CONTENT. Chapter 1 Review of Literature. List of figures. List of tables

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1 Abstract Abbreviations List of figures CONTENT I-VI VII-VIII IX-XII List of tables XIII Chapter 1 Review of Literature 1. Vaccination against intracellular pathogens Role of different immune responses against infections Role of antibodies in intracellular infections Role of T-cell responses in intracellular infections Requirements for successful vaccination Different Strategies for vaccination Heat killed vaccines Live attenuated vaccines Heat or chemically inactivated vaccines Recombinant vaccines DNA vaccines Immunologic adjuvants Classification of adjuvants Properties of adjuvants Selection of an appropriate adjuvant for vaccination Immunological perspective of controlled antigen delivery Balance between immunity versus tolerance Induction of affinity maturation and isotype switching Induction of immune memory Manipulation of Th1/Th2 responses Liposomes as antigen delivery systems Suitability of liposomes as vaccine carriers Immunopotentiation effect of liposomes Strategies for optimization of liposomal 17 adjuvanticity 1.7 Fusogenic liposomes derived from microbial lipids 17-26

2 1.7.1 Virosomes Archaeosomes Yeast lipid liposomes Sphaeroplasts Vaccines against Intracellular pathogens Anti-Listerial Vaccines Killed or Inactivated Vaccines Live Attenuated Vaccines Subcellular and DNA Vaccines Vaccines against M. tuberculosis BCG vaccine Recombinant BCG Boosting BCG with subunit antigens Subunit Vaccines DNA vaccines Therapeutic vaccines 33 Chapter 2 Establishment of prophylactic potential of archaeosome based vaccine against experimental listeriosis in Balb/C mice 2.1 Introduction Material and Methods Chemicals and Reagents Animals Bacterial culture Isolation and SDS PAGE profile of Secretory proteins Western blot analysis Preparation of liposome and determination of 42 entrapment efficiency Size characterization of liposomes Interaction of calcein bearing liposome with 43 macrophages Mode and schedule of immunization Collection of Sera Determination of antigen specific isotypes by ELISA 43-44

3 Measurement of the Anti-Listerial DTH response Splenocyte preparation and culture Determination of IFN-γ, IL-12 and IL-4 by sandwich 45 ELISA Lymphocyte proliferation assay Staining for memory T cell and co-stimulatory 46 surface markers Infection and bacterial burden Statistical analysis Results SDS-PAGE analysis and western blotting of culture 47 supernatant antigens Characterization of liposome Archaeosome encapsulated SAg elicited elevated Th1 4 response Isotype profile Lymphoproliferative response Delayed Type Hypersensitivity Archaeosome encapsulated SAg enhances the expression of costimulatory molecules Induction of memory response upon immunization 55 with archaeosome entrapped antigen Protection studies Discussion Chapter 3 Listeria monocytogenes spheroplast induces cell mediated immune response in immunized mice 3.1 Introduction Material and Methods Chemicals and Reagents Animals Bacterial culture Isolation and SDS PAGE profile of Listeria whole 66 cell protein (LWCP)

4 3.2.5 Western blot analysis Preparation of liposome and determination of entrapment efficiency Preparation of Heat Killed bacteria Preparation of Spheroplast Transmission electron microscopy Mode of immunization Collection of Sera Antigen preparation for ELISA Determination of isotypes by ELISA Splenocyte isolation and culture Determination of IFN-γ, IL-12, and IL-4, by sandwich 70 ELISA Lymphocyte proliferation assay Staining for memory T cell and co-stimulatory 71 surface markers Infection and bacterial burden Statistical analysis Results SDS-PAGE analysis and western blotting of Listeria 73 whole cell protein (LWCP) and antigens Characterization of spheroplast Assessment of Spheroplast entrapped antigens as a vaccine candidate T cell proliferation assay Delayed type hypersensitivity Antibody class switching Expression of co-stimulatory molecules on 79 splenocytes Activation of T-cells leads to generation of memory response Protection studies performed on animals immunized with various formulations

5 3.4 Discussion Chapter 4 Archaeosome based RD antigen can impart long term protection against experimental murine tuberculosis. 4.1 Introduction Material and Methods Reagents and antibodies Culture of Mycobacterium tuberculosis Cloning, expression and purification Isolation of total polar Lipids Preparation of liposome and assessment of antigen 94 entrapment efficiency Animals and immunization Determination of Rv3619c specific isotypes Preparation of lymphocytes and T cell isolation Cytokine assay: Determination of IFN-γ, IL-4 and IL Staining for memory T cell and co-stimulatory surface 96 markers Challenge of animals with Mycobacterium tuberculosis Determination of bacterial load in lung and spleen 97 of immunized Animals Lymphocyte proliferation assay Histopathology Statistical analysis Results Characterization of PCR product of Rv3619c gene BCG produces less, armoury (Th1 cytokines) than archaeosome entrapped antigen Class switching of IgG enhanced by archaeosome 101 mediated antigen delivery Lymphocyte Proliferation assay Flow cytometry reveals increased expression of co-stimulatory and effector markers Protective efficacy of archaeosome entrapped Ag

6 against mycobacterial infection 4.4 Discussion References

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