©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Anchoring of an Immunogenic Plasmodium falciparum Circumsporozoite Protein on the Surface of Dictyostelium discoideum(*)

Christophe D. Reymond (1), Carole Beghdadi-Rais (2), Mario Roggero (2), Elizabeth A. Duarte (2)(§), Chantal Desponds (2), Michel Bernard (2)(¶), Dorinne Groux (1), Hugues Matile (3), Claude Bron (2), Giampietro Corradin (2), Nicolas J. Fasel (2)(**)

From the (1) Institute of Histology and Embryology, University of Lausanne, Rue du Bugnon 9, 1005 Lausanne, the (2) Institute of Biochemistry, University of Lausanne, chemin des Boveresses 155, 1066 Epalinges, and (3) Hoffman La Roche Ltd., Pharma Research Department, Basel 4002, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.


INTRODUCTION

The development of safe and effective vaccine is important to control diseases such as malaria, which kills 1-2 million persons (mostly children) per year. Vaccination with irradiated Plasmodium sporozoites, the infectious stage inoculated by mosquitoes, confers protection against malaria infection in mammals (1-4). This protection is mediated by specific T lymphocytes (helper and/or cytotoxic) and antibodies directed against the immunodominant repeat region of the circumsporozoite protein (CSP),() an abundant surface protein. However, the preparation of sporozoite-based vaccines has been hampered by the difficulty in obtaining sufficient amounts of sporozoites. Thus, it is important to develop heterologous expression systems allowing for large scale production of properly processed recombinant antigens. In previous studies, parts of the CSP were produced in various systems and shown to elicit both antibodies and T cells which mediated partial protection (1, 5) . Production of a complete protein, in either soluble or membrane-bound form, should enhance both immunization and protection potential.

The first 17 amino acids of the CSP, as deduced from the cDNA sequence (6), constitute a putative signal sequence essential for translocation into the parasite endoplasmic reticulum (ER). Two regions (I and II) conserved in different plasmodia species (7) flank a large central repeat domain. Region II is essential for the binding to hepatocytes (8) and is followed by an hydrophobic C terminus possibly involved in membrane anchoring. The Plasmodium falciparum CSP repeat domain, consisting of about 40 Asn-Ala-Asn-Pro (NANP) units, is an immunodominant B cell epitope (6, 9) . The repeat domain, together with carrier molecules, was used as subunit vaccine but conferred only limited protection (10, 11) . Elements important for CD4 and CD8 T cell responses have been mapped outside the repeat region, indicating that the entire CSP or segments thereof could be used to improve vaccine efficacy (1, 5, 12) . Moreover, partial or complete protection against parasite infection was obtained in mice injected with an attenuated strain of Salmonella typhimurium expressing the Plasmodium berghei CSP gene (13, 14) , with a mixture of mastocytoma P815 cells transfected with the Plasmodium yoelii CSP, or the sporozoite surface protein 2 (15) . Full-length or partial P. falciparum recombinant soluble CSP have been produced using different expression systems, but production of a complete CSP was often limited by a low level of expression, or endogenous proteolytic cleavages (16, 17, 18) . The role of the hydrophobic CSP C-terminal domain remains unclear, despite its resemblance to a GPI anchor addition region. No GPI modification of CSP has yet been described in vivo, even though CSP expressed in COS cells seems attached to the membrane by a GPI anchor (19). We, therefore, decided to obtain cell surface expression of CSP and analyze its immunogenicity in mice.

Advances in molecular genetics have allowed to reintroduce in vitro modified genes within D. discoideum, thus enabling heterologous proteins expression (21) . We decided to express the P. falciparum CSP in Dictyostelium due to the resemblance in the high AT content of the two genomes. We choose two Dictyostelium discoideum promoters for expressing CSP which are induced at different developmental stages. The gene encoding the lectin-binding protein discoidin I is expressed either early after starvation, when cells have been grown on bacteria (22) , or during vegetative growth when cells are grown on a semi-synthetic medium (HL-5) (23) . The Dd ras D gene from Dictyostelium, which encodes a protein resembling mammalian ras oncogenes (24) , is not transcribed during growth, and its expression requires starvation and extracellular cAMP, thus allowing essentially conditional expression (25) . In previous work, we showed CSP accumulation in Dictyostelium using such promoters (26) . In this work, we obtained secretion of an almost complete CSP, as well as cell surface expression of a GPI-modified version. Finally, we show in this study that cells presenting CSP at their surface are capable of eliciting antibody response in mice.


MATERIALS AND METHODS

Chemicals and Cell Cultures

Bacterial media and current cloning procedures are described (27) . (NANP) peptide was obtained from Dr. F. Sinigaglia (Hoffman La Roche Ltd., Basel, Switzerland). Dictyostelium cells were cultured in shaking suspensions in HL-5 media and starved in PDF (28) . Electroporation and selection of expressing cells were done as described (21, 29) . For discoidin I promoter-dependent expression, D. discoideum cells were starved for 4 h in shaking suspension (160 rpm) in PDF at a density of 5 10/ml unless otherwise stated (26) . For ras promoter constructs, the cells were starved for 6 h in PDF at about 5 10/ml and transcription was induced by addition of 200 µM cAMP for 1 h unless otherwise stated (25) .

DNA Constructs

To obtain expression of different forms of CSP in D. discoideum, we used the vectors pVEII (discoidin I promoter) (30) and pERI (ras promoter) (26) . The construction of both pEDII-CS and pERI-CS based on the CSP gene isolated of P. falciparum NF54 strain has been described earlier (26) . New constructs are shown in Fig. 1A. The leader peptide of CSP was replaced by a leader peptide of D. discoideum, the CsA leader peptide (31) (Fig. 1B). The natural stop codon of the CSP was changed to UAA by replacing most of the CSP coding region by a DNA fragment amplified using specific oligonucleotides carrying adequate restriction sites. The 3` amplimer contained an in-frame UAA instead of the UAG creating construct pEDII-CS 49. The secreted form of the CSP (CSP-150) was obtained by deleting the 69 nucleotides encoding the last 23 amino acids of the C-terminal segment. The construct pEDII-CS 150 was obtained by replacing most of the CSP coding region by an amplified fragment lacking the C-terminal segment encoding information but carrying useful restriction fragment for insertion. The hepatocyte binding sequence (part of conserved region) (8) is present on the polypeptide. The last amino acid is now a serine (position 310) (32) following the fourth cysteine of CSP. To express a GPI-anchored form, we modified the pERII vector, which contains the ras promoter, by adding a multiple cloning site, the actin 6 termination sequence, and the actin 15 neo cassette.() A DNA fragment encoding the last 40 amino acids of the CsA sequence corresponding to the GPI addition recognition sequence was obtained by polymerase chain reaction amplification and cloned between the EcoRV and XhoI in the multiple cloning site of pERII. A DNA fragment comprising the CSP gene but excluding the N-terminal signal peptide and the C-terminal hydrophobic encoding segments was amplified and inserted into the modified pERII vector between the Asp718 and EcoRV sites, leading to pERIV-CS. The region of fusion between the amino acid sequence CSP and the CsA C-terminal segment is shown in Fig. 1C.


Figure 1: Circumsporozoite protein expression vectors. A, pEDII is derived from pVEII (30) and contains the Tn903 neoR gene between an actin 15 promoter and termination sequences, allowing G418 selection in D. discoideum (dottedline). CSP lacking its first 18 amino acids was fused in-frame to the CsA leader peptide (see panelB) leading to pEDII-CS 2. Replacement of the original P. falciparum UAG to UAA resulted in construct pEDII-CS 49. Removal of the last 23 amino acids comprising the hydrophobic C-terminal peptidic region lead to pEDII-CS 150. pERIV-CS was derived from pERII (see ``Materials and Methods''), which contains the Tn5 neoR gene between actin 15 promoter and termination sequences. The CSP and CsA signal peptides were placed behind the D. discoideum ras promoter fragment 3.1 (25). The GPI anchoring domain of the CsA protein was placed in frame at the C terminus of CSP (see panelC). L, CsA leader peptide; CSP, P. falciparum circumsporozoite surface antigen; DiscoidinI and ras, sequences promoting transcription in Dictyostelium. Arrows indicate transcription start sites. I, II, and III indicate highly conserved domains of the CSP (6). B, the discoidin I promoter in the pEDII series was fused to a D. discoideum contact site A (CsA) synthetic leader peptide keeping the original discoidin I AUG (M). This sequence differs from the original sequence (31, 49) by the replacement of the lysine in position 2 by serine and arginine. An asterisk indicates the probable cleavage site of the CsA leader peptide. The amino acids of the P. falciparum CSP are underlined. C, in pERIV-CS, the CSP hydrophobic domain was replaced by the last 48 amino acids of the CsA protein. An extra proline (P) was added during cloning between the CSP (underlined) and CsA sequences.



Protein Analysis

The proteins from 2 10 cells (per 4-mm-wide slot), boiled in 1 Laemmli buffer for 5 min, were separated by 10% SDS-PAGE (27) . Proteins were electrotransferred onto nitrocellulose (immunoblots). Fifty µg/ml anti-NANP mAb (Sp3E9) (33) was added to the filter and incubated overnight at room temperature. I-Protein A or alkaline phosphatase-conjugated protein A and chemiluminescence reaction (Amersham Corp.) were used to reveal anti-NANP binding. Partially purified CSP was obtained from Triton X-114-soluble proteins (34) loaded on an anti-NANP affinity column (26) . In purity assays, CSP synthesizing D. discoideum cells were pulse-labeled by [S]methionine for 2 h after an initial starvation period of 2 h (35) .

For protein sequencing, 10 liters of cells grown in HL-5 up to 10/ml were starved for 4 h, lysed in 200 ml of Triton X-114 (34) , and phase separated. Four volumes of a solution containing 25 mM Tris-HCl, pH 8.2, 50 mM NaCl, 0.5% Nonidet P-40, and 0.5% deoxycholic acid were added to the detergent fraction, and the samples were passed over an anti-NANP immunoaffinity column. The material was eluted in glycine, pH 2.5, and lyophilized. The sample was loaded on a 10% SDS-PAGE and, after Coomassie staining, the 62-kDa band was eluted from the gel using an elution device from Life Technologies Inc. The eluted material was concentrated using a Centricon filter, lyophilized, and resuspended for amino acid sequencing.

FACS Analysis

Fluorescence-activated cell sorter (FACS) analysis was performed on approximately 1 10 pERIV-CS and control Ax2 cells. After starvation and cAMP induction, the cells were centrifuged at about 1,000 g and resuspended in 200 µl of medium containing 5% fetal calf serum. Cell suspensions were incubated for 45 min at 4 °C with a 1:500 dilution of Sp3E9. Cells were washed by centrifugation through a cushion of fetal calf serum (FCS), resuspended in 110 µl of medium containing 5% FCS and 10 µl of commercial biotin-conjugated donkey anti-rabbit IgG (Amersham). After a 45-min incubation at 4 °C, the cells were washed and resuspended in 100 µl of medium containing 5% FCS. Finally, cells were incubated for 1 h at 4 °C with 10 µl of fluorescein-conjugated streptavidin (Amersham), washed, and analyzed on a fluorescence-activated cell sorter (FACS II System, Becton Dickinson).

GPI-Phospholipase D Assay

The GPI-PLD sensitivity of GPI-anchored CSP was tested by lysing pERIV-CS cells in 20 mM Tris/HCl, pH 7.5, 0.1 M CaCl, 0.008% Triton X-100 by four cycles of freezing and thawing. One or 5 units of GPI-PLD enzyme (Boehringer Mannheim) was added and the extracts incubated for 1 h at 37 °C. Triton X-114 in 1 Tris-buffered saline containing 1 mM EDTA was then added to a final concentration of 1%, and the aqueous and detergent phases were separated. The samples were resolved on a 10% SDS-polyacrylamide gel and analyzed by immunoblotting using the Sp3E9 monoclonal antibody as described previously.

Immunofluorescence Staining

5 10 cAMP-induced starved pERIV-CS cells were preincubated for 5 min in 200 µl of 1% skim milk in 1 PBS and kept at 4 °C. After centrifugation at 1,000 g for 1 min., the cells were resuspended in 200 µl of 50 µg/ml Sp3E9 antibody in 1% skim milk and incubated for 30 min. After centrifugation, the cells were rinsed three times in 1 PBS. The cells were then resuspended in 200 µl of fluorescein isothiocyanate-conjugated anti-mouse antibody (diluted 1:40) (Nordic, DAKO Immunoglobulin) and incubated for 30 min. After three washes in 1 PBS, the cells were mounted for microscopy and observed under a Leitz microscope.

ELISA

Serum and monoclonal antibodies produced against the N-terminal (amino acids 22-125), the NANP repeat peptide, or the C-terminal segment peptides were assayed by ELISA. Briefly, vinyl plates were coated with different peptides, washed, and blocked with 1% bovine serum albumin in PBS. Monoclonal or serum antibodies were serially diluted in 1% bovine serum albumin/PBS containing 0.05% Tween 20. Diluted sera were added to antigen-coated wells and incubated for 1 h at room temperature. Plates were washed with PBS with 0.05% Tween 20, and an appropriate dilution of peroxidase-conjugated, species-specific anti-IgG was added and incubated for 1 h at room temperature. One hundred microliters of peroxidase substrate solution were added to each well, and the A was determined. The end point of ELISA titers for the mice sera was designated to be the serum dilution producing an absorbance value 2 S.D. greater than the average of the control mice.

Immunization of Animals and Analysis of the Antisera

Two 25 µl or 1 50 µl of a 1:1 sonicated mixture of incomplete Freund's adjuvant and 2 10 cells were injected into BALB/c mice either subcutanously or intraperitonally, respectively. After 4 weeks, a boost was performed with an equivalent material, and sera were collected 10 days afterward and analyzed by ELISA. The same antisera were analyzed by immunofluorescence on air-dried, unfixed sporozoites of isolates NF54 using fluorescein-labeled rabbit anti-mouse IgG as second antibody (36) .


RESULTS

A Dictyostelium Leader Peptide Is Required for CSP Expression

The genome of the P. falciparum malaria parasite is particularly rich in adenosine and thymidine resulting in a biased codon usage, reminiscent of the slime mold D. discoideum(37) . Thus, we expressed the Plasmodium CSP gene in D. discoideum under the discoidin I promoter, which was selected to take advantage of its high level of expression both during vegetative growth and early starvation (23) . When merging the complete CSP coding region with the discoidin I promoter up to the AUG (pEDI-CS, Fig. 1), no recombinant protein was detectable, even though we detected CSP mRNA in the transfected cells (data not shown). We questioned the ability of the first 16 amino acids of the CSP to allow translocation through the ER in D. discoideum, since alterations of leader peptide sequences have been shown to affect dramatically the level of protein synthesis in other systems (38) . We thus replaced the CSP leader peptide encoding region by a synthetic oligonucleotide sequence based on the D. discoideum contact site A leader peptide (31) (pEDII-CS, Fig. 1B). Fig. 2A shows the detection of CSP using a monoclonal antibody (Sp3E9) against the NANP repeat motif. A 62-kDa protein was detected in pEDII-CS cells (Fig. 2A, lanec) but not in cells containing either the vector pVEII alone (Fig. 2A, lanea) or pEDI-CS (Fig. 2A, laneb).


Figure 2: Immunoblot analysis of CSP produced in D. discoideum cells. A, cells were transformed with the expression vector pVEII alone (P1, lane a), the expression vector pEDI-CS (C1B, laneb), and the expression vector with the contact site A leader peptide pEDII-CS (CSP, lanec). Stably transformed cells were harvested after 4 h of starvation to ensure optimal transcription from the discoidin I promoter and CSP was revealed by immunoblotting using (NANP) monoclonal antibody Sp3E9. The apparent molecular mass of the CSP (62 kDa) was estimated using molecular mass standards. B, proteins from pVEII-CS cells were analyzed as in A, except that the membrane strips were probed with an anti-NANP monoclonal antibody preincubated for 10 min at room temperature in presence of (NANP) peptide. Numbers at bottom of the figure represent molar ratios of (NANP) to mAb (e.g.1:1 in lanea). Lanee, no (NANP) peptide added. Molecular mass standards were as follows: phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg white ovalbumin (42.7 kDa).



Although both D. discoideum and P. falciparum share an AT-biased preferential codon usage, the CSP stop codon is UAG, whereas UAA is used in the vast majority of the Dictyostelium genes. We replaced the UAG stop codon of the CSP gene by a UAA codon (pEDII-CS 49), which resulted in enhanced CSP accumulation (data not shown). This strain was used in further studies.

To confirm the nature of the produced polypeptide and exclude cross-reactivity with other epitopes, we inhibited the binding of the monoclonal antibody Sp3E9 with synthetic (NANP) peptide. A hundredth equimolar amount of (NANP) peptide was sufficient to observe a significant reduction in the binding of the monoclonal antibody to the 62-kDa CSP (Fig. 2B, lane c), whereas complete inhibition was obtained with a tenth equimolar amount (Fig. 2B, lane b).

These results demonstrate that leader peptides are not interchangeable between P. falciparum and D. discoideum and suggest that the presence of an inadequate leader peptide dramatically affects the expression of heterologous proteins.

Integrity of the CSP Expressed in Dictyostelium

As a first purification step, we took advantage of the presence of a C-terminal hydrophobic segment (32) , which should allow the partitioning of the protein in Triton X-114. Indeed, most of the newly synthesized CSP accumulated in the detergent phase (data not shown). To monitor the subsequent purification steps, cells were pulse-labeled with [S]methionine (35) . Triton X-114-soluble proteins were loaded on an anti-NANP affinity column. After elution a major [S]methionine-labeled protein with an apparent molecular mass of 62 kDa was detected by fluorography only in CSP-synthesizing cells (data not shown).

Considering that in other expression systems, CSP was sensitive to proteolytic degradation, we partially sequenced CSP produced in D. discoideum. Immunopurified CSP was gel eluted and subjected to amino acid sequencing. The N terminus of the protein varied between two independent preparations, but was located within the first 25 amino acids downstream of the signal peptide cleavage site. This indicates the presence of exopeptidase activity rather than of a specific protease-sensitive site in the CSP polypeptide as reported in other expression systems (16, 17, 18) . Due to difficulties encountered during the sequencing of other Dictyostelium proteins isolated in parallel, we suspect the presence of a protected N terminus on the completed size CSP.

Secretion of CSP into the Medium

The presence of the leader peptide, as well as of the C-terminal segment of CSP resembling GPI anchor recognition sequences should have resulted in cell surface expression of modified CSP. However, we only detected CSP within the D. discoideum cells, and not at the cell surface or secreted in the medium (data not shown). We treated pEDII-CS 49 extracts with GPI-PLD (Fig. 3A) to cleave the lipid moiety of the GPI anchors. We used GPI-PLD instead of GPI-PLC, since the GPI anchor of contact site A contains a ceramide in its lipid moiety (39) , which prevents its cleavage by most PLCs. Removal of the lipidic portion of the anchor should have altered its partitioning in Triton X-114. No change in partitioning could be observed upon GPI-PLD treatment (Fig. 3A), indicating that CS-49 is probably not modified by a GPI in D. discoideum.


Figure 3: Intracellular and secreted forms of CSP. A, cells producing CSP-49 (49) or CSP-150 (150), the protein encoded by pEDII-CS 150 (Fig. 1A), were lysed in 1% Triton X-114 and subjected to the GPI-PLD present in human serum (lanes +) or mock-incubated (lanes -) for 5 min at 30 °C in presence of protease inhibitors. The samples were adjusted to 2% Triton X-114 and phase separated. Proteins partitioning into the detergent (TX) or aqueous (Aq) phases were separated by SDS-PAGE, blotted, and reacted with the Sp3E9 antibody. B, cells producing CSP-49 (49) or CSP-150 (150) were centrifuged at 10,000 g and lysed, and then proteins were separated by SDS-PAGE (about 2 10 cells/lane). An equivalent amount of medium supernatant was precipitated by adding four volumes of acetone. After centrifugation at 10,000 g, the pelleted proteins were separated by SDS-PAGE electrophoresis (medium). After transfer to nitrocellulose, multiple strips were cut out of each lane and the CSP was revealed using the Sp3E9 antibody.



The non-processing of GPI anchors resulted in intracellular retention in other systems (40, 41, 42, 43) . We thus deleted the C-terminal 23 amino acids of CSP encompassing the putative GPI anchoring sequence, leaving the last cysteine residue (residue 309) followed by a serine to avoid possible misfolding. The modified coding region was inserted in pVEII and the resulting plasmid (construct pEDII-CS 150) introduced into D. discoideum cells. The CS-150 protein, on the contrary to CS-49, was detected solely in the aqueous phase after Triton X-114 partitioning (Fig. 3A). GPI-PLD treatment had no effect on the protein, as expected. Furthermore, the CS-150 protein was secreted into the culture medium as seen by immunoblotting after acetone precipitation (Fig. 3B). It should be noted that we were unable to precipitate CS-150 from the medium using trichloroacetic acid (10%), indicating the extreme acid solubility of the CSP (data not shown).

Expression of CSP on the Dictyostelium Cell Surface

For the development of a live vaccine, diagnostic test, or hepatocyte binding studies, we wished to express CSP on the surface of D. discoideum. We thus replaced the CSP C-terminal hydrophobic segment by the last 49 amino acids of the contact site A (CsA) polypeptide containing a GPI-anchoring domain (31) . To obtain inducible expression and to avoid possible toxicity problem, we inserted the CSP/CsA fusion gene under the control of the ras promoter (25, 26). The construct was introduced into D. discoideum cells (pERIV-CS) and CSP surface expression was analyzed by immunofluorescence using anti-CSP antibodies (Fig. 4). ras promoter expression is induced by addition of external cAMP in only 20-40% of the cells, which are those differentiating into prestalk cells (24) . Consequently, we observed CSP/CsA in a restricted number of cells (Fig. 4). The staining was clearly restricted to the cell periphery (arrow) and the punctuated appearance indicated the presence of patches of CSP on the cell surface (arrowhead) as seen in the sporozoite of P. falciparum.


Figure 4: Cell surface expression CSP in Dictyostelium. pERIV-CS cells were starved for 6 h and induced for 1 h with 200 µM cAMP. The cells were reacted with anti-(NANP) antibody, followed by fluorescein-conjugated anti-mouse IgG at 4 °C to prevent phagocytosis and pinocytosis. A restricted number of cells are stained, corresponding to the prestalk cells. Prespore cells can be considered as negative control. A, phase contrast. B, fluorescence. The bar represents 50 µm.



This result was confirmed by flow cytometry using different antibodies. First, we used the mAb Sp3E9 directed against the NANP epitope (Fig. 5A). Cells transformed with the vector alone (P1) showed no fluorescence over background, whereas pERIV-CS cells showed a second fluorescence peak corresponding to about 40% of the cells (Fig. 5C). When cAMP addition was omitted, the second peak mostly disappeared, indicating that the expression of CSP was cAMP-inducible. In contrast, no signal could be detected with a mouse antiserum directed against a peptide of the N terminus of CSP corresponding to amino acids 22-125 (data not shown). To exclude that the absence of recognition was due to a proteolytic cleavage of the CSP N terminus, we performed immunoblots. As seen in Fig. 6A, a 69-kDa CSP was detected in pERIV-CS cells by human serum of infected patients (B10), and mouse antisera raised against peptide 22-125 or peptide 289-390. This result suggests that the CSP at the surface of D. discoideum cells adopts a configuration that influences its recognition by specific antibodies.


Figure 5: Surface fluorescence labeling. Labeled cells were analyzed using a fluorescence activated cell sorter. Fluorescence intensity in arbitrary units (x axis) is represented against the number of cells (y axis). PanelA corresponds to pVEII-transfected cells (P1) after cAMP induction, panelB to CSP-expressing cells grown in absence of cAMP, and panelC to CSP-expressing cells in presence of cAMP.




Figure 6: Integrity and phospholipid modification of CSP expressed at the surface of D. discoideum cells. A, total cell proteins were analyzed as described in Fig. 2, except that the human antiserum (B10), a mouse antiserum against residues 22-125 of CSP, or a mouse antiserum against residues 289-390 was used instead of anti-NANP antibody. B, proteins extracted from about 2 10 cells were treated by GPI-PLD with 1 (lanes1) or 5 units for 1 h (lanes5) and analyzed as described in Fig. 3A. Aq, aqueous phase; Tx, Triton X-114 detergent phase. 0, samples not treated by GPI-PLD.



Finally we asked how the CSP might be anchored in the membrane. The CSP/CsA protein has the amphiphilic character expected for a GPI-anchored protein (34, 40) since it partitions in the Triton X-114 detergent phase (Fig. 6B). To confirm the presence of a GPI anchor on the CSP/CsA fusion protein, we lysed the cells by three cycles of freezing and thawing in 0.008% Triton X-100 and treated the cell lysates for 1 h with 1 or 5 units of with GPI-PLD. Removal of the lipidic portion of the CSP/CsA with 5 units of GPI-PLD altered its hydrophobic character and provoked its partitioning into the aqueous phase (Fig. 6B, lane5). Incubation with 1 unit had a reduced effect (Fig. 6B, lane1 and data not shown). These results show the presence of GPI anchored CSP at the surface of Dictyostelium cells, thus indicating that D. discoideum can produce, transport, and process heterologous parasite proteins.

Immune Response to CSP Harboring Dictyostelium Cells

To assess the ability of the GPI-modified CSP to induce an immune response, BALB/c mice were immunized subcutanously or intraperitonally with 2 10 whole cells mixed with incomplete Freund's adjuvant. Ten days after a second injection, the humoral immune response was analyzed by ELISA against synthetic peptides from different regions of the CSP (). Antibodies were detected against the immunodominant NANP repeat region, against the C-terminal non-repetitive region (amino acids 289-390), but not against the N-terminal 22-125 synthetic peptide. Antibody titers did not vary as a result of the route of injection (), and no specific antibodies were detected following injection of live cells in absence of adjuvant (data not shown). Six of these antisera raised against CSP produced in Dictyostelium were further assayed for recognition of P. falciparum air-dried, non-fixed sporozoites. All mice sera positive for D. discoideum CSP were found to bind to the sporozoite as scored by immunofluorescence using fluorescein isothiocyanate-linked secondary anti-mouse IgG antibody. Furthermore, the limiting dilution for air-dried sporozoites resembled that measured using synthetic peptides in an ELISA () with a maximum of 1/5,000 for one serum.


DISCUSSION

With the challenging view of developing sporozoite-like cells with potential as live vaccine or in diagnostic tests, we expressed the CSP of P. falciparum at the surface of D. discoideum cells. D. discoideum shares an AT-biased preferential codon usage with numerous mammalian parasites and has a potential in biotechnology applications (44) . These reasons prompted us to define the various requirements for cell surface expression of a parasite antigen in Dictyostelium and to analyze the immune response toward this cell surface antigen.

As shown in this study, expression of CSP was successful only after replacement of its original signal peptide by a Dictyostelium derived sequence. The CSP signal sequence seems also non-functional in mammalian cells, since it had to be replaced by a mammalian leader peptide for expression in mouse mastocytoma P815 cells (45) . Although the sequences required for a peptide to function as a translocation signal have been well defined (46), it is not clear why some signals are better recognized and why signal peptides are in many instances not interchangeable between organisms (47) .

The CSP produced in D. discoideum showed an apparent molecular mass of 62 kDa (CS-49) either by silver staining, Ponceau, or metabolic labeling. The size of the CSP made in P. falciparum is about 50 kDa (6) , whereas the expected size based on the gene sequence is 43,340 Da. This difference in migration on SDS gels remains unclear, since no putative glycosylation sites or phosphorylation sites can be found on the protein sequence itself. Removal of the potential GPI anchoring site in CS-150, or GPI anchor addition due to the CsA sequence in pERIV-CS did not change the size of the protein to a large extent. These results can be compared to the situation in P. falciparum, where the apparent molecular weight of CSP is also larger than expected for still unknown reasons.

Quantitative estimation by ELISA indicates that CSP may represent between 0.03% and 0.3% of total D. discoideum proteins (data not shown). This is in the range obtained in yeast cells for the CSP of Plasmodium knowlesii(50) but is lower than the amount obtained for CSP produced with baculovirus in insect cells. The level of expression was increased by the replacement of the stop codon UAG with an ochre stop codon to facilitate translation termination. Most codons used by P. falciparum to encode the CSP are also preferred by Dictyostelium with the exception of the arginine codon (AGG) and of the UAG and UGA stop codons. Thus, additional changes in other codons could further improve the yield of the CSP in Dictyostelium.

CSP with its original C terminus did not accumulate at the surface of Dictyostelium cells, even though the protein is present at the surface of sporozoites. Parallel experiments (45) showed that a complete CSP with its C-terminal hydrophobic peptidic segment was not expressed at the surface of mastocytoma cells either. Instead, it was degraded intracellularly and specific peptides were presented in association with major histocompatibility class I molecules. These results are reminiscent of the intracellular retention of non-processed GPI anchored molecules as a result of either non-functional signal sequences or the use of GPI metabolism deficient cell lines (40, 41, 42, 43) . Even though no GPI anchor has been detected on CSP in vivo, the C-terminal hydrophobic region contains all amino acids required for such modification (51, 52, 53, 54, 55) and such post-translational modification has been reported, although at low level, in COS cells (19) . Systematic studies on interspecies requirements are necessary to define the structural requirements for the GPI anchor addition enzyme.

Dictyostelium cells are able to add GPI anchors, which are either cleavable by bacterial phosphatidylinositol-specific phospholipase C (e.g. PsA glycoprotein) (56) or not, due to the presence of a ceramide within the anchor (e.g. contact site A) (39) . Since both GPI-PLC and PLD treatment did not release a soluble form of CSP, we suspected that the CSP hydrophobic region was unable to function as GPI addition sequence in D. discoideum. Accordingly, labeling experiments using either inositol or ethanolamine (data not shown) failed to reveal any CSP modification. We circumvented this problem by removing the hydrophobic domain and fusing CSP to a sequence responsible for the addition of a GPI anchor to the contact site A protein in Disctyostelium (31). As a result, the CSP/CsA fusion protein accumulated at the cell surface. Furthermore, the GPI anchor was then cleavable by GPI-PLD, indicating proper modification of the CSP/CsA fusion protein expressed in D. discoideum. Recent evidence (57) shows that GPI anchoring tends to stabilize proteins at the surface of Dictyostelium cells. The patchy appearance of CSP by immunofluorescence is reminiscent of capping processes (58) . Even though we cannot fully exclude a staining procedure artifact, the patchy appearance may indicate a tendency of CSP to aggregate at the cell surface of D. discoideum, as it does at the parasite surface in P. falciparum.

Raising parasite-reactive antibodies using recombinant proteins or peptides as immunogens has been difficult, possibly due to differences between native and recombinant protein conformations (59) . The GPI-anchored CSP at the surface of Dictyostelium cells adopts a peculiar conformation since the protein is recognized by anti-NANP and by anti-C terminus but not by anti-N terminus antibodies raised against synthetic peptides. In addition, antibodies obtained by immunization with CSP-harboring Dictyostelium cells reacted with the NANP repeat, but not with the N-terminal synthetic peptide. Thus, the NANP repeats are most likely localized at the surface of the CSP produced in D. discoideum, whereas the N-terminal region could either be localized internally and/or be non-immunogenic in a H2 haplotype host. Additional experimental evidence is necessary to define if the protein produced in D. discoideum shows the same conformation as its native P. falciparum counterpart.

In conclusion, our ability to obtain membrane-bound CSP indicates that Dictyostelium is a promising system to express a variety of Plasmodium parasite proteins. The absence of a cell wall, as well as the relatively simple culture conditions (28) , renders D. discoideum suitable for large scale protein preparation of such immunogenic cell surface antigens. Producing a live vaccine using Plasmodium protein harboring Dictyostelium cells can now be envisaged, particularly since Dictyostelium is non-pathogenic and non-toxic, at least in mice as seen during the immunizations performed in this study. Furthermore, the low cost of growing and maintaining this organism may offer economic advantages for producing membrane-bound recombinant proteins.

  
Table: ELISA and immunofluorescence assay (IFA) reactivity of sera of pERIV-CS immunized mice with (NANP), M1(289-390) peptides, and air-dried fixed sporozoites

Sera were obtained 7 days after a single boost of BALB/c mice immunized with 2 10 cells.



FOOTNOTES

*
This work was supported by Swiss National Fund for Scientific Research Grants 31-9208.87 (to C. D. R.) and 31-36343.92 and 31-26320.89 (to N. J. F.), and by Grant FOR-383.90.2 from the Swiss Cancer League (to N. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Fogarty International fellowship.

Supported by RMF-Dictagene S.A.

**
Recipient of a Dr. Max Clotta Medical Research Foundation fellowship. To whom correspondence should be addressed. Tel.: 41-21-692-5732; Fax: 41-21-692-5705.

The abbreviations used are: CSP, circumsporozoite protein; CsA, contact site A; ER, endoplasmic reticulum; G418, geneticin sulfate; PAGE, polyacrylamide gel electrophoresis; PDF, pad dilution fluid; mAb, monoclonal antibody; GPI, glycosyl phosphatidylinositol; AT, adenosine thymidine; FACS, fluorescence-activated cell sorting; PLC and PLD, phospholipases C and D, respectively; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.

C. D. Reymond and D. Groux, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. G. Gerisch for advice, Dr. U. Certa for critical reading of the manuscript, Dr. P. Caspers for the NF54 CSP gene, Dr. A. Noegel for the CsA cDNA clone, Dr. F. Sinigaglia for the (NANP) peptide, Dr. L. Kühn for synthesizing oligonucleotides, M. Rousseaux and S. Pinaud for technical assistance, S. Frütiger for amino acid sequencing, P. Zaech and C. Knabenhans for FACS analysis, and P. Dubied and B. Allegrini for photographic work.


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