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Immunology & immunological preparation

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Immunology & immunological preparation

  1. 1. Immunology & Immunological Preparations 1 By: Bijaya Kumar Uprety
  2. 2. Immunology •Branch of biological science concerned with the study of immunity, •Or concerned with the structure and function of immune system. Immunity •Latin term immunismeaning “exempt”. •Immunity means the state of protection from infectious disease. 2
  3. 3.       Year Name Event 430 B.C –Earliest written reference to the phenomenon of immunity. Thucydides (great historian of the Peloponnesian war). In describing plague in Athens, he wrote during the war, only who had recovered from the plague could nurse the sick because they would not get the disease for the second time. 15thcentury-First record to induce immunity deliberately. Chinese and Turks Dried crust derived from small pox pustules were either inhaled into the nostrils or inserted into small cuts in the skin (technique known as variolation). 1718 Lady Mary Wortley (wife of british ambassador to Constantinople) Performed variolationon her own children after realizing the technique was effective among her native people. 1798 Edward Jenner (physician) Propounded an idea that introducing fluid from a cowpox pustule into people might protect them from smallpox and he tested his idea on eight year old kid which was successful. 3
  4. 4. Louis Pasteur grew the fowl cholera causing bacterium in culture and when this was injected it into chicken they developed cholera but later on when he once again injected them with the same old culture they got ill but recovered later on. He grew fresh culture and tried it on same chickens. They completely recovered. Hence, Hypothesized and proved that aging had weakened the virulence of the pathogen and concluded that the attenuated strain might be administered to protect against the disease. He called this attenuated strain a vaccine. 4
  5. 5. Continue….. Laterextendedhisfindingstootherdiseasesanddemonstrateditispossibletoattenuate,orweakenapathogenandadministerthemtouseitasavaccine. In1881,Pasteurvaccinatedonegroupofsheepwithheat-attenuatedanthraxbacillus(bacillusanthracis) andleftanothergroupofunvaccinatedsheep.Allunvaccinatedsheepdiedwhileotherlived. Thiswasthebeginningsofthedisciplineofimmunology.In1885,hefirstadministeredhisfirstvaccinetoahuman(ayoungboy)againstrabies. 5
  6. 6. Pasteur proved vaccination worked but didn’t know how it worked. In 1890, Emil von Behring and Shibasaburo Kitasato gave first insight into the mechanism of immunity Got nobel prize in 1901. They demonstrated that serum from animals previously immunized to diptheria could transfer the immune state to unimmunized animals. 6
  7. 7. Duringnextdecade,itwasdemonstratedbyvariousresearchersthatanactivecomponentfromimmuneserumcouldneutralizetoxins,precipitatetoxinsandagglutinatebacteriaandactiveagentwasnamedforitsactivityitexhibited:antitoxin,precipitinandagglutininresp. Initiallydifferentserumcomponentwasthoughttoberesponsibleforeachactivitybutduring1930,ElvinKabat(mainlyhim)foundthatgamma-globulin(nowimmunoglobulin,alsoafractionofserum)wasresponsibleforallthese. 7
  8. 8. The active molecules in the immunoglobulin fraction are called antibodies. Because the immunity was mediated by antibodies contained in the fluids (known at that time as humors), it was called humoral immunity. In 1883, Elie Metchnikoff demonstrated that cells also contribute to the immune state of an animal. He hypothesized that cells rather than serum components were major effector of immunity.(term phagocytes was coined and an idea of cell-mediated immunity dvpt). Controversy developed between two concepts. 8
  9. 9. But latter proved that both were correct. Immunity requires both humoraland cellular responses. In 1950, lymphocyte was identified as the cell responsible for both cellular and humoralimmunity and experiments on chicken pioneered by Bruce Click at Mississippi State University indicated that there are two types of lymphocytes. 1.T-lymphocytes derived from thymus mediated cellular immunity. 2.B-lymphocytes from bursa of Fabriciuswere involved in humoralimmunity. Both these systems work hand in hand to protect our body against various foreign attack. 9
  10. 10. Introduction to Immune system Remarkably versatile defense system that protect animals against various invading micro-organisms and cancer. Able to generate enormous variety of cells and molecules capable of specifically recognizing and eliminating large variety of foreign invaders. Invaders human bodyimmune system respondeliminate or destroys the invaders 10
  11. 11. Bodyendowedwithdifferentdefensesystem. Atfirst,externaldefensesystemcomesintoplaywhichincludes,skin,secretionofmucus,ciliaryaction, lavagingactionofbactericidalfluids(e.g.tears), gastricacidandmicrobialantagonism. Ifpenetrationoccurs,bacteriaaredestroyedbysolublefactorssuchaslysozymeandbyphagocytosiswithintracellulardigestion. 11
  12. 12. Functionally, immune response can be divided into two related activities- 1.Recognition –Remarkable for its specificity. 2.Response. Immune system is able to recognize subtle chemical differences that recognize one foreign pathogen from another. Able to discriminate between foreign molecules and the body’s own cells and proteins. 12
  13. 13. Once a foreign organism has been recognized, it recruits a variety of cells and molecules to mount an appropriate response, called effectorresponse, to eliminate or neutralize the organism. The immune response enables the elimination or neutralization of the cells/molecules (pathogens) from the body. Convert initial recognition event variety of effectorresponseseliminate or neutralize particular pathogen. 13
  14. 14. Later exposure to the same foreign organism induces a memory response, characterized by a more rapid and heightened immune reaction that serves to eliminate the pathogen and prevent disease. 14
  15. 15. Types of immunity Two types of immunity: 1. Innate or nonspecific immunity. 2. Acquired or specific immunity. Innate immunity: It is the basic resistance to diseases that an individual has from the time of its birth. Not specific to any one pathogen but rather constitutes a first line of defense. 15
  16. 16. It consists of following four types of defensive barriers: 1.Anatomic barriers 2.Physiologic barriers 3.Endocytosis/phagocytosisbarriers 4.Inflammatory barriers 16
  17. 17. 17
  18. 18. •Endocytosis-Processofcellularingestionofmacromoleculesbyinvaginationofplasmamembranetoproduceanintracellularvesiclewhichenclosestheingestedmaterial. •3types- Phagocytosis(forparticulates), Pinocytosis(liquid), Receptormediatedendocytosis(LDL). •Mostphagocytosis(mostcommon)isdonebybloodmonocytes,neutrophils, andtissuemacrophages. Fig. Steps in phagocytosis of a bacterium. 18
  19. 19. Fig 2 showing the major events in the inflammatory response.[ vasoactive and chemotactic factors i.e kinin and histamine. Additionally , bradykinins which is a type of kinin stimulate pain receptors and fibrin-clot] 19
  20. 20. Acquired Immunity Also known as adaptive immunity. Capable of recognizing and selectively eliminating specific foreign microorganisms and molecules (i.e. foreign antigens). Displays four characteristic attributes: 1.Antigenic specificity 2.Diversity 3.Immunologic memory 4.Self/nonselfrecognition. 20
  21. 21. Components of Acquired Immunity Involves the following two major groups of cells 1.Lymphocytes which includes B and T lymphocytes. 2. Antigen presenting cells (APCs)- Group of B-cells, dendriticcells and macrophages. They express class II MHC molecules on their membranes & They are able to deliver a co-stimulatory signal that is necessary for THcell activation. APC have Class II MHC (major –histocompatibilitycomplex) molecules on their surfaceMHC molecules bind to antigen derived peptidespresent them to lymphocytes immune system activated. 21
  22. 22. B lymphocytes Blymphocytesmaturewithinthebonemarrow;whentheyleaveit,eachexpressesauniqueantigen-bindingreceptoronitsmembrane.Thisantigen-bindingorB-cellreceptorisamembrane-boundantibodymolecule. Antibodiesareglycoproteinsthatconsistoftwoidenticalheavypolypeptidechainsandtwoidenticallightpolypeptidechains.Eachheavychainisjoinedwithalightchainbydisulfidebonds,andadditionaldisulfidebondsholdthetwopairstogether. Theamino-terminalendsofthepairsofheavyandlightchainsformacleftwithinwhichantigenbinds. WhenanaiveBcell(onethathasnotpreviouslyencounteredantigen)firstencounterstheantigenthatmatchesitsmembraneboundantibody,thebindingoftheantigentotheantibodycausesthecelltodividerapidly;itsprogenydifferentiateintomemoryBcellsandeffectorBcellscalledplasmacells. MemoryBcellshavealongerlifespanthannaivecells,andtheyexpressthesamemembrane-boundantibodyastheirparentBcell. Althoughplasmacellsliveforonlyafewdays,theysecreteenormousamountsofantibodyduringthistime.Ithasbeenestimatedthatasingleplasmacellcansecretemorethan2000moleculesofantibodypersecond.Secretedantibodiesarethemajoreffectormoleculesofhumoralimmunity. 22
  23. 23. 23
  24. 24. 24
  25. 25. T lymphocytes Tlymphocytesalsoariseinthebonemarrow.UnlikeBcells,whichmaturewithinthebonemarrow,Tcellsmigratetothethymusglandtomature. Duringitsmaturationwithinthethymus,theTcellcomestoexpressauniqueantigen-bindingmolecule,calledtheT-cellreceptor,onitsmembrane. Unlikemembrane-boundantibodiesonBcells,whichcanrecognizeantigenalone,T-cellreceptorscanrecognizeonlyantigenthatisboundtocell- membraneproteinscalledmajorhistocompatibilitycomplex(MHC) molecules. MHCmoleculesthatfunctioninthisrecognitionevent,whichistermed“antigenpresentation,”arepolymorphic(geneticallydiverse)glycoproteinsfoundoncellmembranes. TherearetwomajortypesofMHCmolecules: ClassIMHCmolecules,whichareexpressedbynearlyallnucleatedcellsofvertebratespecies,consistofaheavychainlinkedtoasmallinvariantproteincalled2-microglobulin. ClassIIMHCmolecules,whichconsistofanalphaandabetaglycoproteinchain,areexpressedonlybyantigen-presentingcells. 25
  26. 26. WhenanaiveTcellencountersantigencombinedwithaMHCmoleculeonacell, theTcellproliferatesanddifferentiatesintomemoryTcellsandvariouseffectorTcells. Therearetwowell-definedsubpopulationsofTcells:Thelper(TH)andTcytotoxic(TC)cells.AlthoughathirdtypeofTcell,calledaTsuppressor(TS)cell, hasbeenpostulated,recentevidencesuggeststhatitmaynotbedistinctfromTHandTCsubpopulations. ThelperandTcytotoxiccellscanbedistinguishedfromoneanotherbythepresenceofeitherCD4orCD8membraneglycoproteinsontheirsurfaces.TcellsdisplayingCD4generallyfunctionasTHcells,whereasthosedisplayingCD8generallyfunctionasTCcells.THcellsgenerallyrecognizeantigencombinedwithclassIImolecules,whereasTCcellsgenerallyrecognizeantigencombinedwithclassImolecules. AfteraTHcellrecognizesandinteractswithanantigen–MHCclassIImoleculecomplex,thecellisactivated—itbecomesaneffectorcellthatsecretesvariousgrowthfactorsknowncollectivelyascytokines.ThesecretedcytokinesplayanimportantroleinactivatingBcells,TCcells,macrophages,andvariousothercellsthatparticipateintheimmuneresponse. 26
  27. 27. DifferencesinthepatternofcytokinesproducedbyactivatedTHcellsresultindifferenttypesofimmuneresponse. UndertheinfluenceofTH-derivedcytokines,aTCcellthatrecognizesanantigen–MHCclassImoleculecomplexproliferatesanddifferentiatesintoaneffectorcellcalledacytotoxicTlymphocyte(CTL). IncontrasttotheTHcell,theCTLgenerallydoesnotsecretemanycytokinesandinsteadexhibitscell-killingorcytotoxicactivity. TheCTLhasavitalfunctioninmonitoringthecellsofthebodyandeliminatinganythatdisplayantigen,suchasvirus- infectedcells,tumorcells,andcellsofaforeigntissuegraft. CellsthatdisplayforeignantigencomplexedwithaclassIMHCmoleculearecalledalteredself-cells;thesearetargetsofCTLs. 27
  28. 28. ANTIGEN-PRESENTING CELLS Activationofboththehumoralandcell-mediatedbranchesoftheimmunesystemrequirescytokinesproducedbyTHcells. ItisessentialthatactivationofTHcellsthemselvesbecarefullyregulated,becauseaninappropriateT-cellresponsetoself- componentscanhavefatalautoimmuneconsequences. ToensurecarefullyregulatedactivationofTHcells,theycanrecognizeonlyantigenthatisdisplayedtogetherwithclassMHCIImoleculesonthesurfaceofantigen-presentingcells(APCs). Thesespecializedcells,whichincludemacrophages,Blymphocytes, anddendriticcells,aredistinguishedbytwoproperties:(1)theyexpressclassIIMHCmoleculesontheirmembranes,and(2)theyareabletodeliveraco-stimulatorysignalthatisnecessaryforTH-cellactivation. Antigen-presentingcellsfirstinternalizeantigen,eitherbyphagocytosisorbyendocytosis,andthendisplayapartofthatantigenontheirmembraneboundtoaclassIIMHCmolecule.TheTHcellrecognizesandinteractswiththeantigen–classIIMHCmoleculecomplexonthemembraneoftheantigen-presentingcell.Anadditionalcostimulatorysignalisthenproducedbytheantigen- presentingcell,leadingtoactivationoftheTHcell. 28
  29. 29. 29
  30. 30. 30
  31. 31. HumoralImmune Responses It is based on antibodies. It can be conferred on nonimmune individuals by administration of serum antibodies from an immune individual. Antibodies act as an effector of humoral response. They bind to the antigens and facilitate their elimination. Elimination could be in various ways. 31
  32. 32. Fig showing the structure of Antibody. 32
  33. 33. 1. By forming clusters through cross-linking of antigen molecules, which are readily ingested by phagocytic cells. 2. By binding of antibodies to a microorganism can activate the complement system, which lyses the mo’s. 3. Antibodies bind to toxins and viral particles, and prevent their subsequent binding to host cells. 33
  34. 34. Cell-mediated Immune Responses Based on T cells, which are a type of lymphocyte. T cells are of the following two types: 1.T helper (TH) 2.T cytotoxic(TC) cells. TH cell interacts with an antigen –MHC II molecule complex present on an APC cytokines secreted cytokines activate B cells, TcCells, and various phagocyticcells. 34
  35. 35. Activatedphagocyticcellsabletokillmo’s(bacteriaandprotozoa). WhenTccellinteractswithanantigen-MHCIcomplex,theTccellproliferatesundertheinfluenceofcytokinesproducedbyactivatedTHcells. TheseTccellsdifferentiateintocytotoxicTlymphocytes(CTLs).TheCTLskillallsuchcellsthatdisplayforeignantigenscomplexedwithMHCImolecules.Suchcellsarecalledalteredself-cells,theyareusuallyvirus-infectedcells,tumorcellsandforeigntissuecells. 35
  36. 36. Thus TH cells and CTLs are effectors of the cell- mediated immune response. 36
  37. 37. Fig 2. Overview of humoral and cell mediated immune responses. 37
  38. 38. Passive Immunization Itistheadministrationofpreformedantibodies(usuallyIgG)eitherintravenouslyorintramuscularly. UsedtoproviderapidprotectionincertaininfectionssuchasdiptheriaortetanusorintheeventofaccidentalexposuretocertainpathogenssuchashepatitisB. Alsousedtoprovideprotectioninimmunecompromisedindividualswhoareunabletoproduceappropriateantibodyresponseorinsomeinstancesincapableofmakinganyantibodyatall(i.e.severecombinedimmunodeficiency). 38
  39. 39. Antibodies given to immune deficient patients are usually IgG-derived from pooled normal plasma and are administered on a continuous basis (ideally every three weeks) as they are continuously catabolized and are effective only for short duration. Preformed antibodies from animals, notably horse are also administered for some diseases but it presents a danger of immune complex formation and serum sickness (if repetitively injected). 39
  40. 40. Active immunization Administration of vaccines containing microbial products with or without adjuvantsin order to obtain long term immunological protection against the offending microbe. 2 types: 1.Systemic Immunization. 2. Mucosal Immunization. 40
  41. 41. Systemic Immunization This is the method of choice at present for most vaccinations. Carried out by injecting vaccine subcutaneously or intramuscularly into the deltoid muscle. Ideally all vaccines given soon after birth but some deliberately delayed. Common egincludes vaccines for measles, mumps, and rubella usually given at the age of 1. If given earlier maternal antibody would decrease their effectiveness. 41
  42. 42. ButcarbohydratevaccinesforPneumococcus, Meningococcus,andHaemophilusinfectionsaregivenatabout2yearsasbeforethisagetheyrespondpoorlytopolysaccharidesunlesstheyareassociatedwithproteincomponentsthatcanacttorecruitTcellhelpfordevelopmentofanti-polysaccharideantibody. 42
  43. 43. Mucosal Immunization Mostoftheinfectiousagentsgainentrytothesystemicsystemthroughmucosalrouteandthelargestsourceoflymphoidtissueisalsopresentatthemucosalsurfaces. Thusrecentvaccinationapproacheshavefocussedonthemucosalrouteasthesiteofchoiceforimmunizationeitherorallyorthroughthenasalassociatedimmunetissue(NALT). Moreover,iteliminatetheneedforpainfulinjectionandallowforself-administrationofcertainvaccinessuchasthoseforimmunizationagainstinfluenza. 43
  44. 44. Adjuvant vaccines and live vector vaccines have been used to target mucosal immune system with some success. Attenuated strains of salmonella can act as a powerful immune stimulus as well as acting as carriers of foreign antigens. This approach has been used to immunize against mucosal surfaces against herpes simplex virus and human papillomavirus. Bacterial toxins, egthose derived from cholera, E. coli etc which posses immunomodulatoryproperties are also being exploited in the dvptof mucosallyactive adjuvants. 44
  45. 45. Table 1. Passive immunization 45
  46. 46. Vaccine Vaccineisapreparationcontainingapathogen(diseaseproducingorganism)eitherinattenuatedorinactivatedstate. Thispreparationisintroducedintoanindividualtoinduceadequateantibodyproductionagainstthepathogeninquestionsothattheindividualbecomesprotectedagainstinfection,atalaterdate,bythatpathogen. Theintroductionofavaccineinanindividualiscalledvaccinationorimmunizationasitleadstothedevelopmentofimmunityinthevaccinatedindividualstotheconcernedpathogen. 46
  47. 47. The immunity is induced by the antigens of pathogen origin present in the vaccine. Conventionally, various vaccines can be broadly classified into two groups: 1. Vaccines containing killed or inactivated pathogens, i.e. most bacteria vaccines and some virus vaccines (e.g. influenza virus inactivated by formalin, rabies virus inactivated by phenol and β-priolactone). 2. Those containing live but attenuated pathogens, e.g., most virus vaccines. 47
  48. 48. Attenuation means a drastic reduction in the virulence of a pathogen which is achieved as follows: Several consecutive passages through an animal, which is not the usual host of the pathogen, e.g., small pox virus in calf. Several passages through cultured cells of the host, e.g., rabies virus in human diploid cell culture, or of a different species, e.g., rabies virus, yellow fever virus in chick embryo cell culture. Selection of less virulent strains of pathogens, e.g., a mutant strain of polio virus. 48
  49. 49. Treatment of the pathogen with some chemicals, e.g., B.C.G. (Bacillusof CalmetteGuerien) vaccine produced by culturing the bacteria on a medium containing bile. Culturing pathogens under unfavourableconditions like high temp, e.g., anthrax vaccine obtained by cultivation of the bacterium (Bacillus anthracis) at 40- 50 0C. In general, inactivation of virus is always coupled with attenuation to minimize the accidental presence of active virulent particles which could cause disease in the vaccinated individuals. 49
  50. 50. Thedifferentvaccinesdifferintheircomposition, efficacyandthedurationofeffectiveprotectiontothevaccinatedindividuals.Theseareoneoftheearliestexamplesofbiotechnologicalinterventioninhumanandanimalhealthcare. 50
  51. 51. Types of various vaccines There are various types of vaccines, 1.Whole-Organism Vaccines (Conventional vaccines) 2. Purified Macromolecules as Vaccines (Conventional ) 3.Subunit Vaccine 4.Recombinant-Vector Vaccines 5.DNA Vaccines 51
  52. 52. Whole organism vaccines Manyvaccinesnowavailableforhumans,andanimalusearemadeusingwholeorganisms(bacteriaorvirus),eitherintheinactivated(killed)formorattenuated(livebutavirulent)form. Examples:Salmonellatyphi(killedbacteria)againstTyphoid,Salmonellaparatyphi(killedbacteria)againstparatyphoid,Vibriocholerae(killedcellsorcellextract)againstcholera,Attenuatedvirusagainstyellowfever,measles,mumps,rubellaandpolio. 52
  53. 53. PreparationandstorageofTyphoid-ParatyphoidAandBVaccine[TAB- Vaccine] Typhoidfever(entericfever)isanacutegeneralizedinfectioncausedbySalmonellatyphi;whereas,paratyphoidfeveriscausedbySalmonellaparatyphiAandSalmonellaparatyphiB. Preparation (1)Thevaccineispreparedbythegeneralprocessandcontainsthefollowingineachmillilitre:Typhoidbacilli(Salmonellatyphi):1000millionParatyphiAbacilli(S.paratyphiA)andParatyphiBbacilli(S.paratyphiB):500or750million. (2)ThesmoothstrainsofthethreeorganismsknowntoproducethefullcomplementofOsomaticantigensshouldbeused.ThisspecificstrainofS. typhimustcontainthevirusassociatedantigens(Vi-antigen). (3)Ithasbeendulyestablishedthatwhentheorganismswerekilledwith75% ethanolandtheresultingvaccinepreservedwith22.5%ethanol,thepotencyofthealcoholtreatedvaccinewasfoundtobealmostdoubletothatoftheheat-treatedvaccine,therebyminimizingthepossibilityofbothlocalandconstitutionalreactionwiththerelativelysmallerdose.Besides,alcoholtreatedvaccinesdidpossessdefinitelyandpredominantlylongerlifeundertheoptimalstorageconditions[viz.,storagebetween2-4°Cwithoutallowingthevaccinetofreeze]. 53
  54. 54. Purified macromolecules as vaccine The purified antigenic portions from the bacterial cell wall, or viral coat protein are used as vaccines, and they can elicit immune reaction. Examples of macromolecule vaccines are: 1.Capsular polysaccharides 2.Surface antigens 3.And inactivated exotoxinscalled toxoids. The macromolecule vaccines are generally safe since they don’t contain live organism. Example is given in next slide. 54
  55. 55. Preparation of Meningococcal Polysaccharide Vaccine TheMeningococcalPolysaccharideVaccineconsistsofoneormorepurifiedpolysaccharidesobtainedfromappropriatestrainsofNeisseriameingitidisgroupA, groupC,groupYandgroupW135thathavebeenadequatelyprovedtobecapableofproducingpolysaccharidesthatareabsolutelysafeandalsocapableofinducingtheproductionofsatisfactorylevelsofspecificantibodyinhumans. Thevaccineispreparedimmediatelybeforeusebyreconstitutionfromthestabilizeddriedvaccinewithanappropriateprescribedsterileliquid.Itmayeithercontainasingletypeofpolysaccharideoranymixtureofthetypes. 55
  56. 56. The various Preparation steps adopted are as stated under : (1)Thepreparationofthevacccineisbasedonaseed-lotsystem.Eachseed-lotissubjectedtomicrobiologicalexaminationbycultureinanappropriatemediaandmicroscopicexaminationofGram-stainedsmears. (2)Thepolysaccharideshowntobefreefromcontaminatingbacteriaisprecipitatedbytheadditionofcetrimoniumbromideandthenpurified. (3)Eachpolysaccharideisdissolvedunderasepticconditionsinasterilesolutioncontaininglactoseoranothersuitablestabilizingmediumforfreezedrying. (4)Thesolutionisblended,ifappropriate,withsolutionofthepolysaccharidesofanyoralloftheothergroupsandpassedthroughabacteria-retentivefilter. (5)Finally,thefiltrateisfreezedriedtoamoisturecontentshowntobefavourabletothestabilityofthevaccine 56
  57. 57. Limitation of Conventional Vaccines Notallinfectiousagentscanbegrownincultureandnovaccineshavebeendevelopedforanumberofdiseases,wheretheinfectiousagentisnonculturable. Productionofanimalandhumanvirusesrequiresanimalcellculture,whichisexpensive. Yieldandrateofproductionofanimalvirusesislow. Extensivelaboratoryprecautionsareneededwhiledealingwithhighlyinfectiousagents. Inspiteofthebestprecautions,somebatchesofvaccinesmaynotbecompletelykilledorattenuated. Attenuatedstrainsmayreverttopathogenicstate,occasionally, andmaycauseactualdiseaseagainstwhichprotectionwassought. Notallinfectiousdiseasearepreventablebytraditionalvaccines(e.g.AIDS). HavelimitedShelf-lifethusrequiresrefrigeration. 57
  58. 58. Vaccines made through recombinant DNA technology RecombinantDNAtechnologycanbebestusedinthefollowingwaysinvaccinedevelopment. 1.Virulencegenescanbedeletedfromtheinfectiousagentretainingtheimmunogenicproperties. 2.Anorganism(non-pathogenic)carryingantigenicdeterminantscanbecreatedbyinsertionofthegenescodingfortheantigenicproteins. 3.Fornon-culturableagents,genesfortheprotein(criticalantigenicdeterminants)canbeclonedandexpressedinanexpressionvector(e.g.E.Colioramammaliancellline). 4.Atargetedcell-specifickillingsystemthatkillsonlytheinfectedcellscanbedesigned.Inthistechnique,genefora‘fusionprotein’isconstructed.First,onepartofthefusionproteinbindstotheinfectedcell.Thentheotherpartkillstheinfectedcell. 58
  59. 59. Subunit Vaccines Forviruses,ithasbeenshownthatspecificproteinfromthecoatorenvelopeisenoughtoelicittheimmuneresponse. Vaccineswithcomponentsofapathogenicorganismratherthanthewholeorganismarecalledsubunitvaccines.rDNAtechnologyisbestsuitedtodevelopsubunitvaccines. Purifiedproteinsaremorestableandarechemicallypreciseandsafefromsideeffects.However,purificationofproteincanbeexpensiveandsometimespurificationcanaltertheconfigurationofproteinandalteritsantigenicity!!! Thesefactorshavetobeassessedbeforemakingaproteinpreparation. OneoftheexampleofsubunitvaccinesdevelopedthroughrDNAtechwillbediscussedintheupcomingslides. 59
  60. 60. Subunit vaccine for foot and mouth disease virus (FMDV) Formalin-killedFMDVwasusedasvaccineearlier.ThegenomeofFMDVissinglestrandedRNA(ssRNA). ThecDNAcomplementarytothisssRNA,8000nucleotidelongisprepared.Itisdigestedwithrestrictionenzymes,andthefragmentsareclonedinE.coli. Referfigure.Whiteboard!!!!!!!!!!!!!!!! 60
  61. 61. Recombinant vector vaccines Genesthatencodemajorantigensofespeciallyvirulentpathogenscanbeintroducedintoattenuatedvirusesorbacteria. Theattenuatedorganismservesasavector,replicatingwithinthehostandexpressingthegeneproductofthepathogen. Anumberoforganismshavebeenusedforvectorvaccines, includingvacciniavirus(itismoststrongcandidateasitisefficientindeliveryandexpressionofclonedgenes), thecanarypoxvirus,attenuatedpoliovirus,adenovirus, attenuatedstrainsofSalmonella,theBCGstrainofMycobacteriumbovisandcertainstrainsofstreptococcusthatnormallyexistintheoralcavity. 61
  62. 62. Vacciniavirushasbeenwidelyemployedasavectorvaccine.Thislarge,complexvirus,withagenomeofabout200genes,canbeengineeredtocarryseveraldozenforeigngeneswithoutimpairingitscapacitytoinfecthostcellsandreplicate. Theprocessofproducingavacciniavectorthatcarriesaforeigngenefromapathogenisoutlinedinfigurebelow. Thegeneticallyengineeredvacciniaexpresseshighleveloftheinsertedgeneproduct,whichcanthenserveasapotentimmunogeninaninoculatedhost. 62
  63. 63. 63
  64. 64. Likethesmallpoxvaccine,geneticallyengineeredvacciniavectorvaccinescanbeadministeredsimplybyscratchingtheskin,causinglocalizedinfectioninthehostcells. AntigengenesintroducedintoanimalcellsthroughvacciniavirusgenomeincluesRabiesvirusGprotein, HepatitisBsurfaceantigen,InfluenzavirusNPandHAproteins,etc. Iftheforeigngeneproductexpressedbythevacciniaisaviralenvelopeprotein,itisinsertedintothemembraneoftheinfectedhostcell,inducingdevelopmentofcell- mediatedimmunityaswellasantibodymediatedimmunity. Similartovacciniavectorvaccinesothervectorvaccineswhichhavebeenrecentlytriedincludecanarypoxvirus. 64
  65. 65. DNA vaccines/ Gene vaccine (Genetic Immunization) PlasmidDNAencodingantigenicproteinsisinjecteddirectlyintothemuscleoftherecipient.MusclecellstakeuptheDNAandtheencodedproteinantigenisexpressed, leadingtobothahumoralandcell-mediatedresponse. TheDNAeitherintegrateintothechromosomalDNAortobemaintainedforlongperiodsinanepisomalform. Itoffersadvantageovermanyoftheexistingvaccinesfewofwhicharelistedbelow: 1.Theencodedproteinisexpressedinthehostinitsnaturalform-thereisnodenaturationormodification.Duetothistheimmuneresponseisthereforedirectedtotheantigenexactlyasitisexpressedbythepathogen. 65
  66. 66. 2. It induces both humoral and cell mediated immunity. 3. DNA vaccines cause prolonged expression of the antigen, which generates significant immunological memory. 4. Refrigeration is not required for handling and storage of the plasmid DNA (thus lowers cost and complexity of delivery). 5. The same plasmid vector could be custom tailored to make variety of proteins, so the same manufacturing techniques can be used for different DNA vaccines, each encoding an antigen from a different pathogen. 66
  67. 67. AnimprovedmethodofadministeringthesevaccinesinvolvescoatingmicroscopicgoldbeadswiththeplasmidDNAandthendeliveringthecoatedparticlesthroughtheskinintotheunderlyingmusclewithanairgun(calledgenegun).Thiswillallowrapiddeliveryofavaccinetolargepopulationswithouttherequirementforhugesuppliesofneedlesandsyringes. TestofDNAvaccinesinanimalmodelshaveshownthesevaccinestobeeffectiveagainstvariousviraldiseasesincludinginfluenzavirus. 67
  68. 68. Antigen-Antibody Interaction Theantigen-antibodyinteractionisabiomolecularassociationsimilartoanenzyme-substrateinteraction. However,itdoesn’tleadtoanirreversiblechemicalalterationineithertheantibodyortheantigen. Theassociationbetweenanantigenandantibodyinvolvesvariousnoncovalentinteractionsbetweentheantigenicdeterminant(epitope)oftheantigenandthevariable-region(VH/VL)domainoftheantibodymolecule,particularlythehypervariableregions,orcomplementarity-determiningregions(CDRs). 68
  69. 69. 69
  70. 70. Thenoncovalentinteractionsthatformthebasisofantigen-antibodybindingincludehydrogenbonds, ionicbonds,hydrophobicinteractions,andvanderWaalsinteractions. Sincetheseinteractionsareindividuallyweak(comparedwithacovalentbond),alargenumberofsuchinteractionsarerequiredtoformastrongAg-Abinteraction. Furthermore,eachofthesenoncovalentinteractionsoperatesoveraveryshortdistance(1angstromor1x10-7mm).HenceastrongAg-Abinteractiondependsonaveryclosefitbetweentheantigenandantibody. Suchfitrequireahighdegreeofcomplementaritybetweenantigenandantibody. 70
  71. 71. Cross-reactivity AlthoughAg-Abreactionsarehighlyspecific,insomecasesantibodyelicitedbyoneantigencancross-reactwithanunrelatedantigen.Suchcross-reactivityoccursiftwodifferentantigensshareanidenticalorverysimilarepitope. Cross-reactivityisoftenobservedamongpolysaccharideantigensthatcontainssimilaroligosaccharideresidues. TheABOblood-groupantigens,forexample,areglycoproteinsexpressedonRBCs.SubtledifferencesintheterminalresiduesofthesugarsattachedtothesesurfaceproteinsdistinguishtheAandBbloodgroupantigens. RBCglycoproteinsugarsattachedtotheterminalendofitsubtledifferenceintheterminalresiduesofthesesugarsdistinguishAandBbloodgroupantigens. 71
  72. 72. Anindividuallackingoneorbothoftheseantigenswillhaveserumantibodiestothemissingantigen(s). Theantibodiesareinducednotbyexposuretoredbloodcellantigensbutbyexposuretocross-reactingmicrobialantigenspresentoncommonintestinalbacteria. Thesemicrobialantigensinduceformationofantibodiesinindividualslackingthesimilarblood-groupantigensontheirRBCs. Theblood-groupantibodies,althoughelicitedbymicrobialantigens,willcross-reactwithsimilaroligosaccharidesonforeignRBCs.Thisprovidesthebasisforbloodtypingtestsandaccountsforthenecessityofcompatiblebloodtypesduringbloodtransfusions.TypeAindividualhasanti-Bantibodies,typeBhasanti-AandtypeOhasanti-A&B. 72
  73. 73. Numerousvirusesandbacteriahaveepitopesidenticalorsimilartonormalhost-cellcomponents.Insomecases,thesemicrobialantigenshaveshowntoelicitantibodythatcross-reactswiththehost-cellcomponents,resultinginatissue-damagingautoimmunereaction. Fore.g.bacteriumStreptococcuspyrogenes,expresscellwallproteinscalledMantigens.Absproducedagainsttheseantigenshaveshowntocrossreactwithseveralmyocardialandskeletalmuscleproteinscausingkidneyandheartdamagefollowingstreptococcalinfections. Somevaccinesalsoexhibitcross-reactivity. 73
  74. 74. 74
  75. 75. 1. Precipitation Reactions Antibodyandsolubleantigeninteractinginaqueoussolutionformalatticethateventuallydevelopsintoavariableprecipitate. Antibodiesthataggregatesolubleantigensarecalledprecipitins. AlthoughformationofthesolubleAg-Abcomplexoccurswithinminutes,formationofthevisibleprecipitateoccursmoreslowlyandoftentakesadayortwotoreachcompletion. FormationofAg-Ablatticedependsuponthevalencyofboth: 1.Abshouldbebivalent;aprecipitatewillnotformwithmonovalentFabfragments. 2.Agmustbeeitherbivalentorpolyvalenti.e.itmusthaveatleasttwocopiesofthesameepitopeorhavedifferentepitopesthatreactwithdifferentantibodiespresentinpolyclonalantisera. 75
  76. 76. A. Precipitation reaction in fluids Precipitation reaction in fluids yields a precipitin Curve. A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amount of antigen to the tubes. At one time this method was used to measure the amount of antigen or antibody present in a sample of interest. Once precipitate is formedeach tube centrifuged to pellet the precipitatesupernatant poured off amount of precipitate is measured. 76
  77. 77. Plottingtheamountofprecipitateagainstincreasingantigenconcentrationsyieldsaprecipitincurve. Thefigurebelowshowsthattheexcessofeitherantigenorantibodiesinterfereswithmaximalprecipitation,whichoccursatequivalencepoint. Maximalprecipitationoccursatequivalencepoint. Asalargemacromolecularlatticeisformedatequivalence, complexincreasesinsizeandprecipitateout. Followfigureanddrawit!!!!itisimportanttodrawfigure. 77
  78. 78. 78
  79. 79. B. Precipitation Reaction in Gels PrecipitationrxningelsyieldsvisibleprecipitinLines. Immuneprecipitatescanformnotonlyinsolutionbutalsoinagarmatrix. Whenantigenandantibodydiffusestowardsoneanotherinagar,orwhenAbisincorporatedintotheagarandantigendiffusesintotheantibodycontainingmatrix,avisiblelineofprecipitationwillform. Asinprecipitationreactioninfluid,visibleprecipitationoccursintheregionofantibodyorantigenexcess. Twotypesofimmunodiffusionreactionscanbeusedtodeterminerelativeconcentrationofantibodiesorantigen, tocompareantigens,ortodeterminetherelativepurityofanantigenpreparation. 79
  80. 80. Twotypesofimmunodiffusionreactionsbothofwhicharecarriedoutsemisolidmediumsuchasagar. 1Radialimmunodiffusion(Mancinimethod):Inthismethod,anAgsampleisplacedinawellandallowedtodiffuseintoagarcontainingasuitabledilutionofantiserum.Asantigendiffusesintotheagar,theregionofequivalenceisestablishedandaringofprecipitation,aprecipitinring,formsaroundthewell. Theareaofprecipitinringisproportionaltotheconcentrationofantigen.Bycomparingtheareaoftheprecipitinringwithastandardcurve(obtainedbymeasuringtheprecipitinareasofknownconcentrationsoftheantigen),theconcentrationoftheantigensamplecanbedetermined. 80
  81. 81. 2. Double immunodiffusion (the Ouchterlony method): In this method both the antigen and antibody diffuse, radially from the wells towards each other , thereby establishing a concentration gradient. As equivalence is reached, a visible line of precipitation, a precipitin line is formed. Please refer figure in next slide!!!!!!!!!!!!!! 81
  82. 82. 82
  83. 83. 2. Agglutination Reaction The interaction between antibody and antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reaction are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens. Just as an excess of antibody inhibits precipitation reactions, such excess can also inhibit agglutination reactions; this inhibition is called prozone effect. 83
  84. 84. Its application a.Hemagglutinationisusedinbloodtyping: AgglutinationreactionsareroutinelyperformedtotypeRBCs. IntypingfortheABOantigens,RBCsaremixedonaslidewithantiseratotheAorBblood-groupantigens. Iftheantigenispresentonthecells,theyagglutinate, formingavisibleclumpontheslide. DeterminationofwhichantigensarepresentondonorandrecipientRBCsisthebasisformatchingbloodtypesfortransfusions. b.Bacterialagglutinationisusedtodiagnoseinfection. c.Passiveagglutinationisusefulwithsolubleantigens. 84
  85. 85. Immuno-assay Techniques Radioimmunoassay ELISA Western Blotting Immunofluorescence Immunoelectron Microscopy. 85
  86. 86. Immunoassays Theexquisitespecificityofantigen-antibodyinteractionshasledtothedevelopmentofavarietyofimmunologicassays,whichcanbeusedtodetectthepresenceofeitherantibodyorantigen. Immunoassayshaveplayedvitalrolesindiagnosingdiseases,monitoringthelevelofthehumoralimmuneresponse,andidentifyingmoleculesofbiologicalormedicalinterest.Theseassaysdifferintheirspeedandsensitivity,somearestrictlyqualitative,otherarequantitative. 86
  87. 87. Radioimmunoassay Oneofthemostsensitivetechniquesfordetectingantigenorantibodyisradioimmunoassay(RIA). Principle:TheprincipleofRIAinvolvescompetitivebindingofradiolabeledantigen(usuallylabeledwithgammaemittingisotopesuchas125Ibutbetaemittingisotopessuchastritium3Harealsoroutinelyusedaslabels)toahigh-affinityantibody.Thelabeledantigenismixedwithantibodyataconcentrationthatsaturatestheantigen-bindingsitesoftheantibody. Thentestsamplesofunlabeledantigenofunknownconcentrationareaddedinprogressivelylargeramounts.Theantibodydoesn’tdistinguishlabeledfromunlabeledantigen,sothetwokindsofantigencompeteforavailablebindingsitesontheantibody. 87
  88. 88. Astheconcentrationofunlabeledantigenincreases,morelabeledantigenwillbedisplacedfromthebindingsites. Thedecreaseintheamountofradiolabeledantigenboundtospecificantibodyinthepresenceofthetestsampleismeasuredinordertodeterminetheamountofantigenpresentinthetestsample.[Note:Todeterminetheamountoflabeledantigenbound,theAg-Abcomplexisprecipitatedtoseparateitfromfreeantigen,andtheradioactivityintheprecipitateismeasured. Astandardcurvecanbegeneratedusingunlabeledantigensamplesofknownconcentration(inplaceoftestsample),andfromthisplottheamountofantigeninthetestmixturemaybepreciselydetermined.] AmicrotiterRIAhasbeenwidelyusedtoscreenforthepresenceofthehepatitisBvirus. 88
  89. 89. 89
  90. 90. Enzyme-Linked Immunosorbent Assay CommonlyknownasELISA(orEIA). ItsprincipleissimilartoRIAbutdependsonanenzymeratherthanaradioactivelabel. Anenzymeconjugatedwithanantibodyreactswithacolorlesssubstratetogenerateacoloredreactionproduct. Suchasubstrateiscalledachromogenicsubstrate. AnumberofenzymeshavebeenemployedforELISA, includingalkalinephosphatase,horseradishperoxidase, andβ-galactosidase.TheseassaysapproachthesensitivityofRIAsandhavetheadvantageofbeingsafeandlesscostly. 90
  91. 91. Types:3types. IndirectELISA:usedindeterminationofserumAbsagainstHIV. SandwichELISA CompetitiveELISA InoneoftheversionofELISAusingchemiluminescence,aluxogenic(light-generating) substrateisusedinplaceofchromogenicsubstrate(usedinconventionalELISA).Itsadvantageisincreasedsensitivity. 91
  92. 92. 92
  93. 93. IndirectELISA AntibodycanbedetectedorquantitativelydeterminedwithanindirectELISA(Figure6-10a).Serumorsomeothersamplecontainingprimaryantibody(Ab1)isaddedtoanantigen-coatedmicrotiterwellandallowedtoreactwiththeantigenattachedtothewell. AfteranyfreeAb1iswashedaway,thepresenceofantibodyboundtotheantigenisdetectedbyaddinganenzyme- conjugatedsecondaryanti-isotypeantibody(Ab2),whichbindstotheprimaryantibody. AnyfreeAb2theniswashedaway,andasubstratefortheenzymeisadded.Theamountofcoloredreactionproductthatformsismeasuredbyspecializedspectrophotometricplatereaders,whichcanmeasuretheabsorbanceofallofthewellsofa96-wellplateinseconds. 93
  94. 94. IndirectELISAisthemethodofchoicetodetectthepresenceofserumantibodiesagainsthumanimmunodeficiencyvirus(HIV),thecausativeagentofAIDS.Inthisassay,recombinantenvelopeandcoreproteinsofHIVareadsorbedassolid-phaseantigenstomicrotiterwells. IndividualsinfectedwithHIVwillproduceserumantibodiestoepitopesontheseviralproteins.Generally,serumantibodiestoHIVcanbedetectedbyindirectELISAwithin6weeksofinfection. 94
  95. 95. SANDWICH ELISA AntigencanbedetectedormeasuredbyasandwichELISA(Figure6-10b).Inthistechnique,theantibody(ratherthantheantigen)isimmobilizedonamicrotiterwell. Asamplecontainingantigenisaddedandallowedtoreactwiththeimmobilizedantibody. Afterthewelliswashed,asecondenzyme-linkedantibodyspecificforadifferentepitopeontheantigenisaddedandallowedtoreactwiththeboundantigen.Afteranyfreesecondantibodyisremovedbywashing,substrateisadded, andthecoloredreactionproductismeasured. 95
  96. 96. COMPETITIVEELISA AnothervariationformeasuringamountsofantigeniscompetitiveELISA(Figure6-10c).Inthistechnique,antibodyisfirstincubatedinsolutionwithasamplecontainingantigen. Theantigen-antibodymixtureisthenaddedtoanantigencoatedmicrotiterwell.Themoreantigenpresentinthesample,thelessfreeantibodywillbeavailabletobindtotheantigen-coatedwell. Additionofanenzyme-conjugatedsecondaryantibody(Ab2)specificfortheisotypeoftheprimaryantibodycanbeusedtodeterminetheamountofprimaryantibodyboundtothewellasinanindirectELISA. Inthecompetitiveassay,however,thehighertheconcentrationofantigenintheoriginalsample,thelowertheabsorbance. 96
  97. 97. Western blotting IndentificationofaspecificproteininacomplexmixtureofproteinscanbeaccomplishedbyatechniqueknownasWesternblotting. Inwesternblotting,aproteinmixtureiselectrophoreticallyseparatedonanSDS- polyacrylamidegel(SDS-PAGE),aslabgelinfusedwithsodiumdodecylsulfate(SDS),adissociatingagent. Theproteinbandsaretransferredtoanylonmembranebyelectrophoresisandtheindividualproteinbandsareidentifiedbyfloodingthenitrocellulosemembranewithradiolabeledorenzymelinkedpolyclonalormonoclonalantibodyspecificfortheproteinofinterest. TheAg-Abcomplexesthatisformedontheband,containingtheproteinrecognizedbytheantibody,canbevisualizedinavarietyofways.Iftheproteinofinterestwasboundbyaradioactiveantibody,itspositionontheblotcanbedeterminedbyexposingthemembranetoasheetofx-rayfilm,aprocedurecalledautoradiography. However,themostgenerallyuseddetectionproceduresemployenzyme-linkedantibodiesagainsttheprotein.Afterbindingoftheenzymeantibodyconjugate, additionofachromogenicsubstratethatproducesahighlycoloredandinsolubleproductcausestheappearanceofacoloredbandatthesiteofthetargetantigen. Thesiteoftheproteinofinterestcanbedeterminedwithmuchhighersensitivityifachemiluminescentcompoundalongwithsuitableenhancingagentsisusedtoproducelightattheantigensite. 97
  98. 98. Westernblottingcanalsoidentifyaspecificantibodyinamixture.Inthiscase,knownantigensofwell-definedmolecularweightareseparatedbySDS-PAGEandblottedontonitrocellulose. Theseparatedbandsofknownantigensarethenprobedwiththesamplesuspectedofcontainingantibodyspecificforoneormoreoftheseantigens. Reactionofanantibodywithabandisdetectedbyusingeitherradiolabeledorenzyme-linkedsecondaryantibodythatisspecificforthespeciesoftheantibodiesinthetestsample. ThemostwidelyusedapplicationofthisprocedureisinconfirmatorytestingforHIV,whereWesternblottingisusedtodeterminewhetherthepatienthasantibodiesthatreactwithoneormoreviralproteins. 98
  99. 99. 99
  100. 100. Immunoprecipitation Theimmunoprecipitationtechniquehastheadvantageofallowingtheisolationoftheantigenofinterestforfurtheranalysis. Italsoprovidesasensitiveassayforthepresenceofaparticularantigeninagivencellortissuetype. Anextractproducedbydisruptionofcellsortissuesismixedwithanantibodyagainsttheantigenofinterestinordertoformanantigen-antibodycomplexthatwillprecipitate. However,iftheantigenconcentrationislow(oftenthecaseincellandtissueextracts),theassemblyofantigen-antibodycomplexesintoprecipitatescantakehours,evendays,anditisdifficulttoisolatethesmallamountofimmunoprecipitatethatforms. Whenusedinconjugationwithbiosyntheticradioisotopelabelling,immunoprecipitationcanalsobeusedtodeterminewhetheraparticularantigenisactuallysynthesizedbyacellortissue. 100
  101. 101. 101
  102. 102. Immunofluorescence In1944,AlbertCoonsshowedthatantibodiescouldbelabeledwithmoleculesthathavethepropertytofluorescence. Ifantibodymoleculesaretaggedwithfluorescentdye, orfluorochrome,immunecomplexescontainingthesefluorescentlylabeledantibodiescanbedetectedbycoloredlightemissionwhenexcitedbylightofappropriatewavelength. Antibodymoleculesboundtoantigensincellsortissuesectionscansimilarlybevisualized. Theemittedlightcouldbeviewedwithfluorescencemicroscope,whichisequippedwithUVlightsource. 102
  103. 103. In this technique (immunofluorescence), various fluorescent compounds in use include Fluorescein, Rhodamine, Phycoerythrin. Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect. In direct staining, the specific antibody (the primary Ab) is directly conjugated with fluorescein. In indirect staining, the primary Ab is unlabeled and is detected with an additional flurochrome-labeled reagent. 103
  104. 104. Immunoelectronmicroscopy Specificityofantibodyhasmadethempowerfultoolsforvisualizingspecificintracellulartissuecomponentsbyimmunoelectronmicroscopy. Inthistechnique, Electron-denselabelconjugatedtoFcportionofaspecificantibodyfordirectstainingorconjugatedtoananti-immunoglobulinreagentforindirectstaining Electrondenselabel(commonlyusedareferritinandcolloidalgold)absorbselectronsitcanbevisualisedwithelectronmicroscopeassmallblackdots. 104
  105. 105. Monoclonal Antibodies MonoclonalAntibodiesareusuallyproducedfromhybridomaclones. Eachhybridomacloneisderivedbythefusionofamyelomacellandanantibodyproducinglymphocyte, andthehybridomacloneproducingthedesiredantibodyisidentifiedandisolated. Hybridomacellsaremass-culturedfortheproductionofmonoclonalantibodieseither(1)invivointheperitonealcavityofmiceor(2)invitroinlargescaleculturevessels. 105
  106. 106. Application When Mabs are used to detect the presence of a specific antigen or of antibodies specific to an antigen in a sample or samples, this constitutes a diagnosic application. Antibodies specific to a cell type, say, tumor cells, can be linked with a toxin polypeptide to yield a conjugate molecule called immunotoxin. This immunotoxin will bind to tumor cell and kills it. Immunopurification. 106
  107. 107. ANTIGENS AND HAPTENS Thetwoterminologiesviz.,antigensandhaptensareintimatelyassociatedwithimmunology;and,henceonemayunderstandandhaveaclearconceptaboutthemasfaraspossible. Antigens Anantigeniseitheracellormoleculewhichwillbindwithpreexitingantibodybutwillnotdefinitelycauseinductionofantibodyproduction. Antigenmayalsobedefinedas—‘amacromolecularentitythatessentiallyelicitsanimmuneresponseviatheformationofspecificantibodiesinthebodyofthehost’. Inabroaderperspectivetheantigen(orimmunogen)isinvariablyregardedastheafferentbranchoftheprevailingimmunesystem,andisanycellormoleculewhichwouldprovokeanimmuneresponseverymuchinanimmunologicallyviableandcompetentindividual.Generally,immunogens(antigens)mustfulfillthefollowingtwocharacteristicfeatures,namely: (a)shouldbelargerthan2000inmolecularweight,e.g.,protein,glycoproteinandcarbohydrates,and (b)mustbeabsolutelyforeigntotheindividualintowhomtheyhavebeenintroducedappropriately. 107
  108. 108. Example:Thebestexampleofan‘antigen’isonesownerythrocytes.Because,theywillnotinduceantibodyformationinoneselfbutwilldefinitelyreactwithanantibodyessentiallycontainedinanimproperlymatchedbloodtransfusion. Quiteoftenanantigenisaprotein,butitcouldalsobeapolysaccharideornucleicacidoranyothersubstance. Importantly,itmayalsobepossiblethataforeignsubstance(e.g.,protein)-notnecessarilybelongingtoapathogenicmicroorganism,mayactasanantigensothatonbeinginjectedintoahost,itmayinduceantibodyformation. Besides,theymayturnouttobeantigenicandtherebycausestimulationofantibodyproduction,incasetheyareintimatelyandlightlygetboundtocertainmacromolecules,forinstance:proteins,carbohydratesandnucleicacids. 108
  109. 109. Haptens Inusualpractice,therelativelysmaller,lessrigidorratherlesscomplexmoleculesusuallyarenotimmunogeneticintheirpurestform,butmaybemadesobysimplylinkingthemstrategicallytoeitherlargerormorecomplexstructures.Consequently,thesmallermoleculesareinvariablytermedashaptens;whereas,thelargermoleculesorcellsareknownascarriers. Haptenmayalsobedefined—‘asasubstancethatnormallydoesnotactasanantigenorstimulateanimmuneresponsebutthatcanbecombinedwithanantigenand,atalatertime,initiateaspecificantibodyresponseonitsown’. Furthermore,smallmolecules(micromolecular),suchas:drugsubstances,thatmayserveas‘haptens’andcannormallybemadeantigenicbycouplingthemchemicallytoamacromolecularsubstancee.g.,protein,polysaccharide,carbohydrateetc.Thehaptenisobtainedfromanon-antigeniccompound(micromolecule)e.g., morphine,carteololetc.,whichisultimatelyconjugated, covalentlytoacarriermacromoleculetorenderitantigenic. 109
  110. 110. Oneofthegoodexampleisofgastrin(hapten)whichisdulycoupledtoalbumin(i.e.,proteincarrier)bytreatmentwithcarbodiimides(CCD),whichcouplefunctionalcarboxyl, amino,alcohol,phosphateorthiolmoieties. Importantly,thehapten-conjugatethusobtainedisnormallysubjectedtoemulsificationinahighlyrefined‘mineraloil’preparationcontaining-killedMycobacterium(CompleteFreund’sAdjuvant),andsubsequentlyinjectedintradermallyeitherinhealthyrabbitsorguineapigsonseveraloccasionsatintervals. Evidently,theserumantibodyshouldhavenotonlyhighdegreeofspecificitybutalsoareasonablystrongaffinityfortheprevailingantigens. 110
  111. 111. Hypersensitivity Immunesystemmobilizesvarietyofeffectormoleculesthatacttoremoveantigenbyvariousmechanisms. Generally,theseeffectormoleculesinducealocalizedinflammatoryresponsethateliminatesantigenwithoutextensivelydamagingthehost’stissue.Undercertaincircumstances,however,thisinflammatoryresponsecanhavedeleteriouseffects,resultinginsignificanttissuedamageorevendeath.Thisinappropriateimmuneresponseistermedhypersensitivityorallergy. Althoughthewordhypersensitivityimpliesanincreasedresponse,theresponseisnotalwaysheightenedbutmay,instead,beaninappropriateimmuneresponsetoanantigen.Hypersensitivereactionsmaydevelopinthecourseofeitherhumoralorcell-mediatedresponses. Hypersensitivitymaybedefinedas—‘anabnormalsensitivitytoastimulusofanykind’. 111
  112. 112. There are four types of hypersensitivity reaction: 1.Type I hypersensitivity (IgEMediated Hypersensitivity) 2.Type II (IgGMediated Hypersensitivity) 3.Type III (Immune complex mediated hypersensitivity) 4.Type IV (Cell Mediated Hypersensitivity) 112
  113. 113. 1. IgE mediated Hypersensitivity AtypeIhypersensitivereactionisinducedbycertaintypesofantigens(suchasforeignserum,vaccine,penicillin,ryegrass,antvenom,beevenom,etc)referredtoasallergens, andhasallthecharacteristicsofanormalhumoralresponse. Thatis,anallergeninducesahumoralantibodyresponsebythesamemechanismsasforothersolubleantigens,resultinginthegenerationofantibody-secretingplasmacellsandmemorycells. WhatdistinguishesatypeIhypersensitiveresponsefromanormalhumoralresponseisthattheplasmacellssecreteIgE.ThisclassofantibodybindswithhighaffinitytoFcreceptorsonthesurfaceoftissuemastcellsandbloodbasophils. MastcellsandbasophilscoatedbyIgEaresaidtobesensitized.Alaterexposuretothesameallergencross-linksthemembrane-boundIgEonsensitizedmastcellsandbasophils,causingdegranulationofthesecells. Thepharmacologicallyactivemediatorsreleasedfromthegranulesactonthesurroundingtissues.Theprincipaleffects—vasodilationandsmooth-musclecontraction—maybeeithersystemicorlocalized,dependingontheextentofmediatorrelease. TheclinicalmanifestationsoftypeIreactionscanrangefromlife-threateningconditions, suchassystemicanaphylaxisandasthma,tohayfeverandeczema,whicharemerelyannoying 113
  114. 114. GeneralmechanismunderlyingatypeIhypersensitivereaction.ExposuretoanallergenactivatesBcellstoformIgEsecretingplasmacells.ThesecretedIgEmoleculesbindtoIgEspecificFcreceptorsonmastcellsandbloodbasophils. (ManymoleculesofIgEwithvariousspecificitiescanbindtotheIgE-Fcreceptor.)SecondexposuretotheallergenleadstocrosslinkingoftheboundIgE,triggeringthereleaseofpharmacologicallyactivemediators,vasoactiveamines,frommastcellsandbasophils.Themediatorscausesmooth-musclecontraction,increasedvascularpermeability,andvasodilation. 114
  115. 115. 2. Antibody Mediated cytotoxic Hypersensitivity TypeIIhypersensitivereactionsinvolveantibody-mediateddestructionofcells. Antibodycanactivatethecomplementsystem,creatingporesinthemembraneofaforeigncell,oritcanmediatecelldestructionbyantibodydependentcell- mediatedcytotoxicity(ADCC).Inthisprocess,cytotoxiccellswithFcreceptorsbindtotheFcregionofantibodiesontargetcellsandpromotekillingofthecells.Antibodyboundtoaforeigncellalsocanserveasanopsonin,enablingphagocyticcellswithFcorC3breceptorstobindandphagocytosetheantibody-coatedcell. Examples : The various examples are as stated below : (i) Transfusion reactions i.e., when blood groups are not matched properly, (ii) Haemolytic disease concerning the newly born babies via Rhesus incompatibility, (iii) Graft destruction or rejection i.e., antibody-mediated ‘graft’ destruction or rejection. (iv) Autoimmune reactions usually directed against the formed elements of the blood, and the kidney glomerular basement membrances, etc. 115
  116. 116. Example1:TransfusionReactionsAreTypeIIReactions Alargenumberofproteinsandglycoproteinsonthemembraneofredbloodcellsareencodedbydifferentgenes,eachofwhichhasanumberofalternativealleles.Anindividualpossessingoneallelicformofablood-groupantigencanrecognizeotherallelicformsontransfusedbloodasforeignandmountanantibodyresponse.Insomecases,theantibodieshavealreadybeeninducedbynaturalexposuretosimilarantigenicdeterminantsonavarietyofmicroorganismspresentinthenormalfloraofthegut.ThisisthecasewiththeABOblood-groupantigens. AntibodiestotheA,B,andOantigens,calledisohemagglutinins,areusuallyoftheIgMclass.AnindividualwithbloodtypeA,forexample,recognizesB-likeepitopesonintestinalmicroorganismsandproducesisohemagglutininstotheB-likeepitopes. IfatypeAindividualistransfusedwithbloodcontainingtypeBcells,atransfusionreactionoccursinwhichtheanti-Biso-hemagglutininsbindtotheBbloodcellsandmediatetheirdestructionbymeansofcomplement-mediatedlysis.Antibodiestootherblood-groupantigensmayresultfromrepeatedbloodtransfusionsbecauseminorallelicdifferencesintheseantigenscanstimulateantibodyproduction.TheseantibodiesareusuallyoftheIgGclass. Theclinicalmanifestationsoftransfusionreactionsresultfrommassiveintravascularhemolysisofthetransfusedredbloodcellsbyantibodypluscomplement.Thesemanifestationsmaybeeitherimmediateordelayed. 116
  117. 117. 117
  118. 118. HemolyticDiseaseoftheNewbornIsCausedbyTypeIIReactions HemolyticdiseaseofthenewborndevelopswhenmaternalIgGantibodiesspecificforfetalblood-groupantigenscrosstheplacentaanddestroyfetalredbloodcells.Theconsequencesofsuchtransfercanbeminor,serious,orlethal. Severehemolyticdiseaseofthenewborn,callederythroblastosisfetalis,mostcommonlydevelopswhenanRh+fetusexpressesanRhantigenonitsbloodcellsthattheRh–motherdoesnotexpress. This most commonly happens when a woman with Rhnegative blood becomes pregnant by a man with Rhpositive blood and conceives a baby with Rhpositive blood. Red blood cells from the baby can leak across the placenta into the woman's bloodstream during pregnancy or delivery. This causes the mother's body to make antibodies against the Rhfactor. If the mother becomes pregnant again with an Rh-positive baby, it is possible for her antibodies to cross the placenta and attack the baby's red blood cells. After birth, an affected newborn may develop kernicterus. This happens when bile pigments are deposited in the cells of the brain and spinal cord and nerve cells are degenerated. Incompatibilities between ABO blood types can also cause this condition. These are less common than those of the Rhfactor and tend to be less severe. 118
  119. 119. 3.ImmuneComplex–Mediated(TypeIII)Hypersensitivity Thereactionofantibodywithantigengeneratesimmunecomplexes.Generallythiscomplexingofantigenwithantibodyfacilitatestheclearanceofantigenbyphagocyticcells. Insomecases,however,largeamountsofimmunecomplexescanleadtotissue-damagingtypeIIIhypersensitivereactions. Themagnitudeofthereactiondependsonthequantityofimmunecomplexesaswellastheirdistributionwithinthebody. Whenthecomplexesaredepositedintissueverynearthesiteofantigenentry,alocalizedreactiondevelops. Whenthecomplexesareformedintheblood,areactioncandevelopwhereverthecomplexesaredeposited. Inparticular,complexdepositionisfrequentlyobservedonblood-vesselwalls,inthesynovialmembraneofjoints,ontheglomerularbasementmembraneofthekidney,andonthechoroidplexusofthebrain.Thedepositionofthesecomplexesinitiatesareactionthatresultsintherecruitmentofneutrophilstothesite.Thetissuethereisinjuredasaconsequenceofgranularreleaseoflyticenzymesfromtheneutrophil. 119
  120. 120. TypeIIIhypersensitivereactionsdevelopwhenimmunecomplexesactivatethecomplementsystem’sarrayofimmuneeffectormolecules.TheC3a,C4a,andC5acomplementsplitproductsareanaphylatoxinsthatcauselocalizedmast- celldegranulationandconsequentincreaseinlocalvascularpermeability.C3a, C5a,andC5b67arealsochemotacticfactorsforneutrophils,whichcanaccumulateinlargenumbersatthesiteofimmune-complexdeposition.Largerimmunecomplexesaredepositedonthebasementmembraneofbloodvesselwallsorkidneyglomeruli,whereassmallercomplexesmaypassthroughthebasementmembraneandbedepositedinthesubepithelium.Thetypeoflesionthatresultsdependsonthesiteofdepositionofthecomplexes. MuchofthetissuedamageintypeIIIreactionsstemsfromreleaseoflyticenzymesbyneutrophilsastheyattempttophagocytoseimmunecomplexes. TheC3bcomplementcomponentactsasanopsonin,coatingimmunecomplexes. AneutrophilbindstoaC3b-coatedimmunecomplexbymeansofthetypeIcomplementreceptor,whichisspecificforC3b.Becausethecomplexisdepositedonthebasement-membranesurface,phagocytosisisimpeded,sothatlyticenzymesarereleasedduringtheunsuccessfulattemptsoftheneutrophiltoingesttheadheringimmunecomplex.Furtheractivationofthemembrane-attackmechanismofthecomplementsystemcanalsocontributetothedestructionoftissue.Inaddition,theactivationofcomplementcaninduce aggregationofplatelets,andtheresultingreleaseofclottingfactorscanleadtoformationofmicrothrombi. 120
  121. 121. 121
  122. 122. TypeIVorDelayed-TypeHypersensitivity(DTH) WhensomesubpopulationsofactivatedTHcellsencountercertaintypesofantigens,theysecretecytokinesthatinducealocalizedinflammatoryreactioncalleddelayed-typehyper-sensitivity(DTH). Thereactionischaracterizedbylargeinfluxesofnonspecificinflammatorycells,inparticular,macrophages. Thistypeofreactionwasfirstdescribedin1890byRobertKoch,whoobservedthatindividualsinfectedwithMycobacteriumtuberculosisdevelopedalocalizedinflammatoryresponsewheninjectedintradermally withafiltratederivedfromamycobacterialculture. Hecalledthislocalizedskinreactiona“tuberculinreaction.” ThecharacteristicofatypeIVreactionarethedelayintimerequiredforthereactiontodevelopandtherecruitmentofmacrophagesasopposedtoneutrophils,asfoundinatypeIIIreaction. Inthistype,sensitizedTH1cellsreleasecytokinesthatactivatemacrophagesorTCcellswhichmediatedirectcellulardamage. Macrophagesarethemajorcomponentoftheinfiltratethatsurroundsthesiteofinflammation. 122
  123. 123. 123
  124. 124. 124
  125. 125. 125
  126. 126. 126
  127. 127. 127
  128. 128. Antibody Production (Immunogen Preparation) Theproductionofspecificantibodyprobesisarelativelystraightforwardprocessinvolvingimmunizationofanimalsandrelianceupontheirimmunesystemstoraiseresponsesthatresultinbiosynthesisofantibodiesagainsttheinjectedmolecule.Evenso,severalfactorsaffecttheprobabilityofinducinganimmunizedanimaltoproduceusefulamountsoftarget-specificantibodies.Antigensmustbepreparedanddeliveredinaformandmannerthatmaximizesproductionofaspecificimmuneresponsebytheanimal.Thisiscalledimmunogenpreparation. 128
  129. 129. Antibodyproductionisconceptuallysimple.However,becauseitdependsuponsuchacomplexbiologicalsystem(immunityofalivingorganism),resultsarenotentirelypredictable.Individualanimals– eventhosethataregeneticallyidentical–willresponduniquelytothesameimmunizationscheme,generatingdifferentsuitesofspecificantibodiesagainstaninjectedantigen.Evenso,equippedwithabasicunderstandingofhowtheimmunesystemrespondstoinjectionofaforeignsubstanceandaknowledgeofavailabletoolsforpreparingasampleforinjection,researcherscangreatlyincreasetheprobabilityofobtainingausefulantibodyproduct. Forexample,smallcompounds(drugsorpeptides)arenotsufficientlycomplexbythemselvestoinduceanimmuneresponseorbeprocessedinamannerthatelicitsproductionofspecificantibodies.Forantibodyproductiontobesuccessfulwithsmallantigens,theymustbechemicallyconjugatedwithimmunogeniccarrierproteinssuchaskeyholelimpethemocyanin(KLH).Adjuvantscanbemixedandinjectedwithanimmunogentoincreasetheintensityoftheimmuneresponse. 129

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