ANTIGENS

IMMUNOLOGICAL PROPERTIES OF ANTIGENS

IMMUNOGENICITY: The ability to induce an immune response. An immunogen is typically also an antigen. The confusing part is that we typically talk about Ags as if they are necessarily immunogens.

ANTIGENICITY: Ags were defined on the basis of their specific interactions with the end products of an immune response. While immunogens will be Ags, not all Ags are immunogens. Eg. Haptens, small molecules, can display antigenicity by being bound by Ab, but can not initiate the production of Ab due to their small size.

ALLEROGENICITY: Ag that induces an allergic response.

TOLEROGENICITY: Ag that induces specific immunologic nonresponsiveness.

FACTORS THAT INFLUENCE IMMUNOGENICITY

Humoral Immunogens:

Proteins >> Polysaccharides >> Lipids or Nucleic Acids

In general lipids and nucleic acids are very poor Immunogens reactive only when complexed with protein or polysaccharides.

Cell Mediated Immunogens:

Only proteins and some lipids and glycolipids serve as Ags for CMI. Proteins are not recognized directly, peptides processed from the protein are seen in association with MHC molecules. Lipids and glycolipids are also recognized complexed with the CD1 MHC-like molecule.

Proteins are not intrinsically immunogenic, as immunogenicity is dependent upon the specific proteins interaction with the individuals immune system.

The 4 Defining Properties of a Protein Immunogen

1. Foreignness: To serve as an immunogen a molecule must be seen as non self. The degree of immunogenicity is dependent upon the degree of foreignness. The greater the phylogenetic distance between species the greater the chance of immunogenicity.

Eg. BSA injected into chickens or goats.

Exceptions:

• Highly conserved molecules like collagen or cytochrome c are more poorly immunogenic even in distant species.
• Some self molecules (Eg. sperm, myolin basic protein, or corneal tissue), that are sequestered from the immune system, will raise an immune response in the animal that they came from.

2. Molecular Size: There is a correlation between size and immunogenicity. The best immunogens are in the range of 100,000 Da., while small molecules 5-10,000 Da are generally poor immunogens. Minimally they must be large enough to be processed.

3. Chemical Heterogeneity: Size alone will not make a good immunogen. Synthetic homopolymers are not immunogenic regardless of size. Large co-polymers can be immunogenic, adding aromatic amino acids increases the chance. Proteins with more complexity in primary structure and those showing secondary, tertiary, and quaternary structure increase immunogenicity.

4. Degradability: Proteins must be degraded to be presented by MHC molecules to activate TH cells. Factors that influence this process affect immunogenicity.

• soluble vs insoluble
• small vs large
• D-amino acids

Lipids as Immunogens

• B cell recognition of lipid/glycolipid components is induced by hapten-carrier models where lipids are complexed to protein carriers resulting in a response that recognizes the lipid component.
• T cell recognition of lipid Ags involves Ag display on the MHC-like molecule CD1.

Biological System

The Host Plays a Role

Genotype:

• MHC- Ag presentation by MHC is required for an immune response. Some Ags are better immunogens in some individuals than in others. This is best seen in inbred mice where some Ags are better immunogens in one strain than in another due to the differences in Ag peptide binding to different MHC molecules.
• B cell and T cell receptor diversity. B- or T-cells with receptors capable of interacting with a specific Ag may not exist or may not come in contact with the antigen.

Dosage and Route:

For each Ag there will be an optimal dosage and route of administration. Too high or too low a dose of Ag leads to immunologic unresponsiveness (Tolerance). Generally repeated exposure is required to raise a strong immune response (Boosters). The route of administration also influences the immune response. Typical routes of Ag exposure are;

• Intramuscular
• Intradermal
• subcutaneously
• Intravenous
• Intraperitoneal
• Mucosal

ADJUVANTS

Adjuvant (L. adjuvare, to help)

Substances that when injected with Ag serve to enhance the immunogenicity of the Ag. This leads to a higher titer and longer lasting immune response.

Mechanism of Adjuvant Action?

• Prolong Exposure to Ag: Alum and Freund's adjuvant bind and ppte. Ag to keep it in the system longer to be slowly released to the system. Can increase the time of exposure from a few days to a few weeks. PPTE also increases the size of the Ag to facilitate phagocytosis.
• Stimulate Immune Response:
  • Freund's complete adjuvant, which contains muramyl dipeptides of the cell walls of heat killed Mycobacteria , stimulate macrophage activity. The increase in IL-1 helps activate TH cells
  • Synthetic polyribonucleotides and bacterial LPS stimulates nonspecific lymphocyte proliferation.
  • Some adjuvants stimulate local chronic inflammation and granuloma formation (Freund's complete).
• Co-stimulatory Signal: Freund's adjuvant, LPS, and other factors up regulate co-stimulatory signal systems. Eg. TH cells when stimulated by Ag require a second co-stimulatory signal. B7, a membrane protein on macrophage, interacts with CD28 on the TH to give this 2nd signal. B7 is up-regulated by these adjuvants.

EPITOPES

EPITOPES: Lymphocytes recognize discrete sites on macromolecules called antigenic determinants or epitopes. Epitopes seen by B cells and T cells differ in fundamental ways.

B cell Epitopes:

• B cells bind Ag directly via cell surface Immunoglobulin (Ig)
• Ag in solution
  • epitopes must be accessible of the protein surface (hydrophilic)
  • epitopes must be flexible and mobile for agglutination (often located on bends and loop structure of protein)
  • B cell epitopes in native protein are therefore generally hydrophilic amino acids that are topographically accessible to Ab.
  • Epitope can be sequential or non sequential (conformational)
• Epitope size defined by the binding site of the Ab.
  • Complementary binding between Ag-Ab limits epitope size and conformation
  • Typically 6-7 amino acids or sugars can fit into the deep pocket structures of linear epitope binding sites
  • Conformational epitopes of globular proteins cover much greater space on flatter surface binding sites of Ab. Epitopes may consist of 15-22 amino acids
• Complex proteins may contain multiple overlapping B cell epitopes. Not all will induce a response IMMUNODOMINANCE

Here is the backbone of lysozyme. The epitope which binds to IgG1-kappa D1.3 is thickened. In

another view (not shown) it can be seen that the epitope forms a relatively flat surface. Notice

how the epitope is discontinuous, that is, made up of loops of the protein which are proximal in

the native structure, but are not sequential in the amino acid sequence. This image uses RasMol's

"group" coloring scheme, in which the backbone is colored in rainbow sequence from one end to

the other. This image was constructed from images in a RasMol "movie" script. The atomic coordinate file employed is 1FDL.PDB.

This page is maintained by emartz@microbio.umass.edu

T cell Epitopes:

T cells do not recognize soluble native Ag but rather recognize Ag that has been processed and whose peptide fragments are presented in association with MHC molecules.

• T cell epitopes are generally oligomeric peptides of 7-20 aa in size
  • Ag binding cleft of MHC defines Ag expression
  • MHC class I nonamers are bound best (9-11 aa)
  • MHC class II typically 11-17 aa
• Ag is seen as part of a trimolecular complex
  • TCR-Ag-MHC
  • MHC binding site of the Ag is called the AGRETOPE, binds via hydrophobic amino acids
  • TCR binding site called the EPITOPE, binds via hydrophilic amino acids
• Ag processing required to generate peptides
  • peptides may be internal
  • must be amphipathic
    • must have hydrophobic regions to bind MHC
    • must have hydrophilic regions to bind TCR
• Immunodominant T cell epitopes determined in part by what set of MHC molecules are expressed by an individual and what TCR are expressed by an individual.

characteristic	B cell	T cell
Inter. Ag	binary, Ag-Ab interact directly	ternary, TCR-Ag-MHC
binding of soluble Ag	yes	no
MHC involvement	no	yes
Ag properties	Protein, Polysaccharides Lipids	Protein Lipids via CD1
Epitope properties	accessible, hydrophilic, mobile,linear or conformational	Internal, denatured, processed, amphipathic, linear, binds MHC

Mitogens:

• Induce cell division in a high percentage of B-cells, T cells or both
• Polyclonal activation, not specific activation-not acting as an immunogen
• Many lectins act as mitogens
  • Con A and PHA are T-cell mitogens
  • Pokeweed mitogen is both a B-cell and T-cell mitogen
• Bacterial LPS via the lipid component acts as B-cell mitogens

Superantigens:

• very potent T cell mitogens
• Binds via Vbeta domains of TCR and cross links MHC-TCR in an Ag independent bond held together by the superantigen.
• Therefore activates sets of T cells according to their Vbeta region and not due to Ag binding sites.
• Can activate as many as 1 out of 5 T cells resulting in high release of cytokines and may cause shock to the body
  • Enterotoxins of Staphylococcus responsible for Toxic-shock syndrome toxin (TSST1) is an example
  • Can stimulate a large number of T-cells but with no second signals this can lead to the down-regulation of the immune system.

T-cell dependent and T-cell independent Ags

• The majority of Ags are T-cell dependent
• T-cell independent Ags are generally carbohydrate Ags of bacteria
  • Large repeats of simple sugar units providing multiple repeating epitopes
    • Eg. Gal-GalNac-Ribulose-Gal-GalNac-Rib-etc
  • Results in the cross linking of multiple surface Ig stimulating an immune response independent of T cell help

Innate response Receptors

Pattern-Recognition Receptors

• Innate response receptors typically recognize structural motifs or patterns common to microbial species but not present on the host, therefore self-reactivity is not an issue
• Targets of these receptors include, carbohydrate structures, some specific proteins or lipid types, and unique nucleic acid motifs.
• Some PRRs are soluble proteins in the blood stream or tissue fluids (Eg. Mannose-binding protein or C-reactive protein), some are membrane surface proteins found on macrophage and dendritic cells (Eg. toll-like receptors or scavenger receptors).