1. Antigens March 4, 2003

2. Part I Introduction Chapter 1. Overview of the Immune System Chapter 2. Cells and Organs of the Immune System Part II Generation of B-cell and T-cell Responses Chapter 3. Antigens

3. 本章大綱 : 1. Immunogenicity Versus Antigenicity 2. Factors That Influence Immunogenicity 3. Epitopes 4. Haptens and the Study of Antigenicity

4. Immunogenicity vs Antigenicity

5. Immunogenicity: the ability to induce a humoral and/or cell-mediated immune response Antigenicity: the ability to combine specifically with Ab and/or cell-surface Ig/TCR

6. - 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. - Haptens are antigenic but incapable, by themselves, of inducing a specific immune response.

7. Factors That Influence Immunogenicity

8. Contribution of the immunogen to immunogenicity: - Foreighness - Molecular size - Chemical composition and heterogenicity - Susceptibility to antigen processing and presentation Contribution of the biological system to immunogenicity: - Genotype of the recipient animal - Immunogen dosage and route of administration - Adjuvants

9. 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.

10. Molecular size - There is a correlation between the size of a macromolecule and its mmunogenicity. - The best immunogens tend to have a molecular mass approaching 100,000 Da. - Generally, substances with a molecular mass less than 5,000 – 10,000 Da are poor immunogens.

12. 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. - polypeptides, polysaccharides, (nucleic acids, lipid)

13. 4 levels of protein organizational structure

14. Susceptibility to antigen processing and presentation - Ability to be processed and presented with an MHC molecules on the surface of an antigen- presenting cell or altered self-cell

15. Processing and presentation of exogenous antigens

16. Processing and presentation of endogenous antigens

17. Contribution of the biological system to immunogenicity: - Genotype of the recipient animal (strains) - Immunogen dosage and route of administration - Adjuvants

18. Immunogen dosage and route of Administration Doses: too low  no response too high  tolerance 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

19. Adjuvants - Latin adjuvare, to help - Substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen.

20. Effects of adjuvants 1. Prolong antigen persistence - slower release of antigen at the injection site 2. Enhance co-stimulatory signals - increased expression of MHC & B7 molecules - secretion of cytokines  increased antigen-presenting ability  maximal activation of T H cells 3. Increase local inflammation - formation of a dense, macrophage-rich mass of cells 4. Stimulate lymphocyte proliferation nonspecifically

22. Contribution of the immunogen to immunogenicity: - Foreighness - Molecular size - Chemical composition and heterogenicity - Susceptibility to antigen processing and presentation Contribution of the biological system to immunogenicity: - Genotype of the recipient animal - Immunogen dosage and route of administration - Adjuvants

23. Epitopes

24. - Immune cells do not interact with, or recognize, an entire immunogen molecules; instead, lymphocytes recognize discrete sites on the macromolecule called epitopes, or antigenic determinants. - The recognition of antigens by T cells and B cells is fundamentally different.

26. T-cell and B-cell epitopes - 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. - Because most T cells recognize antigen only when it is combined with an MHC molecule, T cell epitope, as a rule, cannot be considered apart from their associated MHC molecules.

28. Conformation of the epitope recognized by B cells 1. The ability to function as a B-cell epitope is determined by the nature of the antigen- binding site on the antibody molecules displayed by B cells.

29. 3-D structure of an octapeptide hormone (angiotensin II) complexed with a monoclonal Ab Fab Fragment. Red: angiotensin II Blue: the heavy chain Purple: the light chain

30. Model of interaction between hen egg-white lysozyme (HEL) and Fab fragment of anti-HEL antibody light chain (yellow) a glutamine resiue (red) heavy chain (blue) HEL (green)

33. Ag binding sites for different antibody

34. - The interactions of Ab with Ag are through non-covalent bonds. - 4 types of non-covalent bonds: a. Ionic (or electrostatic) bond Ionic bonds form between surfaces of opposite charge.

35. b. Hydrogen bond Hydrogen bonds form between hydrogen atoms and two other electronegative atoms such as oxygen and nitrogen.

36. c. Van der Waals’ force Van der Waals’ forces occur at very close ranges between two atoms. Fluctuations in the electrical charge within electron clouds can lead to attractive or repulsive forces between atoms, dependent on the distance between them.

37. d. Hydrophobic bond Hydrophobic bond is created by the behavior of hydrophobic subunits in aqueous environments. These tend to be pushed together to minimize the instability they cause in the network of hydrogen- bonded water molecules.

38. 2. The B-cell epitope on native proteins generally are composed of hydrophilic amino acids on the protein surface that are topographically accessible to membrane-bound or free antibody.

39. Ab elicited by immunization with the (T,G)-A-L copolymer react largely with the exposed tyrosine and glutamic acid residues. Anti-(T,G)-A-L Abs do not eact with the A-(T,G)-L copolymer

41. 3. B-cell epitopes can contain sequential or nonsequential amino acids.

42. Sperm whale myoglobulin contains 5 sequential B-cell epitopes 6-8 aa

43. Hen egg-white lysozyme composes one nonsequential (conformational) epitope HEL Red: light chain Blue: heavy chain

44. Antibody to native HEL does not bind to reduced HEL

46. Inhibition of reaction between HEL loop and anti-loop antiserum by natural loop or closed synthetic loop only

47. 4. B-cell epitopes tend to be located in flexible regions of an immunogen and display site mobility. 5. Complex proteins contain multiple overlapping B-cell epitopes, some of which are immunodominant.

48. Properties of T-cell epitopes

50. 1. Antigenic peptides recognized by T cells form trimolecular complexes with a TCR and an MHC molecule.

51. 2. - The antigen-binding cleft on an MHC molecule interacts with various oligomeric peptides (nonamer for class I & 12 –25 residues for class II) that function as T-cell epitopes. - A given MHC molecule can bind a variety of different peptides (broad but selective interaction). 3. Antigen processing is required to generate peptides that interact specifically with MHC molecules.

52. Processing and presentation of exogenous and endogenous antigens

53. 4. T-cell epitopes tend to be on the “inside” of the protein molecule

54. 5. Immunodominant T-cell epitopes are determined in part by the set of MHC molecules expressed by an individual.

55. Correlation of MHC-binding ability and T-cell- activating ability of synthetic peptides

56. TCR and MHC-peptide  TCR  peptide  MHC

57. Haptens and the study of antigenicity

58. Hapten: Small organic molecules that are antigenic but not immunogenic. Carrier: Large molecules that are chemically coupled to haptens yield immunogenic hapten-carrier conjugates.

60. (1)

61. TABLE 3-5 REACTIVITY OF ANTISERA WITH VARIOUS HAPTENS (2)

62. Drug allergies ( 藥物過敏 ) When medicines become immunogens, …………..

63. Penicillin allergy

66. 本章大綱 : 1. Immunogenicity Versus Antigenicity 2. Factors That Influence Immunogenicity 3. Epitopes 4. Haptens and the Study of Antigenicity