Antigens, Tolerogens, and Allergens

Concept of Antigens

• Antigens are substances inducing specific immune responses, reacting with the products of those responses
• The term "antigen" originally meant substances stimulating antibody generation
• The immune system consists of more than just antibodies
• The modern definition of antigen includes all substances recognizable by the adaptive immune system

Tolerogens

• Tolerogens are antigens inducing specific immune non-responsiveness based on their molecular form
• A tolerogen can become an immunogen if its molecular form is altered

Allergens

• Allergens induce allergic reactions
• Allergic reactions can occur after exposure through ingestion, inhalation, injection, or skin contact

Classification of Antigens by Origin

• Antigens can be classified based on their origin

Exogenous Antigens

• Exogenous antigens enter the body from the outside via inhalation, ingestion, or injection
• These antigens are taken into antigen-presenting cells (APCs) through endocytosis or phagocytosis, and then processed into fragments

Endogenous Antigens

• Endogenous antigens are generated within cells due to normal metabolism, viral infections, or intracellular bacterial infections

Autoantigens

• Autoantigens are typically normal proteins or protein complexes (sometimes DNA or RNA) recognized by the immune system in patients with autoimmune diseases
• These antigens do not usually trigger an immune response, but genetic or environmental factors can cause a loss of immunological tolerance

Tumor Antigens

• Tumor antigens are presented by MHC I molecules on tumor cell surfaces
• Tumor-specific antigens (TSAs) are only presented by tumor cells and result from tumor-specific mutations

Characteristics of Antigens

• Immunogenicity: The capacity to stimulate antibody production or cell-mediated immune responses
• Antigenicity: The ability to bind to an antibody

Complete vs Incomplete Antigens

• Complete antigens can induce an immune response and bind to antibodies
• Incomplete antigens have antigenic determinants but cannot induce immune responses because they lack necessary attributes
• Haptens are examples of incomplete antigens

Properties of Antigens

• Foreignness
• Specificity
• High molecular weight, ideally > 750 Da
• Structural stability
• Degradability
• Route of administration
• Host genetics
• Dosage

Antigenic Epitopes

• Epitopes (antigenic determinants) are portions of antigen molecules that physically interact with paratopes on immune response molecules
• Epitopes determine antigen specificity

Types of Epitopes

Linear Epitopes

• Linear epitopes are continuous and found in polysaccharides as well as native and denatured proteins
• Specificity relies on the primary sequence
• Typical length is 5-6 subunits

Conformational Epitopes

• Conformational epitopes are discontinuous, involving multiple subunits far apart in the primary sequence
• They are only found in native globular proteins
• Specificity depends on conformation (3D shape from tertiary and quaternary structures)
• Sequences of up to 16 amino acids in certain protein antigens have been shown to interact with paratope

B Cell and T Cell Epitopes

• B cell epitopes are portions of antigen molecules recognized by B cell receptors
• T cell epitopes are regions of antigen molecules recognized by T cell receptors

Classification of Antigens

• Thymus-dependent antigens (TD-Ag)
• Thymus-independent antigens (TI-Ag)

Superantigens

• Superantigens are potent T lymphocyte mitogens that bind to class II MHC molecules
• They are associated with staphylococcal products, potentially causing enterotoxemias and toxic shock syndrome
• Superantigens (SAgs) are secreted proteins (exotoxins) with high mitogenic activity towards T lymphocytes
• SAgs activate up to 20% of the body's T-cells, compared to 0.001-0.0001% in a normal antigen-induced response
• This causes a non-specific massive immune response

Mitogens

• Mitogens induce mitosis
• Mitogens activate T and/or B cells without help from APCs
• Examples include Lectin (concanavalin A), LPS (lipopolysaccharide), and Staphylococcal protein A (SPA)

Adjuvants

• Adjuvants, from Latin "adjuvans" meaning "to help"
• Adjuvants enhance the pharmacological effect of drugs or increase an antigen's ability to stimulate the immune system

Classification of Adjuvants

• Freund’s adjuvant: Complete Freund's adjuvant (CFA) and Incomplete Freund’s adjuvant (IFA)
• Liposome
• Inorganic compound
• Cytokine
• Biodegradable nanoparticles

Mechanisms of Adjuvants

• Prolonged persistence of immunogen molecules at the injection site
• Enhancement of co-stimulatory signals
• Induction of granuloma formation
• Stimulation of lymphocyte proliferation in a non-specific manner

Other Antigens

• Heterophilic antigen: Common antigen existing in humans, animals, and microbes; e.g., Fossman antigen
• Xenogenic antigen: from a different genus and generic; e.g., pathogenic antigen
• Allogenic antigen: Exists in different individuals; e.g., blood type antigens
• Autoantigen: A pathological term; e.g., sperm antigen
• Idiotypic antigen: An antibody molecule recognized as an antigen by the immune system

Antibodies

• Antibodies are also termed Immunoglobulins
• Antibodies are globulin (gamma) fractions of serum proteins
• Antibodies are generated by the immune system against specific foreign antigens
• Antibodies are a main component of Humoral Immunity (HI)
• Rodney Porter described the structure of antibody molecules and received the Nobel Prize in 1972

Immunoglobulin Classes

• IgM: μ (mu) heavy chains, κ (kappa) or λ (lambda) light chains
• IgG: γ (gamma) heavy chains, κ (kappa) or λ (lambda) light chains
• IgA: α (alpha) heavy chains, κ (kappa) or λ (lambda) light chains
• IgE: ε (epsilon) heavy chains, κ (kappa) or λ (lambda) light chains
• IgD: δ (delta) heavy chains, κ (kappa) or λ (lambda) light chains

Major Immunoglobulin Class Properties

• IgG: 160 kDa, 3% carbohydrate, γ electrophoretic mobility, 4 heavy chain domains, 4 subclasses, 21 day half life, 2 antigen binding valency
• IgM: 900 kDa, 12% carbohydrate, β electrophoretic mobility, 5 heavy chain domains, no subclasses, 5 day half life, 5(10) antigen binding valency
• IgA: 360 kDa, 7% carbohydrate, β-γ electrophoretic mobility, 4 heavy chain domains, 2 subclasses, 6 day half life, 2,4 antigen binding valency
• IgE: 200 kDa, 12% carbohydrate, β-γ electrophoretic mobility, 5 heavy chain domains, no subclasses, 2 day half life, 2 antigen binding valency
• IgD: 160-180 kDa, 12% carbohydrate, γ electrophoretic mobility, 4 heavy chain domains, no subclasses, 3 day half life, 2 antigen binding valency

Immunoglobulin Structure

• Each light chain contains about 214 amino acids in two domains
• Heavy chains of IgG contain about 445 amino acids in 4 domains
• The variation in amino acid sequence is largely restricted to three smaller regions within the entire variable region, these regions are known as hypervariable regions
• Between the hypervariable regions, the amino acid sequences are relatively constant and termed as framework regions
• The hinge region consists of about 12 amino acids located between CH1 and CH2 domains, is rich in hydrophilic and proline residues making the region flexible and accessible to proteolytic enzymes and contains all the interchain disulfide bonds

IgG Properties

• IgG has a molecular weight of 160 kDa
• IgG is secreted as a secondary antibody
• IgG is the major secretory antibody in animals
• IgG is found in the highest concentration in blood
• IgG includes the subclasses IgG1, IgG2, IgG3, and IgG4

IgM Properties

• IgM is a pentamer
• IgM is the primary immunoglobulin
• IgM is available as a second-highest concentration in mammalian serum
• All monomers are bound by a cysteine-rich polypeptide called the J-chain (15 kDa) binding two of the units to complete the circle
• The heavy chain of IgM molecules contains an additional CH4 domain

IgA Properties

• IgA is a major secretory antibody of non-ruminants
• IgA produced on body surfaces can pass through epithelial cells into external secretions or diffuse into the bloodstream
• Most IgA in the intestinal wall is carried into the intestinal fluid
• IgA is transported through intestinal epithelial cells to polymeric Ig receptor (plgR), or secretory component- a 71 kDa receptor
• Secretory components bind covalently to IgA dimers forming secretory IgA (SIgA), protecting IgA from digestion by intestinal proteases

IgE and IgD Properties

• IgE is a monomer with an extra CH4 domain in its heavy chains
• IgE is largely responsible for immunity against parasitic worms and Type-I hypersensitivity reactions
• IgE molecules are generally found bound to the surface of mast cells and basophils
• IgD is primarily a B cell receptor
• IgD molecules don't have a CH2 domain; the CH1 and CH3 domains are separated by a long exposed hinge region
• Because it does not have disulfide bonds, IgD is highly susceptible to proteolytic enzymes

Immunoglobulin Variation

• Isotypes: Structural variations in Ig classes that occur in all animals of a species; also called classes
• Allotypes: Minor inherited sequence variations between proteins of different individuals
• Idiotopes: Unique structural variations in antigen-binding proteins such as Igs and TCRs, formed by variable regions on light and heavy chains; collectively known as isotypes

Affinity and Avidity

• Affinity: The tightness of antigen binding site attachment to an antigen determinant (epitope)
• Avidity: The tightness of binding when several antigen binding sites attach to several antigenic determinants

Mechanisms of Antibody Diversity

• Plasma cells make over a billion different types of antibodies
• Each cell contains 100,000 gene coding polypeptides
• Somatic recombination takes place to code for the many antibodies
• Gene segments are shuffled and recombined ways by B cells as these become immunocompetent
• Information of the newly assembled genes is expressed as B cell receptors and antibodies

Antibody Diversity

• Random mixing of gene segments creates unique antibody genes
• Genes encode for H and L chains and account for the variability in antibodies
• V gene segments, called hypervariable regions, mutate and increase antibody variation
• Plasma cells can switch H chains, making two or more classes with the same V region

Antibody Targets

• Antibodies themselves do not directly destroy antigens, they inactivate and tag for destruction
• Antibodies form antigen-antibody (immune) complexes
• Defensive mechanisms used by antibodies include neutralization, agglutination, precipitation, and complement fixation

Complement Fixation and Activation

• Complement fixation is the main mechanism used against cellular antigens
• Antibodies bound to cells change shape and expose complement binding sites
• This triggers complement fixation and cell lysis
• Complement activation enhances the inflammatory response, uses a positive feedback cycle to promote phagocytosis, and enlists more defensive elements

Other Mechanisms of Antibody Action

• Neutralization: Antibodies bind to and block specific sites on viruses or exotoxins
• Agglutination: Antibodies bind the same determinant on more than one antigen, forming cross-linked antigen-antibody complexes
• Cell-bound antigens are cross-linked, causing clumping (agglutination)
• Precipitation: Soluble molecules are cross-linked into large insoluble complexes