VIVA

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Molecular Interactions Involved In Erythrocyte Invasion By Malaria Parasite Thesis Submitted to Jawaharlal Nehru University for the Award of the Degree of Doctor of Philosophy in Molecular Genetics by International Centre for Genetic Engineering and Biotechnology RICCARDO S. GATTA

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Introduction Malaria parasite biology and life cycle,  Expression of binding domain of P.vivax Duffy-binding protein, Morphology of erythrocyte invasion by Plasmodium spp., Malaria parasite - host interactions,  Recombinant PvRII produced as secreted protein in insect cells,  Mouse anti-PvRII antibodies block erythrocyte binding… Overview: Work In Brief: - functionally active, - immunogenic,

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{after Sherman, 1998} Indian medical texts 1600 BCE Hippocrates Lucretius 400 BCE 95BCE Quinine c 1640 CE Giovanni Maria Lancisi 1716 Charles Louis Alfonse Laveran 1880 Ronald Ross 1897 Malaria TIMELINE- Introduction Identification

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Chloroquine 1934 DDT 1937 WW I I 1939-1945 WHO - ERADICATION 1956 DDT resistance 1960’s WHO - CONTROL 1967 {after Sherman, 1998} Introduction Malaria TIMELINE- Control

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Artemisinins 1979 Gene cloning 1983 Genome sequencing 2002 {after Sherman, 1998} Introduction Malaria TIMELINE- Major Advances

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{after WHO, 2000} Population at risk 40% world-wide ~ 2 billion people Population infected ~ 200 million people ~ 150 million more each year Research focus ...new drugs ...vaccines Fatalities ~ 2 million each year ~ 3000 children under five die each day Malaria: a world-wide burden Introduction

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{From Hoffman, 1996} Parasite Life Cycle – Blood Stage Malaria

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 The target is: Vaccines aim to: 1. Sporogonic or Mosquito Stage, 2. Exo-erythrocytic or Liver Stage, 3. Erythrocytic Stage. - Vaccine-induced host antibodies (Abs) are taken up with the blood meal, - Block sporozoite development, - Target vector directly,  - Abs to sporozoites, - Cellular response: induce both cytotoxic T-cells and IFN-γ,  - Reduce symptoms, - Abs that block merozoite cytoadherance and/or invasion of RBCs, - Abs to antigens on parasitized RBC, - Induce IFN-γ and other cytokines would destroy infected RBCs,   Transmission Blocking Prevent Infection and Disease Reduce Parasitemia and Disease Invasion – Targets Malaria Vaccines

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MSP family, MAEBL and extended family, AMA-1, and SERA {From Bannister et al., 2000} Rhop / RAP complexes DBL-EBP family / PvRBPs Surface molecules Apical organelle localization Blood Stage – Merozoite Malaria Parasite

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{Caramello, 2002} Morphology – Figures Malaria Parasite

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{Caramello, 2002} {WHO, 1998} Morphology – Images Malaria Parasite

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1: Attachment – Reorientation -PvRBPs, -MSP-1 complex, -AMA-1, -MAEBL... {From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000} Morphology Erythrocyte Invasion

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2: Irreversible attachment and junction formation - micronemes - rhoptries... {From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000} {From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000} {From Dvorak et al., 1975} 3: Parasitophorous vacuole and invasion Morphology Erythrocyte Invasion

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Plasmodium spp. have individual invasion specificities Erythrocyte Receptors Erythrocyte Invasion

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Two DBL domains: P. falciparum EBA-175, EBL1, BAEBL, JESEBL, PEBL... Single DBL domain: P. vivax DBP, P. knowlesi DBP (α, β, and γ proteins)… {after Chitnis and Miller, 1994} Erythrocyte Binding Proteins Erythrocyte Invasion

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Erythrocyte receptors - ligands can acts as immunogens to induce invasion blocking Abs. - find parasite ligands, Duffy Antigen Receptor for Chemokines (DARC) Receptor – Ligand Interactions Erythrocyte Invasion

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Baculovirus transfer vector pAcGP67B {From Becton Dickenson, PharMingen, Baculovirus Expression Manual, 2001} pAcR2H PvRII Plasmid for Expression in Insect Cells PvRII Expression

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Recombinant Proteins a) Reinfect for Protein prod. b) Amplify for Virus titre Experimental plasmid DNA only (–ve control) Recombinant Proteins Prepare sufficient high titre virus, (Scale-up accordingly) a) Find best protein producing virus b) Plaque Assay and End Point Dilution Assay. Recombinant Viruses Baculovirus Expression Vector System PvRII Expression

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Agarose Gels – Colony PCR and RD Plasmid Characterisation Metal Affinity Chromatography Protein Characterisation Silver Stain and Mobility Shift Western blot of NiNTA purified PvRII sample for RP-HPLC Reverse Phase HPLC

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{after Camus and Hadley, 1985} PvRII Erythrocyte Binding Assay (EBA) Method Protein Characterisation

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EBA Results Protein Characterisation

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Anti-PvRII Antibodies from Mice Immunogenicity

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ELISA Data for α- PvRII Mouse Sera Immunogenicity

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     OD490nm Determination from ELISA Immunogenicity

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α-PvRII Mouse Sera End Point Titres Immunogenicity

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Plasmid pHVDR22 {Chitnis and Miller, 1994} PvRII expressed on surface of plated mammalian cells with: Plated cells are tested for binding with: - Human Duffy positive RBCs, and - Human Duffy negative RBCs. EBA (on plated cells) Functional Assays

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EBA (on plated cells): Method Functional Assays

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EBA (on plated cells): Results Functional Assays

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- 293T cells andamp; pHVDR22 α-PvRII mouse serum Blocking dependant on antibody dilution 50% blocking at 1:25,000 dilution - Human RBCs Inhibition of Erythrocyte Binding Functional Assays

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Summary PvRII was found to be active in functional assays, Expression with baculovirus in insect cells, Anti-PvRII mouse sera (protein from insect cells), - recognises E.coli produced PvRII, - inhibits erythrocyte binding. PvRII depends on Duffy antigen for RBC invasion, Blood-stage malaria vaccine candidate based on PvRII, PvRII was highly immunogenic,

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Acknowledgements

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