Molecular Interactions Involved In Erythrocyte Invasion By Malaria Parasite Thesis Submitted to

1. CLICK for NEXT 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

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

3. CLICK for NEXT Introduction Identification Malaria TIMELINE- Indian medical texts 1600 BCE Hippocrates Lucretius 400 BCE95BCE Quinine c 1640 CE Giovanni Maria Lancisi 1716 Charles Louis Alfonse Laveran 1880 Ronald Ross 1897 {after Sherman, 1998}

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

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

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

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

8.   CLICK for NEXT CLICK for NEXT CLICK for NEXT  {after Chitnis and Miller, 1994 and Miller and Hoffman, 1998} Malaria Vaccines Invasion – Targets Reduce Parasitemia and Disease Transmission Blocking Prevent Infection and Disease  The target is: 1. Sporogonic or Mosquito Stage, 2. Exo-erythrocytic or Liver Stage, 3. Erythrocytic Stage. Vaccines aim to:  • - 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,

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

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

11. Pv Pf CLICK for NEXT Malaria Parasite Morphology – Images {Caramello, 2002} {WHO, 1998}

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

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

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

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

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

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

18. + Viral DNA pAcR2H Sf cell CLICK for NEXT CLICK for NEXT a) Reinfect for Protein prod. b) Amplify for Virus titre PvRII Expression Baculovirus Expression Vector System a) Find best protein producing virus plasmid DNA only (–ve control) Experimental b) Plaque Assay and End Point Dilution Assay. Prepare sufficient high titre virus, (Scale-up accordingly) Recombinant Proteins Recombinant Proteins Recombinant Viruses a) Reinfect for Protein prod. b) Amplify for Virus titre

19. Mw kDa 43 M E Mw kDa PL M FT W1 W2 E1 E2 E3 E4 R NR Mw kDa ←PvRII 9.416 6.557 4.361 2.027 1.353 1.078 9.416 6.557 4.361 2.027 1.353 1.078 79 47 33 25 Mw kDa Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 47 0.25μg 0.5μg 1.0μg 2.0μg kb kb ←PvRII ←reduced PvRII 79 47 33 25 ←native PvRII Mobility shift of PvRII before and after reduction and alkylation ←PvRII Western blot of elution profile Silver stained elution profile of recombinant PvRII ~1 kb Mw PL M FT W1 W2 - E1 E2 E3 47 ~1 kb . kDa Silver stained SDS-PAGE gel of NiNTA purified PvRII ←PvRII BamHI and NotI RD of transformant with pAcR2H Colony PCR screening for pAcR2H CLICK for NEXT CLICK for NEXT CLICK for NEXT CLICK for NEXT RP-HPLC profile of NiNTA purified PvRII Plasmid Characterisation Protein Characterisation Agarose Gels – Colony PCR and RD Metal Affinity Chromatography Silver Stain and Mobility Shift Reverse Phase HPLC Western blot of NiNTA purified PvRII sample for RP-HPLC

20. Add NaCl Add DBPT CLICK for NEXT CLICK for NEXT CLICK for NEXT CLICK for NEXT Protein Characterisation Erythrocyte Binding Assay (EBA) Method PvRII {after Camus and Hadley, 1985}

21. Hu Hu ←RBC Mw c - Chy - none ←Treatment kDa 47 ←PvRII Erythrocyte binding assay with NiNTA purified PvRII CLICK for NEXT Protein Characterisation EBA Results

22. CLICK for NEXT Immunogenicity Anti-PvRII Antibodies from Mice

23. CLICK for NEXT CLICK for NEXT CLICK for NEXT CLICK for NEXT Immunogenicity ELISA Data for α- PvRII Mouse Sera

24. CLICK for NEXT Immunogenicity OD490nm Determination from ELISA     

25. CLICK for NEXT Immunogenicity α-PvRII Mouse Sera End Point Titres

26. PvuII ApaI HindIII HindIII DL6 ID3 PvRII TM CYT SS PPP HSVgD HSVgD CLICK for NEXT Functional Assays EBA (on plated cells) • PvRII expressed on surface of plated mammalian cells with: Plasmid pHVDR22 {Chitnis and Miller, 1994} • Plated cells are tested for binding with: - Human Duffy positive RBCs, and - Human Duffy negative RBCs.

27. CLICK for NEXT CLICK for NEXT CLICK for NEXT Functional Assays EBA (on plated cells): Method

28. CLICK for NEXT CLICK for NEXT CLICK for NEXT Functional Assays EBA (on plated cells): Results

29. CLICK for NEXT Functional Assays Inhibition of Erythrocyte Binding - 293T cells & pHVDR22 • α-PvRII mouse serum - Human RBCs • 50% blocking at • 1:25,000 dilution • Blocking dependant • on antibody dilution

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

31. CLICK for NEXT Acknowledgements

32. LINK back HOME VIEW again from page 1