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Antigens

Hugh B. FackrellHugh B. Fackrell

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Assigned ReadingAssigned Reading Content Outline Content Outline Performance ObjectivesPerformance Objectives

Key termsKey terms Key ConceptsKey Concepts

Short Answer QuestionsShort Answer Questions

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ASSIGNED READING

Chapter 4 pp 87-106Chapter 4 pp 87-106

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OUTLINE

Immunogenicity vs Immunogenicity vs antigenicityantigenicity

Factors that influence Factors that influence immunogenicityimmunogenicity immunogen immunogen

contributionscontributions biological system biological system

contributions contributions

EpitopesEpitopes B cell epitopesB cell epitopes T cell epitopesT cell epitopes

HaptensHaptens MitogensMitogens

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Definitions

AntigenAntigen - a substance recognized by T - a substance recognized by T and B cell receptorsand B cell receptors

ImmunogenImmunogen - a substance capable of - a substance capable of eliciting immune responseeliciting immune response

HaptenHapten - a substance of low molecular - a substance of low molecular weight, that can bind antibodies but weight, that can bind antibodies but induce response only if covalently induce response only if covalently attached to large carrier attached to large carrier Klein, 1992Klein, 1992

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Definitions (cont)

Antigenic DeterminantAntigenic Determinant: : Specific Specific sequence of amino acids that combine sequence of amino acids that combine with receptorwith receptor

Epitope:Epitope: an antigenic determinant on the an antigenic determinant on the surface of a moleculesurface of a molecule

Cryotope:Cryotope: An antigenic determinant that An antigenic determinant that is buried within a molecule and thus is is buried within a molecule and thus is unavailable in the intact moleculeunavailable in the intact molecule

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Immunogen vs Antigen

ImmunogenImmunogen Any compound that triggers Any compound that triggers

the immune responsethe immune response AntigenAntigen

Any compound that combines Any compound that combines with the products of an with the products of an immune responseimmune response

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Factors Influencing Immunogenicity

immunogen contributionsimmunogen contributions biological system contributionsbiological system contributions

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Chemical natureChemical natureChemical complexityChemical complexityMolecular sizeMolecular sizeConformationConformationForeignessForeigness

Immunogen Contributions

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Effects of Biological System on Immunogenicity

GenotypeGenotype Dose of ImmunogenDose of Immunogen Route of InjectionRoute of Injection AdjuvantsAdjuvants

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Genotypic Influences Immune ResponsivenessImmune Responsiveness

Some strains of inbred mice respond to Some strains of inbred mice respond to specific immunogen (high responders)specific immunogen (high responders)

Other strains do not (low responders)Other strains do not (low responders)Hybrids of above have intermediate Hybrids of above have intermediate

responseresponse Structure of MHC receptors on cell Structure of MHC receptors on cell

membranemembrane

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MHC Receptors

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Dose of Immunogen

Tolerance: Tolerance: SpecificSpecific non response to an non response to an immunogenimmunogen

Dose dependentDose dependentToo low- no effectToo low- no effectLow dose: Tolerize T cellLow dose: Tolerize T cellHigh dose: Tolerize B cellsHigh dose: Tolerize B cells

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Mitcheson Tolerance Exp

log [antigen injected]

%

Response

100-

0-

200-

Low Zone Tolerance

High Zone Tolerance

Immunity

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Tolerogenic vs Immunogenic

ImmunogenicityImmunogenicity antigens first processed by APCantigens first processed by APC APC reacts with lymphocyteAPC reacts with lymphocyte responseresponse

Tolerogenicity Tolerogenicity antigens react directly with lymphocyteantigens react directly with lymphocyte NO responseNO response

Immunogenicity requires co stimulationImmunogenicity requires co stimulation

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Route of Injection

Parenteral injectionParenteral injection other than digestive tractother than digestive tract intravenous, intradermal, sucutaneous, intravenous, intradermal, sucutaneous,

intramuscular, intraperitionealintramuscular, intraperitioneal Exposure to different lymphocyte Exposure to different lymphocyte

populationspopulations intravenous - spleenintravenous - spleen subcutaneous- lymph nodessubcutaneous- lymph nodes

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Adjuvants

Substances that enhance immunogenicity of an antigenSubstances that enhance immunogenicity of an antigenalumalumFreund’s ( contains Freund’s ( contains MycobacteriumMycobacterium))

MechanismsMechanismsprolong life of antigenprolong life of antigenenhance co-stimulatory signalsenhance co-stimulatory signalsnonspecific multiplication of lymphocytenonspecific multiplication of lymphocytegranuloma formationgranuloma formation

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Haptens Antigenic but not immunogenicAntigenic but not immunogenic

small molecules eg DiNitroPhenolsmall molecules eg DiNitroPhenolFlexible molecules eg gelatinFlexible molecules eg gelatin

When linked to aWhen linked to a carriercarrier molecule molecule becomes becomes immunogenicimmunogenic

Hapten- Carrier conjugatesHapten- Carrier conjugatesHapten Hapten immunodominant determinantimmunodominant determinant

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Epitopes

SizeSize SpecificitySpecificity AffinityAffinity

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Construction of Epitope

ContinuousContinuousstretch of contiguous residues endowed with stretch of contiguous residues endowed with

distinctive conformational featuresdistinctive conformational featuresDiscontinousDiscontinous

group of residues that are non contiguous in the group of residues that are non contiguous in the sequence but are brought together by the sequence but are brought together by the folding of the polypeptide chainfolding of the polypeptide chain

Klein 1992Klein 1992

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B cell Epitopes

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Immunoglobulin: B cell receptor

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Antigen - Antibody binding

Hydrogen bondsHydrogen bondsElectrostatic forcesElectrostatic forcesVan der Walls interactionsVan der Walls interactionsHydrophobic interactionsHydrophobic interactions

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T cell Epitopes

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Mitogens

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The EndThe End

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Molecular Dissection ofProteinaceous Antigens

Created Maria SawickiCreated Maria SawickiUpdated Hugh B. FackrellUpdated Hugh B. Fackrell

Dec 1997Dec 1997

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X - Ray Crystallography

Crystallized Antigen or Crystallized Antigen or Antigen - Antibody Antigen - Antibody ComplexComplex

Electron Density Map Electron Density Map of the Molecule of the Molecule

Three Dimensional Three Dimensional ImagesImages

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Construction of Epitope

ContinuousContinuousstretch of contiguous residues endowed with stretch of contiguous residues endowed with

distinctive conformational featuresdistinctive conformational featuresDiscontinousDiscontinous

group of residues that are non contiguous in the group of residues that are non contiguous in the sequence but are brought together by the sequence but are brought together by the folding of the polypeptide chainfolding of the polypeptide chain

Klein 1992Klein 1992

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ANTIGEN RECOGNITION SYSTEMS

ImmunoglobulinsImmunoglobulinsintact antigen is presented with several intact antigen is presented with several

conformational epitopes conformational epitopes T cell receptorsT cell receptors

““processed antigen” is presented and can processed antigen” is presented and can be recognized only in complex with Major be recognized only in complex with Major HIstocompability Complex .HIstocompability Complex .

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Synthetic Peptides

Overlapping EpitopesOverlapping Epitopes ImmunodominanceImmunodominance

Benjamini ,1984Benjamini ,1984

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Prediction of antigenicityRegion accesibilityRegion accesibility

Novotny 1988Novotny 1988Segmental mobilitySegmental mobility

Westhof et al 1984, Tainer et al 1985Westhof et al 1984, Tainer et al 1985Relative hydrophilicityRelative hydrophilicity

Hoop and Woods1983, Hoop 1986Hoop and Woods1983, Hoop 1986Chain terminationChain termination

Thorton and Sibanda 1983Thorton and Sibanda 1983Segmental variability Segmental variability

Jemmerson and Parerson 1985Jemmerson and Parerson 1985

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Epitope MappingCross-reactivity studies

Native Protein and Peptide Native Protein and Peptide FragmentsFragments

Synthetic PeptidesSynthetic Peptides

Closely Related ProteinsClosely Related Proteins

Van Renmortel, 1988Van Renmortel, 1988

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Conformational influencesConformational influencesNative protein derivatives Native protein derivatives Overlapping peptide sequences Overlapping peptide sequences Derivatives of active peptides Derivatives of active peptides Synthetic peptides Synthetic peptides Atassi, 1978Atassi, 1978

Studies of Native Proteins and Peptide Fragments

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The EndThe End

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Performance ObjectivesPerformance Objectives

Key terms, concepts Key terms, concepts

short answersshort answers

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KEY TERMS

Accessibility, antigen, antigenic Accessibility, antigen, antigenic determinants, antigenic site mobility, determinants, antigenic site mobility,

antigenicity, continous (linear) antigenicity, continous (linear) determinants), discontinous determinants), discontinous (conformational) determinants(conformational) determinants

epitopes, agreotopes, haptens, epitopes, agreotopes, haptens, immunodominance, immunogen, immunodominance, immunogen, immunogenicityimmunogenicity

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KEY CONCEPTS

Differentiate between and antigen's Differentiate between and antigen's inductive and reactive abilities.inductive and reactive abilities.

Explain the factors that bestow Explain the factors that bestow immunogenicity on moleculesimmunogenicity on molecules

Describe the discrete and distinctive sites on Describe the discrete and distinctive sites on and antigen that are recognized by a B cell and antigen that are recognized by a B cell ImmunoglobulinImmunoglobulin

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KEY CONCEPTS Describe the discrete and distinctive sites on and Describe the discrete and distinctive sites on and

antigen that are recognized by a T cell receptorantigen that are recognized by a T cell receptor Describe the discrete and distinctive sites on and Describe the discrete and distinctive sites on and

antigen that are recognized by the Major antigen that are recognized by the Major Histocompatability ComplexHistocompatability Complex

Discuss some important characteristics of protein Discuss some important characteristics of protein antigenic determinantsantigenic determinants

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SHORT ANSWER QUESTIONS

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Differentiate between immunogenicity and Differentiate between immunogenicity and antigenicity.antigenicity.

All immunogens are antigens but all antigens All immunogens are antigens but all antigens are not immunogens. Explain with the use of are not immunogens. Explain with the use of specific examples.specific examples.

An entire microbe or molecule does not induce An entire microbe or molecule does not induce and immune response. Explain.and immune response. Explain.

Irrespective of the number of epitopes that a Irrespective of the number of epitopes that a molecule has, the immune response is biased molecule has, the immune response is biased and sees what it wants to see. Explain.and sees what it wants to see. Explain.

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Haptens are usually coupled to a carrier Haptens are usually coupled to a carrier molecule before they are immunogenic. How molecule before they are immunogenic. How can you demonstrate that the selected can you demonstrate that the selected immunoglobulins are specific for the hapten immunoglobulins are specific for the hapten and not for the carrier? and not for the carrier?

As a corollory, would you expect ALL of the As a corollory, would you expect ALL of the antibodies to be specific for the hapten? antibodies to be specific for the hapten? explain.explain.

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Why is the definition of an antigen Why is the definition of an antigen considered to be an operational definition.considered to be an operational definition.

Differentiate between epitopes and Differentiate between epitopes and agreotopes.agreotopes.

Differentiate between epitopes recognized Differentiate between epitopes recognized by B cell receptor and epitopes recognized by B cell receptor and epitopes recognized by T cell receptors.by T cell receptors.