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Transcript
Antigens and Antibody Structure Lecture 2 General Concepts An antigen is any agent capable of binding specifically to components of the immune system, such as the B cell receptor (BCR) and antibodies, or the T cell receptor (TCR). Typically elicit an immune response. Can be foreign or they can be self (autoantigen). Sources of antigens include bacteria, viruses, parasites, pollen, tissue and organ transplants, blood transfusion etc. Antigen vs Immunogen An antigen is any agent capable of binding specifically to components of the immune system such as the BCR or TCR An immunogen is any agent capable of inducing an immune response and is therefore immunogenic Antigenicity is the ability of an antigen to combine with the final products that arise from an immune response such as antibodies and cell surface receptors Immunogenicity is an antigens ability to elicit a humoral (antibody mediated) and or cell mediated immune response All immunogens are antigens but not all antigens are immunogens e.g. haptens Major classes of antigens Complex carbohydrates - components of bacterial cell walls, antigens determining ABO blood groups Lipids- immunogenic when coupled to large carrier proteins Nucleic acids - poor immunogens by themselves. Can be immunogenic when conjugated to protein carriers Proteins - Excellent immunogens Haptens - small antigenic substances. Immunogenic only when coupled to large carrier molecules Various properties of antigens that influence their immunogenicity Foreignness - the more foreign the antigen is to the body the more immunogenic High molecular weight and complexity - proteins of 100kD or more are potent immunogens, while 5kD or less are usually poor immunogens Susceptibility to processing and presentation - if a macromolecule cannot be ingested, processed and presented by APCs, it is a poor immunogen Chemical nature - proteins and polysaccharides make potent immunogens Further requirements for Immunogenicity Immunogen dosage: a. Each immunogen has a certain dose that may be required to elicit an immune response. b. Higher doses do not necessarily induce a stronger response. c. Repeated doses e.g. vaccine boosters may be needed to induce a sufficient immune response Further requirements for Immunogenicity Routes of administration - different routes carry antigens to different organs of the immune system Subcutaneous – Generally elicit the strongest immune responses. Due to their uptake, processing, and presentation to effector Langerhans cells present in the skin, which are among the most potent APCs. Responses take place in draining lymph nodes Intravenous – Antigens are carried first to the spleen Gastrointestinal – Antigens elicit local antibody responses within the intestine. Adjuvants An adjuvant is a substance that when mixed with an immunogen, enhances the immune response of the immunogen Examples: Aluminium hydroxide Aluminium phosphate (Alum) Freund’s complete adjuvant: (Immunogen + killed mycobacteria, oil and water) Adjuvant mechanisms include: Increasing the biological or immunological ½ life of vaccine antigens Increasing the production of local inflammatory cytokines Improving antigen delivery, processing and presentation by APCs Epitopes Epitope (antigenic determinant) –the part of an antigen to which an antibody binds or gives rise to the MHC-binding peptide that is recognized by a TCR. Epitopes for antibodies are exposed on the surface of antigens while T cell epitopes are usually located in the internal regions of the protein T cells do not recognize soluble native antigen but rather recognize antigen that has been processed into antigenic peptides and presented by MHC molecules of APCs. Paratope are small regions of antibodies which bind to epitopes Epitopes Antibody epitope T cell epitope B cell epitopes B cell epitope - can bind to an antigen-binding site on an antibody molecule Linear epitopes – the antibody binds parts of the molecule that are adjacent in the linear sequence (For example a string of amino acids that are in linear sequence in a protein). Conformational or discontinuous epitopes – an epitope that is formed by parts of a protein brought together in the folded protein, but which are separated in the amino acid sequence. B cell epitopes Multivalent antigen – any antigen that contains more than one epitope or more than one copy of an epitope. Antigens can bind in pockets, grooves, or extended surfaces in the binding sites of antibodies. Antigens that stimulate B cells can be T cell dependent or T cell independent T-dependent antigens requires the presence of helper T cells to stimulate antibody production by B cells. T-independent antigens are able to trigger B cells to produce antibodies without the presence of T cells. • • • • • T cell dependent Require T cell help Complex proteins Very common Induce IgG and all other isotypes Much stronger immune response • • • • T cell independent Do not require T cell help Simple polysaccharides Less common Induce IgM T cell dependent antigens • • • • • Require T cell help Complex proteins Very common Induce IgG and all other isotypes Much stronger immune response Antibodies Soluble proteins that recognize antigens (foreign or self) with high specificity. Because of their globular shape, they are called immunoglobulins Expressed as secreted or membrane bound forms. They are highly specific for an antigen General characteristics The function of an immunoglobulin is to: Bind pathogens Make the pathogen a target for cells and molecules of the immune system. General characteristics The variable (V) region (varies greatly from one antibody to the next) and is responsible for binding pathogens. The constant (C) region contains binding sites for receptors on phagocytes and complement proteins and is responsible for recruiting cells and molecules for pathogen destruction. General characteristics Igs are composed of 4 polypeptide chains. 2 identical H chains (heavy polypeptide chains) and 2 identical L chains (light polypeptide chains) Linked by disulfide bond(s) Two types of light chains found in Igs: Either 2 Lambda (λ) L chains or 2 kappa (κ) L chains Basic Antibody Structure Hinge region– flexible region of the antibody where all of the arms of the Y come together, allows the Ig to be flexible for binding to antigen and proteins that mediate immune responses. Fab - contains the antigen binding site. Obtained by papain digestion F(ab’)2 - obtained by pepsin digestion FC region–the portion of the constant region that forms the "stem" of the Y shaped Ig molecule. Hypervariable Regions Hypervariable regions (paratope) – 3 regions of high variability within the V domain, designated HV1, HV2, HV3. Framework regions – the regions of the V domain between the hypervariable domains which are relatively invariant. HV1, HV2, and HV3 are sometimes called complementarity determining regions (CDR1, CDR2, CDR3) because they form the antigen binding site. General characteristics There are 5 types (isotypes) of heavy chains 1) Mu (µ)- IgM 2) Delta (δ)- IgD 3) Gamma (γ)- IgG………(IgG1, IgG2, IgG3 and IgG4) 4) Alpha (α)- IgA ...............(IgA1 and IgA2) 5) Epsilon (ε)- IgE Antibodies of different isotypes have different functions Antibody classes or Isotypes The constant region of the H chain determines the Ig’s class or isotype. There are 5 main isotypes of Igs – IgA IgD IgE IgG IgM Some Ig classes are subdivided into subclasses that are encoded by different constant region genes that are similar in sequence. Subclasses are designated by a number after the isotype that identifies the relative abundance of the subclass within the main isotype, i.e. IgG1 is more abundant than IgG2 which is more abundant than IgG3 in serum. In humans there are two subclasses of IgA and 4 subclasses of IgG. Immunoglobulin Variants Isotype- Large differences in C regions that distinguish each Ig class and subclass. IgM, IgG1, IgG2 etc. Allotype- Small differences in C regions that are encoded by different alleles of isotypic genes (Allelic difference) IgG1a versus IgG1b These differences occur in some but not all members of a species Idiotype- Small differences in V regions within an individual Isotype polymers IgM and IgA can form polymers. Constant regions of IgM and IgA antibodies contain a region essential for polymerization. J chain–small peptide that links the polymerization region of Ig molecules to form a polymer. J chains allow IgA to form dimers and IgM to form pentamers. Neutralization and Opsonization functions of Immunoglobulins Neutralization: Neutralizing antibodies directly inactivate a pathogen or toxin by binding tightly to it and preventing it from interacting with human cells to cause harm. Opsonization: Some antibodies are opsonins, they function in opsonization – the coating of a pathogen with an immune system protein. Opsonization can eliminate pathogens in two ways: By inducing phagocytosis –phagocytes have receptors that bind complement and the Fc (constant ) region of some Igs . By complement mediated cell lysis– antibodies bound to cell surfaces can also lead to direct lysis of the cell by complement. Complement and Antibodies working together IgM 1. When bound to B cells it forms the B cell receptor 2. A soluble form (pentamer) is produced in a primary immune response 3. Eliminates pathogens in the early stages of B cell mediated immunity before there is sufficient IgG 4. Can form a pentamer therefore has high agglutination ability 5. Excellent complement fixing or complement activating antibody Ig G 1. 80% of total Ig, therefore provides the majority of antibody related immunity against pathogens 2. Has a longer half life (23 days) than other antibodies 3. Distributed intravascularly and extravascularly 4. Can pass through the placenta 5. Possesses both anti-viral and antibacterial activity IgA 1. Monomeric IgA functions within fluids and tissues 2.Dimeric form is most common. 3. Dimeric form, protects mucosal surfaces (GI, respiratory and urogenital) Predominant antibody in mucus secretions, saliva, tears, sweat and breast milk. Antibody classes or Isotypes IgD They are present on B lymphocyte surfaces with little known activity IgE 1. Present on mast cells and basophils 2. Binds to allergens and triggers histamine release from mast cells and basophils (involved in allergy) 3. Protects against worms 4. It has the shortest half life of 2 days Antibody classes or Isotypes Elevated levels of IgM usually indicates a recent infection The number of different antibodies that can be produced seems limitless Ig heavy (H) and light (L) chain genes are inherited in germline configuration–families of fragmented gene segments that must be rearranged before the Ig can be expressed. Gene segments: Portions of a gene The DNA sequence encoding a variable region is assembled from two or three gene segments. Light Chain L chain V domains are encoded by 2 gene segments: VL gene segment –makes up most of the V domain JL gene segment –joining gene segment The DNA sequence encoding a variable region is assembled from two or three gene segments. Heavy Chain Heavy chain V domains (VH) are encoded by 3 gene segments: VH gene segment DH gene segment–diversity gene segment JH gene segment There are multiple copies of each type of gene segment in the germline DNA. Generation of Ig diversity in B cells before encounter with antigen Somatic recombination – random recombination of different V,J gene segments in variable region of light chains and V,D, J gene segments in variable region of heavy chains. Primarily responsible for diversity of antibodies Generation of Ig diversity in B cells before encounter with antigen Somatic recombination–the Ig gene rearrangement process where V,D, and J gene segments are cut and spliced. Involves one rearrangement event (V-J) in the L chain gene and 2 rearrangement events (V-D-J) in the H chain gene. Occurs only between gene segments on the same chromosome. Involves enzymes that specifically recognize, bring together, cut and then rejoin DNA. V(D)J recombinase– the set of enzymes needed to recombine V,D and J segments Naive B cells use alternate mRNA splicing to make IgM and IgD. After Ig gene rearrangement the heavy chain locus can be transcribed and functional mRNA is produced by removing introns from the primary transcripts. The same rearranged V region gene can be used with any of the C region genes. Naive B cells– B cells that have not encountered specific antigen – express IgM and IgD. Each B Cell Produces Ig of a Single Antigen Specificity. Allelic exclusion–a process of using genes from only one parental chromosome which assures that each B cell expresses only one type of heavy chain and one type of light chain and therefore antibody of a single specificity. or Ig is first made in a membrane-bound form that is present on the B cell surface. B cell receptor (BCR)–the cell surface complex of an immunoglobulin and 2 other proteins (Igα and Igβ). Igα and Igβ proteins–two invariant transmembrane proteins with long cytoplasmic tails that interact with intracellular signaling molecules when antigen is bound by the BCR. Igs with a transmembrane domain must be associated with Igα and Igβ proteins to be transported to the surface of the B cell. Diversification of Igs after B cells encounter antigens After an encounter with antigen, IgM and IgD isotypes can be produced as secreted antibodies IgM antibodies are produced in large amounts and are important in protective immunity IgD antibodies are produced only in small amounts and have no known effector function All the isotypes of Ig can be made in two forms: 1) membrane bound BCR, or 2) secreted antibody Secreted Igs (antibodies) are produced by an alternate pattern of heavy chain mRNA processing. The membrane anchor at the carboxy terminus of membrane bound Ig is encoded by one exon. The carboxy terminus of secreted Ig is encoded by an alternate exon. During differentiation to a plasma cell mRNA is alternately processed and from that time secreted Igs are produced. Diversification of Igs after B cells encounter antigens Somatic hypermutation – mutations that occur in the V genes of heavy and/or light chains of a B cell. Competes mot effectively for binding to antigen Preferentially selected to mature into antibody producing plasma cells Leads to affinity maturation (progressively higher affinity for antigen) Isotype switching – an individual B cell switches to make an antibody of a different class such as IgG, IgA or IgE. Antigen specificity is the same Effector functions are different Diversification of Igs after B cells encounter antigens Somatic hypermutation Somatic hypermutation – mutations that occur in the V genes of heavy and/or light chains of a B cell. Affects V regions only, not C regions or any other B cell genes. Occurs at a rate of 1 mutation per cell division –1 million times the mutation rate normally seen in genes. Diversification of Igs after B cells encounter antigens Somatic hypermutation Binding of antigen stimulates B cells to divide rapidly and differentiate into plasma cells. Somatic hypermutation gives rise to B cells with mutant Ig on their surfaces. Some of these have a higher affinity for antigen. They will be more likely to bind antigen and generate plasma cells. Somatic hypermutation results in affinity maturation–the production of antibodies with progressively higher affinities during the course of an immune response. Seen particularly after secondary and subsequent exposure to antigen (example: after booster vaccinations). Diversification of Igs after B cells encounter antigens Isotype switching Isotype switching produces Igs with different C regions but identical antigen specificities. IgM is the first Ig produced in an antibody response but has limited mechanisms for fighting pathogens. Antibodies with other effector functions can be generated by isotype switching – a further DNA recombination event that allows the rearranged V region gene to associate with other heavy chain C region genes. Diversification of Igs after B cells encounter antigens Isotype switching Isotype switching is stimulated by cytokines released by T cells during the course of an immune response after antigen stimulation. Switch regions – stretches of repetitive DNA which guide recombination that results in isotype switching. Switch regions lie in introns upstream of all of the heavy chain constant regions except the one that gives rise to IgD. Diversification of Igs after B cells encounter antigens Isotype switching Once one isotype switching event has occurred a portion of DNA has been removed and the switch is not reversible, however a subsequent event may occur involving a downstream C region gene.