgggtggtaatttggaagatgtcgactgcacgactcctgctcaaggaacctctatgtatccctcctgttg SalI Ctgtaccaaaccttcggacggaaattgcaagcttatatccagggacctagtagtcagttatgtcaacacta HindIII atatgggcctaaaattcaggcaactattgtggtttcacatttcttgtctcacttttggaagagaaacagttatagagtatttggtgtcttttggagtgtggattcgcactcctccagcttatagaccaccaaatgcccctatcttatcaacacttccgctcgagcaccaccaccaccaccac XhoI

B

Met D I D P Y K E F G A T V E L L S F L P S D F F P S V R D L L D N A S A L Y R E A L E S P E H C S P H H T A L R Q A I L C W G E L Met T L A T W V G G N L E D V D C T T P A Q G T S Met Y P S C C C T K P S D G N C K L I S R D L V V S Y V N T N Met G L K F R Q L L W F H I S C L T F G R E T V I E Y L V S F G V W I R T P P A Y R P P N A P I L S T L P L E H H H H H H

C

1 - ATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCT - 60 1 - M D I D P Y K E F G A T V E L L S F L P - 20 61 - TCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAACGCCTCAGCTCTGTATCGGGAA - 120 21 - S D F F P S V R D L L D N A S A L Y R E - 40 121 - GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATACTG - 180 41 - A L E S P E H C S P H H T A L R Q A I L - 60 181 - TGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGGTAATTTGGAAGATGTCGAC - 240 61 - C W G E L M T L A T W V G G N L E D V D - 80 241 - TGCACGACTCCTGCTCAAGGAACCTCTATGTATCCCTCCTGTTGCTGTACCAAACCTTCG - 300 81 - C T T P A Q G T S M Y P S C C C T K P S - 100 301 - GACGGAAATTGCAAGCTTATATCCAGGGACCTAGTAGTCAGTTATGTCAACACTAATATG - 360 101 - D G N C K L I S R D L V V S Y V N T N M - 120 361 - GGCCTAAAATTCAGGCAACTATTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAA - 420 121 - G L K F R Q L L W F H I S C L T F G R E - 140 421 - ACAGTTATAGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGA - 480 141 - T V I E Y L V S F G V W I R T P P A Y R - 160 481 - CCACCAAATGCCCCTATCTTATCAACACTTCCGCTCGAG - 519 161 - P P N A P I L S T L P L E X - 180

Figure 3.7: Nucleotide and amino acid sequence of csc-3 gene; A represents, nucleotide sequence alongwith restriction enzyme sites; B: deduced amino acid translation of csc-3gene and C: alignment of DNA sequence to amino acids sequence.

Results

56

A

NcoI

ccAtggacattgatccttataaagaatttggagctactgtggagttactctcgtttttgccttctgacttctttccttcagtacgagatcttctagataacgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaatactgtgctggggggaactaatgactctagctacctgggtgggtggtaatttggaagatgtcgacatggggcagaatctttccaccag SalI caatcctctgggattctttcccgaccaccagttggatccagccttcagagcaaacaccgcaaatccagattgggacttcaatcccaacaaggacacctggccagacgccaacaaggtaggagctggagcattcgggaagcttatatccagggacctagtagtcagttat HindIII gtcaacactaatatgggcctaaaattcaggcaactattgtggtttcacatttcttgtctcacttttggaagagaaacagttatagagtatttggtgtcttttggagtgtggattcgcactcctccagcttatagaccaccaaatgcccctatcttatcaacacttccgctcgag caccaccaccaccaccac XhoI

B

Met D I D P Y K E F G A T V E L L S F L P S D F F P S V R D L L D N A S A L Y R E A L E S P E H C S P H H T A L R Q A I L C W G E L Met T L A T W V G G N L E D V D Met G Q N L S T S N P L G F F P D H Q L D P A F R A N T A N P D W D F N P N K D T W P D A N K V G A G A F G K L I S R D L V V S Y V N T N Met G L K F R Q L L W F H I S C L T F G R E T V I E Y L V S F G V W I R T P P A Y R P P N A P I L S T L P L E H H H H H H

C

1 - ATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCT - 60 1 - M D I D P Y K E F G A T V E L L S F L P - 20 61 - TCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAACGCCTCAGCTCTGTATCGGGAA - 120 21 - S D F F P S V R D L L D N A S A L Y R E - 40 121 - GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATACTG - 180 41 - A L E S P E H C S P H H T A L R Q A I L - 60 181 - TGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGGTAATTTGGAAGATGTCGAC - 240 61 - C W G E L M T L A T W V G G N L E D V D - 80 241 - ATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCACCAGTTGGAT - 300 81 - M G Q N L S T S N P L G F F P D H Q L D - 100 301 - CCAGCCTTCAGAGCAAACACCGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACACC - 360 101 - P A F R A N T A N P D W D F N P N K D T - 120 361 - TGGCCAGACGCCAACAAGGTAGGAGCTGGAGCATTCGGGAAGCTTATATCCAGGGACCTA - 420 121 - W P D A N K V G A G A F G K L I S R D L - 140 421 - GTAGTCAGTTATGTCAACACTAATATGGGCCTAAAATTCAGGCAACTATTGTGGTTTCAC - 480 141 - V V S Y V N T N M G L K F R Q L L W F H - 160 481 - ATTTCTTGTCTCACTTTTGGAAGAGAAACAGTTATAGAGTATTTGGTGTCTTTTGGAGTG - 540 161 - I S C L T F G R E T V I E Y L V S F G V - 180 541 - TGGATTCGCACTCCTCCAGCTTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG - 600 181 - W I R T P P A Y R P P N A P I L S T L P - 200 601 - CTCGAG - 606 201 - L E X - 220

Figure 3. 8: DNA sequence and amino acid sequence of csc-4 gene; A represents, nucleotide sequence alongwith restriction enzyme sites; B: deduced amino acid translation of csc-4 gene and C: alignment of DNA sequence to amino acids sequence.

Results

57

A NcoI

ccatggacattgatccttataaagaatttggagctactgtggagttactctcgtttttgccttctgacttctttccttcagtacgagatcttctagataacgcctcagctctgtatcgggaagccttagagtctcctgagcattgttcacctcaccatactgcactcaggcaagcaatactgtgctggggggaactaatgactctagctacctgggtgggtggtaatttggaagatgtcgacatggggcagaatctttccaccag SalI caatcctctgggattctttcccgaccaccagttggatccagccttcagagcaaacaccgcaaatccagattgggacttcaatcccaacaaggacacctggccagacaagcttatatccagggacctagtagtcagttatgtcaacactaatatgggcctaaaattcaggca HindIII actattgtggtttcacatttcttgtctcacttttggaagagaaacagttatagagtatttggtgtcttttggagtgtggattcgcactcctccagcttatagaccaccaaatgcccctatcttatcaacacttccgctcgag caccaccaccaccaccac

XhoI B Met D I D P Y K E F G A T V E L L S F L P S D F F P S V R D L L D N A S A L Y R E A L E S P E H C S P H H T A L R Q A I L C W G E L Met T L A T W V G G N L E D V D Met G Q N L S T S N P L G F F P D H Q L D P A F R A N T A N P D W D F N P N K D T W P D K L I S R D L V V S Y V N T N Met G L K F R Q L L W F H I S C L T F G R E T V I E Y L V S F G V W I R T P P A Y R P P N A P I L S T L P L E H H H H H

C 1 - ATGGACATTGATCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTCTCGTTTTTGCCT - 60 1 - M D I D P Y K E F G A T V E L L S F L P - 20 61 - TCTGACTTCTTTCCTTCAGTACGAGATCTTCTAGATAACGCCTCAGCTCTGTATCGGGAA - 120 21 - S D F F P S V R D L L D N A S A L Y R E - 40 121 - GCCTTAGAGTCTCCTGAGCATTGTTCACCTCACCATACTGCACTCAGGCAAGCAATACTG - 180 41 - A L E S P E H C S P H H T A L R Q A I L - 60 181 - TGCTGGGGGGAACTAATGACTCTAGCTACCTGGGTGGGTGGTAATTTGGAAGATGTCGAC - 240 61 - C W G E L M T L A T W V G G N L E D V D - 80 241 - ATGGGGCAGAATCTTTCCACCAGCAATCCTCTGGGATTCTTTCCCGACCACCAGTTGGAT - 300 81 - M G Q N L S T S N P L G F F P D H Q L D - 100 301 - CCAGCCTTCAGAGCAAACACCGCAAATCCAGATTGGGACTTCAATCCCAACAAGGACACC - 360 101 - P A F R A N T A N P D W D F N P N K D T - 120 361 - TGGCCAGACAAGCTTATATCCAGGGACCTAGTAGTCAGTTATGTCAACACTAATATGGGC - 420 121 - W P D K L I S R D L V V S Y V N T N M G - 140 421 - CTAAAATTCAGGCAACTATTGTGGTTTCACATTTCTTGTCTCACTTTTGGAAGAGAAACA - 480 141 - L K F R Q L L W F H I S C L T F G R E T - 160 481 - GTTATAGAGTATTTGGTGTCTTTTGGAGTGTGGATTCGCACTCCTCCAGCTTATAGACCA - 540 161 - V I E Y L V S F G V W I R T P P A Y R P - 180 541 - CCAAATGCCCCTATCTTATCAACACTTCCGCTCGAG - 576 181 - P N A P I L S T L P L E X - 200

Figure 3.9: Nucleotide and amino acid sequence of csc-5 gene; A represents, nucleotide sequence alongwith restriction enzyme sites; B: deduced amino acid translation of csc-5 gene and C: alignment of DNA sequence to amino acids sequence.

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3.4. Expression and Purification of Chimeric Proteins

To examine the expression of chimeric DNA constructs (pIJMcsc-1, pIJMcsc-2,

pIJMcsc-3, pIJMcsc-4, pIJMcsc-5), the engineered E.coli strains (E. coli: IJMcsc-1, E.

coli: IJMcsc-2, E. coli: IJMcsc-3, E. coli: IJMcsc-4, E. coli: IJMcsc-5) harboring

respective chimeric constructs were grown overnight in LB media containing kanamycin

(50µg/ml) and inducted by lactose/glucose. The bacterial cells were harvested, lysed and

fractionated into supernatant and inclusion bodies forms as cell pellets (See Materials &

Methods).

3.4.1. SDS-PAGE analysis

The fractions collected or the extracts obtained from transformed E.coli strain and E.coli

strain containing the vector alone were analyzed on SDS-PAGE (Fig.3.10). The

electrophoresis analysis of proteins demonstrated that chimeric constructs encodes for

respective chimeric proteins and expression of proteins was seen both in supernatant and

pellet fractions of E.coli strains containing chimeric constructs. It was observed that the

chimeric proteins were highly expressed in pellet fraction.

The purified extracts / fractions containing the chimeric proteins possessed about 20 kDa

(Wt), 20 kDa, 24 kDa and 23kDa of molecular weight for chimeric plasmids, pIJMcsc-1,

pIJMcsc-2 pIJMcsc-4 and pIJMcsc-5 as judged on 10% SDS-PAGE. The present

purification procedure adopted was not able to purify the chimeric protein expressed by

Plasmid “pIJMcsc-3”. So it was not possible to present the result of chimeric protein

“csc-3” for unknown reasons. The results of other proteins are presented in Figure 3.11.

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59

csc-1 csc-2 csc-3

csc-5 csc-4

Figure 3.10: 10% polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate. M: protein molecular weight standard; lane 1: BL21 (supernatant); lane 2-3: BL21 (DE3)+pET28a (Supernatant and pellet) respectively; lane 4-5: Respective chimeric constructs csc-1,csc-2,csc-3,csc-4, csc-5 (Supernatant and pellet) .

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60

csc-1 csc-2

csc-5 csc-4 Figure 3.11: Purified expressed proteins by talon affinity chromatography. M: protein marker’1-7: purified protein. a). Protein “csc-1” 20kDa, b) protein “csc-2” 20kDa; c) protein “csc-4” 24kDa d) protein “csc-5” 23kDa.

Results

61

3.4.2. Further characterization of chimeric csc-5 harboring PreS1

The csc-5 protein was effectively expressed and purified from E.coli. Western blot

analysis using HBc-specific antibodies and PreS identified the chimeric csc-5 protein at

the expected size of 23kDa (Figure 3.12, 3.13). Thus, the chimeric csc-5 protein was

intact and was therefore considered as suitable for immunogenicity studies.

Figure 3. 12: Western blot analysis of the csc-5 for anti-HBc. Protein samples were run on a 12% SDS-PAGE and blotted onto a nitrocellulose (NC) membrane. The membrane was then probed with a rabbit monoclonal anti-HBc which recognizes amino acid residues of hepatitis B core. lane M: Magic marker; lane 1: negative control; lane 2: E.coli derived HBc reference standard; lane 3-7: 1µg, 0.5µg, 0.25µg and 0.0625 µ E.coli derived purified csc-5 protein.

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62

Figure 3.13: Western blot analysis of the csc-5 for anti-PreS. Protein samples were run on a 12% SDS-PAGE and blotted onto a nitrocellulose (NC) membrane. The membrane was then probed with a mouse monoclonal anti-preS which recognizes amino acid residues of hepatitis B preS. lane M: Magic marker; lane 1: negative control; lane 2-6: 1µ, 0.5µg, 0.25µg, 0.125µg and 0.0625 µg E.coli derived purified csc-5 protein.

3.5. Determination of immunogenicity The immunogenicity of chimeric proteins generated by chimeric plasmids was evaluated

by enzyme linked immunosorbent assay (ELISA) as described in Materials & Methods.

Group of 4 female C57BL/6J mice were immunized subcutaneously at the base of tail

with 10µg of purified chimeric protein (as measured by Bradford assay) in Incomplete

Freunds adjuvant, followed by a booster dose of same antigen in Incomplete Freunds

adjuvants at interval of 4 week. Sera were collected at 2, 4, and 6 week.

3.5.1. Immune response induced by subcutaneous immunization

The immunogenicity of the chimeric proteins presenting csc-5 was evaluated separately

by the subcutaneously administration of 10µg of protein (at 0 week and week 4) to 6-

week old female C57BL/6J mice. One group of 4 mice each was immunized with csc-5.

All preparations were formulated with Incomplete Freunds adjuvants according to the

standard procedure of laboratory (Karolinska Institutet, Sweden), immunization with

Results

63

chimeric csc-5 proteins. Serum samples of immunized and non-immunized mice were

analyzed by ELISA assay.

3.5.1.1. csc-5 protein

The chimeric protein was recognized by both classical anti-HBc antibodies and by a

monoclonal antibody to pre-S1. To investigate whether antibodies could be raised

simultaneously against the HBcAg and PreS1-42. Sera were collected from four mice and

tested by ELISA. Serum samples from four mice showed strong response at week six,

none at week two and one out of four mice at week four. The OD values suggested that

chimeric protein containing HBcAg and PreS1-42 were immunogenic, with the priming

anti-HBc and PreS1-42 of antibodies. The graph was plotted as: Mean anti HBc-Ag and

PreS1-42 against a function of time. The graph shows that higher antibody titers were

observed against both HBcAg and Pres1-42 upto dilution of 1: 2160 as shown by (Figure

3.14). The result shows that HBcAg increase the immune response of the PreS1 carrier

molecule.

Figure 3.14: Mean antiHBcAg and HBsAg (1-42) IgG antibody titer after one (w2, w4) or two (w6) s.c immunizations with 10µg of the chimeric protein (csc-5) in C57BL/6J. Values are given as end-point titers (+SD), also showing the number of mice with detectable antibody titer at each timepoint.

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64

3.6. ELISpot Assay

Groups of four C57BL/6J mice were immunized twice, with10µg and 15µg chimeric

protein (csc-5) and monitored for activation of HBcAg and PreS1-42 specific IFN-ɣ-

producing T cells determined two weeks after the second vaccination (week 6). The

results show that HBcAg and PreS1-42 specific producing T cells were not detectable at

week 2 but became detectable 6 week after vaccination (Figure. 3.15). We found that the

booster dose of 15µg of chimeric protein delivered subcutaneously primed HBcAg and

PreS1-42 specific IFN-ɣ-producing T cells, whereas the first dose failed to do so.

3.6.1. Immunogenicity of chimeric HBcAg expressing PreS1

The immunogenicity of the csc-5 was evaluated by immunization with a comparatively

low dose of csc-5 protein (10µg/dose) in C57BL/6 mice. This revealed that two

immunizations led to a significant anti-HBc and anti-PreS1 response in vivo (Figure

3.14). The major increase in antibody levels appeared two weeks after the second

immunization (Figure 3.14). This strongly suggests that the chimeric csc-5 protein

effectively induces potentially neutralizing antibodies to PreS1, which is one goal with

the vaccine construct

The second feature of the csc-5 protein is that it should also be able to induce T cells to

HBcAg. The ability to prime HBcAg-specific T cells was determined by using an

ELISpot assay that measures the IFN-production by T cells from immunized mice in

vitro. HBcAg-specific IFN-producing T cells were effectively primed by the csc-5

protein in vivo (Figure 3.15). As expected, no HBcAg-specific CTLs were primed, since

the insert was located to the tip of the spike. The spike region needs to be intact for an

effective activation of HBcAg-specific CTLs due to a binding to naïve B cells. However,

this confirms that the csc-5 protein activates both potentially neutralizing anti-PreS1

antibodies and HBcAg-specific IFN-producing T cells, which are the two central

functions needed in a therapeutic vaccine for chronic HBV infections.

Results

65

Figure 3.15: Evaluation of the immunogenicity of two doses (10µg and 15µg) of chimeric protein (csc-5) subcutaneously immunized C57BL/6J mice by enzyme linked immunospot Assay (ELISpot). Group of 4 mice were immunized subcutaneously with two doses of 10µg and 15µg chimeric protein, and IFN-ɣ-production was detected at week 2 and week 6 after first and booster dose immunization.

3.7. Other Chimeric Proteins evaluated for Immunogenicity

3.7.1. csc-1 protein

Both classical anti-HBc antibodies and a monoclonal antibody to pre-S1 were used to

evaluate the chimeric purified protein, csc-1 that contains core and PreS12-32 epitopes.

The results of this evaluation are presented in Figure 3.16. Both antibodies used were

highly reactive with the purified protein and were immunogenic in nature. The graph

(Figure 3.16) represents that higher antibody titer was observed against both core and

PreS up to dilution factor of 1:2160. ELISA analysis demonstrated that the chimeric

protein had specific antigenicity for both anti-core and anti-preS antibody.

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66

Figure 3.16: Antigenicity of csc-1 protein synthesized and purified. ELISA of csc-1 protein (bound to solid support) for monoclonal anti-HBc (Red) antibody and monoclonal anti-preS antibody (Green) with serial dilution of csc-1 protein.

Results

67


To investigate the antigenicity against HBc, we used polyclonal antibodies. The graph

was plotted between mean OD values for anti HBcAg against a function of dilution

factor. The chimeric protein was recognized by anti-HBc antibodies up to dilution of

1:2160 as shown by (Figure 3.17).

Figure 3.17: Antigenicity of csc-2 protein synthesized and purified. ELISA of csc-2

protein (bound to solid support) for monoclonal anti-HBc (Red) antibody with decreasing

concentration of csc-2 protein.


Enzyme linked immunosorbent assay using the purified chimeric protein (csc-4) as

capturing antigen for detecting antiHBcAg antibody was established. Figure 3.18. Shows

the results of assay for detecting the anti-core antibody when OD graph was plotted

between mean anti HBcAg against a function of dilution factor. The graph represents that

higher antibody titer was observed against core up to dilution of 1:2160 as shown by (Fig

3. 18).

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68

Figure 3. 18: Antigenicity of csc-4 protein synthesized and purified. ELISA of csc-4

protein (bound to solid support) for monoclonal anti-HBc (Red) antibody with dilution of

csc-4 protein.

Discussion

Chapter 4

Discussion

69

Discussion

Therapy for chronic infection caused by HBV has improved significantly in the past 15

years with the introduction of small molecule drugs (Yang and Roberts, 2010; Woo et

al., 2010). These are effective at suppressing HBV replication during therapy. However,

the drugs available today can only slightly improve the sustained off-therapy responses

signified by a HBeAg to anti-HBeAg seroconversion above the annual 10% spontaneous

seroconversion rate (Woo et al., 2010). Thus, new therapies that can improve the

sustained off-therapy response rates are needed.

It has been well documented that the seroconversion from HBeAg to anti-HBe is

associated with an activation of the endogenous T cell response to HBV. A highly

attractive approach to improve sustained response rates is therefore therapeutic

vaccination approach whereby the host T cell becomes specifically activated, or

reactivated. This is attractive in particular in the light of the now highly active small

molecule antivirals since these can be use to pre-treat the patient for a longer period.

After 6 to 12 months of effective suppression of HBV replication and reduced presence

of viral antigens, the T-cell tolerance becomes less pronounced. Thus, a combination of

these small molecules with a therapeutic vaccine would hit the virus by two different

mechanisms and may be able help the host to control the infection.

In the present study, an attempt has been made to develop a therapeutic vaccine

candidate that should be able to induce a polyclonal immune response and T-cell

response. To achieve this objective, five chimeric constructs pIJMcsc-1, pIJMcsc-2,

pIJMcsc-3, PIJMcsc-4 and pIJMcsc-5, were developed containing core regions as a

carrier molecule for the epitopes of preS regions (see Figure 4.1).

Discussion

70

Figure 4.1: Construction of chimeras using core and surface region.

Previous studies reported that HBV core protein has much potential to be used for the

production of T cell response in the body. The important Th and CTL epitopes present in

hepatitis B virus core antigen comprising amino acid number 1-20, 50-69, 117-131

(Milich et al., 1987). While amino acids number 18-27(Milich et al., 1987), 84-91 and

88-95 (Ehata et al., 1992) are the good candidate of CTL epitopes.

Core regions encoding amino acid 1-78 and 80-144 was selected as carrier molecule in

this study. One of the reasons for the selecting of this region was the presence of

assembly domain (1-149) and absence of protease sensitive domain that is located at the

protein region 145-153 (Seifer and Standring, 1994). From the point of immunogenicity,

the best place for the insertion of any epitope in core protein is the outer loop of core

protein and at amino acid number 80 (Argos and Fuller, 1988). Further, HBcAg loop

was focused as a target for insertion of the epitope of preS regions. It is well

documented that the major epitopes of preS1 region was conformational and its specific

antibody was virus-precipitating (Alberti et al., 1990; Heermann et al., 1987). It is

documented that incorporation of preS1 (21-47) into prophylactic or therapeutic

vaccines might be a potential strategy to overcome non-responsiveness to HBsAg

vaccines or to develop new therapeutic vaccines against HBV (Milich, 1988).

Discussion

71

Earlier reports (Eyigun et al., 1998; Madalinski et al., 2001; Leroux-Roels et al., 1997)

indicated the effect of vaccines carrying preS1, but the antibody responses against preS1

are very weak and the introduction of preS1 did not lead to obvious improvements in the

present HBsAg vaccines. The region between positions 78 and 83 of HBcAg (Major

Immunodominant region MIR) is surface accessible (Zlotnick et al., 1997; Conway et al.,

1998). Thus the position is an ideal site for foreign epitope inserting. Some reports have

mentioned that the immunogenicity of preS1 epitope could be improved when fused in

MIR (Hui et al., 1999a; 1999b; Xu et al., 1994).

HBc carrier could provide a high level of B cell and T cell immunogenicity for foreign

epitopes. Being a display carrier for foreign epitopes, HBc has more advantages over

other proposed particulate carriers.(Pumpens and Green, 1999). In this study, assembly

domain encoding (1-144) sequence of HBc was cloned and one amino acids was deleted

(amino acid 79) in the MIR in order to minimize the interfering of HBc MIR to foreign

epitope-specific immune response. Based on these advantages, five epitopes of PreS were

inserted into MIR of HBcAg to further improve the immunogenicity of PreS regions.

Full length core gene, full length surface gene and five constructed chimeric genes

(Figure 3.5, 3.6, 3.7, 3.8 and 3.9) were sequenced and amino acids sequences of these

proteins were deduced using ExPasy Program. The results show that this sequence of

constructs belong to genotype D ( ayw serotype) which is most prevalent in South east

Asia and thus good candidate to develop therapeutic vaccine for local people in Pakistan

and for this region.

All five chimeric proteins (csc-1, csc-2, csc-3, csc-4, and csc-5) harboring Hepatitis B

core segments or regions, as a carrier molecule for the epitopes of preS regions were

highly expressed in the form of inclusion bodies and generated 20 kDa, 23kDa and 24

kDa proteins on SDS-PAGE (Figure 3.10). The best immunogenicity results which

stimulate T cell response were found with chimeric protein (csc-5).

Discussion

72

It has been documented that HBcAg aggregates upon expression in E.coli (Cohen and

Richmond, 1982). Core form dimers which can be dissociated by strong denaturing and

reducing conditions during SDS-PAGE. Self assembly is mediated by protein protein

interactions of the core protein domain from the N-terminus to amino acid 144. The

following arginine-rich domain is responsible for nucleic acid binding but it does not play

a role for assembly (Birnbaum and Nassal, 1990). HBcAg has four cysteine residues, thus

a large number of potential disulfide bonding patterns are possible. No intra-chain

disulfide bonding patterns are possible. No intra-chain disulfide bonds exist but two to

three inter-chain disulfide bonds can occur (Zheng et al., 1992). In the present study, the

expressed protein is devoid of arginine rich domain in order to avoid binding with nucleic

acid and solublized by dissolving in denaturing agent 6M guanidine chloride.

To simplify and optimize purification conditions, expressed chimeric proteins has six

histidine residues at the C-terminal allowing the purification of chimeric protein under

denaturing conditions via talon affinity chromatography at pH 7:00. It was found that

after lysis of E.coli cells, chimeras proteins were present in the unsoluble precipitate of

cell lysate. This might be the result of aggregation to unsoluble inclusion bodies.

However resuspension of the precipitated material in 6M guanidine chloride solubulized

the proteins such that the histidine residues were exposed and could bind efficiently to the

talon affinity column.

It has been described that the fusion of foreign sequences to the C-terminus allows in

some cases the detection with specific antibodies indicating the outside localization of the

foreign epitopes (von Brunn et al., 1993; Borisova et al., 1998). Since the HBc capsids

contain holes on the particulate surfaces (Bottcher et al., 1997; Conway et al., 1997).

Crowther et al. (1994) speculated that the foreign polypeptide chains emerged through

the holes thus becoming localized on the surface and accessible to antibodies. Therefore,

it was thought that a C-terminal hexa-histidine tag would be more exposed on the particle

surface than a N-terminal extension described previously as being surface inaccessible

(Karpenko et al., 1997). However, the results showed that the C-terminal hex-histidine

tag could not be reached for purification purpose that the capsid shell as suggested by

Discussion

73

Wingfield et al. (1995) or alternatively not accessible in E.coli expressed misfolded

structures.

By combining denaturation, affinity purification based on His tagged and renaturation

processes, a method applied for the purification of all chimeric proteins had high yields

and to near homogeneity (Figure 2.11). Only four csc-1, csc-2, csc-4 and csc-5 chimeric

proteins were successfully used to investigate their immunogenicity. With the present

protein purification procedure, it was not possible to purify csc-3 protein.

Only, four chimeric proteins (csc-1, csc-2, csc-4 and csc-5) were tested in vitro for

antigenicity and immunogenicity using ELISA. These proteins showed antibody core

response in vitro (Figure 3.14, 3.16, 3.17, and 3.18) and showed antibody response upto

dilution of 2160. In vivo study was performed only for chimeric protein (csc-5). Further,

it was identified by western blot analysis (Figure 3.12 and 3.13) that the preS1 (1-42)

fragment was the major immunogenic domain in the preS1 region in mouse. In the

western blot analysis for csc-5, an additional band of dimer appeared. The observation

suggested disulfide bonds might be responsible for the formation of dimers of chimeric

HBcAg. This conclusion is in agreement with the previous reported results (Zhou and

Standring, 1992; Zheng et al., 1992)

ELISA results (Figure 3.14) indicated that the antigenicity of preS1 (1-42) and core

region were significant. The results obtained were in contrast to study carried out

previously that hepatitis B core antigen gene bearing the 39 amino acid long domain A of

hepatitis B surface antigen within the hepatitis B core antigen immunodominant loop has

demonstrated HBs but not HBc antigenicity and elicited in mice B cell and T-cell

responses against native HBcAg and HBsAg as described by (Borisova, 1993).

Chimeric protein possesses not only antigenic but also immunogenic properties of

inserted sequence. Moreover, the inserted oligopeptide show relative higher antibody titer

while the level of anti-HBc antibodies is markedly lower. Further analysis of the

Discussion

74

antigenicity and immunogenicity of csc-5 confirmed that HBV bound polyclonal anti-

HBc and monoclonal anti-PreS1-42 by an ELISA (Figure 3.14).

These results indicated that insertion of preS1-42 amino acid in the immunodominant

region of hepatitis B core allowed the chimeric protein to maintain its immunogenicity

for both antiHBc and antiPreS epitopes. A previous report of Schodel et al. showed that

fusion of preS2 133-143 to the carboxyl terminus of HBcAg sequence of 1-156 did not

affect its assembly to particles and thus preserved the major HBcAg antibody-binding site

and T cell epitope (Schodel et al., 1992). However, our results have gone further, and

showed that the inserted preS 1-42 amino acid were not only surface accessible as

assayed by the ELISA, but that the chimeric protein also preserved its immunogenicity,

as it can elicit a anti-core and anti-preS response in C57BL/6J mice. This difference from

that of others (Schodel et al., 1992; Prange, et al., 1995), may be from the different length

of preS1 being introduced (Schodel et al., 1992; Hui et al., 1999).

Since HBVcsc-5 has the immunogenic and related characteristics of HBcAg and preS1, it

is hypothesized that csc-5 may be useful as a therapeutic vaccine of chronic HBV

infection. To test this, csc-5 with adjuvant IFA was used to immunize C57BL/6J mice.

Strong specific humoral immune response was induced in C57BL/6J (Figure 3.14) and

both anti-HBc and anti-PreS1 could be detected in sera of mice as early as 4 weeks after

their first immunization. Recently, Reidl et al., 2002 reported that recombinant HBcAg

containing aa 1-44 or aa1-149 induced an immune response which was Th2 biased

compared to with wild type HBcAg, which induced a Th1-biased immune response. They

noted that truncated HBcAg with aa1-144 or aa1-149 has drastically reduced nucleic acid

binding activity (>98%). They hypothesized that Th1-biased response obtained with wild

type HBcAg was facilitated by the trace amount of host bacterial RNA bound to its

arginine-rich carboxyl domain and is critical for HBV infection clearance

Herein, it is evident that a therapeutic vaccine candidate has been developed that should

be able to induce a polyclonal immune response. First, the inactivation of IFNɣ-

producing HBcAg-specific T cells should be highly antiviral since HBV replication is

Discussion

75

highly sensitive to IFNɣ (Guidotti and Chisari, 2006). Importantly, HBcAg-specific T

cells will enter the liver as local IFNɣ-producing T cells, which has been found to be

instrumental for the antiviral effect (Guidotti and Chisari, 2006). Next, we could show

that the chimeric protein (csc-5) effectively activated anti-PreS1 antibodies that have

been shown to neutralizing (Rehermann and Nascimbeni, 2005). Thus, these are the ideal

type of responses to be raised in a HBV chronically infected host. One additional branch

of the host response that may be in a HBV chronically infected host. One additional

branch of the host response that may be required in an optimal vaccine design is CTLs.

However, by introducing the PreS-insert at the tip of the spike we found that the ability of

exogenous csc-5 to prime HBcAg-specific CTLs was lost, which is fully consistent with

our previous data( Figure 3.15) Thus, to effective prime CTLs to the csc-5 protein one

could combine the chimeric protein based vaccine with a genetic (Nystrom et al., 2010).

In conclusion, it is clear that the csc-5 protein can induce HBV-specific antibodies and T

cells, two functions that well csc-5 protein can induce HBV-specific antibodies and T

cells, two functions that well complement the activity of antiviral compounds. Thus the

csc-5 protein may be a future component in a therapy for chronic HBV infections where

the host is gaining a sustained control of the infection.

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Chapter 5

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Appendix

Appendix 2.1. L B Medium ( Luria- Bertani Medium). For one Liter Trypton 10 g Yeast extract 5g NaCl 5g Shake to dissolve solutes. Adjust pH to 7.5 with 1N NaOH. Adjust volume of the solution to 1 liter with deionized water. Sterilize by autoclaving for 20 min at 15 lb/sq. on liquid cycle (Sambrook et al., 1989). Appendix 2. 2. Preparation of lysis buffer. 45mM MgCl2 : 4.28g 1M tris-HCl : 121.1g 450mM NaCl : 26.35g 1.5% Nonidot P-40 : 15ml Make volume with deoinized water upto one liter. Appendix 2.3. TE buffer 10mM Tris HCl ( pH 8.0) 1mM EDTA Appendix 2.4. 50X Tris-acetate EDTA buffer (TAE). Tris Acetate: 242 g Tris base 57.1 ml glacial acetic acid 100 ml 0.5 M EDTA (pH 8.0) Make up the final volume with distilled water to 1 liter. Ampicilin Stock (50 µl/ml) 1g ampicillin dissolves in 20ml distilled water. Filter sterilizes the solution with syringe and covers the aliquots with para-film and store at -20 °C.

Appendix 2.5. 6X loading dye. Dissolve 4g sucrose and 2.5mg of bromophenol blue in a 6ml solution of 10mM Tris

HCl, pH 8.0 and 1mM EDTA (TE buffer). Once dissolve bring up to volume of 10ml

with TE buffer. Store at room temperature.

Appendix 2.6. Amplification conditions for the surface, core, other epitopes and

constructs.

1. Amplification of Hepatitis B virus surface gene, 681bp

HBSF1 (GCG AAG CTT ATG GAG AAC ATC ACA TCA GG Forward) and HBSR1

(GAC CTC GAG CAT CCA ATG ACA TAG CCC ATG Reverse) were used to amplify

target hepatitis B surface antigen gene through PCR. A total reaction volume of 50 µl

was used for PCR containing 10 µl DNA as template extracted from HBV chronic patient

Final concentration of the reagent were, PCR Buffer IX, MgCl2 1.5mM, dNTPs 0.15mM,

two set of primers 25 pmol concentration each per reaction, Taq DNA polymerase 1 unit

per reaction and 10 µl DNA extracted from 100 µl plasma of HBV infected patient as

template for regular PCR reaction negative control was also included in the PCR

amplification reaction to make it more authentic and reliable. The reaction mixture and

temperature profile used for regular is given below.

Reagents concentration Volume used for one reaction

Double deionized distilled water 21.6 µl

Buffer (10X) 5 µl

MgCl2 (25mM) 5 µl

dNTPs (2.5mM) 4 µl

Forward Primer (HBSF1) (25pm/µl) 2 µl

Reverse Primer (HBSR1) (25pm/µl) 2 µl

Taq polymerase (5U/µl) 0.4 µl

Template DNA (from 100µl plasma) 10 µl

Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B virus surface gene

Steps Temperature Time

Lid temperature 105°C

1-Denaturation of DNA 95 °C 5 min (For first cycle only)

2-Denaturation of DNA 95 °C 45 sec

3-Annealing of primers 56 °C 45 sec

4-DNA amplification 72 °C 45 sec

5-GOTO 2 Rep 35 cycles

6- Completion 72 °C 15 min

7- Hold 22°C Enter

--------------------------------------------------------------------------------------------------

2. Amplification of Hepatitis B virus core gene (549bp).

HBCF1 (AGCGAATTCATGGACATTGATCCTTA) Forward and HBCR1 (CAC CTC

GAG CTA ACA TTG AGA TTC CCG Reverse) primers were used to amplify target

hepatitis B core antigen gene. A total reaction volume of 50 µl was used for PCR

containing 10 µl DNA as template extracted from HBV chronic patient.

The reaction mixture and temperature profile used for amplification is mentioned below:


Double deionized distilled water 26.5µl

Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer (HBCF1) (25pm/µl) 1 µl

Reverse Primer (HBCR1) (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B virus core gene


Lid temperature 105oC

1-Denaturation of DNA 94oC 5 min (For first cycle only)

2-Denaturation of DNA 94oC 45 sec

3-Annealing of primers 52oC 45 sec

4-DNA amplification 72oC 45 sec


6- Completion 72oC 15 min

7- Hold 22°C Enter

Amplification of epitopes.

1. Amplification of HBc Ag (1-78) DNA fragment: 234bp.

HBC1-78F (AGC GAA TTC ATG GAC ATT GAT CCT TA Forward) and HBC1-78R

(CGA GTC GAC ATC TTC CAA ATT ACC AC Reverse) primers were used to amplify

target hepatitis B core antigen gene. A total reaction volume of 50 µl was used for PCR

containing 2 µl DNA as template from plasmid pIJM.HBc-Ag.

The reaction mixture and temperature profile used for amplification is described below:



Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer (HBC1-78F) (25pm/µl) 1 µl

Reverse Primer (HBC1-78R) (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B core antigen gene (1-78)









7- Hold 22oC Enter

2. Amplification of HBc Ag (80-144) DNA fragment (195bp).

HBC80-144F (AC AAG CTT ATA TCC AGG GAC CTA GTA GTC Forward) and

HBC80-144R (CA CTC GAG TCG GAA GTG TTG ATA AGA TAG Reverse) primers

were used to amplify target hepatitis B core antigen gene. A total reaction volume of 50

µl was used for PCR containing 2 µl DNA as template from the plasmid pIJM.HBc-Ag.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer (HBc80-144F) (25pm/µl) 1 µl

Reverse Primer (HBc80-144R) (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B core antigen gene (80-144)









7- Hold 22oC Enter

3. Amplification of hepatitis B preS1 region 124-147 DNA fragment: 72bp.

HBPreS124-147F (TTAGTCGACTGCACGACTCCTGCT Forward) and HBPreS124-

147R (TTGAAGCTTGCAATTTCCGTCCGA Reverse) primers were used to amplify


containing 2 µl DNA as template from the plasmid pIJM.HBs-Ag.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer (HBS124-147F) (25pm/µl) 1 µl

Reverse Primer (HBS124-147R) (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B S antigen gene (124-147)









7- Hold 22oC Enter

4. Amplification of hepatitis B preS1 region (1-42) DNA fragment (126bp).

HBPreS1-42F (TTAGTCGAC ATG GGG CAG AAT CTT TC Forward) and HBPreS1-

42R (TTGAAGCTTGTCTGGCCAGGTGTCCT Reverse) primers were used to amplify


containing 2 µl DNA as template from the plasmid pIJMcsc-4.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer HBPreS1-42F (25pm/µl) 1 µl

Reverse Primer HBPreS1-42R (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B PreS1 antigen gene (1-42)









7- Hold 22oC Enter

5. Amplification of hepatitis B preS1 region (12-32) DNA fragment (63 bp).

PreS12-32F (GA GTC GAC GGA TTC TTT CCC GAC Forward) and PreS12-32R (GG

AAG CTT CCA ATC TGG ATT TGC GGT Reverse) primers were used to amplify






Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl

Forward Primer PreS12-32F (25pm/µl) 1 µl

Reverse Primer PreS12-32R (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B PreS1 antigen gene (12-

32)









7- Hold 22oC Enter

6. Amplification of hepatitis B preS1 region (32-53) DNA fragment (66bp).

PreS32-53F (TA GTC GAC TGG GAC TTC AAT CCC A Forward) and PreS32-53R

(TA AAG CTT CCC GAA TGC TCC AGC T Reverse) primers were used to amplify






Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl


Reverse Primer PreS32-53 R (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B PreS1 antigen gene (32-

53)








6- Completion 72°C 15 min

7- Hold 22oC Enter

7. Amplification of hepatitis B preS1 region (1-53) DNA fragment size (159bp).

PreS1-42F (TTAGTCGAC ATG GGG CAG AAT CTT TC Forward) and PreS32-53R

(TA AAG CTT CCC GAA TGC TCC AGC T Reverse) primers were used to amplify


containing 10 µl DNA as template extracted from HBV chronic patient




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl


Reverse Primer PreS32-53R (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B PreS1 antigen gene (1-53)









7- Hold 22oC Enter

------------------------------------------------------------------------------------------------

8. Amplification of chimeric (1-78-12-32-144) DNA fragment.

HBC1-78F (Forward) and HBC80-144R (Reverse) primers were used in order to confirm

the ligation of targeted amplify epitopes in expression vectors. A total reaction volume of

50 µl was used for PCR containing 2 µl DNA as template from plasmid pIJMcsc-1.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl


Reverse Primer (HBC80-144) (25pm/µl) 1 µl



Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B chimeric gene (1-78-12-

32-144)









7- Hold 22oC Enter


HBC1-78F (Forward) and HBC80-144R (Reverse) primers were used to in order to

confirm the liagtion of target amplify epitopes in expression vector (pET28a). A total

reaction volume of 50 µl was used for PCR containing 2µl DNA as template from the

plasmid pIJMcsc-2.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl





Total Volume 50 µl


53-144)









7- Hold 22oC Enter


HBC1-78F (Forward) and HBC80-144R (Reverse) primers were used for the

confirmation of ligated targeted epitopes in pET28a vector. A total reaction volume of 50

µl was used for PCR containing 2 µl DNA as template the plasmid pIJMcsc-4.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl





Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B chimeric gene (1-78-1-53-

144)









7- Hold 22oC Enter


HBC1-78F (Forward) and HBC80-144R (Reverse) primers were used in order to confirm

the liagtion of amplify targeted epitopes in pET28a expression vector. A total reaction

volume of 50 µl was used for PCR containing 2 µl DNA as template from the plasmid

pIJMcsc-5




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl





Total Volume 50 µl

Temperature profile used for PCR amplification of hepatitis B chimeric gene (1-78-1-42-

144)









7- Hold 22oC Enter


HBC1-78F (Forward) and HBC80-144R (Reverse) primers were used to confirm the

ligation of amplify targeted epitopes in expression vector pET288a. A total reaction

volume of 50 µl was used for PCR containing 2 µl DNA as template from the plasmid

pIJMcsc-3.




Buffer (10X) 5 µl

MgCl2 (25mM) 3 µl

dNTPs (2.5mM) 3 µl





Total Volume 50 µl


147-144)






4-DNA amplification 72+C 45 sec



7- Hold 22oC Enter

Appendix 2.7. Preparation of Heat Shock E. coli Competent Cells.

1. A single colony from a freshly grown plate of E. coli is picked and transferred

into 5 ml LB (Appendix 2.1) medium in test tube incubate at 37°C overnight

with vigorous shaking.

2. 2 ml of the overnight culture is taken and diluted to 50 ml in 250 ml flask and

shaken vigorously at 37°C for overnight.

3. 15 ml of the overnight culture is taken and diluted to 200 ml in 1-liter flask and

shaken vigorously at 37°C until the A600 reaches 0.45-0.55.

4. Culture is cooled by placing on the ice for 30 min. The cells are transferred

aseptically to sterile disposable 50 ml propylene tubes.

5. The cells are pelleted by centrifugation at 4000 rpm at 4°C for 5 min and

resuspend in 5 ml of 0.1M MgCl2.

6. The cells are pelleted by centrifugation at 4000 rpm at 4°C for 5 min and

resuspend in 5 ml of 0.1M CaCl2 and kept on ice for 30 min.

7. The cells are again pelleted by centrifugation at 4000 rpm for 5 min and

resuspend finally in appropriate amount of 0.1 M CaCl2 and sterile cold 100%

glycerol.

8. The cells are stored in aliquots of 200 µl at -70°C.

Precautions:

All tips, eppendorf tubes and solutions used here should be cold

Appendix 2.8. Solutions for Auto-induction.

Appendix 2.9. List of Transformants.

Sr. No Name of transformants

Description

1 E.coli: IJM.HBs-Ag Top 10 bacterial cells containing

plasmid(pIJM.HBs-Ag)

2 E.coli: IJM.HBc-Ag To10 bacterial cells containing plasmid

(pIJM.HBc-Ag)

3 E.coli: IJMcsc-1 BL21( DE3) bacterial cell containing plasmid ( pIJMcsc-1)





Solution Composition

1M MgSO4 Dissolved 246.5g of MgSo4.7H2O in ¾ of volume of deoinised water and then make volume up to 1 liter. Filter sterilize through a 0.22 um filter.

5052X5052 Glycerol: 250g, Glucose: 25g, ᾳ-Lactose: 100g and

make volume upto 1 liter with deionised water.

Lactose take take a long time to dissolve so brief

heating in oven may help to dissolve.

20XNPSC( Low

Phosphate buffer)

Na2HPO4: 71g, KH2PO4: 68g, NH4Cl: 53.5g,

Na2SO4:14.2g and make volume up to 1 liter with

deoionised water. Adjust the pH with 10M NaoH and

autoclave.

Correspondence: M. S ä llberg, Karolinska Institutet, Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska University Hospital, Huddinge, SE-141 86 Stockholm, Sweden. Tel: � 46 8 52483803. Fax: � 46 8 58587933. E-mail: [email protected]

(Received 18 February 2011 ; accepted 20 July 2011 )

Introduction

About 350 million people worldwide are chronically infected by hepatitis B virus (HBV), a member of the Hepadnaviridae family [1]. Chronic HBV infection is a major cause of severe liver disease and liver can-cer in Asia and Africa [1]. The virus consists of a partially double-stranded DNA of 3.3 kb in size encoding the viral envelope, core, polymerase, and X proteins [2]. The virus consists of a host-derived outer envelope containing HBV surface antigen (HBsAg) and an icosahedral nucleocapsid assembled from the core antigen carrying the viral genome [2]. The core protein is encoded by the core gene and is composed of 183 or 185 amino acid residues (21 kDa) that spontaneously assemble into virus-like par-ticles (VLPs) [2]. HBV core antigen (HBcAg) can act as both a T cell-dependent and T cell-independent antigen [3]. HBcAg is highly immunogenic and can prime specifi c antibodies, T helper cells (Th), and cytotoxic T cells (CTLs) [4]. This can be utilized by using HBcAg as a carrier for foreign epitopes [5]. In

particular the spacing of the spikes protruding from the HBcAg shell appears to be optimal to cross-link B cell receptors and activate B cells [6 – 8]. Hence, the B cell seems to be the primary antigen-present-ing cell (APC) for HBcAg [8].The envelope proteins of HBV consist of 3 related membrane-bound gly-coproteins designated as the large (L), middle (M), and small (S) proteins. These antigens are known to elicit virus-neutralizing and protective antibodies [4]. In this study we combined the effects of HBsAg with the ability of HBcAg to act as a carrier for HBsAg-derived sequences, since this may constitute an appropriate platform to be used as a therapeutic vaccine.

Materials and methods

Generation of chimeric HBsAg – HBcAg proteins

HBcAg DNA was amplifi ed and cloned into the pET28a vector (Novagen, Darmstadt, Germany) with

SHORT COMMUNICATION

A bi-functional hepatitis B virus core antigen (HBcAg) chimera activates HBcAg-specifi c T cells and preS1-specifi c antibodies

IMRAN RIAZ MALIK 1,2 , ANTONY CHEN 1 , ANETTE BRASS 1 , GUSTAF AHL É N 1 , MOAZUR RAHMAN 2 , MATTI S Ä LLBERG 1 , JAVED ANVER QURESHI 2 & LARS FRELIN 1

From the 1 Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, and 2 Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan

Abstract A major problem in chronic hepatitis B virus (HBV) infection is that treatment with specifi c antivirals is life-long since they rarely induce a sustained response. An attractive option is therefore to combine antiviral therapy with some type of immune stimulator, such as a therapeutic vaccine. Several lines of evidence suggest that a key target for the cellular immune response is the HBV core antigen (HBcAg). However, it may also be of advantage to simultaneously improve the neutral-izing antibody response to the surface (S) region of HBV. We therefore generated chimeric HBcAg particles expressing preS1 residues 1 – 42 at the tip of the spike region. We could show that this chimeric HBcAg – preS1 protein primed both HBcAg-specifi c T cells and antibodies to preS1. This strongly suggests that this may be a viable approach to develop an effective bi-functional therapeutic vaccine as an add-on for the treatment of chronic HBV infections.

Key words: HBV , HBcAg , vaccine , T cell , preS1

Scandinavian Journal of Infectious Diseases, 2012; 44: 55–59

ISSN 0036-5548 print/ISSN 1651-1980 online © 2012 Informa HealthcareDOI: 10.3109/00365548.2011.608711

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56 I. R. Malik et al.

a C-terminal His-tag using NcoI and XhoI digestion (Figure 1a). A chimeric gene was then generated by insertion of the PreS1 – 42 amplicon between residues 79 and 80 of HBcAg using the SalI and HindIII restriction sites (Figure 1a). The size of the chimeric gene was verifi ed by restriction enzyme digestion. The resulting construct produced a His-tagged HBcAg and PreS1 – 42 chimeric protein. The plasmid and cor-responding recombinant protein was referred to as pC42. The pC42 protein was analyzed for integrity by an in vitro coupled rabbit reticulocyte lysate transcrip-tion and translation system (TNT Coupled Reticulo-cyte Lysate Systems; Promega, Madison, WI) as described previously [9].

The pC42 protein was expressed in Escherichia coli strain C41BL21 (Novagen) using standard pro-tocols. Expression of pC42 was carried out by auto-induction in the form of inclusion bodies as described previously [5]. In brief, E. coli cells were harvested by centrifugation, re-suspended, and disrupted by sonication. The crude cell extract was centrifuged, and inclusion bodies were solubilized in denaturing buffer. The solubilized protein was purifi ed using a Talon affi nity column (Clontech, Mountain View, CA). Fractions containing the target proteins were identifi ed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purifi ed pro-tein was collected, dialyzed against phosphate buff-ered saline (PBS) buffer, concentrated, and stored at 4 ° C until use. The protein concentration was deter-mined by the Bradford assay method using bovine serum albumin as standard.

SDS-PAGE and Western blot

Samples were boiled and separated on 4 – 12% SDS-PAGE. Western blots were performed for both HBcAg and PreS1 as described previously [10,11]. Antigens were detected using primary antibodies to HBcAg (Dako, Glostrup, Denmark) and PreS1 (MA18/7 kindly provided by Dr Paul Pumpens, Uni-versity of Latvia, Riga, Latvia) diluted 1:1500 and 1:500, respectively. The membrane was cut and then incubated with enzyme conjugated anti-rabbit and anti-mouse antibodies (Invitrogen, Carlsbad, CA). Proteins were visualized using the ECL Plus Western blotting reagent (GE Healthcare, Uppsala, Sweden) according to the manufacturer ’ s protocol.

Recombinant proteins and synthetic peptides

Recombinant particulate HBcAg encompassing resi-dues 1 – 183 (rHBc) were produced in E. coli and puri-fi ed as described by Billaud et al. [5]. Generation and production of the recombinant HBcAg – PreS1 – 42 chimeric protein is described above. Chicken egg

albumin (ovalbumin, e.g. OVA) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The follow-ing peptide pools were used for stimulation of HBcAg T cell responses in vitro: (1) a peptide pool contain-ing 8 peptides covering the fi rst half of the HBcAg protein, including the HBcAg-CTL epitope MGLK-FRQL (H2-K d ), and (2) a peptide pool containing 7 peptides covering the second half of the HBcAg protein, including the HBcAg-Th epitope VSF-GVWIRTPPAYRPPNAPIL. Peptides were kindly provided by Dr M. Levi, ChronTech Pharma AB, Huddinge, Sweden.

Animals

Inbred C57BL/6J (H-2 b ) mice were obtained from Charles River, Sulzfeld, Germany. All mice were 6 – 12 weeks old at the start of the experiments. All mice were maintained at the Karolinska Institutet in accordance with the regulations of the ethics com-mittee for animal research at Karolinska Institutet.

Determination of immunogenicity

Groups of female C57BL/6J mice were immunized subcutaneously at the base of the tail with 10 μ g of purifi ed pC42 protein in incomplete Freund ’ s adju-vant (e.g. mixed 1:1), followed by a booster dose of the same antigen in incomplete Freund ’ s adjuvant at an interval of 4 weeks. Serum samples were collected at weeks 2, 4, and 6. Samples were analyzed for the presence of hepatitis B virus core antibody (anti-HBc)

Figure 1. (a) Schematic map of the chimeric HBcAg – PreS1 plasmid pC42. (b) Also shown is the Western blot analysis of the pC42 protein using anti-HBc and (c) anti-PreS1. In (b) the fi rst well represents the magic marker (M, from Invitrogen); lane 1, negative E. coli control; lane 2, E. coli-derived HBcAg; and lanes 3 – 6, 1 μ g, 0.5 μ g, 0.25 μ g, and 0.125 μ g, respectively, of purifi ed pC42 protein. In (c) the fi rst well represents the magic marker (M, from Invitrogen); lane 1, negative E. coli control; lanes 2 – 6, 1 μ g, 0.5 μ g, 0.25 μ g, 0.125 μ g, and 0.0625 μ g, respectively, of purifi ed pC42 protein.

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Chimeric HBcAg primes cellular and humoral immunity 57

and anti-PreS1 essentially as described previously, using HBcAg and pC42 as the solid phase antigens [12]. A positive anti-HBc and anti-PreS1 sample had to yield an optical density (405 nm) 3 times that of the pre-immunization sera to be considered positive.

A commercially available ELISpot assay was used to measure the frequency of splenocytes producing interferon-gamma (IFN- γ ). ELISpot assays were per-formed as previously described [12].

Results

Characterization of chimeric HBcAg expressing PreS1

The use of an in vitro transcription and translation system revealed that the pC42 plasmid expressed a protein of the expected size of 24 kDa (data not shown). Also, the pC42 protein was effectively expressed in and purifi ed from E. coli. Western blot analysis using HBcAg- and PreS-specifi c antibodies identifi ed the chimeric pC42 protein at the expected size of 24 kDa (Figure 1, b and c). Thus, the chime-ric pC42 protein was intact and was therefore con-sidered as suitable for immunogenicity studies.

Immunogenicity of chimeric HBcAg expressing PreS1

The immunogenicity of the pC42 protein was evalu-ated by immunization with a comparatively low dose of pC42 protein (10 μ g/dose) in C57BL/6J mice. This revealed that 1 low dose immunization primed undetectable or low levels of anti-HBc and anti-PreS1. However, 2 immunizations primed a signifi -cant anti-HBc and anti-PreS1 response in vivo (Figure 2a). The major increase in antibody levels appeared 2 weeks after the second immunization (Figure 2a). This strongly suggests that the chimeric pC42 protein effectively induces potentially neutral-izing antibodies to PreS1, which is one goal with the construct.

The second feature of the pC42 protein is that it should also be able to induce T cell responses to the HBcAg – PreS1 fusion protein. The ability to prime HBcAg- and PreS1-specifi c T cells was determined by measuring the activation of IFN- γ -producing T cells in immunized versus non-immunized mice using a commercial ELISpot assay. Importantly, Milich et al., have shown that the N-terminal region of PreS1 does not contain any known H-2 b T cell recognition sites. Thus, the addition of PreS1 will not improve the T cell response by adding new T cell epitopes to HBcAg in H-2 b mice [13]. HBcAg – PreS1-specifi c IFN- γ -producing T cells were effec-tively activated after two 10 μ g pC42 protein immunizations in C57BL/6J mice (Figure 2, b and c). As expected, no HBcAg-specifi c CTLs were

primed, since the insert was located at the tip of the spike. The spike region needs to be intact for an effective activation of HBcAg-specifi c CTLs due to a binding to na ï ve B cells [6]. Moreover, no IFN- γ production was detected when recalled using Th peptides but with rHBcAg (Figure 2, b and c). How-ever, our results confi rm that the pC42 protein acti-vates both potentially neutralizing anti-PreS1 antibodies and HBcAg-specifi c IFN- γ -producing T cells, which are the 2 central functions needed in a therapeutic vaccine for chronic HBV infections.

Discussion

Therapy for chronic infections caused by HBV has improved signifi cantly in the past 15 y with the intro-duction of small molecule drugs [1,14]. These are effective at suppressing HBV replication during ther-apy. However, the drugs available today can only slightly improve the sustained off-therapy responses signifi ed by a hepatitis B virus e antigen (HBeAg) to hepatitis B virus e antibody (anti-HBe) seroconver-sion above the annual 10% spontaneous seroconver-sion rate [14]. Thus, new therapies that can improve the sustained off-therapy response rates are needed.

It has been well documented that the seroconver-sion from HBeAg to anti-HBe is associated with an activation of the endogenous T cell response to HBV. A highly attractive approach to improve sustained response rates is therefore therapeutic vaccination whereby the host T cells become specifi cally acti-vated, or re-activated. This is attractive in particular in the light of the now highly active small molecule antivirals, since these can be use to pre-treat the patient for a longer period. After 6 to 12 months of effective suppression of HBV replication and reduced presence of viral antigens, the T cell tolerance becomes less pronounced. Thus, a combination of these small molecules with a therapeutic vaccine would hit the virus with 2 different mechanisms and may help the host to control the infection.

In this study we developed a therapeutic vaccine candidate that should be able to induce a polyclonal immune response. First, the activation of IFN- γ -producing HBcAg-specifi c T cells should be highly antiviral since HBV replication is highly sensitive to IFN- γ [15]. Importantly, HBcAg-specifi c T cells will enter the liver as local IFN- γ -producing T cells, which has been found to be instrumental for the antiviral effect [15]. Next, we could show that the chimeric pC42 protein effectively activated anti-PreS1 antibod-ies that have been shown to be neutralizing [4]. Thus, these are the ideal types of response to be raised in an HBV chronically infected host. One additional branch of the host response that may be required in an opti-mal vaccine design is CTLs. However, by introducing

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58 I. R. Malik et al.

Figure 2. (a) Mean anti-HBc and anti-PreS1 (1 – 42) IgG antibody titre after 1 (weeks 2 and 4) and 2 (week 6) subcutaneous immunizations with 10 μ g of the chimeric protein (pC42) in C57BL/6J mice. Values are given as end-point titres, also given are the number of mice with detectable antibodies at each time point. (b) The number of IFN- γ -producing HBcAg- and pC42-specifi c T cells at week 6 are shown from mice immunized with two 10- μ g doses of pC42 and (c) non-immunized mice. Data are given as the number of IFN- γ -producing spot-forming cells (SFC)/10 6 splenocytes. The dotted line in the immunized and non-immunized groups indicates the ELISpot cut-off set to 50 SFC/10 6 splenocytes.

the PreS1 insert at the tip of the spike we found that the ability of exogenous pC42 to prime HBcAg-specifi c CTLs was lost, which is fully consistent with our previous data. Thus, to effectively prime CTLs to the pC42 protein one could combine the chimeric protein-based vaccine with a genetic vaccine [12].

We have previously shown that lipopolysaccharide (LPS) present in a highly contaminated HBcAg prep-aration had no signifi cant effect on anti-HBc anti-body production in vivo [16]. Importantly, an LPS contamination cannot explain the immunogenicity of HBcAg in mice, either in vitro or in vivo. Coomassie

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Chimeric HBcAg primes cellular and humoral immunity 59

gels made during protein purifi cation showed only 1 major band (data not shown). A much stronger IFN- γ production is shown in pC42 immunized mice than in na ï ve C57BL/6J mice stimulated with the respective pC42 or rHBcAg antigens. Thus, the C42 primed immune responses are antigen-specifi c, and not due to LPS contaminations.

In conclusion, we have shown that the pC42 protein can induce HBV-specifi c antibodies and T cells, 2 functions that complement the activity of antiviral compounds well. Thus, the pC42 protein may be a future component in a therapy for chronic HBV infections where the host is gaining a sustained control of the infection.

Acknowledgements

The study was supported by grants from the Swed-ish Science Council (MS), the Swedish Cancer Soci-ety (MS), the Swedish Society of Medicine (LF), Å ke Wiberg Foundation (LF), Karolinska Institutet (MS, LF), and the Higher Education Committee of Pakistan (IRM).

Declaration of interest: The authors report no confl icts of interest. The authors alone are respon-sible for the content and writing of the paper.

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