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Mucosal Immunization.

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 prevent colonization and infection.

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Mucosal Immunization.

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  1. Mucosal Immunization. Leslie Ann Mitchell, PhD. Gene Therapy Institute, Hadassah Ein Karem

  2. Immune Response. • Innate immunity: • macrophages, NK cells, mucous, lysozyme, cytokines, etc. • Provides first line of defense to prevent colonization and infection. • Determines which antigens will be recognized by B- and T-cells and the nature of the response.

  3. Immune Response. • Acquired immunity: B- and T-lymphocytes. • B-cells: produce antibodies that neutralize viruses and bacterial toxins, block attachment of microbes to host cells, or opsonize microbes for phagocytosis • T-cells: help (CD4+) or cytotoxic (CD8+) functions (elimination of intracellular microbes and viruses)

  4. 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 inducing local immunity.

  5. 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). • Mucosal surfaces are easily tolerized: non-responsiveness has evolved to prevent unwanted inflammatory responses.

  6. Design of Mucosal Vaccines: Important Factors. • Antigen must be protected from enzymatic digestion or acid hydrolysis. • 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.

  7. Pathogen-associated molecular patterns (PAMPs). • Conserved molecular structures found on bacterial and viral pathogens. • Examples: • Gram + and – bacteria: LPS, teichoic acid, peptidoglycans, CpG motifs • RNA virus: dsRNA • Yeasts: mannans

  8. 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, collectins, toll-like receptors (TLRs), mannose binding protein (MBP), serum amyloid P, C’ receptors, CD11b/CD18, DEC205

  9. PAMPs and PPRs: Mechanism of activation. Example: bacterial CpG binds to TLR9 • Binding 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. • Enhances antigen presentation, T-cell activation, increases adaptive immune response.

  10. Mucosal Epithelial Cells. • Interface between pathogen and immune system: • Innate (MHC Class I) and inducible (MHC Class II) antigen presentation function • Induced by pathogens to synthesize and secrete: • Antimicrobial peptides and proteins • Immunoregulatory cytokines • Colony-stimulating factors • Chemokines (recruitment of immune cells)

  11. TH1: IFN-g, TNF-b Cellular immunity vs. intracellular bacteria, small parasites Induction of neutralizing antibodies of the IgG2a subclass (in mice) TH2: IL-4, IL-5, IL-10, and IL-13 Induced by helminth parasites, allergens, immunization with soluble or alum-adsorbed antigens Immunity to extracellular parasites, bacteria Helper function in production of IgA, IgE, and neutralizing IgG to bacterial toxins Helper T-Cell Subsets.

  12. Regulation of T-cell Response. • TH1 vs TH2: cross-regulation by TH cytokines 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.

  13. CD4 T-cell response induced by: Antigens targeted toward MHC Class II processing pathway Live or killed bacteria or viruses Purified protein or peptide antigens with adjuvants such as alum. CD8 T-cell response induced by: Intracellular viruses or bacteria Delivery systems targeting towards MHC Class I pathway: liposomes,ISCOMS, microparticles, naked DNA Targeting T-cell Response in Vaccination Protocols.

  14. Requirements for T-cell Activation. • Antigen recognition through TcR in the context of MHC Class I or II molecules. • Co-stimulatory molecular interactions between T-cell and APC: APC: T-cell: B7-1, B7-2 CD28 CD40 CD40L ICAM-3 LFA-1 LFA-3 CD2

  15. Role of Dendritic Cells (DCs). • 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, increase presentation ability, enhanced expression of MHC Class II and B7. • Maturation influenced by PAMPs.

  16. Immunomodulatory Molecules. • Enhance or disrupt co-stimulation. • Influence direction of DC maturation. • PAMPs (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.

  17. Mucosal Adjuvants and Delivery Systems: Influence on Immune Response.

  18. Mucosal Adjuvants and Delivery Systems: Influence on Immune Response.

  19. Bacterial Toxin Adjuvants. • Vibrio cholera Toxin (CT, CTB, mutants), E. coli heat-labile toxin (LT, mutants), pertussis toxin • Intra-nasal, oral, and systemic routes • Depending upon antigen dose, route can favor TH2 (CT), TH1 (LT) or mixed TH1/TH2 responses.

  20. Monophosphoryl Lipid A (MPL). • Derived from LPS (Gram –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 TH2 to TH1 switch.

  21. 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 responses after parenteral, oral, intra-rectal, or intra-nasal administration with antigen.

  22. Microparticle Delivery Systems: PLG Polymers. • PLG: poly(lactide-co-glycolide) polymers • Target mucosal M-cells in Peyer’s patches when administered orally. • Effective adjuvants for intra-nasal delivery. • Favor TH1 response and facilitate induction of CD8+ CTLs.

  23. Mucosal Delivery Systems: Liposomes. • Artificial lipid bilayers composed of lipids + cholesterol. • 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.

  24. Immune-stimulating 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 membrane). • Stimulate production of IL-12. • Favor TH1 response (CD8+ CTLs).

  25. Chitosan particles. • Chitosan= deacylated chitin. • Enhance adsorption of protein antigens at mucosal surfaces by opening tight junctions. • Systemic immunity: enhance responses to parenterally-administered antigens and nonspecific host resistance. • Favor TH2 responses.

  26. Live Attenuated Vectors. • Concept: antigen delivery as a recombinant gene in live vector mimicks natural infection, giving rise to strongly protective immunity. • Vectors: bacteria (commensals, attenuated Salmonella spp., BCG), viruses (poliovirus, vaccinia, canarypox, etc.) • Replicating vectors enhance immune response. • Induces strong cellular (TH1) response (CTLs) and also antibodies.

  27. 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 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]). • Favors TH1, CTL, IgA responses.

  28. 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 tract • difficult to control dose • risk for development of tolerance to antigen, • risk for breaking tolerance to food antigens if immunomodulatory genes included

  29. Summary. • Mucosal immunization has the potential to induce both local (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. • Immunologic memory must be induced (unknown at present).

  30. Role of Immunomodulatory Molecules on Response.

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