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J. Biol. Chem., Vol. 277, Issue 49, 47533-47540, December 6, 2002

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Serine Repeat Antigen (SERA5) Is Predominantly Expressed among the SERA Multigene Family of Plasmodium falciparum, and the Acquired Antibody Titers Correlate with Serum Inhibition of the Parasite Growth^*

Sayaka Aoki, Jie Li, Sawako Itagaki, Brenda A. Okech^§, Thomas G. Egwang^§, Hiroyuki Matsuoka^¶, Nirianne Marie Q. Palacpac, Toshihide Mitamura, and Toshihiro Horii

From the  Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan, the ^§ Division of Medical Parasitology and Tropical Medicine, Med Biotech Laboratories, Kampala, Uganda, and ^¶ Department of Medical Zoology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan

Received for publication, July 17, 2002, and in revised form, September 17, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Plasmodium falciparum serine repeat antigen (SERA) is one of the blood stage malaria vaccine candidates. The malaria genome project has revealed that SERA is a member of the SERA multigene family consisting of eight SERA homologues clustered on chromosome 2 and one SERA homologue on chromosome 9. Northern blotting and real time quantitative reverse transcription-PCR with five independent parasite strains, including three allelic representative forms of the SERA gene, have shown that all of the SERA homologues are transcribed most actively at trophozoite and schizont stages and that SERA5 (SERA/SERP) is transcribed predominantly among the family. Polyclonal antibodies were raised against recombinant proteins representing the N-terminal portions of four significantly transcribed SERA homologues (SERA3 to -6) in the center of the cluster on chromosome 2. Using these antibodies, indirect immunofluorescence microscopy detected the expression of SERA3 to -6, with similar localization, in all trophozoite- and schizont-infected erythrocytes. We have examined 40 sera from Ugandan adults for their antibody reactivity and found that enzyme-linked immunosorbent assay titer against SERA5 N-terminal domain, but not against other SERA proteins, is positively correlated with the inhibition of in vitro parasite growth by individual sera. Our data confirm the usefulness of the N-terminal domain of SERA5 as a promising malaria candidate vaccine.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Malaria remains a devastating disease worldwide, especially in the tropics. Among four species of human malaria parasites, Plasmodium falciparum is responsible for more than a million deaths annually. The appearance of drug-resistant parasites and insecticide-refractory mosquito vectors has made its control more difficult. It is therefore of increasing importance to develop effective malaria vaccines.

P. falciparum serine repeat antigen (SERA)¹ (1) is an asexual blood stage antigen produced in large amounts, specifically during late trophozoite and schizont stages (2, 3). SERA protein (also called SERP (4) or p126 (5)) is secreted into the lumen of the parasitophorous vacuole after removal of the signal peptide (6). Upon schizont rupture, SERA is processed into a 47-kDa N-terminal, a 50-kDa central, an 18-kDa C-terminal, and a 6-kDa domain (6). The complex of 47- and 18-kDa peptides is associated with merozoite, and the 50-kDa fragment is shed into the culture medium (7, 8). The activity responsible for the primary processing step of SERA to P47 and P73 is sensitive to the serine protease inhibitor diisopropyl fluorophosphate, whereas the activity for the conversion of P56 into P50 is sensitive to cysteine protease inhibitors E-64, leupeptin, and iodoacetoamide (9).

Mouse and rat antibodies against the N-terminal 47-kDa domain have been shown to inhibit the intraerythrocytic proliferation of parasites in vitro (7, 10, 11, 12), but rat antibodies against the central 50-kDa domain have little effect (10). Recombinant proteins corresponding to the 47-kDa domain of SERA conferred protective immunity in Aotus and squirrel monkeys against the parasite challenges (13-16). The epidemiological study in a holoendemic area of Uganda has revealed that increased level of IgG against 47-kDa peptide correlates with lower parasitemias in the peripheral blood and absence of fever in a group of children, but IgG level against 50-kDa peptide does not (17). Thus, the N-terminal domain of SERA is a promising candidate for a malaria vaccine.

It was previously reported that the N-terminal domain of SERA is polymorphic, and according to the amino acid sequences, all of the examined alleles can be grouped into three major allelic families, namely FCR3 type, K1 type, and Honduras-1 type in laboratory strains and field isolates (18, 19).

Knapp et al. (20) have reported that a SERA homologue gene (SERP-H) is located adjacent to the SERA gene, although it does not contain a serine stretch, and that a 130-kDa polypeptide is expressed from the SERP-H gene during schizont stage and localizes in the parasitophorous vacuole. Fox and Bzik (3) have shown that another SERA homologue (designated as SERA3 in the original paper) is located 1.8 kb upstream of the SERA gene and is transcribed at the trophozoite and schizont stages. Recently, the malaria genome project has revealed that these genes belong to the SERA multigene family, consisting of eight open reading frames clustered in tandem on chromosome 2 (21). The eight open reading frames on chromosome 2 are designated as SERA1 to SERA8 in the direction from centromere to telomere. Previously described SERA (SERP or p126), SERP-H and SERA3, correspond to SERA5 (PFB0340c), SERA6 (PFB0335c), and SERA4 (PFB0345c), respectively. A serine repeat is found only at the N-terminal region of SERA5. All members in the SERA multigene family contain a papain protease-like motif, and SERA1 to -5 contain a serine residue instead of a cysteine residue at the putative active nucleophile position, suggesting that they are serine proteases with a typical structure of cysteine protease (3, 22-24).

The evasion and/or prevention of the protective host immune responses are critical for the successful survival of Plasmodium parasites. Genetic polymorphism in a single locus gene or multigene family, frequently found in malaria vaccine candidate genes, may represent genetic backgrounds that function for parasite immune evasion mechanism. To see whether the SERA multigene family, as a vaccine candidate antigen, exhibits antigenic variation, we characterized the expression profile of each member in this family. The data obtained demonstrate that the SERA5 gene is predominantly expressed and co-expressed with adjacent SERA homologue genes (SERA3, -4, and -6) in every single parasite cell with similar localization in trophozoites and schizonts. Moreover, antibody level in an individual human serum against SERA5 N-terminal domain, but not those for other homologues, is correlated with in vitro parasite growth inhibition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Parasites-- FCR3 (25), Honduras-1 (26), K1 (generous gift from Dr. Masatsugu Kimura), 3D7 (27), and Dd2 (28) strains of P. falciparum were maintained in culture according to the methods mainly by Trager and Jansen (29) and modified by Mitamura and co-workers (30, 31). Cultures were maintained in 5% O₂ and 5% CO₂ atmosphere with 3% type O erythrocyte (v/v) in the culture medium containing 10% heat-inactivated human serum. For large scale culture and growth inhibition assay with human serum, 5 mg/ml AlbuMax (Invitrogen) was used in place of 10% human serum. For synchronization, schizont-rich parasites were purified by 63% (v/v) Percoll (Amersham Biosciences) density centrifugation (32) and incubated within 4 h in fresh medium with 3% erythrocyte prior to 5% sorbitol treatment.

Reverse Transcription Semiquantitative PCR-- Total RNA was isolated from 0.075% saponin-treated (Sigma) synchronized parasite cells of Honduras-1, FCR3, K1, Dd2, and 3D7 with TRIZOL^TM reagent (Invitrogen). First strand cDNA was synthesized with the Superscript^TM First-strand Synthesis System for RT-PCR (Invitrogen) using 20 ng of each total RNA. Target cDNAs were amplified by the following primer sets: SERA1/for (5'-AAATTCAGCAATTTGTATGAAATATCC-3') and SERA1/rev (5'-AGAAATAGCATGTGGTTCATAACCTT-3'), SERA2/for (5'-GAAAAACCTGACACCACTACTAGGAT-3') and SERA2/rev (5'-GCAGGTGCTATAAAATCATATTCATC-3'), SERA3/for (5'-GATATGTTTAAAGCAAATGAACATGG-3') and SERA3/rev (5'-AAACTTTTAATGGGTTTGAACCTTCT-3'), SERA4/for (5'-AACTTAAAGCAACCAATAACATCCAT-3') and SERA4/rev (5'-AAATGATATTCGCTAGATTCCTCATC-3'), SERA5/for (5'-CTTAGATAATTATGGGATGGGAAATG-3') and SERA5/rev (5'-GTTGTATCAACATGTACGACACCTTT-3'), SERA6/for (5'-TTGTTAAAATCTCATTCTGACGAAAA-3') and SERA6/rev (5'-CATCAGAATTTTCTTTGTCATCATTT3'), SERA7/for (5'-TAATTGTTCGGATAGAGATTCTGATG-3') and SERA7/rev (5'-TTTTGTAGTCATACGTTGTCTTGGAC-3'), SERA8/for (5'-TACCTGAGAGGAAAATATTCAAACCT-3') and SERA8/rev (5'-GTAAGCTGCTATAACAACACTCGAAG-3').

As an internal control, a primer set (MSP1/for (5'-TTCGTGCAAATGAATTAGACGTAC-3') and MSP1/rev (5'-GGATCAGTAAATAAACTATCAATGT-3')) that annealed to the conserved blocks 3 and 5 of the merozoite surface protein-1 (MSP-1) gene (33) was mixed together with one of the SERA primer sets. All RNA preparations gave no PCR product when reverse transcriptase was omitted from the RT-PCR. The efficacies of PCR primers were confirmed by using genomic DNA (gDNA) as a template. gDNAs were isolated from saponin-treated parasite cells of Honduras-1, FCR3, and K1 with DNAZOL^TM reagent (Invitrogen). The PCR cycle used was as follows: 91 °C (1 min 30 s) followed by appropriate cycles of 91 °C (30 s), 50 °C (30 s), and 58 °C (3 min).

Northern Blotting-- Total RNA from Honduras-1, FCR3, and K1 strains were fractionated on a 1.2% agarose/formaldehyde gel (1 µg/lane) and transferred onto Nytran membrane (Schleicher & Schuell). The membrane was probed with PCR products, which had been amplified by the same set of primers for RT-PCR and labeled with deoxycytidine 5'-triphosphate -³²P (250 µCi/mmol) (PerkinElmer Life Sciences), exposed to a Fuji Film BAS imaging plate, and analyzed with MacBAS 1500 (Fuji Film Co.).

Real Time Quantitative PCR-- Real time quantitative PCR (34, 35) was performed using the ABI PRISM 7700 (PerkinElmer Life Sciences), and results were analyzed with the accompanying software. A 50-µl mixture was formulated with first strand cDNA prepared above using TaqMan^TM PCR Core Reagent Kit (PerkinElmer Life Sciences), the corresponding primer sets, and the appropriate TaqMan probe. Primers and probe sets were as follows: SERA1/for (5'-AGTTGATATGTATGGACCATCAACA-3'), SERA1/rev (5'-ATGGTTTACCTTATCTTCTTGGGA-3'), and SERA1/probe (5'-TGTTCATCAGACGCATTAACCAATTTCA-3'); SERA2/for (5'-CCGCATCTGAGGCAGGA-3'), SERA2/rev (5'-ATCGGTTGATACAGGTAATGCTACA-3'), and SERA2/probe (5'-TCCTTGTTTCGTAATTTTTCCACCCGT-3'); SERA3/for (5'-TCTTACCAACAGAAGGAGATTATTCA-3'), SERA3/rev (5'-ATTTTGTTCTAATAATTTTGCATTTGC-3'), and SERA3/probe (5'-CTGGGCATGTTTCACCAACTTTACTTTG-3'); SERA4/for (5'-CCTCATCAAGCGGACAACAA-3'), SERA4/rev (5'-CTTCTGCCGGTGATGCTTCT-3'), and SERA4/probe (5'-CAACACAAGGACTATCACCAGCAACTGGAG-3'); SERA5/for (5'-TATTCTCTGAAAAGGAAGATAATGAAAACA-3'), SERA5/rev (5'-TGAAGTTCCTGCAGATTCTAATGC-3'), and SERA5/probe (5'-CCTGATCCTGCCGTATCTTGACCGAAT-3'); SERA6/for (5'-TGTAGCTAATTGTTCTAAGAGAAAACCTAT-3'), SERA6/rev (5'-AGGACAAGAATTACCTGCACTTGTA-3'), and SERA6/probe (5'-AAATTCTAATGGATTCGATCCTTCTTCACA-3'); SERA7/probe (5'-TCGTCGGATCGAATCCAGTTGAATTTCTAG-3'); SERA8/for (5'-TCTGTATTTGTTTCTATGGAAGTAACAGA-3'), SERA8/rev (5'-AATACTAAGGCATGATCCGGACTAT-3'), and SERA8/probe (5'-TCACAACTCATCATAACTTTTGTCCCATCA-3'); SERA9/for (5'-ACTGTTCATGGACAAAGTGGAGAA-3'), SERA9/rev (5'-ACAGCTCCTCTGTTCGAATCTTG-3'), and SERA9/probe (5'-TTCAACCTTCACAACTTCGATCTACCGCT-3'); MSP-1/for (5'-ATCCAAATCCTACTTGTAACGAAAATA-3'), MSP-1/rev (5'-TTCTTTCTGCTGCTACCTGAATC-3'), and MSP-1/probe (5'-TGGCATCTGCATCACATCCACC-3').

The first strand cDNA was added to the reaction mixture just prior to thermal cycling. The PCR cycle used was as follows: 50 °C (2 min) and 95 °C (10 min), followed by 60 cycles of 95 ° (15 s) and 60 °C (1 min).

Expression Plasmid Constructs-- RT-PCR was used to prepare cDNA encoding a part of the N-terminal region of SERA3, SERA4, and SERA6 as described in the legend to Fig. 1. NdeI site and BamHI sites were introduced at the end of forward and reverse primers, respectively (restriction enzyme sites are underlined): SERA3/for (5'-GGAATTCCATATGACAACAGTGGACGAGAGTACC-3' and SERA3/rev (5'-CGGGATCCAAATTTAAATGTTTGGTTTTTTCCAG-3'; SERA4/for (5'-GGAATTCCATATGACAACCGCCAGTACTACTCA-3') and SERA4/rev (5'-CGGGATCCGAAATCAAATTTTTTTGTGTCATC-3'); SERA6/for (5'-GGAATTCCATATGGAAGGAAATAAAGTGACTGTGA-3') and SERA6/rev (5'-CGGGATCCTAGTTTAAAATGATATCCTTCAGA-3').

The amplified fragment was digested with BamHI and NdeI and ligated to a BamHI- and NdeI-digested pET15b plasmid vector (Novagen). The resultant plasmid, pET-SE3N, pET-SE4N, or pET-SE6N, encodes the His tag (6 histidine residues) fused to its N-terminal domain of SERA3 (Thr⁶⁷-Phe⁵⁷⁰), SERA4 (Thr⁶⁷-Phe⁵⁵²), or SERA6 (Glu⁹⁷-Leu⁷⁶⁵), respectively. The encoded fusion proteins were designated as His-SE3N, His-SE4N, and His-SE6N.

Expression and Purification of the SERA Recombinant Proteins-- The freshly transformed Escherichia coli BL21(DE3) cells with RIG plasmid (36) and either pET-SE3N, pET-SE4N, or pET-SE6N were grown in LB to a cell density of 1.0 × 10⁸ cells/ml at 37 °C, and then isopropyl--D-thiogalactopyranoside was added to a final concentration of 50 µg/ml. After incubation for an additional 3 h, cells were harvested and stored at 80° until use.

Subsequent operations were carried out at 4 °C or on ice. The frozen cells expressing His-SE3N, His-SE4N, or His-SE6N protein were thawed and suspended in 5 cell paste volumes of buffer A (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 20 mM imidazole). The cells were disrupted by freezing and thawing, followed by repeated treatments with an ultrasonic disrupter (Tomy Seiko model UR-200P). The sonicated mixture was centrifuged at 10,000 rpm for 10 min, and guanidine HCl powder was directly dissolved into the supernatant at a final concentration of 6 M.

Purifications of all three His-tagged fusion proteins (His-SE3N, His-SE4N, and His-SE6N) were performed with the same procedure provided from the Hi-Trap chelating column (Amersham Biosciences). The column (1-ml bed volume) was preloaded with 0.5 ml of 0.1 M NiSO₄ to bind nickel ion and then equilibrated with 5 ml of buffer B (6 M guanidine HCl in buffer A). The 5-15 ml of cell lysate prepared above was applied onto the column. The proteins bound to the resin were further washed with 5 ml of buffer C (6 M urea in buffer A) and then refolded with 10 ml of buffer A. Bound proteins were eluted with buffer D (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 500 mM imidazole). The eluted sample was applied again onto the column, and the whole purification procedure described above was repeated. The eluted fractions from second column chromatography were dialyzed against PBS, prior to thrombin protease treatment (10 units/1 mg of protein). The treated sample was loaded onto the equilibrated Hi-Trap chelating column bound with nickel ion, and flow-through fractions were collected. After removal of the His tag, each recombinant protein was designated as SE3N, SE4N, or SE6N. Each recombinant protein gave a single band with an expected molecular mass as follows: SE3N, 28 kDa; SE4N, 30 kDa; SE6N, 33 kDa. After they were concentrated to 1 mg/ml by Centriprep YM-10 (Millipore Corp.), each purified recombinant protein was used for the custom antibody preparation (Asahi Techno Glass). Purification of total IgG from each serum was performed with a HiTrap Protein G column (Amersham Biosciences) according to the methods described previously (7).

Preparation of recombinant SE47' protein and affinity-purified mouse and rabbit anti-SE47' antibodies was previously described (10).

Expression and Purification of Recombinant Merozoite Surface Protein-1-- Recombinant protein of block 17 in merozoite surface protein-1 (rMSP-1₁₉) was prepared in the silkworm, Bombyx mori, as follows. Genomic DNA of the P. falciparum MAD 20 strain was used for PCR to obtain DNA fragments encoding signal sequence (Met¹-Leu³²) and block 17 (Pro¹⁵⁷¹-Gly¹⁶⁸⁶) of MSP-1 (31). The obtained DNA fragments were connected with spacer nucleotides, GGAATT (encoding Gly-Ile), and then ligated to plasmid pBm030 (37). The constructed plasmid was co-transfected with a wild type of B. mori nuclear polyhedrosis virus into an insect cell line, BmN4 (Funakoshi), and the recombinant virus was purified by plaque assay (37). The purified recombinant virus was injected into silk worms on the first day of the fifth larval instar (5 × 10⁴ plaque-forming units/worm). Hemolymph was collected 4 days later. An affinity purification column was prepared with Affi-Gel 10 (Bio-Rad) and MSP-1 block 17 specific monoclonal antibody 5.2 purchased from the American Type Culture Collection (Manassas, VA). With the affinity column, recombinant MSP-1 was purified from the hemolymph according to the manufacturer's instructions. 10 µg of purified rMSP-1 was obtained from 1 ml of the hemolymph.

ELISA and Western Blot Analyses-- 100 µl of 1 µg/ml each recombinant protein (SE3N, SE4N, SE47', SE6N, or MSP-1₁₉) was used as antigens to coat each well of a 96-well microtiter plate. The second antibody used was biotinylated goat IgG specific to human IgG ( chain) (Vector Laboratories), and color development was conducted using Vectastain ABC kit (Vector Laboratories) with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate. ELISA titers were determined by a cut-off absorbance of 0.2 at 412 nm with a microtiter plate reader (Titertek Multiskan MCC/340 MKII).

For Western blot, Percoll-purified trophozoite and schizont stage parasites were lysed in SDS buffer and loaded onto 8% SDS-polyacrylamide gel. Membrane transfer, primary antibody binding, and horseradish peroxidase-conjugated secondary antibody binding, followed by color development with the DAB substrate kit (Funakoshi, Japan), were according to the methods described (9).

Indirect Immunofluorescence Assay for Localization of SERA Protein-- Percoll-purified trophozoite- and schizont-infected erythrocytes were fixed with 4% paraformaldehyde in PBS on ice for 30 min, spread onto slides, and air-dried. To permeabilize cells, samples were treated with PBS containing 1% Triton X-100 (7). Slides were blocked with PBS containing 3% BSA (buffer E) for 1 h and subsequently reacted with the affinity-purified mouse SE47'-specific IgG and either rabbit -SE3N serum, rabbit -SE4N serum, or rabbit -SE6N serum. All of the rabbit antisera used were diluted at 1:1000, and the concentration of the purified mouse IgG used was 1 µg/ml in buffer E. The slides reacted with two primary antibodies were washed five times with PBS and then incubated in buffer E containing 1000-fold diluted Cy3-conjugated sheep anti-rabbit IgG (Sigma), 100-fold diluted fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Sigma), and 1 µg/ml 4',6'-diamidino-2-phenylindole (Sigma). After five washes with PBS, the slides were mounted with PermaFluor^TM Aqueous Mounting Medium (Immunon^TM). Fluorescence microscopy was performed by using an Axioskop fluorescence microscope (Carl Zeiss). Images were recorded by an AxioCam MRm CCD camera (Carl Zeiss).

Human Serum and Growth Inhibition and Invasion Assay-- Individual sera from Ugandans were collected from 40 healthy adults (age >18 years) living in Atopi Parish, a malaria holoendemic area, located 5 km west of Apac Town, 300 km north of Kampala. Blood samples were obtained with informed consent (and approval by the Uganda National Council for Science and Technology) by venipuncture and collected in Vacutainers containing EDTA. Serum samples were separated into fresh serum vials and stored at 20 °C.

The parasite growth inhibition assay was performed in a 96-well microtiter plate with FCR3, Honduras-1, and K1 parasite strains. Individual Ugandan serum samples were added at 5% (v/v) to the parasite culture containing 3% erythrocyte with 0.3-0.5% trophozoite- and schizont-rich cells and incubated for 24 h. Japanese malaria naive serum was used as control. Parasitized erythrocytes were counted in Giemsa-stained thin smears, and the parasitemia was scored by counting over 5000 erythrocytes in a slide. The growth inhibition (%) is calculated by (A  B)/A × 100, where A and B are control parasitemia (%) and parasitemia from sample (%), respectively. Correlation coefficients (r) were calculated using Pearson's test for pairs of logarithm of ELISA titers to base 2 and the parasite growth inhibition (%). The p values under 0.05 are considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcription Profile of the SERA Multigene Family-- Transcriptional activity of each gene belonging to the SERA multigene family on chromosome 2 was examined by RT-PCR (Fig. 1). Three parasite strains representative of typical SERA5 allelic forms, Honduras-1, K1, and FCR3, as well as two standard strains, Dd2 and 3D7, were used as total RNA templates for RT-PCR. When 20 ng of total RNA was used for the reverse transcriptase reaction, the 40 cycles of PCR could yield all of the expected products corresponding to SERA1-8 genes (data not shown), demonstrating that SERA1-8 genes are active in transcription. To estimate relative transcriptional activities of SERA1-8 genes in each five parasite strains, PCR cycles were reduced to 20. As an internal control, the conserved region of MSP-1, block 3-5, was amplified with corresponding specific primers. As shown in Fig. 2, it appeared that SERA5 was predominantly transcribed among SERA family genes and that the activities were followed by the adjacent SERA4, -3, -6, and -7 without any significant difference among five parasite strains examined. The observed transcription profiles were further confirmed by Northern blot analysis of total RNA prepared from three representative parasite strains (Fig. 3). Northern blot indicates that the SERA5 transcription is severalfold higher than the internal control, MSP-1 gene.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Schematic diagrams of SERA multigene family. SERA1-8 genes are clustered in tandem on chromosome 2, and the SERA9 gene is on chromosome 9. The length of the open reading frame is described in nucleotide numbers (bp) under the gene name. Solid lines represent protein coding regions with nucleotide numbers. Open boxes indicate regions corresponding to recombinant protein constructs. Arrows indicate the positions of intron insertions. Open triangles indicate the positions of primers for RT-PCR, the amplified products of which are used as probes in Northern blotting. Filled triangles indicate the positions of primers for real time quantitative PCR. On SERA7, the same primer sets were used for RT-PCR and real time quantitative PCR. The length of PCR product is described in bp. Numbers in parentheses are lengths of PCR product from gDNA.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Transcription activity of SERA1-8 genes in five parasite strains. RT-PCR was carried out with total RNA (20 ng/reaction) prepared from Percoll-purified trophozoite and schizont-infected erythrocyte as described under "Experimental Procedures." The parasite strains used are indicated. Primer sets used are SERA1 (lane 1), SERA2 (lane 2), SERA3 (lane 3), SERA4 (lane 4), SERA5 (lane 5), SERA6 (lane 6), SERA7 (lane 7), and SERA8 (lane 8). In all of the lanes, the MSP-1 primer set was also included as an internal control. Observed sizes of the products are in accordance with the predicted sizes of mature mRNAs. Lane M shows a 100-bp ladder (Promega).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Northern blot of SERA1-8 genes in three parasite strains. Northern blot analysis of SERA1-8 genes was carried out with total RNA (1 µg/lane). RNA was prepared from Percoll-purified trophozoite- and schizont-infected erythrocyte of parasite strains K1 (K), Honduras-1 (H), and FCR3 (F). The blotted membrane was probed with each of the radiolabeled PCR products of SERA1-8 genes and MSP-1 that were radiolabeled in a single tube to keep a specific radioactivity of both probes constant.

Since we have obtained consistent results with semiquantitative RT-PCR and Northern blot experiments, we carried out real time PCR for more quantitative comparison of the transcriptions among the SERA multigene family. In this experiment, the ninth SERA homologue, which was on chromosome 9 through the genome data base search (available on the World Wide Web at www.PlasmodDB.org), was also included (SERA9). To avoid possible inaccuracies caused by hybridization efficiency of primers used in above described RT-PCR, we prepared the new primer sets for each SERA gene except for SERA7. The new primer sets were designed to amplify the 3'-proximal region of each gene, because oligo(dT)-primed cDNA was used for the real time PCR. Based on the reproducible results from three independent experiments, the transcription profiles of SERA1-8 genes are conserved in all of the parasite strains examined (Fig. 4). These results were consistent with the previous experiments described above. However, SERA9 and MSP-1 gene transcriptions varied from experiment to experiment and in parasite strains as well (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Relative abundance of mRNA for each gene in SERA multigene family. RNA was prepared from Percoll-purified trophozoite- and schizont-infected erythrocyte of parasite strains K1 (open bar), Honduras-1 (hatched bar), and FCR3 (filled bar). Real time quantitative RT-PCR was carried out three times with 20 ng of independently prepared total RNA from each parasite strain using primers and probe sets as described under "Experimental Procedures." The TaqMan probe sequence was designed according to the recommendation of the ABI 7700 analyzer. Relative copy number of mRNA for each SERA gene was standardized, with that of SERA5 set at 100%. (The control real time PCR that was carried out with the same sets of primers and gDNA as template confirmed that all PCRs were performed at comparable efficiency (data not shown).)

The stage specificity of each gene expression may largely affect the expression profile, especially SERA9 and MSP-1 genes; therefore, total RNA prepared from the tightly synchronized parasite cells were subjected to real time quantitative PCR. Ring, early trophozoite, late trophozoite, and schizont stage parasites were harvested, respectively, at 8, 15, 29, and 34 h after reinvasion. Fig. 5 showed the amount of each SERA gene transcript relative to that of the SERA5 gene detected at the late trophozoite stage as 100%. All of the SERA genes were transcribed at late trophozoite and schizont stages but not at ring and early trophozoite stages. The transcription of the MSP-1 gene was mainly at schizont stage.

View larger version (9K): [in this window] [in a new window]   Fig. 5.   Stage-specific expression of each gene in the SERA multigene family. Real time RT-PCR was carried out with 20 ng of total RNA purified from different stages of FCR3 parasites that were synchronized as described under "Experimental Procedures." Ring, early trophozoite, late trophozoite, and schizont stage parasites were harvested at 8, 15, 29, and 34 h after reinvasion, respectively. Columns 1-9 and M represent SERA1-9 and MSP-1 genes, respectively. Relative abundance of mRNA for each SERA gene was calculated by taking that of SERA5 in late trophozoite stage as 100%.

Expression of SERA3, SERA4, SERA5, and SERA6 Proteins-- SERA3, SERA4, SERA5, and SERA6 genes were significantly transcribed in all of the parasite strains examined. To analyze the protein expression by immunological methods, we attempted to produce recombinant proteins as a His-tagged protein and antiserum against each recombinant protein for SERA3, SERA4, or SERA6 gene product. The previously described SE47' protein (10) was used for SERA5. N-terminal regions were chosen to produce the recombinant proteins as shown in Fig. 1, because the amino acid sequence in this region shows less homology to each other among the SERA family. The sizes of recombinant proteins SE3N, SE4N, SE47', and SE6N for SERA3, -4, -5, and -6 are 28, 30, 40, and 33 kDa, respectively. To avoid a possible cross-reaction to histidine-rich proteins of P. falciparum, the His tag of the recombinant proteins was removed by thrombin protease prior to the rabbit immunization. Western blots of trophozoite and schizont stage parasite cells confirmed the expression of SERA3, -4, -5, and -6 (Fig. 6). Western blot with the Honduras-1 strain showed that trophozoite and schizont stage parasites are expressing the full-length SERA3, -4, -5, and -6 proteins with apparent molecular sizes of 110, 120, 120, and 130 kDa, respectively, consistent with the deduced size from cDNA. In reducing condition, SERA4 and SERA5 migrate almost the same, but in nonreducing conditions, they migrate differently. The results of Western blot also confirmed that the prepared antisera were not cross-reactive with other SERA.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of SERA proteins. The parasite cells (Honduras-1) at trophozoite and schizont stages were prepared as described under "Experimental Procedures." Parasite cells were subjected to 8% SDS-PAGE (5 × 106 cells/lane) under reducing and nonreducing conditions. The blotted membrane was reacted with each of the HiTrap Protein G column-purified antibodies. Lane P, preimmune IgG (2 µg/ml); lane 1, anti-SE3N IgG (2 µg/ml); lane 2, anti-SE4N IgG (1 µg/ml); lane 3, anti-SE47' IgG (0.5 µg/ml); lane 4, anti-SE6N IgG (1 µg/ml); lane M, molecular marker.

Localization of the expressed SERA3, -4, -5, and -6 proteins was examined by indirect immunofluorescence assay with trophozoite and schizont stage parasite-infected erythrocytes. The rabbit anti-SE3N, -SE4N, or -SE6N serum and mouse anti-SE47' antibody were used. As shown in Fig. 7, all of the SERA proteins examined were detected in an indirect immunofluorescence assay. They appeared to be similarly localized as SERA5 protein in the parasitophorous vacuole. The similar staining patterns were observed with two other parasite strains, K1 and FCR3 (data not shown). The results also confirmed the previous report that SERA6 was in the parasitophorous vacuole (20). All of the single parasite cells in the field so far inspected were co-stained by anti-SE47' antibody and either anti-SE3N, -SE4N, or -SE6N antiserum, indicating that every single cell expresses all the proteins examined.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   SERA3-6 genes are expressed in single parasites at similar localization. Trophozoite- and schizont (Honduras-1)-infected erythrocytes were purified by Percoll and subjected to immunofluorescence staining with rabbit anti-SE3N, -SE4N, or -SE6N antiserum and mouse anti-SE47' IgG. The secondary antibodies used were Cy3-conjugated anti-rabbit IgG and fluorescein isothiocyanate-conjugated anti-mouse IgG. 4',6'-Diamidino-2-phenylindole was also used to stain parasite nuclei.

Immunogenicity of SERA3, SERA4, SERA5, and SERA6 Proteins-- Since SERA3, SERA4, SERA5, and SERA6 proteins were co-expressed, we intended to compare the human immune responses against each of their N-terminal domains. ELISA titers of individual sera from 40 adults living in a malaria holoendemic area of Uganda were tested with recombinant SE3N, SE4N, SE47', SE6N, and MSP-1₁₉ proteins (Fig. 8). Transcription of the SERA6 gene is least among the SERA genes examined (Figs. 2-4); however, a relatively large population (97%) responded to SE6N, comparable with responders against SE4N and MSP-1₁₉. In contrast, the population responding to SE47' was smaller than that responding to SE4N, SE6N, and MSP-1₁₉ and was comparable with that responding to SE3N, although transcripts from the SERA5 gene are most abundant among the SERA multigene family (Figs. 2-4). The population of high responders (ELISA titer >1000) to SE47' and MSP-1₁₉ were 38 and 80%, respectively. We did not observed any correlations between responses against any two antigens (e.g. SE47' versus MSP-1₁₉ in Fig. 8F), suggesting that the immune response against each antigen is independent.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Antibody titers against SERA5 N-terminal region but not against other SERA protein correlate with serum inhibition of in vitro parasite growth. ELISA titers in 40 individual Ugandan sera were determined against the recombinant SE3N (A), SE4N (B), SE47' (C), SE6N (D), and MSP-1 (E) proteins. The threshold of ELISA titer was 0.2 at 413 nm. The serum inhibition of parasite growth was assayed by adding each serum sample to the parasite culture at a final concentration of 5% (v/v). Parasite growth inhibition (%) was calculated as described under "Experimental Procedures." Parasite strains K1, Honduras-1, and FCR3 were used for the growth inhibition assay, and the mean values are presented with S.D. F, reciprocal ELISA titer plot against recombinant SE47' and MSP-1 proteins. Correlation coefficients (r) and the p values are indicated in parentheses where a p value under 0.05 was obtained. N.S., the correlation was not statistically significant.

It has been reported that the IgG antibody level to SE47' is negatively correlated with a blood parasitemia in a group of children in a malaria holoendemic area (17), suggesting that the serum of a group of children, in which the IgG titer against SE47' is high, retains the antiparasitic activity. We examined whether the serum from 40 Ugandan adults inhibits in vitro parasite growth. Individual human serum was added to the parasite culture to a final concentration of 5%. The averages of the growth inhibition values in three independent experiments with Honduras-1, K1, and FCR3 parasite strains were plotted against ELISA titer to each recombinant protein. As expected, a statistically significant correlation of ELISA titers to SE47' and growth inhibition was observed (r = 0.785, p < 0.0001). However, ELISA titers to neither SE3N, SE4N, nor SE6N were significantly correlated to the serum inhibition of parasite growth. The correlation of anti-MSP-1₁₉ titers was much less significant (r = 0.329, p = 0.038) than that of anti-SE47' titers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Multigene families function to produce a variety of antigenic molecules in the pathogenic organism that can be recognized by the immune system of the vertebrate host. Whether or not the SERA multigene family shows antigenic variation, we have analyzed the expression of the SERA multigene family. Transcriptional profile among the SERA multigene family measured by RT-PCR with limited PCR cycles (Fig. 2), the Northern blot analysis (Fig. 3), and the real time quantitative PCR (Fig. 4) consistently showed that the abundance of SERA5 RNA was always higher than those of other SERA genes and was in 3-5-fold excess of MSP-1 RNA. This observation is in good accord with the previous report that SERA5 cDNA copies were abundant, since 0.5-1.5% of clones are SERA cDNAs in the cDNA library constructed from poly(A) RNA of erythrocytic stage P. falciparum parasites (38).

Almost all of the SERA multigene family genes examined here were most actively transcribed in trophozoite and schizont stages (Fig. 5). The relative abundances of SERA1 to SERA8 transcripts were constant through independent RNA preparations and well conserved in all strains examined; however, those of MSP-1 and SERA9 were not (Figs. 4 and 5). The timing of transcriptional activation of SERA1-8 genes appeared a little earlier than that of the MSP-1 gene (Fig. 5). This fact might influence the observed findings that the relative abundance of MSP-1 RNA was changeable in the RNA preparations, since RNA were prepared from mixtures of different populations of trophozoite and schizont stage parasite cells. The relative abundance of SERA9 RNA was also changeable in RNA preparations, particularly in strains (Figs. 4 and 5). These observations suggest that SERA1-8 genes in a cluster on chromosome 2 might be under the same transcription regulation.

The transcription activities of SERA1, -2, -7, and -8 genes in a peripheral location of the cluster were much lower than those of the centrally located SERA3, -4, -5, and -6 genes (Figs. 2-5). The accompanying paper (39) demonstrated that SERA1, -2, -3, -7, and -8 genes were dispensable by constructing knockout parasites. In addition, sequence analysis of the SERA8 gene from several P. falciparum strains revealed that several stop codons appear in the coding frame for the SERA8 open reading frame based on 8 of 10 strains.² SERA genes in a peripheral location of the cluster might be remnants of gene duplications that probably generated the SERA multigene family.

Since RNA production does not assure protein production, we have prepared antibodies against N-terminal regions of SERA3, -4, -5, and -6 genes that had significant transcriptional activities (Figs. 2-5). In Western blot of the trophozoite and schizont stage parasite cell lysate, the antibodies prepared detected each protein with the expected full-length size (Fig. 6). Although we have constructed a recombinant protein of N-terminal region of SERA9, the hydrophobic nature of the protein did not allow us to do further characterization. Our results are consistent with the previous report that SERA5 and SERA6 proteins were expressed (20). More importantly, it was revealed that SERA3, -4, -5, and -6 proteins were co-expressed in every single parasite cell found under every microscopic field examined by indirect immunofluorescence assay (Fig. 7). The finding that each parasite cell produces all SERA proteins examined demonstrates that a gene expression switching for antigenic variation does not take place in the SERA multigene family.

We also found that SERA3, -4, and -6 proteins were localized in the parasitophorous vacuole of trophozoite and schizont stage parasite-infected erythrocytes similar to SERA5 (Fig. 7). The facts that processing of SERA5 precedes parasitophorous vacuole and/or erythrocyte membrane rupture (8) and that the processed fragment, P50, contains a protease motif suggest a possible role of SERA5 in schizont rupture to release free merozoites. SERA4 was also processed into smaller fragments in the schizont stage, and the N-terminal domain was found in the merozoite (data not shown). Although the processing and localization of processed fragments of SERA6 needs to be studied, these findings and the observation that SERA4, -5, and -6 gene knockout parasites could not be obtained (39) suggest that the existing SERA4, -5, and -6 genes are not for exerting redundant roles, but rather, each gene product plays a distinguishable role in the parasite, possibly as a component in a protease cascade.

Our previous work (17) showed an epidemiological correlation between the antibody titer against the SERA5 N-terminal region and apparent malaria fever and blood parasitemia. Here we have shown that antibody titer against SERA5 positively correlated with the serum inhibition of parasite growth (Fig. 8). This observation is a more direct evidence for the antibodies against SERA5 N-terminal to be protective against malaria and strongly supports the N-terminal domain of SERA5 as a malaria vaccine candidate antigen. SERA3, -4, and -6 are highly immunogenic in humans; however, antibody titers against SE3N, SE4N, or SE6N did not correlate to the serum inhibition of the parasite growth. These recombinant proteins may not represent possible protective epitopes in SERA3, -4, and -6. It was reported that antibodies against MSP-1₁₉ were an important component of the inhibitory activity of immune human sera; however, total MSP-1₁₉ antibodies as measured by ELISA did not correlate to MSP-1₁₉-specific invasion inhibitory antibodies (40). Consistent with their results, total antibody titer against MSP-1₁₉ did not correlate well to the serum inhibition of parasite growth in the present study.

As discussed above, the SERA multigene family does not function as a gene resource for antigenic variation that could be a possible immune evasion mechanism. It is noteworthy that in Ugandans the proportion of high responders to SERA5 was lower than those to recombinant proteins of SERA3, -4, or -6 or MSP-1, although RNA expression of the SERA5 gene was highest among the SERA family and MSP-1 genes. In addition, SERA3, -4, -5, and -6 proteins appeared in a similar location in the parasitized erythrocytes, suggesting that the presentation of these antigens to the human immune system is in a similar way. Human immune response against SERA5 protein might be suppressed by unidentified mechanisms. This possibility, however, does not undermine the value of the SERA5 N-terminal domain as a promising malaria vaccine candidate, since the immune responses to artificial immunization would be very different from those to the pathogen.

	ACKNOWLEDGEMENTS

We thank Dr. Brendan S. Crabb and colleagues for sharing unpublished results prior to publication. We also thank Drs. Masatsugu Kimura (Osaka City University) and Wim G. J. Hol (University of Washington) for providing P. falciparum K1 strain and RIG plasmid, respectively. We thank the scientists and funding agencies comprising the international Malaria Genome Project for making sequence data from the genome of P. falciparum (3D7) public prior to publication of the completed sequence. The Sanger Centre (UK) provided sequence for chromosomes 1, 3-9, and 13, with financial support from the Wellcome Trust. A consortium composed of the Institute for Genome Research, along with the Naval Medical Research Center, sequenced chromosomes 2, 10, 11, and 14, with support from NIAID (National Institutes of Health), the Burroughs Wellcome Fund, and the Department of Defense. The Stanford Genome Technology Center sequenced chromosome 12, with support from the Burroughs Wellcome Fund. The Plasmodium Genome Database is a collaborative effort of investigators at the University of Pennsylvania and Monash University (Melbourne, Australia), supported by the Burroughs Wellcome Fund.

	FOOTNOTES

^* This work was supported by Grant-in-Aid for Scientific Research (A) 13357002 and Grant-in-Aid for Scientific Research on Priority Areas 13226058 (to T. H.) from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. This work also received financial support from the United Nations Developmental Program/World Bank/World Health Organization/Special Program for Research and Training in Tropical Diseases (TDR).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-6-68798280; Fax: 81-6-68798281; E-mail: horii@biken.osaka-u.ac.jp.

Published, JBC Papers in Press, September 18, 2002, DOI 10.1074/jbc.M207145200

² R. Nakajima and T. Horii, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: SERA, serine repeat antigen; gDNA, genomic DNA; RT, reverse transcription; MSP-1, merozoite surface protein-1; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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