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 CLICK for NEXT

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, CLICK for NEXT

{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 CLICK for NEXT

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

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

{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 CLICK for NEXT

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

   {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 CLICK for NEXT CLICK for NEXT CLICK for NEXT

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

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

PfPvPv {Caramello, 2002} {WHO, 1998} Morphology – Images Malaria Parasite CLICK for NEXT

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

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 CLICK for NEXT CLICK for NEXT

 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 CLICK for NEXT

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 CLICK for NEXT

 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 for NEXT CLICK for NEXT CLICK for NEXT

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

Recombinan t Proteins a) Reinfect for Protein prod. b) Amplify for Virus titre Sf cell + Viral DNA pAcR2H Experimental plasmid DNA only (–ve control) Recombinan t Proteins a) Reinfect for Protein prod. b) Amplify for Virus titre 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 CLICK for NEXT CLICK for NEXT

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

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

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

Immunization Schedule Day * Pre- Bleed Priming Injection 1 st Bleed Boost I Injection 2 nd Bleed 3 rd Bleed Boost II Injection 4 th Bleed 5 th Bleed ~250μ l sera 25μg PvRII 250μl cFA ~250μ l sera 25μg PvRII 250μl iFA ~250μ l sera 25μg PvRII 250μl iFA ~250μl sera * Day 0 for immunizations was Experimental Design BALB/c ( ♀ ) mice 5 experimental (25  g PvRII + adjuvant (Freund’s) 3 control mice (adjuvant alone) Anti-PvRII Antibodies from Mice Immunogenicity CLICK for NEXT

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

   OD 490nm Determination from ELISA Immunogenicity CLICK for NEXT

End Point Titres MouseBleed 1 (Day 14) Bleed 2 (Day 38) Bleed 3 (Day 52) Bleed 4 (Day 66) Bleed 5 (Day 76) 10580,0001,200,000480,000640, ,000480,000700,000980, ,000680,000800,0001,100, ,0001,100,00080,000250, ,000900,000340,000300,0001,150,000 * Values obtained from control mice, given adjuvant alone, were used to calculate the baseline for end point titer determination for experimental mice. End point titers were fixed at 2.5 x baseline. α-PvRII Mouse Sera End Point Titres Immunogenicity CLICK for NEXT

PvuIIApaI SS ID3 DL6 PPP TMCYT HSVgD PvRII HindIII 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 CLICK for NEXT

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

Comparative data for COS7 & 293Tcells COS7293T Positive (fluorescent) * 625 Binders per well * * average of three experiments. EBA (on plated cells): Results Functional Assays CLICK for NEXT CLICK for NEXT CLICK for NEXT

- 293T cells & 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 CLICK for NEXT

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, CLICK for NEXT

Acknowledgements CLICK for NEXT

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