Malaria diagnosis
A prompt and accurate diagnosis is the key toeffective malaria management in the field.
The recent introduction of Rapid Diagnosis Tests(RDT) is of considerable interest: Easy to performby any village health worker, they do not requirespecific training or special equipment. Basically,RDTs detect circulating antigens usingmonoclonal antibodies
But …
Specificity et sensitivity could be improved RDTs are not quantitative RDTs display fluctuating inter-batch reproducibility >
need for quality assurance and quality control policy Cost Stability > Performance characteristics affected by
exposure to excess heat and humidity. Shelf live ofRDTs compromised upon long-term storage in fieldconditionsCur
ent
lim
itatio
ns
How to improve RDTs ?by antibodies engineering
Expression and purification of recombinant antibodiesis less expensive, easier to perform, and less timeconsuming than production of conventional mAbs.
Recombinant antibodies offer a stable genetic source> better reproducibility.
[Ac recombinant] AND [therapy]: 14269 references[Ac recombinant] AND [diagnostic]: 25259 references.
Structure and function can be improved by muta-genesis resulting in better stability at elevatedtemperature.
Adv
anta
ges
StrategyValorization of mAbs already existing at IPP and IMTSSA
o Characterization of antibody specificities o Producing recombinant proteins mimicking target Ags o Producing and designing recombinant F(ab) fragmentso F(ab) stabilization by site-directed mutagenesis
PfHRPII PfHSP 72
Mabs YES YES F1110/F1546 G4C17/E5A12
Rec. protein NO YES
Recombinant proteins1- Pf i72HSP (pGEX fusion GST)
z
1 Bacterial pellet 2 Purified fusion Pgex i723 Purified i72
4 Native PfHSP2 Purified fusion Pgex i72
Immune HS anti HSP mab
Strategy for producing recombinant F(ab) antibodies
mARN purification from hydroma cells
First-strand cDNA synthesis
VH-CH1 and VL-CL PCR amplification
Cloning in expression vector
Sequencing and analysis
stra
tegy
Purified recombinant F(ab)
250
150
75
50
37
25
250
150
75
50
37
25
Unreducing Reducing
G4C
17
E5A1
2
F111
0/2
F154
6 /5
F111
0 /3
F154
6 /4
G4C
17
E5A1
2
F111
0/2
F154
6 /5
F111
0 /3
F154
6 /4
* * * * *48KDa
* ** * *24KDa
anti HSP anti HRPII anti HSP anti HRPII
Conclusion 1
Towards a « Home made » RDT discriminating for > No Malaria
> Falciparum malaria or mixed > Malaria other than Pf
Pf Pv
E5A12 IgG1K + + Pan specific Yes
G4C17 IgG2aK + + Pan specific Yes
F1110 IgG1K + - Pf Yes
F1546 IgG1K + - Pf Yes
Rec protein
anti HRP2
anti HSP70
Reactivity Isotype Mab Specificity
Conclusion 2
Cloning and characterization of cDNA encoding variable regions of mabs with speficities for two major target antigens
Expression and in vitro production (better reproducibility and cheaper)
Antibody engineering > improvement of stability and affinity
Recombinant F(ab) fragments
Cloning and sequencing Assembling
CL CH in pPE1 vector
E5A12 q
G4C17
F1110
F1546
Production
Thierry FANDEUR Unité d’Immunologie Moléculaire des parasites / RIIP)
Didier Ménard Unité du Paludisme, Institut Pasteur de Madagascar
Hugues Bedouelle. Unité de Recherche, Prévention et Thérapie moléculaire des Maladies humaines
Thierry Fusai Unité de recherche en biologie et épidémiologie parasitaires, IMTSSA
Halima ZAMANKA. Unité de Parasitologie, Centre de Recherches Médicales et Sanitaire du Niger
Odile PUIJALON Unité d’Immunologie Moléculaire des parasites (Chef d’Unité)
Projet soutenu par le Fond Dédié SANOFI AVENTIS