Unit 2: Antigens; Immunoglobulins (Antibodies) - Subjective Questions

An antigen is any substance (usually a foreign molecule) that can be specifically recognized and bound by the products of an immune response, namely antibodies or T-cell receptors.

Essential properties of an antigen:

• Antigenicity (Reactivity): The ability of a molecule to react specifically with the antibodies or sensitized T-cells it has induced.
• Immunogenicity: The ability to induce a humoral and/or cell-mediated immune response. (Not all antigens are immunogenic, e.g., haptens.)

Key characteristics:

• Antigens are usually macromolecules such as proteins, polysaccharides, lipids, or nucleic acids.
• They possess specific regions called epitopes (antigenic determinants) that are recognized by the immune system.
• They are generally foreign (non-self) to the host, though autoantigens can trigger autoimmune responses.

Thus, every immunogen is an antigen, but not every antigen is an immunogen.

Although the terms are often used interchangeably, there is an important conceptual distinction between the two.

Key points:

• Every immunogen is an antigen, because it can both induce and react with antibodies.
• Not every antigen is an immunogen, because some antigens (like haptens) can only react with antibodies but cannot induce a response on their own.
• A hapten becomes immunogenic only when coupled to a large carrier protein.

Thus, immunogenicity refers to the capacity to provoke a response, while antigenicity refers to the capacity to react with the response products.

The immunogenicity of a substance depends on both the intrinsic properties of the molecule and the biological system it enters.

A. Properties of the Immunogen:

• Foreignness: The immune system responds to molecules recognized as non-self. The greater the phylogenetic distance between the source of the antigen and the host, the stronger the response.
• Molecular Size: Larger molecules are more immunogenic. The most potent immunogens have a molecular weight Da. Molecules below – Da are usually weakly immunogenic.
• Chemical Composition and Complexity: Complex molecules with diverse building blocks are more immunogenic. Proteins are the best immunogens, followed by polysaccharides. Lipids and nucleic acids are poor immunogens alone.
• Molecular Structure & Heterogeneity: Chemical complexity, aromatic amino acids, and structural diversity increase immunogenicity.
• Degradability (Susceptibility to Antigen Processing): Molecules that can be processed and presented by APCs (antigen-presenting cells) via MHC molecules are more immunogenic.

B. Biological System (Host) Factors:

• Genotype of the recipient: MHC genes influence the ability to respond to a given antigen.
• Dose of antigen: An optimal dose is required; too low or too high a dose may cause tolerance.
• Route of administration: Subcutaneous > intramuscular > intravenous in general effectiveness.
• Use of adjuvants: Substances that enhance the immune response to an antigen.

Thus, immunogenicity is a combined function of the molecule's chemistry and the host's biological context.

Epitopes, also called antigenic determinants, are the specific small regions on an antigen molecule that are recognized by and bind to antibodies, B-cell receptors, or T-cell receptors. A single antigen may possess many different epitopes.

Types of Epitopes:

Additional Points:

• B-cell epitopes are often conformational and located on the surface of antigens.
• T-cell epitopes are always linear peptides presented by MHC molecules after antigen processing.
• The portion of the antibody that binds the epitope is called the paratope.

A hapten is a small molecule (low molecular weight) that is antigenic but not immunogenic by itself. It can react with specific antibodies but cannot induce an immune response on its own because of its small size.

Hapten-Carrier Concept:

• When a hapten is chemically coupled to a large carrier protein (e.g., bovine serum albumin, keyhole limpet hemocyanin), the conjugate becomes immunogenic.
• The carrier provides the size and T-cell epitopes required to stimulate helper T-cells.
• The immune response then produces antibodies against:
  • The hapten alone
  • The carrier alone
  • The hapten-carrier junction

Landsteiner's Experiments:

• Karl Landsteiner used small chemical groups such as dinitrophenol (DNP) and aminobenzene derivatives as haptens.
• He demonstrated that the specificity of the antibody response is determined by the chemical structure of the hapten, including subtle differences in position of functional groups (ortho, meta, para isomers).
• These studies established the fine chemical specificity of antigen-antibody interactions.

Examples of haptens: penicillin (can cause allergy after binding host proteins), drugs, and small chemical groups like DNP.

An immunoglobulin (antibody) is a Y-shaped glycoprotein composed of four polypeptide chains held together by disulfide bonds.

Basic Structural Components:

• Four polypeptide chains:
  • Two identical Heavy (H) chains (~50–70 kDa each)
  • Two identical Light (L) chains (~25 kDa each)
• The chains are linked by inter-chain disulfide bonds and intra-chain disulfide bonds.

Regions of the molecule:

• Variable region ( and ): The N-terminal ends that vary greatly and form the antigen-binding site.
• Constant region ( and ): Relatively conserved regions that determine effector functions and class.
• Hypervariable regions / Complementarity-Determining Regions (CDRs): Three per variable domain, forming the actual antigen contact.

Functional Fragments (papain digestion):

• Fab (Fragment antigen-binding): Two fragments, each with one antigen-binding site.
• Fc (Fragment crystallizable): Mediates effector functions such as complement fixation and binding to Fc receptors.

Hinge region: A flexible region between and that allows the arms to move.

Light chain types: Either kappa () or lambda ().

The two identical antigen-binding sites make a basic monomeric antibody bivalent.

Immunoglobulins are classified into five major classes (isotypes) based on the type of heavy chain they possess.

Summary:

• IgG – long-term immunity and neonatal protection.
• IgM – early defense.
• IgA – mucosal/secretory protection.
• IgE – allergy and antiparasitic defense.
• IgD – B-cell activation marker.

IgG is the most abundant immunoglobulin in serum, accounting for about 70–75% of total serum antibodies.

Structure:

• Monomeric structure with two heavy chains and two light chains.
• Molecular weight approximately 150 kDa.
• Four subclasses in humans: IgG1, IgG2, IgG3, and IgG4, differing in hinge region and disulfide bonds.

Functions:

• Neutralization: Neutralizes toxins and viruses.
• Opsonization: Coats pathogens to enhance phagocytosis via Fc receptors.
• Complement Activation: Activates the classical complement pathway (IgG1, IgG3 most effective).
• Antibody-Dependent Cell-mediated Cytotoxicity (ADCC): Facilitates killing of target cells by NK cells.
• Placental Transfer: The only immunoglobulin that crosses the placenta, providing passive immunity to the fetus/newborn.
• Secondary Immune Response: Predominant antibody in the memory/secondary response, produced in large amounts.

Why most versatile:

• Long half-life (~23 days).
• Participates in nearly all antibody-mediated functions.
• Provides both immediate and long-lasting protection.

These combined features make IgG the workhorse of humoral immunity.

IgM is the largest immunoglobulin and the first antibody to appear during an immune response.

Structure:

• Exists mainly as a pentamer in serum (five monomeric units).
• The five units are joined by a J (joining) chain and disulfide bonds.
• Each monomer has two heavy chains and two light chains.
• The pentamer has 10 antigen-binding sites (theoretical valency 10, effective valency ~5 for large antigens).
• Molecular weight approximately 900–970 kDa.
• A monomeric membrane-bound form serves as the B-cell receptor (BCR).

Biological Significance:

• First antibody produced in the primary immune response and in the developing fetus.
• Excellent agglutinator due to its high valency.
• Most efficient activator of the classical complement pathway (a single IgM can initiate complement fixation).
• Confined mainly to the bloodstream because of its large size.

First line of defence:

• Because IgM appears earliest and is a potent agglutinator and complement activator, it provides rapid early protection before high-affinity IgG is produced. Hence it is termed the first line of humoral defence.

Detection of IgM against a pathogen usually indicates a recent or acute infection.

IgA is the second most abundant immunoglobulin in serum and the predominant antibody in secretions.

Structure and Forms:

• Serum IgA: Exists mainly as a monomer.
• Secretory IgA (sIgA): Exists as a dimer joined by a J chain and associated with a secretory component.
• Heavy chain is of the alpha () type.
• Two subclasses: IgA1 and IgA2.

Secretory Component:

• Derived from the poly-Ig receptor on epithelial cells.
• Protects sIgA from proteolytic degradation in the harsh environment of mucosal secretions.

Roles in Mucosal Immunity:

• Found in saliva, tears, colostrum, breast milk, nasal secretions, and the gut and respiratory mucosa.
• Neutralizes pathogens and toxins at mucosal surfaces before they enter the body.
• Prevents adherence of microbes to epithelial cells (immune exclusion).
• Provides passive immunity to newborns through breast milk (colostrum).

Significance:

• Acts as the first line of defence at mucosal surfaces, which are the main entry points for pathogens.
• Does not activate complement strongly (non-inflammatory), suiting its role at delicate mucosal surfaces.

IgE is present in the lowest concentration in serum but plays a crucial role in hypersensitivity and antiparasitic immunity.

Structure:

• Monomeric immunoglobulin with epsilon () heavy chains.
• Has an extra constant domain compared to IgG.
• Binds with high affinity to Fc receptors on mast cells and basophils.

Role in Allergic (Type I Hypersensitivity) Reactions:

• On first exposure to an allergen, IgE is produced and binds to mast cells (sensitization).
• On re-exposure, the allergen cross-links the bound IgE.
• This triggers degranulation and release of mediators such as histamine, leukotrienes, and prostaglandins.
• Results in allergic symptoms: hay fever, asthma, urticaria, and anaphylaxis.

Role in Antiparasitic Immunity:

• IgE coats helminth (worm) parasites.
• Eosinophils bind to the Fc region of IgE via Fc receptors and release toxic granule contents.
• This antibody-dependent cell-mediated cytotoxicity (ADCC) kills the parasites.

Summary:

• IgE is a double-edged sword: protective against parasites but responsible for allergic diseases.

The immune response to an antigen differs greatly between the first and subsequent exposures.

Key Points:

• The rapid and stronger secondary response is due to immunological memory provided by memory B and T cells.
• Affinity maturation through somatic hypermutation increases antibody quality in the secondary response.
• This is the principle underlying vaccination and booster doses.

Thus, the secondary response is faster, stronger, of higher affinity, and dominated by IgG.

Antigenic determinants are specific portions of a molecule that are recognized by the immune system. Immunoglobulins themselves possess antigenic determinants and can act as antigens, giving rise to three categories of variation.

1. Isotype:

• Antigenic determinants located in the constant regions of the heavy and light chains.
• These define the class and subclass (e.g., IgG, IgM) and are identical in all members of a species.
• Determined by the constant region genes.

2. Allotype:

• Antigenic determinants that vary between individuals of the same species.
• Result from allelic differences (polymorphisms) in the constant region genes.
• Inherited in a Mendelian fashion.

3. Idiotype:

• Antigenic determinants located in the variable regions (specifically the CDRs) of the antibody.
• Unique to each antibody's antigen-binding site.
• The set of idiotopes forming the idiotype defines the specificity of the antibody.

Summary Table:

The immune system can generate an enormous repertoire of antibodies (estimated specificities) from a limited number of genes. This is achieved by several mechanisms.

1. Multiple Germline Gene Segments (Combinatorial Diversity of Segments):

• Antibody genes are made up of separate segments: V (Variable), D (Diversity, heavy chain only), and J (Joining) segments.
• There are many copies of each segment in the germline.

2. V(D)J Recombination (Somatic Recombination):

• During B-cell development, one V, one D (for heavy chain), and one J segment are randomly joined.
• Mediated by RAG-1 and RAG-2 enzymes recognizing recombination signal sequences (RSS).
• Enormous combinations possible from random joining.

3. Combinatorial Association of Heavy and Light Chains:

• Any heavy chain can pair with any light chain, multiplying the diversity.

4. Junctional Diversity:

• Imprecise joining of segments and addition/removal of nucleotides.
• N-nucleotide addition by the enzyme Terminal deoxynucleotidyl Transferase (TdT) at the junctions.
• P-nucleotide additions also contribute.

5. Somatic Hypermutation:

• After antigen stimulation, point mutations are introduced into the variable region genes.
• Leads to affinity maturation, selecting B-cells producing higher-affinity antibodies.

Summary: Combinatorial diversity, junctional diversity, and somatic hypermutation together account for the vast diversity of antibodies.

V(D)J recombination is the somatic gene rearrangement process by which functional immunoglobulin genes are assembled from separate germline segments during B-cell development.

Gene Segments Involved:

• Heavy chain: V (Variable), D (Diversity), and J (Joining) segments.
• Light chain: Only V and J segments (no D segment).

Process:

1. For the heavy chain, a D segment first joins to a J segment, then a V segment joins to the DJ complex.
2. For the light chain, a V segment joins directly to a J segment.
3. This produces a complete variable region gene, which is then transcribed with the constant region.

Role of RAG Enzymes:

• RAG-1 and RAG-2 (Recombination Activating Genes) form a complex that initiates recombination.
• They recognize Recombination Signal Sequences (RSS) flanking each gene segment.
• RSS consist of conserved heptamer and nonamer sequences separated by 12- or 23-base pair spacers (the 12/23 rule ensures correct joining).
• The RAG complex introduces double-stranded DNA breaks at the RSS.
• The broken ends are then joined by the Non-Homologous End Joining (NHEJ) DNA repair machinery.

Significance:

• Random combination of segments creates combinatorial diversity.
• Imprecise joining and nucleotide addition (by TdT) create junctional diversity.

Mutations in RAG genes lead to Severe Combined Immunodeficiency (SCID) due to the absence of functional B and T cells.

Enzymatic digestion of an antibody molecule yields functional fragments that helped elucidate antibody structure and function.

Hinge Region:

• A flexible segment located between the and domains of the heavy chain.
• Rich in proline and cysteine residues.
• Provides flexibility, allowing the two Fab arms to move and adjust to bind epitopes at varying distances.
• Contains inter-chain disulfide bonds.

Papain Digestion:

• Cleaves the antibody above the hinge region into three fragments:
  • Two Fab fragments (Fragment antigen-binding): Each contains one light chain and part of a heavy chain (); retains one antigen-binding site (monovalent).
  • One Fc fragment (Fragment crystallizable): Contains the and domains; mediates effector functions (complement fixation, Fc receptor binding, placental transfer).

Pepsin Digestion:

• Cleaves below the hinge region, producing:
  • One F(ab') fragment: Two Fab units joined; bivalent and can still cross-link/agglutinate antigen.
  • The Fc portion is digested into small peptides.

Significance:

• Fab determines antigen specificity and binding.
• Fc determines biological/effector activity and antibody class.
• These fragments are used therapeutically (e.g., F(ab') in antivenoms to reduce immune reactions).

Antigens differ in whether they require helper T-cell assistance to elicit an antibody response by B-cells.

Explanation:

• TD antigens are processed and presented on MHC-II to helper T-cells, which then provide co-stimulatory signals (e.g., CD40-CD40L) and cytokines to B-cells, enabling class switching, affinity maturation, and memory.
• TI antigens have repeating epitopes that can directly cross-link B-cell receptors, activating B-cells without T-cell help, but generate mainly a short-lived IgM response.

This distinction is important in vaccine design (e.g., conjugate vaccines convert polysaccharide TI antigens into TD antigens by linking to a carrier protein).

Valency refers to the number of binding sites available for interaction.

Antigen Valency:

• The number of epitopes on an antigen that can be bound by antibodies simultaneously.
• Large complex antigens are multivalent (many epitopes).
• Haptens are typically univalent (one determinant).

Antibody Valency:

• The number of antigen-binding sites (paratopes) on an antibody.
• IgG, IgD, IgE: valency of 2 (bivalent).
• Secretory IgA (dimer): valency of 4.
• IgM (pentamer): theoretical valency of 10, effective valency ~5 for large antigens.

Influence on Interactions:

• Avidity: The overall strength of binding increases greatly with valency (multivalent binding = high avidity). Even if individual bonds (affinity) are weak, multiple simultaneous bonds create strong, stable binding.
• Agglutination and Precipitation: Multivalent antibodies (like IgM) can cross-link multivalent antigens to form large lattices, essential for agglutination and precipitation reactions.
• Lattice Formation: The zone of equivalence (optimal antigen:antibody ratio) produces maximum lattice/precipitate.

Relationship:

Thus, valency is a key determinant of the stability and functional efficiency of antigen-antibody complexes.

Class switching (isotype switching) is the process by which an activated B-cell changes the class of antibody it produces (e.g., from IgM to IgG, IgA, or IgE) without changing the antigen specificity.

Mechanism:

• The variable region (V(D)J) that determines antigen specificity remains unchanged.
• Only the heavy chain constant region () gene is switched.
• This occurs through Class Switch Recombination (CSR) at specific DNA sequences called switch (S) regions upstream of each constant gene.
• The enzyme Activation-Induced Cytidine Deaminase (AID) initiates CSR.
• Requires T-helper cell signals, particularly CD40-CD40L interaction and specific cytokines:
  • IL-4 → IgE, IgG1
  • IFN- → IgG2/3
  • TGF- → IgA

Biological Importance:

• Allows the immune system to produce antibodies with the same specificity but different effector functions suited to different situations.
• Example: IgG for opsonization and placental transfer, IgA for mucosal defence, IgE for antiparasitic/allergic responses.
• Contributes to a more effective and versatile immune response.

Note: Class switching occurs only in T-dependent responses and typically after the initial IgM response.

B-cells and T-cells recognize antigens differently, and hence the epitopes they respond to differ in nature.

Explanation:

• B-cell epitopes are recognized directly by antibodies on the surface of the intact antigen; therefore, surface-exposed and often conformational regions are important.
• T-cell epitopes are generated after antigen processing by APCs and are presented as short peptides bound to MHC class I (to CD8+ T-cells) or MHC class II (to CD4+ T-cells).

Understanding both types of epitopes is essential for vaccine and epitope-based therapy design.