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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 CLICK CLICK for for NEXT NEXT

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

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Page 1: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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 CLICKCLICKforfor

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Page 2: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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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Page 3: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

{after Sherman, 1998}

Indian medical texts1600 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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Page 4: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Chloroquine1934

DDT 1937

WW I I1939-1945

WHO - ERADICATION 1956

DDT resistance 1960’s

WHO - CONTROL 1967

{after Sherman, 1998}

IntroductionMalaria TIMELINE- Control

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Page 5: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Artemisinins1979

Gene cloning 1983

Genome sequencing 2002

- Search for new drugs- Vaccine development

{after Sherman, 1998}

IntroductionMalaria TIMELINE- Major Advances

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Page 6: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

{after WHO, 2000}{after WHO, 2000}

Population at risk40% 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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Page 7: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

{From Hoffman, 1996}

Parasite Life Cycle – Blood StageMalaria

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Page 8: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

{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

BlockingPrevent Infection

and DiseaseReduce Parasitemia

and Disease Invasion – Targets

Malaria Vaccines

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Page 9: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

MSP family, MAEBL and extended family, AMA-1, and SERA

{From Bannister et al., 2000}

Rhop / RAP complexesDBL-EBP family / PvRBPsSurface molecules Apical organelle localizationBlood Stage – MerozoiteMalaria Parasite

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Page 10: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

{Caramello, 2002}

Morphology – FiguresMalaria Parasite

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Page 11: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Pf

Pv

{Caramello, 2002}{WHO, 1998}

Morphology – ImagesMalaria Parasite

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Page 12: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

1: Attachment – Reorientation

-PvRBPs,

-MSP-1 complex,

-AMA-1,

-MAEBL...

{From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000}

MorphologyErythrocyte Invasion

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Page 13: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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

MorphologyErythrocyte Invasion

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Page 14: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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 ReceptorsErythrocyte Invasion

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Page 15: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Two DBL domains: P. falciparum EBA-175, EBL1, BAEBL, JESEBL, PEBL...

I II III IV V VI

TMSS CYT 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

TMSS CYTFIIFI

Erythrocyte Binding ProteinsErythrocyte Invasion

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Page 16: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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 antigenP. 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 InteractionsErythrocyte Invasion

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Page 17: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Baculovirus transfer vector pAcGP67B

{From Becton Dickenson, PharMingen, Baculovirus Expression Manual, 2001}

pAcR2H PvRII

Plasmid for Expression in Insect CellsPvRII Expression

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Page 18: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Recombinant Proteins

a) Reinfect for Protein prod.b) Amplify for Virus titre

Sf

cell

+

Viral DNA pAcR2H

Experimentalplasmid DNA only (–ve control)

Recombinant 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.

RecombinantViruses

Baculovirus Expression Vector SystemPvRII Expression

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Page 19: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

9.4166.5574.361

2.027

1.353

1.078.~1 kb

Mw 1 2 3 4 5 6 7 8 9kb

Colony PCR screening for pAcR2H

9.4166.5574.361

2.027

1.353

1.078

Mw 1 2 3

~1 kb

kb

BamHI and NotI RD of transformant with pAcR2H

Agarose Gels – Colony PCR and RDPlasmid Characterisation

PL M FT W1 W2 E1 E2 E3 E4Mw kDa

79

47

33

25

←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 ChromatographyProtein Characterisation

Silver stained SDS-PAGE gel of NiNTA purified PvRII

0.25μg 0.5μg 1.0μg 2.0μgMw kDa

79

47

33

25

←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

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Page 20: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Add DBPT

Add NaCl

{after Camus and Hadley, 1985}

PvRII

Erythrocyte Binding Assay (EBA) MethodProtein Characterisation

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Page 21: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Erythrocyte binding assay with NiNTA purified PvRII

47

kDa

←PvRII

Hu Hu ←RBC Mw c - Chy - none ←Treatment

EBA ResultsProtein Characterisation

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Page 22: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Immunization ScheduleDay * -1 0 14 28 38 52 56 66 78

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

~250μl sera

25μg PvRII 250μl cFA

~250μl sera

25μg PvRII 250μl iFA

~250μl sera

~250μl sera

25μg PvRII250μl iFA

~250μl sera

~250μl sera

* Day 0 for immunizations was 24.05.2002

Experimental Design

BALB/c ( ♀ ) mice

5 experimental (25 g PvRII + adjuvant (Freund’s)3 control mice (adjuvant alone)

Anti-PvRII Antibodies from MiceImmunogenicity

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Page 23: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

BLEED 1

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1:40,000

1:80,000

1:160,000

1:320,000

1:640,000

1:1,280,000

1:2,560,000

Background

Dilution

OD

490n

mMEAN Exp Mice

Control

BLEED 2

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1:40,000 1:80,000 1:160,000 1:320,000 1:640,000 1:1,280,000 1:2,560,000 Background

Dilution

OD

490

nm

BLEED 3

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1:40,000 1:80,000 1:160,000 1:320,000 1:640,000 1:1,280,0001:2,560,000Background

Dilution

OD

490

nm

BLEED 4

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1:40,000 1:160,000 1:640,000 1:2,560,000

Dilution

OD

490 n

m

BLEED 5

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1:40,000 1:160,000 1:640,000 1:2,560,000

Dilution O

D490 n

m

ELISA Data for α- PvRII Mouse SeraImmunogenicity

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Page 24: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Bleed 5

0,000

0,050

0,100

0,150

0,200

0,250

0,300

0,350

0,400

1:40,000 1:80,000 1:160,000 1:320,000 1:640,000 1:1,280,000 1:2,560,000 BackgroundDilutions

OD

490n

mMouse 1

Mouse 2

Mouse 3

Mouse 4

Mouse 5

Control

OD490nm Determination from ELISAImmunogenicity

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Page 25: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

End Point Titres Mouse Bleed 1

(Day 14)Bleed 2(Day 38)

Bleed 3(Day 52)

Bleed 4(Day 66)

Bleed 5(Day 76)

1 0 580,000 1,200,000 480,000 640,000

2 0 300,000 480,000 700,000 980,000

3 0 580,000 680,000 800,000 1,100,000

4 0 450,000 1,100,000 80,000 250,000

5 100,000 900,000 340,000 300,000 1,150,000

* 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 TitresImmunogenicity

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Page 26: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

PvuII ApaISS

ID3 DL6

PPP

TM CYT

HSVgD HSVgD

PvRIIHindIII 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

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Page 27: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

EBA (on plated cells): MethodFunctional Assays

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Page 28: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

Comparative data for COS7 & 293TcellsCOS7 293T

Positive (fluorescent) * 6 25

Binders per well * 5.33 30.67

* average of three experiments.

EBA (on plated cells): ResultsFunctional Assays

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Page 29: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

- 293T cells & pHVDR22 - α-PvRII mouse serum

0

20

40

60

80

100

Dilution

Bin

din

g (%

) Blocking dependant on antibody dilution

50% blocking at 1:25,000 dilution

- Human RBCs

Inhibition of Erythrocyte BindingFunctional Assays

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Page 30: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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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Page 31: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

AcknowledgementsCLICKCLICK

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Page 32: Molecular Interactions Involved  In Erythrocyte Invasion By  Malaria Parasite Thesis Submitted to

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