黏膜免疫

1、Mucosal Immunization.,Leslie Ann Mitchell, PhD.Gene Therapy Institute,Hadassah Ein Karem,Immune Response.,Innate immunity: macrophages, NK cells, mucous, lysozyme, cytokines, etc. Provides first line of defense to pr

2、event colonization and infection.Determines which antigens will be recognized by B- and T-cells and the nature of the response.,Immune Response.,Acquired immunity: B- and T-lymphocytes.B-cells: produce antibodies that

3、neutralize viruses and bacterial toxins, block attachment of microbes to host cells, or opsonize microbes for phagocytosisT-cells: help (CD4+) or cytotoxic (CD8+) functions (elimination of intracellular microbes and vir

4、uses),Local Immunity.,Most pathogens enter the body via mucosal surfaces.Local immunity (specific IgA, cellular immunity) is an important first line of defense vs. pathogens.Systemic immunization is ineffective in indu

5、cing local immunity.,Inherent Problems with Mucosal Immunization.,Most protein antigens are poorly immunogenic when delivered via the mucosal route.Immunologic tolerance may be induced (especially via the oral route).M

6、ucosal surfaces are easily tolerized: non-responsiveness has evolved to prevent unwanted inflammatory responses.,Design of Mucosal Vaccines: Important Factors.,Antigen must be protected from enzymatic digestion or acid h

7、ydrolysis.Antigen uptake by mucosal M-cells and epithelial cells must be enhanced.Innate immune system must be stimulated to ensure appropriate adaptive immune response.Immunologic memory must be induced.,Pathogen-ass

8、ociated molecular patterns (PAMPs).,Conserved molecular structures found on bacterial and viral pathogens.Examples:Gram + and – bacteria: LPS, teichoic acid, peptidoglycans, CpG motifsRNA virus: dsRNAYeasts: mannans,

9、Pathogen-associated molecular patterns (PAMPs).,Not present on mammalian cells but interact with non-clonal pattern recognition receptors (PPRs) on macrophages, dendritic cells, epithelial cells.Examples: CD14, collecti

10、ns, toll-like receptors (TLRs), mannose binding protein (MBP), serum amyloid P, C’ receptors, CD11b/CD18, DEC205,PAMPs and PPRs: Mechanism of activation.,Example: bacterial CpG binds to TLR9Binding of microbial molecule

11、s 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.Enhances antigen presentation, T-cell act

12、ivation, increases adaptive immune response.,Mucosal Epithelial Cells.,Interface between pathogen and immune system:Innate (MHC Class I) and inducible (MHC Class II) antigen presentation functionInduced by pathogens to

13、 synthesize and secrete:Antimicrobial peptides and proteinsImmunoregulatory cytokinesColony-stimulating factorsChemokines (recruitment of immune cells),Helper T-Cell Subsets.,TH1:IFN-g, TNF-bCellular immunity vs. i

14、ntracellular bacteria, small parasitesInduction of neutralizing antibodies of the IgG2a subclass (in mice),TH2:IL-4, IL-5, IL-10, and IL-13Induced by helminth parasites, allergens, immunization with soluble or alum-ad

15、sorbed antigensImmunity to extracellular parasites, bacteriaHelper function in production of IgA, IgE, and neutralizing IgG to bacterial toxins,Regulation of T-cell Response.,TH1 vs TH2: cross-regulation by TH cytokine

16、s or by cytokines from subsets of dendritic cells (DC1 and DC2).IL-4 drives TH2 pathway.Must be tightly regulated or immune-mediated hypersensitivity or autoimmune disease will result.,Targeting T-cell Response in Vacc

17、ination Protocols.,CD4 T-cell response induced by:Antigens targeted toward MHC Class II processing pathwayLive or killed bacteria or virusesPurified protein or peptide antigens with adjuvants such as alum.,CD8 T-cell

18、response induced by:Intracellular viruses or bacteriaDelivery systems targeting towards MHC Class I pathway: liposomes,ISCOMS, microparticles, naked DNA,Requirements for T-cell Activation.,Antigen recognition through T

19、cR in the context of MHC Class I or II molecules.Co-stimulatory molecular interactions between T-cell and APC:APC:T-cell:B7-1, B7-2CD28CD40CD40LICAM-3LFA-1LFA-3CD2,Role of Dendritic Cells (DCs).,

20、DCs of two lineages: lymphoid and myeloid differentially influence maturation of TH1 and TH2.Immature DCs: phagocytic, express CCR5 & CCR6, low levels of MHC Class II and B7.Mature DCs: lose phagocytic capacity, in

21、crease presentation ability, enhanced expression of MHC Class II and B7.Maturation influenced by PAMPs.,Immunomodulatory Molecules.,Enhance or disrupt co-stimulation.Influence direction of DC maturation.PAMPs (LPS, Cp

22、G, 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

23、Adjuvants and Delivery Systems: Influence on Immune Response.,Mucosal Adjuvants and Delivery Systems: Influence on Immune Response.,Bacterial Toxin Adjuvants.,Vibrio cholera Toxin (CT, CTB, mutants), E. coli heat-labile

24、toxin (LT, mutants), pertussis toxinIntra-nasal, oral, and systemic routesDepending upon antigen dose, route can favor TH2 (CT), TH1 (LT) or mixed TH1/TH2 responses.,Monophosphoryl Lipid A (MPL).,Derived from LPS (Gram

25、 –ve bacteria).Interacts with TLR4 and CD14 on host cells to activate NF-kB and production of inflammatory cytokines (IFN-g, IL-12), enhances B7-1 expression.Used in combination with QS21 (from Quil A saponin) induces

26、TH2 to TH1 switch.,CpG-ODN Mucosal Adjuvant.,Bacterial-derived, unmethylated: 5’-purine (x2)------pyrimidine (x2)3’Synthetic oligodeoxynucleotides with CpG motifs are potent adjuvants for driving local and systemic TH1

27、responses after parenteral, oral, intra-rectal, or intra-nasal administration with antigen.,Microparticle Delivery Systems: PLG Polymers.,PLG: poly(lactide-co-glycolide) polymersTarget mucosal M-cells in Peyer’s patches

28、 when administered orally.Effective adjuvants for intra-nasal delivery.Favor TH1 response and facilitate induction of CD8+ CTLs.,Mucosal Delivery Systems: Liposomes.,Artificial lipid bilayers composed of lipids + chole

29、sterol.Stable at low pH, resistant to bile and pancreatin (suitable for oral delivery).Also effective for intra-nasal delivery.Effectiveness enhanced by incorporating other adjuvants (CT, MPL) with antigen.,Immune-sti

30、mulating complexes (ISCOMs).,Combination of antigen with terpenoid glycosides or saponins derived from Quillaja saponaria (e.g., QuilA) incorporated into lipid particles.Enhance antigen uptake by APCs (form pores in mem

31、brane).Stimulate production of IL-12.Favor TH1 response (CD8+ CTLs).,Chitosan particles.,Chitosan= deacylated chitin.Enhance adsorption of protein antigens at mucosal surfaces by opening tight junctions.Systemic immu

32、nity: enhance responses to parenterally-administered antigens and nonspecific host resistance.Favor TH2 responses.,Live Attenuated Vectors.,Concept: antigen delivery as a recombinant gene in live vector mimicks natural

33、infection, giving rise to strongly protective immunity.Vectors: bacteria (commensals, attenuated Salmonella spp., BCG), viruses (poliovirus, vaccinia, canarypox, etc.)Replicating vectors enhance immune response.Induce

34、s strong cellular (TH1) response (CTLs) and also antibodies.,DNA Vaccines.,Naked (plasmid) DNA may be injected i.m. or applied to mucosae (nasal, vaginal, salivary gland, gastric).DNA may be combined with other adjuvant

35、s 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])

36、.Favors TH1, CTL, IgA responses.,Edible Vaccines from Transgenic Plants.,Recombinant vaccine antigens expressed in transgenic plants may be used for oral immunization.Problems: inefficient uptake of antigen in GI trac

37、tdifficult to control doserisk for development of tolerance to antigen,risk for breaking tolerance to food antigens if immunomodulatory genes included,Summary.,Mucosal immunization has the potential to induce both loc

38、al (sIgA, CTL) and systemic (IgG, IgA, CTL) immunity.Immune response may be enhanced and modulated selectively by adjuvant or targeted delivery system.Risk for development of tolerance when antigen is delivered orally.