Presentation on theme: "Effector Mechanisms of Humoral Immunity"— Presentation transcript:
1 Effector Mechanisms of Humoral Immunity Jason Cyster, PhDLearning Objectives of lecture:Explain the concept of antibody-mediated neutralizationUnderstand how antibody coating of an antigen opsonizes itDescribe how antibody coating of cells targets them for attackUnderstand the core steps in complement activationAppreciate how complement can opsonize a particleDescribe how complement components act as chemoattractantsBriefly explain formation of the ‘membrane attack complex’Understand how exaggerated complement activation contributes to diseaseExplain how IgA reaches the mucosal surface and protectsDescribe what is meant by ‘passive immunity of the newborn’
2 Features of primary and secondary antibody responses
3 5 Different Antibody Classes -> When B cells switch heavy chain, they keep the same Variable domain (and same light chain) and thus the same antigen binding specificity-> the different heavy chains confer different effector functions
4 Antibody Structure (reminder) Domains at ends of heavy chain and light chain are highly variable and responsible for binding antigenthe gene segments encoding these domains are mutated in GC B cells during antibody affinity maturation
5 The effector functions of antibodies Properties of AntibodiesAntibody binding can compete with the ability of pathogen or toxin to bind its receptor -> neutralization especially important for antibody in mucus secretions (IgA)Most protective effects of systemic antibodies depend on activites of the Fc regions contain binding sites for phagocytes and complement (C’)Fc-dependent functions require Ag recognition by the Fab regions:changes the conformation of the IgM pentamer, exposing the Fc-regions for interaction with C1q;in the case of IgG, C1q can only bind when >2 Fc regions are colocalizedSpecialized Fc receptors (FceR) exist for mediating the effector functions of IgE
6 -> antibody responses can be long-lasting – can exist for years after the initial encounter or vaccination (e.g. ~10 years for tetanus toxoid vaccination)
7 Antibody-mediated opsonization and phagocytosis of microbesIgG cannot bind strongly to the FcR in monomeric form; needs to be prebound to the antigen in a multivalent array to provide sufficient binding strength to remain bound to the FcRs on the phagocytePhagocytic cells (macrophages, neutrophils) express activating Fcg receptors - when crosslinked they transmit signals that promote engulfment and increased bactericidal activity.Therapeutic antibodies such as Rituxan (against B cell surface molecule CD20) also utilize this pathway to promote clearance of targeted (cancer) cells.
8 Antibody-dependent cellular cytotoxicity (ADCC) Note this slide is revision: NK cells are introduced in Dr Defranco’s innate immunity lecture; Dr Abbas mentions the ADCC concept and the role of IgE in eosinophil activationAgain, monomeric IgG does not remain bound, rather the NK cell binds to surfaces that are coated with large numbers of antibody moleculesWorms are also called helminthic parasites or helminths
9 The Complement (C’) System Complement opsonizes antigens for phagocytosis and can promote direct lysis of some bacteriaC’ activation occurs in mulitple diseasese.g. glomerulonephritis, lupus, autoimmune heart disease, RA, age-related macular degeneration, Alzheimer’s diseaseAll the C’ components pre-exist in an inactive form in the blood (mostly made in the liver); C3 is the most abundantEnzymatic (proteolytic) cascade - initial signal strongly amplifiedWhen a C’ component is cleaved, the larger ‘b’ fragment is chemically labile and becomes covalently bound to nearby surfaces; the smaller ‘a’ fragment is soluble and can diffuse awaySome general principalsMight play a role in multiple disease e.g. RA, autoimmune heart disease, age-related macular degeneration, Alzheimer’s disease
10 Various complement components activated to generate Activation of Complement (C’)Lectin PathwayClassical PathwayAlternative PathwayMannose poly-saccharide & MBLAntibody/AntigenComplex & C1Microbial surface-bound C3bMBL is C1-like but activated by binding Mannose-rich polysaccharidesVarious complement components activated to generateThese core pathway features of C’ activation need to be remembered“C3 convertase” (protease)C3C3b + C3a
11 Early steps of complement activation IgM or IgG antibodybNote: because C3 is very abundant, once a C3 convertase forms, lots of C3b is generated and becomes covalently bound to nearby surfacesClassical Pathway: activation of the endogenous protease activity of C1 following binding to IgM or IgG leads to generation of a protease that cleaves C3 (a C3 convertase)Alternative pathway: a low level of C3 is spontaneously activated; when this occurs near a microbial surface the active C3 (C3b) can bind to the surface and then interact with components of the alternative pathway, leading again to formation of a C3 convertaseNote: because C3 is very abundant, once a C3 convertase forms, lots of C3b is generated and becomes covalently bound to the nearby surfaces, opsonizing for phagocytosisb
12 Early steps of complement activation Classical Pathway: activation of the endogenous protease activity of C1 following binding to antigen-bound IgM or IgG leads to generation of a protease that cleaves C3 (a C3 convertase)Alternative pathway: a low level of C3 is spontaneously activated; when this occurs near a microbial surface the active C3 (C3b) can bind and then interact with components of the alternative pathway, leading again to formation of a C3 convertase
13 The late steps of complement activation Key point: C5b catalyzes formation of the Membrane Attack ComplexC9Again, don’t need to remember all the complement components. Key point here is that cleavage of C5 generates C5a and C5b, and C5b catalyzes formation of the Membrane Attack Complex-> Gram negative organisms have a thin peptidoglycan layer and are the most sensitive to the MAC (e.g. Neisseria)
14 Activation of Complement (C’) Lectin PathwayClassical PathwayAlternative PathwayMannose poly-saccharide & MBLAntibody/AntigenComplex & C1Microbial surface-bound C3bMBL is C1-like but activated by binding Mannose-rich polysaccharidesVarious complement components activated to generateNote: this slide summarizes all the core components of the C’ cascade that are useful to rememberThese core pathway features of C’ activation need to be remembered“C3 convertase” (protease)C3C3b + C3aC3b decorates surface; some C3b forms a C5 convertase, generating C5a and C5b;C5b causes formation of theMembrane Attack Complex (C5-C9)
15 The biological functions of complement C5a, C3a (and C4a) also known as Anaphylatoxins > induce mast cell and basophil mediator release (when occurring systemically, can cause anaphylactic shock)C3a and C5a are known as anaphylatoxins because when distributed systemically they can cause anaphylatic shockAnaphylatic shock - bronchial constriction, massive tissue edema and cardiovascular collapseClassic example is ABO mismatched blood transfusion - preexisting antibodies recognize the mismatched blood group, activate the C’ cascade and cause anaphylatic shock
16 Regulation of complement (C’) activation Host cells express membrane anchored C’ regulatory proteins that inactivate complement when it deposits on a host cellSome of these proteins are linked to the membrane via a glycosylphosphatidylinosital (GPI) anchorPlasma contains soluble C1-Inhibitor (C1-INH) protein that limits the extent of C’ activationGeneral point is that there are membrane bound proteins on our own cells that inactivate the complement cascade as soon as it gets started, and there are soluble proteins in the serum that negatively regulate the cascade and prevent it from spreadingGPIGPI: a glycolipid that can be attached to the C-terminus of a protein after translation and mediate attachment to the plasma membraneBy binding to the C5b-8 complex CD59 limits the number of C9 molecules interacting with C5b-8 and the insertion of C9 molecules into the cell membrane. CD59 may prevent the formation of poly-C9 also by binding directly to C9. Adapted from Morgan and Meri, From Antti Väkevä.MASP-1 and MASP-2 proteases in MBL complexes of the lectin pathway are also inactivated. This way, C1-inhibitor prevents the proteolytic cleavage of later complement components C4 and C2 by C1 and MBL. Although named after its complement inhibitory activity, C1-inhibitor also inhibits proteases of the fibrinolytic, clotting, and kinin pathways.
17 Complement in disease Complement overactivity: Complement deficiency 1) Immune complex glomerulonephritisdamage caused by Antigen-Ab complexes deposited in glomerular basement membrane activating C’ and recruiting and activating neutrophilsStrep pyogenes can cause acute glomerulonephritis (AGN)2) Hereditary angioedema (deficiency of C1-INH)severe attacks of edema because the cascade is more easily activated and a lot of C3a, C5a are made3) Paroxysmal nocturnal hemoglobulinuria (deficiency of GPI anchored membrane proteins - includes several C’ regulatory proteins)increased autologous red cell lysisNocturnal because breathing patterns at night alter oxygen content of blood; leads to altered properties of RBC that make them more sensitive to lysisComplement deficiencyC3 - increased risk of infection by many types of bacteriaC5, C6, C7, or C8 - increased risk of Neisseria infectionsStrep pyrogenes can cause AGN (acute glomerulonephritis) - attack rate can be 10-15% following skin infections (e.g. pyoderma). Occurs days after nephritogenic streptococcal infection; C’ levels are low and circulating Ag-Ab ICs are present. In kidney biopsy see Ig and C’ on glomerular basement membrane. May be due to cross-reactive Ags between the strep cell membrane and the glomerular basement membrane or due to the deposition of preformed Ag-Ab complexes on the glomerulus.Nocturnal because breathing patterns at night alter oxygen content of blood; leads to altered properties of RBC that make them more sensitive to lysisStreptococcus pyogenes (pyo=pus-genes=to produce)It is thought that the complement system might play a role in many diseases with an immune component, such as Barraquer-Simons Syndrome, asthma, lupus erythematosus, glomerulonephritis, various forms of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-reperfusion injuries. and rejection of transplanted organs.The complement system is also becoming increasingly implicated in diseases of the central nervous system such as Alzheimer's disease and other neurodegenerative conditions such as spinal cord injuries.Mutations in the complement regulators factor H and membrane cofactor protein have been associated with atypical haemolytic uraemic syndrome. Moreover, a common single nucleotide polymorphism in factor H (Y402H) has been associated with the common eye disease age-related macular degeneration. Polymorphisms of complement component 3, complement factor B, and complement factor I, as well as deletion of complement factor H-related 3 and complement factor H-related 1 also affect a person's risk of developing age-related macular degeneration. Both of these disorders are currently thought to be due to aberrant complement activation on the surface of host cells.
18 The poly-Ig receptor is a special Fc receptor that binds dimeric IgA IgA is our most abundant immunoglobulin, largely secreted across mucous membranes. Humans secrete about 3g/day into the intestine.IgM is also a substrate for the polyIg receptor in humans and mice.Secreted IgA and IgM functions by neutralizing pathogens or their toxins.Note: the J-chain polypeptide that holds the two IgA molecules together as a covalent dimer (and that functions to hold IgM together as a pentamer) is not the same as the joining (J) element that is part of the V-D-J region of the Ig heavy chain!The poly-Ig receptor is a special Fc receptor that binds dimeric IgAThe process of transporting IgA across the cell is known as transcytosisThe IgA released into the gut lumen remains associated with part of the poly-Ig receptor (known as the secretory component) and this provides protection against proteolysis by gut proteases
19 passively transferred maternal IgG Neonatal ImmunityMaternal IgG is transported by the neonatal Fc receptor (FcRn)across the placenta to the fetusfrom colostrum across the newborn gut epitheliumColostrum is the protein-rich fluid secreted by the early postnatal mammary glandConfers passive immunity in the newbornThe duration of protection is 3-4 months (or ~5 IgG half-lives)explains the high incidence of disease after this period by bacteria such as Haemophilus influenzaeHuman milk contains IgAprovides some protection against gut pathogensNeonatal protection is only as good as the titer of IgG (and IgA) in the mother against the specific organismAfter 6 months protection is essentially gone which explains the high incidence of disease caused by bacteria such as Haemophilus influenzae, especially H. flu meningitis. That is why the conjugate vaccine is so important.Neonatal protection is only as good as the titer of IgG in the mother against the specific organism. So child could be infected if mother doesn't have IgG.Passive immunity can sometimes be harmful: if an Rh- mother mounts Ab response against Rh+ newborn due to red cell transfer during birth, those anti-Rh antibodies will be transferred into the blood supply of the next fetus and (if it is Rh+) cause lysis of the red cellsFraction of adult level of serum immunoglobulinspassively transferred maternal IgG100
20 IgG half-lifeFcRn is also present in the adult and involved in protecting IgG from degradationAccounts for the long (3 week) half-life of IgG compared to other Ig isotypesTherapeutic agents that are fused to IgG Fc regions take advantage of this property e.g. Enbrel (TNFR-Fc)Also explains why B cell deficient patients need IVIG injections only every 3-4 weeks
21 Evasion of humoral immunity by microbes Many viruses and bacteria mutate their antigen surface molecules such that they are no longer recognized by the existing antibody-> basis for existence of multiple serotypes of some pathogens (e.g. rhinoviruses, Salmonella enterica, Strep. pneumoniae)-> population builds up immunity to some serotypes and remains susceptible to the othersSome viruses have only a single serotype (e.g. measles, mumps) and this is the basis for the success of the vaccinePathogens that have serotypes – this is because they are evading immunity (e.g. 100 serotypes of rhinoviruses; many serotypes of Strep. Pneumoniae; S. enterica, V. cholera);Popln builds up immunity to some serotypes and remains susceptible to the othersPolysacc capsule more than 85 antigenically distinct types; capsules are virulence factors that interfere with phagoycotsis;Mortality in elderly as well as young (vaccine for at least 5 years)Keep in mind thought that many viruses have only a single serotype (e.g. measles, mumps etc) and this is the basis for the success of the vaccinePneumococcal conjugate vaccine (PCV) is a vaccine used to protect infants and young children against disease caused by the bacterium Streptococcus pneumoniae (pneumococcus).Prevnar is a heptavalent vaccine, meaning that it contains the cell membrane sugars of seven serotypes of pneumococcus, conjugated with Diphtheria proteins. It is manufactured by Wyeth. In the United States, vaccination with Prevnar is recommended for all children younger than 2 years, and for unvaccinated children between 24 and 59 months old who are at high risk for pneumococcal infections.Synflorix is produced by GlaxoSmithKline. It is a decavalent vaccine, meaning that it contains ten serotypes of pneumococcus (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) which are conjugated to a carrier protein.Prevnar 13 is produced by Pfizer. It is a triskaivalent vaccine, meaning that it contains thirteen serotypes of pneumococcus (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) which are conjugated to a carrier protein. Prevnar 13 was approved by the U.S. Food and Drug Administration for immediate release to the public on February 24, It is to be given on the same schedule as was Prevnar.
22 Ig Heavy chain class (isotype) switching Also note that Ig isotypes differ in their serum half-livesIgM ~ 1 weekIgG ~ 3 weeksIgA ~ 1 week (in circulation)IgE ~ 3 daysNeutralizationEosinophil andNeutralizationNeutralization
23 Vaccines Most vaccines work by inducing neutralizing Abs attenuated forms of microbes (treated to abolish infectivity and pathogenicity, but retain antigenicity) are most effectiveroute of administration important e.g. oral administration of Polio vaccine ensures generation of neutralizing IgAimmunization with inactivated microbial toxins generates toxin neutralizing antibodiesLed to world-wide eradication of Small PoxMay soon (?) achieve eradication of PolioMany vaccines still needed - HIV, Malaria, Schistosomes, Mtb etc
24 Classification of Licensed Vaccines This table is included just for interestAwareness of vaccines currently in use and the impact they have had on human health helps highlight the importance of understanding how to induce strong humoral immune responses