110/30/2015 Antigens Antigens Hugh B. Fackrell

210/30/2015 ä Assigned Reading ä Content Outline ä Performance Objectives ä Key terms ä Key Concepts ä Short Answer Questions

310/30/2015 ASSIGNED READING ä Chapter 4 pp

410/30/2015 OUTLINE ä Immunogenicity vs antigenicity ä Factors that influence immunogenicity ä immunogen contributions ä biological system contributions ä Epitopes ä B cell epitopes ä T cell epitopes ä Haptens ä Mitogens

510/30/2015 Definitions ä Antigen - a substance recognized by T and B cell receptors ä Immunogen - a substance capable of eliciting immune response ä Hapten - a substance of low molecular weight, that can bind antibodies but induce response only if covalently attached to large carrier Klein, 1992

610/30/2015 Definitions (cont) ä Antigenic Determinant: Specific sequence of amino acids that combine with receptor ä Epitope: an antigenic determinant on the surface of a molecule ä Cryotope: An antigenic determinant that is buried within a molecule and thus is unavailable in the intact molecule

710/30/2015 Immunogen vs Antigen ä Immunogen ä Any compound that triggers the immune response ä Antigen ä Any compound that combines with the products of an immune response

810/30/2015 Factors Influencing Immunogenicity ä immunogen contributions ä biological system contributions

910/30/2015 ä Chemical nature ä Chemical complexity ä Molecular size ä Conformation ä Foreigness Immunogen Contributions

1010/30/2015 Effects of Biological System on Immunogenicity ä Genotype ä Dose of Immunogen ä Route of Injection ä Adjuvants

1110/30/2015 Genotypic Influences ä Immune Responsiveness ä Some strains of inbred mice respond to specific immunogen (high responders) ä Other strains do not (low responders) ä Hybrids of above have intermediate response ä Structure of MHC receptors on cell membrane

1210/30/2015 MHC Receptors

1310/30/2015 Dose of Immunogen ä Tolerance: Specific non response to an immunogen ä Dose dependent ä Too low- no effect ä Low dose: Tolerize T cell ä High dose: Tolerize B cells

1410/30/2015 Mitcheson Tolerance Exp log [antigen injected] % Response Low Zone Tolerance High Zone Tolerance Immunity

1510/30/2015 Tolerogenic vs Immunogenic ä Immunogenicity ä antigens first processed by APC ä APC reacts with lymphocyte ä response ä Tolerogenicity ä antigens react directly with lymphocyte ä NO response Immunogenicity requires co stimulation

1610/30/2015 Route of Injection ä Parenteral injection ä other than digestive tract ä intravenous, intradermal, sucutaneous, intramuscular, intraperitioneal ä Exposure to different lymphocyte populations ä intravenous - spleen ä subcutaneous- lymph nodes

1710/30/2015 Adjuvants ä Substances that enhance immunogenicity of an antigen ä alum ä Freund’s ( contains Mycobacterium) ä Mechanisms ä prolong life of antigen ä enhance co-stimulatory signals ä nonspecific multiplication of lymphocyte ä granuloma formation

1810/30/2015 Haptens ä Antigenic but not immunogenic ä small molecules eg DiNitroPhenol ä Flexible molecules eg gelatin ä When linked to a carrier molecule becomes immunogenic ä Hapten- Carrier conjugates ä Hapten immunodominant determinant

1910/30/2015 Epitopes ä Size ä Specificity ä Affinity

2010/30/2015 Construction of Epitope ä Continuous ä stretch of contiguous residues endowed with distinctive conformational features ä Discontinous ä group of residues that are non contiguous in the sequence but are brought together by the folding of the polypeptide chain ä Klein 1992

2110/30/2015 B cell Epitopes

2210/30/2015 Immunoglobulin: B cell receptor

2310/30/2015 Antigen - Antibody binding ä Hydrogen bonds ä Electrostatic forces ä Van der Walls interactions ä Hydrophobic interactions

2410/30/2015

2510/30/2015 T cell Epitopes

2610/30/2015 Mitogens

2710/30/2015 The End

2810/30/2015 Molecular Dissection of Proteinaceous Antigens Created Maria Sawicki Updated Hugh B. Fackrell Dec 1997

2910/30/2015 X - Ray Crystallography ä Crystallized Antigen or Antigen - Antibody Complex ä Electron Density Map of the Molecule ä Three Dimensional Images

3010/30/2015 Construction of Epitope ä Continuous ä stretch of contiguous residues endowed with distinctive conformational features ä Discontinous ä group of residues that are non contiguous in the sequence but are brought together by the folding of the polypeptide chain ä Klein 1992

3110/30/2015 ANTIGEN RECOGNITION SYSTEMS ä Immunoglobulins ä intact antigen is presented with several conformational epitopes ä T cell receptors ä “processed antigen” is presented and can be recognized only in complex with Major HIstocompability Complex.

3210/30/2015 Synthetic Peptides ä Overlapping Epitopes ä Immunodominance Benjamini,1984 Benjamini,1984

3310/30/2015 Prediction of antigenicity ä Region accesibility ä Novotny 1988 ä Segmental mobility ä Westhof et al 1984, Tainer et al 1985 ä Relative hydrophilicity ä Hoop and Woods1983, Hoop 1986 ä Chain termination ä Thorton and Sibanda 1983 ä Segmental variability ä Jemmerson and Parerson 1985

3410/30/2015 Epitope Mapping Cross-reactivity studies Native Protein and Peptide Fragments Synthetic Peptides Closely Related Proteins ä Van Renmortel, 1988

3510/30/2015 ä Conformational influences ä Native protein derivatives ä Overlapping peptide sequences ä Derivatives of active peptides ä Synthetic peptides Atassi, 1978 Atassi, 1978 Studies of Native Proteins and Peptide Fragments

3610/30/2015 The End

3710/30/2015 Performance Objectives Key terms, concepts short answers

3810/30/2015 KEY TERMS ä Accessibility, antigen, antigenic determinants, antigenic site mobility, ä antigenicity, continous (linear) determinants), discontinous (conformational) determinants ä epitopes, agreotopes, haptens, immunodominance, immunogen, immunogenicity

3910/30/2015 KEY CONCEPTS ä Differentiate between and antigen's inductive and reactive abilities. ä Explain the factors that bestow immunogenicity on molecules ä Describe the discrete and distinctive sites on and antigen that are recognized by a B cell Immunoglobulin

4010/30/2015 KEY CONCEPTS ä Describe the discrete and distinctive sites on and antigen that are recognized by a T cell receptor ä Describe the discrete and distinctive sites on and antigen that are recognized by the Major Histocompatability Complex ä Discuss some important characteristics of protein antigenic determinants

4110/30/2015 SHORT ANSWER QUESTIONS

4210/30/2015 ä Differentiate between immunogenicity and antigenicity. ä All immunogens are antigens but all antigens are not immunogens. Explain with the use of specific examples. ä An entire microbe or molecule does not induce and immune response. Explain. ä Irrespective of the number of epitopes that a molecule has, the immune response is biased and sees what it wants to see. Explain.

4310/30/2015 ä Haptens are usually coupled to a carrier molecule before they are immunogenic. How can you demonstrate that the selected immunoglobulins are specific for the hapten and not for the carrier? ä As a corollory, would you expect ALL of the antibodies to be specific for the hapten? explain.

4410/30/2015 ä Why is the definition of an antigen considered to be an operational definition. ä Differentiate between epitopes and agreotopes. ä Differentiate between epitopes recognized by B cell receptor and epitopes recognized by T cell receptors.