Lecture 3: Recognition of Antigen by B-cells and Antibodies

Basic classification of lymphocytes

• B-lymphocytes (B-Cells) → produce antibodies and attack extracellular pathogens - have BCRs

• T-lymphocytes (T-Cells) → attack intracellular pathogens - have TCRs

  • They have receptors on their surface that can recognise foreign molecules, e.g. those on pathogens (antigens)

Origin of B-Cells

• Originate and differentiate in the adult bone marrow

• Migrate to the lymphoid tissue such as spleen, and mainly the lymph nodes where they become specialised and form plasma B-cells

Origin of T-Cells

• Originate in bone marrow

• Differentiate to mature T-cells in the thymus before doing their function

• Migrate to the lymphoid tissue such as spleen, and mainly the lymph nodes

Adaptive Immune System

• It is much more specifc than innate immunity and is driven by

  • B cells = B cell receptor (BCR)

    • (membrane-bound antibody).

  • T cells = T cell receptor (TCR)

B-Cell Receptor (BCR)

• an antibody that is still bound to the membrane – Y-shaped with a membrane domain and adaptor proteins with intracellular domains that can signal the cell

• This is important for modulating gene expression, adhesion or survival.

• Its signalling determines the fate of antigen-encountered B cells.

  • Differentiate into plasma B cell

Fate of Antibodies Produced By B-Cell

• Antibodies are either:

  • (i) Attached to B-cell surface as B-cell receptors (BCRs)

  • (ii) Secreted into plasma (not attached)

• BCRs are essential for cell signalling and differentiation

• When a BCR binds an antigen:

  • The B-cell proliferates

  • Some cells become plasma cells that secrete antibodies

  • Others become memory B-cells

Main Function of Antibodies

• Bind foreign antigens encountered by the host

• Mediate effector functions to neutralise or eliminate foreign invaders

Structure of Antibody

• Antibody (Ab) = Immunoglobulin (Ig)

• Structure studied using proteases like papain

• Papain cleaves at disulphide bonds between heavy chains, producing:

  • Two Fab (Fragment antigen-binding) regions → bind antigen (N-terminal end)

  • One Fc (Fragment crystallisable) region → does not bind antigen, involved in immune functions (C-terminal end)

Antibody Structure: Heterodimers

• They have:

  • two identical light chains (L-chain) - smaller

  • two identical heavy chains (H-chain) – bigger

• Chains are bound together by disulfide bonds

Antibody Structure: Immunoglobulins

• They have immunoglobulin domains that give rise to the final structure of the Ab

• They have a hinge region that allows flexibility in movement to allow binding to antigens

• The antigen binding site of Abs is located at the N-terminus

Antigen

• A (general) molecule recognised by an antibody – not PAMPs (they are shared by many pathogens)

• Usually big molecules (>4000Da)

• Proteins (most common)

  • Carbohydrates (Polysaccharides)

  • Lipids

  • DNA

Antigenic Epitope

• The parts of the antigen recognised by antibodies -small fragments that are recognised by the Ab

  • e.g. a virus is an antigen; the Ab recognises a small part of the virus

• Each antigen has hundreds → between 10¹¹ - 10²⁵

Antigenic Epitope of SARS-COV2

• Antibodies generated by infection/ vaccine bind to the spike protein of Sars and not the whole virus

Antigen-Antibody Interaction

• Antibody interacts with a specific binding site which is determined by the hypervariable regions of the antibody

  • antigen binding site must be complementary in shape to the antigenic epitope

• Hypervariable regions form the complementary determining region (CDR), allowing for specific recognition

• CDR regions undergo gene rearrangement, enabling the production of diverse antibodies to recognise many epitopes

• Binding requires shape complementarity between the CDR (Ab) and epitope – they must fit well together to have an effect

  • it is specific

Variable vs Constat Regions of Antibodies

• Variable regions are located at the N-terminal end (last domain)

  • They vary between antibodies and determine antigen specificity

• Constant (C) domains remain unchanged across antibodies

  • Provide structural support and effector function

• Each antibody has a unique antigen-binding site shape due to its variable region

Conformational Epitopes

• Antigenic epitopes with a 3D shape

• Structural, formed by protein folding but may not be a continuous sequence of amino acids.

• Most antibodies recognise these

Linear Epitopes

• Recognise amino acids in sequence (2D) → Continuous sequence of amino acids.

• Rare in nature; seen when proteins are denatured (e.g. Western blots)

  • Less common.

Antibody Recognition of Antigens & Epitopes

• Different antigens/pathogens can share similar surface structures (epitopes), allowing the same antibody to bind to multiple antigens.

• An antibody may recognise antigens 1, 2, and 3 if they all share a common epitope.

• Some antibodies are specific to unique epitopes on a single antigen.

• During infection, the immune system produces many different antibodies, each targeting different epitopes on the antigen.

Antigen-Antibody Interactions: Affinity and Avidity

• How strongly an antibody binds to an antigen – want it to bind as strongly as possible so that it is bound for as long as possible to induce the effector function

Non-Covalent Forces

• Weak forces that are involved in antigen-antibody binding

  • Hydrogen bonding

  • Electrostatic interaction

  • Van der Waal forces

  • Hydrophobic interactions

• The antigenic epitope must be very close to the Ab to bind and maximise these interactions

Antigen-Antibody Interactions: Specificity

• Different antibodies bind different antigenic epitopes

Affinity

• The strength of the binding between an epitope and an antibody-antigen binding site.

  • Interactions are weak unless the two binding molecules are close together

  i.e. need a good fit between antigenic epitope & antibody binding site 

Cross-Immunity

• When an antibody to one antigen provides protection against another.

• This can happen in two ways:

  1. The antibody binds specifically to the same epitope found on different antigens.

  2. The antibody has weaker affinity but still binds sufficiently to similar epitopes.

• Even low-affinity binding to an epitope is mportant for cross-immunity.

Cross-Reactivity

• The same antibodies could bind different antigenic epitopes

• It is good as it can allow one antibody to recognise a variety of pathogens

  • Affinity for the cross-reacting epitope is less than for the original epitope

• It is problematic if an Ab recognises self-proteins via cross-reactivity and low affinity, which may result in an autoimmune response

Avidity

• The overall strength of an antibody-antigen complex

• If an antibody uses both binding sites to bind to 2 epitopes on the same particle this increases the total binding strength

Affinity vs Avidity

• Affinity refers to the strength of binding between a single antibody binding site and an epitope.

• Avidity is the overall strength of binding when multiple binding sites are involved.

  • High affinity, low avidity – antibody binds strongly to one epitope, but only one site is occupied.

  • High affinity, high avidity – antibody binds strongly and both binding sites are engaged.

How do antibody levels in the body compare to the amount needed for protection?

• Number of antibody specificities = 10¹¹

• Volume of blood = 5000 ml (5L)

• Protective antibody level = 10 ng/ml

• Amount of antibody required:

  • Calculation: 10¹¹ x 5000 ml x 10 ng = 5 x 10¹⁵ ng = 5000 kg

  • Comparison: This is far more than the 70 kg body weight!

• The body doesn't produce enough antibodies for constant protection. Antibodies are mainly generated after infection or vaccination, reaching levels that provide protection.

Antibody Production After an Infection

• First infection – initial immune response, innate immunity; after day 7 adaptive immunity; after 2 weeks recovery

• Following re-infection protective immunity present – little to no symptoms – peak of Ab production

• Overtime immunological memory is generated – provides long-term protection – sharper and higher peak of Ab production

Types of Antidbody Response

• Acquired – Abs produced  after exposure to the pathogen                

• Specific - for a specific pathogen – go through a process

• Memory -  2^nd response bigger and faster – longer protection

Key Features of Antibody Structure

• Constant domain: Provides structural stability.

• Variable domain: Responsible for antigen specificity.

• Antigen binding site: Located at the N-terminus of the antibody.

• Fc (crystallizable fragment): Allows the antibody to interact with pathogens or antigens. Known as the effector domain.

Humoral Immune Respnse

• Mediated by antibodies in serum

• Antibodies have many effector functions

  • Neutralisation

  • Opsoninatio

  • Activation of Complement

  • Antibody dependent cell-mediated cytotoxicity (ADCC)

Neutralisation

• Antibodies prevent pathogens or proteins from binding to their target.

• Mechanism: Antibodies bind to epitopes on pathogens, preventing them from adhering to surfaces or entering cells.

• Protection: This is crucial for preventing viral infections, as it stops the virus from entering cells and replicating.

• Vaccines: Many vaccines, such as COVID-19 vaccines, induce this response by preventing spike proteins from binding to cell receptors, thereby preventing infection.

Opsinosiation

• A pathogen tagged by antibodies is phagocytosed by phagocytic cells such as macrophages or neutrophils.

• Pathogen taken up by phagocytic cell and is placed in a phagolysosome where the pH is low and full of proteases that will destroy the pathogen

• The fc-domain of the antibody binds the Fc-receptor on the phagocyte to trigger phagocytosis – triggers the uptake of the pathogen into the phagocytic vesicle

Complement Activation

• Antibodies attached to the surface of a pathogen activate the first protein in the complement system.

• This can lead to two things:

  1. Complement proteins (C5b) deposit on the pathogen's surface, leading to pore formation, altering osmolality, with fluid entering the pathogen, and causing the pathogen to lyse (burst).

     1. mediated by Membrane attack complex

  2. Complement receptors on phagocytes recognise the deposited complement proteins, inducing phagocytosis. The antibody's Fc region binds to receptors on the phagocyte, triggering receptor oligomerization, which facilitates pathogen uptake into phagolysosomes for destruction.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

• Host cells infected by viruses can express viral proteins on their surface, which helps the virus evade detection.

• Antibodies specific to the virus recognise and bind to these viral proteins.

• NK cells (innate immune cells) and macrophages have Fc receptors (FcR) that can bind to the antibody bound to the infected cell.

• Upon binding, NK cells release perforin (a lytic enzyme) and granzymes that mediate extracellular killing and induce cell death by apoptosis, leading to the removal of infected cells without phagocytosis.

• This process is key for eliminating intracellular pathogens, especially viruses, by indirectly targeting infected cells for destruction.

Immunotherapies

• Can generate antibodies against certain abnormal proteins expressed by cancer cells, triggering the toxicity-dependent effects to kill specific target cells

• This can be done by directly triggering the tumour cell to be coated with the Ab, ADCC/NK cell response, with it binding to the receptor of the Ab generated to target these cells, which may induce phagocytosis

Agglutination- Blood Typing:

• Ability of an antibody to immunoprecipitate a soluble or non-soluble antigen.

  • Agglutination occurs where blood cells precipitate and break – used to identify blood types

• A and B transferases are encoded by the ABO gene 

• Blood groups: A, B, O

• Group A express the A transferase antigen; Anti-B Abs in serum – if receive type B blood – attack B-transferase as if it was a threat

Blood Group Genotypes:

• OO:    O

• AA, AO: A

• BB, BO:            B

• AB:     AB

Antibody Classes

• They are determined by different C regions in the heavy chain of the antibody.

  • There are 5 types

• Each has unique structural and functional characteristics that allow them to perform specific functions.

• Functions:

  • Bind to Fc receptors.

  • Activate complement.

  • Regulate secretion.

• An antibody can be of a different class but still have the same antigen-binding specificity.

• Different antibody classes are identified based on their Fc receptor.

IgG Antibody

• The most abundant

• 4 classes

• Ig_1 IS THE MOST COMMON

IgM Antibody

• Earliest antibody made after antigen contact

• Pentamer via J chain and disulphide bonds

  • Up to 10 binding sites

IgD Antibody

• Rare

  • function in serum is unclear

IgA1 Antibody

• Serum and major Ig in Mucosa

• 2 subclasses

• Dimeric structures in secretions → 2 Ig molecules joined together by the J chain

IgE Antibody

• Lowest present in serum

  • Bind to mast cells

  • Important in Allergy and worm infection

IgM Polymer

• Forms a pentamer with 5 Ig_’s binding through disulfide bonds and a J-chain (15kDa peptide chain)

• It is able to bind an epitope more strongly due to the formation of a complex that is able to bind more domains

IgA Polymer

• Forms a dimeric structure with the assistance of the J-chain, which is key for its function

Immunoglobulin A

• Produced by B cells in the bone marrow and then moves to secondary lymphoid tissues.

• Another major site of production is Mucosal Associated Lymphoid Tissue (MALT), such as in the gut.

  • ~approximately 5g/day produced

• Predominantly found in external secretions like saliva, tears, and mucus.

  • It serves an important role at membrane surfaces, which are the main entry points for pathogens.

  • Helps prevent pathogen entry by acting as a barrier.

• a dimer in secretions, joined by a J chain.

IgA Entry Into The Gut

• IgA antibodies form a dimer.

• A molecule called the poly Ig receptor is found on the basolateral side of intestinal epithelial cells.

  • This receptor attaches to IgA, and is internalized into the cell.

  • It is then transported to the lumen of the gut.

Secretory Component

• Part of the poly Ig receptor is cleaved, and the remaining portion becomes the secretory component (SC).

• SC protects IgA from being destroyed by gut enzymes.

• SC allows IgA to be transported into the gut.