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. 2010 Jul-Aug;2(4):416-27.
doi: 10.4161/mabs.12438. Epub 2010 Jul 1.

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Elisabeth Ravaoarisoa  1 , Halima Zamanka  2 , Thierry Fusai  3 , Jacques Bellalou  4 , Hugues Bedouelle  4 , Odile Mercereau-Puijalon  4 , Thierry Fandeur  2
Affiliations

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2

Elisabeth Ravaoarisoa et al. MAbs. 2010 Jul-Aug.

Abstract

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.

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Figures

Figure 1
Figure 1
Reactivity of mAbF1546 and mAbF1110 with P. falciparum as assayed by western blots and indirect immunofluorescence. The western blot assayed the reactivity of mAbF1546 (lane 1) and mAbF1110 (lane 2) with P. falciparum crude antigenic extracts, subjected to electrophoresis under denaturing conditions. Apparent MW (kDa) are shown on the left side of the immunoblot. The indirect immunofluorescence staining patterns for mAbF1546 (part 1) and mAbF1110 (part 2) were performed on air-dried blood stages of P. falciparum. Antigenic preparations were obtained from 3D7 parasites.
Figure 2
Figure 2
Reactivity of mAbF1546 and sandwich ELISA with mAbF1546 and mAbF1110. Microtiter plates were coated with either mAbF1546 (A) or mAbF1110 (B) as capture antibody at a 10 µg.mL−1 in PBS, then reacted with decreasing concentrations of soluble PfHRP2 containing fractions equivalent to 500–0.25 IRBC µL−1 prepared by hypotonic lysis (SCHIZ-H20) or mild denaturation with 0.05% SDS in PBS (SCHIZ-0.05% SDS) of IRBC. Bound antigen was finally revealed using biotin labeled mAbF1546 and avidin peroxydase as indicated in the Materials and Methods section. The negative control (C neg) consists of equivalent preparations of uninfected human erythrocytes treated by 0.05% SDS and assayed in similar conditions.
Figure 3
Figure 3
Alignment of the deduced consensus amino-acid sequences of the light chain (VL-CL) and heavy chain (VH-CH1) from anti-PfHRP2 hybridomas secreting mAbF1110 and mAbF1546. Differences in amino-acid residues are boxed in black and residues of the same group are shaded in gray. Dashes indicate gaps introduced by ClustalW for optimization of the alignment.
Figure 4
Figure 4
Nucleotide and deduced amino-acid sequences for the genes encoding the variable domains of mAbF1546 and mAbF1110. Differences between these sequences and the closest germline nucleotide sequences and corresponding amino acid sequences are shown. In (A), the VL domains from mAbF1546 and mAbF1110 are compared with the VL germline sequence IGKV3-5*01. The J gene region is also indicated. In (B), the VH domain of mAbF1110 is compared with the germline sequence IGHV1-S22*01, with detailed D and J gene regions. In (C), the VH domain of mAbF1546 is aligned with the germline sequence IGHV3-8*02, with the D and J genes.
Figure 4
Figure 4
Nucleotide and deduced amino-acid sequences for the genes encoding the variable domains of mAbF1546 and mAbF1110. Differences between these sequences and the closest germline nucleotide sequences and corresponding amino acid sequences are shown. In (A), the VL domains from mAbF1546 and mAbF1110 are compared with the VL germline sequence IGKV3-5*01. The J gene region is also indicated. In (B), the VH domain of mAbF1110 is compared with the germline sequence IGHV1-S22*01, with detailed D and J gene regions. In (C), the VH domain of mAbF1546 is aligned with the germline sequence IGHV3-8*02, with the D and J genes.
Figure 5
Figure 5
FabF1546-H6 and FabF1110-H6 productions in E. coli. Samples were fractionated by SDS-PAGE on 7.5–15% polyacrylamide gels (Biorad) and stained with Coomassie Blue. Molecular markers (kDa) are shown in lane M. Crude periplasmic extracts from recombinant HB2151 strains, producing recombinant Fab-H6 fragments, were subjected to electrophoresis under non-reducing conditions (lanes 1 and 2). The affinity-purified recombinant Fab-H6 were also subjected to electrophoresis under non-reducing conditions (lanes 3 and 4) or reducing conditions (lanes 5 and 6). Lanes 1, 3 and 5, FabF1546-H6; lanes 2, 4 and 6, FabF1110-H6.
Figure 6
Figure 6
Reactivity of the recombinant MalE-PfHRP2 protein in the commercial CareStart Malaria Combo test. Crude soluble fraction prepared from non-transfected HB2151 control cells (lane 1) and crude soluble fraction from HB2151 (pER1) cells expressing MalE-PfHRP2 grown with IPTG (lane 2).
Figure 7
Figure 7
Reactivity of parental mAbF1546 and recombinant FabF1546 to a crude extract of P. falciparum antigenic extract in western blots. A crude P. falciparum antigenic extract (lanes 1 and 2) and the periplasmic fluid of induced HB2151 (pER1) expressing MalE-PfHRP2 (lanes 3 and 4) were subjected to SDS-PAGE on a 7.5–15% polyacrylamide gel run under non reducing conditions. They were probed with the parental mAbF1546 (lanes 2 and 4, ascitic fluid at a 1/1,000 dilution) and the recombinant FabF1546-H6 (lanes 1 and 3, periplasmic fraction at a 1/10 dilution).
Figure 8
Figure 8
Binding specificities of the recombinant FabF1546-H6 as determined by ELISA on parasite and recombinant PfHRP2. In part A, microplate wells were coated with a crude P. falciparum soluble extract (protein concentration adjusted to 20 µg.mL−1 with PBS) and reacted with two-fold dilutions of a periplasmic extract of HB2151(pF1546) from 1/10 to 1/10,240. In part B, wells were coated with various concentrations of affinity-purified MalE-PfHRP2 protein, from 1.5 ng.mL−1 to 20 µg.mL−1, and reacted with a periplasmic extract of HB2151 (p1546) at a 1/10 dilution. Bound antibodies or fragments were detected with an alkaline phosphatase-conjugated Fab-specific anti mouse IgG. Doted lines correspond to the technical background level (mean blank values + 2SD).
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