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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. - PowerPoint PPT Presentation
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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 CLICKCLICKforfor
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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 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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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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Artemisinins1979
Gene cloning 1983
Genome sequencing 2002
- Search for new drugs- Vaccine development
{after Sherman, 1998}
IntroductionMalaria TIMELINE- Major Advances
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{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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{From Hoffman, 1996}
Parasite Life Cycle – Blood StageMalaria
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{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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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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{Caramello, 2002}
Morphology – FiguresMalaria Parasite
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Pf
Pv
{Caramello, 2002}{WHO, 1998}
Morphology – ImagesMalaria 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}
MorphologyErythrocyte 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
MorphologyErythrocyte Invasion
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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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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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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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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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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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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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Add DBPT
Add NaCl
{after Camus and Hadley, 1985}
PvRII
Erythrocyte Binding Assay (EBA) MethodProtein Characterisation
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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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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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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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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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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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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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EBA (on plated cells): MethodFunctional Assays
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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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- 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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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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AcknowledgementsCLICKCLICK
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