BIOT 307Kuby, 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 antigen-

binding 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: poorChemical 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 immunoregulationEVIDENCE - Mice• Inbred strains of mice respond differently to Ag• F1 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-DR4 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

ADJUVANTS = substances administered with Ag enhance immunogenicityUses: low immunogenicity OR only small amounts of Ag

availableMechanism of action: 1. Ligands for TLR on DCs and MΦ2. Prolong Ag persistence “depot effect”3. Enhance costimulatory molecules4. Increase local inflammation5. Stimulate nonspecific lymphocyte stimulation

ADJUVANT TYPES

HUMAN USE• Alum – aluminum potassium sulfate – APPROVED• MF59® – water in oil emulsion, Th1 and Th2 –

APPROVED in EU• AS04 - IN DEVELOPMENT: molecular mechanism based adjuvantsANIMAL USE ONLY• Freund’s incomplete adjuvantNOT 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 non-covalent 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

HAPTENShaptens, 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