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Natalie McCloskey

This thesis is presented to the University of London for the degree of Doctor of Philosophy in the Faculty of Medicine

September 1996

Immunobiology Unit Institute of Child Health

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ABSTRACT

T h e four human IgG subclasses display greater than 90% sequence homology and their production is antigen restricted, with lgG1 and lgG 3 being preferentially elicited against protein antigens and lgG2 against carbohydrate antigens. Although the titre of a particular subclass is clearly important in overcoming infection, it is becoming apparent that the functional affinity (avidity) of such a response is also crucial. T hese studies were therefore initiated to investigate aspects of antibody avidity of the four human IgG subclasses.

Utilising V region identical m ouse-hum an IgG subclasses avidity differences w ere found by solid-phase avidity E L IS A and biospecific interaction analysis (BIA). lgG 4 was consistently of highest avidity and furtherm ore this reflected differences in dissociation rate constants. Following removal of the constant region such differences w ere abolished suggesting an involvement for the constant region in the control of human IgG subclass avidity.

Serum IgG subclass avidity specific for pneumococcal polysaccharide serotypes 3 , 6 , 19 and 23 w ere m easured by solid-phase avidity ELISA following pneumococcal vaccination. A complex pattern of avidity em erged with serotype 3 specific antibodies generally being of higher avidity. Significant differences were obtained comparing serum antibodies from children below 2 years of age with children more than 2 years of age. Such differences may contribute to the higher incidence of bacterial infection observed in children less than 2 years of age.

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cytokines on antigen specific total IgG avidity utilising solid-phase avidity assays developed throughout this thesis. Preliminary evidence presented in this thesis suggest that the cytokines IL-6, IL-10, T N F a and IFNy do not influence antibody avidity.

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ACKNOWLEDGEMENTS

There are m any individuals who have been influential throughout this thesis. Primarily, I would like to express my gratitude towards my supervisor Dr. David Goldblatt, for his constant enthusiasm, motivation and patience. I would also, like to thank Prof. M ac Turner for his unfailing attention to detail and our m any lively discussions.

On a lighter note the individuals within the division of Cell and M olecular Biology have m ade my time spent at the Institute of Child Health a most entertaining and social occasion. In particular I would like to thank Richard, Yem i and Dom for putting up with me in the laboratory. To Linda I would just like to say - w hat do you call a raver in a filing cabinet ?, to Paula, I am grateful to her for not allowing m e to starve, and thanks go to Helen for m any enjoyable hours in the Rugby and various restaurants. I would also like to thank David M. for taking m e to see som e great films and to Kate and Helen for giving m e the respect I deserve (I).

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CONTENTS

A b s tra c t... 2

A ckn o w le d g em en ts... 4

List of Figures and T a b les... 9

Abbreviations... 13

C H A P T E R 1 : G E N E R A L IN T R O D U C T IO N T h e immune system ... 16

Cells of the immune system ... 16

B cell developm ent... 20

Generation of diversity... 25

Immunoglobulin isotype switching... 27

Affinity maturation... 33

Immunoglobulin: the mediator of humoral immunity... 36

Immunoglobulin G: structure and function... 39

The IgG subclasses... 41

Gm allotypes... 45

Structure-function relationships of the IgG subclasses... 46

Human IgG subclass restriction... 48

Avidity of the human IgG subclasses... 52

Aims of the thesis... 55

C H A P T E R 2: M A T E R IA L S A N D M E T H O D S G eneral reagents... 58

Buffers and solutions... 61

M ethods... 64

General ELISA... 64

Purification of chimeric monoclonal antibodies... 6 6 Coupling of antigens to poly L-lysine... 67

Coupling of NIP hapten to B SA... 67

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C H A P T E R 3: T H E A V ID IT Y O F M O U S E -H U M A N C H IM E R IC V

R E G IO N ID E N T IC A L M O N O C L O N A L IgG S U B C L A S S E S

Introduction... 72

The avidity of naturally occurring IgG subclasses... 7 2 IgG subclass avidity associated with disease states... 7 3 The influence on avidity of immunoglobulin structure remote

from the V region... 7 4 B72.3 mAbs... 7 5 Anti-NIP mAbs... 7 6 Solid-phase avidity ELISAs... 7 6 Aims of the Study... 8 0

M ethodology... 81 Results... 84

Purification of 8 7 2 .3 ... 8 4 Antigen specific ELISA... 91

Solid-phase avidity ELISA; Competitive binding... 9 4 Thiocyanate elution... 9 8

Discussion... 104

C H A P T E R 4: B IN D IN G K IN E T IC S O F M O U S E -H U M A N C H IM E R IC V R E G IO N ID E N T IC A L IgG S U B C L A S S E S

Introduction... 1 0 9 Biosensor technology... 1 1 0 Surface plasmon resonance... I l l BIAcore™ Biosensor... I l l Kinetic and affinity analysis... 1 1 3 Aims of the study... 1 1 4

M ethodology... 1 1 5

Results... 1 1 7 Preparation of B72.3 chimeric IgG subclasses F(ab')2 fragments 1 1 7

BIAcore™ Analysis... 1 2 8

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C H A P T E R 5: T H E A V ID IT Y O F IgG S U B C L A S S E S S P E C IF IC F O R P N E U M O C O C C A L P O L Y S A C C H A R ID E S

Introduction... 151

Streptococcus pneumoniae... 1 5 1 Imm une responses to pneumococcal polysaccharides... 1 5 2 Factors influencing the anti-pneumococcal polysaccharide response... 1 5 4 Pneumococcal polysaccharide vaccines... 1 5 7 Aims of the study... 1 5 9

M ethodology... 160 Results... 162

Antibodies to pneumococcal polysaccharides from standard serum 1 6 2 Competitive Binding ELISA... 1 6 4 Thiocyanate Elution ELISA ... 1 7 1 Response of children immunised with Pneumovax... 178 Discussion... 187

C H A P T E R 6: T H E E F F E C T O F V A R IO U S C Y T O K IN E S O N S P E C IF IC IN V IT R O IgG A V ID IT Y

Introduction... 195

The use of adjuvants to enhance the immune response... 195

The influence of cytokines as adjuvants upon the immune response.... 196

The effect of cytokines upon antibody affinity... 196

Somatic hypermutation... 198

Cytokines... 199

Aims of the study... 20 2 M ethodology... 20 3 Results... 20 7

IgG subclass profile of standard serum antibody to influenza virus 20 7

The detection of influenza specific IgG in tonsillar cell cultures 20 8

The effect of cytokines on influenza specific IgG production... 20 9

Development of avidity ELISA ... 211

The avidity of influenza specific IgG produced from T M C s ... 21 5

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C H A P T E R 7: G E N E R A L D IS C U S S IO N

Analysis of antibody avidity... 22 6 Antibody binding kinetics... 23 0 IgG subclass avidity differences... 231 T h e role of antibody avidity in protection... 23 4 T h e control of somatic hypermutation... 23 7 Concluding remarks and future perspectives... 239

A P P E N D IC E S

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List of Figures

1.1 Cells involved in the immune system 1.2 Hum an B cell development

1.3 Proposed mechanisms involved in directing V (D )J recombination 1.4 M echanism s and control of immunoglobulin isotype switching 1.5 G e n e organisation of the human immunoglobulin genom e 1.6 Domain structure of immunoglobulin G

1.7 G en e organisation of human and mouse IgG subclasses 1.8 Domain structure of the human IgG subclasses

3.1 10% reducing S D S -P A G E of B 72.3 mAbs

3 .2 10% reducing S D S -P A G E of anti-N IP mAbs (a, b and c) 3 .3 7 .5 % non-reducing S D S -P A G E of purified IgG subclasses 3.4 Mucin specific ELISA of purified B72.3 IgG subclasses 3.5 N IP specific ELISA of purified anti-N IP IgG subclasses

3.6 Dose-response curves for B72.3 Ig G I, lgG2, lgG3, lgG4 binding to mucin 3.7 Dose-response curves obtained for anti-N IP lgG2, lgG3 and lgG 4 binding to N IP -B S A at epitope densities of NIP^g-BSA, NIP^g-BSA and NIPg-BSA 3.8 Competitive binding curves of B 72.3 mAbs binding to mucin

3.9 Competitive binding curves of anti-N IP mAbs binding to NIP^g-BSA 3 .1 0 Dose-response curves for B72.3 mAbs

3.11 Dose-response curves for anti-NIP mAbs

3 .1 2 Am m onium thiocyanate elution ELISA using ammonium thiocyanate from 0.1 M to 0.6M to disrupt antigen-antibody binding

3 .1 3 Am m onium thiocyanate elution ELISA using ammonium thiocyanate from 0.1M to 2M to disrupt antigen-antibody binding

4.1 Optical configuration of the BIAcore™ Biosensor

4 .2 10% non-reducing S D S -P A G E of pepsin digested B 72.3 Ig G I and lgG3 4 .3 10% non-reducing S D S -P A G E of pepsin digested B 72.3 lgG2 and lgG4 4 .4 S u p erd ex-200 column calibration

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4 .6 Elution profile of B 72.3 lgG2 mAb digested with pepsin 4 .7 Elution profile of B 72.3 lgG3 mAb digested with pepsin 4 .8 Elution profile of B 72.3 lgG4 mAb digested with pepsin

4 .9 10% non-reducing S D S -P A G E of B72.3 Ig G I and lgG 4 pepsin digested fractions eluted from S u perdex-200

4 .1 0 10% non-reducing S D S -P A G E of B 72.3 lgG2 pepsin digested fractions eluted from S u perdex-200

4.11 7 .5 % non-reducing S D S -P A G E of B72.3 lgG3 pepsin digested fractions eluted from Superdex-200

4 .1 2 Preconcentration of mucin to a sensor chip 4 .1 3 Immobilisation of mucin to a sensor chip

4 .1 4 Preconcentration of NIP^g-BSA to a sensor chip 4 .1 5 Immobilisation of NIP^g-BSA to a sensor chip

4 .1 6 Sensorgram s illustrating the specificity of the mucin sensor chip 4 .1 7 Sensorgram s illustrating the specificity of the NIP^g-BSA sensor chip 4 .1 8 O verlay of mucin binding sensorgrams produced for Ig G I and lgG 2 B 72.3

mAbs

4 .1 9 O verlay of mucin binding sensorgrams produced for B 72.3 mAbs lgG3 and lgG4

4 .2 0 O verlay of mucin binding sensorgrams obtained for B 72.3 IgG subclasses 4.21 O verlay of mucin binding sensorgrams obtained for B72.3 peptic F (a b' ) 2

fragm ents

4 .2 2 O verlay of NIP binding sensorgrams produced for lgG 2 and lgG 3 anti-N IP mAbs

4 .2 3 O verlay of NIP binding sensorgrams produced for lgG 4 anti-N IP mAb 4 .2 4 O verlay of NIP^g-BSA binding sensorgrams

5.1 Dose-response curves for standard serum 5.2 Competitive binding curves

5.3 Polysaccharide serotype 3 competitive binding curves 5.4 Competitive binding curves

5.5 Competitive binding curves

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5.7 Mobility of pneumococcal polysaccharide serotype 6B 5.8 Mobility of pneumococcal polysaccharide serotype 19F 5.9 Mobility of pneumococcal polysaccharide serotype 23F 5 .1 0 Mobility of pneumococcal polysaccharide serotype 3 5.11 Dose-response curves of standard serum lgG 2 antibodies 5 .1 2 Thiocyanate elution binding curves

5 .1 3 M ean avidity indices produced for serum IgG specific for pneumococcal polysaccharides serotypes 3, 6B, 19F and 2 3 F

5 .1 4 M ean avidity indices produced for serum Ig G I and lgG2 specific for pneumococcal polysaccharides serotypes 3, 6B, 19F and 2 3 F 5 .1 5 M ean avidity indices produced for serum IgG antibodies specific for

pneumococcal polysaccharide serotype 3

5 .1 6 M ean avidity indices produced for serum IgG antibodies specific for pneumococcal polysaccharide serotype 2 3 F

6.1 Influenza specific IgG subclass response from standard serum 6.2 IgG total standard curve

6 .3 T h e effect of cytokines, T N F a , IFNy, IL-6 and IL-10 upon influenza specific IgG production by E B cells

6.4 Dose-response curves obtained for standard serum IgG

6 .5 Dose-response curves obtained for influenza-lysine specific standard serum IgG

6 .6 Am m onium thiocyanate elution ELISA using ammonium thiocyanate from 0 .5 M to 6M to disrupt serum derived antibody-antigen binding

6 .7 T h e avidity of influenza specific IgG produced from T M C s, m easured by thiocyanate elution ELISA

6 .8 Competitive binding curves

6 .9 Influenza specific IgG from two different tonsils 6 .1 0 Competitive binding curves

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List of tables

1.1 Physico-chemical properties of human immunoglobulin classes 1.2 Biological properties of human immunoglobulin molecules

1.3 Gm allotypes of human immunoglobulins adapted from Lefranc and Lefranc (1990)

1.4 Biological and structural properties of IgG subclasses

2.1 Antigen specific ELISA procedures: variations from general method 3.1 Applications of solid-phase avidity ELISA procedures

3 .2 Avidity indices derived for B72.3 mAbs measured by competitive binding EL IS A

3 .3 Avidity indices derived for anti-NIP lgG2, lgG3 and lgG 4 mAbs m easured by competitive binding ELISA

3 .4 Avidity indices derived for B 72.3 mAbs m easured by thiocyanate elution EL IS A

3 .5 Avidity indices derived for anti-NIP lgG2, lgG3 and lgG 4 mAbs m easured by thiocyanate elution ELISA

4.1 Optimal conditions for the pepsin digestion of B 72.3 mAbs

4 .2 Apparent rate constants of intact chimeric B72.3 IgG subclass proteins and peptic F(ab' ) 2 fragments studied

4 .3 Apparent rate constants of intact chimeric anti-N IP subclass proteins studied

5.1 Relative antibody avidity indices

5.2 M ean avidity indices for serum antibodies specific for pneumococcal polysaccharide serotypes m easured by thiocyanate elution ELISA 5 .3 Num bers of patients responding to Pneum ovax with serotype specific

Ig G I and lgG2 antibodies

6.1 Avidity indices obtained for standard serum IgM and IgG antibodies binding to influenza-lysine

6.2 Avidity indices produced for influenza specific IgG obtained from T M G preparations incubated with influenza virus, with or without various cytokines

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ABBREVIATIONS

A b antibody

A b s absorbance

A E T S-2-aminoethylisothiouronium bromide A F C antibody forming cell

A g antigen

A P C antigen presenting cell B C R B cell receptor

B IA biospecific interaction analysis

0 constant

C D cluster of differentiation

C O R complimentarity determining region C I L cytotoxic T lymphocyte

C W P S cell wall polysaccharide

D diversity

D N A deoxyribonucleic acid E B V epstein barr virus

E L IS A enzym e linked immunosorbent assay F ab fragm ent antigen binding

Fc fragm ent crystalline

FC S foetal calf serum FD C follicular dendritic cell

F P L C fast protein liquid chromatography

H heavy chain

Hib Haem ophilus influenzae type b H IV human immunodeficiency virus

IFN interferon

IG F insulin growth factor

igG Immunoglobulin G

IL interleukin

J joining

K equilibrium constant

ka association rate constant kd dissociation rate constant KLM keyhole limpet hemocyanin

L light chain

m 0 m acrophage

m A b monoclonal antibody

M H C major histocompatibility complex

M P O myeloperoxidase

N IP 4-hydroxy-3-iodo-5-nitrophenylacetyl

N K natural killer

P B E F pre B cell colony enhancing factor P B S phosphate buffered saline

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P B S -T w -B S A phosphate buffered saline/0.5% T w e e n 2 0 /1 % BSA P R P poiyribosylribitolphosphate

PS polysaccharides

R N A ribonucleic acid

RU resonance unit

S switch region

S C F stem cell factor

S D S -P A G E sodium dodecyl sulphate-polyacrylam ide gel electrophoresis sig surface immunoglobulin

S P R surface plasmon resonance (pL surrogate light chain

T C R I cell receptor

Tdep T dependent

T G Fp transforming growth factor p Th T helper lymphocyte

Th1 T helper lymphocyte type 1 Th2 I helper lymphocyte type 2

^ind T independent

T M C tonsil mononuclear cell T N F tumour necrosis factor Ts T supressor lymphocyte

V variable

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General Introduction

T h e immune system ... 16

Cells of the immune system ... 16

B cell developm ent... 20

Generation of diversity... 25

Immunoglobulin isotype switching... 27

Affinity m aturation... 33

Immunoglobulin: the mediator of humoral immunity... 36

Immunoglobulin G: structure and function... 39

The IgG sub classes... 41

Gm allotypes... 4 5 Structu re-function relationships of the IgG subclasses... 4 6 Human IgG subclass restriction... 48

Avidity of the human IgG subclasses... 52

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INTRODUCTION

The Immune System

T h e immune system provides an individual with protection from infection by pathogens such as viruses, bacteria and parasites. The quality of the response depends upon the ability of the host to discriminate between self and non-self antigens and subsequently to elicit an appropriate immune response with which to elim inate potential pathogens. T h e immune system must, therefore, be carefully controlled since a breakdown in regulation m ay lead to disease.

Cells of the Immune System

Cellular (cell mediated), humoral (antibody m ediated) and innate immunity work together to protect an individual from invasive pathogens. Imm une responses are m ediated primarily by lymphocytes and phagocytes, which arise from a mulipotent haematopoeitic stem cell(s).

Cellular immunity involves the cell mediated killing of pathogens by immune cells, such as neutrophils, m acrophages (mcj)), natural killer (NK) cells,

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T Lymphocytes

T lymphocytes originate in the bone marrow, develop and differentiate in the thymus and then travel through the circulation to primary and secondary lymphoid tissues. It is within such tissues that, by virtue of the T cell receptor (T C R ), they recognise antigen expressed on an antigen presenting cell (ARC), in association with M H C molecules. The T C R consists of an antigen binding portion formed by heterodimers of a and p or y and ô polymorphic polypeptide chains. T h e T C R itself does not m ediate cellular signalling pathways; these are elicited by an associated molecule, CD 3, which consists of a complex of polypeptides. T lymphocytes are distinguished from other cells by the characteristic cell surface marker, C D 3 man, whilst in the mouse, Thy1 is a characteristic

marker.

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B Lym phocytes

B lymphocytes develop in the bone marrow and foetal liver. M ature B lymphocytes are responsible for immunoglobulin production which, when m em brane bound, forms the characteristic B cell receptor (BCR). B lymphocytes also express M H C class II molecules and therefore may act as A P C s, presenting antigen to C D 4 + T lymphocytes. In addition, B lymphocytes have on their surface a variety of other markers, such as Fc receptors and C D 40, which are essential for cell activation and proliferation.

T h e B cell receptor is specific for antigen and for the majority of antibody responses T cell help is also required. Protein antigens, which depend directly on T cell help in the form of cell-cell contact or cytokines, are referred to as T cell dependent (T^ep) antigens. Those antigens which do not require direct T cell help are designated T cell independent (Tj^d) and are primarily carbohydrates. T^d antigens are thought to stimulate B lymphocytes directly. Following interaction with antigen, providing that they receive the appropriate signals, B lymphocytes clonally expand and differentiate into antibody forming cells (A FC ). Furthermore, long lived B lymphocytes, referred to as m emory cells, may be produced and these retain the ability to produce specific antibody in response to subsequent antigen exposure.

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Pluripotent haem atopoietic stem cell

M yeloid stem cell

Lymphoid stem cell

Thymocyte B FU -E

M egakaryocyte

Platelets

Erythrocyte

C F U -E C F U -B C F U -G M

Basophil

Eosinophil

Monocyte

ro /P re B cell

Neutrophil

M em ory B cell

M acrophage

C P U = colony form ing unit BFU = blast form ing unit

P lasm a cell

(antibody producing)

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B Cell Development

Lymphocyte developm ent proceeds through various stages characterised by specific gene rearrangem ents and cell surface m arker expression (Tonegaw a, 1983; Alt et al. 1987), resulting in mature B and T cells expressing antigen specific immunoglobulin or TC R . It has been estimated that once the immune system is established there are approximately 5 x 10^^ cells in the human B cell compartment, 10% of which are regenerating precursors and 9 0 % resting m ature B cells (Relink and Melchers, 1993).

Different schem es exist for the nomenclature of B cell progenitors and precursors. Throughout this discussion the following nom enclature will be used: pro B cell, pre B-l cell, pre B-ll cell, immature and mature B cell (Figure 1.2)

Pro B cells

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Pro B cell

Pro B-ll cell

Pre B-1

pre BCR

C034

CD10 CD19

x<pL

CD19

germline V

h

V

l

Dh- Jh

Pre B-ll

pre BCR

Immature B cell

BCR

CD19 C019 CD19

Vh- Dh- Jh

V.-J.

Mature B cell

mig (BCR)

CD19

cp

BCR = B cell receptor

Figure 1.2

Human B cell development

Diagram showing gene rearrangements and immunoglobulin expressions during human B cell developmental stages, adapted from (Guelpa Fonlupt et al. 1994)

o

m

O

a c

3 O

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P re B cells

B cell developm ent is characterised by sequential immunoglobulin gene rearrangem ents, which begin in the heavy chain locus of pre B-l cells, in the Dh

to Jh segm ents. In the mouse, pre B-l cells may be defined as having long term proliferative capacity on stromal cells in the presence of 11-7 and have undergone □hJh rearrangem ents, whilst retaining germline L chain genes.

Bone marrow derived human B cell precursors grow in the presence of IL-7 similarly to m ouse pro/pre B-l cells (Melchers et al. 1995). The cell-cell contact betw een human precursor B cells and stromal cells has been identified as C D 4 9 d /C D 2 9 - C D 106, and this interaction has furthermore been dem onstrated to enhance IL-6 production which promotes B cell growth (Jarvis and LeBien, 1995). Such signals presumably enable the pro/pre B-l cells to proliferate and it is thought that loss of stromal contact stimulates pro/pre B-l cells to further differentiate.

In mice, large pre B-ll cells have at least one of their IgH loci VhJhDh rearranged. Expression of this pH chain cannot occur alone and is expressed in association with the surrogate L chain (cpL), a heterodimer of the protein products

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T h e pre B cell receptor appears to differentially signal to prom ote allelic exclusion (productive rearrangem ent of only one allele) and proliferation of pre B-II cells. At present, candidate ligands for the pre B cell receptor remain elusive (M elchers et al. 1995). Human B lineage cells express the cpL chain throughout

pro and pre B cell developm ent but cell surface expression is confined to the pre B stages (Lassoued et al. 1993).

Im m ature B cells

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The role o f cytokines

Various cytokines have been implicated in the control of B cell developm ent. Growth and differentiation of pro and pre B cells is influenced by IL-7 (N am en et al, 1988), stem cell factor (S C F ) (Billips et al, 1992), insulin growth factor (IG F -1 ) (Gibson et al, 1993), pre B cell growth stimulatory factor (P B S F) (N ag a saw a et al, 1994) and pre B cell colony-enhancing factor (PB E F) (Sam al et al, 1994). Factors that may inhibit B cell developm ent have also been described and include IL-4 (Rennick et al, 1987), IL-1 (Dorshkind, 1988), T G F p (Lee et al, 1989), IFNy (Garvy and Riley, 1994), oestrogen (M edina et al, 1993) and IL-3 (Hirayam a et al, 1994).

The role o f tyrosine kinases

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Generation of Diversity

T h e primary repertoire of immunoglobulin specificity is large and is estim ated to exceed 1 million (Tonegawa, 1983). It is generated without exposure to exogenous antigen, by virtue of gene rearrangem ents, with additional diversity m ediated by the imprecise joining of gene segments. The antigen binding portion of the immunoglobulin is formed by the recombination of V, (D) and J segments, V (D )J recombination is thus responsible for the generation of diversity.

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Following recombination the V(D)J segment is expressed with a downstream C\i gene segment to form a productive heavy chain. Light chain genes undergo similar recombination but lack the D segment and therefore diversity is greatest in the heavy chain gene. As mentioned earlier productive pH chains are expressed prior to pL chains at the pre B cell stage. The expression of productive L chains allows cytoplasmic IgM expression at the pre B-ll cell stage. Membrane expression then occurs at the mature B cell stage.

7bp 9bp

9bp 7bp

i

R A G -1 /2

O th e r proteins e.g X R C C 5 ,S C ID

Jun ctional D ivers ity (T d T )

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Immunoglobulin Isotype Switching

During an immune response a B cell may switch from expressing pH or ôH

chains to the production of another Ch: y, e or a , whilst still retaining the V region, thus ensuring that antigen specificity is maintained. Isotype (class) switching enables a B cell to produce an antibody with the appropriate effector functions required to eliminate a particular pathogen. Associated with the isotype switch is deletion of the DMA of the previous Ch chain and thus a B cell cannot switch back to expressing pH or ÔH chains.

T h ree models have been proposed to be responsible for the DMA deletion event (reviewed extensively by Harriman et al. 1993).

(i) recombination between homologues

During mitosis four DMA strands are produced with only one allele being productive, these four strands could cross over at sites of homology upstream of the Ch gene giving rise to a daughter cell producing the V region joined to another Ch region. No evidence as yet has been presented to support this model, although it cannot be totally ruled out and probably occurs at low frequency. (ii) unequal sister chromatid exchange

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(iii) looping out and deletion

M any lines of evidence accum ulated in previous years have pointed to this model being responsible for isotype switching. In this model intervening D N A is looped out and excised as a ‘switch circle’ containing Cp. and the Ch switch region to

which the cell has switched (Figure 1.4). Switch circles have been identified from m ouse spleen cells stimulated using the alkaline lysis method (M atsuo ka et al. 1990). So called “switch regions” located 5' to the Ch genes except for CÔ facilitate homologous recombination and/ or focus the putative ‘switch recom binase' enzym e (Figure 1.4).

Switch (S) Regions

M ouse S regions are tandem ly repeated sequences 5' of the Ch genes, including several distinct motifs. Sp consists of (G A G C T )n (G G G G T ) w h ere n= 1-7, repeated approximately 150 times. O ther S sequences contain multiple copies of these pentanucleotides as well as pentamers, A C C AG , G C A G C and T G A G C and the heptam eric repeat (C /T )A G G T T G (Coffman et al. 1993). S a and Ss tandem ly repeated motifs are 4 0 and 80bp respectively and Cy are 4 9 or 52bp. Th ese S regions are particularly recombinogenic, with S\x being 100 fold more

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The control o f isotype switching b y cytokines

Class switch recombination is preceded by the production of sterile (non­ productive) germline transcripts from the gene to be expressed (S taven e zer-Nordgren and Sirlin, 1986). Transcription of germline Oh R N A is initiated with I exons located 5' to each S region, continues through the S region and term inates

3' to the Ch gene, as shown in Figure 1.4 (G auchat et al. 1990; Gerondakis, 1990; Rothman et al. 1990). T h e importance of I exons in class switch recombination is demonstrated in mice which lack lyl and are unable to produce Ig G I (Thyphronitis et al. 1993). T h e preceding transcriptional activity of the Ch

locus is believed to m ake the region accessible to factors that m ediate class switch recombination, such as the putative switch recombinase. This hypothesis is referred to as the accessibility model (Stavenezer-N ordgren and Sirlin, 1986). Accessibility can be induced by creating (i) nucleosom e free D N a s e I hypersensitivity sites for recom binase and R N A polym erase access (Barton and Vietta, 1990; Ford et al. 1992), or (ii) déméthylation of DNA. The production of germ line transcripts has been dem onstrated to be under the control of cytokines, including IL-4, IL-13, IFNy and TG F p . Secondary signals provided by IL-5, IL-6,

cognate T-B interactions m ediated by C D 4 0 -C D 4 0 L and C D 5 8 -C D 2 are then required to proceed to class switch recombination.

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to lgG 2a (Snapper at al. 1988), w hereas T G Fp promotes switching to IgA (Coffm an et al. 1989). All these studies w ere performed in vitro, but recent in vivo

analyses have supported the role of IL-4 in stimulating murine Ig G I and IgE (Nakanishi et al. 1995).

T h e m echanisms underlying cytokine driven class switching are unclear at present but presumably involve specific binding regions, such as the IL-4 response elem ent identified 5' to the s switch site in mice (Ichiki et al. 1993). Interestingly, cholera toxin, a potent immunogen and adjuvant, has also been dem onstrated to promote the formation of germline y1 transcripts synergistically with IL-4 in vitro in murine LPS stimulated B cells. T h e m echanism s responsible w ere found to include increased intracellular cAMP, which augm ents germline transcript production (Lycke, 1993).

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With regard to the human IgG subclasses, studies by Fujieda et al. (1 9 9 5 ) have demonstrated switching of human B cells to Ig G I, lgG3 and lgG 4 but not lgG 2 by IL-4 and C D 40 mAb. Kotowicz and Callard, (1993 ) have demonstrated increased production of Ig G I, lgG2 and lgG3 by EB V stimulated human B cells using IL-4. Additionally, Kawano et al. (1995 ) have dem onstrated increased production of human IgG subclasses using IL-6.

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VDJ S/i

D N A

VDJ C/i

cyto kin es eg . IL -4 , IF N y

ly Sy Cy

R N A

g e rm lin e /s te rile tra n s c rip t

p u tative 'sw itch re c o m b in a s e '

V D J SyW Sy Cy

DNA

truncated n -y switch region

D e ie te d sw itch circle R e ta in e d g e n o m ic D N A

(34)

Affinity Maturation

During an immune response antibody affinity increases with time following initial exposure to antigen. This phenomenon is referred to as affinity maturation. Affinity maturation is due to somatic hypermutation of the variable region which physically occurs within structures called germinal centres in secondary lymphoid tissues.

Som e mutations in the immunoglobulin V region give rise to higher affinity antibodies and B cells expressing lower affinity or self-reactive antibodies die by apoptosis. In contrast to Burnet’s theory of positive and negative selection followed by clonal expansion (1957), it is now widely accepted that all B cells are programmed to die by apoptosis, unless rescued by a positive signal such as the C D 4 0 -C D 4 0 L interaction, in addition to sig receptor cross-linking.

Following antigen stimulation mature B cells migrate to primary follicles of secondary lymphoid organs, including the spleen and tonsils, w here they proliferate extensively to form germinal centres (N euberger and Milstein, 1995). T h e germinal centre is divided into two main compartments, the dark and the light zone. B cells are activated in the T cell rich zones outside the follicles and on average only 3 B cell blasts colonise each follicle. Proliferating B cell blasts (centroblasts) are located in the dark zone giving rise to non-proliferating centrocytes in the follicular dendritic cell (FD C ) rich light zone.

(35)

but such antigens may receive non-T cell help, and the true 7^^ nature of the antigen needs to be clarified.

Hum an tonsillar B cell/FDC adhesion is mediated by C D 5 4 and VC A M -1 on F D C binding to C D 1 1 a /C D 1 8 and V L A -4 respectively on centrocytes (Koopm an et al. 1991; Holder et al. 1992). The analysis of such interactions has been ham pered by the poor identification of the true FD C and the small numbers of F D C which can be purified. The recent reports of FDC-like human cell lines FD C -1 (Clark et al. 1995) and HK (Kim et al. 1995) should help to clarify these reactions. Nevertheless, it is known that the anti-lg cross linking signal is not enough to rescue B cells from apoptosis. Additional signals m ediated by C D 4 0 -C D 4 0 L (M aclennan and Gray, 1986), B 7.1:-C D 28/-C T L A .4 (Han et al. 1995), C D 2 0 engagem ent (Holder et al. 1995) and possibly C D 1 9 and C D 2 2 cross-linking are required (Chaouchi et al. 1995).

(36)

Is som atic hypermutation random o r directed ?

T h e pattern of somatic hypermutation of V genes has been extensively analysed in anti-hapten responses. Previously, somatic hypermutation w as thought to be random, the existence of mutational hotspots being due to antigenic selection. Analysis of replacem ent versus silent mutations has dem onstrated that somatic hypermutation is non-random (Berek and Milstein, 1988; W eiss et al. 1992). The location of such intrinsic hotspots have been identified by studying immunoglobulin passenger transgenes (transgenes that do not contribute to expressed antibody). In mouse and man a major intrinsic hotspot is in the second base of serine codons A G C /T , the existence of these unusual serine codons in the C D R s may favour local targeting to the complementarity determining regions

responsible for antigen binding (Neuberger and Milstein, 1995). T h e recent sequencing of the whole human Vh locus (Cook and Tomlinson, 1995) and the analysis of transgenes should enable somatic mutation to be analysed in more detail. A recent study by Yelam os et al. (1995) has shown that replacem ent of a

Vk region with non-lg human p globin sequences resulted in hypermutation of the

hum an p globin gene, thus the V region itself is not required to direct

hypermutation. T h e mechanisms responsible for somatic mutation remain enigmatic, but are believed to involve some form of error prone D N A repair process (Rajewsky, 1996). Various cis-acting elem ents have now been identified in vitro. T h e murine IgK intron and 3' enhancers appear to be critical (Betz et al.

(37)

Immunoglobulin: The Mediator of Humoral Immunity

Immunoglobulins comprise a four polypeptide chain unit of two identical light (L) chains and two identical heavy (H) chains which are cross linked by interchain disulphide bonds and stabilised by non-covalent interactions. G reat progress has been m ade since the identification in the 1 9 5 0 ’s of five immunoglobulin classes (isotypes) in man characterised by specific heavy chains: p, y, 6, a and c. In man immunoglobulin genes are located on chromosome 14 and in mice on chrom osom e 12 (Figure 1.5)

The L chains are folded into two globular domains while the heavy chains are folded into four or five domains depending on the immunoglobulin class/isotype. Som e of the physio-chemical properties of human antibodies are illustrated in Tab le 1.1

Table 1.1

Physico-chemical properties of human immunoglobulin classes

IgG

IgM

IgA

IgD

IgE

Heavy chain

y1-4 p a 1 -2 Ô 8

Molecular weight (kDa)

146 950 160 175 190

Sedimentation constant

7 8 19 8 7 8 7 8 8 8

No. of H chain domains

4 5 4 4 5

% carbohydrate

2-3 12 7-11 9 -1 4 12

Half life (days)

23 5.8 5.1 2 .8 2 .5

(38)

8

60

26

35

cy1

cy4

m

cy3

ce c a 2 cy2

30

18

23

10

CO -vj

Figure 1.5

Gene organisation of the human immunoglobulin genome

(39)

T h e N-terminal domains of both the heavy and light chains are highly variable (V domains), whilst the remaining domains are relatively constant (C domains), and are characteristic of the immunoglobulin class. The domains consist of a p barrel structure with seven (C domain) or nine (V domain) strands of p sheet stabilised by intrachain disulphide bonds. The variability of the V domain is clustered into hypervariable loops, sometimes referred to as complimentarity determining regions (C D R s). The hypervariable loops are brought together by the folding of the V domain and the heavy and light chains generate a surface of six hypervariable loops, which determine antibody specificity. This region has been called the paratope. The residues surrounding the hypervariable loops are term ed fram ework residues and together with the rest of the V region constitute the fragm ent antigen binding (Fab) portion of the antibody molecule. The fragm ent crystalline (Fc) distal to the Fab region mediates the effector functions of antibodies, some of which are summarised in Tab le 1.2

Tab le 1.2 Biological properties of human immunoglobulin molecules

IgM

IgG

IgA

IgD

IgE

Complement fixation

++

+

- -

-Platelet binding

-

+

- -

-Mast cell/basophil sensitisation

- - - -

+

Transplacental transfer

-

+

- -

-Opsonisation capacity

-

+

+

-

-Sensitisation for K cells

-

+

- -

-Binding to lymphocytes

T

T,B

T.B

-

T,B

Binding to mononuclear cells

+/-

+

- -

-Binding to neutrophils

+

+

+

-

-Binding to protein A

+/ - + / - " -

(40)

Immunoglobulin G is the major isotype in normal human serum, accounting for 7 0 -7 5 % of total serum immunoglobulin. IgG is mounted early in the secondary response to antigenic challenge and its affinity is seen to increase during an im m une response. Clinical deficiency of IgG is also associated with susceptibility to infection. IgG is therefore an important mediator of the humoral immune system and much research has focused upon elucidating the structure and function of IgG over the past three decades.

Immunoglobulin G: Structure and Function

IgG is a glycoprotein composed of two heavy and two light chains linked together via interchain disulphide bonds and non-covalent interactions (Porter, 1962). The light chains are solely associated with the Fab portion of the molecule and the heavy chains span both the Fab and Fc portions. A single disulphide bond connects the light and heavy chains w hereas the number of inter heavy chain bonds differs from subclass to subclass. The IgG molecule has been shown by crystallography to be composed of domains, with each domain having a characteristic folding pattern called the immunoglobulin fold. The immunoglobulin fold consists of two surfaces of anti-parallel p sheet stabilised by a disulphide bridge. This is a characteristic structure shared by the m em bers of the immunoglobulin supergene family, which include M H C and T cell receptor molecules.

(41)

between the Ch2 and Ch3 domains produces the Fc fragment (Figure 1.6). The domain pairing is stabilised by non-covalent interactions, except for the 0^,2 domains, which have two N-linked branched carbohydrates interposed between them. The Fab fragment domain faces are also predominantly hydrophobic and therefore efficient domain pairing in the Fab fragment requires the removal of these faces from the aqueous environment.

The Fab arms are linked to the Fc region by virtue of a length of polypeptide, called the hinge. The hinge region is susceptible to proteolytic attack by enzymes such as papain and pepsin. Papain cleaves the IgG molecule N-terminal to the intrachain disulphide bridge, producing two Fab fragments, whereas peptic cleavage C-terminal to the disulphide bridges results in a divalent

F(ab' ) 2 fragment and various Fc cleavage products including pFc'.

p a p a in e p s in

In t e r h e a v y c h a in d is u lp h id e b o n d s

C a r b o h y d r a t e

W

}

Fab

Hinge

Fc

(42)

The IgG Subclasses

(43)

Mouse

V» ~ 2 0 0 Dh( 1 - 3 0 ) J„ (1-6)

Ch

H um an

95 V„

6 y3 y l y2b y 2a £ a

Y e

g ene duplication (~ 75 million years ago)

y e a y e a

□h(10-20) Jh( 6 )

41H1H-Ô y3 y l life a l y2 y4 e a 2

Figure 1.7

Gene Organisation of Human and Mouse IgG subclasses

(44)
(45)

Waving

Bending

ir.

Rotation

C„2

C„3

Fc wagging

Ig G I

C„1,

lgG2

I

-88

-Î I

1

C„2,

C „ t

il-C„3'

igG4

igG 3

Figure 1.8

Domain Structure of the Human IgG Subclasses

(46)

Gm Allotypes

Gm allotypes are antigenic determinants present on the constant regions of y heavy chains which obey M endelian laws of inheritance. Since their first description by Grubb (1956 ) m any Gm allotypes have been characterised, shown in T ab le 1.3. Two systems of nomenclature exist for the classification of Gm allotypes, the World Health Organisation (W H O ) recom m ends the numeric system, although both alphabetical and numerical systems are found in the literature

Table 1.3

Gm allotypes of human immunoglobulins adapted from Lefranc and

Lefranc, (1990)

Subclass

Domain

Alphabetical

Numeric

IgGI

Ch3 G 1m (a) G 1m (1)

Ch3 G1m (x) G 1m (2) ChI G1m (f) G 1m (3)

ChI G 1m (z) G 1m (17)

lgG2

Ch2 G 2m (n) G 2m (23)

lgG3

Ch3 G3m (b°) G 3m (11)

Ch2 G 3m (b ') G 3m (5) Ch3 G3m(b") G 3m (13) Ch2 G3m(b") G 3m (14) Ch3 G3m(b®) G 3m (10) Ch3 G3m(c^) G 3m (6) Ch3 G3m(c®) G 3m (24) Ch2 G 3m (g) G 3m (21) Ch2 G 3m (u) G 3m (26) Ch3 G3m (v) G 3m (27) Ch3 G3m (s) G 3m (15)

C^3 G3m (t) G 3m (16)

Ch3 G 3m (g5) G 3m (28)

(47)

typed as G 2m (n )’. A relationship between Gm allotype status and immune response to certain bacterial organisms, for exam ple Haem ophilus influenzae, Streptococcus pneum oniae and Meningococci, have been found. Polysaccharide specific antibody titre has been shown to be related to G 2m (n) allotype (discussed in more detail in chapter 6). It is clear that Gm allotype exerts an effect on IgG subclass concentration, although the mechanisms underlying this relationship are as yet unresolved.

S tru c tu re -F u n c tio n R e la tio n s h ip s o f th e IgG S u b c la s s e s

It is within the Fc portion of the immunoglobulin molecule that effector functions are m ediated, but there are important differences between the subclasses (Table 1.4)

T a b le 1.4 Biological and structural properties of IgG subclasses

IgGI

lgG2

IgGS

lgG4

Heavy chain

y l y 2 y 3 y 4

Molecular weight of intact protein (kDa)

1 4 6 1 4 6 1 7 0 1 4 6

Hinge aa length

1 5 1 2 6 2 1 2

Light chain

k

:X

2 . 4 1.1 1 .4

8

Inter-heavy chain disulphide bonds

2 4 11 2

Serum levels (mg/ml)

5 - 1 2 2 - 6 0 .5 - 1 0 .2 - 1

Half-life (days)

2 1 - 2 3 2 0 - 2 3 7 2 1

Complement fixation

+ + + + +

-Specific anti-CHO responses

+ + + + + - - /+

Specific anti-protein responses

+ + - + +

-/+

Protein A binding

+ + -

+

Protein G binding

+

+

+

+

FcyRI (CD64) interaction

+++

-/+

++++

++

FcyRII (CD32) interaction

+++

+ +

++++

++

(48)

C1q binding is mediated by the motif G lu 318-X -L ys320-X -L ys322. T h e context of the binding motif is of importance since the IgG subclasses differ in their ability to promote com plem ent mediated cell lysis. Human Ig G I and lgG 3 are very effective, lgG2 is effective only at high concentrations and lgG 4 is not effective (G ud dat et al. 1993; Bruggemann et al. 1987; Riechmann et al. 1988; Valim and Lachm ann, 1991). Recent studies of hinge deleted lgG3 molecules has revealed that the long flexible hinge is not itself a prerequisite for C lq binding but that the inter H chain disulphide bond is essential for com plem ent activation (Brekke et al. 1995).

Differences between the human IgG subclasses have been dem onstrated for protein A binding, with Ig G I, lgG2 and lgG 4 binding w hereas lgG 3 does not. The critical residue for protein A binding is H is435, which in the lgG 3 of Caucasians is replaced by Arg. In oriental races lgG3 having the G 3m (st) allotype is commonly found and this is associated with a histidine residue at position 4 3 5 and hence with protein A binding.

Another major effector system is the recognition of antibody coated target cells by cells expressing Fc receptors. Fc receptors m ediate a variety of functions including phagocytosis (monocytes, macrophages, neutrophils) and antibody d ependent cellular cytotoxicity (monocytes, macrophages, lymphocytes). Fey

(49)

g en e and are m embers of the Ig gene superfamily (Fridman et al. 1992). FcyR binding has been shown to be mediated by the extrem e N-terminal portion of the Ch2 domain. In particular, Leu235 has been demonstrated to be a critical residue in such binding (W oof et al. 1986; Canfield and Morrison, 1991).

Th e human IgG subclasses differ in their ability to bind Fc receptors. Hum an FcyRI is expressed on monocytes and m acrophages and its expression on neutrophils may be induced by IFNy in vitro (Perussia et al. 1983). The binding of the IgG subclasses differ with lgG3 exhibiting the highest affinity (Burton, 1985). lgG2 is thought to be incapable of binding to FcyRI (W alker et al. 1989), but m ay bind with very low affinity (Van Den Herik Oudijk et al. 1994). FcyRII is expressed on virtually all haematopoetic cells, w hereas FcyRIII expression is restricted to m acrophages, NK and m ast cells (W einshank et al.

1988; Daeron et al. 1990).

FcyR m ediate a wide spectrum of activities, related to cell type. For exam p le receptors expressed by macrophages m ediate phagocytosis, endocytosis, antigen presentation and killing of IgG sensitised cells, a function also shared with NK cells (Fridman et al. 1992).

Human IgG Subclass Restriction

(50)

extended by m any investigators and it is now well recognised that antibody patterns are generally predictable, with Ig G I, lgG3 and/or lgG4 being directed against T^ep protein antigens and lgG2 being produced in response to Tj^d polysaccharide antigens.

Support for a predominantly lgG2 response directed against polysaccharide antigens was demonstrated following immunisation with

(51)

It is also becoming clear that certain IgG subclasses are associated with d isease states. A predominance of lgG1 and lgG3 directed against m yeloperoxidase has been described in association with renal vasculitis (Esnault et al. 1991). Interestingly, infection with Schistoma m ansoni is associated with antibodies of all four subclasses, but w hereas Ig G I and lgG3 m ediate killing of the parasite, lgG4 antibodies have been shown to block such killing mechanisms (Khalife et al. 1989). The route of antigen delivery has also been shown to affect the isotype profile with Ig G I and lgG3 predominating following natural hepatitis B infection (Persson et al. 1988), but Ig G I and lgG2 predom inate following hepatitis B vaccination (Borzi et al. 1992). In contrast, Ig G I and lgG 4 antibodies have been shown to dominate in hepatitis B-immunoglobulin complexes (Sallberg et al. 1991) and these authors propose that this is due to lgG3 com plexes being effectively cleared from the circulation and therefore not being detectable in the serum.

T h e role of lgG4 is even more unclear than that of the other subclasses. It is known not to bind complement and binds poorly to FcyRs. lgG4 production has been dem onstrated following secondary or tertiary immunisation and after chronic antigenic stimulation (Aalberse et al. 1983; Bird et al. 1990). lgG 4 anti­ egg antibodies have also been shown to be correlated with infection by

(52)

the functional significance of the lgG4 subclass remains unclear. Taken together these studies suggest that human IgG subclass restriction exists but that the exact subclass elicited depends on the specific antigen, route of entry and dose.

The ontogeny o f the hum an IgG subclasses

(53)

Avidity of The Human IgG subclasses

Antibody binding affinity is defined as the strength of the interaction betw een a m onovalent antibody and monovalent antigen; w hereas the bivalent binding of an antibody to a complex antigen is referred to as functional affinity or avidity (Hornick, 1972). The affinity of an antibody is determined by the “fit” of the antigen in the binding groove of the Fab portion of the antibody formed by the heavy and light chain variable regions. T h e binding forces involved in the interactions between antigens and antibodies are of a non-covalent, purely physicochemical nature, the primary forces being Liftshitz-van der W aals, electrostatic and polar (hydrogen bonds) in nature (van Oss, 1994). T h e extent to which each of the primary bonds contribute varies between different interactions and thus determ ine antibody affinity. The affinity of an antibody plays a crucial role in determining its biological activity, with high affinity antibody being superior (Stew ard, 1981). In the assessm ent of the immune response to vaccines and natural infection it is important to characterise an antibody in terms of affinity as well as titre.

(54)

antibody was of low affinity. Furthermore the lgG4 response displayed restricted affinity heterogeneity. Persson and co-workers subsequently dem onstrated affinity differences between serum IgG subclasses specific for hepatitis B surface antigen and pneumococcal polysaccharide derived from vaccinated adults. Employing affinity purification followed by globulin precipitation, IgG subclasses directed towards hepatitis B could be affinity ranked in the order Ig G I > lgG 2 > lgG 3 > lgG4, w hereas the response to pneumococcal polysaccharide was restricted to Ig G I and lgG2, with lgG2 being of highest affinity (Persson et al. 1988). Th ese findings were confirmed and extended by W en et al. (1 9 9 0 ), who also dem onstrated Ig G I and lgG4 specific for hepatitis B surface antigen from naturally infected individuals. These authors found that individuals with chronic hepatitis had high levels of low affinity Ig G I, w hereas asymptomatic carriers had significantly lower levels and higher affinity. IgG subclass affinity differences associated with disease have subsequently been dem onstrated for other system s such as high affinity anti-m yeloperoxidase (M P O ) Ig G I and lgG3 associated with renal vasculitis (Esnault et al. 1991). High affinity Ig G I and lgG3 have also been shown to be associated with recent streptococcal infections (Falconer et al. 1993) and high affinity lgG 3 specific for the organism M. catarrhalis has also been described (Goldblatt et al. 1991).

(55)

Coincidentally there appeared to be loss of high affinity Ig G I and the authors suggested a preferential switch with time from high affinity Ig G I to high affinity lgG 4 (D e v e y et al. 1990).

(56)

Aims of the work described in this Thesis

During the past few decades much progress has been m ade towards elucidating structural and functional properties of the human IgG subclasses. As outlined earlier the four human IgG subclasses display greater than 9 0 % sequence homology and their production is antigen restricted, with Ig G I and lgG 3 being preferentially elicited against protein antigens and lgG2 against carbohydrate antigens. However, many questions still remain unanswered. Although the titre of a particular subclass is clearly important in overcoming infection, it is becoming increasingly clear that the functional affinity (avidity) of such a response is also crucial. This study was therefore initiated to investigate aspects of antibody avidity of the four human IgG subclasses. T h e primary aim w as to develop suitable assays for the m easurem ent of serum antibody avidity and various E L IS A based methods w ere explored for each system studied.

(57)

T h e delay in ontogeny of the human lgG 2 subclass has been suggested to play a role in the higher incidence of bacterial infections in infants. Despite its pivotal role, little work has been published investigating the avidity of such responses. Utilising pneumococcal antigen specific ELISAs, the titre and avidity of serum IgG subclasses specific for pneumococcal polysaccharides from children vaccinated with Pneum ovax was investigated. This approach was employed in order to explore the relationship between age and IgG subclass

avidity.

(58)

General Materials and Methods

General reagents... 58 Buffers and solutions... 61 M ethods... 64

Figure

Figure 1.1 Cells Involved In The Immune System
Figure 1.2 Human B cell developmentDiagram showing gene rearrangements and immunoglobulin expressions during human B cell developmental stages, adapted from (Guelpa Fonlupt et al
Figure 1.3 Proposed mechanisms involved in directing V(D)J recombination
Figure 1.4 Mechanisms and Control of Immunoglobulin Isotype Switching
+7

References

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