453 Lecture 3 - Antigens

Chemical Composition and Heterogeneity

-Long chains of a single amino acid fail to elicit immune response, chains of 2 AAs increase, 3 even further, etc -Protein structure: more complex primary structure = good, same for 2ndary, tert, quarternary structures; more extensive = better immunogen

Binding of Peptides to MHC Molecules

-MHC I molecules bind peptides 8-11 AAs in length -MHC II molecs bind peptides 12-25 AA long -Not specific interaction for either MHC I or II -Each can bind a spectrum of peptides -May be due to agretopes shared btwn peptides

Immunodominant T Cell Epitopes Dictated by Antigens Bound by MHC

-MHC can bind many antigens, not all -Only antigens capable of being complexed by MHC can stimulate T cells -Therefore, spectrum of peptides bound by MHC dictates dominance of T cell antigens

Factors Affecting Immunogenicity

-Many compounds are immunogenic, some are better than others --Best immunogens are proteins and polysaccharides --Nucleic acids and lipids can be immunogens if conjugated (linked) to proteins or polysaccharides -Cell Mediated Immune (CMI) system can only use proteins and lipids, Processed into smaller fragments (peptides), Presented to CMI in context of MHC molecs -Both the immunogen and the biological system contribute to how strong the immune response is

Molecular Size

-Small molecs can be too small to elicit a response, <1kDa is poor, can make stronger by conjugating w/ larger immunogen -Best immunogens around 100kDa -Larger immunogens still elicit response, may not be very specific ---Very large antigens have large number of EPITOPES ---Cause generation of too many antibodies, likely to cross react w/ other immunogens

Antigens on Native Proteins

-Some epitopes will be exposed on surface of proteins, others buried deep inside of protein -Only antigens on surface are accessiible for interaction w/ B cells -Surface of proteins = hydrophilic environment, therefore B cell epitopes on native proteins are typically hydrophilic surface exposed proteins --Accessible for binding to membrane bound antibodies, binding to soluble antibodies

Properties of T Cell Antigens

-T cells do not recognize native protein, only proteins that have been broken down into ANTIGENIC PEPTIDES -Bound and presented by MHC molecules, forms trimolecular complex upon recognition between peptide, MHC, and TCR

Immunogen Dosage and Route

-TOLERANCE: Too much/too little induces immunological unresponsiveness -Single dose insufficient, usually requires repeated administration of immunogen (BOOSTERS) -Must have correct route: INTRAVENOUS, INTRADERMAL, SUBCUTANEOUS, INTRAMUSCULAR, INTRAPERITONEAL -IV immunization results in antigen processing in the spleen -Immunization into tissues results in antigen processing in lymph nodes

How Many Epitopes Can a Protein Have?

-The larger the protein, the more epitopes it can possess -Usually # of epitopes on a protein is more than that which is recognized by animal's immune system -Immune system capable of recognizing all determinants, but only recognizes preferentially specific epitopes -Antigenic determinants overlap, some determinants more antigenic than others

Aspects of Immunogen Affecting Immunity (4)

1) Foreignness 2) molecular size 3) chemical composition & heterogeneity 4) susceptibility to antigen processing and presentation

Contribution of Biological System to Immunogenicity (3)

1) Genotype of Recipient animal 2) Immunogen Dosage and route of administration 3) Adjuvants

Sequential Epitopes

AAs are next to each other in primary protein structure -Can fragment protein to yield multiple epitopes and antibodies will still bind

Freunds Incomplete Adjuvant

Antigen in aqueous solution, oil, and emulsifier (mannide manooleate) Oil surrounds antigen, retards dispersion from injection site (prolonging antigen persistence)

Haptens

Antigenic but not Immunogenic -Haptens are compounds that are too small to elicit immune response -Can cause response if conjugated to large carrier molecule (BSA) -Generates 3 types of antibodies: anti-haptens, anti-carriers, anti-hapten/carriers

Freunds Complete Adjuvant

Contains heat killed mycobacteria, muramyl dipeptide in cell activates mphage, more phagocytic, higher MHC II, better antigen presentation, more IL-1 produced (enhances co-stimulatory signal)

2 distinct interaction sites of T cell antigens

Epitope - interacts w/ TCR AGRETOPE - interacts w/ MHC molecule -T cell epitopes must be viewed in terms of ability to bind MHC and TCR

Genotype of Recipient Animal

GENOTYPE dictates strength of response -2 mice strains immunized w/ same anitigen, one strain makes strong antibodies, other didn't, progeny of cross produced intermediate response -Due to differences in MHC molecules, different MHC molecs have different spectrums of peptides they can present -Also genes encoding B cell and T cell receptors

Conformational Epitopes

Non-sequential AAs in proximity b/c secondary, tertiary, and quarternary structures -Clevage or denaturation can destroy epitope, antibody can no longer bind

Evidence Supporting B Cell Antigen Hypothesis

Picture: Tyr and Glu facing outwards = accessible to B cells, robust antibody response Tyr and Glu facing IN = Tyr and Glu not accessible to B cells, NO anti-TyrGlu antibody response

Adjuvants (misc.)

Polyribonucleotides and lipopolysaccharides stimulate non-specific proliferation of lymphocytes, better chance of antigen induced clonal selection

Immunodominant

Preferentially recognized by immune systems

Epitopes

aka ANTIGENIC DETERMINANTS -The part of an antigen that is recognized by the immune system -Very defined sites on molecule -Often short stretches of amino acids -T cell and B cell recognition of antigenic determinants fundamentally different --B cells capable of binding to soluble antigens (cell free) --T cells can only interact w/ antigens presented by MHC

Evidence Supporting Conformational Epitopes

hen egg white lysozyme (HEL) identified, one end product used to compete w/ HEL to antibody, one has a disulfide bond that occured naturally in HEL, other had same primary amino acid structure but no disulfide bond -3 competitors in antibody binding assay: --Wild-type HEL, closed epitope loop, open epitope loop (did not work)

Alum in adjuvants

stimulates local chronic inflammatory response, causes infiltration of phagocytes and granulocytes, forms granuloma

Granuloma

tumor-like mass of macrophage

Adjuvants

-ADJUVANTS enhance immunogenic potential of immunogens when mixed and injected w/ it -Used if immunogen has low immunogenicity or only have small amounts of it

What is an Antigen?

-An ANTIGEN is any substance capable of eliciting an immune response -A broad spectrum of biologically active molecules can serve as an antigen: proteins, lipids, polysaccharides, nucleic acids -Synthetic man-made compounds are also capable of eliciting an immune response

Epitope and Protein Flexibility

-Analysis of numerous epitopes found that epitopes tend to be the most flexible region of the protein -Proposed that flexibility allows an induced fit of antigen into membrane associated and soluble antibody

Properties of B Cell Epitopes

-Antibodies interact w/ antigens using weak non-covalent interactions -Strong binding due to complementary antigen-antibody binding site shapes -Places interacting groups in proximity to each other -Shape of antigen dictates shape of antigen binding site of antibody that will bind to it --Small epitopes tend to bind to antibodies that have deep narrow clefts --Large epitopes involve interactions between large protein faces epitope and antibody

Susceptibility to Antigen Processing

-Antigen must be susceptible to degradation to elicit response -Th cells interact w/ antigen bound by MHC class II, internalized by APC and B cells, digested to smaller pieces in phagolysosome -Tc cells interact w/ antigen bound by MHC I molecs -Proteins degraded by proteasome in cytosol --Transported to ER and bound by MHC I --Antigens resistant to either form of degradation cause poor immune response

Antigen Processing Required for Generation of Peptides

-Antigens have to be broken down into small fragments to be presented to T cells by MHC -Exogenous antigens presented by MHC II -Endogenous antigens presented by MHC I -Epitopes presented to T cells often derived from internal regions of mature proteins -Would not be accessible for humoral immune response

Cross Reaction

-Discovered through use of haptens -Attaches to carrier protein (BSA)

Antigen Antibody Interaction for Globular Proteins

-Entire surface of globular proteins can serve as source for epitopes -Usually hydrophilic AAs that protrude from surface provides interaction point -Often results in complementary protrusions and depressions

Immunogenicity vs. Antigenicity

-IMMUNOGENICITY: capacity to induce humoral or cell mediated immune response -ANTIGENICITY: ability to combine w/ final products of immunogenic response --can bind to activated T cell through interaction w/ TCR, can bind to antibodies produced & secreted by B cells, can't elicit immune response on their own -HAPTENS: very small antigenic molecules -All immunogens are anitgens, not all antigens are immunogens

Foreignness

-Immune system capable of distinguishing self from non-self -Substance must be significantly different from anything found in host in order to elicit an immune response ---Effector cells acquire ability during maturation ---Any antigen not encountered during maturation perceived as foreign -Strength of response depends on degree of foreignness -Ex. bovine serum albumin not immunogenic to cattle, somehwhat immunogenic to goats, extremely immunogenic to mice

Explanations for adjuvants

Unknown mechanism: 1) Prolong antigen persistence 2) Enhance co-stimulatory signals (cytokines) 3) Induce granuloma formation 4) Stimulate lymphocyte proliferation no-specifically?