MucosalImmunization黏膜免疫.pptx

Mucosal Immunization.Leslie Ann Mitchell,PhD.Gene Therapy Institute,Hadassah Ein KaremImmune Response.nInnate immunity:nmacrophages,NK cells,mucous,lysozyme,cytokines,etc.n Provides first line of defense to prevent colonization and infection.nDetermines which antigens will be recognized by B-and T-cells and the nature of the response.Immune Response.nAcquired immunity:B-and T-lymphocytes.nB-cells:produce antibodies that neutralize viruses and bacterial toxins,block attachment of microbes to host cells,or opsonize microbes for phagocytosisnT-cells:help(CD4+)or cytotoxic(CD8+)functions(elimination of intracellular microbes and viruses)Local Immunity.nMost pathogens enter the body via mucosal surfaces.nLocal immunity(specific IgA,cellular immunity)is an important first line of defense vs.pathogens.nSystemic immunization is ineffective in inducing local immunity.Inherent Problems with Mucosal Immunization.nMost protein antigens are poorly immunogenic when delivered via the mucosal route.nImmunologic tolerance may be induced(especially via the oral route).nMucosal surfaces are easily tolerized:non-responsiveness has evolved to prevent unwanted inflammatory responses.Design of Mucosal Vaccines:Important Factors.nAntigen must be protected from enzymatic digestion or acid hydrolysis.nAntigen uptake by mucosal M-cells and epithelial cells must be enhanced.nInnate immune system must be stimulated to ensure appropriate adaptive immune response.nImmunologic memory must be induced.Pathogen-associated molecular patterns(PAMPs).nConserved molecular structures found on bacterial and viral pathogens.nExamples:nGram+and bacteria:LPS,teichoic acid,peptidoglycans,CpG motifsnRNA virus:dsRNAnYeasts:mannansPathogen-associated molecular patterns(PAMPs).nNot present on mammalian cells but interact with non-clonal pattern recognition receptors(PPRs)on macrophages,dendritic cells,epithelial cells.nExamples:CD14,collectins,toll-like receptors(TLRs),mannose binding protein(MBP),serum amyloid P,C receptors,CD11b/CD18,DEC205PAMPs and PPRs:Mechanism of activation.Example:bacterial CpG binds to TLR9nBinding of microbial molecules to TLRs transduces signals through a common adaptor(MYD88)and NFkB resulting in the generation of cytokines(IL-1,IL-12,TNF-a)and activation molecules(B7)on lymphocytes.nEnhances antigen presentation,T-cell activation,increases adaptive immune response.Mucosal Epithelial Cells.nInterface between pathogen and immune system:nInnate(MHC Class I)and inducible(MHC Class II)antigen presentation functionnInduced by pathogens to synthesize and secrete:nAntimicrobial peptides and proteinsnImmunoregulatory cytokinesnColony-stimulating factorsnChemokines(recruitment of immune cells)Helper T-Cell Subsets.nTH1:nIFN-g,TNF-bnCellular immunity vs.intracellular bacteria,small parasitesnInduction of neutralizing antibodies of the IgG2a subclass(in mice)nTH2:nIL-4,IL-5,IL-10,and IL-13nInduced by helminth parasites,allergens,immunization with soluble or alum-adsorbed antigensnImmunity to extracellular parasites,bacterianHelper function in production of IgA,IgE,and neutralizing IgG to bacterial toxinsRegulation of T-cell Response.nTH1 vs TH2:cross-regulation by TH cytokines or by cytokines from subsets of dendritic cells(DC1 and DC2).nIL-4 drives TH2 pathway.nMust be tightly regulated or immune-mediated hypersensitivity or autoimmune disease will result.Targeting T-cell Response in Vaccination Protocols.nCD4 T-cell response induced by:nAntigens targeted toward MHC Class II processing pathwaynLive or killed bacteria or virusesnPurified protein or peptide antigens with adjuvants such as alum.nCD8 T-cell response induced by:nIntracellular viruses or bacterianDelivery systems targeting towards MHC Class I pathway:liposomes,ISCOMS,microparticles,naked DNARequirements for T-cell Activation.nAntigen recognition through TcR in the context of MHC Class I or II molecules.nCo-stimulatory molecular interactions between T-cell and APC:APC:T-cell:B7-1,B7-2CD28CD40CD40LICAM-3LFA-1LFA-3CD2Role of Dendritic Cells(DCs).nDCs of two lineages:lymphoid and myeloid differentially influence maturation of TH1 and TH2.nImmature DCs:phagocytic,express CCR5&CCR6,low levels of MHC Class II and B7.nMature DCs:lose phagocytic capacity,increase presentation ability,enhanced expression of MHC Class II and B7.nMaturation influenced by PAMPs.Immunomodulatory Molecules.nEnhance or disrupt co-stimulation.nInfluence direction of DC maturation.nPAMPs(LPS,CpG,dsRNA)or host cell molecules(CD40L,IL-1,TNF-a)modulate DC maturation and subsequent TH response.e.g.LPS drives DC1 maturation and TH1 response;PC-GP(nematodes)drives DC2 maturation and TH2 response.Mucosal Adjuvants and Delivery Systems:Influence on Immune Response.TH1 Response(Cytotoxicity)TH 2 Response(Antibody Production)Adjuvant or Delivery System-+SolutionAlumChitosanCT or LT(+/+)CT or LT(mut)PLGQuil A/QS21QS21+MPLMucosal Adjuvants and Delivery Systems:Influence on Immune Response.TH1 Response(Cytotoxicity)TH2 Response(Antibody Production)Adjuvant or Delivery System+IL-12Live vectorsNaked DNACpG-ODNBacterial Toxin Adjuvants.nVibrio cholera Toxin(CT,CTB,mutants),E.coli heat-labile toxin(LT,mutants),pertussis toxinnIntra-nasal,oral,and systemic routesnDepending upon antigen dose,route can favor TH2(CT),TH1(LT)or mixed TH1/TH2 responses.Monophosphoryl Lipid A(MPL).nDerived from LPS(Gram ve bacteria).nInteracts with TLR4 and CD14 on host cells to activate NF-kB and production of inflammatory cytokines(IFN-g,IL-12),enhances B7-1 expression.nUsed in combination with QS21(from Quil A saponin)induces TH2 to TH1 switch.CpG-ODN Mucosal Adjuvant.nBacterial-derived,unmethylated:5-purine(x2)-pyrimidine(x2)3nSynthetic oligodeoxynucleotides with CpG motifs are potent adjuvants for driving local and systemic TH1 responses after parenteral,oral,intra-rectal,or intra-nasal administration with antigen.Microparticle Delivery Systems:PLG Polymers.nPLG:poly(lactide-co-glycolide)polymersnTarget mucosal M-cells in Peyers patches when administered orally.nEffective adjuvants for intra-nasal delivery.nFavor TH1 response and facilitate induction of CD8+CTLs.Mucosal Delivery Systems:Liposomes.nArtificial lipid bilayers composed of lipids+cholesterol.nStable at low pH,resistant to bile and pancreatin(suitable for oral delivery).nAlso effective for intra-nasal delivery.nEffectiveness enhanced by incorporating other adjuvants(CT,MPL)with antigen.Immune-stimulating complexes(ISCOMs).nCombination of antigen with terpenoid glycosides or saponins derived from Quillaja saponaria(e.g.,QuilA)incorporated into lipid particles.nEnhance antigen uptake by APCs(form pores in membrane).nStimulate production of IL-12.nFavor TH1 response(CD8+CTLs).Chitosan particles.nChitosan=deacylated chitin.nEnhance adsorption of protein antigens at mucosal surfaces by opening tight junctions.nSystemic immunity:enhance responses to parenterally-administered antigens and nonspecific host resistance.nFavor TH2 responses.Live Attenuated Vectors.nConcept:antigen delivery as a recombinant gene in live vector mimicks natural infection,giving rise to strongly protective immunity.nVectors:bacteria(commensals,attenuated Salmonella spp.,BCG),viruses(poliovirus,vaccinia,canarypox,etc.)nReplicating vectors enhance immune response.nInduces strong cellular(TH1)response(CTLs)and also antibodies.DNA Vaccines.nNaked(plasmid)DNA may be injected i.m.or applied to mucosae(nasal,vaginal,salivary gland,gastric).nDNA may be combined with other adjuvants or delivery systems:liposomes,ISCOMs,cationic lipids,CT,microaggregated albumin conjugates,PLG microparticles,vectors(live attenuated bacteria Shigella or replication-defective viruses Semliki Forest virus).nFavors TH1,CTL,IgA responses.Edible Vaccines from Transgenic Plants.nRecombinant vaccine antigens expressed in transgenic plants may be used for oral immunization.nProblems:ninefficient uptake of antigen in GI tractndifficult to control dosenrisk for development of tolerance to antigen,nrisk for breaking tolerance to food antigens if immunomodulatory genes includedSummary.nMucosal immunization has the potential to induce both local(sIgA,CTL)and systemic(IgG,IgA,CTL)immunity.nImmune response may be enhanced and modulated selectively by adjuvant or targeted delivery system.nRisk for development of tolerance when antigen is delivered orally.nImmunologic memory must be induced(unknown at present).Role of Immunomodulatory Molecules on Response.