A Novel 70-kDa Triton X-114-Soluble Antigen ofPlasmodium falciparumThat Contains Interspecies-Conserved Epitopes

EXPERIMENTAL PARASITOLOGY ARTICLE NO.

83, 322–334 (1996)

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A Novel 70-kDa Triton X-114-Soluble Antigen of Plasmodium falciparum That Contains Interspecies-Conserved Epitopes HUI WEN MA,* PRATIMA RAY,* VIJAYA DHANDA,† PRADEEP K. DAS,† SEEMA PALIWAL,‡ NARESH SAHOO,* KAILASH P. PATRA,† LALIT K. DAS,† BALWAN SINGH,* AND FRED A. S. KIRONDE*,1 *Malaria Group, International Centre for Genetic Engineering and Biotechnology, NII Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India; †Vector Control Research Centre, Medical Complex, Indira Nagar, Pondicherry 605 006, India; and ‡University of Delhi, Delhi 110 006, India

MA, H. W., RAY, P., DHANDA, V., DAS, P. K., PALIWAL, S., SAHOO, N., PATRA, K. P., DAS, L. K., SINGH, B., AND KIRONDE, F. A. S. 1996. A novel 70-kDa Triton X-114-soluble antigen of Plasmodium falciparum that contains interspecies-conserved epitopes. Experimental Parasitology 83, 322–334. In order to identify novel conserved integral membrane and other membraneassociated proteins of Plasmodium falciparum, lgt11-P. falciparum DNA library phages were immunoscreened with convalescent-phase mouse sera and rabbit antiserum against Triton X114-soluble proteins of P. falciparum. One recombinant phage clone, L857, reacted with both of the antibody probes. Insert DNA (857 bp long) in L857 was 69% dA / dT rich and hybridized to a fragment of 1800 bp from mung bean nuclease-digested P. falciparum genomic DNA. The cloned parasite DNA did not show notable sequence homology with any known protein gene. The L857-encoded polypeptide, p34 (Mr 34 kDa) was expressed in bacteria, fused to glutathione S-transferase (GST). The fusion peptide, GST-p34 (Mr 62 kDa), was recognized by immune serum against Triton X-114-soluble antigens of P. falciparum and was reactive with anti-P. falciparum, anti-Plasmodium yoelii, and anti-GST sera. Rabbit antiserum raised against the fusion peptide recognized a 70-kDa protein from lysates of P. falciparum cells and a putative homologous 100-kDa protein from lysates of P. yoelii. The rabbit serum anti-fusion peptide antibodies bound to acetone-fixed P. falciparum-infected erythrocytes and, in immunofluoresent antibody tests, produced a punctate pattern of fluorescence suggesting that the 70-kDa native protein is associated with an apical organelle of the parasite. q 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Integral membrane protein; interspecies conserved antigen; Plasmodium falciparum; Plasmodium yoelii; apical organelle; Apicomplexa; protective; punctate; Triton X-114; merozoite; novel; kb, kilobases; IMP, integral membrane protein; GST, glutathione S-transferase; MBNase, mung bean nuclease; CSP, circumsporozoite protein; PVM, parasitophorous vesicular membrane; PV, parasitophorous vesicle; bp, base pairs; kDa, kilodaltons; NBT, nitroblue tetrazolium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; IPTG, isopropylthio-b-D-galactoside.

INTRODUCTION Invasion of host cells is a vital ingredient part of a parasite life cycle and is associated The nucleotide sequence reported in this paper has been submitted to the EMBL Data Bank with Accession No. X91661. 1 To whom correspondence should be addressed at ICGEB, Aruna Asaf Ali Marg, New Delhi 110 067, India.

with several generically conserved morphological and functional characteristics. In invasive zoite forms of Apicomplexan parasitic protozoa (Fawcett et al. 1984; Aikawa et al. 1978; Pohl et al. 1989; Scholtyseck and Mehl-

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322 0014-4894/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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horn 1970) three major types of vesicular organelles are present in a well-defined anterior or apical region. Considered central to invasion, these shared apical organelles include the rhoptries, dense granules (also called microspheres), and micronemes. The dense granules may additionally occur in central and peripheral regions of the zoite cytoplasm. In Plasmodium falciparum, the erythrocytic merozoite specially contains large tear-shaped paired rhoptries, numerous intermediate-sized spherical dense granules, and smaller tubelike micronemes (Bannister et al. 1975; Perkins 1992). Up to 32 oval-shaped merozoites inhabit a matured erythrocyte (schizont), such that immunofluorescent antibody (IFA) examination of schizonts shows merozoite surface proteins as grape-like fluorescent patterns while organellar-associated molecules give a multiple-dot type of fluorescence. Studies of the invasion process in Toxoplasma, Eimeria, Sarcocystis, and Plasmodium parasites demonstrate that during invasion, the rhoptries, dense granules, and micronemes secrete their contents, assisting in parasite penetration and formation of a new parasitophorous vesicular membrane (PVM). The contents of rhoptries are believed to be membranous (Etzion et al. 1991; Bannister et al. 1986; Stewart et al. 1986; Sam-Yellowe 1990), and a few described antigens of the rhoptry organelles and micronemes are either integral membrane proteins (IMPs) or lipophillic in nature (Howard and Pasloske 1993; Fine et al. 1984; Smythe et al. 1988). It is therefore interesting that several membrane-associated proteins of P. falciparum zoites have been implicated to play a role in the invasion process and are potential candidates for incorporation in an anti-malarial vaccine (Peterson et al. 1989; Smythe et al. 1988; Howard and Pasloske 1993; Holder and Blackman 1994). However, extensive diversity and antigenic variation associated with proteins of the invasive and other stages of Plasmodium (Howard 1984; McBride et al. 1982) frustrate the development of a widely

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effective vaccine against malaria (Cox 1991). Accordingly, it is advantageous to identify new additional antigens containing protective oligopeptide domains that are conserved, presumably for common function (Del Portillo et al. 1991; Waters et al. 1990), such as invasion. Significantly, recent studies (Ray et al. 1994) showed that: 1. The serum antibody immunoglobulin G of mice convalescent from Plasmodium yoelii infection cross-reacts with at least 15 P. falciparum polypeptides, 8 of which are presumed integral membrane proteins, and 2. the convalescent-phase mouse anti-P. yoelii serum antibodies inhibit intraerythrocytic growth of P. falciparum and invasion of the red cells in vitro. These results suggest that P. falciparum antigens recognized by anti-P. yoelii antibodies contain conserved and potentially protective epitopes. Here, we report selection of recombinant gene clones that express interspecies-conserved putative integral membrane proteins of Plasmodium. Phage clones of lgt11-P. falciparum DNA libraries were co-immunoscreened with convalescent-phase mouse anti-P. yoelii sera (Ray et al. 1994) and immune serum from a rabbit immunized with Triton X-114 phase separated proteins of P. falciparum (Smythe et al. 1988; Ray et al. 1994; Bordier 1981). By this approach, we have identified a novel conserved lipophilic 70-kDa antigen of P. falciparum. Immunofluorescence tests with immune antisera against a recombinant fusion peptide comprising part of the antigen gave punctate patterns of immunofluorescence in schizonts, suggestive of antigens located in the apical organelles of the parasite. The antifusion peptide serum also reacted with an analogous 100-kDa protein from P. yoelii. MATERIALS

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Parasites, parasite lysate antigens, and immune antisera. The isolate (FCD3) of P. falciparum used in this work was initially obtained from blood of a malaria patient in New Delhi (India), adapted to in vitro culture (Trager and Jensen 1979), and kindly gifted to us by Dr V. K. Bhasin of Delhi University. Blood stages of P.

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falciparum were synchronized by lysis in 5% sorbitol (Lambros and Vanderberg 1979); two sequential rounds of sorbitol treatment, 35 hr apart, produced more than 97% synchronized cultures. The rodent malaria parasites, P. yoelii nigeriensis (Yoelii et al. 1975), were obtained from Dr G. P. Dutta of Central Drug Research Institute, Lucknow, India, and were maintained by serial passage in BALB/c mice (H-2d). Convalescent-phase mouse antiyoelii serum was obtained by repeated P. yoelii infection of BALB/c mice and cure (Ray et al. 1994; Kironde et al. 1991). Parasite lysate antigens were prepared as previously described (Kironde et al. 1991) Human anti-P. falciparum sera were obtained from donors resident in Koraput district of Orissa, a malaria endemic area in India (Rajagopala et al. 1990). IMPs of P. falciparum were isolated by phase separation in Triton X-114 (Smythe et al. 1988; Ray et al. 1994; Bordier 1981). The protocol of Smythe and co-workers (1988) which is optimized to exclude nonintegral membrane proteins was strictly followed. Parallel control experiments with solutions containing known proteins showed that the Triton X-114 fraction did not contain water-soluble proteins, as shown previously (Ray et al. 1994) A preparation enriched in IMPs of P. falciparum was immunized in rabbits (Harlow and Lane 1988) to raise anti-IMP sera. Parasite and human DNA, P. falciparum DNA library, nucleotide sequencing, and other cloning methods. Genomic DNA from parasites and normal human lymphocytes was extracted by standard described methods (Maniatis et al. 1982) Libraries of P. falciparum genomic DNA were constructed in lgt11 as described (Snyder et al. 1987). P. falciparum DNA was digested with either Sau3AI or EcoRI. The DNA fragments of 0.5 to 5 kb were isolated and prepared for ligation to EcoRI-digested arms of lgt11. Recombinant lgt11 phages were packaged into infectious particles in vitro and amplified in Escherichia coli Y1090 (r0m/) cells (Promega). Immunoscreening of expression libraries was conducted with antisera diluted 1 in 300. Nucleotide sequences of DNA were determined by the chain termination method (Sanger et al. 1977) using either synthetic oligonucleotide primers or unidirectionally digested fragments of the DNA (Henikoff 1987). Nucleic acid probes were labeled by the random hexanucleotide procedure (Feignberg and Vogelstein 1983). Parasite DNA was digested with mung bean nuclease (MBNase), electrophoresed, transferred to nitrocellulose, and probed by hybridization as described (Vernick et al. 1988; Southern 1975). P. falciparum DNA was digested with MBNase in a reaction mixture containing 33% formamide, whereby the nuclease precisely cuts the parasite DNA between the genes (Vernick et al. 1988). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of proteins and Western blot assays (Laemmli 1970; Towbin et al. 1979) were conducted as specified (Ray et al. 1994; Kironde et al. 1991)

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In the Western immunoblot assays, normal serum, preimmune sera, anti-P. falciparum, anti-P. yoelii, and antiIMP sera were each used at 1 in 1000 dilution, while anti-fusion peptide serum was used diluted 1 in 20,000. Preabsorption of antisera with immunogens or with red blood cell membranes was carried out according to established procedures (Harlow and Lane 1988). Expression and purification of glutathione S-transferase fusion. Cloned parasite DNA (L857) was subcloned into pGEX 4T-1 plasmids (Pharmacia) which were then used to transform E. coli ABLE K cells (Stratagene). The L857 encoded polypeptide was produced in fusion with glutathione S-transferase (GST) and the fusion product was affinity purified on glutathione–agarose as described (Smith and Johnson 1988). Primers. The following primers were used in polymerase chain reaction (PCR) amplifications. Forward primer, 5*-GATCAAAATGAAACTGGAACAAAAAAACC-3*; reverse primer, 5*-GATCCACGTTTTACATCTGATTTTG-3*. Immunofluorescent antibody tests (IFA). IFA tests were carried out as follows. Parasitized erythroctyes were applied to microscope slide wells, air-dried, and fixed either in 90% acetone, 10% methanol or in 1% formalin, 99% phosphate-buffered solution (PBS). Wells containing fixed or unfixed cells were then sequentially incubated with primary serum antibody and secondary fluoresceinlabeled reagents, mounted in PBS/glycerol, and observed with a fluorescent microscope, as described (Taylor et al. 1981). In some assays, ethidium bromide was used as a counterstain (10 mg/ml, 30 sec) to delineate nuclear staining. The primary antibody reagent was rabbit GST-p34specific antiserum diluted 1 in 25; the secondary antibody reagent was fluorescein-labeled goat serum IgG (diluted 1 in 60) antibody to rabbit immunoglobulin G.

RESULTS Identification of Reactive Clones and Analysis of Cloned Parasite DNA Two hundred thousand recombinant phage plaques from each of the lgt11 libraries of Sau3AI- and EcoRI-digested P. falciparum DNA were immunoscreened with convalescent-mouse anti-P. yoelii sera. Three hundred thousand plaques from the two libraries were immunoscreened with rabbit antiserum against Triton X-114-soluble proteins of P. falciparum. Ten phage plaques were found to be reactive with anti-P. yoelii sera while the rabbit anti-IMP serum identified 6 positive plaques. When the positive clones were retested with

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both the anti-P. yoelii and the anti-IMP sera, we found that a clone, L857, from the library of the Sau3AI-digested DNA had been selected independently by each of the two antibody probes, suggesting that L857 encodes a portion of a putative integral membrane protein which is shared between P. yoelii and P. falciparum. To obtain adequate amounts of L857 insert DNA for hybridization, sequencing, and other experiments, lgt11-L857 phage DNA was digested with EcoRI and the released insert (857 bp) was subcloned and amplified in the plasmid pBluescript II (Stratagene). The L857 insert was radiolabeled and used (see Fig. 1) to probe undigested, MBNasedigested P. falciparum genomic DNA, and randomly sheared human lymphocyte genomic DNA. The results show that L857 insert DNA hybridized both to undigested P. falciparum DNA (lane 1) and to a 1.8-kb fragment of MBNase-digested parasite DNA (lane 2). The insert did not hybridize to human DNA (lane 3). Together, these results suggest that the cloned insert DNA is derived from a P. falciparum gene of about 1800 bp. The nucleotide sequence of L857 insert DNA and the deduced amino acid sequence are shown in Fig. 2. Each of the insert DNA strands was sequenced at least thrice. Typical of malarial parasite genes (Weber 1987) the insert contains a high dA / dT content of 69%. Searches of the databases (EMBL 39 and SWISS-PROT 29) revealed no significant sequence homology with any known protein. The primary sequence (Fig. 2) shows that the L857-encoded polypeptide contains seven repeats of 19 amino acids (TKKPSKYTMNLDSPLLKGS) each, reminiscent of the repeated sequences commonly found in other described malarial proteins (Howard 1984; Howard and Pasloske 1993; Cox 1991). The repeats are not consecutive. Rather, they are separated by comparable but nonidentical spacers of 17 amino acids each. These spacer sequences might constitute degenerate repeats. In order to confirm accuracy of the cloned DNA, we

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FIG. 1. Probing of MBNase-digested P. falciparum DNA with L857 insert. Undigested (lane 1), mung bean nuclease-digested (lane 2), P. falciparum genomic DNA and human lymphocyte sheared DNA (lane 3) were electrophoresed, transferred onto nitrocellulose, and probed with 32P-labeled L857 insert DNA. Sizes (kb) of the DNA fragments are shown on the left side.

carried out PCR analysis using genomic parasite DNA as template and 20-mer oligonucleotide primers (see Materials and Methods) which hybridize to the two termini of L857 insert DNA (Fig. 2). A DNA fragment of the correct length (approximately 860 bp) and containing different predicted restriction enzyme sites was obtained. Expression of p34 by pGEX 4T-1 Figure 3a shows the SDS – PAGE analysis and Coomassie staining of proteins from lysates of nonrecombinant and recombinant IPTG-induced (0.5 mM IPTG, 2 hr) trans-

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FIG. 2. Nucleotide sequence of DNA encoding a fragment (34 kDa) of the interspecies-conserved 70kDa antigen of P. falciparum. DNA sequence and protein translation, underneath, are shown. The major repeat sequence is underlined.

formant E. coli cells. A thick band of 28 kDa corresponding to GST can be seen in lane 1 (IPTG induced nonrecombinant pGEX), whereas a comparable band is absent in lane 3 (IPTG induced recombinant pGEX). Conversely, a 62-kDa dense band corresponding to a fusion peptide is visible in lane 3 but is missing in lane 1. The affinity-purified 28-kDa GST molecule (lane 2) and the purified 62-kDa fusion peptide (lane 4) are also shown. We designated the polypeptide encoded by L857 insert DNA as p34 and the recombinant fusion peptide as GSTp34. It can be seen (lane 4) that the predictable sum of the 28-kDa GST carrier protein and the deduced L857-encoded polypeptide

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(34 kDa) agrees with the observed size (62 kDa) of the fusion peptide band (lane 4). To analyze the fusion peptide further (see Fig. 3b), nitrocellulose-bound GST-p34 and GST were probed with rabbit anti-fusion peptide and human and mouse anti-parasite sera. The anti-fusion peptide serum (lane 1) and the two anti-parasite sera (lanes 4 and 5) recognized the fusion peptide. On the other hand, while anti-fusion peptide sera also recognized purified GST (lane 2), both the anti-P. falciparum (lane 3) and the anti-P. yoelii (lane 6) sera did not detect the GST molecule. These results demonstrate that binding of GST-p34 by anti-P. falciparum and anti-P. yoelii antibodies is specific to

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FIG. 3. (a) Coomassie staining of SDS–PAGE analysis of expressed fusion product, GST-p34. Lane 1, soluble portion of lysates from IPTG-induced bacteria transformed with nonrecombinant plasmid, pGEX 4T-1; lane 2, affinity-purified GST; lane 3, soluble portion of bacteria transformed with the recombinant plasmid, pGEX 4T-1/ L857; lane 4, affinity-purified GST-p34. (b) Western blot analysis of GST-p34. Affinity-purified GST-p34 was probed with anti-GST (lane 1), anti-P. falciparum (lane 4), anti-P. yoelii (lane 5). Purified GST was probed with anti-GST (lane 2), anti-P. falciparum (lane 3), and antiP. yoelii (lane 6) sera. Normal rabbit and normal human sera did not react with purified GST-p34 or GST.

p34, the parasite-associated moiety in the fusion peptide.

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uninfected red blood cells (results nor shown). These results demonstrate that pf70 is a parasite antigen which is detected by p34-specific serum antibodies. The molecular weight (70 kDa) of pf70 (Fig. 4) is consistent with the size of a protein encoded by a 1.8-kb fragment of MBNase-digested DNA (see Fig. 1), since the enzyme cleaves P. falciparum DNA between the genes (Vernick et al. 1988). Thus, L857 represents nearly half of the total gene encoding pf70. It has been shown previously (Ray et al. 1994) that while several antigens shared between P. yoelii and P. falciparum are synthesized throughout the erythrocyte cycle of the parasite, others are mainly present at particular morphological stages. In order to determine the stage during which pf70 is expressed, parasite cultures were synchronized with sorbitol (Lambros and Vanderberg 1979). At different times (0, 12, 18, 24, 36, 40 hr) after the second of two cycles of sorbitol lysis, 20 ml of para-

Antisera against GST-p34 Recognized a 70-kDa Polypeptide in Lysates of P. falciparum Cells To identify the native protein related to p34, rabbit GST-p34 specific antiserum was used to probe separated antigens from lysates of unsynchronized P. falciparum cells. Figure 4a shows that the antiserum specifically recognized a 70-kDa P. falciparum antigen (lane 1). We designated the native molecule, which is recognized by the GST-p34-specific antiserum, as pf70. Unabsorbed anti-GST sera (lane 2) and anti-fusion peptide serum preabsorbed with GST-p34 (lane 3) did not recognize the 70-kDa polypeptide, showing that, in the GST-p34-specific antisera, only antibodies specific to p34 bind to pf70. In addition, preabsorption of the anti-fusion peptide serum with GST failed to abolish the serological detection of pf70, and GST-p34-specific antiserum did not recognize antigens from lysates of

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FIG. 4. Western blot analysis of P. falciparum antigens with antisera against GST-p34. (a) Antigens in lysates of nonsynchronized parasite cells were separated by SDS– PAGE (30 mg loaded for each lane), transferred to nitrocellulose, and probed with unabsorbed rabbit antiserum against GST-p34 (lane 1), anti-GST (lane 2), and immunogen-absorbed GST-p34-specific antisera (lane 3). (b) Blots of antigens from parasites harvested 1, 12, 18, 24, 36, and 40 hr after the second of two cycles of sorbitol lysis (lanes 1 to 6, respectively) were probed with rabbit antisera against GST-p34. After addition of NBT-BCIP substrate solution, colors in lanes 3 to 6 developed in 5 min. However, the membrane (carrying distinct lanes for all bloodstages) was incubated for 30 min so as to allow for the slower development of color in the ring-associated lanes (lane 1 and 2).

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site culture was harvested, and parasite antigen extracts were prepared and subjected to SDS–PAGE, loading 30 mg of extract protein in each lane of the separating gel. The resolved antigens were then blotted onto nitrocellulose and probed with antiserum against GST-p34. Immunoblots (Fig. 4b) showed that pf70 occurs predominantly in the schizont stages (lanes 5 and 6) but is also detectable in the rings (lanes 1 and 2) and trophozoites (lanes 3 and 4). Immunoblot assays processed with different batches of parasite culture consistently showed that the substrate (NBTBCIP) colors for schizont (lanes 5 and 6) and trophozoite (lanes 3 and 4) stages developed much more quickly than the colors for the ring stages (lanes 1 and 2), indicating that rings contain less pf70 than trophozoite and schizont stages (see Fig. 4 legend). Subcellular Localization of pf70 Immunofluorescent antibody tests (Fig. 5) showed faint single-dot fluorescences on the early and late ring stages (Figs. 5a and 5b), while early (Fig. 5c) and late (Fig. 5d) trophozoites showed a more diffused localization of the antigen, with fuzzy or reticular-like strings joining the dots. A punctate pattern, showing multiple distinct dots of fluorescence, was seen on the schizont stages (Figs. 5e and 5f), suggestive of antigens located on organelles in the bodies of free aggregated merozoites or merozoites inside a schizont. (McBride et al. 1982; Schofield et al. 1986; Howard et al. 1984). The patterns in the schizont smears (Figs. 5e and 5f) did not show dot-to-dot threads of fluorescence seen in trophozoite preparations (Figs. 5c and 5d). The fluorescence in the schizonts was clearly distinct from nuclear components counterstained with ethidium bromide. Uninfected erythrocytes incubated with GST-p34-specific antibodies did not show any fluorescence. In addition, the fluorescence-specific antibodies were effectively removed when anti-fusion peptide serum was preabsorbed with GST-p34. Surface

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FIG. 5. Immunofluorescence localization of pf70 on P. falciparum cells. P. falciparum-infected red cells were harvested and processed for IFA at 1 and 12 hr (early and late rings: a and b), 18 and 24 hr (early and late trphozoites: c and d), and 36 and 40 hr (mid and late schizont stages: e and f), respectively, after the second of two rounds of sorbitol lysis. Bars, 10 mm.

IFA tests, where fresh suspensions of unfixed schizont-infected erythrocytes were used as previously described (Schofield et al. 1986), did not show fluorescence, indicating that antigenic determinants of the expressed fragment (p34) of pf70 are intracellular. Positive control tests in which unsynchronized parasites were incubated with human anti-P. falciparum sera (results not shown) showed distinct multiple patterns of fluorescence (McBride et al. 1982), as expected. Solubility of pf70 in Triton X-114 At least eight Triton X-114-soluble polypeptides from P. falciparum cell lysates cross-

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react with anti-P. yoelii sera and are the targets of immune response during natural infection (Ray et al. 1994). To determine the detergent solubility of pf70, we subjected Triton X-114 phase purified proteins of P. falciparum to SDS–PAGE and blotted the separated polypeptides onto nitrocellulose (Fig. 6a: lanes 1, 3, and 4). The blots were probed with rabbit antiserum against GST-p34 (lane 1), anti-IMP (lane 3), and anti-GST serum (lane 4). As a control for the aqueous phase fraction, another strip of nitrocellulose was blotted with parasite lysate that was presorbed thrice with Triton X-114. The control strip was then also probed with antiserum against GST-p34 (lane 2). Results show that while anti-IMP sera detected many antigens in the Triton X-114 fraction (lane 3), the anti-fusion peptide serum recognized only a major 70-kDa polypeptide and a minor 50-kDa band, perhaps a degradation product (see lane 1). The aqueous control fraction did not contain any antigens reactive with the anti-fusion peptide serum (lane 2), and the anti-GST antibodies did not detect any polypeptide in the Triton X-114 fraction (lane

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FIG. 7. Immunoblot of P. yoelii antigens with antisera against GST-p34. Extract proteins of P. yoelii cells were subjected to SDS–PAGE, blotted, and probed with unabsorbed (lane 1), immunogen-absorbed (lane 2) or GSTp34-specific sera or with unabsorbed anti-GST sera (lane 3).

4). Further, the fusion peptide (GST-p34) was specifically recognized by rabbit anti-IMP serum (Fig. 6b, lane 1). SDS–PAGE and immunoblot tests of undissolved membraneous precipitates (insoluble in water and Triton X-114) from P. falciparum cells showed that strikingly large quantities of pf70 remain unextracted from the insoluble precipitate even after several detergent phase separations (results not shown). Antiserum against GST-p34 Recognized a 100-kDa Polypeptide from Lysates of P. yoelii Cells

FIG. 6. (a) Probing of putative integral membrane proteins of P. falciparum proteins with antisera against GSTp34. Triton X-114 phase resolved parasite antigens were electrophoresed, blotted onto nitrocellulose, and probed with antiserum against GST-p34 (lane 1), anti-IMP (lane 3), and anti-GST (lane 4) sera. Aqueous control (lane 2): antigens from parasite lysates phase partitioned with Triton X-114 solution were probed with antiserum against GST-p34. (b) Probing of the fusion peptide with antiIMP sera. The fusion peptide, GST-p34, was probed with rabbit anti-IMP (lane 1) and preimmune serum (lane 2).

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The selection of clone L857 and binding of the fusion peptide by anti-P. yoelii serum antibody (Fig. 3b, lane 5) suggested that an analogue of pf70 occurs in P. yoelii. Further, anti-P. yoelii antibodies affinity purified on antigens of P. falciparum cell lysates were previously found to recognize several polypeptides of P. yoelii in the molecular weight range of 85 to 110 kDa (Ray et al. 1994). To

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identify the P. yoelii analogue of pf70, antigens from lysates of P. yoelii cells were probed with antiserum against GST-p34. Figure 7 (lane 1) shows that a 100-kDa P. yoelii antigen is clearly recognized by anti-fusion peptide serum. We designated the 100-kDa antigen recognized by GST-p34-specific antisera in P. yoelii lysates as py100. When lysates of P. yoelii-infected red cells were phase partitioned with Triton X-114 solution, we detected py100 only in the detergent and insoluble fractions (results not shown). Similar to pf70 (Fig. 6), the aqueous phase did not contain any detectable quantities of py100. Binding of anti-GST.p34 antibodies to P. yoelii antigens was effectively abolished by preabsorption of the antiserum with the immunogen, GST-p34 (lane 2). Anti-GST serum was nonreactive with P. yoelii antigens (lane 3). Corroborating the detergent solubility results, IFA tests of P. yoelii-infected red blood cells showed multiple-dot-type patterns of fluorescence in the schizont-stage smears (results not shown), similar to the findings for P. falciparum. DISCUSSION By coordinate probing of P. falciparum DNA libraries with anti-P. yoelii sera and rabbit anti-IMP serum, we identified a clone (L857) encoding a 34-kDa fragment (p34) of an interspecies-conserved putative integral membrane protein (Mr 70 kDa) which we named pf70. Antisera against p34 recognized a cross-reactive 100-kDa antigen of P. yoelii and, in IFA tests with P. yoelii-infected red cells, gave dot-type or punctate immunofluorescence patterns similar to results for P. falciparum, showing that pf70 and the 100-kDa P. yoelii molecule may be analogous proteins. Pf70 is apparently a novel P. falciparum antigen; we found no sequence homology between p34 and any other known protein. The solubility characteristics of pf70 (see Results and Fig. 6a) and the reactivity of GST-p34 with the rabbit anti-IMP sera (Fig. 6b) suggest

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that the novel antigen is tightly associated with the cell membranes. Determination of the full sequence of pf70 will help to elucidate the observed lipophilicity of the antigen and its precise association with the membranes. Smythe and co-workers (1988) recently reported selection of clones that express IMPs by probing with human anti-parasite antibodies which were affinity purified on immobilized integral membrane antigens of P. falciparum. The method described here improves on that approach in at least two ways. First, a more potent postimmunization antiserum reactive with the IMPs of the parasite is used here; this antiserum clearly recognizes a larger number of antigens than human postinfection sera (see Fig. 6 and Smythe et al. 1988). Second, our method facilitates the differentiation of cloned putative IMPs into two types, namely, interspecies-conserved and nonconserved. Conserved antigens are specially important, since they may contain invariant oligopeptides constituting targets of either broadrange vaccine immunity (Saul et al. 1992a; Tanner et al. 1995) or potent curative drugs (Land et al. 1995; Francis et al. 1994). The existence of different epitopes common to P. yoelii and P. falciparum was earlier suggested by the binding of monoclonal anti-P. yoelii antibodies to P. falciparum cells (Taylor et al. 1981). More recently, our laboratory showed (Ray et al. 1994) that anti-P. yoelii antibodies inhibit the growth of P. falciparum and that of about 15 polypeptides of P. falciparum apparently shared between the two species, at least 8 are soluble in Triton X-114. On the other hand, anti-P. falciparum antibodies cross-react with at least 10 P. yoelii polypeptides (Mr 15–120 kDa), a result (Ray et al. 1994) consistent with the detection of the 100kDa P. yoelii antigen by pf70-specific antibodies (Fig. 7). The stage specificity, size, and solubility of pf70 in Triton X-114 (Figs. 4 and 6) imply that pf70 is probably the same as the 76-kDa P. falciparum molecule previously shown (Ray et al. 1994) to cross-react with anti-P. yoelii sera.

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At present, we do not know the biological function and precise organellar location of pf70. However, additional initial studies have indicated that the cloned partial fragment (p34) of the antigen: 1. induces antibodies that block P. falciparum merozoite invasion and 2. generates a protective immune response against P. yoelii infection in mice (Kironde, Ma, and Sahoo, manuscript in preparation). Conservation of the antigen (Fig. 7), likely apical location indicated by the IFA patterns (Fig. 5), and the above-mentioned inhibition test results suggest that pf70 may be important in merozoite invasion or schizont rupture and merozoite release. Based on the IFA patterns observed in the schizonts (Figs. 5e and 5f) we presume pf70 to be located in structures within the anterior apical region of merozoites (McBride et al. 1982; Schofield et al. 1986; Howard et al. 1984). Although detectable amounts of pf70 are retained by the parasite ring stages after merozoite invasion, Western blot (Fig. 4) and IFA tests (Fig. 5) suggest that the antigen is concentrated in the schizonts and free merozoites. Apicomplexan protozoa contain vesicular organelles which are believed to extrude secretory contents when the parasite enters into or exits from the host cell cytoplasm (Aikawa et al. 1978; Perkins, 1992; Fawcett et al. 1983; Entzeroth 1985). P. falciparum rhoptry-associated antigen 1 (RAP-1), 42-kDa RAP-2, and 110-kDa RAP-3 have all been found in the membraneous material which is thought to be secreted at initial stages of invasion (Bushell et al. 1988; Sam-Yellowe et al. 1988; Saul et al. 1992b; Ridley et al. 1990; Brown et al. 1991). Ring-infected erythrocyte surface antigen (RESA), a 155-kDa molecule believed to be involved in invasion and located in the merozoite dense granules, has been reported to be released into the PVM after merozoite attachment and invagination of the host cell membrane (Culvenor et al. 1991; Torii et al. 1989). On the other hand, two molecules, located in the micronemes and considered to function as parasite receptors for invasion (the circum-

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sporozoite protein of malarial sporoziotes and the Duffy receptor of P. knowlesi merozoites), are also believed to be exocytosed after merozoite attachment whereby they assist in junction formation (Adams et al. 1990; Fine et al. 1984). Determination of whether pf70 is actually located in the anterior truncated conical end (conoid) of the merozoite or occurs in one of the apical vesicular organelles and is secreted during invasion will help to clarify the biological role of pf70. The apparent interspecies conservation of pf70 and py100 (shown by serological crossreactivity, similar IFA patterns, cross-inhibition, and solubility in Triton X-114) constitutes one of few such examples of antigens whose invariable cross-reactive determinants conceivably reflect the preserved morphological and functional characteristics of invasion in Apicomplexan parasites. Cross-reactive epitopes between pf70 and py100 might mediate a critical step of merozoite invasion or release from schizonts. Other similarly depictive examples include the cross-reactive rhoptry antigens of Babesia species (Dalrymple et al. 1995), rhoptry apical merozoite antigen 1 (AMA 1) molecules (Peterson et al. 1989, 1990; Waters et al. 1990; Marshall et al. 1989), and microneme CSP and CSP-related proteins of malarial sporozoites and erthrocyte stages (Fine et al. 1984). Analysis of total rhoptry and microneme proteins indicates that the apical complex organelles may contain up to 60 or more distinct polypeptides (Etzion et al. 1991; Pohl et al. 1989). Presumably, the limited total of known interspecies-conserved apical-organelle antigens is due to the meager proportion (about 5 to 10) of molecules (from these organelles) which have been cloned and sequenced so far (Peterson et al. 1989; Saul et al. 1992b; Ridley et al. 1990; Brown and Coppel 1991; Keen et al. 1990; Suarez et al. 1994). It is reasonable to expect that as more apical secretory molecules are identified and characterized, valuable conserved sequence motifs involved in key steps of invasion will be recognized. Complete sequencing of pf70

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and py100 genes may allow structural analysis of the antigen and identification of conserved functional domains in these putative homologues. At any rate, as for AMA-1 (Waters et al. 1990; Marshall et al. 1989; Peterson et al. 1990), the interspecies homology of pf70 implies that the molecule contains conserved domains in different strains of P. falciparum, an advantage which may avoid the problem of antigenic variation (Howard 1984; McBride et al. 1982) confronting many potential vaccine candidates (Cox 1991; Tanner et al. 1995). REFERENCES ADAMS, J. H., HUDSON, D. E., TORII, M., WARD, G. E., WELLEMS, T. E., AIKAWA, M., AND MILLER, L. H. 1990. The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63, 141–153. AIKAWA, M., MILLER, L. H., JOHNSON, J., AND RABBEGE, J. 1978. Erythrocyte entry by malarial parasites, a moving junction between erythrocyte and parasite. Journal of Cell Biology 77, 77–82. BANNISTER, L. H., BUTCHER, G. A., DENNIS, E. D., AND MITCHELL, G. H. 1975. Structure and invasive behaviour of Plasmodium knowlesi merozoites in vitro. Parasitology 71, 483–491. BANNISTER, L. H., MITCHELL, G. H., BUTCHER, G. A., AND DENNIS, E. D. 1986. Lamellar membranes associated with rhoptries in erythrocytic merozoites of Plasmodium knowlesi, a clue to the mechanisms of invasion. Parasitology 92, 291–303. BORDIER, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 256, 1604–1607. BROWN, H. J., AND COPPEL, R. L. 1991. Primary structure of a Plasmodium falciparum rhoptry antigen. Molecular and Biochemical Parasitology 49, 99–110. BUSHELL, G. R., INGRAM, L. T., FARDOULYS, C. A., AND COOPER, J. A. 1988. An antigenic complex in the rhoptries of Plasmodium falciparum. Molecular and Biochemical Parasitology 28, 105–112. COX, F. E. G. 1991. Malaria vaccines-progress and problems. TIBTECH 9, 389–394. CULVENOR, J. G., DAY, K. P., AND ANDERS, R. F. 1991. Plasmodium falciparum ring-infected erythrocyte surface antigen is released from merozoite dense granules after erythrocyte invasion. Infection And Immunity 59, 1183–1187. DALRYMPLE, B. P., CASU, R. E., PETERS, J. M., DIMMOCK, C. M., GALE, K. R., BOESE, R., AND WRIGHT, I. G. 1995. Characterization of a family of multi-copy genes

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