BIOT 307 Kuby Ch 3 Antigens March 2013

BIOT 307 Kuby, Ch. 3, Antigens March, 2013

General Introduction • Specificity due to recognition of antigenic determinants or epitopes • Epitopes = immunologically active regions that bind to: – Ag-specific membrane receptors on lymphocytes – Secreted antibodies • Multiple epitopes can be found on single macromolecule

ANTIGENS = SUBSTANCES RECOGNIZED BY • immunoglobulin receptor of B cells • T- cell receptor when complexed with MHC • Host determines whether B or T cell’s antigenbinding receptor actually induces immune response • B and T cell recognition are different

Immunogenicity and Antigenicity • Immunogenicity = ability to induce humoral and/or cell- mediated immune response • Antigenicity = ability to combine specifically with the final products of the above responses (i. e. , antibodies and/or cell-surface receptors). • Haptens, small molecules, are antigenic but incapable of inducing specific immune response

FACTORS INFLUENCING IMMUNOGENICITY • Type of molecule: – Protein and polysaccharides immunogenic – Lipids and nucleic acids must be conjugated with another molecule, i. e. , like above

PROPERTIES DETERMINING IMMUNOGENICITY Foreignness or non-self • Non-self antigens are eliminated by recognition and response • lymphocytes that recognize self antigens are eliminated by inactivation • Antigens that are more foreign are more immunogenic – Exceptions: • Collagen and cytochrome c – similar in all species • Corneal tissue and sperm - kept apart from immune system

Molecular Size • > 100, 000 Da: excellent • < 5, 000 -10, 000: poor Chemical Composition and Heterogeneity • Homopolymers: poor • Protein structure: 1’, 2’, 3’, 4’ all contribute • Lipids serve as haptens Ab against lipids useful – leukotrienes, steroids, vitamins

Susceptibility to Ag processing and presentation • Interaction of T-cells with processed Ag • Processing by: MΦs, neutrophils • Presentation via APCs (MΦs, DCs, B cells, mast cells) • Larger molecules more easily phagocytosed

HOST CONTRIBUTES TO IMMUNOGENICITY • Genes in MHC • Genes that encode B and T-cell receptors • Genes involved in immunoregulation EVIDENCE - Mice • Inbred strains of mice respond differently to Ag • F 1 generation – intermediate • Backcross analysis mapped differences to MHC subregion

HOST CONTRIBUTES TO IMMUNOGENICITY EVIDENCE – Humans • HLA determines immune response to some pathogens • African-Americans respond poorly to interferon-α • HLA-DR 4 associated with high responsiveness to antigens specific to M tuberculosis but not to antigens shared with other mycobacteria (p = 0. 0005)

HOST CONTRIBUTES TO IMMUNOGENICITY Immunogen dosage • Dose response – Not enough to activate lymphocytes or tolerance – Too much tolerance Example: capsular polysaccharide – O. 5 mg: no IR – 0. 0005 mg: Ab response IMPORTANCE OF BOOSTERS: clonal proliferation

HOST CONTRIBUTES TO IMMUNOGENICITY Immunogen route of administration • Routes: experimental Ag via intravenous (IV), intradermal (ID), subcutaneous (SC), intramuscular (IM), intraperitoneal (IP) • Influences which immune organs, tissues and cells – IV spleen – Subcutaneously local lymph nodes

HOST CONTRIBUTES TO IMMUNOGENICITY Immunogen route of administration • Routes: vaccine Ag via intramuscular (IM), oral, subcutaneous (SC), • Influences which immune organs, tissues and cells – SC and IM local lymph nodes – Oral MALT

ROLE OF ADJUVANTS = substances administered with Ag enhance immunogenicity Uses: low immunogenicity OR only small amounts of Ag available Mechanism of action: 1. Ligands for TLR on DCs and MΦ 2. Prolong Ag persistence “depot effect” 3. Enhance costimulatory molecules 4. Increase local inflammation 5. Stimulate nonspecific lymphocyte stimulation

ADJUVANT TYPES HUMAN USE • Alum – aluminum potassium sulfate – APPROVED • MF 59® – water in oil emulsion, Th 1 and Th 2 – APPROVED in EU • AS 04 IN DEVELOPMENT: molecular mechanism based adjuvants ANIMAL USE ONLY • Freund’s incomplete adjuvant NOT USED EVEN IN ANIMALS • Freund’s complete adjuvant

EPITOPES • immune cells do not interact with, or recognize, entire immunogen molecule • Lymphocytes recognize discrete sites on immunogen molecule called epitopes, or antigenic determinants. • Epitopes are the immunologically active regions of an immunogen that bind to antigen-specific membrane receptors on lymphocytes or to secreted antibodies • Studies with small antigens reveal that B and T cells recognize different epitopes on same antigenic molecule

B-CELL EPITOPES • Hydrophilic amino acids on protein surface – Protruding regions – Interior is hydrophobic; must be denatured to be open to Ab • These recognize membrane bound or free Ab

B-CELL EPITOPES

B-CELL EPITOPES

• 15 - 22 amino acids on Ag contact Ab • 75– 120 hydrogen bonds as well as by ionic and hydrophobic interactions.

Conformational Epitope

• Ag-Ab binding due to weak non-covalent interactions operating over short distances • Precise complementary shapes increase noncovalent bonding • Smaller ligands such as carbohydrates, small oligonucleotides, peptides, and haptens often bind within deep pocket of Ab

• B-cell epitopes tend to be located in flexible regions of an immunogen and display site mobility

• Complex proteins contain multiple overlapping B-cell epitopes • Only some are immunodominant • Determined with monoclonal antibodies (MAbs)

• More potential antigenic sites than number recognized by immune system – Varies from species to species – Within species, individuals can • recognize different epitopes as immunogenic and • mount immune responses that are stronger (immunodominant) against different epitopes

T CELL EPITOPES • Antigen processing is required to generate peptides that interact specifically with MHC molecules • Epitope is not conformational rather linear • Epitopes recognized by T cells are often internal • given MHC molecule can selectively bind variety of different peptides

• Antigen processed into antigenic peptides are presented in combination with MHC molecules • Antigenic peptides recognized by T cells form trimolecular complexes with T-cell receptor and MHC molecule

HAPTENS haptens, small organic molecules that are antigenic but not immunogenic Become immunogenic when linked to carrier molecule, e. g. , large protein drugs, peptide hormones, and steroid hormones

USE OF HAPTENS • Configuration plays major role in determining whether it can react with a given antibody

• drugs, peptide hormones, and steroid hormones