![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
H:S Blodbank KI2033, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark,1 Department of Pharmacology, The Danish University of Pharmaceutical Sciences, DK-2100, Copenhagen, Denmark,2 Laboratoire de Parasitologie Biomedicale, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France3
Received 17 June 2005/ Returned for modification 28 July 2005/ Accepted 27 February 2006
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An invariable
structural feature of all reported MSP-3 sequences is the presence of
three regions each of which contains three, four, or five conserved
heptad repeat units. Previously published structural analyses suggest
that the heptad repeat regions have an amphipathic alpha-helical
secondary structure. A coiled-coil bundle conformation including these
regions is a theoretical possibility supported by experimental data
(24). The C-terminal part
of MSP-3 contains a leucine zipper-like domain possibly implicated in
dimerization and the formation of tetramers in vivo
(5). MSP-3 contains a
96-amino-acid predicted globular region of high amino acid complexity.
The region comprises amino acid residues 166 to 261 numbered according
to the D10 sequence (GenBank accession number
L07944) positioned C-terminal to the second
putative 
-helix
(18). Naturally occurring
antibodies affinity purified on the C-terminal part of this globular
region (MSP-3 amino acid residues 194 to 257
[MSP-3194-257]) have been shown to exert a strong
inhibition in ADCI assays
(27). A recent phase I
vaccine trial using a long synthetic peptide spanning this region of
MSP-3 has shown promising results in terms of raising both humoral and
cellular responses (1).
Functional studies of the long synthetic peptide-induced responses have
shown encouraging results in vitro in ADCI assays and in vivo in an
immunocompromised BXN mouse model
(13). Furthermore, an
exceptional degree of conservation in this region makes it a prominent
vaccine candidate
(39).
Additionally, this region shows complete homology with a sequence of 11 amino acids (MSP-3220-230) from the MSP-6 antigen (MSP-6182-192), except for a valine-to-alanine substitution at position 229 of the MSP-3 sequence (49) as well as high homology with two additional antigens. The latter antigens and their resemblance to MSP-3 and MSP-6 have been described and designated H101 and H103 recently (31). It has been shown that naturally occurring antibodies affinity purified on MSP-6-derived peptides cross-react with MSP-3-derived homologous peptides and exert an ADCI effect in vitro, thereby confirming the biological relevance of these homologies (40).
Recombinant antibodies would be excellent tools to elucidate the role of isotype and fine specificity of anti-MSP-3 antibodies. All previous work has relied on either polyclonal bulk antibody or affinity-purified antiparasitic antibody. In contrast to the polyclonal antibody preparations used so far, specificity and isotype of recombinant antibodies can be controlled at will by use of DNA techniques.
To clarify the role of anti-MSP-3 antibodies, cross-reactivity with MSP-6, and functional properties in naturally occurring malaria immunity, we decided to clone antibodies directed against the target antigen fragment MSP-3194-257 (27). The present paper describes the isolation and characterization of three distinct antibodies directed to MSP-3194-257 as well as functional in vitro studies in the ADCI assay of one of these antibodies. The amino acid residues of the antigen fragments used in this study are numbered according to their position in the P. falciparum D10 clone of isolate FC27/PNG amino acid sequence (GenBank accession numbers L07944 [MSP-3] and AY518888[MSP-6]).
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The 3D7 clone (NF54 strain) of P. falciparum was propagated in human red blood cells (10). Lars Hviid, University Hospital, Copenhagen, Denmark, kindly provided parasites for specificity studies.
Recombinant MSP-3194-257 (MSP-3 DG210) antigen (27) was produced as a six-His-tagged protein in E. coli and purified by immobilized metal affinity chromatography. Recombinant antigen was kindly provided by Michael Theisen, Statens Serum Institute, Copenhagen, Denmark.
The peptides MSP-3190-217 (H-VEKDYERAKNAYQKANQAVLKAKEASSY-OH), MSP-3211-237 (biotin-AKEASSYDYILGWEFGGGVPEHKKEEN-OH), MSP-3230-257 (biotin-PEHKKEENMLSHLYVSSKDKENISKENE-OH), and MSP-6182-192 (biotin-ILGWEFGGGAP-OH) were chemically synthesized (Schafer-N, Denmark).
Negative control recombinant antibody. Recombinant Fab to the Haemophilus influenzae type b capsular polysaccharide (19) (HibCP) was used as a negative control (a kind gift from Lotte Hougs, University Hospital Copenhagen). Vector, host, and production conditions for the control antibody were identical to the antimalarial antibodies (26).
Purified recombinant anti-rhesus D (RhD) (15) of the immunoglobulin G1 (IgG1) and IgG3 isotypes was also used as a negative control. Mammalian cloning vectors were kindly provided by Lars Norderhaug (26). Allotypes of heavy chain constant regions, hosts, and production conditions for the control recombinant antibodies (rAbs) were identical to those of the antimalarial Abs.
PIAG. Positive control IgG (PIAG) was purified from a serum pool obtained from 30 adult Africans living permanently in Garitenga, Burkina Faso, where malaria is holoendemic. Donors were free of clinical symptoms and of heavy parasitemia and thus regarded as immune individuals (2). The IgG was extracted by ion-exchange chromatography on DEAE-Sephadex (Amersham Biosciences, Buckinghamshire, United Kingdom); protein concentration was determined to be 20 mg/ml with the bicinchoninic acid protein determination reagents (Sigma, St. Louis, MO), and the level of malaria-specific Ab was determined by immunofluorescence assay (2) with an endpoint titer of 1:52,000.
NIG. Negative control IgG (NIG)was prepared as described above for PIAG from a commercially available pool from more than 1,000 healthy French blood donors (Biotransfusion CRTS, Lille, France). The immunofluorescence test was negative at a dilution of 1:200.
Sampling of peripheral blood leukocytes followed by PCR amplification and cloning of antibody genes. A total of 100 ml of peripheral blood was collected with informed consent from each of 13 adults living in the village of Toubakouta, near Dielmo in an area of Senegal where malaria is holoendemic, with approximately 200 infectious bites per year (41). Donors were aged 19 to 27 years and were selected on the basis of permanent residency in the area and acquisition of immunity based on the absence of recorded malaria attacks for several years. Total leukocytes were separated by simple centrifugation, resuspended in 6 M guanidinium HCl, and stored in liquid N2 (17). RNA was isolated from the samples by acid phenol extraction according to the procedure of Chirgwin et al. (7). mRNA was converted to cDNA as described by Ørum et al. (29).
Amplification
of antibody genes was carried out as separate primary and secondary
reactions for variable heavy (VH) chain region genes and for
entire light chain genes (V
-C and
V
-C), respectively, as
described in detail previously
(15). Amplification
products from the secondary extension PCR were separated on
low-melting-point agarose followed by digestion with agarase. The
extended VH amplificates were digested with restriction
enzymes NheI and ApaI in two separate reactions.
The phagemid
used in this work was pFAB73HHUI developed from pFAB4H
(15) and described by
Engberg et al. (17).
Briefly, the vector harbors a truncated version of the M13 phage
envelope protein pIII coding gene gIII. By cloning Fab encoding genes
into pFAB73HHUI, the plasmid will encode a Fab-
pIII protein.
pFAB73HHUI was digested with restriction enzymes NheI and ApaI. The
VH amplificates were ligated into the plasmid and
electroporated into E. coli Top10 Tet using an E.
coli pulser set (Bio-Rad). Transformed bacteria were plated on LB
agar (34) containing 50
mg/liter carbenicillin, 12.5 mg/liter tetracycline, and 2% glucose.
Plates were incubated overnight at 37°C. Colonies were washed
off the plates and used to start a liquid culture, which was eventually
used for preparation of vector DNA with QIAGEN columns (QIAGEN GmbH,
Hilden, Germany). The extended V-C and
V-C products were digested with
restriction enzymes AscI and SfiI. The V-C
and V-C genes were subsequently
cloned into SfiI- and AscI-digested pFAB73HHUI harboring VH
genes.
Production of phage and panning procedure. A 50-ml culture of the library in LB medium with 50 mg/liter carbenicillin, 12.5 mg/liter tetracycline, and 2% glucose was superinfected with VCSM13 helper phage (Stratagene) at an optical density at 600 nm (OD600) of 0.8. A multiplicity of infection of 100 was used and the mixture was incubated at 37°C with gentle shaking (50 rpm) for 1 h. Then the culture was diluted into 950 ml of medium as above but without glucose and incubated at 30°C overnight. After a 15-min spin at 10,000 x g, phage in the supernatant was precipitated with polyethylene glycol 6000 and sodium chloride at final concentrations of 4% and 0.5 M, respectively. The supernatant was incubated for 1 h on ice and centrifuged for 30 min at 12,000 x g. Precipitated phage was resuspended in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA; Sigma) and used immediately. The total number of phage (34) was 1.3 x 1013.
Biotinylated MSP-3194-257 was coupled to Dynabeads M-280 streptavidin (cat. no. 112.05; Dynal, Norway) as described by the manufacturer. After the antigen-coated beads were incubated with phage in an end-over-end mixer for 1 h, beads were captured with a magnetic particle concentrator (MPC-6; Dynal cat. no. 120.02) for 5 min and then washed six times in 10 ml of PBS with 0.05% Tween 20 for 2 min. After the last wash beads were resuspended in 1 ml of PBS with 1 mg of trypsin (Worthington), and phage was eluted during a 1-h incubation at 37°C. Then a volume of 3 ml of exponentially growing Top10 with an OD600 of 1 was added, and the mixture was incubated at 37°C for 30 min to allow eluted phage to attach to E. coli. The bacteria were finally plated on LB agar (34) containing 50 mg/liter carbenicillin, 12.5 mg/liter tetracycline, and 2% glucose and incubated overnight at 37°C. Colonies were washed off the plates with LB medium and stored as a glycerol stock at –80°C or grown for production of phage or DNA as described.
Subsequent to the first panning round, three series of
pannings were carried out in parallel, to a total of four rounds. In
the first series, the number of beads was reduced by a factor of 5 for
each subsequent panning round. In the second and the third series, the
number of beads was reduced by a factor of 10 and 20, respectively.
After the fourth panning round, single colonies from all three series
were grown in sterile microtiter plates (NUNC cat. no. 163320) for
production of
Fab-pIII.
Nucleotide sequence analysis. DNA sequencing of selected colonies was performed with the Sanger dideoxy method (35) using PRISM AmpliTaq FS and a Big Dye Terminator Cycle sequencing kit (cat. no. 4303152) from PE Biosystems according to the manufacturers' instructions. Reactions were analyzed on an ABI Prism 310 genetic analyzer (PE Applied Biosystems). The following primers were used: (forward) 5'-CTTGGAGGAGGGTGCC-3' and (reverse) 5'-CTCGAGAAGGAGACAGTC-3' for the variable heavy chain and (forward) 5'-TGGCGGGAAGATGAAGAC-3' and (reverse) 5'-CACACAGGAAACAGCTATGA-3' for the variable light chain. Sequences were aligned with Ig germ line genes by the ImMunoGeneTics database (http://imgt.cines.fr; Marie-Paule Lefranc, initiator and coordinator, Montpellier, France).
Production, detection, and semiquantitation of Fab. E. coli cells infected with phagemid harboring heavy and light chain genes were grown in LB medium supplemented with 1% glucose, 50 µg/ml carbenicillin, and 12.5 µg/ml tetracycline overnight. A new culture was started in LB medium with 50 µg/ml carbenicillin and 12.5 µg/ml tetracycline. Isopropyl-ß-D-thiogalactopyranoside was added at an OD600 of approximately 0.5 to a concentration of 50 µM, and the culture was grown overnight. Bacteria were spun down, and supernatants were used for further study.
Detection and semiquantitation were carried out with a sandwich enzyme-linked immunosorbent assay (ELISA) by use of a capture antibody (goat antihuman Fab; Sigma I5260) and a detection antibody (goat anti-human Fab conjugated to alkaline phosphatase; Sigma A8542). For the present purposes, dilutions of Fab-containing supernatants yielding the same level of reactivity were assumed to contain the same amounts of Fab.
In order to obtain purified and concentrated Fab,
we modified the pFab73H vector harboring the antibody genes by cutting
out the pIII-coding gene with the EagI restriction enzyme.
This leads to the production of Fab molecules with a six-His tag. Fab
with a six-His tag was purified by chromatography with
Ni-nitrilotriacetic acid-coupled agarose (cat. no. 30210; QIAGEN) as
described previously
(19).
Production of intact recombinant IgG1.
Intact RAM1-IgG1, RAM2-IgG1,
RAM3-IgG1, and RAM1-IgG3 were produced in Chinese hamster ovary cells
(CHO) essentially as previously described
(26). In brief,
VH and V genes were amplified by PCR.
Primers were designed to introduce restriction enzyme sites making
amplificates compatible with the vector system for eukaryotic
expression of intact IgG1 or IgG3 as previously described
(26). CHO cells were
transfected with a mixture of two plasmids encoding each of the
immunoglobulin chain genes, and limiting dilution was used for
isolation of good producers.
Protein was precipitated with 70% ammonium sulfate from cleared CHO culture supernatant, and the pelleted protein was resuspended in water. Subsequently, the buffer was changed to 70 mM sodium acetate, pH 5.0, by dialysis. Recombinant IgG was purified using DEAE Sepharose FF (Amersham Biosciences), the flowthrough was applied to an ABx column (J. T. Baker, Phillipsburg, NJ), the column was washed with 50 mM bicine, pH 8.5, and the IgG was finally eluted with a gradient of 50 mM bicine supplemented with 0.5 M NaCl, pH 8.5. Fractions containing Ab were pooled and precipitated with 70% ammonium sulfate, resuspended in water, and purified on a Superdex 200 column (Amersham Biosciences). Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining, and protein concentration was determined spectrophotometrically.
ELISA. ELISA plates (Maxisorb; NUNC 4-39454; Denmark) were coated with 200 ng per well of purified recombinant MSP-3194-257 in PBS, blocked with PBS-1% BSA, and used for standard ELISA with undiluted supernatant or supernatant diluted in PBS-1% BSA as previously described (14, 15). After a washing step, goat anti-human Fab conjugated to alkaline phosphatase (Sigma cat. no. A8542) was applied as a detection antibody. Finally, p-nitrophenyl phosphate (Sigma phosphatase substrate tablets, cat. no. 104-105) was used as a substrate. Color development was measured as the OD405 minus the OD490.
Similar experiments were performed using intact IgGs in ELISA wells coated with the synthetic peptides MSP-3190-217, MSP-3211-237, MSP-3230-257, or MSP-6182-192. In this ELISA goat anti-human IgG (Fc-specific) conjugated to alkaline phosphatase (Sigma cat. no. A9544) was used as a secondary antibody.
All ELISAs were done in triplicates.
Competition studies.
To wells coated
with MSP-3194-257 and containing 50 µl of a
fixed quantity of Fab-pIII was added 50 µl of various
dilutions of competition antigen, MSP-3194-257.
Coating of wells with MSP-3194-257 and detection of
bound antibody were carried out as above. RAM1, RAM2, or RAM3
Fab-pIII were used in amounts yielding OD405 minus
OD490 values of approximately 1 in ELISA on a coating of
MSP-3194-257. The concentration of competition
antigen ranged from 2 nM to 200 mM. Each dilution of competition
antigen was tested in triplicate.
Using the same setup, dilutions
of RAM3 Fab without pIII were used to compete the binding of
RAM1, RAM2, and RAM3 Fab-pIII. In this experiment, bound
Fab-pIII was specifically detected with a mouse anti-pIII
(cat. no. PSKAN 3; MoBiTech, Germany) followed by an anti-mouse
alkaline phosphatase conjugate (cat. no. D314; Dako,
Denmark).
Immunofluorescence microscopy. Parasitized red blood cells enriched for late developmental stage parasites were obtained by magnet-activated cell sorting of an in vitro culture of P. falciparum clone 3D7 as previously described (45). Cells were washed twice in PBS and sedimented on a Superfrost Plus slide (cat. no. J1800AMNZ; Menzel GmbH & Co. KG), washed once in PBS, and fixed in 1% formaldehyde (Sigma cat. no. F1635) in PBS for 10 min, followed by methanol for 10 min. All handling was performed at room temperature. The slides were blocked for 2 h with PBS-3% BSA, incubated with RAM1, RAM2, or RAM3 IgG1 overnight, washed three times in PBS-0.05% Tween 20, and subsequently incubated with a goat anti-human Fc (cat. no. F9544; Sigma) at a 1/50 dilution in PBS-3% BSA for 30 min. The slides were washed three times as described above and incubated with an Alexa Fluor 488 chicken anti-goat IgG (Molecular Probes cat. no. A21467) at a 1/100 dilution in PBS-3% BSA containing propidium iodide (20 mg/liter) for 30 min. Slides were examined with a Zeiss LSM 510 confocal laser scanning microscope. To confirm the binding of RAM1-3 to the late-stage parasites only, similar and other fixing methods have been used with nonenriched parasite cultures including all blood cycle parasite stages.
Affinity studies by surface plasmon resonance. The affinities of the antibodies were determined by surface plasmon resonance on a Biacore X instrument (Biacore AB, Uppsala, Sweden). MSP-3194-257 in 50 mM NaAc, pH 5.5, was immobilized on an EDC/NHS [N-ethyl-N'-(3-dimethyl aminopropyl)-carbodiimide hydrochloride/ (N-hydroxysuccinimide)]-activated CM5 sensor chip as described by the manufacturer (Biacore AB), yielding a surface density of approximately 50 resonance units. Purified Fab fragments were diluted in the recommended HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.05% surfactant P-20, pH 7.4; Biacore AB) and analyzed at 25°C using a flow rate of 10 µl per minute. Bound Fabs were dissociated using HBS buffer at a flow rate of 10 µl per minute. Association and dissociation rate constants were calculated from a minimum of four sensorgrams generated by the Biacore control software using the curve-fitting BIAevaluation software, version 3.0 (Biacore AB) and the 1:1 Langmuir model.
Flow cytometry. P. falciparum culture enriched for late developmental stage parasites by magnet-activated cell sorting as described previously (45) was permeabilized and fixed by incubation in 32% ethanol for 30 min on ice. Parasites were then washed in PBS with 1% BSA, incubated overnight with Fab, washed twice with PBS-1% BSA, and incubated for 30 min with FITC-conjugated goat anti-human Fab (Sigma F5512) diluted 1:25. Cells were analyzed in a Coulter EPICS-2 flow cytometer. HibCP-specific Fab was used as control in identical concentrations. Fixed infected and fixed noninfected red cells were stained with propidium iodide (20 mg/liter) and compared to enable gating of infected red cells.
Immunoblotting. Parasitized red blood cells, at late trophozoite and schizont stage, were obtained by magnet-activated cell sorting as previously described (45). The purified cells containing late-stage parasites were solubilized on ice in 2% Triton X-100 by ultrasound four times for 15 s each time and incubated for 2 h on ice before being mixed with a reducing sample buffer, heated to 100°C for 10 min, and subjected to SDS-PAGE in a morpholinepropanesulfonic acid-buffered 4 to 12% gradient gel (NOVEX, San Diego, CA). Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon, Millipore, Molsheim, France). Strips were incubated for 3 h with RAM1-IgG1, RAM2-IgG1, RAM3-IgG1, or anti-HibCP-IgG1 as a negative control. Protein G conjugated to alkaline phosphatase (cat no. 32391; Pierce Chemical Co., Rockford, IL) was used as a detecting conjugate. Finally, CSPD [disodium 3-(4-methospiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate] (cat. no. CD100R; Tropix Inc., Bedford, MA,) chemiluminescence substrate was added, and light emission was detected with an Alpha Innotech FluorChem 8000 system (San Leandro, CA).
Parasite culture for ADCI. The P. falciparum 3D7 clone derived from the NF54 strain, the NF54 strain, and the Uganda Palo Alto strain were cultured in AB+Rh+ erythrocytes in RPMI 1640 medium supplemented with hypoxanthine, 0.5% albumax (Gibco, Invitrogen Corporation, Carlsbad, CA), sodium bicarbonate, HEPES, penicillin, and streptomycin. Parasites were synchronized by alternate sorbitol treatment and plasmagel flotation, and mature parasites were used in the ADCI assay.
ADCI assay. The ADCI assay was done essentially as described using previously published techniques (2, 20). A synchronized P. falciparum culture containing mature schizonts adjusted to 0.5% parasitemia with a final hematocrit of 2% in RPMI medium was added to wells containing a suspension of 2 x 105 monocytes. In addition to test Ab, the following controls were used simultaneously in each plate: (i) parasite culture without monocytes, (ii) culture with monocytes, (iii) culture with NIG, (iv) culture with monocytes and NIG, (v) culture with PIAG, (vi) culture with monocytes and PIAG, (vii) culture with anti-RhD-IgG1, (viii) culture with monocytes and anti-RhD-IgG1, (ix) culture with anti-RhD-IgG3, and (x) culture with monocytes and anti-RhD-IgG3. At 48 h and 72 h an additional 50 µl of RPMI medium containing 0.5% albumax, penicillin, and streptomycin was added to each well. At the end of the assay (96 h), the parasitemia in each well was determined, both by counting more than 50,000 erythrocytes on Giemsa-stained film under the microscope and by flow cytometry analysis after staining with hydroethidine. Determination of parasitemia by flow cytometry with a FACSCalibur (Becton-Dickinson, San Jose, CA) was done as previously reported (50). The CellQuest program (Becton-Dickinson) was used to determine the percentage of parasitemia. Flow cytometry and microscopy gave similar results.
The specific growth inhibition index (SGI) was calculated as 100 x {1 – [(% parasitemia with monocytes and test Ab/% parasitemia with test Ab)/(% parasitemia with monocytes and NIG/% parasitemia with NIG)]}.
Nucleotide accession numbers. DNA sequences for all of the antibodies have been submitted to the GenBank database with the following accession numbers:AY543586 (RAM1 VH), AY543587 (RAM1 VL), AY882577 (RAM2 VH), AY882578 (RAM2 VL), AY882579 (RAM3 VH), and AY882580 (RAM3 VL) (15a).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Selection of antigen binding Fabs. To obtain specific and preferably high-affinity antibodies, an antigen-reducing panning strategy was used. The phage stock produced from the first panning eluate was used in three separate panning series (series a to c). Each series had its own reduction factor for the number of beads used in subsequent pannings. The three different factors (5, 10, and 20) of antigen reduction were employed to cover the range of antigen reduction likely to be productive (21). The eluate from the third and fourth panning steps of each series was screened for the presence of Fab producers and antigen binders in ELISA. After the third panning there were no Fab producers among the 93 colonies screened (31 from each series). After the fourth panning, the eluate from the series employing the highest antigen reduction factor (20) was nonproductive, whereas reduction factors of 5 and 10 resulted in nine and eight binders, respectively (Table 1). Seven clones in each of these two series were identical and designated RAM1 (recombinant anti-MSP-3 no. 1). A reduction factor of 5 yielded two identical clones designated RAM2, and one clone designated RAM3 was recovered from the series with reduction factor 10. Antigen binding Fabs were analyzed by DNA sequencing of the genes encoding the VL and VH regions. Three distinct sets of antibody genes were found, of which all light chains were kappa light chains.
|
Competition ELISA.
Competition of Fab-pIII
reactivity with the coating antigen MSP-3194-257 was
done with soluble MSP-3194-257 in an ELISA.
Reactivity of all three clones could be competed with soluble protein,
thus confirming that the clones reacted with
MSP-3194-257 in solution and not with a plastic or
denaturation-dependent conformation (Fig.
1A).
|
Affinity determination of Fabs by Biacore. The Biacore results confirmed the binding of Fabs to the MSP-3194-257 antigen. The on and off rates of the three Fabs were determined by the curve fitting BIAevaluation software, version 3.0. On this basis the affinity constant (KD) of the three clones was estimated to be approximately 35, 20, and 46 nM for RAM1, RAM2, and RAM3, respectively. Despite the similarities in affinity, RAM1 had faster kinetics than RAM2 and RAM3. RAM1 had a higher on rate (kon = 1.5 x 105 M–1s–1) than RAM2 and RAM3 (kon = 3.3 x 104 M–1s–1 and 2.1 x 104 M–1s–1, respectively). Also, RAM1 had a higher off rate (koff = 5.2 x 10–3 s–1) compared to the off rates of RAM2 and RAM3 (koff = 6.6 x 10–4 s–1 and 9.6 x 10–4 s–1, respectively).
Immunofluorescence microscopy. Specificity for the parasite was demonstrated by immunofluorescence and confocal microscopy (Fig. 2). By fixing 3D7 parasites to microscope slides by formaldehyde and methanol treatment, we were able to specifically detect red blood cells containing parasites, as demonstrated by propidium iodide staining of DNA. Staining with RAM1, RAM2, or RAM3 IgG using an anti-human-Fc fluorescein isothiocyanate (FITC) conjugate revealed that red blood cells containing parasites were recognized by the cloned antibodies (Fig. 2); extensive studies have shown no reaction with an isotype control using the same labeling technique (data not shown). Furthermore the RAM1, RAM2, or RAM3 reacted only with late stages of the parasite cycle (schizont) showing "grape-like" structures indicative of merozoite surface labeling and free merozoites (Fig. 2). In numerous immunofluorescence microscopy assays, using several fixing methods and unpurified malaria cultures, early stages (ring and trophozoite) did not react with the Ab (data not shown). These findings are in accordance with previous findings locating MSP-3 to the late schizont stage and the merozoite surface (22, 27).
|
|
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Roeffen et al. (32) and Sowa et al. (44) have previously published the isolation by phage display of human recombinant ScFv fragments against the gametocyte stage antigen Pfs48/45 and a variant of PfMSP-1, respectively. The ScFv libraries used in those studies were made from the peripheral blood lymphocytes of patients with acute malaria, i.e., not from individuals with immunity to malaria, as in the present study. Previous attempts to produce human anti-MSP-3 antibodies using Hu-SPL-SCID mice have not been successful in obtaining stable production of such antibodies (3).
Our panning procedure was designed to enable isolation preferentially of high-affinity binders by gradually reducing the amount of antigen used in subsequent rounds of panning. The panning strategy was established through preliminary optimizations and was inspired by previously published theoretical work (21). The basic notion was that good binders are present in very low frequency in the initial stock. To recover a minimum number of rare binders, a high initial concentration of antigen is needed to ensure Fab-antigen interaction. In subsequent rounds of panning, binders are assumed to be present with increasing frequency. Accordingly, the amount of antigen should be reduced for each panning, thereby ensuring competition between binders and consequently avoiding the survival of mediocre binders. Antigen concentration was decreased by reducing the number of antigen-coated magnetic beads. This procedure concomitantly reduced the matrix area for each panning, thus reducing transfer of deletion mutants by nonspecific adherence to matrix. During the panning procedure deletion mutants are believed to have a selective advantage due to a shorter replication time, and, if transferred through the washing steps, they will dominate the eluate (36). Our results indicate that deletion mutants, which are not being strained by Fab production, have a tendency to overgrow the phage population if there is not a strong selective advantage of Fab production during the panning procedures (Table 1). Our strategy of reducing the panning matrix area and the amount of antigen successfully introduced an advantage to the antigen-binding clones.
Surface
plasmon resonance measurements showed that all three antibodies had
affinities (KD) in the nanomolar range. Previous
work on an invasion-inhibiting anti-circumsporozoite antibody has
reported a KD of approximately 300 nM
(kon, 
4 x
103; koff, 1.2 x
10–3)
(51), and HIV
neutralizing antibodies have been shown to have a
KD of 4.6 nM (kon, 8.4
x 104; koff, 3.9
x 10–4)
(8), showing that the RAM
antibodies described here indeed have affinities within a biologically
relevant range. RAM1 had the fastest kinetics of the three; i.e., RAM1
was rapidly binding to the antigen and was also released from the
antigen more quickly than the other two clones. The overrepresentation
of RAM1 could point to either the on rate as being an important factor
for selection in this specific panning procedure or the possibility
that the productivity and subsequent display of Fabs by this clone were
superior. The fact that RAM1 has been isolated from two parallel
panning series indicates that this clone was either one in a few with
the properties necessary to be selected in this particular setup or
that it was overrepresented in the initial donor-derived mRNA and
thereby in the constructed library. Antigen-coated magnetic beads of
2.8 µm were chosen due to practical considerations, but in fact
they closely mimicked the natural presentation of MSP-3 on the
merozoite surface. The fast reaction kinetics of RAM1 could be the
optimal kinetics for a fast interaction of the immune system with the
released merozoite being accessible for only a few
minutes.
Sequencing of the three clones and the alignment studies showed that the antibodies isolated in this study contain variable regions of human origin and that for at least RAM1 and RAM3 little somatic hypermutation has taken place. In addition, it revealed that RAM2 was unusual compared to known humane germ line genes. We cannot at this time sort out whether the sequence differences found represent somatic mutation, undescribed germ line genes present in African populations, or artifacts introduced during library construction.
The flow cytometry technique used in this study is novel in the context of malaria parasites. Untreated late-stage parasite culture did not react with the antibodies, probably due to lack of access to the parasite residing inside the schizonts. Flow cytometry was therefore done with ethanol-permeabilized and fixated schizont-infected red cells. RAM1 and RAM2 demonstrated specific binding to this parasite preparation. In contrast, no reactivity of RAM3 was detectable. One reason for the lack of binding of RAM3 could be that its epitope is not conserved after the fixation and permeabilization procedure used.
The three clones had different patterns of reactivity with MSP-3 peptides in Western blotting and in flow cytometry, despite the fact that they were all selected on the same panning antigen, MSP-3194-257. Differences in SDS-PAGE mobility of MSP-3 full-length recombinants have been assigned to major conformational differences including complex tertiary structures (18). Recent work on the conformation and polymerization of MSP-3 molecules has suggested an elongated form of the molecule and the generation of intramolecular cross-linking, making the molecules more compact and faster migrating on SDS-PAGE gels (5). Such intramolecular cross-linking could be the basis for the diverse mobilities of MSP-3 in gels. In an initial work on MSP-3, McColl et al. suggested that the MSP-3 antigen present on the merozoite surface (44 kDa) is the result of proteolytic processing of a larger MSP-3 precursor (62 kDa) (22), an observation recently confirmed and specified by Pearce et al. The latter study also confirmed the presence of a low-abundance band of 52 kDa (30). The 44-kDa band seen by McColl et al. corresponds most likely to the low-molecular-weight band (48 kDa) seen in our study with RAM1. The reactivity of RAM2 and RAM3 with a band of approximately 62 kDa, though not with the 48-kDa band, could indicate the existence of epitopes in the precursor protein that are accessible for only RAM2 and 3 but not for RAM1. In contrast, RAM1 may react with only MSP-3, having made the suggested intramolecular cross-linking (Fig. 4). This explanation fits well with the observation that only RAM 2 and RAM3 react with the probably linear, short MSP-6182-192 peptide (Fig. 5). The apparent binding of both RAM2 and RAM3 with bands of lower molecular masses cannot be explained from known data of the MSP-3 antigen. These molecules could be proteolytic degradation products derived from either MSP-3 or MSP-6, in accordance with the fact that these antibodies react with the MSP-6182-192 peptide (Fig. 5) or other MSP-3 homologs, i.e., H101 and H103 (31).
The competitive effect of RAM3 on both RAM1 and RAM2 (Fig. 1), in spite of the different peptide reactivity (Fig. 5), illustrated the overlapping nature of the epitopes, as expected with a 62-amino-acid fragment used for the panning procedure. The reaction patterns obtained in this study are unexpectedly diverse, given the monoclonal nature of the antibodies, and need further elucidation. Recent functional studies of affinity-purified anti-MSP-3 antibodies have confirmed the importance of fine specificity to MSP-3 (39). The complex conformation of this antigen could therefore pose a challenge in terms of raising an immune response leading to protective antibodies in terms of fine specificities and isotypes, as recently seen in MSP-3 vaccine studies (1, 13).
In order to
assess the functional properties of the RAM1 antibody, the variable
chain genes isolated were recombined with the allotypes of the most
prevalent constant region genes found in African populations. The
C
1 allotype, G1m(a,z), with a frequency close to 100% in
Africa (11), and the most
prevalent African C3 allotype, G3m(b), were chosen to study
the functional properties of the two cytophilic subclasses IgG1 and
IgG3. The haplotype G1m(a,z);G3m(b) is found in 68% of Nigerians
(11).
Results from
the ADCI assay showed that the IgG3 version of RAM1 induced 1.6 times
stronger inhibition than the IgG1 version over a broad range of
concentrations (Fig. 6).
This finding is in accordance with previously reported
sero-epidemiological studies linking clinical immunity with specific
IgG3 responses against several different target antigens: MSP-3, GLURP
R0 and R2 (28,
42,
48), MSP-2
(46), and MSP-1 Block 2
(6). However, in other
settings, IgG1 was found to be associated with protection: a high
IgG1-to-IgG3 ratio in Kenyan children is associated with inhibitions in
ADCI (38) and a
sero-epidemiological study also from Kenya demonstrated association of
protection from severe malaria with possession of elevated
schizont-specific IgG1 levels relative to IgG2 and IgG4 levels
(25). The
diverging associations are explained by the fact that Ab is
biologically active in cooperation with monocytes. The merozoite-Ab
complex interacts with FcRIIA on the monocyte and induces
release of antiparasitic substances
(4). FcRIIA is
polymorphic, and distinct functional phenotypes are associated with the
various alleles. In one study
(47), serum with
dominance of IgG3 induced the highest phagocytosis with cells of the
genotype FcRIIA-Arg/Arg131, whereas serum with
dominance of IgG1 induced the highest phagocytosis with cells of the
genotype FcRIIA-His/His131. In Kenya,
possession of the genotype FcRIIA-Arg/Arg131 per se
is associated with protection from high-density infection
(37).
Only the addition of monocytes in ADCI initiated the antiparasitic action, thus substantiating previous reports stating that the effect of anti-MSP-3 is elicited in cooperation with monocytes (4, 2).
In conclusion, the present study takes another step to "close the circle" from the transfer of antibody-based immunity in the clinic, defining the functional properties associated with the effect, isolating an involved antigen (MSP-3), and finally to constructing defined recombinant antibodies with functional properties comparable to those of the antibodies initially found in malaria-immune individuals. The definitive step would be the validation of the antiparasitic effect in a clinical setting.
|
ACKNOWLEDGMENTS |
|---|
We thank Betina Poulsen for excellent technical assistance.
This work was in part supported from the European Union STD Program (contract number TS3*-CT94-0317), the Novo Nordisk Research Foundation, and the Toyota Foundation, Denmark. M.H.D. has been supported by a fellowship from the Danish MRC; L.K.N. and R.L. have been supported by scholarships from the Research Foundation of H:S.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| 1. | Audran,
R., M. Cachat, F. Lurati, S. Soe, O. Leroy, G. Corradin, P. Druilhe,
and F. Spertini. 2005. Phase I malaria vaccine trial
with a long synthetic peptide derived from the merozoite surface
protein 3 antigen. Infect. Immun.
73:8017-8026. |
| 2. | Bouharoun-Tayoun,
H., P. Attanath, A. Sabchareon, T. Chongsuphajaisiddhi, and
P. Druilhe. 1990. Antibodies that protect humans
against Plasmodium falciparum blood stages do not on their own
inhibit parasite growth and invasion in vitro, but act in cooperation
with monocytes. J. Exp. Med.
172:1633-1641. |
| 3. | Bouharoun-Tayoun, H., G. Noun, P. Druilhe, C. Nakhle, N. Haddad, and S. Chamat.2004 . Plasmodium falciparum: production of human antibodies specific for the MSP-3 protein in the Hu-SPL-SCID Mouse.Exp. Parasitol. 108:47-52.[CrossRef][Medline] |
| 4. | Bouharoun-Tayoun,
H., C. Oeuvray, F. Lunel, and P. Druilhe. 1995.
Mechanisms underlying the monocyte-mediated antibody-dependent killing
of Plasmodium falciparum asexual blood stages. J. Exp.
Med.
182:409-418. |
| 5. | Burgess,
B. R., P. Schuck, and D. N. Garboczi.2005
. Dissection of merozoite surface protein 3, a
representative of a family of Plasmodium falciparum surface
proteins, reveals an oligomeric and highly elongated molecule.J. Biol. Chem.
280:37236-37245. |
| 6. | Cavanagh,
D. R., C. Dobano, I. M. Elhassan, K. Marsh, A.
Elhassan, L. Hviid, E. A. T. G. Khalil,
T. G. Theander, D. E. Arnot, and J. S.
McBride. 2001. Differential patterns of human
immunoglobulin G subclass responses to distinct regions of a single
protein, the merozoite surface protein 1 of Plasmodium
falciparum. Infect. Immun.
69:1207-1211. |
| 7. | Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299.[CrossRef][Medline] |
| 8. | Cleveland,
S. M., T. D. Jones, and N. J.
Dimmock. 2000. Properties of a neutralizing
antibody that recognizes a conformational form of epitopeERDRD in the gp41 C-terminal tail of human immunodeficiency
virus type 1. J. Gen. Virol.
81:1251-1260. |
| 9. | Cohen, S., I. A. McGregor, and S. Carrington. 1961. Gamma-globulin and acquired immunity to human malaria.Nature 192:733-737.[CrossRef][Medline] |
| 10. | Cranmer, S. L., C. Magowan, J. Liang, R. L. Coppel, and B. M. Cooke. 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro.Trans. R. Soc. Trop. Med. Hyg. 91:363-365.[CrossRef][Medline] |
| 11. | de Lange, G. G. 1991. Allotypes and other epitopes of immunoglobulins. Baillieres Clin. Haematol. 4:903-925.[Medline] |
| 12. | Druilhe,
P., and S. Khusmith. 1987. Epidemiological correlation
between levels of antibodies promoting merozoite phagocytosis of
Plasmodium falciparum and malaria-immune status.Infect. Immun.
55:888-891. |
| 13. | Druilhe, P., F. Spertini, D. Soesoe, G. Corradin, P. Mejia, S. Singh, R. Audran, A. Bouzidi, C. Oeuvray, and C. Roussilhon. 2005. A malaria vaccine that elicits in humans antibodies able to kill Plasmodium falciparum. PLOS Med. 2:e344.[Medline] |
| 14. | Dziegiel,
M., M. B. Borre, S. Jepsen, B. Hogh, E. Petersen, and J.
Vuust. 1991. Recombinant Plasmodium
falciparum glutamate rich protein; purification and use in
enzyme-linked immunosorbent assay. Am. J. Trop. Med.
Hyg.
44:306-313. |
| 15. | Dziegiel, M., L. K. Nielsen, P. S. Andersen, A. Blancher, E. Dickmeiss, and J. Engberg. 1995. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D. J. Immunol. Methods 182:7-19.[CrossRef][Medline] |
| 15a. | Dziegiel, M. S. H., R. Lundquist, and L. K. Nielsen. September 2005. Recombinant anti-Plasmodium falciparum antibodies. United Kingdom patent 2378949. |
| 16. | Edozien, J. C., H. M. Gilles, and I. O. K. Udeozo. 1962. Adult and cord-blood gamma-globulin and immunity to malaria in Nigerians. Lancet 2:951-955. |
| 17. | Engberg, J., P. S. Andersen, L. K. Nielsen, M. Dziegiel, L. K. Johansen, and B. Albrechtsen. 1996. Phage-display libraries of murine and human antibody Fab fragments.Mol. Biotechnol. 6:287-310.[Medline] |
| 18. | Hisaeda, H., A. Saul, J. J. Reece, M. C. Kennedy, C. A. Long, L. H. Miller, and A. W. Stowers. 2002. Merozoite surface protein 3 and protection against malaria in Aotus nancymai monkeys.J. Infect. Dis. 185:657-664.[CrossRef][Medline] |
| 19. | Hougs,
L., L. Juul, A. Svejgaard, and T. Barington. 1999.
Structural requirements of the major protective antibody to
Haemophilus influenzae type B. Infect. Immun.
67:2503-2514. |
| 20. | Khusmith,
S., and P. Druilhe. 1983. Cooperation between
antibodies and monocytes that inhibit in vitro proliferation of
Plasmodium falciparum. Infect. Immun.
41:219-223. |
| 21. | Levitan, B. 1998. Stochastic modeling and optimization of phage display. J. Mol. Biol. 277:893-916.[CrossRef][Medline] |
| 22. | McColl, D. J., A. Silva, M. Foley, J. F. Kun, J. M. Favaloro, J. K. Thompson, V. M. Marshall, R. L. Coppel, D. J. Kemp, and R. F. Anders. 1994. Molecular variation in a novel polymorphic antigen associated with Plasmodium falciparum merozoites. Mol. Biochem. Parasitol. 68:53-67.[CrossRef][Medline] |
| 23. | McGregor,
I. A. 1964. The passive transfer of human
malarial immunity. Am. J. Trop. Med. Hyg.
13:237-239. |
| 24. | Mulhern, T. D., G. J. Howlett, G. E. Reid, R. J. Simpson, D. J. McColl, R. F. Anders, and R. S. Norton. 1995. Solution structure of a polypeptide containing four heptad repeat units from a merozoite surface antigen of Plasmodium falciparum.Biochemistry 34:3479-3491.[Medline] |
| 25. | Ndungu, F. M., P. C. Bull, A. Ross, B. S. Lowe, E. Kabiru, and K. Marsh. 2002. Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2. Parasite Immunol. 24:77-82.[CrossRef][Medline] |
| 26. | Norderhaug, L., T. Olafsen, T. E. Michaelsen, and I. Sandlie.1997 . Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.J. Immunol. Methods 204:77-87.[CrossRef][Medline] |
| 27. | Oeuvray,
C., H. Bouharoun-Tayoun, H. Gras-Masse, E. Bottius, T. Kaidoh, M.
Aikawa, M. C. Filgueira, A. Tartar, and P. Druilhe.1994
. Merozoite surface protein-3: a malaria protein
inducing antibodies that promote Plasmodium falciparum killing
by cooperation with blood monocytes. Blood
84:1594-1602. |
| 28. | Oeuvray,
C., M. Theisen, C. Rogier, J. F. Trape, S. Jepsen, and P.
Druilhe. 2000. Cytophilic immunoglobulin responses to
Plasmodium falciparum glutamate-rich protein are correlated
with protection against clinical malaria in Dielmo, Senegal.Infect. Immun.
68:2617-2620. |
| 29. | Orum,
H., P. S. Andersen, A. Oster, L. K. Johansen, E.
Riise, M. Bjornvad, I. Svendsen, and J. Engberg. 1993.
Efficient method for constructing comprehensive murine Fab antibody
libraries displayed on phage. Nucleic Acids Res.
21:4491-4498. |
| 30. | Pearce, J. A., A. N. Hodder, and R. F. Anders. 2004. The alanine-rich heptad repeats are intact in the processed form of Plasmodium falciparum MSP3.Exp. Parasitol. 108:186-189.[Medline] |
| 31. | Pearce, J. A., K. Mills, T. Triglia, A. F. Cowman, and R. F. Anders. 2005. Characterisation of two novel proteins from the asexual stage of Plasmodium falciparum, H101 and H103. Mol. Biochem. Parasitol. 139:141-151.[CrossRef][Medline] |
| 32. | Roeffen,
W. F., J. M. Raats, K. Teelen, R. M.
Hoet, W. M. Eling, W. J. van Venrooij, and
R. W. Sauerwein. 2001. Recombinant human
antibodies specific for the Pfs48/45 protein of the malaria parasite
Plasmodium falciparum. J. Biol. Chem.
276:19807-19811. |
| 33. | Sabchareon,
A., T. Burnouf, D. Ouattara, P. Attanath, H. Bouharoun-Tayoun, P.
Chantavanich, C. Foucault, T. Chongsuphajaisiddhi, and P. Druilhe.1991
. Parasitologic and clinical human response to
immunoglobulin administration in falciparum malaria.Am. J. Trop. Med. Hyg.
45:297-308. |
| 34. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 35. | Sanger, F., G. M. Air, B. G. Barrell, N. L. Brown, A. R. Coulson, C. A. Fiddes, C. A. Hutchison, P. M. Slocombe, and M. Smith.1977 . Nucliotide sequence of bacteriophage phi X174 DNA.Nature 265:687-695.[CrossRef][Medline] |
| 36. | Seehaus, T., F. Breitling, S. Dubel, I. Klewinghaus, and M. Little.1992 . A vector for the removal of deletion mutants from antibody libraries. Gene 114:235-237.[CrossRef][Medline] |
| 37. | Shi, Y. P., B. L. Nahlen, S. Kariuki, K. B. Urdahl, P. D. McElroy, J. M. Roberts, and A. A. Lal. 2001. Fc gamma receptor IIa (CD32) polymorphism is associated with protection of infants against high-density Plasmodium falciparum infection. VII. Asembo Bay Cohort Project. J. Infect. Dis. 184:107-111.[CrossRef][Medline] |
| 38. | Shi, Y. P., V. Udhayakumar, A. J. Oloo, B. L. Nahlen, and A. A. Lal. 1999. Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites. Am. J. Trop. Med. Hyg. 60:135-141.[Abstract] |
| 39. | Singh, S., S. Soe, J. P. Mejia, C. Roussilhon, M. Theisen, G. Corradin, and P. Druilhe. 2004. Identification of a conserved region of Plasmodium falciparum MSP3 targeted by biologically active antibodies to improve vaccine design.J. Infect. Dis. 190:1010-1018.[CrossRef][Medline] |
| 40. | Singh,
S., S. Soe, C. Roussilhon, G. Corradin, and P. Druilhe.2005
. Plasmodium falciparum merozoite surface
protein 6 displays multiple targets for naturally occurring antibodies
that mediate monocyte-dependent parasite killing. Infect.
Immun.
73:1235-1238. |
| 41. | Smith,
T., G. Killeen, C. Lengeler, and M. Tanner. 2004.
Relationships between the outcome of Plasmodium falciparum
infection and the intensity of transmission in Africa.Am. J. Trop. Med. Hyg.
71:80-86. |
| 42. | Soe,
S., M. Theisen, C. Roussilhon, K. S. Aye, and P. Druilhe.2004
. Association between protection against clinical
malaria and antibodies to merozoite surface antigens in an area of
hyperendemicity in Myanmar: complementarity between responses to
merozoite surface protein 3 and the 220-kilodalton glutamate-rich
protein. Infect. Immun.
72:247-252. |
| 43. | Soe, S., S. A. Khin, A. Htay, W. Nay, A. Tin, S. Than, C. Roussilhon, J. L. Perignon, and P. Druilhe.2001 . Premunition against Plasmodium falciparum in a malaria hyperendemic village in Myanmar. Trans. R. Soc. Trop. Med. Hyg. 95:81-84.[CrossRef][Medline] |
| 44. | Sowa, K. M., D. R. Cavanagh, A. M. Creasey, J. Raats, J. McBride, R. Sauerwein, W. F. Roeffen, and D. E. Arnot. 2001. Isolation of a monoclonal antibody from a malaria patient-derived phage display library recognising the Block 2 region of Plasmodium falciparum merozoite surface protein-1. Mol. Biochem. Parasitol. 112:143-147.[CrossRef][Medline] |
| 45. | Staalsoe, T., H. A. Giha, D. Dodoo, T. G. Theander, and L. Hviid. 1999. Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry 35:329-336.[CrossRef][Medline] |
| 46. | Taylor, R. R., S. J. Allen, B. M. Greenwood, and E. M. Riley. 1998. IgG3 antibodies to Plasmodium falciparum merozoite surface protein 2 (MSP2): increasing prevalence with age and association with clinical immunity to malaria. Am. J. Trop. Med. Hyg. 58:406-413.[Abstract] |
| 47. | Tebo, A. E., P. G. Kremsner, and A. J. F. Luty. 2002. Fc gamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro. Clin. Exp. Immunol. 130:300-306.[CrossRef][Medline] |
| 48. | Theisen,
M., D. Dodoo, A. Toure-Balde, S. Soe, G. Corradin, K. K.
Koram, J. A. L. Kurtzhals, L. Hviid, T. Theander,
B. Akanmori, M. Ndiaye, and P. Druilhe. 2001.
Selection of glutamate-rich protein long synthetic peptides for
vaccine development: antigenicity and relationship with
clinical protection and immunogenicity. Infect. Immun.
69:5223-5229. |
| 49. | Trucco, C., D. Fernandez-Reyes, S. Howell, W. H. Stafford, T. J. Scott-Finnigan, M. Grainger, S. A. Ogun, W. R. Taylor, and A. A. Holder.2001 . The merozoite surface protein 6 gene codes for a 36-kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex. Mol. Biochem. Parasitol. 112:91-101.[CrossRef][Medline] |
| 50. | van der Heyde, H. C., M. M. Elloso, W. J. vande, K. Schell, and W. P. Weidanz. 1995. Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.Clin. Diagn. Lab. Immunol. 2:417-425.[Medline] |
| 51. | Wohlhueter, R. M., K. Parekh, V. Udhayakumar, S. N. Fang, and A. A. Lal. 1994. Analysis of binding of monoclonal antibody to a malarial peptide by surface-plasmon resonance biosensor and integrated rate equations. J. Immunol. 153:181-189.[Abstract] |
This article has been cited by other articles:
| |||||||||
, RAM1; •, RAM2; and 
,
RAM3.
