PHAR 504: Antigens and Antigenicity (Lectures 2 & 3)
Antigens (Ags) and Antigenicity
Introduction to Antigens
Relevance: This knowledge is fundamental to understanding:
Vaccines
Antibodies
Immune-mediated diseases
Drug Design: Knowledge of epitopes is crucial for developing therapeutic antibodies and other biologics targeting specific antigens.
Definition of an Antigen (Ag):
Typically, a foreign substance that can be specifically bound by an antibody (Ig) or a B-cell receptor (BCR).
Generally triggers an immune response in the body.
Ideally, antigens should be non-self (foreign).
Recognition of self-antigens (autoantigens) is problematic and can lead to autoimmune diseases.
Neoantigens: Mutant proteins not encoded by the human genome.
Produced by cancer cells.
May be leveraged for tumor control (e.g., in cancer immunotherapies).
Epitopes / Antigenic Determinants
Definition: The specific part of an Ag that stimulates an immune response and is recognized by immune components.
Specifically recognized by Immunoglobulins (Igs), B cell receptors (BCRs), or T cell receptors (TCRs).
Typically small regions on the surface of an antigen, often consisting of a few amino acids or sugar residues (due to glycosylation).
Myoglobin Example: Illustrates that not all parts of a protein are antigenic; specific regions (epitopes) are recognized.
A single antigen can possess multiple distinct epitopes on its surface, each capable of interacting with different B cell receptors or antibodies.
Types of Epitopes:
Linear Epitopes (Continuous):
Composed of amino acids that are contiguous (in a continuous sequence) in the primary amino acid sequence.
Antibodies will recognize a denatured (i.e., linearized) protein because the epitope's structure is maintained even after denaturation.
Conformational Epitopes (Discontinuous):
Composed of amino acids that are NOT contiguous in the primary amino acid sequence.
Their structure depends on the protein's 3D folding, bringing distant amino acids into proximity.
Antibodies will NOT recognize a denatured (linearized) protein because the specific 3D arrangement of amino acids recognized by the antibodies no longer exists.
Relevance in Pharmacy: Most antibodies (approximately $90\%$ ) recognize conformational epitopes, as these more accurately represent the antigen's native state in the body, which is crucial for drug development, vaccines, and diagnostic tools.
Protein Folding and Conformational Epitopes:
Proteins have several levels of structure (primary, secondary, tertiary, quaternary).
Tertiary ($3^ ext{o}$) and Quaternary ($4^ ext{o}$) structures are referred to as the “native” state of the protein.
Protein structure directly influences antigenicity and the identity of epitopes.
This is critically important in immunizations because antigens on an infectious pathogen will be in their native, folded state.
Characteristics of a Good Antigen
"Foreignness": Must be distinguishable from host (self) molecules to elicit an effective immune response without autoimmunity.
Size: Larger molecules generally tend to be better antigens.
Chemical Complexity: Good antigens have a complex chemical structure and heterogeneity (composed of different components rather than a single, simple structure).
Example: Proteins and protein complexes are typically excellent antigens due to their diverse amino acid composition and complex folding.
Solubility: While not always strictly necessary, soluble antigens are often more easily recognized and processed by the immune system, leading to better presentation.
Degradability: The antigen must be processable by antigen-presenting cells (APCs). This allows for effective fragmentation and presentation of antigenic peptides to T cells.
Antigenic Complexity Impact on Antigenicity
Proteins: Very good antigens.
Composed of different amino acids, providing high potential for chemical diversity.
Can have many different epitopes on a single protein.
Polysaccharides:
Complex polysaccharides with considerable chemical diversity can be good antigens.
Simple polysaccharides with little chemical diversity are usually poor antigens.
Lipids and Nucleic Acids: Generally have little chemical diversity and are relatively poor antigens.
Haptens: Small molecules that, by themselves, cannot elicit an immune response. They only become immunogenic when attached to a large carrier molecule, such as a protein.
SARS-CoV-2 Spike Protein: An Example of Antigens and Epitopes in Action
The Spike (S) protein is crucial for viral entry into host cells by binding to the hACE-2 receptor.
Receptor Binding Domain (RBD): A key region within the S1 subunit of the spike protein that directly interacts with the host receptor.
Therapeutic Antibodies: Monoclonal antibodies (mAbs) like Imdevimab, Casirivimab, Etesevimab, and Bamlanivimab target the RBD to prevent viral binding and/or fusion with host cells.
Glycosylation: The SARS-CoV-2 Spike Protein is heavily glycosylated (modified with oligosaccharides).
While proteins are good antigens, oligosaccharides generally have poor antigenic potential relative to proteins.
Glycosylation can significantly impact the recognition of the spike protein by host antibodies and therapeutic mAbs, potentially shielding epitopes and affecting immune evasion or therapeutic efficacy.
Antigen Processing and Presentation
Antigen Processing: Before an antigen can be presented to T cells, it must be processed. This involves transforming proteins into smaller antigenic peptides.
Presentation: Processed antigens are presented on the cell surface via MHC (Major Histocompatibility Complex) proteins.
MHC Class I: Presents endogenous antigens (from within the cell, e.g., viral proteins, cancer proteins) to killer T-cells.
MHC Class II: Presents exogenous antigens (from outside the cell, e.g., bacteria) to helper T-cells.
T-cell Recognition: T-cell receptors (TCRs) bind specifically to the presented antigenic peptides.
Co-receptors: and molecules bind to corresponding MHC molecules, stabilizing the interaction and contributing to T-cell activation.
Antigen-Antibody (Ag-Ig) Interactions
This knowledge is essential for understanding various aspects of pharmacology, immunology, and diagnostic testing.
Immunoglobulin (Ig) Structure Review
Basic Structure: Composed of two identical Heavy Chains and two identical Light Chains, joined by disulfide bonds.
Regions:
Fab (Fragment Antigen Binding): The "arms" of the antibody, responsible for antigen binding.
Each Ig has two identical Fab regions.
Contains the Ag-Binding Site.
Fc (Fragment Constant): The "stem" of the antibody, mediates effector functions (e.g., binding to immune cells, complement activation).
Complementarity Determining Regions (CDRs):
Also known as Hypervariable Regions.
These are specific regions within the variable domains of both heavy and light chains.
CDRs physically interact with epitopes on an antigen.
Their unique structure determines the antibody's specificity for a particular epitope.
Antigen Binding: An Ig has two identical Ag-binding sites, allowing it to bind up to two molecules of the SAME antigen (if they present the same epitope).
Forces Mediating Ag-Ig Interactions
Ag-Ig interactions are noncovalent.
Relative Strength of Intermolecular Interactions (Increasing Strength):
Van der Waals (London forces)
Dipole-Dipole Interactions
Hydrogen Bonding
Ionic Bonding
Specific Noncovalent Forces in Ag-Ig Interactions:
Hydrogen Bonding: Interactions between a hydrogen atom covalently bound to an electronegative atom and another electronegative atom.
Ionic Bonds (Salt Bridges): Formed between oppositely charged amino acid side chains (e.g., amine groups and carboxyl groups).
Hydrophobic Interactions: Occur between nonpolar side chains, leading to the exclusion of water molecules from the interaction site. Often contributes significantly to binding stability.
Van der Waals Forces (London Dispersion Forces, "packing"): Weak, short-range attractive forces that arise from temporary fluctuations in electron distribution, creating transient dipoles. These are maximized with close molecular fit.
Ag-Binding Properties of an Ig
Specificity: The ability of an antibody to specifically bind to a single epitope, attributed to the unique structure of its CDRs.
Affinity: The intrinsic strength of the interaction between a single Ag-binding site on an antibody and its corresponding epitope on an antigen.
Affinity varies widely among different antibodies and antigens.
Factors influencing affinity: pH, temperature, and solvent conditions can affect the strength of these interactions.
High-affinity Ig-Ag interactions: Characterized by a close fit with minimal space between molecules. This close fit maximizes opportunities for multiple intermolecular interactions (ionic, H-bonds, hydrophobic, Van der Waals).
Result in stronger, longer-lasting, and more specific binding.
Can withstand a wider range of pH and higher temperatures.
Poor-affinity interactions: Result from a less optimal fit, leading to fewer intermolecular interactions, large gaps between molecules, and potential steric clashes (where molecules are too close and repel).
Avidity: The overall strength of binding between an antibody and an antigen, considering all binding sites. It reflects the cumulative strength of multiple affinities.
Influenced by the affinity of the antibody for its antigen AND the valency of both the antibody and the antigen.
Avidity is generally higher when multiple binding sites are engaged simultaneously (e.g., a bivalent antibody binding to two epitopes on an antigen).
Valency: Determines the number of Ag or Ig molecules that can be bound.
All Igs are multivalent (e.g., IgG is bivalent, IgM is decavalent if all sites are accessible).
Antigens are multivalent if they present multiple copies of the same epitope.
Cross-Reactivity: Occurs when an antibody directed against one antigen (AgA) successfully binds with another, different antigen (AgB).
This happens when AgB shares structural similarities (e.g., a common epitope or very similar epitopes) with AgA.
Example: An antibody generated against epitope Y on AgA might also interact with a similar epitope on AgB, even if AgB lacks other epitopes found on AgA (X, Z).
Implications:
Can be a source of false positives in Ig-based assays (e.g., a diagnostic test might indicate the presence of one pathogen when another, cross-reactive pathogen is present).
Bad for therapeutic antibodies: An antibody intended to target a specific antigen (e.g., on a cancer cell) would be a poor choice if it cross-reacts with an antigen on healthy tissues, potentially causing off-target side effects.
Immune Reactions to Antigens
Immunogenicity: The capability of an antigen to induce (generate) an immune response.
Distinction from Antigenicity: An antigen is anything that can bind specifically to immune system components (Ig, BCR, TCR), but it doesn't necessarily cause an immune response. An immunogen causes an immune response.
Significance for Biologics: Critical in the context of biotherapeutic drugs, which are protein-based therapies.
The immune system can develop antidrug antibodies (ADAs) against these biologics.
ADAs may reduce the efficacy of the treatment, prolong treatment duration, or cause adverse reactions.
Clinical management of immunogenicity involves ongoing monitoring and adapting treatment plans to minimize adverse immune responses and ensure drug efficacy.
Superantigens: Certain bacterial toxins (e.g., staphylococcal enterotoxin) that cause a drastic over-response by the immune system.
Mechanism: Rather than being processed into smaller fragments and presented in the peptide-binding groove, superantigens directly bind to conserved regions of MHC Class II receptors outside the peptide-binding groove.
They then crosslink these MHC Class II molecules to T cell receptors (TCRs) on multiple T cells.
Superantigens can also bind to parts of the variable heavy (VH) domain on antibodies and stimulate B cells nonspecifically.
Indiscriminate Activation: Their binding does not depend on the specificity of the receptors, leading to massive, indiscriminate activation of T cells (activating $5\% - 20\%$ of the total T cell population, as opposed to $<0.01\%$ for typical antigen presentation).
Clinical Implications: Implicated in severe conditions such as toxic shock syndrome and rheumatic fever due to the cytokine storm caused by widespread immune cell activation.
Adjuvants and Immunogenicity:
Adjuvant: A substance that enhances the immune response to an antigen when mixed with it, but does not itself cause an immune response.
Use in Vaccines: Often used in vaccines to create a stronger and longer-lasting immunity, which is crucial for effective vaccination.
Mechanisms of Action (Differ between adjuvants):
Activating innate immunity directly.
Causing tissue damage and the release of DAMPs (Damage-Associated Molecular Patterns), which alert the immune system.
Trapping the antigen at the injection site, allowing for prolonged exposure to immune cells.
Historical Note: The first adjuvant (insoluble aluminum salts, or "alum") was licensed in the 1920s. Since then, only a limited number of additional adjuvants have been licensed.
Ag-Ig Formation of Visible Aggregates
These interactions are foundational to many diagnostic procedures and therapeutic monitoring techniques.
Antibody Hinge Region:
Provides flexibility to the antibody structure, allowing the "arms" (Fab regions) to rotate and spread.
This flexibility is crucial for:
Effectively "coating" a single pathogen or cell surface with multiple epitopes.
Crosslinking two separate antigen particles or cells (e.g., in agglutination).
Allowing both arms to bind to surfaces presenting more than one epitope, enhancing avidity.
Types of Visible Aggregate Formation:
Precipitation:
Occurs when soluble antigen (e.g., protein, peptide, oligosaccharide) binds with antibody.
Forms a cross-linked latticework of alternating antibody and antigen molecules.
Applications: Used in serological tests to detect and quantify immunoglobulins and other soluble proteins.
Agglutination:
Occurs when insoluble particulate antigen (e.g., cell, virus, bacterium, or antigen-coated latex beads) binds with antibody.
Crosslinking of these particles by antibodies leads to visible clumping (agglutination).
Applications: Widely used in diagnostic tests, such as blood typing (detecting surface antigens on red blood cells) and the detection of pathogens.
Example: Hemagglutination assays detect viruses or bacteria by observing the clumping of red blood cells.
Ig-based Reactions and Assays: Lab and Home Tests
Indirect Coombs Test (Direct Antiglobulin Test):
Used for transfusion compatibility testing, specifically to detect anti-Rh Ig (antibodies against Rh antigen) in a patient's serum.
Procedure:
Known Rh-positive red blood cells ($Rh^+$ RBCs) are incubated with patient serum.
If anti-Rh Igs are present in the patient's serum, they will bind to the Rh antigen on the surface of the RBCs.
After washing, anti-human IgG (an antibody that binds to human IgG) is added.
If patient Ig is bound to the RBCs, the anti-human IgG will crosslink the RBCs, resulting in a visible agglutination (clumping) as a positive readout.
Lateral Flow Assay (LFA) / Immunochromatographic Assay:
A rapid, simple, and inexpensive diagnostic tool.
Principle: Detects the presence of a target analyte (antigen or antibody) in a liquid sample as the sample flows along a test strip via capillary action. The analyte interacts with reagents (e.g., labeled antibodies) to produce a visible signal.
Advantages: Portable, no specialized equipment required, can be stored at room temperature, making them suitable for point-of-care testing and use in resource-limited settings.
Applications: Used in various fields, including:
Medical Diagnostics: Pregnancy tests, COVID-$19$ antigen tests, rapid strep tests.
Veterinary Medicine.
Food Safety and Environmental Monitoring.
"Sandwich" LFA: Typically used for large analytes that possess multiple epitopes, allowing one antibody to capture the analyte and another (labeled) antibody to bind to a different epitope on the same analyte, forming a "sandwich" complex that generates a signal.