1. MBBS Batch – 17 Remya APPLIED MICROBIOLOGY OF THE GIT

2. Learning outcomes Describe mucosa associated lymphatic tissue (MALT) with special reference to Gut associated lymphatic tissue (GALT) Describe the basic mechanisms and steps of mucosal immunity including role of M cells, secretory IgA, circulating mucosal immune system. List the requirements of oral protective vaccines and strategies for mucosal immunization. List currently used / newer oral vaccines.

5. The first line of defence 1) Microorganisms- stick onto the surface - destroying surface cells - enter inside the body. Obviously the body does not want this to happen so it protects itself by:- a) Sampling what is outside and producing an immune response b) Preventing adhesion

6. 1. Outside of the body :- dry surfaces (skin) wet surfaces ( gastro intestinal, respiratory and urogenital tracts) 2 The wet surfaces - “bug friendly” - especially to the thin epithelia of the GI and respiratory tracts 3. The skin and other stratified epithelia - patrolled by APC and lymphocytes of all classes. Both circulate between the epithelial layer and lymph nodes.

7. Adhesion of bacteria and other lower organisims is made difficult by:- 1 A mechanical barrier - The wet surfaces of the body are protected by a barrier of mucus which makes a barrier between the bacteria and the actual cell surfaces 2. Specific defences - Microorganisms must adhere to cells if they are not to be carried out of the body. IgA class antibodies coat the microorganism preventing adhesion

8. Lymphoid tissue associated with the lymphatic system is concerned with immune functions in defending the body against the infections and spread of tumors. The central or primary lymphoid organs generate lymphocytes from immature progenitor cells - thymus and the bone marrow. Secondary or peripheral lymphoid organs are the sites of lymphocyte activation by antigen.

9. Secondary lymphoid tissue provides the environment for the foreign or altered native molecules (antigens) to interact with the lymphocytes - lymph nodes, and the lymphoid follicles in tonsils, Peyer's patches, spleen, adenoids, skin, etc. that are associated with the MALT Encapsulated lymphatic glands (and organs) & noncapsulated lymphoid tissues Noncapsulated lymphoid tissues- MALT, GALT, tonsils

10. The Mucosal Immune System 1) The secretory immune system - prevents infectious organisms from adhering to the wet surfaces of the body 2) The T cells - concentrate in the gut wall 3) Interactions between the mucosal and the systemic immune systems may produce tolerance; minimise the risk of allergic reactions to food allergens.

11. Mucosa Associated Lymphoid Tissue - MALT Lymphoid tissue associated with human mucosa. MALT is scattered along mucosal linings, measuring roughly 400m 2 It is the most extensive component of human lymphoid tissue.

12. MALT Very diverse Acts as a physical barrier. Alert and trigger the defense when antigens try to enter the body. They are the places where antigens will make the first contact with the pathogens. Thus the direct reaction of the immune system is mandatory.

14. Mucosa Associated Lymphoid Tissue - MALT Two major components of MALT: BALT (Bronchial Associated Lymphoid Tissue) GALT (Gut Associated Lymphoid Tissue) Minor components of MALT Nose-associated lymphoid tissue (NALT) Vulvovagina -associated lymphoid tissue (VALT) Skin associated lymphoid tissue (SALT) is not mucosal but has the same characteristics of the MALT

15. Sampling the Outside of the Body Sampling is carried out by the O-MALT (organised mucous-associated lymphoid tissue). This is found in (GALT) Present as aggregated follicles such as Peyer's patches in the intestine. A specialised epithelium sometimes called a lymphoepithelium which lacks goblet cells but has M-cells

16. MALT The M-cells form a basket into which penetrate B and T cells. The B-cells are mainly "naive" and the T cells helper. Just below the M-cell is an area rich in macrophages and antigen presenting cells. In the Peyer's patch this area of differentiation overlies an area rich in what appear to be IgA memory cells M-cells are armed with a series of "take up" devices.

17. M cell Microfold cells are cells found in the follicle-associated epithelium of the Peyer's patch. Receptor mediated endocytosis, fluid phase endocytosis, capable of phagocytosis Both viruses and bacteria have "learnt to exploit" the uptake. Eg: reovirus, poliovirus, mouse mammary tumour virus, Salmonella, Yersinia and Shigella A few chemicals may also invade the Peyer’s patch. Peyer’s patches have been used as a route for vaccines.

18. stimulate B cell growth and increase immunoglobulin secretion stimulate immune response, mediators of fever and of the acute phase response GALT Activated B cells immune response is amplified

19. GALT Peyer’s patches Appendix Pathogenic micro-organisms and other antigens enter the intestinal tract. Encounter macrophages, dendritic cells, B-lymphocytes, and T-lymphocytes found in Peyer's patches and other gut-associated lymphoid tissue (GALT). Peyer's patches - specialized cells which sample antigen directly from the lumen - deliver it to antigen-presenting- cells (APCs) located in a unique pocket-like structure on their baso-lateral side.

20. B-cells and memory cells are stimulated upon encountering antigen in Peyer's patches. These cells then pass to the mesenteric lymph nodes where the immune response is amplified. Activated lymphocytes pass into the blood stream via the thoracic duct and travel to the gut where they carry out their final effector functions. The appendix shows to be mainly lymphoid tissue - the only known function of the appendix is a lymphatic one.

21. Organized lymphoid follicles at specific mucosal sites (O-MALT) Occur in tissues of digestive, respiratory and genital mucosal surfaces Light germinal centers Dark adjacent areas populated by B and T lymphocytes and antigen-presenting cells Site of antigen sampling and generation of effector and memory cells

22. M-Cells Follicle-associated epithelium Dome region Germinal Center Parafollicular region LUMEN Lymphoid Follicle (Microfold cells) O-MALT (Organized lymphoid follicles )

24. Diffuse MALT (D-MALT) Lamina propria lymphocytes (primarily B cells) is the major site of Ig synthesis Lamina propria: the layer of connective tissue underlying the epithelium of a mucous membrane Derived from O-MALT and represent effector and memory cells from cells stimulated by antigen Intraepithelial lymphocytes (IELs) Plasma cells producing dimeric IgA Antigen-presenting cells (macrophages and dendritic cells)

25. IgA IgA are found in large number in the subepithelium of gut respiratory tree urino-genital tract also the mammary glands late in pregnancy IgA normally exists as a polymer of between 2-4 IgA monomers. (joined by J-chain). Polymeric IgA is passed across the epithelia layer into, for example the lumen of the gut

26. IgA molecules do not cause inflammation because they are not recognised by phagocyte nor do they fix complement. IgA molecules function by coating potentially infectious material preventing it from adhering to the walls of the vessel. IgA deficiency in children is a not-uncommon condition. There are few clinical signs or associations apart from some increase in atopy. However certain bacteria (eg Neisseria gonorrheae) have evolved a specific IgA protease, so clearly they have found the system a limit to growth

27. TRANSPORT OF IgA ACROSS THE EPITHELIUM

28. Movement of IgA Plasma Cells and Transfer of IgA Activated IgA plasma cells do not remain in the O-MALT but head off to populate the subepithelia of the wet surface of the body and associated glands. Secreted IgA binds to its receptor (secretory component) on the basal surface of the epithelial cells and is transported across the cells by endocytosis and released at the far side by proteolysis. The secretory component helps anchor the IgA in the mucous layer

29. Lymphocytes activated in one mucosal site migrate via the lymphatic vessels and the blood to other mucosal sites

30. Mucosal T Cells If a section of gut is examined under the microscope we see small, mononucleate, cells inserted between the enterocytes. Until recently these have been dismissed as curiosities but firstly it was found that the cells form a discrete subset of T cells.

31. And their role Their role is not fully understood but it is suggested that because of their ability to recognise antigen without the need for processing that they allow an immediate if weaker response to material penetrating the intestinal wall

32. WHY MUCOSAL IMMUNIZATION? Mucosal surface is major portal of entry for pathogens Mucosa contains highest concentration of lymphocytes Prevent both initial stages of disease (colonization and infection by pathogens) and block its development Can be administered orally or nasally Provides both mucosal immunity and systemic immunity Systemic immunization no mucosal immunity block disease development once the pathogen has crossed the mucosal barrier into the normally sterile systemic environment

33. Requirements of Protective Vaccines Block adherence of microorganism to host Facilitate clearance from host Neutralize toxin Must recognize “virulence” epitopes Must be immunogenic Must not induce autoimmune disease Should induce long-lasting immunity Must induce the type of response that is effective to eliminate pathogen

34. Rational Strategies for Mucosal Immunization Requirements Safe taken orally Long-lasting due to continued maintenance of memory Survive in gastric and intestinal environments Must escape normal clearance mechanisms Must compete for inclusion within M-Cell transport Must arrive intact to antigen-processing cells Must induce dimeric sIgA reactive with cell surface

35. Strategies for Delivery of Vaccine Into O- MALT Inert particulate carriers Biodegradable copolymers Immune-stimulating complexes Live vaccine vectors (recombinant) Vaccinia virus Salmonella Mycobacterium bovis

36. Strategies for Enhancing Mucosal Immune Response Co-delivery with cytokines Co-immunogens (Cholera toxin) Peptides presented with potent T-cell epitopes

37. Newer vaccine development CMIS Oral/Nasal Vaccines Rectal/Vaginal Vaccines NALT GALT Mucosal Lamina Propria Direct antigens to “M” cells. Produce systemic IgA response Prevent pathogen attachment mucosal surfaces Mucosal Immunization

38. The “Common Mucosal Immune System”

39. Currently used / newer oral vaccines Rotarix Proper Name: Rotavirus Vaccine, Live, Oral : For the prevention of rotavirus gastroenteritis caused by G1 and non-G1 types (G3, G4, and G9), for use in infants 6 weeks to 24 weeks of age. RotaTeq Proper Name: Rotavirus Vaccine, Live, Oral, Pentavalent For prevention of rotavirus gastroenteritis in infants and children caused by the serotypes G1, G2, G3, and G4 when administered as a 3-dose series to infants between the ages of 6 to 32 weeks.

40. Adenovirus Type 4 and Type 7 Vaccine, Live, Oral : Indicated for active immunization for the prevention of febrile acute respiratory disease caused by Adenovirus Type 4 and Type 7 Vivotif Proper Name: Typhoid Vaccine, Live, Oral Ty21a : For immunization of adults and children greater than 6 years of age against disease caused by Salmonella typhi. Oral polio vaccine (OPV) – Sabin strain live-attenuated vaccine

41. Dukoral Proper name: WC-rBS, Cholera vaccine, monovalent inactivated vaccine, killed whole cells of V. cholerae O1 plus additional recombinant cholera toxin B subunit. Shanchol Proper name: BivWC, bivalent inactivated vaccine, killed whole cells of V. cholerae O1 and V. cholerae O139. only available in Vietnam used against cholera and traveler's diarrhea.

42. Newer oral vaccines under study….. Mucosal AIDS vaccine ! - HIV-neutralizing sIgA in the genital tract and HIV-reactive T cells in the cervix that confers resistance to infection Autoimmunity Immunocontraceptives: approximately 30% of human infertility is associated with the presence of anti-sperm antibodies in mucous secretions of men and/or women and sperm-reactive T cells in men only----design of a “vaccine” capable of inducing an immune response against sperm antigens in either partner.

43. Thank you………. Man the barrier! strategic defences in the intestinal mucosa. Cathryn Nagler-Anderson Nature Reviews Immunology 1, 59-67 (October 2001) doi:10.1038/35095573 Regulation of IgA synthesis at mucosal surfaces. Sidonia Fagarasan, Tasuku Honjo. http://dx.doi.org/10.1016/j.coi.2004.03.005 http://www.sciencedirect.com/science/article/pii/S0952791504000421 Nature Medicine 11, S45 - S53 (2005) Published online: ; | doi:10.1038/nm1213 Mucosal immunity and vaccines Jan Holmgren1 & Cecil Czerkinsky2