1. ANTIGENS (Haptens and Immunogens) Although a substance that induces a specific immune response is usually called an antigen, it is more appropriately called an immunogen. The antigens are of two types; immunogens and haptens. Immunogens: Are any foreign substances which can stimulate a specific immune response when introduced into a host, in the form of antibody production (humoral) and/or specific reactive T lymphocytes (cellular), and bind to them specifically.

2. Haptens: Are low molecular weight substances which can not induce an immune response, but can bind specifically to antibodies or lymphocytes (i.e., lack immunogenicity). However, when coupled to a larger carrier protein or tissues it becomes immunogen (i.e., can induce immune response). Examples: simple chemicals and drugs as penicillin (may bind to serum albumin), aspirin, or cosmetics and soap.

3. Epitopes or Antigenic Determinants: Despite the fact that potent antigens are relatively large molecules, only small parts of the antigen can stimulate production or bind to antibody (or lymphocytes). These parts are called “epitopes” or “antigenic determinants (AD)”. Each may be composed of 4-7 amino acids, or monosaccharide residues. The numbers of epitopes (valency) on an antigen vary with molecular size. Examples: human albumin has 6 epitopes, and thyroglobulin has 43 AD which means that they can stimulate and/or bind 6 or 43 different antibody specificities or specific lymphocytes respectively.

4. Characteristics of Epitopes (AD): 1. Accessibility:Epitopes should be exposed to the immune system, and they are hydrophilic (in contact with the body fluid). Hidden parts do not work as AD, however, when exposed they do (become AD). Therefore, partial digestion by phagocytic cells can enhance immunogenicity. 2. Electrical Charge: The AD are usually negatively charged since they are hydrophilic. Hence, the antibodies (positively charged) can bind to them.

5. 3. Genetic constitution of the host: The different hosts may vary in their response to the same AD; some are strong responders, while others are weak or none responders. Example: Insulin has 2 AD (A & B); mouse 1 responds to A, but mouse 12 responds to B.

7. Specificity of Epitopes: There are two types of specificities: 1. Sequential: This type of specificity depends on the sequences of amino acids or sugar residues of epitopes. 2. Superstructure or configuration (3-dimentional structure): The immune system depends on this type of specificity more than the sequential one.

8. Factors of immunogenicity: 1. Foreignness: For a substance to be immunogenic it must be foreign to the host in which it is introduced. The immune system normally can distinguish between self and non-self components (antigens). If self tolerance is broken, auto-immune disorders may appear. 2. Molecular size (weight): Small molecules such as amino acids, pure nucleic acids or monosaccharides are usually not immunogenic. As a rule, molecules with a M.W. of less than 5,000- 10,000 have no or only weak immunogenicity.

9. 3. Chemical complexity (nature): The most potent immunogens are proteins. Some polysaccharides of high M.W. are immunogenic. Also, aromatic A.A. contribute more to immunogenicity than non-aromatic ones. Furthermore, immunogenicity is proportional to the numbers of tyrosine molecules in the substance. Therefore, the more complex the structure of the substance, the stronger immunogenicity will be produced.

10. 4. Degradability: For a substance to be immunogenic it has to be susceptible to partial enzymatic degradation that occurs during processing by antigen presenting cells (APC) such as macrophages or dendritic cells. It has been found that peptides composed of D-amino acids, which are resistant to enzymatic degradation, are not immunogenic, whereas, their L-isomers are susceptible to enzymes and are immunogenic.

11. 5. Host genetic constitution: Human beings vary in their response to the same substance; some are strong responder and others are weak ones. Example : penicillin may cause allergy in some individuals.

12. 6. Methods of administration of antigen: A. Dosage: Immunological tolerance can result if very high or very low doses of certain antigens are administered. The number of doses also affects the outcome of the immune response (I.R). Repeated booster doses stimulate strong I.R. (e.g., booster doses of vaccines).

13. B. Route of administration: Subcutaneous (Sc) injection of antigens (Ag) is more stimulant than I.V. or I.M ones. The Sc are carried to local L.N., while the I.V. to the spleen, hence, stimulate different lymphoid cells populating these tissues and subsequently is reflected on the type and magnitude of the I.R. Also, the Sc injected antigens are cleared slower by the immune system (RES) than the I.V. ones.

14. C. Adjuvants (Adj): These are substances that, when mixed with an antigen before its administration will increase the I.R. to that antigen. There are 3 types of Adj: I. Freund’s complete Adj (killed tubercle bacilli + mineral oil + detergent + water). II. Freund’s incomplete Adj (same as I, but with out T.B. bacilli). Types I & II are used in animals only since they produce granuloma. III. Aluminum potassium sulfate (Alum) which can be used for humans (e.g., desensitization of allergy; toxoid vaccine against diphtheria). Note : Other microbial Adj include B. pertussis, BCG (attenuated Mycobacterium), & Cryptosporidium parvum.

15. Mechanism of action of adjuvants: 1. Depot effect; by stimulating a granuloma around the antigen, thus slowing its absorption, i.e., prolonging antigen persistence and increasing stimulation of immune cells. 2. They activate macrophages, thus increasing phagocytosis, antigen processing & presentation, and cytokines secretion such as IL-3. 3. They enhance co-stimulatory signal molecules leading to maximal activation of T- cells. 4. They stimulate non-specific proliferation of lymphocytes.

16. Hapten-Carrier Phenomenon: Haptens have usually one epitope (haptenic AD) which is recognized by B cells. These B cells do not respond by antibody production because there is no T cells help response (i.e., no an other AD recognized by T- helper cells). However, if the hapten is conjugated to a carrier protein (e.g., albumin), the carrier will be recognized by T-helper cells as a second epitope (immunogenic AD) and consequently provide the necessary help (cytokines) to the B cells in order to synthesis and produce antibodies (Ab).

17. Note: Antigens that have 2 AD can stimulate antibody production because 1 AD will be recognized by B cells and the other by T- helper cells (help B cells to produce Ab).

18. Antigen-Antibody Binding: The binding of antigen to antibody can be described as “ Lock-Key”. The efficiency of the antibodies in binding to antigens could be: 1. High affinity antibody: When the antigen and antibody “ fit exactly”. 2. Low affinity antibody: When there is “poor fit” between the antigen and antibody. This is produced in response to unrelated or partially related antigens which bear one or more AD in common. These are called “heterophile antibodies” and “heterophile antigens” and the phenomenon is known as “cross-reactivity”.

20. Applications of cross-reactivity : 1. In vivo: Some infections of auto- immune nature, e.g., acute rheumatic fever in which antibodies to Strep. pyogenes M antigen cross react with cardiac muscle proteins leading to rheumatic carditis and valve destruction. 2. In vitro: Several laboratory tests used for diagnosis are based on detection of heterophile antibodies in patient’s sera:

21. A. Paul Bunnel test: Used in the diagnosis of infectious mononucleosis caused by Epstein Barr virus (EBV). Sera of these patients contain heterophile antibodies that can agglutinate (aggregate) sheep RBC. B. Cold agglutinins: Sera of patients infected with Mycoplasma pneumoniae agglutinate human RBC in the cold (low temperature). C. VDRL: Antibodies in sera of syphilitic patients (reagin antibodies) cross react with an alcoholic extract from beef heart muscles (cardiolipin antigen).

22. Types of Antigens: 1. Bacterial Antigens: These may be: A. Soluble antigens exotoxins, enzymes, or haemolysins, etc… B. Cellular antigens as capsular (K), flagellar (H) & somatic (O) antigens. 2. Viral Antigens: These may be: A. Protein viral antigens. B. Soluble viral antigens as nucleoproteins of influenza or mumps viruses. 3. Human tissue antigens: A. Blood group antigens (ABO). B. MHC (HLA) antigens. 4. Superantigens.

23. Superantigens (SAg): The SAg are antigens that activate multiple clones of T cells. These include a class of bacterial toxins, e.g., Staphylococcal enterotoxins, toxic shock syndrome toxin, group A Streptococcal pyogenic toxin A, and some viral SAg as nucleoprotein of Rabies virus. They are active at very low concentrations and cause a large percent. of T-cell activation irrespective of their specificities causing the release of large amounts of cytokines (IL-1,2 &TNF). This method of stimulation is not specific and does not lead to acquired immunity (no memory ; exhaustive & refractory to further stimulation). SAg can bind both MHC class II and T-cell receptor beta (TCR-beta chain). This explains the wide spread of the clinical manifestations of the diseases caused by such an antigenic stimulation.

24. 5. Thymus Independent Antigens : These antigens can stimulate B cells without the cooperation of the T-helper cells (CD4). These antigens are composed of repeated units of monosaccharides or polysaccharides, or some bacterial endotoxins & cell walls. These antigens stimulate the production of IgM (mainly) and IgG2. They generate no memory cells.