Chronic hepatitis B virus infection in Polynesians

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CHRONIC NTPETTUS B VIRUS II{F-ECTION

IN

POLYI\IESIAI\XS

A

thesis submitted

in

partial fuifilment of the requirements for the degree of

Doctor of Philosophy

at the

University of Auckland

by William Abbott FRACP

27th November 199'4

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ABBREVIATIOT{S

SUMIvIARY OF FTGURES

SUMI{ARY OF TABLES ABSTRACT

CHAPTER

1.

INTRODUCNON.

Section

1.1.

Polynesia.

1.1.1. Early History.

1,.1.2. Polynesians in New Zealand.

Section

1.2.

Polynesian Disease Susceptibility.

1,.2.7. Racial Differences

in

Disease Susceptibility.

1,.2.2. Risk Factors for Disease

in

polynesians.

section

1.3.

Methods

for

Differentiating _Genetic and Non4enetic Influences on Disease Risk.

1.3.1. Family Studies.

1.3.2. Gene Flow Studies.

1.3.3. Chromosome Studies.

1.3.4. Linkage Studies.

1.3.5. Animal Studies.

Section

1.4.

The Hepatitis B Virus ^(HBV).

7.4.1. Diseases Caused by the Hepatitis B Virus.

1.4-2- chronic Hepatitis B Virus Infections

in

^polynesia.

1.4.3. Risk Factors for Chronic Hepatitis B Virus

Pages

i-iii iv-v

vi - vii viii

- x

xi

1-42 2-3 2-3

3

4-t3 4-tL

11-13

14-24 74-77 t7 -22

22

22-24

24

25-38 25-26

27

Section 1.5.

Section 1..5.

Infection

in

Polynesia.

1.4.4. Hepatitis B Virus Structure.

1.4.5. The Immune Response to the Hepatitis B Virus.

1.4.6. Mechanisms of Chronic Hepatitis B Virus Infection.

Polymorphic Immune System Genes and ^Disease Susceptibility.

The T Cell Receptor.

28-30 31-33 34,35 36-38

38-40 40-42 40-41

4t-42

42

43-56

43

M-56

M

M-46

46-47

48-55

55 56 1.6.1. T Cell Receptor Structure.

1.6.2. Antigen Recognition by the T Cell Receptor.

Section

1.7.

Summary

CHAPTER 2. MATERIALS

AND

METHODS.

Section

2.1

Materials.

Section

2.2

Methods.

2.2.1 Hepatitis B Virus Serology.

2.2.2 Restriction Fragment Length polymorphisms.

2.2.3 Single Strand Conformational polymolphisms.

2.2.4 TCR p Chain Variable Regron Gene Assay.

2.2.5 Stimulation of PBMC by SEA.

2.2.6 Stimulation of PBMC by Engerix B.

CHAPTER 3. REPRODUCIBILITY AND VALIDATION OF THE TCR

VARIABLE REGION GENE

ASSAY.

57 -

ffi

Section 3.1. Reproducibility of the

Assay.

ST

Section 3.2. Validation _{of the}

Assay.

57 -

ffi

CHAPTER 4. POLYMORPHISN4S FOR USE

IN

GENE FLOW STUDIES.

Section

4.1.

Introduction.

Section

4.2.

Subjects.

Section

4.3.

Protocol and Statistics.

Section

4.4.

Results.

Section4.5.

Discussion.

CHAPTER

5.

GENE FLOW FROM EUROPEANS TO POLYNESIANS IS ASSOCIATED WITH PROTECTION FROM CHROMC HEPATITIS B VIRUS INFECTION.

Section

5.1.

Introduction.

Section

5.2.

Description of Subjects.

Section

5.3.

Protocol and Statistics.

Section

5.4.

Results.

Section5.5.

Discussion.

6t-69

61

61, - 62 62

u-67

58-69

CHAPTER 6. THE INFLUENCE OF

A

RECOMBTNANT.HBsAg,VACCINE ON THE REPERTOIRE OF T CELL RECEPTOR _P

CHAIN

VARIABLE REGION GENES

IN

PERIPHERAL BLOOD.

Section

6.1.

Introduction.

section

6.2.

subjects Recruited for the Immunisation study.

Section

6.3.

Experimental Design.

Section

6.4.

Results.

Section6.5.

Discussion.

70-87 70-71 7t -74

75

76-87 82-87

88

-

107 88

89-91

92-93

93

-

704 105

-

107

CHAPTER

7.

THE INFLUENCE OF

MA]ERNAL

HEPATMS B VIRUS STATUS ON CORD BLOOD T CELL RECEPTOR BETA CHAIN VAT{IABLE

CFIAPTER

S.

^{THE EFFECT} OF

A

CIIROMC HEPATITIS B VIRUS INFECTION ON PERIPHERAL BLOOD T CELL RECEPTOR BETA

CHAIN

VARIABLE REGION GENE REPERTOIRE

IN ADULTS.

124 - 1,U REGION GENE REPERTOIRE.

Section

7.1.

Introduction.

section

7.2.

subjects for Analysis of Vp Reperroire

in cord

Blood.

Section

7.3.

Experimental _Design and Statistics.

Section

7.4.

Results.

Section

7.5.

Discussion.

Section

8.1.

Introduction.

section

8.2.

subjects for the study of

Vp

Repertoire in Adults.

Section

8.3.

Experimental _Design and Statistics.

Section

8.4.

Results.

Section8.5.

Discussion.

CHAPTER

9.

CONCLUSIONS.

Section

9.1.

Gene Flow Studies

Section

9.2.

Associations _between HBV proteins and

vp

Repertoire

REFERENCES.

APPENDICES

A,

B, C

108

-

123 108 - 109 109

-

110 1,10

-

172

tt1 - tlg

720

-

123

724 125

-

726 727 127

-732

133

-

134

135

-

¹⁴¹

136

-

139 t39

-

147

ACKNOWLEDGEMENTS

This Ph.D. project would not have been possible without considerable advice and assistance from a number of people.

I

am particularly grateful

to my

supervisor, Professor fohn Marbrook

for

his interest

in

the project, constructive criticism and ^general scientific wisdom.

This

project was initiated

by Dr. Lloyd

Cairns and

the

laboratory

work

was conducted in the laboratories of ^the Human Immunology SouP run

initially

by Dr.

Cairns and more recently by Dr ^{Paul Tan,} Dr. Arie Geursen and Dr. Margot Skinner.

The interest they have shown in this work and their ^generous financial assistance are greatly appreciated. In addition I would like to thank Arie ^Geursen for teaching me

the

molecular techniques necessary

to

^complete

this work. I

received valuable assistance

from

the technicians

in

the group. John ^Peake performed

a

number of

restriction fragment length polymorphism and single strand

conformational polymorphism assays. Lana Perko isolated three new ^T cell receptor cDNA dones for

use in the assay of T cell receptor

I

^{chain variable} gene usage and her organisational abilities were invaluable for the smooth running of the laboratory. Bulk preparation

of

plasmids

for

use

in the p

_chain assay was assisted

by

Sarah Farmiloe, Louisa Forbes, |ohn ^Peake, Lana Perko, Laura Townsend and Shining ^Yuan.

The assay of T cell receptor p chain variable ^{gene usage} which was vital for the completion

of

this

work

was developed

by

Dr.

lohn

^Fraser

in

the Department of Molecular Medicine. He isolated and generously supplied us

with

15 of the cDNA clones used

in

the assily. There were many other individuals

in

the Department of Molecular Medicine who contributed ^ideas and suggestions ^to this work, in particular Lois Duncan for help with administrative matters and Euphemia Leung for help

with

Page

i

technical problems.

I

would also like to thank the following people:

Dr. Neil Vande'Water kindly supplied the

VIII

insert for an RFLP analysis.

Alistair Stewart, foanna Stewart and Elizabeth Robinson provided assistance

with

statistics. Leo Foliaki and Nonumalo Lurch translated patient recruiting material into the Tongan and Samoan languages.

The staff of the National Womens and St. Helens Hospital antenatal clinics who patiently tolerated disruptions to their routine while I was recruiting study subjects, and the staff of the National Womens Hospital deliverysuites who collected 162 cord blood samples. Kevin Townend provided epidemiological data on National Womens Hospital patients.

Sandor Milne of the Hepatitis Foundation in Whakatane gave valuable support

to

the Hepatitis B immunisation study and

I would like to

thank the principals, nurses and students

of

De La Salle College, Kelston Boys High School, Onehunga

High

School

and Mt.

Roskill Grammar

who

also made this study possible. Iohn Waldon of the Hepatitis Foundation assayed our samples for markers of hepatitis B

virus infection.

Dr. feff Featherston arranged the hepatitis B immunisation study conducted at the Auckland Fire Service and fan Wills R.N. provided valuable assistance with recruiting and immunisation of eligible subjects. I am very grateful to the fire senvice personnel who gave blood for the study.

Dr.

Ward Friesen and fonette

Suridge of

the Geography Dept., University of Auckland advised on the map of ^polynesia.

Lastly,

I would like to

thank Professor fames Watson

for

the

opportunig

of

Page

ii

shrdying

for

a Pli.D. deglee

in

the Deparh,ent of

Moldr

lr,iledicine at the salne tiure acknowledging ^the high qualittr and diversity of the scientific

qlvironnerrt

_that he rreated iin the depadr-rent.

I

also app-reeiate the use sf the laboratony facilities at Gsresis.

This

researctt

was patfially

f,tnded _-bJ

a gfant

fronn

the

Auckland Medtcal Rer$ar.& Founda,tion

Pagp

ii[

ABBREVI.ATIONS.

AF

allele frequenry

anti-HBc

antibody to HBV core protein

anti-HBe

antibody to HBV e protein

anti-HBs

antibody to HBV envelope protein

bp

^{base pairs}

cDNA

complementary

DNA

CDR

complementarity determining region

CI

confidence interval

CTL

cytotoxic T lymphocyte

dd

^{double distilled}

DEPC

^diethyl pyrocarbonate

DHBV

duck hepatitis B virus

GSHV

ground squirrel hepatitis virus

HBcAg

^{HBV core protein}

HBeAg

^{HBV e protein}

HBsAg

^{HBV small envelope protein}

HBV

^{hepatitis B virus}

HLA

^{human leucocyte antigen}

kb

^kilobases

kD

kilodaltons

I MW

liter

molecular weight

Page

iv

I

MHC

major histocompatibility complex

nm

^nanometer

NZ

^{New Zealand}

ORF

^open reading frame

PBMC

peripheral blood mononuclear ^cells

PBS

phosphate buffered saline

Pre-Sl

^{HBV latge envelope protein}

Pre.S2

^HBV middle*ized envelope protein

RFLP

restriction fragment length pollurorphism

sern

standard error of the mean

SDS

sodium dodecyl sulphate

SEA

staphylococcal enterotoxin

A

Tcr

^{T cell receptor}

pg

^microgram

pl

^microlitre

Vp

T cell receptor p dtain variable region ^gene

WHV

woodchuck hepatitis virus

Page

v

SUMIVIARY OF FIGURES

TITLE FOLLOWING PAGE

2.

79.

21..

31.

u.

ffi.

78.

104.

115.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9,

Figure 10.

Figure 11.

Figure 12.

Map _{of the} Polynesian Islands.

Results of a Hypothetical Non-Ecologic Study.

Results of a Hypothetical Case Control Study.

The Hepatitis B Virus Genome.

Time Course of a Typical Acute HBV Infection.

RNA Mixing Experiment.

Non-ecologic Study Showing an Association Between the Percent of European Genes and HBsAg ^prevalence

in

Europeans and Five Polynesian Ethnic Groups.

Comparison of the Percent of European Genes

in

anti-HBs and HBsAg Positive Subjects from Five Polynesian Ethnic Groups.

Design of the anti-HBV Immunisation Experiments.

Changes

in

Percent of Y p7.4 Transcripts in ^pBMC Following Immunisation of High School pupils.

Changes

in

Percent otYgT.4Transcipts

in

^pBMC Following Immunisation of Firemen.

Maternal HBsAg Status and the percent of yp7.4 Transcripts in Cord Blood PBMC.

Maternal HBeAg Status and the percent of VpS.l Transcripts in Cord Blood PBMC.

81.

92.

97.

Figure 13.

Page

vi

118.

Figlue

14.

Comparison of the Percent of VF6.3 Trarucripts

in

Cord Blood and Lvfaternal PBMC,

Figure

15.

Comparfuon

sf

the Percent of VF5.9 Transcripts in Cord

Blsod and Maternal ^{PBMC. L32"}

Page

vii

SUMMARY OF TABLES

TITLE

_PAGE

Table

1.

^Diseases Reported to Have a High Frequency

in Polynesians.

6.

Table

2.

^Diseases Reported to Have a Low Frequency in

polynesians.

_T.

Table

3.

^Diseases Reported to Have a Higher Frequenry

in

Maori than New Zealand

Europeans.

^g.

Table

4.

^Diseases Reported to Have a Lower Frequency

in

Maori than New Zealand

Europeans.

^g.

Table

5.

^Diseases Reported to Have Different Frequencies in

Different polynesian Ethnic

Groups:

^g.

Table

6.

coefficients of variation for the Tcr 0 chain V Region

Usage

Ar*y.

_5g.

Table

7.

Age and sex of the subjects Recruited

for

the study

of T Cell Receptor Locus Allele

Frequencies.

6g.

Table

8.

Comparison of Allele Frequencies of Tcr variable Region

RFLP's in New Zealand caucasians, Maoris and

samoans.

^66.

Table

9.

Comparison of Allele Frequencies of

rcr

constant Region

RFLP's in New Zealand caucasians, Maoris and

samoans.

67.

Table

10.

HBsAg Positive subjects used in the Non-Ecologic and Case Control Studies.

Table

11.

HBsAg Negative _subjects used

in

the Non-Ecologic and Case Control Studies.

Table

12.

^The Percent of European Genes

in

Five polynesian Ethnic Groups.

72.

73.

Page

viii

77.

Table

13.

case Control srudy comparing the Percent of European Genes

in

HBsAg Positive and anti-HBs Positive

Polynesian Subjects.

Table

14.

Description of High school Pupils Recruited for the anti-HBV Immunisation Study.

Table

15.

Description of Firemen Recruited

for

the anti-HBV Immunisation Study.

Table

15.

^Changes in the

vp

Repertoire of PBMC ar 48hrs and 22 hrs

80.

90.

91.

after Immunisation of High school Pupils

with

Engerix

B.

^96.

Table

1Z

Firemen study. changes in

vp

Repertoire of pBMC at 28

hrs after Engerix B

Immunisation.

99.

Table

18.

Firemen Study. Changes in

vp

Repertoire of pBMC at s2

hrs after Engerix B

Immunisation.

^100.

Table

19.

^Firemen study. changes in

vp

Repertoire of pBMC at ^g

days after Engerix B

Immunisation.

lg2-

Table

20.

Firemen Study. Changes in

v0

Repertoire

in

PBMC Cultured

for Six Days

with

Dialysed Engerix

B.

^103.

Table

21.

Description of subjects Recruited for Analysis of Vp

Repertoire

in

PBMC from Cord

Blood.

^111.

Table

22.

Significance of the Differences

in

Cord Blood Vp

Repertoire Between Neonates of HBsAg positive and HBsAg

Negative Polynesian

Mothers. ll4-

Page

ix

Table

23.

Significance of the Differences

in

Cord Blood Vp

Repertoire Between Neonates of HBeAg Positive and HBeAg

Negative Polynesian

Mothers.

^116.

Table

24.

Comparison of the Cord Blood Vp Repertoires of Neonates

HBsAg Negative Polynesian and European

Mothers. tl9.

Table

25.

Description of the Pregnant women Recruited for Analysis

of

Vp

Repertoire

in

PBMC at 6 Months

Post-Partum.

^126.

Table

26.

significance of the Differences in

vp

Repertoire Between

HBsAg Positive and HBsAg Negative

Adult

Polynesian

Women.

l?f..

Table

27.

Significance of the Differences

in vp

Repertoire Between

HBeAg Positive and HBeAg Negative

Adult

Polynesian Women. 130.

Table

28.

significance of the Differences

in vp

Repertoire Between

Adult

Polynesian and European

Women.

^131.

Table

29.

Significance of the Differences

in vp

Repertoire Between Six Month Post-Partum Women and the Cord Bloods of

Their

Babies.

_lg?-

Page x

ABSTRACT.

Genetic differences between Polynesians and Europeans may contribute

to

the many differences

in

disease frequenry that occur between these races. This thesis describes studies that were designed to determine

if

genetic influences contribute to the high prevalence of chronic hepatitis B virus (HBV) infection in Polynesians.

Alleles suitable

for

gene

flow

measurements were identified and used

to find

associations between European genes

and

indices

of

chronic

HBV

infection

in

Polynesians. An ecologic study found a negative association between European genes

and the

prevalence

of

chronic

HBV

infection

in five

Polynesian ethnic groups (p<0.05).

In

a case control study, there was a positive correlation (r=0.90, p=0.04) between

the

percent

of

European genes

in a

Polynesian ethnic

goup and

the difference

in

the percent of European genes between the HBV caniers and the anti- HBs positive controls

in

that ethnic Soup. Thus, the possibility that genetic factors are a major cause

of

the high prevalence

of

chronic HBV infection

in

Polynesians cannot be exduded. A twin study or candidate gene studies

will

be necessary to take this work further.

We attempted to identify candidate genes in the Tcr _F chain variable region (VF) locus that might contain polymorphisms that influence the human in aiao immune response to the HBV. VF7.4 was associated

with

the immune response to a vaccine containing recombinant HBsAg

in

two separate studies (p<0.0001 and p=6.95). The frequency of.Yp7.4 was low

in

the cord blood of neonates born to HBsAg positive mothers (p=0.001). The frequency of Vp5.1 was high

in

the cord blood of neonates

of

HBeAg positive mothers (p<0.001). Thus the Vpz.4 and

vpS.l

genes

may

be

preferentially used

in

the human immune response

to

the HBV

and may

be an appropriate place to seek polymorphisms that influence the immune response to the HBV and susceptibility to a chronic HBV infection.

Page xi