Antigens and Epitopes (MDSC 321-14) PDF

ANTIGENS So…. What can the immune system “see” Infection (bad “foreign”) Gut Flora (good “foreign”) Cancer...

ANTIGENS So…. What can the immune system “see” Infection (bad “foreign”) Gut Flora (good “foreign”) Cancer (bad “self”) Autoimmunity (good “self”) can see infection, can also see normal gut ora, this doesnt cause disease, its a part of our barrier, but its still not us can be foreign and good can also have cancer, not foreign but bad autoimmunity, IS sees our tissue, its good tissue iS can see anything and its largely based on shape, if it has shape, can recognize it, whether its good or bad is based on context ANTIGENS Whereas the innate immune system recognizes conserved pathogen associated molecular patterns (PAMPS), the adaptive immune system recognizes antigens. innate saw broad general patterns, adaptive sees smething precise, these precise things are antigens antigen = piece of something that is recognized by adaptive IS ANTIGENS Antigens, what are they? some of these terms were invented in immunology before we knew big picture ANTIGENS Antigen (Ag): Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (or the T-cell receptor complexed with MHC). any molecule that speci cally recognized by an antibody or immunoglobulin (larger collective term of anjtibody, binds at the end of antibody arm, what ab sticks to) tiny piece of target that is recognized either by immunogloublin for b cells or by the t cell receptor by t cells ANTIGENS IS can recognize any shape, when we think of immunoglob, can recognize metal ions, as long as it has 3d structure, antibody can stick to it enzymes --> lock and key idea, same with antibody antibody arm has 3d binding pocket, if target can t in it, it will stick dont have immune responses against food we eat, our poretins, not bc we cant see thwm if u have peanut allergy, antibodies see peanuts, antibody can do it, in most of us it doesnt just bc u have shape doesnt mean ur good at building immune response have subgroup of antigens knowns as immunogens, these can elecit immune response Antigen (Ag): Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (or the T-cell receptor complexed with MHC). But not all molecules induce immunity – what’s up with that? Immunogens……. ANTIGENS What is an immunogen? ANTIGENS Immunogen: A molecule that induce a specific immune response. adaptive immune response adaptive = speci c, di erentiate s b/w closely related molecules instead of pamp, have molecule that elcits speci c immune response, this is immunogen Antigen: Any molecule that can interact (specifically) with the immunoglobulin (Ig) receptor of B-cells (T-cell receptor complexed with MHC). ANTIGENS All immunogens are antigens. Not all antigens are immunogens. group of antigens that generate immune response = immuogen this is what matters, cant focus on how IS sees eveyrthing, focus onw hat it actually reacts to these are immunogens  allows us to understand mols in a context ex. proteins tructure such as agella, is a ligand for TLR, this is a PAMP ANTIGENS ◦ at the same time, if u grow bacteria in dish and use lab techniques, and use antibodies to stain bands on gel, now have antibodies binding same protein that was PAMP ◦ if its recognized by antibody, now its antigen ◦ put agella into vaccine and injected it and give it to b cells in vivo, will generate immune response ‣ now its not just antigen, its immunogen ‣ exact same protein can be neither an antigen or immunogen if it binds to TLR, if we use it on bench when we're not generating immune resposne, now its antigen but not immunogen bc doesnt drive response ‣ put it in vaccine and give it to somebody and they make Flagellin antibodies, now this mol is immunogen AND antigen ◦ terms are context related, have antibodies that see dna, ex. of lupus ‣ dna recognized by TLR as well ‣ same mol can be PAMP and antigen and immunogen ‣ some antigens are good at being immunogen, something about them that makes IS angry ‣ all of these can be seen by IS but some make it angry Ligand for macrophages in culture - TLR5 (PAMP) Immunogen – No Antigen - No Western blot - detected by antibodies Immunogen – No Antigen - Yes Ligand for B-cells in vivo Immunogen – Yes Antigen - Yes ANTIGENS What makes a good immunogen? ANTIGENS - immunogenicity Humoral Immunogens (B-cells) – Proteins >> Polysaccharides >> Lipids or Nucleic Acids Cell Mediated Immunogens (T-cells) – Proteins, some lipids, some glycolipids. – Proteins are not recognized directly, peptides processed from the protein are seen in association with MHC molecules, lipids with an MHC-like molecule CD1 ‣ b cell making antibodies, can see anthing ‣ have preference tho, like proteins most ‣ makes sense, if we're seeing 3d shape, proteins are complex, then sugars wqhich are repeating steuctural units, and the.n lipids or NA which are repeating ‣ lots of diversity nad structures in protein, as u go down this preference, mol get more simpke ‣ more complicated = more likely by b cells ‣ t cells only see proteins with some exceptions, more speicifcally see only peptides (piece of proteins), 8-15 a.a, t cell does not ever see target directly, has to be shown with MHC ‣ b cells see anytghin with 3d shape, t cells only see petide and only when its presented by MHC  ANTIGENS – 4 properties of an immunogen 1. Foreignness – To serve as an immunogen, a molecule must be seen as “non- self”. The degree of immunogenicity is dependent upon the degree of foreignness. The greater the phylogenetic distance between species typically the greater the chance of immunogenicity. – E.g. Bovine Serum Albumin injected into chickens or goats. ‣ foreigness = if its not me, wont like it probably, not alway ex. foods, but in general, if its di from me, im gonna see that as a problem, and build IR against it ‣ if its a self protein, hopei ly we've taught IS about it, reason why we dont have autoimmuntiy is bc we're toleraized to our cells ‣ more di = more likely to get response Exceptions ‣ exception = across species, theres mol that are well conserved ex, chicken dna looks like human dna, molecular strucutre is same just di pattern, phopshate backbone, NA ‣ have strucutal proteins ex. collagen, our collagen is not di than chicken, cow , goat, sheep ‣ inject cow collagen into mouse, look s like its mouse collagen, mouse wont see it as foreign ‣ hard to make IR agaisnt it then – Highly conserved molecules like collagen or cytochrome c may not be immunogenic even in distant species. – Some self molecules, normally sequestered from the immune system, will raise an immune response (E.g. sperm or lens tissue) ‣ also have immune privleged body sites, antigen or immunogen is in the animal that they came from. physically separated from immune system ‣ ex. interior comparmtnet of eye, can inject things into space immediately in front of lens and wont reject it ‣ IS cant get in there, doesnt matter how foreign it is, it will live there ‣ cna be in theory recognized but if it cant contact IS, wont matter  ANTIGENS – 4 properties of an immunogen 2. Molecular Size – There is a correlation between size and immunogenicity. – The best immunogens are in the range of 100,000 Da. – Small molecules 5-10,000 Da are generally poor immunogens. – Minimally they must be large enough to be processed. ‣ size = antigens are sometimes too small, antigens have to be big enough to have structure and shaoe but t cells have to se something on mhc, target has to be big enough for dendritic cell to eat it digest it rpocess it and put it back on MHC ‣ if its too small, cell cant phagocyorse that ‣ goldlocks zone, too big, cant eat it, too small, cant show it ‣ protwein thats big enough to chew up into big peptides and put onto mhc is good  ANTIGENS – 4 properties of an immunogen 3. Chemical Heterogeneity – Size alone will not make a good immunogen. – Synthetic homopolymers are not immunogenic regardless of size. – Large co-polymers can be immunogenic, adding aromatic amino acids increases the chance. – Proteins with more complexity in primary structure and those showing secondary, tertiary, and quaternary structure increase immunogenicity. ‣ if it has unique surfaces and di erences in chem residues on surface, IS will like it ‣ can be like foreign mol and its right size and entirely hydrophobic, wont see it, theres no contrast or di erences, no heterngoetity on surface ‣ wont nd unique shape to grab onto ‣ needs to have some structure ‣ proteins have primary aa sequence, fold into helixes and hseets, make seprate peptides, etc. ‣ lots of 3d structures, lots of di erences, lots of heterogenity = good immunogens  ANTIGENS – 4 properties of an immunogen 4. Degradability – Proteins must be degraded to be presented by MHC molecules to activate T-cells. Factors that influence this process affect immunogenicity. insoluble > soluble (more likely to be phagocytosed & processed) large > small (more processing, more epitopes) L-amino acids > D-amino acids (works with processing enzymes) ‣ target has to be able to be degraded ‣ if we make target with wrong a.a = d-a.a, enzymes cant take amide bonds apart, can onternalize it but cant shop it i up, cant put it on mhc, not on mhc, t cells wont see it ‣ need to be able to process it ‣ insoluble better thaj spoluble bc can phagocyrose it, chunks cell can eat and digest ANTIGENS if we can apply theese pricniples to antigen, can turna ntigen into immunogen do this with vaccines, picka. target, might not be one that IS wants to respond to, might be not insoluble, not right size, but vaccines still work this is bc we take target/antigen we want and make it more immunogenic How can we make an antigen more immunogenic? ANTIGENS - adjuvants From the Latin - adiuvare (to help) Substances that when injected with Ag serve to enhance the immunogenicity of the Ag. This leads to a higher antibody titer and longer lasting immune response. Are not specific to an antigen but can be used with many different antigens add something to it, in vaccines, add an adjuvant this is a chemical or compound or mix we add to antigen to make it more immunogenic and more appealing to IS doesnt change antigen, antigen is still antigen, immune system will still see antigen doesnt in uence IR, makes target more appealing to IS not speci c to target, if u get u shot or tetanus, have same antigen, IS will make antibodies to u or tetanus, still see right target but these reprogram ther way body sees antigen ANTIGENS – adjuvant mechanism A. Stimulate Immune Response Freund's complete adjuvant, containing muramyl dipeptides from the cell walls of heat killed Mycobacteria , stimulate macrophage activity. The increase in IL-1 helps activate Th (helper) cells Synthetic polyribonucleotides and bacterial LPS stimulates nonspecific lymphocyte proliferation. stimulate innate IR innate helps adaptive --> need to be able display antigen, phagocytose and put it on mHC, need to provide help to immune system, costimulatory signals exist (ex. cytokines), in ammation = enhance how we can get antigens into lymph node so IS can see it things we can do to stimualte IR do this by including things in adjuvant such as Tol like receptor ligands, not speci c, wont get IS to recognzie LPS, but activate innate cells, make macs phagoytsoe better, present antigen better, provide costimulatory help Some stimulate local chronic inflammation and granuloma formation (Freund's complete).  ANTIGENS – adjuvant mechanism B. Prolong Exposure to Ag Alum and Freund's adjuvant bind and precipitate the Ag to keep it in the system longer and allow for slow released of Ag. Can increase the time of exposure from a few days to a few weeks. Precipitation also increases the size of the Ag to facilitate ex. u shot, are made up 2 proteins HA and NA, no u ivrus or dead virus, no viral rna, just 2 proteins and adjuvant if u inject just 2 proteins, will get lost in uid between ur cells, be cleared from body phagocytosis. invented adjuvants that keep proteins avaialble for few days, do this by causing them to form aggregates, to rpecipiate, to form gelatinous mass, this ball stays under skin for a few days in doing this, create depo e ect, all cells in area can nibble awayand clear the debris, and doing so, are able to pick up antigen better if u leave it in jelly ball, macs need ot get rid of it, clear material from tissue, so whill pagocytose it preipatitin gets it to be bigge r= optimal size rpolonges exposure, keeps it for few days to couple weeks, allwos IS to get time and nd it geenrates help signal, by activating IS, gett beteter phag, better cell presentation, also get help, get signal that hey this thing i ate is dangeorus, need ot respond to it instead of ignore it adjuvant changes thhis context, if u put HA under the skin without adjuvant, body goes so what, theres no infection or virus, why do i care? now put adjuvant as well as HA, adjuvant has TLR ligands, body gets angry, gets in ammation, tells IS that this important, need to respond to it, IS is based on no go or go signals, in absence of help, even if T cell sees it, immune system wont respond. we have to get help signals  ANTIGENS – adjuvant mechanism C. Co-stimulatory Signal Th cells when stimulated by Ag require a second co-stimulatory signal. Freund's adjuvant, LPS, and other factors up regulate co-stimulatory signal systems. ANTIGENS – adjuvant mechanism havent changed antigen or target,but by putting adjuvant there, body is more prone to nd it and respond to it signalling through TLR ex. adjuvant a, activates IS, better phagocytosis and better antigen presentation depo e ect will enhance phagocytosis, will precipitate it and keep it in tissue so get better presentation bc its uptaking (adjuvant b) adjuvant c, activated cells will express mol the t cells are looking for for help, the context that says this a problem, need to respond Nature Reviews Microbiology 5, 505-517 (July 2007) ANTIGENS – epitopes dont see entire antigen, sees a piece of antigen ANTIGENS – epitopes Lymphocytes do not recognize an entire antigen Lymphocytes recognize small, discrete sites on macromolecules called antigenic determinants or epitopes. Epitopes seen by B-cells and T-cells differ in several fundamental ways. antigen = forest, oen spot on entire protein that adaptive IS is seeing is one large yellow tree dont see entire forest, see one tree = epitope these are small structural piece in larger protien or sugar that binds to t cell recpeotr or binds to immunoglob binding sites (end of arms) the epitopes on a given protein that t cell and b cell sees are pundamentally di , see them in di ways  ANTIGENS – epitopes not created equal, IS will nd one or tow favs, can make antibodies for these surfaces, but will only ahve antibodie that will see 2-3 of these epitopes, these are optimal, on surface, exibel, right size, etc., these are called immunodominant, will dominate immune response ◦ optimizing for the thing that is bound and recognized best, if ur a pathogen, will do best to make sure immunodiominant epitopes are not important to me, might even put out on bacterial surface a very good epitope, bc uf they stick to that, wont impede function, set up decoys, set up things IS wants to se but dont bllock my function ◦ pathogens have spots that are intentionally immunodominant so IS fails to recognize it ◦ one of the classic examples = HIV, has GP 120, large glycoprotein, b cells love it, its sugars, protein, 3d shape, outside of virus, ts every criteria but most of that protein doesnt do shit for HIV, doesnt stick to target, doesnt allow infection, the stem does that holds up big protein (thats how it gets into cells), but by having juicy globular protein on top, IS ignores stem, we never block it, as a result, HIV can keep evading immunity ◦ when we're making vaccines, wanna avoid what paothgen wants us to do,instead getting a better and more intelligent vaccine design, wanna ure out what we need to target Predicted HPV L1 Protein B-cell Epitopes ProImmune.com each antigen can have multiple epitopes this is HPV viral protein, predicted 3d model of the surface, protein itself could be target of vaccines, IS sees discrete bindign sites on protein each shaded area = di spot antobdy is know to bind, each is di antibody one will bind dark blue and wont stick to anything else, doenst know anything else about protein even within own immune response, can make 12 di antibodies that see 12 di parts of same protein, parts cna also be overlapping, only one antibodiy at a time will stick but these are just surfaces, if surface has unqie shape antibodiy can dock to, can bind to it multiple eptioes on given antigen ANTIGENS – epitopes B-cell epitopes see proteins, sugar, nuecleuci acid, see 3d shapes, see anything, this is truly the lego bloc, it needs to t together otherwise it doesnt stick  ANTIGENS – epitopes (B-cells) B-cells bind Ag directly via cell surface immunoglobulin (Ig) Ag can be almost anything – sugars, lipids, proteins, nucleic acids, heavy metals … For Ag in solution – Epitopes must be topographically accessible on the native molecular surface (hydrophilic) (exceptions) – Epitopes must be flexible and mobile for agglutination (often located on bends and loop structure of protein) – Epitopes can be sequential or nonsequential (conformational) rules b cell have to follow: typically epitopes have to be outside of protein, b cell cant get to middle of protein, b cell sees outside surface, needs to be accessible to outside world --> large globular protein, lots of a.a buried in middle, b cell cant get there, sees full protein, sees outside surface of that, needs to be accessible to outside world epitope has to be exible, need to stick in bidning pocket, b cells love side chaisn and loops, sugar side chains, a.a that have side chain, they like extra shapes that they can bend and t into binding pockets can be iether sequential or nonsequential, b cell can recognize 3d shape of soemthing or if u stretched protein out, can see a.a sequence, doesnt speci cally matter most b cells see conformational shape, but if u streteched protein out, could generate antibodies b cell only cares about seeing shape or 3d structure  ANTIGENS – epitopes (B-cells) Epitope size defined by the binding site of the Ab. Complementary binding between Ag-Ab limits epitope size. Typically 6-7 amino acids (aa) or sugars can fit into the deep pocket structures of linear epitope binding sites. Conformational epitopes of globular proteins cover much greater space on flatter surface binding sites of Ab. Conformational epitopes may consist of 15-22 aa. Complex proteins may contain multiple overlapping B-cell epitopes. Not all epitopes induce a response IMMUNODOMINANCE size of bindign pocket determiens how big epitope can be 6-7 a.a why can a virus excape imune response? just has to change one of those a.a, only 6 a.a and one changes, 20% of surface has changed, these are speci c binding sites, theyre not tolerable to change, very speci c binding sites true 3d shape that has contributions from 2 di loops on protein, maybe it gets bigger, 15-20 aa. proteins are in hundreds of aa, this is still tiny IS will nd one or two favorite binding sites, could make ab against all surfaces, if u watch IS, by the end of it, will have ab that only see 2-3 epitopes, theres a preference, these are optimal, exible, right size, on surface, these are IMMUNODOMINANT, these dominate IS, optimizing for the thing that is bound and recognized best ANTIGENS – epitopes (B-cells) ◦ orangw and blue make up arms of antibodiy ◦ purple petide is sititng ina ntigen bindign pocke,t this is epitope, this is piece of larger protein b cell sees ◦ this is entirely determiend by how it ts http://proteopedia.org/wiki/images/6/64/Rituxan2.jpg ANTIGENS – epitopes (B-cells) ◦ look at protein structure, can have round socket at top of protein structure, target is gonna t into pocket, entire determiend by how well side chaisn t into space ◦ cylinder, lies through groove ◦ get entirely at surfaces that bind basically at end of ab arm ◦ targets with internal surface ex. enzymes that have catalytic site inside and domains on ab arm, if they t into that space, cant allow it to stick ◦ better the physical t, the better these things hang on  ANTIGENS – epitopes (B-cells) Linear (sequential) vs. Conformational (non-sequential) ◦ can see linear or conformational, this explaiosn how it sees protien or sugar as structure rather than sequence ◦ sees shape, shape can be represented by multiple loops from dif fparts of protien that end up in same 3d shape, antibody may be binding to all of these loops, loops together maing a surface, ab might be binding to little bit of loop 1, 2 and 3 ◦ if u took protein apart, those antiboides wont stick anymore bc surface is gone ◦ at the same time, pieces of proteins can be continuous a.a sequences = linear epitope ◦ if ab sticks to linear epitope, even if u unfold sequence, can still see same ab sequence ◦ there are both the linear epitopes, sequential, all on one piece of molecule or conformational that have di pieces of mol that end up in 3d space Arch Dis Child 2004;89:238-243 doi:10.1136/adc.2002.013250  ANTIGENS – epitopes (B-cells) Linear (sequential) vs. Conformational (non-sequential) ◦ type of epitope determines how it will work in lab ◦ linear epitopes,when we unfold it or chop it up, ab still sees red domain bc in tact ◦ if ab sees red domain based on 3d folded shape, then when we unfold or cut it into small pieces, red domain is gone, piece is missing, ab is not gonna work anymore Epitome - Database of Structurally inferred Antigenic Epitopes in Proteins ANTIGENS – epitopes (B-cells) http://xray.bmc.uu.se/kurs/BSBX2/practicals/practical_2/practical_2.html ◦ can be primary a.a, a helix, peptide, or multi domain proteins ◦ if it has a shape, itll bind ANTIGENS – epitopes T-cell epitopes  ANTIGENS – epitopes (T-cell) Recognize only protein (and some glycolipid) epitopes T-cells do not recognize soluble native Ag Recognize only Ag that has been processed and whose peptide fragments are presented in association with Major Histocompatibility Complex (MHC) molecules. T-cell epitopes are generally oligomeric peptides of 7-20 amino acids in size ─ Ag binding cleft of MHC defines Ag expression ─ MHC Class I typically 9-11 aa ◦ require antigen presenting cells ◦ only see peptides, some glycoproteins, piece sof a protein ◦ have to be displayed on MHC, t cell is seeing peptide but also MHC, mHC = pedestal that has protein sititng on it ◦ will se eepdestal and prtotien ─ MHC Class II typically 11-17 aa ◦ will see both arget and sself protien holding it ◦ 2 avors of t cells, killer and helper ◦ see 2 di types of MHC ◦ have slighlty di shpapes, bind d peptides, one is clsoed pocket, 9-11 a.a, other one is a hotdog in a bun, can ahng out ends a biit and is lot more oeixlbe in length  ANTIGENS – epitopes (T-cell) Ag processing required to generate peptides Ag is seen as part of a trimolecular complex TCR-Ag-MHC Peptides may be internal and must be amphipathic ─ must have hydrophobic regions to bind MHC ─ must have hydrophilic regions to bind TCR ◦ 3 moelcule complex that comes together, TCR, MHC, and peptide ◦ all have to itneract ◦ peptides must have both hyodrtphobic and philic domains, if enitrely philic, and put that on mhc, will wash o with any uid in cell ◦ part that sitkcs ot mhc will sitkc to hydropbihc parts of mhc, wont let go, wont expose phobic parts to extracell env, stuck there ◦ rest of the peptide has to be good with aq env, has to be philic ANTIGENS – epitopes (T-cell) ◦ x has to stya stuck to mhc, t cell will see mhc and peptide ANTIGENS – epitopes (T-cell) MHC I MHC II ◦ 1 = shows stu to killer cells, cancer and viruses ◦ mhc 2 = what phags have, shows t cells outside world, eat, process,a ndput it on mhc ◦ have di shapes ◦ mhc has closed nest and ends ◦ mhc 2 = peptides can hang out ◦ if peptide is doing 2 jobs, sticking to mhc and presenting to TCR, there must be 2 surfaces to that epitope ◦ t cell epitopes have functional domains  ANTIGENS – epitopes (T-cell) MHC binding site of the Ag is called the AGRETOPE, binds via hydrophobic amino acids TCR binding site called the EPITOPE, binds via hydrophilic amino acids Immunodominant T cell epitopes are determined in part by what set of MHC molecules are expressed and what TCR are expressed by an individual. ◦ agretope = sitcks to mhc, hydrophibc, will t into the next/hot dog pocket of mhc, shape selection that binds to mhc ◦ other is the epitope, piece t cells see ◦ t cell has agretope and eptipoe ◦ b has only eptiope ANTIGENS – epitopes (B & T-cell) B-cells T-cells Membrane Ig and Membrane TCR, Antigen interaction antigen antigen, MHC Soluble Antigen? Yes No Additional Molecules No MHC, CD4/CD8 Required Chemical nature of Protein, lipid, Protein antigen polysaccharide, Accessible, Accessible or hydrophilic, mobile, Epitopes internal, linear, sequential or amphipathic conformational ◦ b cells = membrane immunoglob and antigen come together for b cell to reocgnize, for t, its peptide, mhc, and tcr, and target ◦ b cell cant ssee thinhfs insie of protien but t cell does bc mac eats it and processes it