1. VACCINOLOGY (BIO-301) Credit Hrs 3(2-1) Dr. Aneela javed

2. Part 2: Principles of Vaccine Design Immunologic Memory: T and B Cells memory Antigen Processing and Presentation by MHC Class I, II, and Nonclassical Molecules Understanding the Mucosal Immune System for Better Mucosal Vaccine Design Part 2: Principles of Vaccine Design Immunologic Memory: T and B Cells memory Antigen Processing and Presentation by MHC Class I, II, and Nonclassical Molecules Understanding the Mucosal Immune System for Better Mucosal Vaccine Design PART 1 : INTRODUCTION Definition History Types Combination vs single vaccine PART 1 : INTRODUCTION Definition History Types Combination vs single vaccine Course Contents Part 3: ANIMAL MODELS FOR VACCNE TESTING Utility of Mouse Models in Vaccine Design and Development, Utility of Nonhuman Primate Models for Vaccines, Part 3: ANIMAL MODELS FOR VACCNE TESTING Utility of Mouse Models in Vaccine Design and Development, Utility of Nonhuman Primate Models for Vaccines, VACCINE PRODUCTION

3. most of the highly successful vaccines have been made empirically, with little or no immunological insight Recent advances in innate immunity have offered new insights about the mechanisms of vaccine-induced immunity and have facilitated a more rational approach to vaccine design. the failure to develop vaccines against global pandemics such as infection with human immunodeficiency virus (HIV) despite decades of effort has underscored the need to understand the immunological mechanisms by which vaccines confer protective immunity Part 2: Principles of Vaccine Design Immunologic Memory: T and B Cells memory Antigen Processing and Presentation by MHC Class I, II, and Nonclassical Molecules Understanding the Mucosal Immune System for Better Mucosal Vaccine Design Part 2: Principles of Vaccine Design Immunologic Memory: T and B Cells memory Antigen Processing and Presentation by MHC Class I, II, and Nonclassical Molecules Understanding the Mucosal Immune System for Better Mucosal Vaccine Design

7. 1.distinct subsets of helper T cells, such as T H 1, T H 2 and T H 17, are effective at protecting against different pathogens 2.Follicular helper T cells (T FH cells) produce interleukin 21 (IL-21) and help with the differentiation of B cells and generation of memory B cells. 3.In addition, differentiating memory CD4 + and CD8 + T cells can be subcategorized into central memory and effector memory cell subsets, each with a distinct functionality. 1.distinct subsets of helper T cells, such as T H 1, T H 2 and T H 17, are effective at protecting against different pathogens 2.Follicular helper T cells (T FH cells) produce interleukin 21 (IL-21) and help with the differentiation of B cells and generation of memory B cells. 3.In addition, differentiating memory CD4 + and CD8 + T cells can be subcategorized into central memory and effector memory cell subsets, each with a distinct functionality.

8. Pattern recognition receptors (PRRs) : proteins expressed by cells of the innate immune system to identify pathogen-associated molecular patterns (PAMPs), which are associated with microbial pathogens or cellular stress, as well as damage-associated molecular patterns (DAMPs), which are associated with cell components released during cell damage. 1.(PAMPs) and include bacterial carbohydrates (such as lipopolysaccharide or LPS, mannose), 2. nucleic acids (such as bacterial or viral DNA or RNA), bacterial peptides (flagellin, Xa21), 3. peptidoglycans and lipoteichoic acids (from Gram positive bacteria), 4.N-formylmethionine, lipoproteins and fungal glucans. 5.Endogenous stress signals are called danger-associated molecular patterns (DAMPs) and include uric acid.

9. The innate immune system can sense microbes through pattern-recognition receptors (PRRs), such as 1. the Toll-like receptors (TLRs), which are expressed by various cells, including dendritic cells (DCs) 2.the C-type lectin-like receptors 3.the cytosolic Nod-like receptors 4.Cytosolic RIG-I-like receptors sense viral nucleic acids There are many subsets of functionally distinct DCs, and it is now clear that the DC subset, as well as the nature of the PRR, have a key role in determining the magnitude and quality of adaptive immune responses. The innate immune system can sense microbes through pattern-recognition receptors (PRRs), such as 1. the Toll-like receptors (TLRs), which are expressed by various cells, including dendritic cells (DCs) 2.the C-type lectin-like receptors 3.the cytosolic Nod-like receptors 4.Cytosolic RIG-I-like receptors sense viral nucleic acids There are many subsets of functionally distinct DCs, and it is now clear that the DC subset, as well as the nature of the PRR, have a key role in determining the magnitude and quality of adaptive immune responses.

10. 1. dimers, homodimers, TLR2 forms heterodimers 2.endosomal TLRs comprising TLR3, TLR7, TLR8 and TLR9 recognize nucleic acid derived from viruses as well as endogenous nucleic acids in context of pathogenic events. 3.Activation of these receptor leads to production of inflammatory cytokines as well as type I interferons (interferon type I) to help fighting viral infection.

11. TLRs may also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4's recognition of LPS, which requires MD-2. CD14 and LPS-Binding Protein (LBP) are known to facilitate the presentation of LPS to MD-2.

12. When activated, TLRs recruit adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in signaling. These proteins are known as 1. MyD88, 2.Tirap (also called Mal), 3.Trif, and 4.Tram (toll-like receptor 4 adaptor protein). TLR SIGNALING

14. MyD88-dependent pathway utilized by every TLR except TLR3. Its primary effect is activation of NFκB and Mitogen-activated protein kinase MyD88 then recruits IRAK 4, IRAK1 and IRAK2. IRAK kinases then phosphorylate and activate the protein TRAF6, which in turn polyubiquinates the protein TAK1, as well as itself in order to facilitate binding to IKK-β. On binding, TAK1 phosphorylates IKK-β, which then phosphorylates IκB causing its degradation and allowing NFκB to diffuse into the cell nucleus and activate transcription and consequent induction of inflammatory cytokines. [23]NFκBMitogen-activated protein kinaseIRAK1IRAK2TRAF6TAK1IKK-βIκB [23] MyD88-dependent pathway utilized by every TLR except TLR3. Its primary effect is activation of NFκB and Mitogen-activated protein kinase MyD88 then recruits IRAK 4, IRAK1 and IRAK2. IRAK kinases then phosphorylate and activate the protein TRAF6, which in turn polyubiquinates the protein TAK1, as well as itself in order to facilitate binding to IKK-β. On binding, TAK1 phosphorylates IKK-β, which then phosphorylates IκB causing its degradation and allowing NFκB to diffuse into the cell nucleus and activate transcription and consequent induction of inflammatory cytokines. [23]NFκBMitogen-activated protein kinaseIRAK1IRAK2TRAF6TAK1IKK-βIκB [23]

15. TRIF-dependent pathway[edit]edit Both TLR3 and TLR4 utilize the TRIF-dependent pathway, which is triggered by dsRNA and LPS, respectively. For TLR3, dsRNA leads to activation of the receptor, recruiting the adaptor TRIF. TRIF activates the kinases TBK1 and RIPK1, which creates a branch in the signaling pathway. The TRIF/TBK1 signaling complex phosphorylates IRF3 allowing its translocation into the nucleus and production of Interferon type I. Meanwhile, activation of RIPK1 causes the polyubiquitination and activation of TAK1 and NFκB transcription in the same manner as the MyD88-dependent pathway. [23]dsRNALPSTLR3TRIFTBK1RIPK1IRF3Interferon type I [23]. TRIF-dependent pathway[edit]edit Both TLR3 and TLR4 utilize the TRIF-dependent pathway, which is triggered by dsRNA and LPS, respectively. For TLR3, dsRNA leads to activation of the receptor, recruiting the adaptor TRIF. TRIF activates the kinases TBK1 and RIPK1, which creates a branch in the signaling pathway. The TRIF/TBK1 signaling complex phosphorylates IRF3 allowing its translocation into the nucleus and production of Interferon type I. Meanwhile, activation of RIPK1 causes the polyubiquitination and activation of TAK1 and NFκB transcription in the same manner as the MyD88-dependent pathway. [23]dsRNALPSTLR3TRIFTBK1RIPK1IRF3Interferon type I [23].

17. TLR signaling ultimately leads to the induction of thousands of genes. TLRs constitute one of the most pleiotropic yet tightly regulated gateways for gene modulation

18. ReceptorLigand(s) [27] [27] Ligand location [27] [27] Cell types [27] [27] TLR 1 multiple triacyl lipopeptideslipopeptides Bacteria  monocytes/macrophages monocytesmacrophages  a subset of dendritic cellsdendritic cells  B lymphocytes B lymphocytes TLR 2 multiple glycolipidsglycolipidsBacteria  monocytes/macrophages  neutrophils [28] [28]  Myeloid dendritic cells [29] Myeloid dendritic cells [29]  Mast cells Mast cells multiple lipopeptidesBacteria multiple lipoproteinslipoproteinsBacteria lipoteichoic acid Gram- positive bacteria HSP70Host cells zymosanzymosan (Beta-glucan)Beta-glucanFungi Numerous others TLR 3 double-stranded RNAdouble-stranded RNA, poly I:Cpoly I:C viruses  Dendritic cells  B lymphocytes

19. TLR 4 lipopolysaccharide Gram- negative bacteria  monocytes/macrophages  neutrophils [28] [28]  Myeloid dendritic cells [29] [29]  Mast cells  B lymphocytes [30] [30]  Intestinal epithelium Intestinal epithelium several heat shock proteinsheat shock proteins Bacteria and host cells fibrinogenhost cells heparan sulfateheparan sulfate fragmentshost cells hyaluronic acidhyaluronic acid fragmentshost cells nickel Various opioid drugsopioid TLR 5 flagellinBacteria  monocyte/macrophages  a subset of dendritic cells  Intestinal epithelium [[profilin [31] ]] [31] Toxoplasma gondii

20. TLR 6multiple diacyl lipopeptidesMycoplasma  monocytes/macrophages  Mast cells  B lymphocytes TLR 7 imidazoquinoline small synthetic compounds monocytes/macrophages Plasmacytoid dendritic cells [29] B lymphocytes loxoribineloxoribine (a guanosine anal ogue)guanosine bropirimine single-stranded RNARNA viruses TLR 8 small synthetic compounds; single-stranded RNA  monocytes/macrophages  a subset of dendritic cells  Mast cells

21. TLR 9 unmethylated CpG Oligodeoxynucleotide DN ACpG Oligodeoxynucleotide Bacteria, DNA viruses monocytes/macrophages Plasmacytoid dendritic cells [29] [29] B lymphocytes TLR 10unknown TLR 11Profilin Toxoplasma gondii [32]  monocytes/macrophages  liver cells liver  kidney kidney  urinary bladder epithelium urinary bladderepithelium TLR 12Profilin Toxoplasma gondii [34] [34]  Neurons [35] [35]  plasmacytoid dendritic cells  conventional dendritic cells  macrophages TLR 13 [36][37] [36][37] bacterial ribosomal RNA sequence “CGGAAAGACC” Virus, bacteria  monocytes/macrophages  conventional dendritic cells

22. Cell typeTLR Monocyte1,2,4,5,6,7,8,9 Macrophages Dendritic cells Conventional DC Plasmacytoid DC B cells Neutrophils Mast cells Intestinal epithelium

26. Cellular immunity Maximizes the killing efficacy of the macrophages proliferation of cytotoxic CD8 + T cells.CD8 promotes the production of opsonizing antibodies. (IgG, IgM and IgA but not IgE antibodies) Humoral immune system. Humoral immune system Stimulates B-cells into proliferation,B-cells induce B-cell antibody class switching,antibody class switching Increasesneutralizing antibody production (IgG, IgM and IgA as well as IgE antibodies)antibody

28. T h 17 helper cellsT h 17 helper cells mediate host immunity against extracellular bacteria and fungi. T h 17 helper cellsT h 17 helper cells mediate host immunity against extracellular bacteria and fungi. The regulatory T cells (Tregs), formerly known as suppressor T cells, maintain tolerance to self- antigens, and abrogate autoimmune disease.

29. IMMUNE MEMORY

33. T cell-dependent activation 1.The T cell is stimulated to produce autocrines, resulting in the proliferation or differentiation to effector or memory T cells. 2.A certain portion of the resulting effector T cells then activate specific B cells through a phenomenon known as an Immunological synapse. 3.Activated B cells subsequently produce antibodies that assist in inhibiting pathogens until 1.The T cell is stimulated to produce autocrines, resulting in the proliferation or differentiation to effector or memory T cells. 2.A certain portion of the resulting effector T cells then activate specific B cells through a phenomenon known as an Immunological synapse. 3.Activated B cells subsequently produce antibodies that assist in inhibiting pathogens until

34. T cell-independent activation

35. 1.An advantage of forgoing T cell involvement is that an expedited immune response can be mobilized, 2. germinal center formation, isotype switching and affinity maturation do not occur during this form of activation. 3.Also, because almost all memory B cells are derived from germinal centers, effective antibody-mediated memory is 4.against polysaccharide capsules of encapsulated bacteria 1.An advantage of forgoing T cell involvement is that an expedited immune response can be mobilized, 2. germinal center formation, isotype switching and affinity maturation do not occur during this form of activation. 3.Also, because almost all memory B cells are derived from germinal centers, effective antibody-mediated memory is 4.against polysaccharide capsules of encapsulated bacteria

36. Live attenuated vaccines such as those against smallpox or yellow fever are the most successful vaccines ever made and can confer lifelong memory, whereas nonliving vaccines induce protection of much shorter duration and require booster vaccination to maintain protective immunity. Thus, a single dose of the smallpox vaccine maintains serum antibody titers for more than 50 years and cellular immunity is also maintained for decades. engagement of multiple innate pathogen recognition receptors is key to generating T-cell responses of different magnitudes and functional profiles [6, 12–14], suggesting that, the overall breath, functionality and duration of acquired immune responses are dictated by early innate signals triggered by live attenuated vaccines [6, 12–14]. engagement of multiple innate pathogen recognition receptors is key to generating T-cell responses of different magnitudes and functional profiles [6, 12–14], suggesting that, the overall breath, functionality and duration of acquired immune responses are dictated by early innate signals triggered by live attenuated vaccines [6, 12–14].