Antigens & Epitopes (MDSC 321 Midterm)

MDSC 321 - Midterm No. 2 Antigens & Epitopes Antigens ✽ Immune system can see: ○ Infection (foreign bad) ○ Gut flora (foreign good) ○ Cancer (self bad) ○ Autoimmunity (self good) ○ Good or bad depends on context ○ Adaptive immune syst...

MDSC 321 - Midterm No. 2 Antigens & Epitopes Antigens ✽ Immune system can see: ○ Infection (foreign bad) ○ Gut flora (foreign good) ○ Cancer (self bad) ○ Autoimmunity (self good) ○ Good or bad depends on context ○ Adaptive immune system recognizes antigens ○ Antigen: any molecule that can interact (specifically) with immunoglobulin (Ig) receptor of B-cells (or T-cell receptor complexed with MHC) ○ Lock and key model ○ Immunogen: molecule inducing specific immune response (antigen subgroup) ○ All immunogens antigens ○ Not all antigens immunogens ✽ Flagellin Immunogen Antigen Ligand for macrophages in No No culture - TLR5 (PAMP) Western blot - detected by No Yes antibodies Ligand for B-cells in vivo Yes Yes Immunogenicity ✽ Humoral Immunogens (B-cells) ○ Proteins >> Polysaccharides >> Lipids or Nucleic Acids ✽ Cell Mediated Immunogens (T-cells) ○ Proteins, some lipids, glycolipids ○ Proteins not recognized directly, peptides processed from protein seen together with MHC molecules, lipids with an MHC-like molecule CD1 (only peptide from MHC) ✽ 4 Properties of an immunogen Foreignness ○ To serve as an immunogen, a molecule must be seen as nonself. Degree of immunogenicity dependent upon foreignness degree. Greater the phylogenetic distance between species = the greater the chance of immunogenicity  ○ E.g. bovine serum albumin injected into chickens or goats ○ Exceptions: highly conserved molecules like collagen or cytochrome c may not be immunogenic even in distant species ○ Some self molecules, normally sequestered from immune system, will raise immune response (e.g., sperm/lens tissue - in eye) in animal they came from Molecular Size ○ Correlation between size and immunogenicity - big enough for macrophage to digest ○ Best immunogens in range of 100 000 Da ○ Small molecules (5-10000 Da) generally poor immunogens ○ Minimally, must be large enough to be processed Chemical Heterogeneity ○ Size alone not enough ○ Synthetic homopolymers not immunogenic regardless of size - all hydrophobic no specific structure ○ Large copolymers can be immunogenic, adding aromatic amino acids increases chance ○ Proteins with more complexity in primary structure and those showing secondary, tertiary, and quaternary structure increase immunogenicity Degradability ○ Proteins must be degraded to be presented by MHC molecules to activate T-cells. Factors influencing this affect immunogenicity ○ Things cells can digest with antigen presenting cell Insoluble > soluble (more likely to be phagocytosed and processed) Large > small (more processing, more epitopes) L-amino acids > D-amino acids (works with processing enzymes) Adjuvants From Latin - adivare (to help) Substances that when injected with Ag serve to enhance immunogenicity of AG → leads to higher antibody titer and longer lasting immune response Doesn’t change antigen itself, immune system still recognizes right target Not specific to antigen, can be used with many different antigens ✽ Stimulate Immune Response ○ Freund’s complete adjuvant, containing muramyl dipeptides from cell walls of heat killed mycobacteria, stimulates macrophage activity. Increase in IL-1 helps activate Th (helper) cells ○ Synthetic poly ribonucleotides and bacteria LPS stimulates nonspecific lymphocyte proliferation ○ Some stimulate local chronic inflammation and granuloma formation (Freund’s complete) ○ Can include TOLL like ligands ✽ Prolonged Exposure to Ag ○ Alum and Freund’s adjuvant bind and precipitate the Ag to keep in system longer and allow for slow release of Ag, form aggregates, little ball of snot ○ Cells nearby can nibble away at it, become better at picking up antigen ○ Can increase time of exposure from a few days to a few weeks ○ Precipitation also increases size of Ag to facilitate phagocytosis  ○ Co-stimulatory Signal ○ Cells when stimulated by Ag need second costimulatory signal ○ Freund’s adjuvant, LPS, and other factors upregulate costimulatory signal systems ○ Adjuvant with TLR ligands, immune system get angry, will respond to it, without help signals, T-cell will see but not respond Epitopes Lymphocytes don’t recognize entire antigen Recognize small, discrete sites on macromolecules called antigenic determinants/epitopes Whole protein = forest, epitope = 1 tree Epitopes seen by B-cells and T-cells differ in several fundamental ways Each antigen can have multiple epitope ✽ B-cell Epitopes ○ 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 native molecular surface (hydrophilic) (on the outside) (exceptions) - Epitopes must be flexible and mobile for agglutination (often located on bends and loop structures of protein) (side chains) - Epitopes can be non/sequential (linear or conformational) (3D shape or amino acid sequence) ○ Epitope size defined by binding site of Ab - Complementary binding between Ag-Ab limites epitope size - Typically 6-7 amino acids (AA) or sugars can fit into deep pocket structures of linear epitope binding sites - v specific, tiny sample - Conformational epitopes of globular proteins cover much greater space on flatter surface binding sites of Ab - Confirmation epitopes may consist of 15-22 aa ○ Complex proteins may contain multiple overlapping B-cell epitopes ○ Not all epitopes induce a response immunodominance - Not all created equally, antibodies will only see a few of the epitopes, optimal ones - Damn pathogens can set up decoys that don’t block functional - purple = epitope, part immune system sees - Depending on how it fits - better the physical fit, better they hang on - If on loops, once unfolded, antibody won’t see anymore ✽ T-cell Epitopes ○ Recognize only protein (and some glycolipid) epitopes ○ T-cells don’t recognize native Ag ○ Both protein and holder - pedestal ○ Recognize only Ag that has been processed and whose peptide fragments are presented in association with Major Histocompatibility Complex (MHC) molecules ○ T-cell epitopes generally ogliometric peptides of 7-20 amino acids in size - Ag binding cleft of MHC defines Ag expression - MHC Class I typically 9-11 aa - MHC Class II typically 11-17 aa ○ Ag processing required to generate peptides ○ Ag seen as part of trimolecular complex TCR-Ag-MHC ○ Peptides may be internal and must be amphipathic - Must have hydrophobic regions to bind MHC - would just wash away - Must have hydrophilic regions to bind TCR - not stuck to MHC ○ MHC binding site of AG called agretope, binds via hydrophobic amino acids ○ TCR binding site = epitope, binds via hydrophilic amino acids ○ Immunodominant T cell epitopes determined in part by what set of MHC molecules expressed and what TCR expressed by an individual ○ MHC II - outside world, like hotdog in bun, peptide can out the ends ○ MHC 1 - self, cancer, enclosed, nest B-cells T-cells Antigen Interaction Membrane IG and antigen Membrane TCR, antigen, MHC Soluble Antigen Yes No Additional Molecules required No MHC, CD4/CD8 Chemical Antigen Nature Protein, lipid, polysaccharide Protein Epitopes Accessible, hydrophilic, mobile, accessible/internal, linear, sequential or conformational amphipathic, can be on the inside Antigens - Ag-Ab interactions Ag-Ab interactions, like enzyme-substrate interactions, involve highly specific, reversible binding between molecules ○ Tighter it binds, less reversible it is, better fit, lock and key Specificity determined by multiple low affinity non-covalent bonds requiring specific fit between Ag and Ab Can fall of if specificity not good enough The bonds include ○ Ionic bonds (electrostatic) ○ Hydrogen bonds ○ Van der Waals interactions ○ Hydrophobic bonds Rely on bonds to different extents, makes perfect match Affinity Strength of sum total of non covalent interactions between single Ag binding site on Ab and single epitope Dictated by Ag/Ab fit [𝐴𝑏−𝐴𝑔] 𝐴𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 𝐶𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝐾𝑎 = [𝐴𝑏][𝐴𝑔] ○ Where [Ag-Ab] concentration of Ag-Ab complexes ○ [Ab] concentration of free Ab ○ [Ag} concentration of free Ag ○ Low affinity Ab-Ag binding → ka = 104-105 M-1 ○ High affinity Ab-Ag binding → Ka = 1010-1011 M-1 M ○ Numerator big = good stick, small numerator = bad stick ○ In the old days = measured with diffusion, semi-permeable membrane, antigen can move through membrane, put radioactive tag on antibody, both sides of membrane will have same amount of radiation  ○ Antibody can’t move across, reach eq with free antigen, A also has trapped antibody, bigger difference = more bound by antibody Avidity Strength of multiple interactions between multivalent antibody and antigen High avidity can compensate for low affinity ○ E.g. IgM usually has lower affinity than AgG but higher avidity of IgM from its multivalency enables it to bind Ag effectively How many arms are engaged, 2 weak arms, but one can still hang on Affinity vs Avidity Affinity: strength of binding between single Ag epitope and Ab ○ Dictated by Ag/Ab fit Avidity: function of combined strength of binding affinity of Ab/Ag valency ○ Dedicated by affinity and number of Ab/Ag bindings Cross-reactivity Different Ags may share common epitopes ○ Ab specific for common epitope will bind both Ag ○ Can still stick to similar shapes, e.g. flu Epitope may be different but share common chemical properties ○ E.g. similar charges in amino acids ○ Ab will bind 2 different epitopes Things we design may look like us, once antibody cleared, antigen might stick to us ABO Blood groups Anti-A antibodies Originated from influenza virus, whose epitopes similar to α-D-N-galactosamine on the A glycoprotein Sugar residues from flu look like blood antigens - still attack other proteins on flu tho Anti-B antibodies Glycoproteins on Gram-negative bacteria like E. coli that resemble α-D-galactose on the B glycoprotein Ag precipitation Precipitation happens when Ag-Ab interactions result in formation of lattice structure Lattice formation requires at least bivalent Ab (2 arms) and at least  bivalent/polyvalent Ag (multiple binding sites) Precipitin reactions can be measured by adding increasing amounts of Ag to constant Ab concentration Excess in either Ab/Ag inhibits precipitation Maximal precipitation achieved in zone of optimal Ag to Ab concentration called zone of equivalence Not to be confused with equivalent amounts of Ag and Ab Too little, antibodies fight, too much, they don’t have to share, in middle, will share with another antibody = crystal structure Immunoglobulin Antibodies can recognize and bind >108 different Ag in specific fashion Theories Selective (Side Chain) Theory Put for in 1900 by Paul Ehrlic to explain specificity of Ab response Ab producing cells express multiple “side chains” of various Ag specificities → side chain theory Engagement of one of those “side chains” with Ag results in production and secretion of many more side chains with identical Ag specificity Cool but how do you generate enough diversity in Ig repertoire Instructional Theory Developed in 30s/40s to account for enormous diversity within Ig repertoire Ag itself had role in generating Ig specifiti Ag serves as template around which Ig molecule was folded and mutated → Ig took shape complementary to that of Ag Few genes would need to account for diversity as single molecule could assume many specificities Okay but how do you account for specificity before Ag exposure? Two Gene Theory Proposed by Dyer and Bennett (1965) as solution to problem of selective and instructional models 2 genes encode Ig molecule → one for variable region and one for constant region Many different copies of variable gene (accounts for diversity) but only a few constant genes (one for each isotype)  Few constant genes could be combines with any of the numerous variable genes to generate an Ig repertoire with huge diversity Confirmed by Tonegawa and Hozumi (1976) Analysis of Igk genes from myeloma and embryonic cells Myeloma - all white blood cells the same - rna extracted must be represent the glued together ones, embryonic hasn’t rearranged dna yet (what it should like) Everything found in one small piece of DNA while in embryonic, had two parts demonstrated DNA differences Igk DNA of myeloma cells was “rearranged” as compared to “germline” DNA of embryonic cells Additional sequencing identified separate constant gene for each Ig isotype and subclass Gene coding for variable region was found to be made up of multiple gene segments Light Chain Variable Region (κ or λ) ○ Comprised of variable (V) and joining (J) gene segment Heavy Chain Variable Region ○ Comprised of variable (V), joining (J), and diversity (D) gene segments Start with heavy chain, bring 3 separate gene pieces together, both rearranged peptides have to work Figure Kappa and lambda are interchangeable (in humans at least) Kappa light chain locus, variable genes has little leader sequence, has multiple copies in genome, about 40 variable domains for kappa Joining ones 1-5 And single constant gene To make working kappa light chain have to bring joining and constant together Same with heavy chain, have multiple variable domains for heavy, have additional diversity genes, plus cluster of joining genes, and single constant genes for each type of antibody Rearrangement Diversity Generation Heavy Chain 46V Genes x 23D genes x 6J genes = 6438 different H chains Light Chain Κ chain: 38V genes x 5J genes = 190 combinations λ chain: 33v genes x 5J genes = 165 combinations  355 different L chains Total heavy and light chain combinations 6348 x 355 = 2.55 x 106 different immunoglobulin molecules Junctional Diversity Aren’t clean joinings, edges are fuzzy, junctions correspond to binding pocket → more pocket diversity Although combinatorial mechanisms generate bulk of Ig repertoire in mouse and man, additional Ig variation created through junctional diversity Variation in V(D)J joint contributes greatly to Ig diversity as this sequence corresponds to the third hypervariable loop of the V domain - most important part in binding pocket Junctional diversity generated at the time of rearrangement by 3 principle mechanisms; ○ Junctional flexibility ○ P-nucleotide addition ○ N-nucleotide addition (only heavy chain) Junctional flexibility refers to imprecise joining of the V(D)J gene segments, don’t use proper end of gene Through endonuclease activity several nucleotides may be deleted from cut ends of the gene segments being jointed, nibble a bit, lose some, new amino acid sequence Process results in shortened V, D or J and often produces “nonproductive” or out of frame coding joints - damn only ⅓ of rearrangements work P-nucleotide addition Cutting of of hairpin loop at end of gene segment is imprecise - don’t want to leave sticky ends out - loops them back Resolution of hairpin often results relocation of nucleotides from one strand to the other in a reverse sequence Nucleotides deficient from donor strand are filled in complementary to extended strand Resulting palindromic sequence referred to as P-nucleotides Light and heavy chain, more new nucleotides N-nucleotide addition Only on heavy chain Up to 15 nucleotides may be added to the end of gene segments through action of terminal deoxynucleotidyl transferase (TdT) Nucleotides non templated - called N-nucleotides TdT expressed during H chain rearrangement and downregulated before light chain recombination N-nucleotides predominantly feature of VDJ joint of H chains (although some found in L chain VJ joints)  B cells produced continually throughout life Ig rearrangement begins early in B-cell development Ig rearrangement tightly regulated in sequential, stepwise fashion Stages of B cell development defined by progression of ig rearrangement events Failure to successfully rearrange both H and l chain genes results in blockage of B-cell development → will die Rearrangement initiated by RAG complex ○ Recombination Activating Gene - 1 (RAG-1) ○ Recombination Activating Gene - 2 (RAG-2) ○ Entire adaptive immunity system depends on these guys RAG complex recognizes borders of gene segments and facilitates cleavage of DNA Deficiency in either RAG gene results in Severe Combined Immune Deficiency (SCID) phenotype Other necessary components include standard DNA repair enzymes including DNA ligase IV and DNA-dependent protein kinase (DNA-PK) Treat extra J as intron and splice it out Ig Gene Rearrangement and B-cel Development H chain genes rearrange first Initial H chain products paired with surrogate light chain and expressed on surface of cell as Pre-B-cell Receptor, stabilizes heavy chain, proves heavy chain works Surrogate light chains = product of two conserved genes (Vpre-B and λ5) scaffold, chaperone proteins Signaling through Pre-B-cell Receptor induces cell to begin L chain recombination L chain genes rearrange only after successful H chain recombination Pro B as soon as it starts rearranging stuff When rearranging, expressing RAG, when done turn off Express TdT when rearranging heavy chain How does the cell know where to cut germline DNA? Make chromosome immediately after these genes are constant Gene segments flanked by short conserved sequences → recombination Signal Sequences (RSS) RSS made of ○ Conserved heptamer (7bp) ○ 12/23 pb non conserved spacer sequence ○ Conserved nonamer (9bp) Rearrangement always occurs between RSS with 12 bp spacer and RSS with 23 bp spacer → 12/23 rule Helps RAG know orientation  Each cell has 2 of each chromosome - 1 parental/maternal Each chromosome capable of rearrangement and thus producing own Ig specificity But each B-cell produces Ig of only one specificity, how? Only one chromosome rearranges at a time - allelic exclusion Allelic Exclusion Successful rearrangement of one allele for each chain inhibits rearrangement of other alleles → allelic exclusion Inhibition of rearrangement potentially due to signaling through Pre-B cell receptor (stops H chain rearrangement) and BCR (stops L chain rearrangement) Go for light chain also stop signal for heavy Tries again with another chromosome, try again with different allele, can’t afford to waste successful heavy chain Figure slide 1 ○ Can swap out stem piece - change flavour of antibody ○ Have to start as IgM, general purpose ○ G, E, A more specific, come later ○ Help activate complement ○ Some are completely useless lmao - dedicated antibodies for different jobs ○ Flavours = isotypes, have their own gene, not gonna ask organization ○ Differing sugar, not touching binding pockets ○ Separate gene pieces, so can change stem, don’t edit VDJ, just change IgM to something else, splice onto one of the other constant genes with different enzymes ○ Some exceptions, IgG always with IgM ○ Special signal domains, constants have unique signals ○ Before each constant region = switch region, but not delta (can’t be IgD), where to cut DNA ○ Primary RNA - functional ○ But if you want different one, have to physically translocate, bend DNA, partner switch mew with what you want - make dimer, cut DNA in the middle of switch regions, in between is thrown away, open ends attached ○ Hybrid switch, constant epsilon - IgE ○ Happened at DNA level - permanent change, cell can’t go back - but undergo another class switch with one further down ○ Remove intervening DNA ○ Separate machinery so you don’t VDJ again ○ Patients only make IgM, usually IgG most abundant, mutation in AID, can’t undergo class switch ○ Constant region does the doing - effector functions. Same for all of isotype ○ Chains identical, just attach 2, very stable ○ Proteinases cut, target binding from effector stem, fragment of antigen binding, fragment of crystallization, can activate cell, make smaller for immunotherapies - labelling ○ Other one with pepsin, can crosslink, precipitate - create agglutination complexes, FC missing, can’t activate complement  ○ Another antibody could recognize pocket at antigen ○ 70s - anti idiotype - how do you stop antibodies from binding to yourself, make another antibody that will bind to that one? Bruh blocking inception IgG - high affinity, stick well Subclasses- hinge region differs - disulfide bonds IgM - best at activating complement, activates complement as soon as it sticks IgD - oddball, what does it even do? No known receptors, regulates how you activate B-cell IgA - mucosal membranes, monomeric (in blood, minor) and dimeric, we make the most of everyday, critical piece for barriers Where antibodies are sourced based on J chain IgE - allergies, sees antigen, causes mast cells to degranulate, sticks to mast cells as soon as it’s made Since it’s caused by single type - can tolerize you, class switch again, use see allergy and make IgE - but if you make IgA, don’t get allergies no more Immunoglobulins activate B-cells, express on surface Immunoglobulin - Isotypes Ig molecules with differing C regions are generated through isotype class switch recombination Rearranged VDJ elements are spliced to various Ig C gene segments alpha, epsilon, gamma, mew, delta) Figure, figure, figure Isotype class switch involves DNA rearrangement event Switch region immediately 5’ to the desired Ig C gene segment fused to mew switch region Further class switching may occur to any C genes located downstream of the first class switch target Enzyme activation-induce cytidine deaminase (AID) required for class switch AID induces single strand breaks or “nicks” in DNA DNA repair mechanisms recognize breaks and excises intervening DNA Deficiency in AID results in hyper-IgM syndrome Figure Immunoglobulin - Structure Comprised of 4 polypeptides linked by S-S bonds 2 identical H chains, 2 identical L chain N terminal variable regions → Ag binding Antibodies as a Tool Immunoglobulins - Complement Need 2 FC regions close together, bind and activate first complement, C1q and everything starts Immunoglobulins - Neutralization High affinity Ig (IgG, IgA) bound bacteria, viral particles or toxins prevent their binding to target cells - can’t bind to our cells Coating bacteria/virus with Ab will inhibit ability to infect host tissues Most toxins must bind cell receptor to mediate detrimental effects → coating toxin with Ig protects host Binding of viral particles to cell receptors Internalization of viral particles Fusion of viral and cellular membranes Coating of virus with soluble Ig prevents cellular binding and fusion Just because it’s bound, doesn’t mean it’s neutralized - have to bind peaks Immunoglobulins - Fc receptors Several cell surface receptors can bind Ig Fc, several families Ig binds via its CH2 domain Chains determine what cells do FcR binding can lead to cellular inhibition, activation and phagocytosis, induction of target killing and granule release Ig is bound by α chain of FcR Signally mediated through second molecule, 𝛾 chain except (Fc𝛾RII) In most cases, Ig must be bound to Ag to stably associate with FcR (exception FcεR1) ITM’s inhibitory / ITAM activates/turns on Ab-dependent cell-mediated cytotoxicity (ADCC) Coating of target with soluble Ig Binding of NK cell to target via Fc𝛾RII Crosslinking of FcR induces targeted granule release (perforin + granzyme) Death of target cell Steering granule release Granulocyte Degranulation Typically, has to to bind antibody first, Fc region undergoes conformational change Mast cells, basophils and activated eosinophils express high affinity IgE receptor (FcεR1) - wait for cell to engage antigen Soluble IgE stably associates with FcR in absence of Ag binding Thus cells are present precoated in polyclonal Ab, ready to engage in variety of Ag  Crosslinking of FcR bound IgE Ag induces degranulation Granules release histamine and other proinflammatory mediators B-cell Activation Lifespan Conventional B-cells continuously produced throughout life in bone marrow Most IgM+ IgD+ B cells (mature B-cells) that are newly produced have short half-life of 3-4 days Must encounter antigen to survive, damn, dies by neglect Everything from region will filter and concentrate into lymph drain node Lymph constantly filtering - B cell walks through from blood and looks through Especially for naive B cells, if you don’t, drain out into efferent - cycles through again until it recognizes or dies Germinal centres - follicles Primary follicle - resting B cells clustered around dense network of processes of follicular dendritic cells (FDC) - only hang out in lymph node and spleen - still display antigen to innate immune system Secondary follicle (germinal centre reaction) ○ T cell dependent ○ Ag trapped by FDC ○ B cells proliferate ○ Affinity maturation, try to modify B cell receptor every generation, gets better at recognizing antigen ○ B cells with low affinity for Ag die by apoptosis ○ Class-switch recombination ○ Plasma cells and memory cells are produced Germinal centres - affinity maturation Germinal centre reaction produces high affinity B-cells to specific Ag Higher affinity is produced through affinity maturation process → involves introduction of random nucleotide changes into DNA of rearranged Ig variable genes These random nucleotide changes are part of hypermutation - sometimes it gets better/worse If they’re better - survivor signal / if worse - gone, select against Hypermutation mediated by AID and is Ag dependent  High affinity B-cell clones selected to survive/lower affinity clones die via apoptosis Comprised of dark zone (dividing centroblasts) and light zone (selection and maturation of centrocytes - check affinity) ○ Continuous loop - if successful, go back to replicate some more Hypermutation occurs in rapidly dividing centroblasts Mutation occur in centroblast V region genes (CDRs) - every second cell will acquire mutation in receptor at each cell division Strong enrichment of mutation in CDR’s, nucleotide sequence ideal for AID - hotspots, limits mutations in structural pieces, just want to change binding pockets Non dividing centrocytes with high affinity receptors selected for survival Centrocytes will die unless sIg binds to Ag (Ag on surface of FDC) Cells with highest affinity for Ag will be best at competing for Ag on FDC - low affinity cells die by apoptosis Antibodies - the Ultimate Tool Can be used to: label/detect targets ○ Cells ○ Molecules (soluble, solid phase) ○ Tissues ○ Pathogens ○ DNA Cross link molecules Block molecules activate/modulate/kill cells Direct molecules to specific targets Radial Immunodiffusion Holes you punch in gel, put in liquid, well, antibody diffuses into gel, when it recognizes, get the ring precipitate, antigen in well Different size precipitant, bigger = had to diffuse farther to reach zone, started out as more concentration, had to dilute out, has more target Allows for quantification of antigen ***GOOD EXAM QUESTION*** Assay gives same basic info as ELISA But ELISA is faster, more sensitive, often commercially available, so why do RAI? Cheaper, only need 1 antibody (ELISA needs capture/detection AB), gives idea of how many different antigens share epitope “Low tech” Thing in well one that moves Low Immunodiffusion (Ouchterlony) Both Ab/AG diffuse out of wells - put both in well and they both move, get straight lines Recognition of epitopes on antigens by Ab will result in precipitant formation Gain concentration, target identity, target/Ab complexity Wells cut into agar gel, then sample put into well Ag and Ab diffuse out of wells, where they meet = make immune complex that precipitates Antigen multivalent (has more than one antigen where antibodies bind) --? Makes lattices of Ag and Ab. ○ Increasing amount of antigen added to Ab in solution ○ Low concentration = all antibody in the precipitate ○ As more Ab added, protein precipitated increases until zone of equivalence - then precipitation decreases Intersect = non identity continuous line = full identity, antigens identical, specific for antibody in serum ○ spur at one end = partial identity, 2 Ag share at least one epitope  ○ Making basic assays better ○ Easier, safer - quantifying with light (fluorescence) ○ Less sample needed ○ More sensitive, more specific ○ Fewer false positives ○ Faster, more robust ○ Cheaper ○ Higher throughput, automated ELISA enzyme-linked immunosorbent assay Gold standard High sensitivity (pico gram) Automated Not radioactive Lots of samples due to 96 well plates Based on enzyme linked secondary Ab (chromogenes: perioxidase, alkaline phosphatase) - how much antibody sticking to the first antibody End product detected via spectrophotometer (color proportional to Ag-Ab binding) Polyclonal - mix of multiple antibody specificities that recognize different sites (epitopes) on target antigen, easy to make, immunize with target, give booster and then draw blood, blood has antibodies that see target - good enough to use, when you use all of it, you need to make more, might be different second time around, goats have more blood, might see other targets, low affinity Monoclonal - single antibody specificity derived from single, immortal B cell line, choose the best one every well has one kind of antibody, then use another antibody that sees spike and binds to different spot detection antibody sees target - want to know how much it binds - has enzyme or smth on it that changes colour, measures amount of colour is there indirect one , secondary gives higher sensitivity, multiple copies sticking to one antibody, picogram levels, adaptable cover wells in target, measure your own antibodies pregnancy tests are assays, also in covid tests more specific, detergent and gel - single protein suspension, proteins get separated based on size and charge, then transfer to membrane, map of all proteins in sample, then add antibody for  interested antibody → size, shape, complexity Antibody Engineering Efficacy of therapeutic monoclonals often limited for number of reasons ○ Therapeutic Ab antigenic (humans see mouse antibodies as foreign and mount immune response against therapeutic antibody) ○ Too short/too long half-life - might be toxic ○ Poor penetration into tissues (tumours) ○ undesired/inefficient effector functions By mutating/engineering antibody genes in hybridoma cell, one can often reduce/eliminate these issues making antibodies against antibody, antibody itself is antigen, foreign or activate complement Chimeric Ab Can “clone” rearranged VJ and VDJ elements and fuse “variable domains” to constant domains of human Ab gene - transfect back into immortal cell line for production of “recombinant” chimeric Ab proteins Minimal Ag Binding Whole Ab molecule doesn’t recognize target Ag - only 3 complementary determining regions (CDRs) on each heavy/light chain contact and bind Ag Specificity of given Ab entirely determined by 6 regions alone - no need to clone whole variable region, only need to copy 6 CDR regions Humanized Ab Can “clone” CDRs from rearranged mouse heavy and light genes and splice sequences into rearranged human heavy/light chains - transfect back into immortal cell line for production of “recombinant” humanized Ab proteins just CDR is mouse, see as self, all loops are different in humans anyway Antibodies - Therapeutics Highly specific, less side effects, collateral damage → don’t accumulate where you don’t want them  Customizable - can relatively easily retarget therapy to a different cancer, pathogen or keep up with evolving “bugs” Efficient - les drug needed Innovative - bispecific, soluble Fab fragments, antibody-drug conjugates Harness immune system - may replace antibiotics within context multidrug resistant pathogens Science Communication Practice of informing, educating, sharing wonderment, raising awareness of science related topics Science communicators/audiences ambiguously defined, expertise and level of science knowledge varies with each group Science communication may generate support for scientific research/study, inform decision making, including political and ethical thinking Why? Disseminate findings (knowledge users, decision makers, scientists) Solicit input (other opinions, experiences) Make technical knowledge accessible to broader public Inform people of best practice Engage public in what we do Garner support for research activities Now live in info-driven society Social media allows rapid dissemination of info, fact and fiction Public wants to be self-informed Daily decisions made on what people “read online” Training Good at writing scientific manuscripts, presenting at conferences Build on expertise Build relationship with media Participate in science communication activities, community outreach Develop social media presence Contribute to science advocacy Goals Be careful to present fact vs hypothesis/theory Communicate so audience understands Don’t talk down to people Don’t make stuff up Be aware of personal biases Avoid hype Avoid fear Accountability (avoid being anonymous) Integrity How message received is directly linked to your integrity Misleading data, bias, references If given the reason to doubt audience will You are the “expert” responsible for your message Vaccines 12th century  Chinese: inhaled ground crusts from smallpox pustules Turks: inserted crusts from smallpox pustules into cuts in skin Both methods confer smallpox immunity Jenner Called material vaccine - Latin cow (vacca) →vaccination Published findings in 1798 Within 5 years, paper published in languages Within decade, vaccine was used throughout the world Pasteur - Father of “modern” vaccines How to actually weaken pathogen 1879 - discovered pretreatment with attenuated cholera bacteria conferred protection to chickens 1881 - Tested an Anthrax vaccine on sheep - 100% of vaccinated sheep lived, 100% of controls died 1885 - developed rabies vaccine for dogs 1886 - treated Joseph Meister with rabies vaccine Immunity - What is it? Can group immunity into 4 general silos Natural immunity (milk) ○ Immune system not doing anything, evolutionarily derived Induced (artificial) passive immunity (anti-venom) ○ Using antibodies of someone else Natural active (induced) immunity (infection) Induced active immunity (vaccination) Passive Immunity Achieved by preformed immunoglobulins from previously infected/immunized individuals. Equine serum used occasionally, IV injections Provides immediate protection Dissipates after few weeks/months Doesn’t generate memory, you didn’t make them, immunity is lost Passive Immunity - natural Occurs naturally with transfer of maternal antibodies to fetus through placental circulation and breast milk Immune system not doing anything, evolutionarily derived Infants protected for about 6 months Passive Immunity - induced Nonspecific antibody (immunoglobulin) Contains mixture of antibodies reflecting previous exposures of the plasma donors to various antigens Used to prevention or attenuation of illnesses for which there is no specific immunoglobulin preparation IVIg - intravenous immunoglobulin Active Immunity Immunization: gaining protective immunity through exposure to a pathogen (infection, vaccination) →do something and it works, did generate immune response Vaccination: intentionally exposing an individual to pathogen (part of pathogen) with intention of generating protective immunity →try giving immune response  Although goal of vaccination is to immunize ○ Doesn’t always work ○ Administration (injection) ○ Patient genetics - everyone is different, making for most common type of genes ○ Batch quality ○ Pathogen changes Why vaccinate vs immunize ○ Safer ○ Many are non-communicable ○ More controlled ○ Better tested ○ Can’t use “let it burn” mentality for every case ○ Different kinds of infection, intensity of disease when infected Vaccines Any preparation intended to produce immunity to disease by stimulating production of antibodies (WHO) Preparation of killed microorganisms, living attenuated organisms or living fully virulent organisms that is administered to produce or artificially increase immunity to a particular disease (Merriam-Webster) Vaccines most effective and cost-saving tools for disease prevention, preventing untold suffering and saving tens of thousands of lives and billions of dollars in healthcare costs each year (CDC) Only smallpox has ever been eradicated Components of a vaccine 1. Antigen (specific target seen by Adaptive Immune System) Specific target you want immunity against Live, attenuated pathogen/killed pathogen Individual molecules derived from pathogen Chemically modified pathogen molecules DNA coding for pathogen molecules 2. Adjuvant 3. Route and Dose Live Attenuated Killed Subunit or Toxoid weakened/less virulent Whole killed pathogens Specific molecules pathogen (lots of potential isolated from pathogen Actually infect host antigens) Unable to infect host Robust immune response Unable to infect host Weaker, short lived Long lasting memory Weaker, short lived humoral (Ab) immunity Measles, mumps immunity Often requires multiple Often requires multiple boosters boosters Diphtheria, tetanus Polio, influenza Vaccines - Live Attenuated Attenuated microbes multiple in recipient, leading to more robust and long-lasting immune response Elicit strong cellular and humoral responses  Confer lifelong immunity with only one/two doses Possibly that attenuated vaccine strain that could revert to active pathogen, e.g., vaccine-associated poliomyelitis occurs in one out of every 750k doses of live polio vaccine Can’t be given to health professionals or immunocompromised individuals Need to be refrigerated to stay potent Difficult to produce live attenuated vaccines in bacteria because of greater number of genes and thus much more difficult to control Vaccines-Killed Contain inactivated organisms or parts of them e.g. hepatitis A, influenza, polio Have advantage that they pose no risk of vaccine associated infection Inactivation by heat/chemical process/radiation Vaccines are stable and don’t require cold chain, easier to distribute Provide weak/short lived immune response; require several doses, booster shots Components often consist of surface molecules of pathogens that mediate host cell invasion e.g. hemagglutinin and neuraminidase of influenza virus Vaccines-Subunit Consist of antigens (targets) that best stimulate the immune system or specific toxins that mediate disease Methods of preparation ○ Chemical extraction from pathogen ○ Recombinant DNA technology ○ Chemically synthesized Elicit antibody but not cytotoxic T lymphocytes (CTL; T cell) responses Best results with antigens that are conserved e.e.g surface antigen of hepatitis B Major problem for pathogens that are antigenically diverse and variable, e.g. P. falciparum Toxoid Used when bacterial toxin is main cause of illness e.g. diphtheria, tetanus Produced by chemically altering the natural toxin/engineering bacteria to product harmless variants of toxin New Vaccines Trojan horse Engineered DNA/RNA Attach targets from highly virulent pathogen No pathogen to surface of weak pathogen Inoculate with gene encoding pathogen Actually infect host target Robust, long lasting Engages innate immune receptors (RIG-1, No exposure to virulent pathogen TLR9) Engineered viruses Engineered to be “optimal” can target non immunodominant epitopes Vaccines - Engineered Also known as recombinant vector Introduce genes encoding microbial antigens into non-cytopathic/attenuated virus/bacteria and infect individuals Generate true infection Elicit full complement of immune responses, including strong CTL responses  E.g. VSV-EBoV (VSV with Ebola G protein) Vaccines - DNA/RNA Gene for antigen of interest cloned into bacteria plasmid that is engineered to increase bacteria of inserted gene in mammalian cells, no vector Blood, virus vector, bacterial vector, just inject with the plasmid After being injected, plasmid enters host cell where it remains in nucleus Using host cell’s protein synthesis machinery, plasmid DNA directs synthesis of protein it encodes Microbial protein may be presented in context of MHC to elicit T cell responses Elicit designer’s immune responses to proteins other than surface or envelope structures that tend to undergo antigenic variation Zika and dengue virus - protect from one but die from another - dengue was sticking to a weird loop, normally wouldn’t survive without the loop, but we’re just making a vaccine Easy to produce and purify Stability Low cost Potential for genomic integration into host chromosomal DNA and induce mutagenesis and insertional carcinogenesis →why we don’t use DNA vaccines in humans, long term DNA changes Potential for induction of anti-ds-DNA/antibodies against nuclear antigens, leading to autoimmune diseases Covid - mRNA Vaccines New approach to vaccination in humans No pathogen is involved - only genetic sequence coding for viral protein and the body’s own cells make the target protein Gene sequences can be optimized/modified to enhance expression, mutate key amino acids, etc Currently use the whole viral Spike protein - lots of sites for immune recognition - resistant to escape by new virus variants Generates both antibody and T cell immunity mRNA sequence embedded in lipid nanoparticle Lipid nanoparticle allows mRNA to enter patient’s cells and contribute to overall immune activation “Nanoparticle” has short half life and rapidly breaks down patients body (different than carbon or plastic nanoparticles) Vaccine mRNA not stable and breaks down in the patient’s cells (just like your own mRNA, no risk to patient’s DNA) Figure: mRNA instructions to make spike protein → wrapped in lipid nanoparticles, can fuse to membrane and enter cytoplasm → machinery reads and produces spike protein → released to B cells, B cell makes antibodies, some spike displayed on MHC molecules, show T cells, build killer T cells → RNA degrades, spike proteins not made anymore, left with memory B and T cells and protective Ab Advantages Disadvantages Engineer specific immune targets (gene Difficult to make (lipid nanoparticles) sequences) Ultracold storage (limits distribution) Easy to reformulate for new viral strains Need 2 doses (logistics, supply) (new vaccine in weeks if needed)  Elicits both antibody and T cell immunity Can deliver whole proteins, parts of proteins or multiple targets to generate “designer” immune response Case Study - Covid Disease caused by infection with SARS-CoV-2 virus, first appeared 2019, reached pandemic status in early 2020 Within 11 months of ID of SARS-CoV-2 genetic sequence, had multiple approved human vaccines Often people who recover from infection have some degree of immunity Proposed approach for Covid-19 - let people get infected and recover ○ Can’t control “dose” ○ Low risk group might infect others in high risk ○ Higher the virus levels = more difficult to protect high risk group ○ Who is high risk ○ Unclear how long protection will last ○ Emergence of new viral variants Virus ID in december 2019 → genetic sequence made public (Jan 2020), similar to SARS, MERS (draw from experience) → several companies working on coronavirus platforms, new tech, cleared trials → multiple governments and agencies commit to grand challenge, supporting many angles, encouraging stakeholders to come together → massive public support Vaccine Adjuvant Compound added to antigen to make more immunogenic Creates depot effect (retains antigen at sight) Promotes uptake of antigen (phagocytosis) Activates innate immunity (costimulation) Usually mix of oils, metal salts (alums), microbial cell wall components, nucleic acids, preservative (mercury) Retain antigen at injection site, slow release Precipitate antigen - better phagocytosis Activate leukocytes - alum interacts with phagocyte membranes - microbial component, DNA - bind immune receptors → activation Modulate and enhance type of immune response induced by Ag alone Improve uptake of Ag ○ Alum (gel like matrix) Depot effect Increases innate immune cells Triggers lysosomal damage and activation of NLRP3 inflammasome ○ Oil and water emulsions ○ Liposomes ○ microspheres/nanoparticles Enhance immune response by targeting Ag to immune cells, enhancing phagocytosis/activating APC ○ Monophosphoryl lipid A (MPL) ○ CpG ○ Flagellin Route and Dose ROUTE Subcutaneous and intramuscular - slow absorption, transport to local lymph nodes - systemic immunity intranasal/oral - rapid absorption, involvement of mucosal lymphoid tissues - mucosal immunity DOSE Response to prime boost regimen 5-10x higher than to either alone T cell memory can be strongly induced using prime-boost strategies, boosters better than one large dose (tolerance) Figure: Second response will be faster and better → want to replace initial parts with the vaccine Physically how the vaccine does it Get a shot → mix of adjuvant and antigen in shot → antigen drains through lymphatics, lymph node → sticky so they’ll keep at injection site → macrophages can eat/dendritic cells → internalize and migrate to lymph node → inside lymph node: antigens coming in as free antigens, taken up by macrophages, DC and B cells → antigen loaded DC also coming in → educate lymphocytes → antigen presented → activate killer T cells/helper T cells → make a whole bunch of killer T cells → some grow up to be memory/CD4’s make cytokines → T cells help B cells, isotype class switching → make a lot of antibodies, become plasma cells → some also become memory → ultimate goal to make memory cells and plasma cells → can get holistic immune response