1. Antigens

2. Objectives What is an antigen? What type of molecules are antigens? Can any particle/foreign body that enters the cell be an antigen? Properties and features of antigens that make them immunogens. To understand that antigens can be recognized by both B and T cells. The structure of antibodies that facilitates this interaction. To be familiar with the fact that there are different classes of antibody molecules and they have slightly different structures that determine function. With so many antigens how do we generate so many antibodies, TCR that recognize them?

3. ANTIGENS 1.A foreign substance or component of an organism. 2.Antigens are substances that induce a specific immune response and subsequently react with the products of a specific immune response. 3.Any substance against which an antibody is produced.

4. Antigenicity The ability to bind antibody or T cell receptor.

5. Immunogenicity The capacity to stimulate the production of antibodies or cell-mediated immune responses.

6. Antigen vs. Immunogen Antigen – Any substance that can bind to an antibody or T cell receptor Immunogen – Any substance that can elicit an immune response – All immunogens are antigens – Not all antigens are immunogens, i.e. haptens

7. Tolerogen - An antigen that invokes a specific immune non-responsiveness due to its molecular form. If its molecular form is changed, a tolerogen can become an immunogen.

8. Allergen - An allergen is a substance that causes the allergic reaction. The (detrimental) reaction may result after exposure via ingestion, inhalation, injection or contact with skin.

9. Antigens can be classified in order of their origins Exogenous antigens Exogenous antigens are antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. By endocytosis or phagocytosis, these antigens are taken into the antigen-presenting cells (APCs) and processed into fragments.

10. Types of Antigens Exogenous Antigens 1- Bacterial antigens: a- Antigens related to bacterial cells - Somatic antigen (O): part of cell wall gm –ve bacter. - Capsular antigen: usually polysaccharide - Flagellar Ag (H) : a protein made of flagellin - Fimbrial Ag: surface antigens in fimbriated bacilli b- Antigen secreted by bacteria: - Exotoxins - Enzymes 2- Viral antigens: a- protein coat viral antigens b- Soluble antigens (soluble nucleoproteins as in influenza)

11. Endogenous antigens Endogenous antigens are antigens that have been generated within the cell, as a result of normal cell metabolism, or because of viral or intracellular bacterial infection.

12. Types Of Antigens Endogenous antigens Human tissue antigens: a- Blood group antigens: A, B and Rh antigens b- Histocompatibility antigens: Glycoprotein molecules on all nucleotide cells: - Major histocompatibility complex antigens (MHC) - Human leucocyte antigen (HLA)

13. Antigen Binding And Recognition Molecules Antigens are recognized by and bind to: 1) B-cell receptors (BCR) : - These are membrane-bound immunoglobulins (IgM and IgD) on B-cells - BCRs can be secreted in plasma as antibodies 2) T-cell receptors (TCR) - α and β chains anchored to T-cells - There is a groove which binds small peptides presented by MHC on surface of APCs 3) MHC molecules They are essential for presentation of peptides so that they can be recognized and bind to TCRs

14. Table from Cellular and Molecular Immunology Chapter 4 page 76

15. CHEMICAL NATURE OF IMMUNOGENS Proteins The vast majority of immunogens are proteins. These may be pure proteins or they may be glycoproteins or lipoproteins. In general, proteins are usually very good immunogens. Polysaccharides Pure polysaccharides and lipopolysaccharides are good immunogens. Nucleic Acids Nucleic acids are usually poorly immunogenic. However, they may become immunogenic when single stranded or when complexed with proteins. Lipids In general lipids are non-immunogenic, although they may be haptens.

16. Additional Antigen Types We will discuss the following when we talk about T cells: – Major Histocompatibility Complex (MHC) – Super antigens

17. Immunogens or Antigens Haptens: - Low molecular weight substances - These substances not immunogenic by itself - If couple to a larger carrier molecule (albumin, globulins), they become immunogenic - Examples : simple chemicals and drugs: penicillin, sulphonamid, aspirin, cosmetic, t ranquillizers, neomycin skin ointment

18. T-independent and T-dependent Antigens Just another way to group antigens.

19. T-independent Antigens T-independent antigens are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help In general, polysaccharides are T- independent antigens. The responses to these antigens differ from the responses to other antigens.

20. Properties of T-independent antigens Polymeric structure These antigens are characterized by the same antigenic determinant repeated many times Polyclonal activation of B cells Many of these antigens can activate B cell clones specific for other antigens (polyclonal activation). – Type 1 T-independent antigens are polyclonal activators – Type 2 are not. Resistance to degradation T-independent antigens are generally more resistant to degradation and thus they persist for longer periods of time and continue to stimulate the immune system.

21. T-dependent Antigens T-dependent antigens are those that do not directly stimulate the production of antibody without the help of T cells. Proteins are T- dependent antigens. Structurally these antigens are characterized by a few copies of many different antigenic determinants.

22. SUPERANTIGENS When the immune system encounters a conventional T-dependent antigen, only a small fraction (1 in 10 4 -10 5 ) of the T cell population is able to recognize the antigen and become activated (monoclonal/oligoclonal response). However, there are some antigens which activate a large fraction of the T cells (up to 25%). These antigens are called superantigens Examples of superantigens include: Staphylococcal enterotoxins (food poisoning), Staphylococcal toxic shock toxin (toxic shock syndrome), Staphylococcal exfoliating toxins (scalded skin syndrome) and Streptococcal pyrogenic exotoxins (shock). Although the bacterial superantigens are the best studied there are superantigens associated with viruses and other microorganisms as well. The diseases associated with exposure to superantigens are, in part, due to hyper activation of the immune system and subsequent release of biologically active cytokines by activated T cells.

23. Superantigens (SAgs) * They activate multiple clones of T-lymphocytes * Bacterial toxins: Staph. aureus toxic shock syndrome toxin (TSST) and enterotoxins Strpt. pyogenes pyrogenic toxin A * They have the ability to bind both class II MHC molecules and TCR β chain * They act as a clamp between the two, providing a signal for T-cell activation

24. Superantigens (SAgs) * They are active at very low concentration causing release of large amounts of cytokines * The massive T-cell activation and release of large amounts of cytokines cause systemic toxicity * This method of stimulation is not specific for the pathogen * It does not lead to acquired immunity i.e no memory

26. Conventional Ag

27. Incomplete antigens have antigenic determinants, but cannot induce immune responses because they lack one or more of the important attributes needed for this function (one example of an incomplete antigen is a hapten, which is an artificial monovalent epitope)

28. Autoantigens An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease.

29. These antigens should under normal conditions not be the target of the immune system, but due to mainly genetic and environmental factors the normal immunological tolerance for such an antigen has been lost in these patients.

30. Common Experimental Antigens

31. Major Histocompatibility Complex Antigens (MHC) * MHC has an important function in presentation of antigens to T- cells * Helper T-cells recognize foreign antigens on surface of APCs, only when these antigens are presented in the groove of MHC II molecule * Cytotoxic T-cells will only recognize antigens, on the surfaces of virus infected cells or tumor cells only when these antigens are presented in the groove of Class I molecule (MHC restriction)

32. Mitogen An agent that induces mitosis. Here means to activate T cells and/or B cells without help from APCs. E.g Lectin, for example, concanavalin A (ConA), LPS(lipopolysaccharide), Staphylococcal protein A(SPA)

33. Immunogenicity of Antigens Determined by – Foreignness – Molecular Size – Chemical Composition – Degradability Influenced by – Host genotype – Dose and route of administration – Presence of adjuvants

34. Foreignness The immune system normally discriminates between self and non-self such that only foreign molecules are immunogenic.

35. Size There is not absolute size above which a substance will be immunogenic. However, in general, the larger the molecule the more immunogenic it is likely to be.

36. Chemical Composition In general, the more complex the substance is chemically the more immunogenic it will be. The antigenic determinants are created by the primary sequence of residues in the polymer and/or by the secondary, tertiary or quaternary structure of the molecule.

37. Physical form In general particulate antigens are more immunogenic than soluble ones and denatured antigens more immunogenic than the native form.

38. Degradability Antigens that are easily phagocytosed are generally more immunogenic. This is because for most antigens (T-dependant antigens, see below) the development of an immune response requires that the antigen be phagocytosed, processed and presented to helper T cells by an antigen presenting cell (APC).

39. Contribution of the Biological System Genetic Factors – Some substances are immunogenic in one species but not in another. – Some substances are immunogenic in one individual but not in others (i.e. responders and non-responders). – The species or individuals may lack or have altered genes that code for the receptors for antigen on B cells and T cells or they may not have the appropriate genes needed for the APC to present antigen to the helper T cells. Age Usually the very young and the very old have a diminished ability to mount and immune response in response to an immunogen.

40. Method of Administration Dose The dose of administration of an immunogen can influence its immunogenicity. There is a dose of antigen above or below which the immune response will not be optimal. Route Generally the subcutaneous route is better than the intravenous or intragastric routes. The route of antigen administration can also alter the nature of the response Adjuvants Substances that can enhance the immune response to an immunogen are called adjuvants. The use of adjuvants, however, is often hampered by undesirable side effects such as fever and inflammation.

41. Summary of Factors influencing Immunogenicty 1-Foreigness : Foreign substances are immunogenic 2- Molecular size: High molecular weight increase immunogenicty 3- Chemical structure complexity: High complexity increase immunogenicty 4- Route of administration: Parenteral routes are more immunogenic to oral route

42. Antigenic epitopes Epitope,or,Antigenic determinants, are the portions of antigen molecules that physically interact with paratopes (combining sites) of immune response molecules and therefore actually "determine" antigen specificity FeFe

43. Epitopes Discrete sites on immunogens recognized by antibodies or T cell receptors Macromolecules contain many epitopes with different immunogenicities Immunogenicity can vary between individuals and populations. Some epitopes are immunodominant

44. Epitopes Overlapping Non-overlapping Linear Conformational

45. Antigenic epitopes

46. Types of Epitopes 1. Linear epitopes continuous and found in polysaccharides as well as in both native (nondenatured) and denatured proteins, especially fibrillar proteins. specificity depends upon primary sequence. typical size is 5-6 subunits in length.

47. Conformational epitopes Discontinuous (involve multiple subunits, often located far apart in the primary sequence of the antigen molecule) and are thus found only in native (globular) proteins.

48. Specificity depends upon conformation, or three-dimensional shape, which is a combination of tertiary and quaternary structure... supported by primary and secondary structure, of course.

49. Typical size is hard to pinpoint, but sequences of up to 16 amino acids in certain protein antigens have been shown to interact with their complementary paratope.

50. Two different epitopes B cell epitope, a portion of antigen molecule that is recognized by B cell receptors. T cell epitope, the region of antigen molecules that are recognized by T cell receptors.

53. Classification of Antigens Thymus-dependent antigen(TD-Ag) Thymus-independent antigen(TI-Ag)

55. DETERMINANTS RECOGNIZED BY THE INNATE IMMUNE SYSTEM Let’s not forget we have the innate immune system too that recognizes antigens. Determinants recognized are different. Innate immune system recognize broad molecular patterns found in pathogens but not in the host. The broad molecular patterns recognized by the innate immune system have been called PAMPS (pathogen associated molecular patterns) and the receptors for PAMPS are called PRRs (pattern recognition receptors). A particular PRR can recognize a molecular pattern that may be present on a number of different pathogens enabling the receptor to recognize a variety of different pathogens.

56. Adjuvant Adjuvant: The Latin "adjuvans" means to help, particularly to reach a goal. An adjuvant is a substance that helps and enhances the pharmacological effect of a drug or increases the ability of an antigen to stimulate the immune system.

57. Classification of Adjuvant Freund’s adjuvant – Complete Freund’s adjuvant(CFA) – Incomplete Freund’s adjuvant(IFA) Liposome Inorganic compound Cytokine Biodegradable nanoparticles

58. Mechanisms of adjuvants Prolonged persistence of immunogen molecules at the site of injection. Enhancement of co-stimulatory signals. Induction of granuloma formation. Stimulation of lymphocyte proliferation in a non-specific manner.