Recombinant Human Antibodies Specific for the Pfs48/45 Protein of the Malaria Parasite Plasmodium falciparum*

Will F. G. RoeffenDagger §, Jos M. H. Raats||, Karina TeelenDagger , Rene M. A. Hoet**, Wijnand M. ElingDagger , Walther J. van Venrooij, and Robert W. SauerweinDagger

From the Dagger  Department of Medical Microbiology, University Medical Center St. Radboud, 6500 HB Nijmegen, The Netherlands,  Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands, and ** Dyax B.V., 6202 AZ Maastricht, The Netherlands

Received for publication, January 22, 2001, and in revised form, February 12, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one VH family-derived germ-line gene (VH1) and two VL family segments (VL2 and VKI).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmodium malaria is transmitted to the Anopheles vector when mosquitoes ingest blood that contains gametocytes. Gametocytes of Plasmodium falciparum synthesize Pfs230 and Pfs48/45, which are expressed on the surface of macrogametes and zygotes and a target for transmission-blocking immunity. Antibodies in the ingested bloodmeal can bind to sexual forms in the mosquito gut and prevent oocyst development (1-3). A panel of murine and rat monoclonal antibodies (mAbs)1 has been produced against Pfs48/45 and has recognized at least five different epitopes. Some of these mAbs showed transmission-blocking activity (3-6).

Transmission-blocking vaccines directed against sexual stage-specific antigens are designed to arrest the development of sporogonic stages inside the mosquito, thereby reducing the infectivity of the mosquito and thereby prohibiting the spread of the disease. Major obstacles in developing a transmission-blocking vaccine are the production of correctly folded Pfs48/45 because of the conformational nature of the Pfs48/45 epitope. A large panel of anti-Pfs48/45 mAbs was needed for a number of reasons: 1) to elucidate the relationship between Pfs48/45 and Pfs230 and 2) to measure and characterize anti-Pfs48/45 antibodies in experimental and field sera.

Phage display antibodies offer a method for the production of high affinity single chain variable fragment (scFv) derivatives of human antibodies of "natural host" origin (for reviews, see Ref. 7). Our objective was to produce human mAbs against Pfs48/45, a sexual stage antigen of P. falciparum. For this study combinatorial phage display libraries were constructed using B-lymphocytes from P. falciparum gametocyte carriers with transmission-blocking immunity. Subsequently, phages were selected from these libraries by panning on Pfs48/45 antigen and phages bound to the antigen eluted by competition with mAbs (8). This method resulted in human scFv antibodies directed against epitope III of Pfs48/45.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parasites-- Mature P. falciparum gametocytes (NF54 strain) were produced in a semi-automated system as described previously (12). Gametocytes were isolated as described previously (13). The purified gametocytes were (a) used directly, in immunofluorescence assays (see below), or (b) stored at -70 °C until used.

Gametocyte antigens were extracted using 25 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 1.0% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each DNase and RNase. Insoluble debris was pelleted by centrifugation (16,000 × g for 5 min at room temperature), and the supernatant was stored at -20 °C until used for Western blot analysis, for immobilization of Pfs48/45 in the phage selection procedure, or in ELISAs.

Monoclonal Antibodies-- Anti-Pfs48/45 murine mAbs 32F3 (IgG1; epitope Ia) and 32F1 (IgG2a; epitope IIb) and rat mAbs 85RF45.1, 85RF45.2b, 85RF45.3, and 85RF45.5 (recognizing epitope I, IIb, III, and V of Pfs48/45) have been described previously (3, 6).

mAb P5D4 recognizes the C-terminal vesicular stomatitis virus glycoprotein (VSV-G)(14). Labeling of mAb P5D4 with horseradish peroxidase (HRP) was performed using the periodate method with a molar input HRP/IgG ratio of 4 (15). The labeled mAb was dialyzed against PBS, supplemented with thimerosal (0.01%) and fetal calf serum (1%), and stored at -20 °C.

Phage Libraries and Selection Procedures-- In this study, two human phage display combinatorial immune antibody libraries were used. The scFv library was derived from RNA obtained from lymphocytes of (a) 10 gametocyte carriers from Cameroon after pooling of the lymphocytes (Cam; 80 × 106 cells); and (b) a Dutch expatriate (Spa; 45 × 106 cells) living in Cameroon for more than 30 years with regular attacks of clinical malaria. The scFv library was made essentially as described by Marks et al. (16) and Hoet et al. (9).

We used a two-step cloning procedure whereby the heavy and light chain repertoires were cloned sequentially in the phagemid vector pHENIX containing the VSV-G tag (14).2 Library diversity was analyzed by PCR and fingerprinting (BstNI digestion).

The phage libraries were panned for binders using immunotubes (Nunc, Maxisorp) coated with a Nonidet P-40 extract of gametocytes (500,000 parasite equivalents/tube). Elution of antigen binding clones was performed by competition with a mixture of four rat mAbs (concentration of 60 µg/ml each) (recognizing epitope I, IIb, III, and V of Pfs48/45) for 90 min. The eluted phages were then allowed to infect Escherichia coli TG1 host cells to amplify selected phage binding to Pfs48/45. After amplification phages were selected for two additional rounds using the same protocol. An aliquot of each of the polyclonal phages obtained after each round of selection was stored at 4 °C until required.

After each round of selection, 96 single clones were screened for binding to Pfs48/45 by ELISA. Clones of interest were characterized by: (a) PCR-fingerprinting using the restriction enzyme BstNI, (b) competition ELISA, (c) sequencing, (d) immunofluorescence, and (e) Western immunoblot (see below). Periplasmatic soluble scFv antibodies and phage antibodies of different clones were produced as described by Marks et al. (16).

ELISA Screening-- Pfs48/45-specific ELISA was performed using a two-site ELISA as described previously (10). Briefly, microtiter plates were coated with 50 µl of anti-Pfs48/45 mouse mAbs recognizing different epitopes (10 µg/ml) in PBS for 30 min. After washing and incubation with parasite extract (200,000 parasite equivalents/well), 50 µl of bacterial culture supernatant containing scFv or phage antibodies were applied. Bound scFvs were detected by HRP-labeled mouse mAb P5D4 and bound phages by HRP-labeled anti-M13 mouse mAb using 3,3',5,5'-tetramethylbenzidine (TMB, Sigma). Adding H2SO4 after 20 min stopped the reaction, and the optical density was measured at A450 nm (Titertek Multiskan MCC/340).

Epitope recognition of Pfs48/45 by phage or soluble scFv was carried out by a competition ELISA as described previously (10). Briefly, Pfs48/45 was captured from antigen extract in microtiter plates. After washes with PBS, wells were incubated with a mixture of 30 µl of test sample and 30 µl of HRP-labeled anti-Pfs48/45 mAb (recognizing various epitopes of Pfs48/45) for 120 min and detected using 3,3',5,5'-tetramethylbenzidine as described above.

Competition of (a) scFv with phage antibodies or (b) scFv with serum antibodies from a malaria patient was done as follows. After incubation with antigen, the wells were incubated with: (a) 30 µl of periplasmatic scFv antibodies (1:2 diluted with PBS containing 0.1% milk) and 30 µl of phage antibodies (1:3 diluted with PBS containing 0.1% milk) for 120 min; and (b) 30 µl of serum dilutions (ranging from 1/20 to 1/10, 240 diluted with PBS containing 0.1% milk) and 30 µl of soluble scFv antibody fragments (1:2 diluted with PBS containing 0.1% milk) for 120 min. Bound scFvs were detected as described above.

Sequencing-- The sequencing of selected clones was carried out using the CEQ Dye Terminator Cycle sequencing kit (P/N 608000, Beckman), and the products were analyzed on a CEQ 2000 Dye Terminator Cycle sequencer (Beckman). The sequences of VH and VL genes were compared with the sequences present in the V BASE Sequence Directory (17) to determine the closest germ-line counterpart.

Immunofluorescence Assay (IFA)-- An indirect IFA was done with a mix of cultured asexual and sexual stage parasites (NF54 isolate of P. falciparum) air-dried on multispot slides and incubated with 20 µl of culture supernatant (1:2 diluted with PBS) for 30 min. The slides were rinsed with PBS and incubated with 20 µl of mAb anti-VSV-G (P5D4) (10 µg/ml in PBS) for 30 min. After being washed with PBS the slides were incubated with AlexaTM-conjugated goat anti-mouse IgG (Molecular Probes; diluted 1:200 in PBS containing 0.05% Evans Blue) for 30 min. The slides were rinsed, washed, mounted with a mixture of 90% glycerol and 10% Tris-HCl (pH = 9.0) under a coverslip, and examined under ultraviolet illumination with a Leitz microscope. Specific green fluorescence of sexual stage parasites was scored as a positive reaction.

For surface IFA analysis, gametocytes (see above) were allowed to undergo gametogenesis for 30 min by resuspension at a 10% hematocrit in fetal calf serum at 27 °C. 108 live gametes were mixed with 10 µl of packed normal human erythrocytes/ml of PBS. From this suspension, 106 gametes (100 µl) were incubated with 100 µl of bacterial culture supernatant containing scFv antibody fragments for 30 min, washed with PBS, and incubated for a further 30 min with 25 µl of the anti-VSV-G mAb P5D4 (10 µg/ml in PBS). After washing with PBS, the gametes were incubated with 25 µl of AlexaTM-conjugated goat anti-mouse IgG (Molecular Probes; diluted 1:200 in PBS containing 0.05% Evans Blue) for 20 min. After washing the gametes with PBS and resuspending them in 50 µl of PBS, the fluorescence of the membrane of intact gametes was observed under UV illumination at a magnification of ×400.

Blotting Procedures-- For an indication of scFv expression levels in bacterial culture supernatants, nitrocellulose filters were placed in a dot-blotting apparatus (Schleicher & Schuell), and bacterial culture supernatant was applied. Filters were dried and blocked with PBS containing 5% milk for 20 min, bound scFv was detected using anti-VSV-G and alkaline-phosphatase-conjugated rabbit anti-mouse (Dakopatts; 1:1,000 dilution). Dots were visualized using nitro blue tetrazolium/bromochloroindolyl phosphate (NBT/BCIP).

For the detection of scFv binding to Pfs48/45 on Western immunoblots, gametocyte extract was fractionated on SDS-polyacrylamide gels (Novex; NuPAGETM 4-12% Bis-Tris gel) under nonreducing conditions conducted by Laemmli's procedure (18) at 200 V for 35 min. Molecular sizes were estimated with SeeBlueTM Pre-stained Standard (Novex). Proteins were electroblotted to polyvinylidene difluoride membranes in NuPAGE transfer buffer (Novex) for 60 min at 30 V. Nonspecific binding sites of membrane strips used were blocked with PBS containing 0.1% milk. After primary antibody incubation, the strips were washed with PBS and incubated with HRP-labeled mAb P5D4 (2 µg/ml in PBS containing 0.05% Tween 20 and 0.1% milk). Strips were washed again with PBS and developed with 3,3'-diaminobenzidine (Sigma).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two combinatorial human scFv antibody libraries were constructed using B cells from P. falciparum gametocyte carriers essentially as described by Hoet et al. (9). After a primary and a second amplification of heavy and light chain genes using PCR and digestion of these products, both VH and VL genes were purified by Wizard PCR prep (Promega) and ligated sequentially in the pHENIX vector. The diversity of the resulting libraries was more than 108 individual clones (Table I). Library quality was analyzed by PCR for full-length inserts (>75%) and dot-blot analysis for percentage of clones producing soluble antibodies (Table I).

                              
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Table I
Characteristics of the immune combinatorial human scFv antibody libraries
Library size is expressed as colony-forming units/µg vector. Induction is the number of positive clones versus total clones on a dot-blot after isopropyl-1-thio-beta -D-galactopyranoside induction.

The phage libraries were incubated with a Nonidet P-40 extract of gametocytes immobilized to the wall of immunotubes. After washing, the bound phages were eluted by competition with a mixture of four rat mAbs recognizing epitopes I, IIb, III, and V of Pfs48/45 at a concentration of 60 µg/ml each. Displaced phages were used for infection of bacteria, grown, combined, and subjected to further selection rounds (in a total of three rounds). After the second and third rounds, phagemid particles were precipitated by polyethylene glycol. No reactivity was found in the Pfs48/45-specific ELISA before selection. The enrichment of the anti-Pfs48/45-specific phages during selection was from an OD value of 0.09 before selection up to OD = 0.97 after three rounds of selection for the Spa library and from OD = 0.10-0.67 for the Cam library as detected in the Pfs48/45-ELISA. The phage concentration applied was the same for each biopanning (1012 colony-forming units/ml). The number of phages bound to the antigen increased during selection from 0.9 to 6.2 × 107 colony-forming units/ml for the Cam library and from 5.3 to 42.0 × 107 colony-forming units/ml for the Spa library.

After the third round of selection, 96 clones from each library were analyzed by PCR for full-length inserts, and isopropyl-1-thio-beta -D-galactopyranoside induction was performed to obtain soluble scFv expression for ELISA analysis. 53% of the clones from the Spa library and 60% of the Cam library expressed soluble scFv as analyzed by the dot blot technique. The percentage of clones after the third selection with full-length inserts was 33% for both libraries (data not shown). 30 ELISA-positive clones from each library were subjected to fingerprinting. The Spa library gave eight different fingerprint patterns, whereas the Cam library yielded six different fingerprint patterns, which differed from those obtained from the Spa library (data not shown). The positive clones (from both libraries) in the competition-ELISA competed for epitope III of Pfs48/45, whereas no competition was found for epitopes I, IIb, and V of Pfs48/45 (data not shown).

Eleven clones selected from the two human libraries were further analyzed by sequence analysis (Fig. 1) and grouped as depicted in Table II. A comparison with the sequences of germ-line VH genes shows that the clones use a VH1, VH3, or VH4 family-derived germ-line segment. Alignment with the VL germ-line sequences showed that these clones use a VL2 or VKI family segment. Most mutations are found in the clones derived from germ-line gene DP15 of the VH1 family.


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Fig. 1.   Protein sequences of anti-Pfs48/45 antibody fragments selected from two patient-derived libraries. A comparison of the VH and the VL chains is shown. FR, framework region. CDR, complementarity-determining region. Asterisks indicate sequence identity with the germ-line. For references for germ-line genes see "Materials and Methods."

                              
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Table II
Reactivity of various clones in the ELISA and immunofluorescence assay and the V-gene family, germ-line (derivation) of antibody fragments selected from patient-derived libraries
OD, optical density value in the competition ELISA for epitope III of Pfs48/45; (S)IFA: -, negative; +/-, weak positive; +, positive; ++, strong positive. K*, could be determined as germ-line but the sequence was not complete. NS, the sequence analysis failed. Mut, number of amino acid mutations compared with nearest germ-line sequence. Clones beginning with an S were selected from the Spa library, and those beginning with K from the Cam library.

These clones were analyzed further by Western blot analysis, immunofluorescence assay, and competition ELISA. Western blotting was performed with soluble scFv using sequential Nonidet P-40 and SDS extracts from gametocytes. Six clones of the Spa library and two clones of the Cam library stained the typical Pfs48/45 bands doublet, whereas one clone of the Cam library weakly stained a 230-kDa protein band (Fig. 2). Two clones (SG3 and KH9) that were negative on Western immunoblot were also negative in the competition ELISA (Table II). Four clones (SF3, SF5, SG10, and SB12) were selected from the Spa library recognizing Pfs48/45 and an additional 30-kDa protein, whereas clones KB6 and KG2 without the 30-kDa reaction on immunoblot were selected from the Cam library. Clones SC8 and SF1 with a weak reaction on immunoblot are both members of the VL2 family with different mutations compared with the germ-line gene DPL11.


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Fig. 2.   Western blots of scFvs from 11 clones with gametocytes. Sequential Nonidet P-40 and SDS extracts from gametocytes were electrophoresed on 10% acrylamide gels under nonreducing conditions and transferred onto polyvinylidene difluoride sheets, strips of which were incubated with an scFv antibody (1:5 diluted in PBS containing 0, 1% milk) and stained with HRP-labeled anti-VSV-G. On the first and last strip, a mixture of two mouse mAbs (mAb 32F3, anti-Pfs48/45, and mAb 63F2A2, anti-Pfs230) was used as a positive control.

Antibody reactivity against sexual stage parasites of isolate NF54 was determined by immunofluorescence analysis. Clear green fluorescence of the membrane of intact live gametes (surface-IFA) was seen for the clones positive for Pfs48/45 on the Western immunoblot, whereas clone KC4 (weak positive for Pfs230 on immunoblot) was negative in both the IFA and the surface IFA (Table II).

Six clones from the Spa library and two clones of the Cam library showed significant competition for epitope III of Pfs48/45 in the competition ELISA, whereas no competition was found for epitopes I, IIb, and V of Pfs48/45. All clones positive in the competition ELISA were positive for Pfs48/45 on the Western immunoblot (Fig. 2). Clone SG10 has a weak reactivity in the competition ELISA and also a weak reactivity in the IFA (Table II). The competition ELISA-positive clones and the uniformity of the VH and VL genes encoding the different scFvs suggested that these clones were directed against the same or related epitopes of Pfs48/45. To explore this possibility, we produced scFv and phage antibodies from 11 clones to test the capacity of phages to replace scFvs after binding to Pfs48/45. The percentage inhibition of binding of scFvs to Pfs48/45 by phage antibodies of clone SB12 is depicted in Fig. 3. The percentage inhibition of scFvs binding to Pfs48/45 by phage antibodies from clones KC4, SG3, and KH9 was comparable (data not shown). Phages of clone KC4 did only compete with bound scFv of KC4, and no competition was found with the other clones. The same pattern was also seen for clone SG3 and KH9. Clones SB12 to SG10 showed competition, suggesting specificity for epitope III of Pfs48/45, whereas the clones for Pfs48/45-unrelated antigen did not compete. These results show that phage antibodies from the eight competition ELISA-positive clones (Table II) were capable of inhibiting the interaction of scFv antibody fragments to Pfs48/45 by more than 65%. Collectively, these experiments support the notion that all clones are directed against the same or closely spaced epitopes.


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Fig. 3.   SB12 phage antibodies inhibit scFvs binding to Pfs48/45 of other clones. Native Pfs48/45 was captured to the plates, and after incubation with scFvs of various clones, 109 colony-forming units of phage antibodies of SB12 were added to each well for inhibition. Bound phage antibodies were detected by HRP-labeled mouse anti-M13. Inhibition of the scFvs of each of the 11 clones by phage antibodies of clone SB12 was determined and presented as percentage inhibition of binding in the absence of scFvs. Incubation with a nonrelevant scFv served as the inhibition control.

The serum of the malaria patient from which the immune library (Spa) was constructed was tested for its ability to compete with anti-Pfs48/45 antibody fragments for binding to Pfs48/45 in comparison with a blood bank donor serum. The patient serum was able to inhibit the binding of scFvs from clones KG2, SB12, and SF5 to Pfs48/45, whereas the serum from the negative blood bank donor was unable to inhibit binding (Fig. 4). The patient serum competes strongly with the three clones, starting inhibition at a reciprocal dilution of 320 for clone SF5, 640 for clone KG2, and 1280 for clone SB12. The other clones (SF3, KB6, SF1, and SC8) gave comparable results (data not shown). Clone SG10, with the lowest competition titer for epitope III of Pfs48/45 (Table II) started inhibition at a reciprocal dilution of 80. 


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Fig. 4.   Competition of the binding of scFv clones to solid-phase Pfs48/45 by antibodies from a malaria patient. The serum of the malaria patient from whom the immune library was constructed (Spa) was used as a positive serum for competition with clones KG2, SB12, and SF5. Serum from a blood bank donor was used as the negative serum.

Furthermore, 48 serum samples of gametocyte carriers were analyzed in the competition ELISA with mAb 85RF45.3 and scFv clone SB12 as competitor. 16 (33%) of 48 gametocyte carriers were able to compete with both labels in the Pfs48/45 competition ELISA at dilutions varying from 1/20 to 1/640 (Table III). Five (10%) serum samples were positive with mAb 85RF45.3 as competitor but negative with clone SB12 in the competition ELISA. The low titer serum samples (>1/160) seem to be lower with clone SB12 as competitor in comparison with the mAb 85RF45.3. These results imply that the antibody fragments selected from the two different libraries recognize similar epitope regions on Pfs48/45 as anti-Pfs48/45 antibodies present in malaria patients' sera.

                              
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Table III
Reactivity of 48 sera from P. falciparum gametocyte carriers in the competition ELISA for Pfs48/45 (epitope III) with rat mAb 85RF45.3 and scFv clone SB12 as competitor


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study we employed a novel strategy for generating recombinant human monoclonal antibody fragments from peripheral B cells from malaria immune patients. The selection of recombinant human monoclonal antibody fragments specific for the Pfs48/45 antigen from phage display libraries was carried out successfully by competitive elution with mAbs specific for this antigen. This competitive elution technique between a mAb and phage antibody for binding an epitope is based on the principle of a Pfs48/45 two-site ELISA (10).

The libraries from malaria patients used in this study had at least 108 independent clones with >75% full-length insert ratios. Despite the mix of four mAbs (recognizing epitopes I, IIb, III, and V), used for competitive elution, only phage antibodies directed to epitope III of Pfs48/45 were selected. However, the serum of the malaria patient of library SpA reacted with all epitopes of Pfs48/45 as measured by the competition ELISA with the anti-Pfs48/45 rat and mouse mAbs (data not shown).

ScFvs derived from all clones of a VH1 germline gene inhibited binding of rat mAb 85RF45.3 recognizing epitope III of Pfs48/45, whereas scFvs of VH3 and VH4 families did not influence binding in competition ELISAs nor reactivity in the immunofluorescence assay. Interestingly, clone KH9 was negative in the competition and immunofluorescence assays, whereas clone SC8 was positive in these tests. They are both members of the same VL2 family but differ in their VH region (clone KH9 uses a VH4 and clone SC8 uses a VH1 family segment). The same pattern can be seen for clone SG3 (negative in different tests) and clones KG2 and KB6 (positive in different tests). They use the VKI family segment but differ in the VH region. Most likely, the reactivity to Pfs48/45 is not dependent on the VL gene. The selected scFv recognize the same epitope III but differ in their amino acid sequence and possibly in the fine specificity of their interaction with this epitope.

We have previously described (11) a fair agreement between transmission-blocking activity using a feeder assay with NF54 P. falciparum parasites and reactivity in Pfs48/45 competition ELISAs using mouse mAbs in sera from gametocyte carriers from Cameroon. These data show that overall the C45 ELISA for epitope III is a good marker for a comparison with transmission-blocking activity in serum samples. Also, Table III shows a good correlation between reactivity of scFv with the sera from gametocyte carriers in comparison with the rat mAb 85RF45.3. However, transmission-blocking activity was found only for mouse mAbs 82C4.A9 and 81D3.D2 (both are epitope III-specific and of the IgM isotype) (5). All other mouse and rat mAbs against epitope III of Pfs48/45 with an IgG isotype show no transmission-blocking activity (5, 6). In a pilot experiment, four scFv clones did not block the transmission of the parasite (data not shown). For a comparison with the blocking mouse mAbs, it is better to make Fab fragments, diabodies, or tetrabodies. In further studies, the transmission-blocking capacity of the selected clones will be studied by making these fragments. Also different selection methods will be performed to get scFvs to other epitopes.

For Pfs48/45 vaccine development, it is important to study anti-Pfs48/45 antibody profiles in endemic sera after natural infections. So far, the possibility for such study was limited because of high background reactivity when using mouse and rat mAbs to capture antigen. This problem could be resolved in part by using F(ab')2 fragments; however, different methods failed so far to produce and purify F(ab')2 fragments of these anti-Pfs48/45 mAbs. Because scFvs lack the cross-reactive mouse and rat antibody domains, we anticipated that high background could be avoided. Preliminary data suggest that the background can be circumvented by these scFv preparations.

In conclusion, the successful selection of human scFv against Pfs48/45 paved the way for future selection of more recombinant antibodies with possible transmission-blocking activity. This option would emphasize the importance of this molecule as a vaccine candidate. Malaria patient-derived phage antibody display libraries can thus be used to isolate specific immunological reagents of human origin, which is useful in both the characterization of the parasite's antigenic composition and the human hosts' immune response to malaria infection.

    ACKNOWLEDGEMENTS

We are grateful to Marga van de Vegte-Bolmer for parasite cultures.

    FOOTNOTES

* The Dutch Ministry for Development Co-operation (DGIS/SO) Contract NL002701 supported this research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| A research fellow of the Royal Netherlands Academy of Arts and Sciences (KNAW).

§ To whom correspondence should be addressed: UMC St. Radboud, Dept. of Medical Microbiology, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31-243619186; Fax: +31-243614666; E-mail: W. Roeffen@mmb.azn.NL.

Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100562200

2 J. M. H. Raats, unpublished results.

    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; scFv, single chain variable fragment; VL, variable light chain: VH, variable heavy chain; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; IFA, immunofluorescence assay; VSV-G, vesicular stomatitis virus glycoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Kumar, N., and Carter, R. (1984) Mol. Biochem. Parasitol. 13, 333-342[Medline] [Order article via Infotrieve]
2. Rener, J., Graves, P. M., Carter, R., Williams, J. L., and Burkot, T. A. (1983) J. Exp. Med. 158, 976-981[Abstract]
3. Vermeulen, A. N., Ponnudurai, T., Beckers, P. J. A., Verhave, J. P., Smits, M. A., and Meuwissen, J. H. E. Th. (1985) J. Exp. Med. 162, 1460-1476[Abstract]
4. Carter, R., Graves, P. M., Keister, D. B., and Quakyi, I. A. (1990) Parasite Immunol. (Oxf.) 12, 587-603
5. Targett, G. A. T., Harte, P. G., Eida, S., Rogers, N. C., and Ong, C. S. L. (1990) Immunol. Lett. 25, 77-82[Medline] [Order article via Infotrieve]
6. Roeffen, W., van As, J., Teelen, K., van de Vegte-Bolmer, M., Eling, W., and Sauerwein, R. (2001) Exp. Parasitol. 97, 45-49[CrossRef][Medline] [Order article via Infotrieve]
7. Burton, D. R., and Barbas, C. F. (1994) Adv. Immunol. 57, 191-280[Medline] [Order article via Infotrieve]
8. Meulemans, E. V., Slobbe, R., Wasterval, P., Ramaekers, F. C. S., and van Eys, G. J. J. M. (1994) J. Mol. Biol. 244, 353-360[CrossRef][Medline] [Order article via Infotrieve]
9. Hoet, R. M. A., Raats, J. M. H., de Wildt, R., Dumontier, H., Muller, S., van den Hoogen, F., and van Venrooij, W. J. (1998) Mol. Immunol. 35, 1045-1055[CrossRef][Medline] [Order article via Infotrieve]
10. Roeffen, W., Lensen, T., Mulder, B., Teelen, K., Sauerwein, R., VanDruten, J., Eling, W., Meuwissen, J. H. E. Th, and Beckers, P. J. A. (1995) Am. J. Trop. Med. Hyg. 52, 60-65[Medline] [Order article via Infotrieve]
11. Roeffen, W., Mulder, B., Teelen, K., Bolmer, M., Eling, W., Targett, G. A. T., Beckers, P. J. A., and Sauerwein, R. (1996) Parasite Immunol. (Oxf.) 18, 103-109
12. Ponnudurai, T., Lensen, A. H. W., Leeuwenberg, A. D. E. M., and Meuwissen, J. H. E. Th. (1982) Trans. R. Soc. Trop. Med. Hyg. 76, 812-818[Medline] [Order article via Infotrieve]
13. Roeffen, W., Beckers, P. J. A., Teelen, K., Lensen, T., Sauerwein, R. W., Meuwissen, J. H. E. Th., and Eling, W. (1995) Exp Parasitol. 80, 15-26[CrossRef][Medline] [Order article via Infotrieve]
14. Kreis, T. E. (1986) EMBO J. 5, 931-941[Abstract]
15. Wilson, M. B., and Nakane, P. K. (1978) Immunofluorescence and Related Staining Techniques , pp. 215-224, Elsevier/North Holland Biomedical Press, Amsterdam
16. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., and Winter, G. (1991) J. Mol. Biol. 222, 581-597[Medline] [Order article via Infotrieve]
17. Tomlinson, I. M., Williams, S. C., Ignatovich, O., Corbett, S. J., and Winter, G. (eds) (1996) V Base Sequence Directory , MRC Centre for Protein Engineering, Cambridge, UK
18. Laemmli, U. K. (1970) Nature 227, 680[Medline] [Order article via Infotrieve]


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