2. Pamela A. Kozlowski 1 Marian R. Neutra 2 1 Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA. 2 Children’s Hospital, Enders Boston, Massachusetts, USA. Published in: Nature Immunology, February 2006

3. ABSTRACT Most infectious agents enter the body at mucosal surfaces and therefore mucosal immune responses function as a first line of defence. Protective mucosal immune responses are most effectively induced by mucosal immunization through oral, nasal, rectal or vaginal routes, but the vast majority of vaccines in use today are administered by injection. As discussed in this review, current research is providing new insights into the function of mucosal tissues and the interplay of innate and adaptive immune responses that results in immune protection at mucosal surfaces. These advances promise to accelerate the development and testing of new mucosal vaccines against many human diseases including HIV/AIDS.

4. CONTENTS  Mucosal Surface  Epithelial cells  Immune Effector Mechanism  Adaptive Immune protection at mucosal Surface  Induction of Mucosal Immune System  Mucosal Immunization  Challenges in vaccine design  Conclusion

5. COMPARISON BETWEEN PARENTERAL VACCINESMUCOSAL VACCINES  Poor inducers of mucosal immunity  Rapid generation of Abs etc  Measurable immune response  So, high approval ratio from FDA  Efficiently induce it  Slightly delayed  Indeterminate  Only a few have been approved including rotavirus, typhoid and cholera

6. MUCOSAL SURFACES  ENORMOUS SURFACE AREA:  INNATE DEFENCES AT MUCOSAL SURFACES:  delicate epithelial membrane  associated glands produce enzymes like proteases  lymphoid and Antigen Presenting Cells are also present in the mucosal surface

7. FUNCTIONS OF EPITHELIAL CELLS  So, epithelial cells of the intestine function as;  Sensors that detect antigens microbial components through PRRs (like TLRs).  Send cytokine and chemokine signals to underlying mucosal cells, such as dendritic cells (DCs) and macrophages, to trigger innate, nonspecific defences and promote adaptive immune responses  Induce tolerance against nonthreatening nutrients and the normal intestinal flora that could lead to mucosal inflammation

8. MULTIPLE IMMUNE EFFECTOR STRATEGIES Pathogen infection of Mucosal Membrane Block adherence &Invasion Ag capture by DCs Opsonization Cell Mediated Killing ADCC Neutralization Entrapment &Clearance Mucosal Immunity

10. ADAPTIVE IMMUNE PROTECTION  Four principal players in adaptive immune mechanism at the mucosal membrane;  IgA  IgG  Modification in Antigen uptake mechanism  Cyotoxic T lymphocytes

11. Adaptive Immune Protection sIgAIgG Uptake Mech. CTLs Immune exclusion Hindrance ADCC Neutralize the pathogen prevent systemic infection M-cells Presentation Clearance of infection

12. Block adherence &Invasion Ag capture by DCs Opsonization Cell Mediated Killing ADCC Neutralization Entrapment &Clearance

13. INDUCTION OF MUCOSAL IMMUNE RESPONSES Steps included in the induction/ initiation of mucosal immune response are; i. Antigen Sampling ii. Focusing the Immune response

14. 1. ANTIGEN SAMPLING  The first step is the antigen sampling at mucosal surfaces.  The pathogens are identifies and exposed to the regional lymh nodes, causing the activation of the immune system.  The diagram represents the sampling of the antigen by the immune tissue at various locations.

15. MUCOSAL Ag SAMPLING IN GUT  There are organized mucosal inductive sites at various locations within the mucosal tissue like in the gastrointestinal tract;  There are aggregates of mucosal lymphoid follicles which form the Peyer’s patches in the ileum,  These mucosal lymphoid follicles causes the differentiation to a specialized form of epithelium called follicle-associated epithelium (FAE)  These FAE contain Microfold cells (M cells)  The M cells form pockets into which lymphocytes migrate  These lymphocytes deliver samples of foreign material directly into the pocket and to underlying DCs.

17. FAE Deliver samples of foreign material to the pockets Attract the lymphocytes and DCs by chemokinesis DC High density of phagocytic cells at the site of entry ↑local Ag sampling &↓systemic infection Peyer’s Ag to draining lymph node Interface with systemic immune system

19. FUNCTIONS OF THE FOLLICLE ASSOCIATED EPITHELIUM 1. Chemokine release 2. Cytokine release 3. DC activation

20. 2. FOCUSING THE MUCOSAL IMMUNE RESPONSE  The activated B and T cells upregulate the expression of tissue-specific adhesion molecules and chemokine receptors that function as ‘homing receptors’ to guide the lymphocytes back to the mucosa through recognition of endothelial counter-receptors in the mucosal vasculature.  This broad recognition system explains why mucosal immunization at one site can result in the secretion of specific IgA antibodies in other mucosal or glandular tissues; this is referred to as the ‘common mucosal immune system’.

21. There Are Receptor-mediated Recognition Systems That Serve To Focus The Immune Response At The Site Where An Antigen Or Pathogen Was Initially Encountered IgA+ B cells (intestinal inductive sites ) Bloodstream Migrate back due to homing receptors Signal Generation Upregulation of adressins T Cell activation

22. MUCOSAL IMMUNIZATION  Mucosal immunization routes also can induce  the production of serum IgA and IgG  Activation of mucosal DCs and migration to spleen and lymh nodes  Memory CD8+ T cells

23. WHAT IS THE BEST IMMUNIZATION ROUTE?  CHOICE OF MUCOSAL IMMUNIZATION ROUTE DEPENDS UPON:  consideration of the species- humans, non humans etc  the nature of the vaccine- peptide, VLPs, conjugate etc  the expected site of challenge- respiratory, gut, rectal etc

24. VACCINATION STRATEGY  Optimal protection is likely to require both mucosal and systemic immune effectors, and the most effective mucosal vaccine strategies might be prime–boost combinations that involve both mucosal and systemic delivery.  Experimentally, mucosal immunization priming with systemic boosting has been found to be quite effective.

25. CHALLENGES IN MUCOSAL VACCINE DESIGN PROBLEMSOLUTION HOST DEFENCES Diluted in the mucosal secretionsLarger dose ( but increasing the dose can further complicate the situation) So, particulate vaccines may be used to overcome this as they have greater adherence and lesser clearance Proteolytic degradation etc ALERTING THE IMMUNE SYSTEMUse of adjuvants (list discussed later) BREACHING THE EPITHELIAL BARRIERS Various mucosal vaccines have different mechanisms to overcome this barrier (discussed in table below)

26. BREACHING THE EPITHELIAL BARRIER AGENTSMECHANISMGOAL Protein,Peptide, DNA, live ↑ adherence, entry into epithelial cells ↑Ag uptake, ↑Immune Response ParticulateAgs with micro. can pass through the M cells Easy transport, taken up by mucosal DCs, Provide Ag depot VLPs, Bacterial vesicles Size provides an advantage for crossing the M cells ↑Innate activity, ↑Immune Response

27. ADJUVANTS EMPLOYED IN MUCOSAL VACCINES  The best-known mucosal adjuvants are  the secreted enterotoxins of V. cholerae and E. coli,  Immunomodulatory cytokines-IL-12, granulocyte/macrophage colony-stimulating factor (GM-CSF) or a combination of both  TLR Ligands- CpG-containing oligonucleotides, flagellin and bacterial porins.

28. RISKS ASSOCIATED  The mucosal vaccines have some safety and acceptability issues;  Might reflect local inflammation, such as mild enteritis-like symptoms  Possibility of retrograde transport to the brain through olfactory nerves (live attenuated adenovirus). 3/ 4

30. CONCLUDING REMARKS  Much has been learned from animal studies about the attributes of effective mucosal vaccines and the immune effectors that could function together to prevent and control mucosal transmission of HIV and other mucosally transmitted diseases.  The current challenge is to apply this knowledge to vaccine design and to carry out collaborative, comparative clinical trials that systematically monitor all parameters of the immune response — humoral and cellular, mucosal and systemic — in serum, local secretions and mucosal tissues.  Available data indicate that mucosal HIV vaccines should be particulate or live vectored, include components that alert the innate immune system, and include immunogenic, conserved forms of the envelope protein gp41 as well as gp120.  Mucosal HIV vaccines would ideally be administered as part of a prime–boost strategy that induces both mucosal and systemic immunity. Much work remains to be done, but current research continues to clarify the concepts and provide the tools that are needed to exploit the full potential of mucosal vaccines.