Ebook Comprehensive textbook of hepatitis B: Part 1

(BQ) Part 1 book Comprehensive textbook of hepatitis B has contents: Epidemiology of hepatitis B virus, transmission and prevention of hepatitis B, host immunity and clinical outcome in HBV-Infection,.... and other contents.

Textbook of Hepatitis B

Mamun-Al-Mahtab (Shwapnil)

Assistant Professor Department of Hepatology Bangabandhu Sheikh Mujib Medical University

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Textbook of

Hepatitis B

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Comprehensive Textbook of Hepatitis B

© 2011, Jaypee Brothers Medical Publishers

All rights reserved No part of this publication should be reproduced, stored in a retrieval system,

or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editors and the publisher.

This book has been published in good faith that the material provided by contributors is original Every effort is made to ensure accuracy of material, but the publisher, printer and editors will not be held responsible for any inadvertent error(s) In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only.

First Edition : 2011

ISBN 978-93-5025-081-5

Typeset at JPBMP typesetting unit

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Ahmed, Ayesha Mahtab, SK Muizuddin Ahmed, Syeda Ashrafun Nesa, Syeda Rowshanara Khatun; wives, Dr Nuzhat Choudhury Mahtab, Shamsunnehar Akbar, Sayeda Rahman; children, Musarrat Mahtab (Shukonnya), Mashrur Mahtab (Shurjo), Tasmin Akbar, Dr Shahrin Afroz (Rimi), Tahmidur Rahman and Obaidur Rahman; friends, Md Helal Uddin and others and also people with different opinions and beliefs The publication of this book was possible only due to immense help and cooperation from thousands of patients with HBV and other liver diseases; their entity and sufferings led us to adapt

a path that should be followed, not one that is accepted by most in this field.

Department of Gastroenterology and

Hepatology, Sir Ganga Ram Hospital

New Delhi, India

Anil Arora

Hepatology Services

Sir Ganga Ram Hospital

New Delhi, India

Ashish Kumar

Institute of Liver and Biliary Sciences

(ILBS), New Delhi, India

Asian Center for Liver Diseases and

Transplantation, Gleneagles Hospital

Singapore

Fu-Sheng Wang

Research Centre for Biological

Therapy, Beijing 302 Hospital

Beijing, China

Gholam Ali Ghorbani

Baqiyatallah University of MedicalSciences, Tehran, Iran

Gokhan Kabacam

Department of GastroenterologyAnkara University School of MedicineAnkara, Turkey

Hamama-tul-Bushra Khaar

Gastroenterology and HepatologyDivision, Department of MedicineHoly Family Hospital

Rawalpindi, Pakistan

Hasan Ozkan

Professor of GastroenterologyAnkara University School ofMedicine, Ankara, Turkey

Hyo-Suk Lee

Liver Research InstituteSeoul National UniversityCollege of Medicine, Korea

Jian Sun

Hepatology UnitDepartment of Infectious DiseasesInstitute of GastroenterologyNanfang Hospital

Southern Medical University, China

Asian Center for Liver Diseases and

Transplantation, Gleneagles Hospital

Singapore

Kaushal Madan

Department of Hepatology

Institute of Liver and Biliary Sciences

(ILBS), New Delhi, India

Khalid Mumtaz

Assistant Professor of Medicine

Aga Khan University Hospital

Muhammad Umar

Gastroenterology and HepatologyDivision, Department of MedicineHoly Family Hospital

Neeraj Saraf

Department of Gastroenterology andHepatology, Sir Ganga Ram HospitalNew Delhi, India

Pankaj Tyagi

Department of GastroenterologyRam Manohar Lohia HospitalNew Delhi, India

Peter Karayiannis

Department of MedicineImperial CollegeLondon, UK

Piyawat Komolmit

Department of MedicineChulalongkorn UniversityBangkok, Thailand

Puja Sakhuja

Department of Pathology

GB Pant Hospital, New Delhi, India

R Murali

Department of Gastroenterology

Sanjay Gandhi Postgraduate Institute

of Medical Sciences, Lucknow, India

Saeed S Hamid

Professor of Medicine

Faculty of Health Sciences

The Aga Khan University

Karachi, Pakistan

Seyed-Moayed Alavian

Baqiyatallah University of Medical

Sciences, Tehran, Iran

Shiv Kumar Sarin

GB Pant Hospital

Department of Molecular Medicine

Jawaharlal Nehru University

Institute of Liver and Biliary Sciences

(ILBS), New Delhi, India

SM Fazle Akbar

Principal Investigator

Department of Medical Sciences

Toshiba General Hospital

Tokyo, Japan

Ulus Salih Akarca

Professor of MedicineEge University Medical FacultyDepartment of GastroenterologyBornova, Izmir

Turkey

Vivek A Saraswat

Professor of GastroenterologySanjay Gandhi Postgraduate Institute

of Medical SciencesLucknow, India

Yusuf Bayraktar

Gastroenterology ClinicDepartment of Internal MedicineHacettepe University

Faculty of Medicine AnkaraTurkey

Zheng Zhang

Research Centre for BiologicalTherapy, Beijing 302 HospitalBeijing, China

Hepatitis B is a major global health problem The infection and the clinicaloutcomes are very varied and often difficult to diagnose and manage Hepatitis B

is one of those diseases where every year, one or more treatment guidelines andrecommendations are published While a lot of uniformity in the approach todiagnosis and management has evolved, many major issues remain to be addressedand answered

It is a very worthy move by Dr Mamun-Al-Mahtab to compile the latestinformation from a galaxy of global experts in the field of hepatitis B I am veryimpressed by the selection of topics, which instead of being generic are, in fact,issue based The basic biology of the virus has been described by Prof Karayiannis,

a well-known HBV virologist The information, despite being comprehensive, isquite easy to understand This would indeed also be very useful for the researchfellows working in the field of hepatitis B Role of HBV genotypes and theirrelevance in relation to treatment and disease progression is elegantly described

by Prof Campos The submissions on markers of HBV infection and thecontroversial term of ‘carriers’ would be found stimulating by the readers In fact,

it would be evident from the chapter of Dr Madan that the new and universallyaccepted terminology, “Incidentally Detected Asymptomatic HBsAg-PositiveSubject (IDAHS)”, well serves the clinicians instead of the misnomer term carrier,which should be discarded

The chapters by Profs Acharaya, Lee and Wang gel together very well to give

a clear picture of the gigantic disease burden and the manner in which the disease

is still existing and is likely to spread further There is an interesting data to showhow the virus is transmitted and leads to immune tolerance The chapter on hepatitis

B in children is interesting to read and addresses the concerns of pediatriciansand clinicians handling newborns and their mothers It also stimulates need forfurther work in this direction In her illustrative chapter, Prof Sakhuja has givendetailed account of the spectrum of histoptahological lesions present in chronichepatitis B patients Since histopathology is very essential for assessing the severity

of the disease and the outcome of treatment, readers would find this chapter veryhelpful

Over the years, the spectrum of hepatitis B viral infection has changed fromHBeAg-positive to HBeAg-negative Generally, this is a later stage of HBVinfection and despite a lower viral load, the disease is more progressive, severeand difficult to treat This important issue has been addressed by Dr Mahtab in avery informative chapter The clinical issues, the molecular basis and the approach

to such patients would be found very useful by the readers of the book Two specialsituations , hepatitis B in dialysis patients and in pregnant women are complicated

and require special attention Prof Bayrakhtar and Dr Wai have made special efforts

to give updated information on these two special situations The readers wouldfind practical tips and the rationale behind, to handle various clinical dilemmas.The complications of chronic HBV infection are protean These include,reactivation of chronic hepatitis B, acute-on-chronic liver failure due to HBV anddevelopment of hepatocellular carcinoma Comprehensive write-ups on these areasare not readily available and the present volume would serve to be a treasure forthe readers

Coinfection of HBV with HCV and HIV is not uncommon In fact, very often,

we fail to screen patients for multiple viral infections It is heartening to see latestinformation on these difficult areas from Prof Ozkan and Dr Anil Arora.The availability of new antivirals, their mechanisms of action and the efficacyare major learning points for all clinicians and researchers These topics havebeen analytically dealt with by Profs Akarca, Hou, Saraswat and Heathcote andprovide useful information and practical tips to the reader to manage a given patientwith chronic hepatitis B The concern of emergence of drug resistance and itsprevention and management are other areas of learning which this book provides.The emerging trends of sequential therapy and combination therapy in themanagement of chronic hepatitis B give new hopes in the management of chronichepatitis B

Liver transplantation is now a reality and is a hope for survival for a largenumber of patients with end-stage chronic liver disease due to hepatitis B.Drs Wai and Tan have lucidly described the approach to such patients, includingtheir post-transplant management

The field of hepatitis B is rapidly evolving and most of the information hasemerged from Asia and Far East The clinical and basic research data from Chinaand the Indian subcontinent is the basis of most of the guidelines for management

of hepatitis B in the world In fact, recently, many investigators and policy makers

in the West are also relooking at the mammoth problem of chronic HBV infection

We hope that the difficulties of a lack of infectious cell lines and animal modelwould also be overcome in the near future The readers of this book, students,clinicians and researchers alike, would find this book as a storehouse of availableknowledge and use this to surge further into the mechanisms of HBV persistenceand carcinogenesis It would be a great service to humanity, if the disease burdencould be reduced in the next 5 decades and the disease is eliminated in the twenty-first century I would be delighted to see many more editions of this book in theyears to come

SK Sarin

Professor and HeadDepartment of Hepatology, andDirector, Institute of Liver and Biliary Sciences (ILBS)

New Delhi, India

Dynamic changes are taking place in control and management of different infectiousdiseases due to development of insights about epidemiology and pathogenesis ofthese diseases and also because various antimicrobial agents and vaccines havebecome available Smallpox is a matter of history and has been completelyeradicated from the world Many infectious diseases are also going to be eradicated

or effectively controlled in near future However, these realities are not applicable

to hepatitis B virus (HBV) The virus was detected in 1960s A potent vaccineagainst the HBV was developed in the early 1980s, which is regarded one of themajor successes in the field of vaccinology The World Health Organization (WHO)has also recommended the universal use of HBV vaccines around the world andmost of the countries of the world accepted the notion

After all these developments, about 2 billion people or about one-third ofglobal population are infected with the HBV and about 350 million of these peopleare chronically infected with this virus About 75–80% of chronic HBV carriersreside in developing countries of the world Chronic HBV-infected subjects areprone to develop chronic hepatitis and its complications, such as liver cirrhosisand liver cancer In most of the developing countries, the main cause of livercancer is HBV

Although the HBV infection has been effectively controlled in most developedcountries of the world but this has not been reflected in developing countries.There are many factors for these discrepancies It seems that the recommendationsprovided by developed and rich countries are not applicable in the context ofsocioeconomic aspects of developing countries of the world In addition, some ofthe recommendations may be counter-productive for control of the HBV infection

in developing countries In fact, incomplete treatment of chronic HBV-infectedsubjects of developing countries and lack of proper follow-up system wouldbecome a formidable problem for mankind in future Another problem to controlthe HBV in developing countries lies in the fact that the medical curricula ofdeveloping countries do not provide adequate importance about HBV infection

In addition, new information about various aspects of the HBV are rarely updatedand communicated to physicians in these countries

Comprehensive Textbook of Hepatitis B is probably the second reference book

about HBV infection, published from a developing country harboring millions ofchronic HBV carriers The book contains a total of 28 chapters These chaptershave been written by specialists of respective fields Many of these authors arefrom Asia Pacific Region, who have practical experiences about scopes andlimitations regarding control and management of HBV to their countries Inaddition, clinical information about HBV has been provided with updated research

developments about the virus Thus, the readers of the book would be able todecide how the information of the laboratory benches may be applied to developevidence-based control and management strategies against HBV.

We hope that the book would be used as a reference book by medical students,postgraduate students, medical teachers and scientists of different fields of biology,

so that new concepts would help to formulize proper control and managementstrategies against HBV

We would like to express our gratitude to the authors whose invaluablecontribution helped to compile the book We would also like to offer thanks toour institutions in Bangladesh and Japan that helped us to develop insights aboutHBV and the diseases it causes Also, our sincere gratitude is to thousands ofpatients who were our real instructors in this field

Mamun-Al-Mahtab

SM Fazle Akbar Salimur Rahman

1 Hepatitis B Virus: General Features and Molecular Biology 1

4 Host Immunity and Clinical Outcome in HBV-Infection 25

Fu-Sheng Wang, Zheng Zhang

Muhammad Umar, Hamama-tul-Bushra Khaar

SM Fazle Akbar, Mamun-Al-Mahtab, Morikazu Onji

Koji Yano

Seyed-Moayed Alavian, Gholam Ali Ghorbani

Mamun-Al-Mahtab, SM Fazle Akbar

Kaushal Madan, Pankaj Tyagi

13 Hepatitis B Virus Related Acute-on-Chronic Liver Failure 120

Ashish Kumar, Shiv Kumar Sarin

14 Hepatitis B Virus Related Hepatocellular Carcinoma 133

17 HBV and HCV Coinfection 161

Hasan Ozkan, Gokhan Kabacam

18 Hepatitis B and Delta Virus Coinfection and Superinfection 165

Khalid Mumtaz, Saeed S Hamid

Anil Arora, Naresh Bansal

20 Hepatitis B Virus Related Chronic Liver Disease

Yusuf Bayraktar, Ali Shorbagi

Neeraj Saraf, Amit Basnotra

Puja Sakhuja

Jose D Sollano

Ulus Salih Akarca

25 Resistant Hepatitis B Virus Infection: Pathogenesis, 276 Implications and Management

R Murali, Vivek A Saraswat

26 Management of Hepatitis: Pre and Post Liver Transplant 294

Desmond Wai, Kai-Chah Tan

Leung WD, Jenny Heathcote

Jian Sun, Jinlin Hou

The virus was visualized under the electron microscope for the first time in 1970

by Dane and Almeida and their respective colleagues, who described the infectiousform of the virus or Dane particle, and its nucleocapsid core, respectively Thiswas soon followed by the characterization of the virus genome, the proteins itencodes as well as their function The setting-up of diagnostic assays for variousmarkers of infection at the same time facilitated the definition of the serologicalprofiles that characterize acute and chronic infection with the virus

Outbreaks of hepatitis among shipyard workers in Bremen and inmates of anasylum for the mentally insane in Merzig were described towards the end of the19th century, following vaccination against smallpox The condition was referred

to at the time as serum hepatitis and the link with its causative agent, namelyHBV, was not made until the mid-1960s Studies, performed by Krugman andcolleagues at the Willowbrook State School for the mentally handicapped childrenduring this time established that hepatitis A virus and HBV were the causativeagents for infectious and serum hepatitis respectively The presence of Australiaantigen in the sera of patients with leukemia had been described a few years earlier,but its connection with HBV and hepatitis B surface antigen (HBsAg) in particular,

as it is now known, did not become apparent until later studies performed by theteams of Blumberg and Prince These studies set the groundwork for the subsequentserological tests for the diagnosis of HBV and allowed detailed investigationsinto the epidemiological and virological aspects of infection, and connection ofthe virus with the development of hepatocellular carcinoma (HCC) in highprevalence areas of the world

The molecular biology of the virus was unravelled in the years that followedwith the development of genetic engineering techniques that allowed the cloning

of the viral genome, study of the function of its proteins and revealed the fascinatingmechanism of its replication strategy The polymerase chain reaction allowed thespeedy amplification and sequencing of virus isolates that led to the identification

of its genotypes, quasispecies and virus mutants, using bioinformatic tools

A landmark in a historical context was the development and introduction of

a plasma-derived prophylactic vaccine in the early 1980s, following its extensive

evaluation by Smuzness and colleagues in chimpanzees and the gay community

in the United States of America This vaccine was soon replaced by its recombinantcounterpart, and more than 25 years on, the vaccine has proved extremelyefficacious at reducing HBV prevalence rates in many countries where the virus

is endemic Almost concurrently, interferons were introduced for the treatment ofchronic HBV carriers in an attempt to stem the progression to cirrhosis and HCC.Nowadays, apart from interferon which most treatment guidelines recommendthat it be used for up to a year, longer term treatment is possible with nucleos(t)ideanalogs that inhibit DNA synthesis and the reverse transcriptase (rt)/DNApolymerase enzyme of the virus

TAXONOMY AND CLASSIFICATION

Hepatitis B virus is the prototype member of the hepadnaviridae, a family of

hepatotropic DNA viruses, which are divided into two genera Other than HBV,

the orthohepadnavirus genus includes members that infect rodents such as

woodchucks (woodchuck hepatitis virus, WHV) and ground squirrels (groundsquirrel hepatitis virus) These viruses have about 70% sequence homology withHBV but do not appear to infect man or other primates In contrast, HBV istransmissible to chimpanzees, whilst HBV-like isolates have been obtained fromprimates such as gibbons, gorillas, orangutans, woolly monkeys, as well aschimpanzees More distantly related viruses and with almost no sequence homology

to HBV are those of the genus avihepadnavirus which infect birds such as ducks

(duck hepatitis B virus, DHBV), herons, storks and geese In the absence of a cellculture system for the propagation of HBV, the chimpanzee, woodchuck and duckanimal models have proved invaluable over the years, in the study of the naturalhistory of infection, molecular biology of the virus, and the testing for efficacy ofvaccines and antiviral drugs

VIRION STRUCTURE

Under the electron microscope virus preparations from serum appear pleomorphic.Three types of particle are evident—the infectious virions or Dane particles whichmeasure about 42 nm in diameter and have an electron dense core, and two types

of subviral particles devoid of nucleic acid that consist entirely of HBsAg Thelatter are the 22 nm spheres and filaments which outnumber the virions by100-10000-fold The infectious virion has an outer envelope which consists ofHBsAg in a lipid bilayer that fully enwraps the nucleocapsid core of the virus(Fig 1.1) The latter consists of the hepatitis B core protein (HBcAg) and enclosesthe DNA genome of the virus together with its rt/DNA polymerase

GENOME ORGANIZATION/TRANSCRIPTION

The DNA genome of the virus is a relaxed circle in shape, partially double strandedand of a total length of around 3200 base pairs This compact nature of the genome

makes HBV the smallest known DNA virus pathogenic to man The genome ispartially double stranded since the positive strand is incomplete, whilst the negativestrand is nicked The genome contains 4 wholly or partially overlapping openreading frames (ORFs) (Fig 1.2) and what is more, all its regulatory elements

Fig 1.1: Structure of the hepatitis B virion A Electron micrograph of HBV purified from

plasma showing the infectious Dane, and the spherical and filamentous subviral particles.

B Structure of the virion and its components (Reprinted from the Encyclopedia of Virology, Third Edition, Karayiannis and Thomas “Hepatitis B Virus: General Features”, p350-60, 2008)

Fig 1.2: Genome organization of the virus showing the various open reading frames

(ORFs) and some of the regulatory elements (A) The transcripts encoding for the various viral proteins are co-terminal but of various lengths as shown (B) The color coding is the same for the ORFs in both panels (Reprinted from the Encyclopedia of Virology, Third Edition, Karayiannis and Thomas “Hepatitis B Virus: General Features”, p350-60, 2008)

such as enhancers, promoters, encapsidation and replication signals lie within theseORFs The S ORF encodes for the HBsAg proteins, the P ORF for the rt/DNApolymerase and the X ORF for the X protein which is thought to be a transactivator

of viral and cellular genes with a role in hepatocarcinogenesis Finally the C ORFencodes for the HBcAg or core protein which forms the nucleocapsid of the virus

As explained below, an additional nonstructural protein is produced duringreplication, and this protein is known as hepatitis B e-antigen (HBeAg).The viral proteins are translated from the appropriate mRNAs, the synthesis(transcription) of which is carried out in the nucleus by the host RNA poly-merase II Following infection as described later, the viral genome is delivered tothe nucleus where it undergoes repair The incomplete strand is completed andthe ends of each of the two strands are ligated, leading to the formation of acompletely double stranded molecule known as covalent closed circular DNA(cccDNA), capable of operating as a mini-chromosome and a prerequisite stepfor successful gene transcription Each gene of HBV has one or more promotersregulating its activity and these promoters are in turn regulated by one or bothviral enhancer elements (En1 and En2), located upstream of the basal core promoter(Fig 1.2) Moreover, transcription is dependent to a large extent upon liver-enrichedtranscription factors All of the transcripts terminate at a common polyadenylationsignal and consist of the subgenomic mRNAs that encode for the surface and

X proteins, and two longer than genome length ones (3.5 kb) known as thePreC-mRNA and the pregenomic RNA (pgRNA) The synthesis of both of theseRNAs is under the control of the basal core promoter located in the X ORF Due

to the circular nature of the cccDNA template, these mRNAs have a terminalredundancy so that the sequences at the beginning of the RNA (5’) are repeated

at the end (3’) They arise as a result of the RNA polymerase by-passing thepolyadenylation signal during its first passage but not doing so during the second.The Pre-C mRNA encodes for HBeAg, whilst the pgRNA does so for both thecore and polymerase proteins Moreover, the latter also serves as the template forreverse transcription into DNA during replication of the virus

ENVELOPE PROTEINS

The S ORF encodes three envelope glycoproteins produced by differential initiation

of translation at each of three in-frame initiation codons, which are known as thelarge (L), middle (M) and small (S) HBsAgs The proteins are in fact translatedfrom three separate transcripts and are coterminal (Fig 1.2) Thus the C-terminus

of both the L and M proteins is shared in its entirety with the complete amino acidsequence of the more abundant S protein, and is referred to as the S domain The

M protein has at its N-terminus an additional 55 amino acids encoded by thePre-S2 region, whilst the L protein includes apart from the Pre-S2 domain another

125 amino acids at its N-terminus encoded by the Pre-S1 region (Fig 1.2) Allthree proteins are glycosylated whilst the L and S proteins may be present in an

unglycosylated form in particles also They are type II transmembrane proteinsweaving in and out of the lipid bilayer membrane of the endoplasmic reticulum(ER), where they are inserted during their synthesis The proteins form multimersstabilized through disulfide bridges through cysteine residues in the S domain.All three of them form the outer envelope of the Dane particle, but the ratios varywith the S protein representing around 70% of the HBsAg content of the virion.The main virus neutralizing epitope is contained in the S domain and is oftenreferred to as the Major Hydrophilic Region or the “a” determinant The extent

of this region remains undefined, but it is thought to lie between amino acidpositions 100–160 of the S domain and to constitute a conformational cluster ofepitopes, through the formation of disulfide bridges between cysteine residues.Virus neutralizing epitopes also exist in the Pre-S1 and Pre-S2 domains ThePre-S1 protein is thought to contain between amino acid positions 23–47 the regionresponsible for virus interaction with the hepatocyte receptor Moreover, it isthought that that Pre-S1 protein is also involved in binding the nucleocapsids thatare formed within the cytoplasm This entails that the Pre-S1 domain of a fraction

of the L protein be maintained on the cytosolic side of the ER Finally, the Lprotein undergoes myristylation of its N-terminus, a process which is not requiredfor efficient virus assembly but is required for infectivity and membraneassociation

CORE PROTEIN

The C ORF contains two in-frame initiation codons and can, therefore, encodefor two separate translation products from two different mRNAs Initiation oftranslation from the first initiation codon borne by the PreC-mRNA results insynthesis of the precore protein consisting of 29 amino acids from the precoredomain whilst the remainder is identical with the core protein This proteinconstitutes the precursor of HBeAg, a soluble nonstructural protein and marker

of active virus replication A signal peptide at the N-terminus of the protein tethers

it to the ER membrane, so that the rest is localized within the ER lumen Action

by a signal peptidase within the lumen results in the removal of the signal peptide(first 19 amino acids), whilst further proteolytic processing at the C-terminus ofthe protein leads to the loss of an additional 23 amino acids from that end Whatremains of the protein is the HBeAg This is thought to have a tolerogenic effect

on the immune response to the virus, which characterizes the first phase of chronicinfection, particularly in those exposed to the virus in early childhood (immunetolerant phase) Loss of HBeAg and development of the corresponding antibody(anti-HBe) is one of the primary goals of antiviral therapy

The nucleocapsid or core protein (HBcAg) is synthesized through usage ofthe second initiation codon present in the pgRNA The core protein has the capacity

to dimerize through disulfide bonding, followed by self assembly into theicosahydral structure that constitutes the viral nucleocapsid The latter consists of

240 copies of the core protein or 120 dimers, with surface projections as revealed

by X-ray crystallography The tips of these projections bare a major B-cell epitopethat is recognized by anti-HBc antibodies

RT/DNA POLYMERASE

The P ORF covers almost ¾ of the viral genome and encodes for the polymerase

of the virus As already mentioned, the pgRNA encodes for both the core andpolymerase proteins of the virus The P ORF initiation codon lies in the distal part

of the core gene, and thus the two ORFs overlap, but they are not in frame.Preferential binding of ribosomes to the 5’ end of the pgRNA leads to translation

of the C ORF and accumulation of the core protein This is not surprising asmultiple copies of the core protein are required for capsid formation Translation

of the P ORF is, therefore, is less efficient compared to that of the C ORF Atsome stage though, polymerase synthesis occurs followed by binding of the protein

to the 5’ end of its own pgRNA This event arrests further synthesis of the coreprotein and initiates the process of encapsidation

The polymerase has three functional domains which starting at the N-terminusinclude the terminal protein involved in DNA priming, the rt/DNA polymerasedomain and the RNAse H domain that occupies the C-terminus of the protein and

is involved in pgRNA degradation There is also a spacer region of unknownfunction situated between the terminal protein and the rt domain Molecular studieshave shown that this region can be deleted without a detrimental effect onreplication competence The rt domain contains the characteristic YMDD motif(Tyrosine-methionine-aspartic acid-aspartic acid) mutations which have been

associated with resistance to nucleoside analogs such as lamivudine Host cellfactors including chaperones from the heat shock protein family, two of which areHsp70 and Hsp90, are thought to be instrumental in aiding encapsidation,stabilization and activation of the polymerase.

REPLICATION STRATEGY

As stated already the pgRNA has a terminal redundancy which duplicates thenucleotide sequence at its 5’ end of this RNA up to the polyadenylation signal,and encompasses the direct repeat 1 (DR1) region to the beginning of the C ORF.The intervening sequences which happen to overlap with the precore region form

a secondary structure known as the epsilon (ε) or encapsidation signal It is formedthrough intramolecular base pairing of palindromic sequences that lead to theformation of a structure consisting of a lower stem, an upper stem, a side bulgeand an apical loop (Fig 1.3) Once synthesized, the polymerase engages through recognition of its conformational structure Concurrently with this event,

as its name implies, it effects the encapsidation of the pgRNA/polymerase complexinto the nucleocapsid particle, where all subsequent steps in virus nucleic acidsynthesis take place Although the PreC-mRNA is only slightly longer than thepgRNA, only the latter is encapsidated Of the duplicated elements in the pgRNA

Fig 1.3: Replication strategy of the virus A Primer synthesis; B Translocation and binding

to DR1; C Synthesis of the (–)-DNA strand; D Preservation of the RNA primer fragment from RNase H degradation E (+)-DNA strand synthesis (Reprinted from the Encyclopedia

of Virology, Third Edition, Karayiannis and Thomas “Hepatitis B Virus: General Features”, p350-60, 2008)

mentioned above, two, namely the at the 5’ and the DR1 at the 3’ ends of theRNA are crucial elements in the successful replication of the viral nucleic acid.The interaction between the polymerase and # sets in motion the events thatinitiate reverse transcription of the pgRNA to the negative (–)-DNA strand of therelaxed circular HBV-DNA The side bulge of the # structure (Fig 1.3) serves asthe template for the synthesis of a 3-4 nucleotide long DNA primer The synthesis

of the primer is primed by the N-terminal domain of the polymerase encompassingthe terminal protein The primer is covalently attached to the polymerase through

a phosphodiester linkage between dGTP and the hydroxyl group of a tyrosineresidue at position 63 of the terminal protein This event involves the # structure

at the 5’ end of the pgRNA, and is then followed by the translocation of thepolymerase-primer complex to the 3’, where it hybridizes with the DR1 regionwith which it is homologous Elongation of the bound primer, and thus synthesis

of the (–)-DNA strand, occurs by reverse transcription of the pgRNA template Inthe process, the pgRNA template is degraded by the action of the RNase H domain

of the polymerase However, the RNase H degradation stops short of the 5’ of thepgRNA, thus preserving an RNA oligomer consisting of 11-16 ribonucleotidesthat encompass the DR1 sequence at this end, still attached to the cap structure

A second translocation event then takes place which allows the DR1 RNAprimer to bind to DR2 located at the 5’ end of the newly synthesized (–)-DNAstrand This translocation is assisted by the circularization of the (–)-DNA strandwhich is brought about by long-distance interactions between segments of thisstrand which are not directly part of the donor and acceptor elements involved inthe translocation events In any rate, as the polymerase is covalently attached tothe 5’ end of the (–)-DNA strand, the latter is effectively circularized during itssynthesis In spite of this, the long-distance interactions are then necessary to bringinto juxtaposition the DRI and DR2 sites The RNA primer containing DR1hybridizes with DR2 with which it is homologous, initiating (+)-DNA strandsynthesis and using the (–)-DNA strand as template The polymerase extends theprimer a short distance towards the end of the 5’ end of the (–)-DNA strand andthen a template exchange occurs that allows the (+)-DNA strand synthesis toproceed along the 3’ end of the complete (–)-DNA strand, effectively circularizingthe genome As already mentioned, DNA synthesis occurs within the nucleocapsidand this is facilitated through pores allowing entry of nucleotides Once thematuring nucleocapsid is enveloped by budding through the endoplasmic reticulummembrane, the nucleotide pool within the capsid cannot be replenished, hencethe incomplete nature of the (+)-DNA strand

VIRUS LIFE CYCLE

The life cycle of the virus begins with its attachment to the appropriate hepatocytereceptor (Fig 1.4), which still remains unknown The virion is internalized probably

by endocytosis, the envelope is removed and the nucleocapsids are then released

in the cytosol They are transported to the nuclear pores through which therelaxed circular HBV-DNA gains access to the nucleoplasm There, the relaxedcircular HBV-DNA is converted to cccDNA by an unknown mechanism, whichhowever, entails the removal of the covalently attached polymerase, thecompletion of the (+)-DNA strand and the ligation of the ends of the two DNAstrands to form a complete circle The cccDNA constitutes the transcriptionaltemplate for viral mRNA synthesis by the host RNA polymerase II The newlysynthesized transcripts are transported to the cytoplasm where they are translatedinto the relevant proteins Following pgRNA encapsidation and synthesis of bothDNA strands, the mature nucleocapsids bud through the ER membrane, wherethe surface proteins are inserted, into the lumen, acquiring in the process theirouter envelope The virions released into the ER lumen are then transported tothe hepatocyte cell surface within vesicles, and are released into the circulation.Some of the mature cores may be recycled back to the nucleus and this allowsthe build up of the cccDNA pool within hepatocytes thus increasing thetranscriptional potential of the virus Virus persistence relies on the cccDNAharboring hepatocytes, the destruction of which is one of the unmet goals in asubstantial number of patients undergoing antiviral therapy.

Fig 1.4: Diagrammatic representation of the life cycle of the virus (Reproduced with

permission from New Eng J Med 2004;350:1118-29) (For color version see Plate 1)

SUBTYPES AND GENOTYPES

The S protein sequence shared by all three envelope proteins as already

mentioned contains the a determinant region which constitutes the major

immunogenic epitope of the virus In addition, within this region there aresubtypic specificities originally detected by antibodies The presence of lysine

(K) or arginine (R) at position 122 confers d or y specificity respectively Similarly, specificities w and r are conferred by the presence of K or R at position 160 Moreover, the w subdeterminant can be further divided into w1–w4 specificities.

There are nine subtypes of the virus depending on the presence of other subtypic

determining amino-acids elsewhere in the a determinant region and the main four are adw, ayw, adr and ayr.

Nucleotide sequencing studies followed by phylogenetic tree analysis haverevealed that virus isolates can be separated based on sequence divergence of

>8% into eight genotypes designated A–H, with characteristic geographicaldistribution More detailed description of these genotypes, including likelydifferences in relation to pathogenesis and response to antiviral treatment, is given

in another chapter of this book

VARIANTS

Although a DNA virus, HBV replicates through an RNA intermediate which isreverse transcribed, and this step in the replication cycle of the virus is prone toerrors by the RNA polymerase II and the viral reverse transcriptase It is estimatedthat the HBV genome evolves at a rate of 1.4–3.2 × 10–5 nucleotide substitutions/site/year As a result of these substitutions, the virus circulates in serum as apopulation of very closely related genetic variants, referred to as quasispecies.Although a lot of these variants would have mutations that would be deleterious

to the virus, as a result of constraints imposed by the overlapping ORFs, somewould be advantageous, either offering a replication advantage, or facilitatingimmune escape (see later chapters)

Finally, genomic analyses have revealed the presence of stable genomic variantsthat arise during the natural progression of chronic infection These variants haveeither reduced levels (core promoter variants) or complete abrogation of HBeAg(precore variants or stop codon variants, G1896A) production These variants areselected at the time of, or soon after seroconversion to anti-HBe, and becomedominant during the reactivation phase (see later chapters) The most commonprecore mutation is the G1896A substitution, which creates a premature terminationcodon in the precursor protein from which HBeAg is produced The doublemutation affecting the core promoter region (A1762T, G1764A) is thought to result

in decreased transcription of the precore mRNA, with a knock on effect on HBeAgproduction, whilst pgRNA production remains the same or is even upregulated It

is now apparent that additional mutations in this region may contribute to thisphenotype.

Finally, in recent years, other variants have been described which arise inresponse to vaccination These variants evade the immune response, are notrecognized by neutralizing antibodies and may fail to be detected by existingdiagnostic tests In addition, isolates resistant to nucleos(t)ide analogs areresponsible for treatment failure in chronic HBV carriers (see later chapters)

CONCLUSION

Since the discovery of the virus, we have learned a lot about its structure, molecularbiology and its replication mechanism There are still however aspects of its biologythat remain unknown, and one hopes that in the years to come more speedy progresswill be made in this direction The ultimate goal will be the design of more effectivedrugs that will reduce significantly, if not eliminate the chronic carrier pool

FURTHER READING

1 Bruss V Hepatitis B virus morphogenesis World J Gastroenterol 2007;13:65-73.

2 Carman WF, Jazayeri M, Basune A, Thomas HC, Karayiannis P Hepatitis B surface antigen (HBsAg) variants In: Thomas HC, Lemon S, Zuckerman AJ (Eds): Viral Hepatitis, 3rd edn, London, Blackwell Publishing 2005;225-41.

3 Ganem D, Prince AM Hepatitis B virus infection—natural history and clinical consequences N Engl J Med 2004;350:1118-29.

4 Karayiannis P, Carman WF, Thomas HC Molecular variations in the core promoter, precore and core regions of hepatitis B virus, and their clinical significance In: Thomas HC, Lemon S, Zuckerman AJ (Eds): Viral Hepatitis, 3rd edn, London, Blackwell Publishing 2005;242-62.

5 Nassal M Hepatitis B viruses: reverse transcription a different way Virus Res 2008;134:235-49.

6 Seeger C, Mason WS Hepatitis B virus biology Microbiol Mol Biol Rev 2000;64: 51-68.

The prevalence of HBV infection varies widely, with rates ranging from 0.1%

to 20% in dif ferent parts of the world “High” prevalence (hepatitis B surfaceantigen [HBsAg] positivity rates > 8%) regions where the viral infection is highlyendemic include the Far East, parts of the Middle East, sub-Saharan Africa, andthe Amazon basin In these regions, serologic evidence of prior HBV infection(anti-hepatitis B core antigen [anti-HBc] or anti-HBs positivity) is present in thevast majority of individuals Japan, the Indian subcontinent, parts of Central Asiaand the Middle East, Eastern and Southern Europe, in addition to parts of SouthAmerica, are all areas with “intermediate” (2 to 7% HBsAg-positive) prevalence

of chronic HBV infection “Low” prevalence (< 2% HBsAg-positive) regionsinclude the United S tates, Northern Europe, Australia, and the southern part ofSouth America Overall, 45% of the world population resides in “high” prevalenceregions, resulting in the enormous global burden linked with the infection.Migration patterns of individuals from high to low endemic areas have had asignificant impact on the epidemiology of hepatitis B For example, immigrationfrom parts of Southeast Asia has led to an increase in the prevalence of chronichepatitis B in the United S tates

Universal screening of donors with HBsAg has reduced the risk for transmitted HBV Some countries with low prevalence have added tests for anti-HBc to detect chronic carriers with low-level viremia who do not have detectableHBsAg These tests have decreased infection rates to approximately 2.5 to 15.3per million units of blood in low-prevalence areas The United States, Canada,Japan, Australia, and some European countries perform the more sensitive nucleicacid tests in addition to these two serologic tests The importance of this incrementalyield and clinical benefit of nucleic acid tests over serologic tests in these low-prevalence areas, in addition to the need for doing serologic tests as well as nucleicacid tests, is unknown

transfusion-Anti-HBc cannot be used as a screening test in high-prevalence areas due tothe fact that up to 90% of adults have serologic evidence of either past or ongoinghepatitis B infection Thus, combined HBsAg and anti-HBc screening woulddisqualify most volunteer blood donors In such countries, HBsAg alone is currentlyused for screening In high-prevalence areas, however, occult hepatitis B is present

in 3 to 30% of individuals who are anti-HBc-positive and HBsAg-negative

It has been estimated that HBsAg-negative, HBV-DNA-positive blood carriesabout a 10% risk of transmitting HBV to susceptible recipients Strategies of donorscreening using only HBsAg, the risk for transfusion-transmitted HBV infection

in Taiwan was recently estimated to be 100 per million population is at least7–40 times higher than in low-prevalence areas The nucleic acid tests wasestimated to be 20 times greater in high-prevalence areas than in low-prevalenceareas, where it is now used, making it more cost-effective per infection prevented

in these countries In conclusion, anti-HBc screening is more cost-effective thannucleic acid tests in low-prevalence areas, although nucleic acid testing does have

a potential role However, nucleic acid tests vary in sensitivity and specificity andare very costly in most countries in high-prevalence areas Development of newerHBsAg assays, with improved sensitivity, may help solve this problem in the future.Another possible mode of transmission for occult or subclinical HBV is fromorgan or tissue donation It has been estimated that undetected viremia at the time

of donation may be more common among tissue donors than blood donors Thesimplest way to prevent post-transplant infection is to exclude all anti-HBc-positiveindividuals as potential donors, however , this approach may be impractical inhigh-prevalence areas where the majority of the population has been previouslyexposed Therefore, the adding of nucleic acid testing to strategies already existing

in the screening of tissue donors may be the best option for reducing the risk fortransmission

Fig 2.1: Worldwide prevalence of HBV (Source: www.who.net)

HBV is classified into 8 genotypes (A–H) on the basis of diver gence of 8%

or more in the nucleotide sequence Genotype A is prevalent in northwesternEurope, North America, and Africa; genotypes B and C predominate in Asia;genotype D is prevalent worldwide, but predominantly in the Mediterranean area;genotype E is prevalent in Africa; genotype F is prevalent in the aboriginalpopulations of South America; genotype G predominates among HBV carriers inFrance, Georgia, the United States, the United Kingdom, Italy, and Germany; andgenotype H is prevalent in the Amerindian populations of Central America (Fig.2.1).All 8 HBV genotypes are found in the United States, with genotypes A and

C being the most common Genotype A is most common among white and blackpatients, whereas genotypes B and C are most common among those of Asianorigin

4 Kim WR, Benson JT, Therneau TM, et al Changing epidemiology of hepatitis B in

a US community Hepatology 2004;39:811-6.

5 Liu CJ, Lo SC, Kao JH, et al Transmission of occult hepatitis B virus by transfusion

to adult and pediatric recipients in Taiwan J Hepatol 2006;44:39-46.

6 Wang JT, Lee CZ, Chen PJ, Wang TH, Chen DS Transfusion-transmitted HBV infection in an endemic area: the necessity of more sensitive screening for HBV carriers Transfusion 2002;42:1592-7.

7 Busch MP Should HBV DNA NAT replace HBsAg and/or anti-HBc screening of blood donors? Transfus Clin Biol 2004;1 1:26-32.

8 Allain JP Occult hepatitis B virus infection: implications in transfusion Vox Sang 2004;86:83-91.

3 Transmission and

Prevention of Hepatitis B

Hyo-Suk Lee

INTRODUCTION

Viral hepatitis presenting as epidemic jaundice was first described by Hippocrates

in the 5th century BC After 2,500 years after the first clinical description, thehepatitis B virus (HBV) was discovered by Dr Baruch Blumberg in 1965, andwas subsequently visualized by electron microscopy in 1970 This discovery ofHBV was followed by the development of hepatitis B immunoglobulin (HBIG),and then in 1981, by the development of safe and effective HBV vaccines that arecomposed of highly purified HBV surface antigen (HBsAg) This has made HBVinfection preventable and provided gradual impact on the epidemiology of HBVinfection

TRANSMISSION

HBV is present in the body fluids including serum, semen, cervical secretion, and

to a lesser extent in tear, sweat, breast milk, and urine of individual infectedindividuals HBV is an importunate virus that can survive outside the human bodyfor a prolonged period and is easily transmitted through contact with infectedbodily fluids Air-borne, water-borne, or vector-borne transmission of HBV hasnot been reported

The main mode of HBV transmission varies markedly throughout the worldwith association to the prevalence of HBV-infection In highly endemic regions,i.e Southeast Asia and Africa, where >8% of the population has chronic HBVinfection and lifetime infection is >60%, the major route of transmission is perinatal(vertical) transmission and horizontal spread from one child to another duringpreschool years About half of the patients infected with HBV in the world live

in these high-risk areas In intermediate-risk areas, such as Middle East,Mediterranean countries, northern Africa, Japan, Singapore and the Indiansubcontinent, where the HBsAg carrier rate is about 3–5% and the lifetime risk

of infection is 20–60%, the major mode of transmission is similar to that of risk areas However, in areas of low endemicity including western Europe, Northand certain part of South America, Australia and New Zealand, where the carrierrate is approximately 0.1–2% and the lifetime risk of HBV-infection is <20%, the

high-main mode of transmission is sexual contact, intravenous drug use, or occupationalexposure.

Vertical Transmission

Vertical transmission represents the transmission of infection from an HBV carriermother to the neonate This mode of transmission is most problematic, since itaccounts for the majority of new infections in the world today Furthermore, up

to 95% of infected neonates become chronic HBV carrier due to their immatureimmune system and immunologic tolerance to the virus By contrast, only 1–5%

of adults infected with HBV become chronic carrier

Exposure to infected maternal blood during passage through the birth canal,maternal-fetal transfusion at the time of delivery and postnatal close mother-babycontact are the major modes of perinatal (vertical) HBV transmission Intrauterinetransmission and transmission during amniocentesis are uncommon There is noevidence of HBV transmission by breastfeeding, although HBsAg can be detected

in breast milk Therefore, infants born to HBsAg-positive mother can be fed after their vaccination series

breast-The risk of vertical transmission correlates to the HBV replication level, whichcan be estimated by using maternal serum HBV level Consequently, the risk forHBV infection in infants born to hepatitis B e-antigen (HBeAg)-positive mothers(in highly replicative phase with high serum level of HBV DNA) is 85–90%,which is much higher than those born to HBeAg-negative mothers (about15–20%) without neonatal passive-active immunization In terms of HBV genetics,HBV genotypes influence mutations which inhibit the translation, or down-regulatethe transcription of HBeAg synthesis It is well described that HBeAg-seroconversion or -loss is significantly delayed in patients infected with genotype

C (40.2–47.8 years of age) than in those with genotype A, B, D, and F (<20 years

of age) by about 10–30 years These findings implicate that the East and east Asian countries where the genotype C is predominant, e.g Korea, China,Japan, Thailand, and Hong Kong, have higher risk for vertical transmission sincemany of the women infected with HBV may be HBeAg-positive at the time ofchildbirth, which may explain the distinctly high prevalence of chronic hepatitis

South-B in these countries

Horizontal Transmission

Sexual Transmission

Sexual contact still accounts for the most important mode of HBV transmission

in developed countries In the United States, about 50% of acute HBV-infectionsamong individuals whose data on risk factors were available, was associated withsexual activity Chronic hepatitis B is prevalent in men who have sex with men aswell as in heterosexuals with multiple sex partners The risk of HBV infection via

sexual transmission increases with multiple sex partners and history of sexuallytransmitted diseases (STDs).

TRANSFUSION

Before the 1970s when significant amount of blood products came from paid blooddonors and serologic screening tests for HBV were not standard procedures, therisk of HBV infection from transfusion was as high as 50% However, theintroduction of blood donor screening with HBsAg and exclusion of paid blooddonors significantly reduced the incidence of transfusion-associated HBV-infection

to 0.6% in the United Kingdom Nevertheless, it should be emphasized that negative, but anti-hepatitis B core antibody (anti-HBc)-positive blood which wouldhave passed the screening, could still lead to HBV infection In the Unites States,anti-HBc is used for transfusion donor screening as well as HBsAg It should benoted, however, that anti-HBc screening is controversial because of its lowspecificity

HBsAg-PERCUTANEOUS TRANSMISSION

Percutaneous transmission usually occurs among intravenous drug users who sharecontaminated syringes and needles In the United States, about 16% of new HBVinfections are related to intravenous drug use The risk of HBV transmission inintravenous drug users proportionally increases with the frequency of injection,the years of drug abuse, and sharing of drug paraphernalia Reuse of contaminatedneedles for acupuncture, tattoos, and body piercing is also associated withtransmission of hepatitis B Sharing contaminated toothbrushes and razors mayalso cause percutaneous HBV transmission

NOSOCOMIAL INFECTION

HBV is the most common virus transmitted by blood-borne transmission in thehealthcare setting Patient-to-patient or patient-to-healthcare personnel transmissiongenerally occurs via contaminated instruments or needle stick injury However,transmission to patient from healthcare worker is rare Although seropositivityfor HBeAg of the source patient is closely related to the risk of acquiring HBVinfection after needle stick injury, HBV transmission can occur from HBeAg-negative individual with high HBV DNA level and precore mutation As verticaltransmission rarely occurs with maternal serum HBV DNA level <107 copies/mL,transmission of HBV via needle-stick injury is thought to be unlikely to occur atserum HBV DNA level <107 copies/mL

Patients with end-stage renal disease on hemodialysis may be infected throughcontaminated dialysis machines, blood transfusions, and interpersonal horizontaltransmission in the dialysis unit Availability of HBV vaccines has reduced theincidence of HBV-infection among these patients from 3% in 1980 to 0.1% in

1993 in United States However, impaired antibody response to vaccine in dialysispatients and the presence of occult HBV-infection (HBsAg-negative but HBVDNA-positive) remained obstacles to further control of HBV transmission in thesepatients.

ORGAN TRANSPLANTATION

Currently, all organ donors are routinely screened for HBsAg HBV transmissioncan occur after transplantation of extrahepatic organs, such as kidneys and evencornea, from HBsAg-positive organ donor However, most posttransplantationhepatitis B is seen among seronegative liver recipients over extrahepatic organrecipients or hepatitis surface antibody (anti-HBs) positive recipients

PREVENTION

Although there are a number of drugs that have antiviral activities against HBV,such as pegylated interferon, entecavir, lamivudine, telbivudine, adefovir andtenofovir, their effectiveness for prevention of hepatic decompensation andhepatocellular carcinoma (HCC) has significant limitations Furthermore, long-term use of oral nucleos(t)ide analogues can cause antiviral-resistant HBV mutationand, consequently, can result in the development of multidrug-resistant mutant,which can worsen the clinical course of chronic hepatitis B Therefore, primaryprevention of HBV-infection is of utmost importance to control the rate of HBV-infection and, subsequently to reduce morbidity and mortality from HBV-relatedcomplications including liver cirrhosis and HCC

Preventive measures for HBV infection can be categorized into three parts:(i) blockade of transmission routes from infected to uninfected individulas,(ii) postexposure prophylaxis, and (iii) active immunization with vaccination.Though these preventive measures, especially vaccination, HBV-infection is nowconsidered preventable, and we aim to eradicate HBV in the future as we havewith smallpox

BLOCKADE OF TRANSMISSION ROUTES

One strategy to prevent HBV infection is the interruption of transmission routesfrom HBV-infected individuals to uninfected persons

Sexual transmission of HBV can be prevented by monogamous sexualrelationships and safe sex practice including use of condoms in individuals withmultiple partners Percutaneous inoculation can be reduced by public healtheducation and the use of disposable syringes, needles, and equipments

To minimize the transfusion-associated transmission of HBV, screening HBc and nucleic acid testing (NAT) can be considered in addition to HBsAg forscreening blood donors However, the practical values of anti-HBc and NAT arenot clear In low prevalence areas, anti-HBc is not cost effective due to the low

anti-incidence of transfusion-related HBV-infection In high prevalence areas, as much

as 22% of the donor population can be excluded through screening with unspecificanti-HBc test and the implementation of NAT is hampered by the cost

Nosocomial HBV infection can be also prevented by HBV vaccination of healthcare personnel, and aseptic techniques and standard precautions can preventexposure to blood-borne pathogens

POSTEXPOSURE PROPHYLAXIS

The second measure to prevent HBV-infection is prophylaxis after exposure toinfected blood or bodily fluids It is recommended for all unvaccinated personsafter exposure to HBsAg-positive source Postexposure vaccination is indicatedfor any percutaneous, ocular, or mucous membrane exposure and the first dose ofHBV vaccine along with 0.06 mL/kg of HBIG should be administered at the sametime in different sites as early as possible, preferably within 12 hours of exposure

If the exposed individual is a known nonresponder (anti-HBs <10 mIU/mL) toprevious vaccinations, two doses of HBIG with one month’s interval or one dose

of HBIG plus initiation of revaccination is recommended If response to vaccination

is unknown, anti-HBs titer should be checked and if anti-HBs is <10 mIU/mLgive him/her one dose of HBIG and booster dose of vaccine should be given Theindividuals with protective anti-HBs titer (>10 mIU/mL) do not requirepostexposure prophylaxis These postexposure prophylaxis recommendations can

be applied for individuals who are wounded from explosives and other similarmass-casualty events

VACCINATION

HBV Vaccine

The development of safe and efficacious HBV vaccine is one of the mostremarkable medical achievements of the twentieth century Vaccincation remainsthe mainstay of prevention against HBV infection In 1970, scientists noticed thatboiling of HBV-infected serum for 1 minute destroyed the infectivity of HBV.Interestingly, the boiled material had antigenicity And from this discovery theydeveloped inactivated vaccine, the progenitor of HBV vaccine we know today.Currently, three different classes of HBV vaccines are available based on thesource from which they are derived from (plasma, yeast, and mammalian cells)

In 1982, the first commercially available plasma-derived inactivated vaccine whichwas composed of purified HBsAg was introduced The plasma-derived vaccinenow comprises about 80% of all vaccine produced due to relatively low productioncost However, this vaccine has concerns for transmitting blood-borne infectionsand is rarely used in developed countries today Next, the yeast-derivedrecombinant vaccines was produced by cloning HBV S gene in yeast cells in themid 1980s Presently, two yeast-derived recombinant vaccines (Recombivax HB

and Engerix-B) which do not contain thimerosal (an organic mercurial preservativethat have potential risk of abnormal neurodevelopment) are available Finally, themammalian cell-derived recombinant vaccine (Hepacare and Hepagene) have alsobeen developed These vaccines contain pre-S2 and/or pre-S1 region in addition

to HBsAg, and show enhanced immunologic response compared to the derived recombinant vaccines Another candidate vaccine which can beadministered as a nasal spray has been developed and its clinical trial is now inprogress Currently available HBV vaccines are very safe and highly efficacious(>90%)

yeast-HBV vaccine is administered intramuscularly in the deltoid muscle of adultsand in the anterolateral thigh of neonates and infants HBsAg-specific helper Tcells and T cell-dependent B cells are induced to produce neutralizing antibodyagainst “a” epitope of HBsAg as early as 2 weeks after the first injection of HBVvaccination The vaccines generally attain an anti-HBs titer >100 mIU/mL andthis titer provide perfect protection against HBV infection Lower anti-HBs titer(10–100 mIU/mL) is also usually seroprotective as well This protective levels ofanti-HBs (>10 mIU/mL) can be achieved by HBV vaccination in greater than90% of individuals However, anti-HBs response after vaccination can be blunteddue to obesity, smoking, injection to other sites (e.g buttock), chronic liver disease,presence of HLA-B8,SC01, and DR3 alleles, absence of HLA-A2 allele, andcompromised host immunity (e.g immunosuppression, chemotherapy, chronickidney disease, and advanced liver disease) Overall, about 5 to 8% of individualsreceiving HBV vaccine fail to achieve detectable anti-HBs titers Doubling thedose of vaccination may be beneficial to the individual with low or absent response

to HBV vaccination Response rate can also be improved by intradermal injection,although it is not widely used due to concerns regarding technical difficulty Forpeople who do not respond after a full series of vaccination, the recommendation

is to repeat another series of vaccination If an individual still does not respondafter the second series of vaccination, further vaccination is usually not effectiveand therefore, not recommended

Although anti-HBs titer decreases to nonprotective levels in 25–50% ofindividuals who have achieve protective level of anti-HBs after vaccination over5–10 years’ period, a booster vaccination is not recommended to immunocompetentadults and children since strong immunologic memory induced by HBV vaccinationcan prevent infection even in subjects with low or undetectable anti-HBs titers.The sole indication for booster immunization is for patients undergoinghemodialysis, in whom anti-HBs titer should be evaluated annually and a boosterdose should be administered if the titer is <10 mIU/mL

HBV vaccine is usually given intramuscularly in three doses at 0, 1, and 6months The recommend dosage is presented in Table 3.1 For patients onhemodialysis or immunocompromised patients, higher doses of vaccine arerequired: such as 40 μg of Engerix-B, Recombivax HB, or Hepavax-gene TF

The safety of HBV vaccination has been well established Common side effectsinclude local reaction over the injection site, fever, malaise, headache, arthralgiaand myalgia HBV vaccines exhibit no known teratogenic effects and can beadministered during pregnancy Because of concerns for abnormal neuro-development due to mercury exposure, HBV vaccines containing the preservativethimerosal are not recommended in developed countries However, there is noevidence of adverse events from the amount of mercury exposure in children withroutine HBV immunization schedule and the risk of not vaccinating children farexceeds the theoretical risk with thimerosal exposure Another concern about HBVvaccination is the development of HBV escape mutants which have mutation inthe HBV genome encoding HBsAg, rendering them escapable from anti-HBsneutralization The escape mutants were reported worldwide, especially in highlyendemic areas However, the prevalence of escape mutation is currently low.Based upon the proven benefit and safety of HBV vaccine, the WHO hasrecommended that all countries continue their hepatitis B vaccine programs.

be completed with either monovalent HBV vaccine or a combination vaccinecontaining HBV vaccine The second dose should be administered at age 1 or 2months The final dose should be administered no earlier than age 24 weeks Infantsborn to HBsAg-positive mothers should be tested for HBsAg and anti-HBs aftercompletion of the HBV vaccine series, at age 9 through 18 months (generally atthe next well-child visit).”

Vaccination of these neonates is very cost-effective with an estimated cost peryear of life saved of $164 HBV vaccine and HBIG are given at the same time attwo different sites within 12 hours of delivery, and two additional doses of HBV

Table 3.1: HBV vaccines and recommended dosage

Vaccine brand Age-group (yr) Dose ( μg) Volume (mL)

vaccine are given at 1–2 months and 6-12 months This passive-activeimmunization has a protective efficacy of 95% that is significantly greater thanthat of vaccine alone (75–80%) However, the vaccination programs that includehigh-risk groups have been proven to have very little impact on the incidence ofchronic HBV-infection Therefore, universal vaccination of all neonates withoutregard to mothers’ HBsAg status is required for global eradication of HBV-infection The integration of HBV vaccine into national immunization programswas recommended by the Global Advisory Group of the Expanded Program onImmunization in 1991 and endorsed by the World Health Organization (WHO) in

1992 One hundred and seventy-one of the 193 member countries of WHO hadintroduced universal HBV vaccine into their national immunization program as

of 2007 As a result, overall global HBV vaccination coverage of infants was60% in 2006 However, the estimated vaccination coverage strikingly varies acrossthe regions: i.e 28% in Southeast Asia, 49% in Africa, 74% in Europe, 78% inEastern Mediterranean region, 85% in East Asia, and 86% in America; and 61–63% in developed or developing countries, 50% in least developed countries Incountries accepting universal HBV vaccination, especially in high-risk countries,HBsAg-positive rate was significantly reduced For examples, childhood carrierrates were decreased from 6–14% to 0% in Alaska during 16-year period, from6.2 to 1.9% in Indonesia during 6-year period, from 12.0 to 2% in the Philippinesduring 10-year period, and from 10 to 0% in Hong Kong during 18-year periodafter implementation of the universal HBV vaccination program In Taiwan, wherethe coverage of HBV vaccination is 84–94%, HBV carrier rate of children <15year-old was decreased from 9.8 to 1.2% after 20 years of universal vaccinationprogram These findings indicate that the universal neonatal vaccination candecrease vertical and horizontal transmission of HBV infection in countries withhigh prevalence Vaccination of newborns of HBsAg-positive mothers is the mostimportant first step toward the eradication of chronic hepatitis B The incidence

of HCC among children was reduced by more than 50% (from 0.79 cases per100,000 between 1981 and 1986 to 0.36 cases between 1990 and 1994) and theannual incidence of HCC among children aged 6–9 years declined from 0.5/100,000 for those born in 1974–1984 to 0.1/100,000 for those born in 1986–

1988 in a landmark study from Taiwan As a result, HBV vaccination is nowconsidered as the first vaccine that can prevent cancer

VACCINATION OF ADULT

If a child was not vaccinated during the neonatal period, catch-up vaccination can

be performed to immunize him/her before they reach adolescence According toRecommended Adult Immunization Schedule—United States, 2009 provided byCDC, the indications of HBV vaccination in adult are categorized as follows:

1 Medical indications: Persons with end-stage renal disease, including patients

receiving hemodialysis; persons with human immunodeficiency virus (HIV)infection; and persons with chronic liver disease

2 Occupational indications: Healthcare personnel and public-safety workers

who are exposed to blood or other potentially infectious body fluids

3 Behavioral indications: Sexually active persons who are not in a long-term,

mutually monogamous relationship (e.g persons with more than 1 sex partnerduring the previous 6 months); persons seeking evaluation or treatment for aSTD; current or recent injection-drug users; and men who have sex with men

4 Other indications: Household contacts and sex partners of persons with chronic

HBV-infection; clients and staff members of institutions for persons withdevelopmental disabilities; international travelers to countries with high orintermediate prevalence of chronic HBV-infection; and any adult seekingprotection from HBV-infection

This recommendation mentions that all adults in the following settings should

be vaccinated: STD treatment facilities; HIV testing and treatment facilities;facilities providing drug-abuse treatment and prevention services; healthcare settingtargeting services to injection-drug users or men who have sex with men;correctional facilities; end-stage renal disease programs and facilities for chronichemodialysis patients; and institutions and nonresidential daycare facilities forpersons with developmental disabilities Prevaccination screening for anti-HBcwith/without HBsAg and anti-HBs is cost-effective only in countries where theHBV-infection prevalence exceeds 30%

The first viral disease eradicated with vaccination was smallpox The globaleradication of smallpox was officially announced in 1979, about 200 years afterthe first injection of smallpox vaccine to human and 13 years after launching theIntensified Smallpox Eradication Program by WHO Smallpox was successfullyeradicated because it was a human-specific disease, with no subclinical cases sothat effective surveillance was relatively easy, and because there were effectivevaccines With worldwide universal vaccination, we aimed to eradicate HBV aswell As with smallpox, human are the only known reservoir for HBV and effectivevaccines are available However, HBV-infection is different from smallpox, mostimportantly in that HBV-infection has an asymptomatic carrier state and withchronic infection persistently or chronically Therefore, careful laboratory screening

is required on large number of infants and older people, most of whom areasymptomatic for surveillance Global elimination of HBV requires countries toparticipate in programs with long-term commitment, over decades, to outlast theexisting cases of chronic HBV-infection Accordingly, the eradication of HBVmay take much longer than it did for smallpox

In summary, HBV vaccine provides great benefit not only for the prevention

of infection but also for the prevention of the sequalae of chronic infection such as HCC Therefore, HBV vaccine is the first vaccine that can preventcancer in the natural history of the disease To achieve global eradication of HBV,worldwide universal vaccination programs must be urgently established with globalcooperation commitment

3 Betancourt AA, Delgado CA, Estevez ZC, Martinez JC, Rios GV, Aureoles-Rosello

SR, et al Phase I clinical trial in healthy adults of a nasal vaccine candidate containing recombinant hepatitis B surface and core antigens Int J Infect Dis 2007;11:394- 401.

4 Blumberg BS, Alter HJ, Visnich S A “New” Antigen in Leukemia Sera JAMA 1965;191:541-6.

5 Buster EH, van der Eijk AA, Schalm SW Doctor to patient transmission of hepatitis

B virus: implications of HBV DNA levels and potential new solutions Antiviral Res 2003;60:79-85.

6 CDC Recommended adult immunization schedule—United States, 2009 MMWR 2008;57.

7 CDC Recommended immunization schedules for persons aged 0 through 18 years— United States, 2009 MMWR 2009;57.

8 Livingston SE, Simonetti JP, McMahon BJ, Bulkow LR, Hurlburt KJ, Homan CE,

et al Hepatitis B virus genotypes in Alaska Native people with hepatocellular carcinoma: preponderance of genotype F J Infect Dis 2007;195:5-11.

9 Ni YH, Huang LM, Chang MH, Yen CJ, Lu CY, You SL, et al Two decades of universal hepatitis B vaccination in taiwan: impact and implication for future strategies Gastroenterology 2007;132:1287-93.

10 Wasley A, Grytdal S, Gallagher K Surveillance for acute viral hepatitis—United States, 2006 MMWR Surveill Summ 2008;57:1-24.

11 Young MD, Schneider DL, Zuckerman AJ, Du W, Dickson B, Maddrey WC Adult hepatitis B vaccination using a novel triple antigen recombinant vaccine Hepatology 2001;34:372-6.

12 Yuen MF, Sablon E, Yuan HJ, Wong DK, Hui CK, Wong BC, et al Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma Hepatology 2003;37:562-7.