1. 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 R ICCARDO S. G ATTA

2. 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,

3. {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

4. 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

5. Artemisinins 1979 Gene cloning 1983 Genome sequencing 2002 - Search for new drugs - Vaccine development {after Sherman, 1998} Introduction Malaria TIMELINE- Major Advances

6. {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

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

8.    {after Chitnis and Miller, 1994 and Miller and Hoffman, 1998}  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

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

10. {Caramello, 2002} Morphology – Figures Malaria Parasite

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

12. 1: Attachment – Reorientation -PvRBPs, -MSP-1 complex, -AMA-1, -MAEBL... {From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000} Morphology Erythrocyte Invasion

13. 2: Irreversible attachment and junction formation - micronemes - rhoptries... {From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000} {From Dvorak et al., 1975} 3: Parasitophorous vacuole and invasion Morphology Erythrocyte Invasion

14.  Plasmodium spp. have individual invasion specificities Erythrocytes / treatment P.vivax P.knowlesi P. falciparum Human Duffy +ve + + + Human Duffy –ve. Human Neuram. Rhesus Rhesus Chymo. - - + ND + ND - + - + + - Erythrocyte Receptors Erythrocyte Invasion

15. Two DBL domains: P. falciparum EBA-175, EBL1, BAEBL, JESEBL, PEBL... I II III IV V VI TMSSCYT RII Single DBL domain: P. vivax DBP, P. knowlesi DBP (α, β, and γ proteins)… {after Chitnis and Miller, 1994} I II III IV V VI TMSSCYT FIIFI Erythrocyte Binding Proteins Erythrocyte Invasion

16.  Erythrocyte receptors {From Tournmouille, 1997} - ligands can acts as immunogens to induce invasion blocking Abs. - find parasite ligands, Parasite Ligands (Region II) Erythrocyte Receptors P. vivax RII ONLY Human Duffy antigen P. knowlesi α-RII Rhesus / human Duffy Ag P. falciparum F2 Sialic acid / glycophorin A Sialic acid (rhesus RBC) β-RII Rhesus RBC (unknown) γ-RII External Internal Duffy Antigen Receptor for Chemokines (DARC) Receptor – Ligand Interactions Erythrocyte Invasion CLICK HERE for PART2