Table of Content CHAPTER-1... 1 1 INTRODUCTION... 1 CHAPTER-2... 4 2 REVIEW OF LITERATURE... 4 2.1 History of Filariasis... 4 2.1.1 Discovery of Symptoms (1588-1592)... 4 2.1.2 Discovery of Microfilariae (1863 and 1866)... 4 2.1.3 Discovery of Adult Worm (1876)... 5 2.1.4 Discovery of Filarial Life Cycle (1877)... 5 2.1.5 Discovery of Transmission (1900)... 5 2.2 Causative agent... 7 I. Wuchereria bancrofti... 7 II. Brugia malayi... 7 III. Brugia timori... 8 2.3 Life cycle of B. malayi... 9 2.3.1 Development in intermediate host (mosquito)... 9 2.3.2 Development in definitive host (humans or rodents)... 9 2.3.3 Periodicity... 10 2.4 Epidemiology... 11 2.4.1 Global Distribution... 11 2.4.2 Current situation in India... 12 2.4.3 Economic burden of disease... 13 2.4.4 Disability adjusted Life years (DALY)... 14 2.4.5 Hold in endemic countries... 15 2.5 Symptoms and pathology... 15 2.6 Development of the disease... 16 2.6.1 The pre-patent period (biological incubation period)... 16 2.6.2 The patent (symptom-less) period... 16 2.6.3 The acute or allergic stage... 16 2.6.4 The chronic manifestations... 17 2.7 Categories of filarial subjects in filaria endemic area... 17 2.7.1 Mf carriers... 17 2.7.2 Symptomatic... 17 2.7.3 Endemic normal... 18 2.7.4 Other forms of filariasis... 19 2.8 Pathogenesis... 20 2.9 Disease management... 22 2.9.1 Diagnosis... 22
2.9.1.1 New advances in diagnosis... 23 2.9.2 Treatment and control... 26 2.9.3 Global Programme to Eliminate Lymphatic Filariasis (GPELF)... 29 2.10 Vector control... 31 2.11 Drug targets... 34 2.12 Newer targets from Brugia genome... 38 2.13 Vaccine development... 39 2.14 Vaccine targets... 40 2.15 Immune response against filarial infection... 41 2.15.1 Host-parasite interactions... 41 2.15.2 Humoral immune responses... 41 2.15.3 Cellular immune responses... 43 2.16 Molecular basis of immune responses... 43 2.17 Cells involved in inflammatory response... 46 2.18 Immune modulation: in vivo mechanisms... 50 2.19 Role of DIM-1 protein... 51 CHAPTER-3... 54 3 MATERIALS AND METHODS... 54 3.1 Animals... 54 3.2 Parasite... 54 3.3 Animal models... 54 3.4 Maintenance of B. malayi infection... 56 3.4.1 Rearing and breeding of A. aegypti colony... 56 3.4.2 Feeding of mosquitoes on mf positive M. coucha... 56 3.4.3 Isolation of L3 from mosquitoes... 57 3.4.4 Inoculation of L3 to M. coucha/jird and assessment of the infection... 58 3.5 Cloning... 60 3.5.1 Gene selection... 60 3.5.2 Design of primer for the gene... 60 3.5.3 mrna isolation... 61 3.5.3.1 Isolation of adult parasites... 61 3.5.3.2 mrna isolation by Trizol method... 61 3.5.4 cdna synthesis... 62 3.5.5 PCR amplification of gene... 66 3.5.5.1 Elution of amplified DNA (gene product) from the gel... 67 3.5.6 Cloning of DNA in TA vector... 67 3.5.6.1 Ligation of PCR product and TA vector... 68 3.5.6.2 Transformation in DH5α competent strain of E.coli... 69 3.5.6.3 Confirmation of positive colony by colony PCR... 70 3.5.6.4 Sequencing of gene... 70
3.5.7 Sub cloning of DIM-1 TA clone in expression vector (ptriex-4)... 71 3.5.7.1 Isolation of plasmid DNA from transformed E. coli... 71 3.5.7.2 Expression vector (ptriex-4)... 71 3.5.7.3 Restriction digestion of plasmid of TA clone and plasmid of expression vector (ptriex-4)... 72 3.5.7.4 Elution of digested plasmid from the gel... 73 3.5.7.5 Ligation of digested expression vector with amplified product... 73 3.5.7.6 Transformation... 74 3.5.7.7 Confirmation of clone... 74 3.5.7.8 Isolation of plasmid from transformed DH5α cells... 74 3.5.8 Over expression of DIM-1 in expression cell (BL21-DE3 cells)... 74 3.5.8.1 Transformation of isolated plasmid in BL21-DE3 cells... 74 3.5.8.2 Confirmation of clones by colony PCR... 74 3.5.8.3 Expression of proteins from transformed E. coli BL21-DE3 cells... 75 3.5.9 Checking of protein solubility... 75 3.5.10 Purification of recombinant DIM-1 of B. malayi (rdim-1bm) by Ni NTA-column (affinity chromatography)... 79 3.5.11 Protein estimation by Bradford method... 82 3.5.12 Western blotting for transfer of resolved proteins on nitro cellulose paper (NCP)...83 3.5.13 Confirmation of r-protein with anti-histidine antibody in western blot... 85 3.5.14 Bacterial endotoxins test for recombinant DIM-1... 85 3.6 Sequence and phylogenetic analysis... 87 3.7 In silico modeling studies... 87 3.8 Immuonolocalization of DIM-1 in B. malayi adult parasite by confocal microscopy... 88 3.9 Reactivity of antibodies to rdim-1bm with resolved fractions of mf, L3, L4 and adult worm stages by immunoblotting... 89 3.9.1 Isolation of parasites (adult and L4) and preparation of parasite extract... 89 3.9.2 Isolation of L3 and preparation of parasite extract... 90 3.9.3 Isolation of mf and preparation of mf extract... 90 3.9.4 Fractionation of different stages of B. malayi by SDS-PAGE... 90 3.9.5 Immunoblotting of resolved component of mf, L3, L4 and BmA with sera of rdim- 1bm immunized M. coucha...90 3.10 Immune responses of rdim-1bm immunized animals... 91 3.10.1 Grouping and immunization of animals... 91 3.10.1.1 Immunization and experimental schedule... 91 3.10.2 Assessment of infection status... 92 3.10.3 Weekly body weight... 94 3.10.4 Rectal temperature... 94 3.10.5 Hematology (at autopsy)... 94
3.10.5.1 Collection of Blood... 94 3.10.5.2 Total leukocyte count (TLC)... 95 3.10.5.3 Differential leukocyte count (DLC)... 95 3.10.5.4 Packed cell volume (PCV)... 95 3.10.5.5 Erythrocyte sedimentation rate (ESR)... 95 3.10.5.6 Haemoglobin concentration (Hb)... 95 3.10.6 Humoral mediated immune response... 96 3.10.6.1 Determination of IgG antibody and subtypes... 96 3.10.6.2 Determination of specific IgE... 96 3.10.6.3 Determination of specific IgM... 97 3.10.6.4 Determination of specific IgA... 97 3.10.7 Cell mediated immune response... 97 3.10.7.1 Nitric oxide production by macrophages... 97 3.10.7.2 Cellular proliferation (lymphocyte transformation test; LTT)... 99 3.10.7.3 Cytokine determination by ELISA... 99 3.10.7.4 Macrophage function test... 100 3.11 Histopathology of lymph nodes...100 3.12 Statistical analysis...101 CHAPTER-4... 102 4 RESULTS... 102 4.1 Cloning of DIM-1... 102 4.1.1 Amplification of DIM-1 gene... 102 4.1.2 Sub cloning of amplified product in ptz57r/t vector... 102 4.1.3 Sub cloning of gene in the expression vector (ptriex4)... 103 4.1.4 Over expression and purifiaction of rdim-1bm... 104 4.1.5 In silico DIM-1bm sequence analysis... 105 4.1.6 Molecular modeling studies... 108 4.1.7 Confocal microscopy to localize rdim-1bm in adult B. malayi... 109 4.1.8 Reactivity of rdim-1bm with resolved fractions of various stages of B. malayi... 110 4.2 NO release, cellular proliferation, proinflammatory (TNF -α, IFN- and IL-6) and antiinflammatory (IL-10) cytokine production in splenocytes of normal M. coucha stimulated with rdim-1bm... 111 4.2.1 Nitric oxide (NO) release... 111 4.2.2 Cellular proliferation response... 111 4.2.3 Cytokines release... 112 4.3 Effect of repeated exposures of M. coucha to rdim-1bm... 113 4.3.1 3-Dose immunization schedule (standard immunization protocol for vaccination) 113 4.3.1.1 In-life observations... 113 4.3.1.1.1 Behavior... 113 4.3.1.1.2 Body weight... 113
4.3.1.1.3 Rectal temperature... 114 4.3.1.1.4 Organ weight... 115 4.3.1.2 Hematological parameters... 115 4.3.1.3 Parasitological responses... 117 4.3.1.3.1 Effect of rdim-1bm on L3 induced infection... 117 4.3.1.4 Immunological responses... 118 4.3.1.4.1 Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA......118 4.3.1.5 Effect on cell mediated immune responses... 119 4.3.1.5.1 Lymphocyte transformation test (LTT)... 119 4.3.1.5.2 Macrophage phagocytic activity test (MAT)... 120 4.3.1.5.3 NO release... 120 4.3.1.5.4 Cytokine release... 120 4.3.2 3- Week immunization schedule (non-standard procedure)... 123 4.3.2.1 In-life observations... 123 4.3.2.1.1 Behavior... 123 4.3.2.1.2 Body weight... 123 4.3.2.1.3 Rectal temperature... 124 4.3.2.1.4 Organ weight... 125 4.3.2.2 Hematological parameters... 125 4.3.2.3 Parasitological responses... 127 4.3.2.3.1 Effect of rdim-1bm on L3 induced infection... 127 4.3.2.4 Immunological responses... 128 4.3.2.4.1 Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA...128 4.3.2.5 Effect on cell mediated immune responses... 129 4.3.2.5.1 Lymphocyte transformation test (LTT)... 129 4.3.2.5.2 Macrophage phagocytic activity test (MAT)... 130 4.3.2.5.3 NO release... 130 4.3.2.5.4 Cytokine release... 130 4.3.3 6- Week immunization schedule (non-standard procedure)... 134 4.3.3.1 In-life observations... 134 4.3.3.1.1 Behavior... 134 4.3.3.1.2 Body weight... 134 4.3.3.1.3 Rectal temperature... 135 4.3.3.1.4 Organ weight... 136 4.3.3.2 Hematological parameters... 136 4.3.3.3 Parasitological responses... 138 4.3.3.3.1 Effect of rdim-1bm on L3 induced infection... 138 4.3.3.4 Immunological responses... 139
4.3.3.4.1 Effect on humoral responses: IgG and its subclass, IgE, IgM and IgA... 139 4.3.3.5 Effect on cell mediated responses... 140 4.3.3.5.1 Lymphocyte transformation test (LTT)... 141 4.3.3.5.2 Macrophage phagocytic activity test (MAT)... 141 4.3.3.5.3 NO release... 141 4.3.3.5.4 Cytokine release... 142 4.3.4 Histopathological studies...144 CHAPTER-5... 145 5 DISCUSSION... 145 CHAPTER-6... 155 6 SUMMARY... 155 CHAPTER-7... 162 7 BIBLIOGRAPHY... 162 LIST OF PUBLICATIONS...195