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The parasite Plasmodium falciparum causes the majority of malaria deaths, taking its greatest toll on young children. Over time, individuals living in areas endemic for malaria acquire a nonsterile immunity to P. falciparum. Antibodies, in conjunction with cellular immune responses, appear to play an important role in the development of this immunity. In now classical experiments, Cohen and colleagues demonstrated that the passive transfer of purified antibodies from pooled human immune serum was beneficial to children with life-threatening falciparum malaria infections (8). Using adult Thai recipients, Druilhe and coworkers confirmed Cohen's results (2). They also showed that some pooled immunoglobulins from human immune sera, as well as immunoglobulins from mice or rabbit immune sera, inhibit parasite growth in vitro when tested in combination with normal human monocytes. This inhibition occurred even in cases where the separate use of immunoglobulins or monocytes was ineffective (2). Thus, antibodies are important elements in the development of a protective immune response to malaria parasites, and the identification and characterization of epitopes bound by inhibitory antibodies are one desirable focus in vaccine development efforts.

Proteins localized to the rhoptries, specialized organelles of the invasive P. falciparum merozoite stage, have been a focus of intense interest as potential vaccine candidates (16). One of these proteins, rhoptry-associated protein 1 (RAP-1), which is processed into molecules of 86, 82, 70, and 67 kDa, forms heterooligomeric protein complexes with the rhoptry proteins RAP-2 and RAP-3 (4, 5, 7, 13, 28). Monoclonal antibodies (MAb) that bind epitopes in RAP-1 or RAP-2 and inhibit invasion in vitro have been identified (9, 22, 28). Several of the inhibitory anti-RAP-1 MAb map to the linear sequence N₂₀₀TLTPLEELYPT₂₁₁ located in the amino (N-)-terminal one-third of RAP-1 (9). This epitope, inhibitory B-cell epitope 1, is here termed iB-1 (Fig. 1).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Schematic diagrams of P. falciparum RAP-1, the recombinant protein A-2-2, and recombinant fusion proteins MBP-RAP-11-294, p82T1.24, p82T1.24HindIII, p82T1.24del6, and p82T1.24del11. The locations of structural features of RAP-1 are indicated (including those of the octameric repeats, iB-1, iB-2, and the Cys-containing region). Within the recombinant constructs, the termini of the RAP-1 fragments, the approximate lengths of the fusion partners MBP and trpE, and the locations of the 6-His residues are provided. The locations of BamHI (B) and HindIII (H) restriction sites are shown; a second HindIII site, located in the vector DNA of the p82T1.24 construct and used to construct p82T1.24HindIII, is not shown. The RAP-11-294 fragment is found in A-2-2, MBP-RAP-11-294, and p82T1.24.

The potential role of iB-1 and other RAP-1 epitopes in the development of a protective, anti-P. falciparum immune response has not been explored. Owl and squirrel monkeys can be productively infected with P. falciparum, and antibodies from immune monkeys can inhibit P. falciparum proliferation in vitro in the absence of monocytes or other effector immune cells (6, 24). Furthermore, monkeys, in contrast to humans, rapidly develop immunity to subsequent P. falciparum challenge. Thus, these nonhuman primates provide models for studying protective immune responses after immunization or after P. falciparum infection. In the squirrel monkey model, monkeys immunized with parasite-derived RAP-1, RAP-2, and RAP-3 and challenged with a heterologous strain of P. falciparum developed a delayed and relatively low parasitemia compared to that of control animals (26). However, while the prechallenge immune sera contained anti-RAP-1 antibodies, there was no correlation between antibody concentrations and peak parasitemias in the protected animals (26). Antibodies binding particular epitopes in RAP-1, such as iB-1, may have played a role in protecting these animals, but the RAP-1 epitopes recognized by the prechallenge antibodies were not mapped. The present study was initiated to further characterize anti-RAP-1 and anti-iB-1 antibody responses in animal models and to identify additional inhibitory anti-RAP-1 MAb.

Monkey immune sera screened for anti-RAP-1_1-294 and anti-iB-1 antibodies. Owl monkeys were experimentally infected with P. falciparum to examine the relationship between immunity to P. falciparum and the anti-RAP-1 and anti-iB-1 antibody responses. Six naïve male splenectomized owl monkeys (Aotus nancymai, karyotype 1) were infected with blood-stage P. falciparum FVO (Vietnam Oak Knoll) isolate parasites by William Collins (Centers for Disease Control and Prevention). Splenectomized monkeys were used to promote their susceptibility to subsequent reinfection. The animals were drug treated (20 mg of quinine and 50 mg of mefloquine) 7 to 10 days after the first infection when the parasitemia exceeded 75,000 parasites/µl (~2% parasitemia), and blood was drawn for serum 4 weeks after infection (Fig. 2A). The monkeys were reinfected with FVO-parasitized erythrocytes (RBC) 102 days after the initial infection (Fig. 2B). The monkeys were drug treated 16 days after secondary infection, and blood was drawn for serum 15 days later. Even with splenectomy, the monkeys exhibited an enhanced ability to control a secondary infection, as indicated primarily by a prolonged prepatent period (Fig. 2B).

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Parasitemias of six owl monkeys following two experimental infections with P. falciparum. Parasitemias were determined from daily blood smears of individual monkeys. (A) After the first infection, monkeys were drug cured on day 7 (AI1707 and AI447), day 8 (AI1709 and AO510), day 9 (AI1708), and day 10 (AI1711). (B) All monkeys were drug cured 16 days after a second P. falciparum infection.

View larger version (55K): [in this window] [in a new window]   FIG. 3.   The binding of owl monkey antibodies to the recombinant RAP-1 fragment A-2-2, the peptide iB-1, and the unrelated peptide Ova323-339. (A) Scatter plots of antibody reactivities to the three antigens. The means of triplicate ELISA determinations of normal monkey serum (), serum after the first infection with P. falciparum (), and monkey serum after a second infection () (diluted 1:1,000) on the recombinant and peptide antigens are shown. The peptide FL-160#3 (not shown) produced results nearly identical to those of Ova323-339. (B) Comparison of anti-A-2-2 and anti-iB-1 antibody responses in individual monkeys. Shown are reactivities of individual monkey sera, obtained after a second P. falciparum infection, to recombinant protein A-2-2 (black) and peptide iB-1 (white). Serum samples 1 to 6 are from monkeys AI1707, AI1708, AI1709, AI1711, AI1447, and AO510, respectively. pep, peptide.

Mouse antibodies to RAP-1_1-294 within a recombinant RAP-1 fusion protein. A majority of the anti-RAP-1 inhibitory MAb that have been identified map to the iB-1 peptide sequence (9, 28). With the goal of identifying additional inhibitory antibodies that bind within the region RAP-1_1-294, mice were immunized with the fusion protein maltose binding protein (MBP)-RAP-1_1-294. This protein consists of coding regions of the Escherichia coli MBP and RAP-1_1-294 with a 6-His tag (Fig. 1) and was expressed in greater relative yield than was A-2-2 (results not shown). The plasmid DNA expressing MBP-RAP-1_1-294 was constructed by blunting the SphI site of pA-2-2 with T4 DNA polymerase, excising the RAP-1_1-294 insert with BamHI, and ligating it into the XmnI and BamHI sites of the bacterial expression vector pMAL (New England Biolabs [NEB]). A 6-His tag sequence was added at the XbaI/PstI sites of pMAL, located 3' of the RAP-1_1-294 sequence, using a synthetic adapter prepared from the oligonucleotides 5'-CTAGA TCTCATCACCATCACCATCACTAACTGCA-3' (upper strand) and 5'-GTTAGTGATGGTGATGGTGATGAGAT- 3' (lower strand). A clone expressing MBP-RAP-1_1-294 was identified after induction with 2 mM isopropyl--D-thiogalactopyranoside. The fusion protein was shown to be sequentially purified on amylose beads (NEB) and Ni⁺ chelating resin (Qiagen), indicating that the entire coding region was translated, including the 3' His codons. Large-scale purification was accomplished by binding in batch to amylose beads after releasing protein from bacteria disrupted by freeze-thawing and sonication in column buffer (10 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA). Protein was eluted from the amylose with 10 mM maltose in column buffer. SDS-PAGE analysis suggested that the partially purified MBP-RAP-1_1-294 preparation contained full-length as well as partial-length products (not shown). These products are probably produced from a combination of premature termination of translation and proteolysis of the translation products. To isolate full-length MBP-RAP-1_1-294 from this mixture, protein was dialyzed and solubilized in 8 M urea, bound to Ni⁺-chelate beads, and eluted in batch with 250 mM imidazole in 8 M urea-100 mM NaH₂PO₄-10 mM Tris-HCl (pH 6.3) buffer. The purified MBP-RAP-1_1-294 was essentially homogeneous for an ~80-kDa band by SDS-PAGE (not shown). Purified, dialyzed protein was concentrated with Aquacide (Calbiochem) and redialyzed against PBS. Protein concentration was determined by a Coomassie protein assay (Pierce). The cleavage site for Factor Xa provided in the pMAL vector was not accessible in this fusion protein.

View larger version (83K): [in this window] [in a new window]   FIG. 4.   Binding of MAb 1D6 to recombinant RAP-1 on immunoblots. Nitrocellulose blots of E. coli pATH lysate containing trpE (lanes a to c) and the recombinant trpE-RAP-1 fusion proteins p82T1.24 (lanes d to f) and p82T1.24HindIII (lanes g to i) were probed with polyclonal anti-MBP-RAP-11-294 mouse serum (lanes a, d, and g), 1D6 (lanes b, e, and h), and MAb 2D9 (lanes c, f, and i). Positive binding reactions are shown in lanes d to g and i. The polyclonal antiserum and MAb 2D9 also bind smaller molecules (lanes d, f, and g); these represent truncated forms of RAP-1 present in the bacterial lysate, probably resulting from incomplete translation and/or from proteolysis in the bacteria. The different binding specificities of MAb 1D6 and 2D9 (compare lanes e with f and lanes h with i) indicate that MAb 1D6 recognizes an epitope 3' of the internal HindIII site in RAP-1 and that MAb 2D9 recognizes an epitope 5' of the HindIII site.

View larger version (81K): [in this window] [in a new window]   FIG. 5.   Binding of mouse antibodies to the iB-1 peptide. ELISA were performed with plate-bound peptide iB-1. Shown are the means of triplicate determinations of preimmune (open bars) and immune (gray bars) sera diluted 1:2,000 from two mice and determinations with MAb 1D6, 2D9, and 2.15 diluted 1:1,000 (black bars). Mouse no. 1 was immunized with MBP-RAP-11-294 in Freund's adjuvant, and mouse no. 2 received MBP-RAP-11-294 in Ribi adjuvant.

Inhibition of parasite invasion in vitro by the anti-RAP-1 MAb 1D6. MAb 1D6 and 2D9 were tested by an in vitro RBC invasion assay to determine whether they inhibit RBC invasion. The knob-expressing Honduras I/CDC isolate of P. falciparum was used for these inhibition studies. Since MBP-RAP-1_1-294 contains Honduras-derived RAP-1 sequence, this provides a homologous system for testing these anti-RAP-1 antibodies. The parasites were cultured at 37°C in mixed gas with RTNGGS, an RPMI-based growth medium with 10% A⁺ human serum, and A⁺ or O⁺ human RBC as described elsewhere (14). Synchronous parasite cultures were produced and maintained with regular treatments of sorbitol and/or sedimentation in modified gelatin (18, 23, 29). Assays were performed in duplicate or triplicate wells of a microtiter plate with schizont-stage parasitized RBC at 0.5 to 3% starting parasitemia and 5% hematocrit in RTNGGS in a final volume of 100 µl per well. Cultures were supplemented with either no antibody or purified IgG. IgG was purified from ascites on protein A-Sepharose, concentrated with Aquacide, extensively dialyzed against PBS followed by serum-free parasite culture medium, and then filter sterilized. Control mouse IgG was purchased (Sigma). The protein concentration of the IgG solutions were determined by a Coomassie protein assay prior to use. The effects of the purified IgG preparations on parasite reinvasion were assessed by counting rings in thin blood smears after 24 h in culture. MAb 1D6 (125 µg/ml) inhibited parasite invasion by 61% relative to the control culture in the in vitro assay shown in Table 1 (experiment 1). This inhibition was statistically significant compared to medium alone, as judged by a two-tailed Student's t test. The inhibition was not a nonspecific effect of IgG1 MAb, since MAb 2D9 appeared to increase invasion relative to medium alone. This increase might occur if antibody promotes binding of merozoites to RBC or increases adventitious protein-protein interactions. The invasion inhibition by 125 µg of 1D6 IgG/ml is consistent with inhibitory concentrations of other anti-RAP-1 antibodies (9, 22, 28). At 10 µg/ml, MAb 1D6 no longer inhibited invasion, relative to control IgG, while some enhancement of invasion was still observed with 2D9 (Table 1 [experiment 2]). Thus, MAb 1D6 inhibits parasite invasion of RBC and also fails to bind iB-1, suggesting that this mAb identifies iB-2, a second inhibitory epitope in RAP-1.

Binding of MAb 1D6 to the iB-2 epitope in RAP-1. The epitope iB-2, as defined by MAb 1D6, was shown to be located between residues 124 and 294 of RAP-1 and to be distinct from iB-1. To further localize iB-2, nested deletions from the C terminus of RAP-1_1-294 were produced, and the resulting recombinant proteins were probed with the MAb. These expression clones were prepared from p82T1.24 (Fig. 1) by linearizing the plasmid at the 3' end of the RAP-1 coding region with BamHI and digesting DNA with BAL 31 nuclease (BMB) for 1, 2, and 3 min. Pooled DNAs from the three time points were agarose gel purified, blunted with T4 DNA polymerase (BMB) and deoxynucleoside triphosphates, and self-ligated. Nitrocellulose blots containing truncated RAP-1 proteins expressed from the resulting clones (11) were analyzed as described above. Mouse polyclonal anti-RAP-1_1-294 antibodies were used to confirm protein expression (not shown), and separate blots were incubated with MAb 1D6. Clones p82T1.24del6 (del6) and p82T1.24del11 (del11) were found to delineate the MAb 1D6 epitope. 1D6 bound a protein in clone del6 (Fig. 6, lane c) as well as in the parental clone p82T1.24 (lane a), but did not bind other deletion products (Fig. 6, lane d; data not shown). Clone del11 had the longest RAP-1 insert (restriction mapping data not shown) that failed to bind 1D6 (Fig. 6, lane d). DNA sequences of the RAP-1 inserts of the del11 and del6 plasmid DNAs were obtained after subcloning SspI-SspI restriction fragments generated from the plasmids into pBluescript. These analyses showed that the RAP-1 open reading frame of del11 terminated at Asp237 while that of del6 terminated at Leu262 (Fig. 1). These data map iB-2 between amino acids 238 and 262 in RAP-1, a site C terminal to the iB-1 sequence at amino acids 200 to 211 (Fig. 7). These results confirm our iB-1 peptide ELISA data, which suggested that iB-1 and iB-2 are distinct epitopes.

View larger version (75K): [in this window] [in a new window]   FIG. 6.   Deletion mapping of the epitope for MAb 1D6. Deletion constructs were prepared by BAL 31 nuclease digestion at the BamHI site of p82T1.24. Bacterial lysates with fusion proteins expressed from full-length p82T1.24 (lane a) and parental pATH vector (lane b) and from deletion clones del6 (lane c) and del11 (lane d) were screened by immunoblotting with MAb 1D6.

View larger version (30K): [in this window] [in a new window]   FIG. 7.   Amino acid sequence in the region of the N terminus of p67 including the locations of iB-1 and iB-2. An alignment of deduced amino acid sequences of the RAP-1 gene from P. falciparum laboratory isolates (Sierra Leone, Tanzania I/CDC, K1, and Honduras I/CDC) and the wild-type isolate India D (GenBank accession numbers L10322, L10323, M32853, M80807, and U41074, respectively) is given (11, 12, 27). The locations of the iB-1 and iB-2 epitopes are underlined, and the cleavage site responsible for the formation of p67 from the p82 precursor is marked (25). Sites of amino acid differences in this region are highlighted in boldface. Both Ala and Val were observed at X (position 184) in different genetic clones derived from the Sierra Leone isolate.