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INFECTION AND IMMUNITY, Aug. 1997, p. 3430–3437 0019-9567/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 65, No. 8

Development of Two Monoclonal Antibodies against Plasmodium falciparum Sporozoite Surface Protein 2 and Mapping of B-Cell Epitopes YUPIN CHAROENVIT,1* VICTORIA FALLARME,1 WILLIAM O. ROGERS,1 JOHN B. SACCI, JR.,1,2 MANJIT KAUR,1 JOAO CARLOS AGUIAR,1,3 LEO F. YUAN,1 GIAMPIETRO CORRADIN,4 ELLEN ANDERSEN,5 BENJAMIN WIZEL,1† RICHARD A. HOUGHTEN,6 AGGREY OLOO,7 PATRICIA DE LA VEGA,1 AND STEPHEN L. HOFFMAN1 Malaria Program, Naval Medical Research Institute, Bethesda, Maryland 20889-56071; Department of Microbiology & Immunology, University of Maryland at Baltimore, Baltimore, Maryland 212012; Pan American Health Organization, Washington, D.C. 200373; The Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland4; Tropical Medicine Department, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-47995; Torrey Pines Institute for Molecular Studies, San Diego, California 921216; and Vector and Control Research Centre, Kenya Medical Research Institute, Kisumu, Kenya7 Received 28 October 1996/Returned for modification 18 December 1996/Accepted 24 April 1997

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 mg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine. The logic for including Plasmodium falciparum sporozoite surface protein 2 (PfSSP2) (27), also known as thrombospondin-related anonymous protein (TRAP) (26), in experimental P. falciparum human malaria vaccines is based primarily on the fact that the PfSSP2/TRAP rodent malaria homologue, Plasmodium yoelii SSP2 (PySSP2) (5, 10, 28), is the target of protective CD81 T lymphocytes that eliminate malaria-infected hepatocytes. Immunization with recombinant P815 mastocytoma cells expressing PySSP2 induces PySSP2specific CD81 cytotoxic T lymphocytes (CTL) and protects BALB/c mice against challenge with sporozoites, and this protection is absolutely dependent on CD81 T lymphocytes (14). Furthermore, adoptive transfer of a PySSP2 CD81 CTL clone protects naive BALB/c mice against malaria (15). Humans

immunized with radiation-attenuated P. falciparum sporozoites (37, 38) and naturally exposed to malaria (1) develop CD81 CTL against PfSSP2/TRAP. Recently it has been shown that immunization of A/J mice with an 18-amino-acid PySSP2 peptide induces sterile protective immunity against malaria that is dependent on CD41 T lymphocytes and gamma interferon (35). Thus, there has been great emphasis on inducing PfSSP2/ TRAP T-cell responses that protect against malaria by eliminating infected hepatocytes expressing PfSSP2/TRAP peptides in association with major histocompatibility complex molecules (11). However, there is reason to believe that antibodies to PfSSP2/TRAP may contribute to protection. Antibodies to PfSSP2/TRAP partially inhibit sporozoite invasion and development into hepatocytes in vitro (20, 27), and both proteins include a sequence called region II (7) or region II1, amino acids 348 to 367, that binds to hepatocytes (24, 31) and is thought to mediate the first interaction of sporozoites with hepatocytes in vivo. To investigate the potential for anti-PfSSP2 antibodies to inhibit sporozoite invasion of hepatocytes, we produced and characterized several monoclonal antibodies

* Corresponding author. Mailing address: Malaria Program, Naval Medical Research Institute, 12300 Washington Ave., Rockville, MD 20852. Phone: (301) 295-1177. Fax: (301) 295-6171. E-mail: [email protected]. † Present address: Department of Cellular Biology, The University of Georgia, Athens, GA 30602. 3430

B-CELL EPITOPES OF P. FALCIPARUM SSP2/TRAP

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(MAbs) to PfSSP2, identified their epitopes, produced branched-chain polymer–multiple-antigen peptide vaccines based on these epitopes, immunized mice, and evaluated the inhibitory capacity of these antisera. MATERIALS AND METHODS Animals. Female, 4- to 6-week-old mice of three inbred strains, A/J, C57BL/6, and BALB/c ByJ (Jackson Laboratory, Bar Harbor, Maine) and one outbred strain, CD1 (Charles River Laboratory, Wilmington, Mass.), were used. The experiments reported herein were conducted according to the principles set forth in reference 13a. Sporozoites. All experiments were conducted with sporozoites of the NF54 strain of P. falciparum raised in Anopheles stephensi mosquitoes. Sporozoites from dissected salivary glands suspended in Dulbecco’s modified Eagle medium (Gibco, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum were used in the inhibition-of-sporozoite-invasion (ISI) (12) assay and in the inhibition-of-liver-stage-development assay (ILSDA) (18). Sporozoites isolated by a discontinuous gradient (21) in Medium 199 (Gibco) without serum were used in the indirect fluorescent-antibody assay (IFA). Production of MAbs. Crude recombinant PfSSP2/TRAP, including all except the first 145 amino acids (27), was used as an immunogen for the production of MAbs. Briefly, BALB/c ByJ mice were immunized intraperitoneally (i.p.) or intramuscularly three times at 2-week intervals with 5 mg of crude insoluble protein emulsified in 50 ml of TiterMax (CytRx Corp., Atlanta, Ga.) or in Freund’s adjuvants (Gibco). When Freund’s adjuvants were used, Freund’s complete adjuvant was used in the first immunization and Freund’s incomplete adjuvant was used in subsequent immunizations. One week after the third immunization, spleen cells were isolated from the mice and fused with X63.Ag8.653 myeloma cells as previously described (5). Culture supernatants from hybridoma lines were screened by IFA for the presence of antisporozoite antibodies. Positive hybrids were cloned by limiting dilution, and a series of hybridoma clones secreting MAbs to sporozoites were obtained. Antibody isotypes of the MAbs were determined by a double-diffusion-in-gel method and confirmed by standard enzyme-linked immunosorbent assay (ELISA) with the ISOTYPE Ab-STAT kit (SanStat Medical Corporation, Menlo Park, Calif.), according to the manufacturer’s specifications. On the basis of their reactivity with sporozoites, three MAbs, PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM), were selected for further study. PfSSP2.1 and PfSSP2.2 were purified from ascitic fluids by staphylococcal protein A affinity chromatography (8). PfSSP2.3 was partially purified from ascitic fluid by 50% ammonium sulfate precipitation. IFA. The IFA was performed as previously described (5). Briefly, hybridoma supernatants, mouse sera, and purified MAbs were incubated for 30 min at 37°C with P. falciparum sporozoites that were air-dried on wells of multispot antigen slides at 5,000 sporozoites/well. The slides were washed three times with phosphate-buffered saline (PBS) and further incubated for 30 min with fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulins (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). The slides were washed three times with PBS, mounted with 30% glycerol in PBS, and examined with an Olympus UV microscope. Epitope mapping. A total of 558 sequential octapeptides overlapping by seven amino acids were synthesized based on the sequence of 574 amino acids (27) (GenBank accession no. M94013) of PfSSP2 by using the method of Geysen et al. (9). Amino acids 361 to 369 were omitted, as they comprised the same sequence as amino acids 352 to 360. These octapeptides were synthesized on blocks of 96 polypropylene pins (Cambridge Research Biochemicals, Valley Stream, N.Y.). The synthesis was carried out in a 96-well plate. Each well carried amino acids dictated by the synthesis schedule. The amino acids used were pentafluorophenyl ester of 9-fluorenyl-methoxycarbonyl derivatives. To monitor the synthesis, the tetrapeptides PLAQ and GLAQ were synthesized simultaneously as positive and negative controls. The pins were blocked for 1 h at 37°C in PBS containing 1% bovine serum albumin and 0.05% Tween 20 (pH 7.2) and then incubated overnight at 4°C with supernatant MAbs (2 to 10 mg/ml of MAb) diluted 1:100 with the same buffer or incubated with PBS alone. The pins PLAQ and GLAQ were incubated with commercially available murine MAbs (Cambridge Research Biochemicals) directed against PLAQ and GLAQ. The pins were washed four times with PBS containing 0.05% Tween 20 and 0.05% sodium azide (pH 7.2) and then incubated for 1 h at 37°C with 1 mg of alkaline phosphatase-labeled goat anti-mouse IgG or IgM (Kirkegaard & Perry, Inc., Gaithersburg, Md.) per ml and washed once more in this buffer. Color reaction was developed with a commercially available phosphatase kit (Kirkegaard & Perry). Optical density (OD) readings were taken in a Dynatech micro-ELISA auto reader, model MR5000, at 410 nm. Readings obtained from peptides incubated with PBS were then subtracted from the readings obtained from the corresponding peptides incubated with the MAbs. Linear peptides. Linear peptides of 15 to 27 amino acids in length were used in the ELISA to confirm the results of epitope mapping of the MAbs. These peptides were NDKSDRYIPYSPLSP, (DRYIPYSP)3, (DRYI)6, FLVGCHPS DGKCNLY, (CHPSDGKCN)2, HPSDGKCNLYADSAW, SEDRETRPHGR NNEN, (RETRPHGR)3, and (TRPHGR)4. The (CHPSDGKCN)2 peptide was

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synthesized by a stepwise solid-phase method (19). The 15-amino-acid peptides, FLVGCHPSDGKCNLY, HPSDGKCNLYADSAW, and NDKSDRYIPYS PLSP, were synthesized by the solid-phase “tea bag” method (13). Peptides (DRYI)6, (DRYIPYSP)3, (TRPHGR)4, and (RETRPHGR)3 were purchased from Multiple Peptide Systems, San Diego, Calif. Conjugation of peptides to KLH. The peptides NDKSDRYIPYSPLSP and (CHPSDGKCN)2 were conjugated to keyhole limpet hemocyanin (KLH) by the method of Reichlin (23). The conjugated peptides were dialyzed for 24 h at 4°C against four changes of 500 ml of PBS before use in the immunization study. MAPs. MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 were used in this study. MAP4 (DRYIPYSP)3P2P30 consists of a central lysine core and four branched-chain peptides, each containing 3 copies of DRYIPYSP conjugated to two T-helper epitopes, P2 (QYIKANSKFIGITE) and P30 (FNN FTVSFWLRVPKVSASHLE), from tetanus toxin (33). MAP4 (CHPSDG KCN)3P2P30 consists of a central lysine core and four branched-chain peptides, each containing 3 copies of CHPSDGKCN conjugated to P2 and P30. These multiple-antigen peptides (MAPs) were synthesized as previously described (34). Immunization studies. (i) Immunization with KLH-conjugated peptides. Groups of five BALB/c mice were immunized subcutaneously at the tail base three times at 3-week intervals with 50 ml of 50 mg of KLH-conjugated NDKS DRYIPYSPLSP or (CHPSDGKCN)2 emulsified with Freund’s adjuvants or TiterMax at a 1:1 ratio. When Freund’s adjuvants were used, the first dose was antigen emulsified in Freund’s complete adjuvant and the subsequent doses were antigen in Freund’s incomplete adjuvant. Mice immunized with adjuvants alone served as controls. Serum samples were collected from the mice 3 weeks after each immunization. These sera were tested by IFA against air-dried P. falciparum sporozoites and by ELISA against unconjugated NDKSDRYIPYSPLSP and (CHPSDGKCN)2. The sera were also tested by ISI assay in HepG2.16 hepatoma cells to determine their inhibitory effects on sporozoite invasion. (ii) Immunization with MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSD GKCN)3P2P30. The immunogenicity of these MAP vaccines was evaluated in three inbred strains of mice, A/J (H-2a), C57BL/6 (H-2b), and BALB/c ByJ (H-2d), and one outbred strain, CD1, in TiterMax adjuvant. Groups of 10 mice were immunized subcutaneously at the tail base three times at 3-week intervals with 25 ml of antigen-adjuvant mixture (1:1 ratio) containing 50 mg of MAP4 (CHPSDGKCN)3P2P30 or MAP4 (DRYIPYSP)3P2P30. Mice immunized with TiterMax emulsified in PBS (1:1 ratio) served as the controls. Serum samples collected from the mice 3 weeks after each immunization were tested by IFA against P. falciparum air-dried sporozoites and by ELISA against the linear peptides. These sera were also tested by ISI assay to determine their inhibitory effect on P. falciparum sporozoite invasion of the HepG2.16 hepatoma cells. (iii) Immunization with a recombinant vaccinia virus expressing full-length PfSSP2. To determine whether or not mice immunized with PfSSP2 (vP1155) induced antibodies to the B-cell epitopes defined by the MAbs, groups of three BALB/c ByJ and C57BL/6 mice were immunized i.p. or intradermally (i.d.) three times at 3-week intervals, with 107 PFU of vaccinia viruses expressing full-length PfSSP2 (vP1155) (39) or a vector control vaccinia (vP933). Serum samples collected from the mice 3 weeks after the third immunization were tested by standard ELISA against 2 mg of (DRYIPYSP)3 and (CHPSDGKCN)2 per ml. ELISA. Nine peptides were used as solid-phase antigens in the ELISA. These peptides were NDKSDRYIPYSPLSP, (DRYIPYSP)3, (DRYI)6, FLVGCHPS DGKCNLY, (CHPSDGKCN)2, HPSDGKCNLYADSAW, SEDRETRPHGRN NEN, (RETRPHGR)3, and (TRPHGR)4. Briefly, 50 ml of the optimal concentration (2 to 10 mg/ml) of each peptide was coated on wells of Immunolon II micro-ELISA plates (Dynatech Laboratory Inc., Chantilly, Va.) and incubated for 6 h at room temperature. The wells were washed three times with PBS containing 0.05% Tween 20 (washing buffer) and incubated overnight at 4°C with 100 ml of 5% nonfat dry milk (blocking buffer). After being washed three times with a washing buffer, the wells were incubated for 2 h with 50 ml of different concentrations (0.001 to 100 mg/ml) of the MAbs or immunized mouse sera diluted in PBS. The wells were washed three times, incubated for 1 h with peroxidase-labeled goat anti-mouse IgG or IgM (Kirkegaard & Perry), and then washed again three times. The wells were incubated for 20 min with 100 ml of a solution containing ABTS substrate [2,29-azino-di-(39-ethyl-benzthiazoline sulfonate)] (Kirkegaard & Perry) and H2O2. Color reaction was measured in a Dynatech micro-ELISA auto reader, model MR5000, at OD410. All reaction steps except blocking were performed at room temperature. The means 6 standard deviations of the OD readings of quadruplicate assays were recorded, and the ELISA OD units were determined. An OD unit was the reciprocal of the serum dilution at which the OD reading was 0.5. Inhibition ELISA. MAb PfSSP2.1 was tested by inhibition ELISA with peptides (DRYI)6 and (DRYIPYSP)3 as inhibitors (4). Briefly, a fixed concentration (2 mg/ml) of the MAb was preincubated for 2 h at 37°C with varying concentrations (0.001 to 200 mg/ml) of (DRYI)6 or (DRYIPYSP)3. The peptide-antibody mixtures were then tested by standard ELISA as above, with (DRYIPYSP)3 as a solid phase. ILSDA. MAbs PfSSP2.1 and PfSSP2.2 were tested by ILSDA for the inhibitory effect on P. falciparum liver-stage parasite development as previously described (18). Briefly, human hepatocytes suspended in a complete medium were seeded in eight-chamber Lab-Tek plastic slides (Nunc, Inc., Naperville, Ill.) at 105 cells/chamber. After incubation for 24 h at 37°C in an atmosphere of 5% CO2– 95% air, the medium was removed and 25 to 100 mg of PfSSP2.1 or PfSSP2.2 per

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FIG. 1. IFA patterns of monoclonal and polyclonal antibodies. MAbs PfSSP2.1 (IgG1), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were tested against air-dried P. falciparum sporozoites at 2 mg/ml. Polyclonal sera induced by MAP4 (DRYIPYSP)3P2P30 or MAP4 (CHPSDGKCN)3P2P20 were tested at a 1:100 serum dilution. The MAbs (A) and polyclonal sera (B) produced the same IFA pattern, showing bipolar staining. This pattern is different from that produced by NFS1, a MAb directed against PfCSP which reacted with the antigens on the entire surfaces of the sporozoites (C).

ml and 6 3 104 sporozoites suspended in a complete medium were simultaneously added to hepatocyte cultures. After 3 h of incubation, excess MAbs and unattached sporozoites were removed from the cultures by washing them with medium, and the cultures were further incubated for a total of 6 days. The cultures were then fixed for 10 min in cold methanol and stained by a detecting MAb (NFS1; 100 mg/ml) and fluorescein isothiocyanate-labeled goat anti-mouse IgG. QGPS1 (IgG1), a mouse MAb directed against the P. yoelii circumsporozoite protein (CSP) major repeat, (QGPGAP)n (12), was used as a negative control. NFS1, an IgG1 MAb directed against the P. falciparum CSP major repeat, (NANP)n, was included in a second experiment as a positive control. The number of liver schizonts in each culture was counted with an Olympus UV microscope, and the mean number of liver schizonts in triplicate cultures was recorded. Percent inhibition was calculated based on the numbers of schizonts in cultures to which the control MAb had been added. ISI. The ISI assay was performed by the method previously described (12). Briefly, eight-chamber Lab-Tek tissue culture glass slides (Nunc, Inc.) were coated for 24 h at 4°C with 0.1 ml of entactin-collagen IV-laminin cell attachment matrix (Upstate Biotechnology Inc., Lake Placid, N.Y.). The matrix was removed and 5 3 104 HepG2.16 hepatoma cells in 0.1 ml of a modified Eagle medium (Gibco) supplemented with 10% fetal bovine serum were added to each chamber. The hepatoma cell cultures were incubated for 72 h at 37°C in an atmosphere of 5% CO2 in air to form a monolayer culture. The medium was removed, and the mixtures of 50 ml of varying concentrations of MAbs, or different dilutions of immunized mouse sera, and 50 ml of a suspension containing 3 3 104 P. falciparum sporozoites were added to the monolayers and further incubated for 3 h. The cultures were washed three times with PBS to remove unattached sporozoites, fixed for 10 min with cold, anhydrous methyl alcohol, washed three more times with PBS, and stained with a detecting MAb (NFS1; 100 mg/ml) and peroxidase-labeled goat anti-mouse IgG (Kirkegaard & Perry). After staining, the slides were air dried, mounted with 24- by 60-mm glass coverslips by using permount, and examined by phase contrast microscopy. The mean numbers of sporozoites invading hepatoma cells in triplicate cultures in the presence of the tested and control antibodies were compared, and percent inhibitions were calculated. Detection of antibodies against peptides (DRYIPYSP)3 and (CHPSDGKCN)2 in sera of humans living in malaria-endemic areas. Sera from 28 individuals living in Irian Jaya, Indonesia, and sera from 33 individuals living in western Kenya were tested against 2 mg of (DRYIPYSP)3 and (CHPSDGKCN)2 per ml by standard ELISA with horseradish peroxidase-conjugated goat anti-human IgG and IgM as detecting antibodies. The negative controls were sera from 14 healthy individuals living in the United States, who had no known history of malaria infection. The OD readings in wells not coated with antigen were subtracted from the readings in the presence of antigen, and mean OD readings 6 standard deviations of quadruplicate assays were calculated. Mean OD readings of sera from individuals living in malaria-endemic areas that were greater than mean OD readings plus 2 standard deviations of sera from the negative control individuals were considered positive.

RESULTS AND DISCUSSION Production and characterization of the MAbs. A total of 79 hybridoma clones secreting antibodies against P. falciparum

sporozoites were produced in six fusion experiments. These clones secreted antibodies of either IgG1, IgG2a, or IgM isotype as determined by gel double diffusion and confirmed by standard ELISA. The MAbs with the strongest IFA reactivity against air-dried sporozoites, designated PfSSP2.1 (IgG1), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM), were selected for further study. The immunofluorescence staining pattern, stage and species specificity, and biological activity of these MAbs were similar to those of previously described polyclonal sera raised against the same recombinant PfSSP2 protein used to generate the MAbs (27). All MAbs produced the same pattern of IFA reactivity, showing a spotty, bipolar staining on air-dried P. falciparum sporozoites (Fig. 1A). This pattern was the same as that seen with polyclonal antibodies raised against recombinant PfSSP2 (Fig. 1B) and differed from that of the anti-CSP MAb, which reacted with antigen on the entire surface of the

TABLE 1. Inhibition of P. falciparum liver-stage parasite development in human hepatocyte cultures by MAb PfSSP2.1 Concn (mg/ml)

No. of parasites in triplicate cultures (mean 6 SD)

% Inhibition

100 50 25

26, 24, 30 (26.7 6 2.5) 22, 40, 35 (32.3 6 9.0) 55, 45, 38 (46.0 6 8.5)

41.6 20.6 0

100 50 25

50, 41, 46 (45.7 6 3.7) 38, 34, 50 (40.7 6 6.8) 39, 40, 34 (37.7 6 2.6)

100 50

5, 6, 7 (7.0 6 0.8) 5, 5, 12 (7.3 6 3.3)

QGP-S1 (IgG1)

100 50

59, 44, 47 (50.0 6 6.5) 24, 31, 23 (26.0 6 3.6)

NFS1b (IgG1)

100

0, 0, 0 (0.0)

MAb

Experiment 1 PfSSP2.1 (IgG1)

QGP-S1a (IgG1)

Experiment 2 PfSSP2.1 (IgG1)

a b

86.0 71.9

100

MAb directed against the P. yoelii CSP major repeat (control). MAb directed against the P. falciparum CSP major repeat (control).

B-CELL EPITOPES OF P. FALCIPARUM SSP2/TRAP

VOL. 65, 1997 TABLE 2. Epitope mapping of MAbs PfSSP2.1, PfSSP2.2, and PfSSP2.3 Amino acid sequencea

Peptide position

ELISA OD410

PfSSP2.1 (IgG1)

PNDKSDRY NDKSDRYI DKSDRYIP KSDRYIPY SDRYIPYS DRYIPYSP RYIPYSPL

420–427 421–428 422–429 423–430 424–431 425–432 426–433

0.074 2.355 2.382 2.309 2.462 2.475 0.363

PfSSP2.2 (IgG2a)

GCHPSDGK CHPSDGKC HPSDGKCN PSDGKCNL

204–211 205–212 206–213 207–214

0.172 1.316 1.305 0.005

PfSSP2.3 (IgM)

DRETRPHG RETRPHGR ETRPHGRN TRPHGRNN RPHGRNNE

463–470 464–471 465–472 466–472 467–473

0.046 2.281 1.030 1.980 0.108

MAb

a Underlined sequences reacted strongly with the indicated MAb, as discussed in the text.

sporozoites (Fig. 1C). Like the polyclonal sera, these MAbs reacted in IFA with sporozoites and early (24- to 48-h) exoerythrocytic-stage parasites but not with late (4- to 6-day) exoerythrocytic-stage parasites, infected erythrocytes, or sporozoites of Plasmodium vivax, P. yoelii, or Plasmodium berghei (data not shown). They are therefore species and stage specific. Sharma et al. recently detected a 78-kDa protein in infected erythrocyte extracts by using antisera raised against a peptide derived from PfSSP2/TRAP (30). However, we have been unable to detect the presence of PfSSP2 at any time later than 2 days after infection of the hepatocytes. Finally, like the polyclonal anti-PfSSP2 serum (27), one of the MAbs, PfSSP2.1 (IgG1), was able to suppress liver-stage parasite development in the ILSDA. Two ILSDA experiments were performed with

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freshly isolated hepatocytes from two different donors. In the first experiment, PfSSP2.1, at 100 mg/ml, inhibited 42% (P , 0.005 [Student’s two-tailed t test]) of P. falciparum liver-stage parasite development (Table 1), while PfSSP2.2 (IgG2a) had no significant inhibitory effect (data not shown). PfSSP2.3 (IgM) was not tested by ILSDA because of the difficulty in purification and instability of this MAb. In the second experiment, PfSSP2.1 had a higher inhibitory effect (86%) (P , 0.001 [Student’s two-tailed t test]) (Table 1). This inhibitory effect did not reach the level produced by NFS1, a MAb directed against the P. falciparum CSP major repeat (100%), but the results were consistent with previous findings that antisera raised against recombinant PfSSP2/TRAP partially blocked invasion of hepatoma cells (20) and inhibited liver-stage parasite development in human hepatocyte cultures (27). Epitope mapping. We next defined the epitopes recognized by the MAbs in order to construct peptide vaccines based on the relevant B-cell epitopes. Epitope mapping by the method of Geysen et al. (9) demonstrated that PfSSP2.1, PfSSP2.2, and PfSSP2.3 recognized three distinct epitopes which included DRYI, HPSDGKC, and TRPHGR respectively (Table 2). Standard ELISAs against peptides containing these amino acid sequences confirmed the results of peptide mapping. PfSSP2.1 reacted strongly with two peptides containing DRYI sequences, NDKSDRYIPYSPLSP and (DRYIPYSP)3; however, it did not react with (DRYI)6 (Fig. 2A). To demonstrate that this negative result was not due to the inability of (DRYI)6 to coat the plates, we performed an inhibition ELISA with (DRYI)6 and (DRYIPYSP)3 as the inhibiting agents. The results demonstrated that preincubation of PfSSP2.1 with (DRYI)6 did not alter the binding of this MAb to (DRYIPYSP)3, while preincubation with (DRYIPYSP)3 inhibited this binding (Fig. 2D). These results suggest either that DRYI alone is not a minimal epitope for MAb PfSSP2.1 or that the conformation adopted by the six tandem repeats of DRYI was not appropriate for binding of the MAb. PfSSP2.2 reacted strongly with all three peptides containing the HPSDGKC sequence, (CHPSDGKCN)2, FLVGCHPSDGKCNLY, and HPSDGKCNLYADSAW), with the strongest reactivity

FIG. 2. ELISA of PfSSP2 MAbs. Varying concentrations of MAbs were tested by standard ELISA against 10 mg of peptides per ml containing their epitopes, as described in Materials and Methods.

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route. Two of the three C57BL/6 mice immunized with vP1155, via either route, developed moderate levels of antibodies to (DRYIPYSP)3 as determined by ELISA (Fig. 3). Neither BALB/c mice immunized with vP1155 nor control mice developed detectable levels of antibody to (DRYIPYSP)3. None of the mice immunized with vP1155 or vP933 developed antibodies to (CHPSDGKCN)2 (data not shown). Previous work has shown that volunteers immunized with irradiated sporozoites (27) and West African adults and children (29) have antibodies to recombinant PfSSP2/TRAP. We found that residents of malaria-endemic areas had antibodies to the (DRYIPYSP)3 and (CHPSDGKCN)2 epitopes. Sera from 28 individuals living in Irian Jaya, Indonesia, 33 individuals living in western Kenya, and 14 healthy control individuals living in the United States were tested by ELISA against the (DRYIPYSP)3 and (CHPSDGKCN)2 peptides. Twelve of 28 Irianese and 9 of 33 Kenyans had low levels of antibody to (DRYIPYSP)3 compared to the levels in the healthy control individuals (Fig. 4A). Thirteen of 28 Irianese and 10 of 33 Kenyans had antibodies to (CHPSDGKCN)2, most at low levels, except Kenyan subject K26, who had a high reactivity (OD reading of 0.911 6 0.002) to this peptide (Fig. 4B). These results clearly demonstrate that humans living in P. falciparum

FIG. 3. ELISA reactivity of sera from C57BL/6 mice immunized with vaccinia virus expressing full-length PfSSP2 to (DRYIPYSP)3. Varying concentrations of recombinant vaccinia-PfSSP2 (vP1155)-immunized mouse sera were tested against 2 mg of (DRYIPYSP)3 per ml. Two of three mice immunized i.p. with vP1177 (A) and two of three mice immunized i.d. (B) developed antibodies to (DRYIPYSP)3. Mice immunized with the control construct (vP933) did not develop antibody to this peptide.

against (CHPSDGKCN)2 (Fig. 2B). MAb PfSSP2.3 (IgM) reacted with peptides containing the TRPHGR sequence [(TRPHGR)4, (RETRPHGR)3, and SEDRETRPHGRNNEN] only at the highest concentration (100 mg/ml) of the MAb tested (Fig. 2C). The PfSSP2.2 epitope includes a conserved cysteine, but the epitopes for these three MAbs are not otherwise located in sequences strongly conserved between PySSP2 and PfSSP2 (10, 25–28). These epitopes do not overlap the previously defined region II-like domain which has been shown to be involved in sulfatide binding (3, 20, 22, 24), the previously reported peptide sequences recognized by MAbs directed against TRAP (24), or any of the previously defined human or murine CTL epitopes (1, 37–39). Since MAbs PfSSP2.1, PfSSP2.2, and PfSSP2.3 were generated from a recombinant protein lacking the first 145 amino acids (27), epitopes defined by these MAbs do not include the DGSGS (amino acids 55 to 59 in PfSSP2) motif commonly found in the Mg21 binding site (DXSXS) of adhesive molecules in the a-subunit “A domain” of various integrins (16, 32). Given the inhibitory effect of PfSSP2.1 in the ILSDA, its epitope, at least, must be expressed on the surfaces of live sporozoites. Recombinant PfSSP2 vaccinia-induced and naturally occurring antibodies to (DRYIPYSP)3 and (CHPSDGKCN)2. We next determined whether immunization with recombinant vaccinia expressing PfSSP2 or natural exposure to malaria induced antibodies to the B-cell epitopes defined by these MAbs. Groups of three BALB/c and C57BL/6 mice were immunized three times at 3-week intervals with 107 PFU of vaccinia viruses expressing full-length PfSSP2 (vP1155) or with vaccinia viruses containing no malaria gene (vP933 control) via the i.p. or i.d.

FIG. 4. Sera from individuals living in malaria-endemic areas contain antibodies to peptides (DRYIPYSP)3 and (CHPSDGKCN)2. Sera (1:200 dilution) from 14 healthy individuals, 28 individuals living in malaria-endemic areas in Irian Jaya, Indonesia (I), and 33 individuals living in western Kenya (K) were tested against 2 mg of peptides (DRYIPYSP)3 and (CHPSDGKCN)2 per ml. (A) Sera from 12 Irianese and 9 Kenyans were positive against (DRYIPYSP)3. (B) Sera from 13 Irianese and 11 Kenyans were positive against (CHPSDGKCN)2. The mean plus 2 standard deviations of the OD readings of the 14 healthy individuals (no history of exposure to malaria) is indicated by the dotted line. OD readings greater than this value were considered positive.

B-CELL EPITOPES OF P. FALCIPARUM SSP2/TRAP

VOL. 65, 1997 TABLE 3. Antibody levels in sera of mice after the third immunization with MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax Immunogen and mouse strain

IFA titera

ELISA OD unitsb against: (DRYIPYSP)3 (CHPSDGKCN)2

MAP4 (DRYIPYSP)3P2P30 A/J (H-2a) C57BL/6 (H-2b) BALB/c ByJ (H-2d) CD1 (outbred)

5,120 5,120 5,120 5,120

181,000 181,000 56,000 205,000

,1,000 ,1,000 ,1,000 3,500

MAP4 (CHPSDGKCN)3P2P30 A/J (H-2a) C57BL/6 (H-2b) BALB/c ByJ (H-2d) CD1 (outbred)

5,120 2,560 5,120 5,120

,1,000 ,1,000 ,1,000 ,1,000

363,000 218,000 107,000 .512,000

,10

,1,000

,1,000

TiterMax control (all strains) a

Against air-dried P. falciparum sporozoites. OD unit, the reciprocal of the serum dilution at which the OD reading against the homologous peptide is 0.5. b

malaria-endemic areas develop an antibody response to the PfSSP2 B-cell epitopes defined by MAbs PfSSP2.1 and PfSSP2.2. Immunogenicity in mice immunized with peptides containing PfSSP2 B-cell epitopes. We next investigated the immunogenicity of peptide vaccines based on these epitopes and the biological activity of antibodies induced by these vaccines. BALB/c mice immunized with KLH-conjugated NDKSDRYIPYSPLSP or (CHPSDGKCN)2 in Freund’s adjuvants developed moderate levels of antibodies to P. falciparum sporozoites, with IFA titers of 1024 and 512 respectively. These mice also developed antibodies to the homologous peptide with 50,000 and 45,000 ELISA OD units, respectively. Mice immunized with KLH-conjugated (CHPSDGKCN)2 in TiterMax developed lower levels of antisporozoite antibodies (IFA titer, 128) than mice immunized with this peptide in Freund’s adjuvants; however, the levels of antipeptide antibodies were the same (50,000 OD units). None of the control mice immunized with KLH in Freund’s or TiterMax developed antisporozoite or antipeptide antibodies. We have previously reported (36) that mice immunized with MAP4 (QGPGAP)4P2P30 in TiterMax developed very high levels of antibodies to P. yoelii sporozoites (IFA titers, 5,000 to 10,000) and to (QGPGAP)2 (ELISA OD units, .100,000) and that these mice were protected against sporozoiteinduced infection. In this study, we evaluated the immunogenicity of MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDG KCN)3P2P30 in three inbred mouse strains (A/J, C57BL/6, and BALB/c ByJ) and one outbred mouse strain (CD1) with TiterMax as an adjuvant. The results are summarized in Table 3. All mice immunized with MAP4 (DRYIPYSP)3P2P30 developed a strong antibody response to P. falciparum sporozoites (IFA titer, 5,120) and to peptide (DRYIPYSP)3 in the following order: CD1 . A/J 5 C57BL/6 . BALB/c (2.1 3 105, 1.8 3 105, 1.8 3 105, and 5.6 3 104 OD units, respectively). Mice immunized with MAP4 (CHPSDGKCN)3P2P30 also developed strong antibodies to sporozoites (IFA titers ranging from 2,560 to 5,120) and to peptide (CHPSDGKCN)2, with ELISA titers in the following order: CD1 . A/J . C57BL/6 . BALB/c (5.1 3 105, 3.6 3 105, 2.2 3 105, and 1.1 3 105 OD units, respectively). Biological activity of polyclonal sera. To evaluate the biological activity of antibodies induced by immunization with the

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defined B-cell epitopes of PfSSP2, we examined the ability of polyclonal sera to prevent hepatocyte invasion by P. falciparum sporozoites by ISI assay. Sera collected from BALB/c mice 3 weeks after the third immunization with linear peptide conjugated to KLH partially inhibited P. falciparum sporozoite invasion of HepG2.16 human hepatoma cells (data not shown). Sera collected from C57BL/6, BALB/c ByJ, A/J, and CD1 mice 3 weeks after the third immunization with MAP4 (DRYIP YSP)3P2P30 or MAP4 (CHPSDGKCN)3P2P30 were also tested in this assay, in three separate experiments. Only sera from A/J mice immunized with MAP4 (DRYIPYSP)3P2P30 or MAP4 (CHPSDGKCN)3P2P30 consistently produced an inhibitory effect on P. falciparum sporozoite invasion, with the average level of inhibition being 53 and 62%, respectively (Table 4). The inhibitory effects produced by sera from other strains of mice were not consistent, varying from an average of 8 to 31% (data not shown). It is possible that these sera failed to induce more substantial inhibition in the ISI assay because they do not include functionally important epitopes in the region II1 sulfatide binding domain (3, 10, 20) or the integrinlike A domain (16, 32); however, polyclonal sera raised against partial recombinant PfSSP2/TRAP proteins which included either region II (27) or region II and the A domain (20) had only modest inhibitory effects in the ILSDA and ISI assay, respectively. In summary, we have generated and characterized three MAbs specific for PfSSP2/TRAP and identified the B-cell epitopes that they recognize. The IFA staining pattern, stage and species specificity, and ability to inhibit liver-stage parasite

TABLE 4. Inhibition of P. falciparum sporozoite invasion of HepG2.16 hepatoma cells by seraa of A/J mice immunized with MAP vaccines in TiterMax MAP vaccine or control

Expt 1 MAP4 (DRYIPYSP)3P2P30 MAP4 (CHPSDGKCN)3P2P30 Adjuvant control NFS1c (100 mg/ml) Medium control Expt 2 MAP4 (DRYIPYSP)3P2P30 MAP4 (CHPSDGKCN)3P2P30 Adjuvant control NFS1c (100 mg/ml) Medium control Expt 3 MAP4 (DRYIPYSP)3P2P30 MAP4 (CHPSDGKCN)3P2P30 Adjuvant control NFS1c (100 mg/ml) Medium control

No. of sporozoites invaded in triplicate cultures (mean 6 SD)

% Inhibitionb

63, 79, 74 (72.0 6 6.7) 76, 51, 51 (59.3 6 11.8)

39.2 49.9

101, 121, 133 (118.3 6 13.2) 18, 16, 1 (11.7 6 7.6) 143, 137, 204 (161.3 6 30.3)

92.8

3, 9, 8 (6.7 6 2.6) 9, 5, 3 (5.7 6 2.5)

66.1 71.2

14, 21, 24 (19.7 6 4.2) 4, 3, 5 (4.0 6 0.8) 41, 40, 42 (41.0 6 0.8)

90.2

4, 5, 6 (5.0 6 0.8) 2, 7, 3 (4.0 6 2.2)

54.5 63.6

8, 13, 12 (11.0 6 2.2) 7, 4, 4 (5.0 6 1.4) 58, 60, 71 (63.0 6 5.7)

92.1

a Sera collected from mice 3 weeks after the third immunization were tested at a 1:50 serum dilution. b Percent inhibition by immunized mouse sera was always calculated based on the number of sporozoites that invaded in the presence of sera from mice immunized with the adjuvant. The average percent inhibition, in three experiments, of sera from MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDG KCN)3P2P30 was 53 and 62%, respectively. c MAb directed against the P. falciparum CSP major repeat (positive control).

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CHAROENVIT ET AL.

development are similar to those of polyclonal sera raised against recombinant PfSSP2/TRAP (27). Immunization with recombinant vaccinia expressing PfSSP2/TRAP or natural exposure to malaria induced antibodies to two of these epitopes. Immunization with peptide vaccines based on the sequence of two of these B-cell epitopes induced antisporozoite antibodies which provided partial inhibition of sporozoite invasion of hepatocytes, in contrast to the profound inhibitory effect of MAbs or antisera against PfCSP (17, 18). Although the modest inhibitory activity of these MAbs and the antisera induced by immunization with the defined B-cell epitopes provided little support for a single-component PfSSP2/TRAP vaccine designed to induce antibodies that prevent sporozoite invasion of hepatocytes, it is possible that antibodies to PfCSP and PfSSP2/TRAP may be additive or synergistic in their biological activity, and further studies will be required to determine if this is the case. Regardless, the MAbs described here and peptides recognized by these MAbs are valuable reagents that will be useful in the development of PfSSP2/TRAP as a component of multivalent malaria vaccines. ACKNOWLEDGMENTS This work was supported by the Naval Medical Research Development and Command (Work Unit no. 611102A.S13.00101-BFX.1431 and 612787A.870.00101.EFX.1432). We thank Christopher Paul for providing P. falciparum-infected mosquitoes; Esteban Abot for excellent technical assistance; Angela Appiah for performing ISI assays; Dorina Maris for statistical analysis; and the National Institutes of Health, Research and Development, and the Ministry of Health, Jakarta, Indonesia, for facilitating access to specimens of serum. REFERENCES 1. Aidoo, M., A. Lalvani, C. E. M. Allsopp, M. Plebanski, S. J. Meisner, P. Krausa, M. Browning, S. Morris-Jones, F. Gotch, and D. A. Fidock, et al. 1995. Identification of conserved antigenic components for a cytotoxic T lymphocyte-inducing vaccine against malaria. Lancet 345:1003–1007. 2. Ak, M., J. H. Bower, S. L. Hoffman, M. Sedegah, A. Lees, M. Carter, R. L. Beaudoin, and Y. Charoenvit. 1993. Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites. Infect. Immun. 61:2493–2497. 3. Cerami, C., F. Kwakye Berko, and V. Nussenzweig. 1992. Binding of malarial circumsporozoite protein to sulfatides [Gal(3-SO4)b1-Cer] and cholesterol3-sulfate and its dependence on disulfide bond formation between cysteines in region II. Mol. Biochem. Parasitol. 54:1–12. 4. Charoenvit, Y., W. E. Collins, T. R. Jones, P. Millet, L. Yuan, G. H. Campbell, R. L. Beaudoin, J. R. Broderson, and S. L. Hoffman. 1991. Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. Science 251:668–671. 5. Charoenvit, Y., M. F. Leef, L. F. Yuan, M. Sedegah, and R. L. Beaudoin. 1987. Characterization of Plasmodium yoelii monoclonal antibodies directed against stage-specific sporozoite antigens. Infect. Immun. 55:604–608. 6. Cowan, G., S. Krishna, A. Crisanti, and K. Robson. 1992. Expression of thrombospondin-related anonymous protein in Plasmodium falciparum sporozoites. Lancet 339:1412–1413. 7. Dame, J. B., J. L. Williams, T. F. McCutchan, J. L. Weber, R. A. Wirtz, W. T. Hockmeyer, W. L. Maloy, J. D. Haynes, I. Schneider, D. Roberts, G. S. Sanders, E. P. Reddy, C. L. Diggs, and L. H. Miller. 1984. Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 225:593–599. 8. Ey, P. L., S. J. Prowse, and C. R. Jenkin. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15:429–436. 9. Geysen, H. M., R. H. Meloen, and S. J. Barteling. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 81:3998–4002. 10. Hedstrom, R. C., J. R. Campbell, M. L. Leef, Y. Charoenvit, M. Carter, M. Sedegah, R. L. Beaudoin, and S. L. Hoffman. 1990. A malaria sporozoite surface antigen distinct from the circumsporozoite protein. Bull. W. H. O. 68(Suppl.):152–157. 11. Hoffman, S. L., E. D. Franke, M. R. Hollingdale, and P. Druilhe. 1996. Attacking the infected hepatocyte, p. 35–75. In S. L. Hoffman (ed.), Malaria vaccine development: a multi-immune response approach. American Society

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2793. 37. Wizel, B., R. Houghten, P. Church, J. A. Tine, D. E. Lanar, D. M. Gordon, W. R. Ballou, A. Sette, and S. L. Hoffman. 1995. HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J. Immunol. 155:766–775. 38. Wizel, B., R. A. Houghten, K. C. Parker, J. E. Coligan, P. Church, D. M. Gordon, W. R. Ballou, and S. L. Hoffman. 1995. Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2. J. Exp. Med. 182:1435–1445. 39. Wizel, B., W. O. Rogers, R. A. Houghten, D. E. Lanar, J. A. Tine, and S. L. Hoffman. 1994. Induction of murine cytotoxic T lymphocytes against Plasmodium falciparum sporozoite surface protein 2. Eur. J. Immunol. 24:1487– 1495.