chapter 4 antigens and antibodies oct 17, 19 & 24, 2006 complementarity of interacting surfaces...

Chapter 4 Antigens and Antibodies

Oct 17, 19 & 24, 2006

Complementarity of interacting surfaces of Ab and Ag

AbAg

你需要學習的課題 :

1. 「好」的免疫原 (immunogen) 有些什麼特質？2. 什麼叫做抗原決定區 (epitope) ？3. B-cell epitope 有何特性？*********************************************************

4. 抗體分子的基本構造及各部位的名稱。5. 抗體的種類、特性及功能。6. 單株抗體與多株抗體。

Outline

1. Immunogenicity versus antigenicity 2. Epitopes **********************************************************

3. Basic structure of antibodies (Abs) 4. Ab-binding site 5. Ab-mediated effector functions 6. Ab classes and biological activities 7. Antigenic determinants on immunoglobulins (Ig) 8. The B-cell receptor 9. The Ig superfamily10. Monoclonal Abs

Immunogenicity vs. Antigenicity

Immunogenicity 免疫性 :

the ability to induce an Ab and/or cell-mediated immune response

Antigenicity 抗原性 :

the ability to combine specifically with Ab and/or cell-surface receptors (Ig/TCR)

- Although a substance that induces a specific immune response is usually called an antigen, it is more appropriately called an immunogen.

- Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true.

- Some small molecules, called haptens, are antigenic but incapable, by themselves, of inducing a specific immune response. In other words, they lack immunogenicity.

DNP: dinitrophenol

{(bovine serum albumin)

Landsteiner’s work demonstrated both the specificity of the immune system for small structural variations on haptens and the enormous diversity of epitopes that the immune system is capable of recognizing.

Factors That Influence Immunogenicity

Intrinsic properties of an immunogen:

- Foreignness- Molecular size- Chemical composition and heterogeneity- Susceptibility to antigen processing and presentation

The biological system:

- Genotype of the recipient animal- Immunogen dosage and route of administration- Adjuvants

Foreignness

- Generally, the greater the phylogenetic distance between two species, the greater the structural (and therefore the antigenic) disparity between them.

- Some macromolecules (e.g., collagen and cytochrome c) were highly conserved throughout evolution and therefore display very little immunogenicity across diverse species lines.

- Conversely, some self-components (e.g., corneal tissue and sperm) are effectively sequestered from the immune system, so that if these tissues are injected even into the animal from which they originated, they will function as immunogens.

Molecular size

- There is a correlation between the size of a macromolecule and its immunogenicity.

- The best immunogens tend to have a molecular mass >100,000 daltons (Da).

- Generally, substances with a molecular mass less than 5,000 – 10,000 Da are poor immunogens.

Chemical composition and complexity

- Synthetic homopolymers tend to lack immunogenicity regardless of their size.

- All 4 levels of protein organization – primary, secondary, tertiary and quaternary – contribute to the structural complexity of a protein and hence affect its immunogenicity.

Four levels of protein organizational structure

For Ab (B cell) responses:

Proteins are the most potent immunogens, with polysaccharides ranking second. Lipids and nucleic acids of an infectious agent generally do not serve as immunogens unless they are complexed with proteins or polysaccharides.

For T cell responses:

Only proteins and some lipids (glycolipids and phospholipids) serve as immunogens.

Susceptibility to antigen processing and presentation

- The development of both Ab-mediated and T-cell-mediated immune responses requires interaction of T cells with Ag that has been processed and presented together with MHC molecules.

- Large, insoluble macromolecules generally are more immunogenic than small, soluble ones because the larger molecules are more readily phagocytosed and processed.

- Molecules that cannot be degraded (e.g., polymers of D-amino acids) and/or cannot be presented with MHC molecules are poor immunogens.

The biological system contributes to immunogenicity

- Genotype of the recipient animal

- Immunogen dosage and route of administration

- Adjuvants

Immunogen dosage and route of administration

Doses: too low no response, too high tolerance

Exposure: repeated administration (booster) over a period of time is usually more effective

Routes: orally ( 從口入的 ) parenterally ( 非從口入的 )

- intravenous (iv) : into a vein - intradermal (id) : into the skin - subcutaneous (sc) : beneath the skin - intramuscular (im) : into a muscle - intraperitoneal (ip) : into the peritoneal cavity

為什麼 Ag 的量、接觸次數 及 路徑

與免疫反應的強度有關？

Adjuvants 佐劑

- Latin adjuvare, to help

- Substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen.

Effects of adjuvants1. antigen persistence is prolonged - slower release of antigen at the injection site e.g., alum 明礬 [AlK(SO4)2],

2. costimulatory signals (p. 159) are enhanced - increased expression of B7 molecules on APC maximal activation of TH cells

3. local inflammation is increased4. nonspecific proliferation of lymphocytes is stimulated

- formation of a dense, macrophage-rich mass of cells called a granuloma 肉芽腫 e.g., incomplete Freund’s adjuvant (IFA), complete Freund’s adjuvant (CFA)

1. 在實驗動物中製備抗體時，經常使用 CFA (complete Freund’s adjuvant) ，其作用機轉 為何？

2. 為何局部發炎反應能增強 Ab 反應？

Epitopes

- Lymphocytes do not interact with, or recognize, entire immunogen molecules; instead, they recognize discrete sites on the macromolecule called epitopes, or antigenic determinants.

- epitopes: immunologically active regions of an immunogen, that bind to Ag-specific membrane receptors on lymphocytes or to secreted Abs.

T-cell and B-cell epitopes

- The recognition of antigens by T cells and B cells is fundamentally different.

- Because B cells bind antigen that is free in solution, the epitopes they recognize tend to be highly accessible sites on the exposed surface of the immunogen.

- T-cell epitopes are peptides combined with MHC molecules. Thus, there is no requirement for solution accessibility such as B-cell epitope.

Properties of B-cell epitopes

1. B-cell epitopes on native proteins generally are composed of hydrophilic a.a. on the protein surface that are topographically accessible to membrane-bound or free Ab.

2. B-cell epitopes may be composed of sequential or nonsequential amino acids.

Sperm whale myoglobulin contains 5 sequential B-cell epitopes

Hen egg-white lysozyme (HEL) composesone nonsequential (conformational) epitope

Contact with Ab light chain

Contact with Ab heavy and light chains

Contact with Ab heavy chain

Ab to native HEL does not bind to reduced HEL

3. B-cell epitopes tend to be located in flexible regions of an immunogen and display site mobility.

- site mobility of epitopes maximizes complementarity with the Ab’s binding site

4. Complex proteins contain multiple overlapping B-cell epitopes, some of which are immunodominant.

- Most of the surface of a globular protein is potentially immunogenic.

- Some epitopes, called immunodominant, induce a more pronounced immune response in a particular animal than other epitopes.

Basic structure of Abs

Electrophoresis of immune serum (Tiselius & Kabat, 1939)

Immune sera

after reactionwith Ag

globulin (G)

Immunoglobulin (Ig): IgG, IgM, IgA, IgE, IgD

Antibody (Ab)

Ab molecules contain 4 peptide chains - A dimer of heterodimers

V: variableC: constant

: IgM :IgG :IgA : IgD : IgE

(H)

(L)

50-55 kDa

22 kDa

100 kDa

→ numerous small peptides

150 kDa

Fab fragment: antigen binding

45 kDa

Fc fragment: crystallizable

50 kDa

Antibody to the Fab fragment could react with both the H and L chains, whereas antibody to the Fc fragment reacted only with the H chain.

Fab consists of portions of an H and a L chain. Fc contains only H chain components.

- A heterogeneous spectrum of antibodies in the serum -globulin fraction

- Multiple myeloma: a cancer of Ab- producing plasma cells

- Myeloma protein: 95% of the serum Ig

- Bence-Jones proteins: the excess light chains in the urine.

- MOPC: mineral-oil induced plasmacytoma in mice

Heavy and light chains are folded into “domains”

(IgG, IgD, IgA)

(IgM, IgE)

Associations between domains of an Ab molecule

2 Ag-binding sites:

Immunoglobulin Domains - each contains about 110 a.a. residues and an intrachain disulfide bond that forms a loop of 60 a.a.

Variable-Region Domains - hypervariable regions: (15% - 20% of the variable domain)

= complementarity-determining regions (CDR) CDR1, CDR2, CDR3

- framework regions (FR)

- diversity in the VH domain is concentrated in CDRs

Variability of a.a. residues in the VH and VL domains

CDRs bind Ags

number of different amino acids at a given positionVariability = _____________________________________________________ frequency of the most common amino acid at a given position

Conformational changes may be induced by antigen binding

─ : after binding to the Ag

─ : before binding to the Ag

CDRs of L chain : L1, L2, L3

CDRs of H chain : H1, H2, H3

Fab

Ab-mediated effector functions

- Antibodies generally do not kill or remove pathogens solely by binding to them.

- While V regions bind to Ag, the CCH H regionregion is responsible for a variety of collaborative interactions with other proteins, cells, and tissues that result in the effector functions of the Ab responses.

Ab-Mediated Effector Functions

- Opsonization ( 調理作用 ) is promoted by Ab

- Abs activate complement ( 補體 ) (chapter 7)

- Antibody-dependent cell-mediated cytotoxicity (ADCC) kills cells (p. 366 chapter 14)

- Some Abs can cross epithelial layers by transcytosis

Ab promotes opsonization through abinding to Fc receptors (FcR)on phagocytes

Ab activates complement-mediated cytolysis or promotes opsonization through a binding to C receptors on phagocytes

Antibody-dependent cell-mediated cytotoxicity (ADCC)

(Figure 14-15)

Transcytosis

- movement of Ab across epithelial layer

- delivery of IgA to the mucosal surfaces of the respiratory, gastrointestinal, and urogenital tracts, as well as its export to breast milk

- transplacental transport of IgG from mother to fetus

Secretory IgA in Breast Milk

Bind to microbes in baby’s digestive tract and thereby prevent their attachment to the walls of the gut and their subsequent passage into the body’s tissues.

Ab classes and biological activities

5 major classes of secreted antibody

IgG

- most abundant in serum

- 80% of total serum Ig

- 4 IgG subclasses

4 subclasses of human IgG

- size of the hinge region- no. & position of the interchain -S-S- bond- IgG1>IgG2>IgG3>IgG4 in serum conc.- 90% - 95% homologous in DNA sequences- varied effectiveness in placenta transfer and C activation

IgM Pentamer

- monomer on the membrane & pentamer in secretion

- 5% - 10% serum Ig - 1st Ab in neonates - 1st Ab in primary response

- more efficient in agglutination & C fixation

- J (joining) chain is required for polymerization of the monomers to form pentameric IgM

- also present in mucosal surfaces

IgA Dimer

- 10% - 15% of total serum Ig

- monomers, dimers, trimers and tetramers in serum - predominant in external secretions, e.g., breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts

Secretory IgA

Dimers and tetramers in secretion with a secretory component

Formation of Secretory IgA

Transcytosis

IgE

- potent biological activity

- extremely low conc. in serum

- mediates the immediate hyper- sensitivity reactions

- responsible for the symptoms of hay fever, asthma, hives, and anaphylactic shock

Allergen cross-linkage of receptor-bound IgE on mast cells

- induces degranulation, causing release of substances that mediate allergic manifestations

IgD

- 0.2% of total serum Ig

- together with IgM, is the major membrane-bound Ig on mature B cells

- thought to function in the activation of B cells

- no biological effector function has been identified

Antigenic determinants on Igs

3 Antigenic Determinantsof Immunoglobulins

Isotypes

Allotypes

Idiotypes

Isotypic Determinants

- constant-region determinants that collectively define each H-chain class and subclass, and each L-chain type and subtype within a species

Allotypic Determinants

- differences in amino acids in C regions, which occur in some, but not all, members of a species

Idiotypic Determinants

- The unique amino acid sequence of the V regions of a given Ab. In some cases, an idiotype is the actual antigen-binding site

B-cell Receptor

Fc receptors bound to Fc regions of Abs

neonatal Fc receptor

Fc receptors - essential for many of the biological functions of Abs

- movement of Abs across cell membranes, e.g., the transfer of IgG from mother to fetus across the plancenta

- passive acquisition of Ab by many cell types, including B and T lymphocytes, neutrophils, mast cells, eosinophils, macrophages, and natural killer cells (a linker between Ab molecules and various types of cells)

The Ig superfamily

The Ig superfamily -1

The Ig superfamily -2

Monoclonal Abs

Clonal Selection of B Lymphocytes

Production of monoclonal antibodies (mAb)

Questions

• How to predict whether a molecule is a “good” immunogen?

• What are the differences between B-cell epitopes and T-cell epitopes?

• Draw a schematic diagram of a typical IgG molecule and label each of the chains, bonds, regions, sites, fragments and domains.

• How does Ab kill or remove pathogens?

• What is the principal of making monoclonal antibody?