Recombinant Fab fragments specific for the pfHRPII andpfHSP72 : implications for

malaria diagnosis

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

Commercially available RDTs

Bell D et al., Nat Rev Microbiol. 2006 Sep;4(9 Suppl):S7-20. Review.

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

Recombinant proteins2- Pf HRPII (pMAL protein fusion MBP)

Pmal Cl1 Pmal Cl3 C -

Monoclonal antibodies

WB on Pf extracts

IFI

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

cDNA amplification

VL-CL and VH1-CH for HRPII mAbs

CL

CH

VL-CL and VH1-CH for HSP mAbs

CL

CH

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

Milijaona RANDRIANARIVELOJOSIA IPMDenise MATTEI IPPThierry et Micheline BLISNICK IPP

Projet soutenu par le Fond Dédié SANOFI AVENTIS