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Infection and Immunity, November 1999, p. 5604-5614, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

CD4+ T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from a Plasmodium yoelii 17-Kilodalton Hepatocyte Erythrocyte Protein

Yupin Charoenvit,1,* Victoria Fallarme Majam,1,2 Giampietro Corradin,3 John B. Sacci Jr.,1,4 Ruobing Wang,1,2 Denise L. Doolan,1,5 Trevor R. Jones,1 Esteban Abot,1,2 Manuel E. Patarroyo,6 Fanny Guzman,6 and Stephen L. Hoffman1

Malaria Program, Naval Medical Research Center, Bethesda, Maryland 20814-50551; Henry M. Jackson Foundation, Rockville, Maryland 208522; Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland3; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 212014; Pan American Health Organization, Regional Office of the World Health Organization, Washington, DC 200375; and Instituto de Immunologia, Hospital San Juan de Dios, Universidad Nacional de Colombia, Bogota, Colombia6

Received 17 March 1999/Returned for modification 1 June 1999/Accepted 9 August 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8+ T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4+ T cells and gamma interferon (IFN-gamma ). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4+ T cells and IFN-gamma . The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8+ T cells. Data represented here demonstrate that CD4+ T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4+ T-cell responses, which will complement efforts to induce protective antibody and CD8+ T-cell responses.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

There are a number of approaches to malaria vaccine development (13, 15, 20). One approach is to induce immune responses that prevent malaria parasites from emerging from the liver into the bloodstream and thereby preclude the development of clinical symptoms of malaria, which manifest during the erythrocytic cycle. Work in this area has focused on inducing antibodies that block sporozoite invasion of hepatocytes and CD8+ T-cell responses that eliminate infected hepatocytes. The rationale for antibody-mediated protection is based on the observation that passive transfer of monoclonal antibodies (MAbs) (1, 3, 32) and polyclonal antibodies (10, 30) against the repeat region of the major sporozoite surface protein, the circumsporozoite protein (CSP), protects mice and monkeys against sporozoite challenge. Efforts to elicit protective CD8+ T-cell responses are based on the observations that immunization with radiation-attenuated sporozoites protects mice against sporozoite challenge. This protection is absolutely dependent on CD8+ T cells (8, 25, 26, 31). Adoptive transfer of a CD4+ T-cell clone that recognizes an epitope on the Plasmodium yoelii CSP protects mice against sporozoite challenge (22). Immunization of mice with a multiple-antigen peptide (MAP) containing four copies of 14 amino acids (aa) from P. berghei CSP protein (aa 57 to 70) protected BALB/c mice against P. berghei sporozoite challenge (19). Recently, we demonstrated that immunization of A/J mice with an 18-aa synthetic linear peptide from P. yoelii sporozoite surface protein 2 (SSP2) in the adjuvant TiterMax protects mice against sporozoite challenge in a CD4+ T-cell- and gamma interferon (IFN-gamma )-dependent manner (29). This peptide includes the B-cell epitope recognized by a MAb derived by cloning cells from a mouse immunized with radiation-attenuated sporozoites (2, 12, 23). These were the only examples of active induction of CD4+ T-cell- and IFN-gamma -dependent protection by a short linear synthetic peptide in malaria and, to the best of our knowledge, in all of the infectious diseases.

Accordingly, we attempted to identify additional peptides which could induce CD4+ T-cell and IFN-gamma -mediated protection. Recently, we reported the discovery, cloning, and characterization of a 17-kDa P. yoelii protein expressed in hepatocytes and erythrocytes, designated hepatocyte erythrocyte protein 17 (HEP17) (4, 7). We demonstrated that a MAb directed against this protein eliminated infected hepatocytes in culture and delayed the onset and density of blood-stage parasitemia in vivo (4) and that immunization with a DNA plasmid expressing HEP17 induces CD8+ T-cell-dependent protective immunity in mice (9). We now identify the B-cell epitopes of MAbs against the HEP17 protein and report that immunization of one strain of inbred mice and one strain of outbred mice with synthetic linear peptides corresponding to these epitopes in TiterMax adjuvant elicits CD4+ T-cell-dependent and IFN-gamma -dependent protection.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mouse strains. Female 6- to 8-week-old inbred A/J (H-2a), C57BL/6 (H-2b), and BALB/cByJ (H-2d) mice (The Jackson Laboratory, Bar Harbor, Maine) and outbred CD1 mice (Charles River Laboratory, Wilmington, Mass.) were used. The experiments reported herein were conducted according to the principles set forth in Guide for the Care and Use of Laboratory Animals (21).

Parasites. P. yoelii 17XNL (nonlethal strain) clone 1.1 was used. Sporozoites dissected from salivary glands of P. yoelii-infected Anopheles stephensi mosquitoes or P. yoelii-infected mouse erythrocytes were suspended in medium 199 containing 5% normal mouse serum for intravenous (i.v.) challenge. In vivo and in vitro liver-stage parasites were prepared as previously described (4) for immunofluorescence antibody test (IFAT).

Peptides. Twenty-nine sequential 15-mer peptides overlapping by 10 aa and derived from the 151 amino-terminal residues of the 162-residue protein (7) were synthesized by previously described methods (16, 18, 27). Briefly, the peptides were synthesized on p-methylbenzhydrylamine resin (Bachem California, Torrance, Calif.), using t-butyloxycarbonyl solid-phase peptide synthesis. Optimum coupling reaction time was set to 1 h and monitored by the qualitative ninhydrin test. Peptides were eluted from the resin by treatment with low and high concentration of HF for 2 h at 0°C and 1 h at -20°C, respectively, using Anisol as the scavenger. The final products were washed 10 times with 10 ml of ethyl ether and extracted with 10% acetic acid. After evaporation of the acid, the peptides were placed in reducing conditions, purified by high-pressure liquid chromatography on reverse-phase columns, and freeze-dried. These peptides were used as solid-phase antigens for epitope mapping. Two MAPs (Fig. 1), MAP4(SFPMNEESPLGFSPE)3P2P30 and MAP4(GFSPEEMEAVASKFR)3P2P30, and four linear peptides, (SFPMNEESPLGFSPE)3, (GFSPEEMEAVASKFR)3, SFPMNEESPLGFSPE, and GFSPEEMEAVASKFR, were used as immunogens. Linear peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 were used as solid-phase antigens in the enzyme-linked immunosorbent assay (ELISA).


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  		FIG. 1.   Schematic diagram of MAP4(SFPMNEESPLGFSPE)3P2P30 and MAP4(GFSPEEMEAVASKFR)3P2P30. Each MAP contains a central lysine core and four branched chains of three copies of a B-cell epitope (SFPMNEESPLGFSPE) or (GFSPEEMEAVASKFR) from P. yoelii HEP17 protein, conjugated to two T-helper epitopes, P2 (QYIKANSKFIGITE) and P30 (FNNFTVSFWLRVPKVSASHLE) from tetanus toxin (28).

Antibodies. For epitope mapping, two MAbs, designated Navy yoelii liver stage 2 (NYLS2, immunoglobulin M [IgM]) and Navy yoelii liver stage 3 (NYLS3, IgG1), produced as previously described (4) were used. They recognize a 17-kDa P. yoelii protein expressed in liver- and blood-stage parasites but do not recognize sporozoites. For depletion studies, purified rat immunoglobulin anti-CD4+, anti-CD8+, and anti-IFN-gamma MAbs were used. The purified rat immunoglobulin was purchased from Rockland Immunochemicals Inc. (Gilbertsville, Pa.). The anti-CD4+ MAb GK 1.5 (rat IgG2a; ATCC [American Type Culture Collection] TIB-207) (6) and the anti-CD8+ MAb 2.43 (rat IgG2b; ATCC TIB-210) (24) were obtained from ATCC (Manassas, Va.). The anti-IFN-gamma MAb XMG-6 (rat IgG1) (5) was a gift from Fred D. Finkelman (University of Cincinnati College of Medicine, Cincinnati, Ohio). All MAbs were purified from ascitic fluids by 50% ammonium sulfate precipitation, and antibody concentrations were measured by optical density (OD).

Epitope mapping. Epitopes for NYLS2 and NYLS3 MAbs were determined by ELISA as previously described (3), with the slight modification that 29 overlapping synthetic HEP17, 15-mer peptides were used as solid-phase antigens. Briefly, 50 µl of each peptide (10 µg/ml) in phosphate-buffered saline (PBS) was added to wells of Immunolon II 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 µl of 5% nonfat dry milk in PBS (blocking buffer). After three washes with washing buffer, the wells were incubated for 2 h with 50 µl of a 1:20 dilution of supernatant NYLS2 or NYLS3 MAb diluted in PBS containing 3% nonfat dry milk (diluting buffer). The wells were washed three times, incubated for 1 h with peroxidase-labeled goat anti-mouse IgG or IgM (Kirkegaard & Perry, Gaithersburg, Md.) diluted 1:2,000 in diluting buffer, and then washed again three times. The wells were incubated for 20 min with 100 µl of a solution containing ABTS substrate [2,2'-azino-di-(3 ethylbenzthiazoline sulfonate); Kirkegaard & Perry] and H2O2. Color reaction was measured in a micro-ELISA automated reader (Dynatech MR5000) at an OD of 410 nm. All reaction steps except blocking were performed at room temperature. Means ± standard deviations (SD) of the OD readings of quadruplicate assays were recorded.

Active immunization. Three inbred mouse strains (A/J, C57BL/6, and BALB/c ByJ) and one outbred strain (CD1) were used for MAP vaccine immunizations. A/J mice were used for linear peptide immunization. For immunization with MAP vaccines and 45-aa linear peptides, groups of 8 to 20 mice were immunized subcutaneously (s.c.) at the base of the tail, two or three times at 3- or 6-week intervals, with 25 µg of MAP4(SFPMNEESPLGFSPE)3P2P30, MAP4(GFSPEEMEAVASKFR)3P2P30, and linear peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 in TiterMax adjuvant (CytRx Corp., Norcross, Ga.). For immunization with 15-aa peptides, group of 12 A/J mice were immunized s.c., two times at 3-week intervals, with 25 and 50 µg of SFPMNEESPLGFSPE or GFSPEEMEAVASKFR in TiterMax. Control mice received adjuvant alone. Sera collected from mice 10 days after the last immunization (4 days before challenge) were used to assess antibody levels and isotypes. Mice were challenged 14 days after the last immunization with 100 P. yoelii sporozoites or 200 infected erythrocytes. Parasitemia levels were determined by microscopic examination of Giemsa-stained thin blood smears prepared from mice at 3, 5, 7, 9, 11, and 14 days postchallenge. Mice were considered protected if all blood smears were negative for parasites, since more than 10 years of experience has shown that mice that were negative on day 14 do not develop blood-stage parasitemia.

Passive immunization. Sera were collected from groups of 40 A/J mice 10 days after the second immunization with 25 µg of linear peptide (SFPMNEESPLGFSPE)3 or (GFSPEEMEAVASKFR)3 in TiterMax or from TiterMax control mice. Antibodies were purified from sera on protein A-Sepharose 4B (Sigma Chemical Co., St. Louis, Mo.) by affinity chromatography (11) and used in passive immunization studies as previously described (4). Briefly, groups of six A/J mice were injected i.v. with 100 P. yoelii sporozoites. At 1, 24, and 36 h after sporozoite inoculation, mice were injected i.v. with purified antibodies from peptide-immunized or TiterMax-immunized mice, at a dose of 2.5 mg in 0.2 ml of PBS per mouse. Blood smears were examined daily to assess parasitemia levels from days 3 through 14 after sporozoite inoculation. One hundred grid fields (2 × 104 erythrocyte counts) were examined from each smear, and the frequency of infection and percentage of parasitemia were calculated.

Antibody analysis. Serial dilutions of pooled sera from MAP vaccine- or linear peptide-immunized mice or from TiterMax control mice were analyzed by IFAT against liver-stage parasites grown in culture (in vitro liver schizonts) or against liver-stage parasites taken from the livers of mice that had been infected with P. yoelii sporozoites (in vivo liver schizonts), as previously described (4). Fluorescein-labeled goat anti-mouse IgG (H+L [heavy plus light chain]) (Becton Dickinson, San Jose, Calif.) was used as the detecting antibody. Sera were also analyzed by ELISA as described above, using (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 (each at 0.5 µg/ml) as solid-phase antigens and peroxidase-labeled goat anti-mouse IgG (H+L) (Kirkegaard & Perry) as the detecting antibody. The assay was performed in quadruplicate, and the ELISA titer was reported as an OD1.0 unit (the reciprocal of the serum dilution at which the mean OD reading was 1.0).

Isotype determination. Antibody isotypes were determined by standard ELISA as described above, using a relevant linear peptide [(SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3] as a solid-phase antigen and heavy-chain-specific, horseradish peroxidase-labeled goat anti-mouse immunoglobulins (Fisher Biotech, Pittsburgh, Pa.) as detecting antibodies.

ILSDA. Polyclonal antibodies were tested by inhibition of liver-stage development assay (ILSDA) for the inhibitory effect on P. yoelii liver-stage parasite development as previously described (4, 17). Briefly, mouse hepatocytes were seeded in eight-chamber Lab-Tek plastic slides (Nunc, Inc, Naperville, Ill.) at 105 cells in 300 µl of culture medium per chamber and incubated for 24 h in an atmosphere of 5% CO2 in air. The medium was removed, and 7.5 × 104 P. yoelii sporozoites suspended in 50 µl of medium were added to the cultures and incubated for 3 h. The cultures were washed with medium and incubated for 44 h with sera (final dilution of 1:20 in medium) or purified antibodies (final concentration of 100 µg/ml in medium) from vaccine-immunized or TiterMax control mice. The cultures were then washed with PBS, fixed with ice-cold methanol, and immunostained with NYLS3 MAb and fluorescein isothiocyanate-labeled goat anti-mouse IgG (H+L) (Kirkegaard & Perry). The numbers of liver schizonts in each culture were counted in an Olympus fluorescence microscope, and the mean number of liver schizonts in triplicate cultures was recorded. Percentage of inhibition was determined based on the number of schizonts in cultures to which TiterMax control sera had been added.

Depletion. To identify the specific T-cell subsets or cytokines involved in peptide-induced protection, groups of 10 A/J mice were immunized s.c. two times at 3-week intervals with (SFPMNEESPLGFSPE)3 or (GFSPEEMEAVASKFR)3 in TiterMax. Immunized mice were then treated with purified rat immunoglobulin control or a specific MAb (anti-CD4+ T cells, MAb GK 1.5; anti-CD8+ T cells, MAb 2.43; or anti-IFN-gamma , MAb XMG-6). For the treatment with purified rat immunoglobulin control, mice received an intraperitoneal (i.p.) injection of 1.0 mg of rat immunoglobulin in 0.5 ml of PBS on days -7, -6, -5, -4, -3, -2, -1, 0, and +2 (relative to sporozoite challenge). For CD4+ T-cell depletion, mice received an i.p. injection of 1.0 mg of anti-CD4+ MAb in 0.5 of PBS on days -7, -6, -5, -4, -3, -2, -1, 0, and +2. For CD8+ T-cell depletion, mice received an i.p. injection of 0.5 mg of anti-CD8+ MAb in 0.5 ml of PBS on days -6, -5, -4, -3, -2, -1, and 0. For anti-IFN-gamma treatment, mice received an i.p. injection of 1.0 mg of anti-IFN-gamma MAb in 0.5 ml of PBS on days -5, -4, -3, -2, and 1.5 mg on days -1, +2, and +4. Additional control groups were immunized untreated and TiterMax-immunized mice. Mice were challenged i.v. on day 0 with 100 P. yoelii sporozoites. Blood smears were examined as described above. To confirm the efficiency of depletion, groups of two mice that received anti-CD4+ and anti-CD8+ MAbs or rat immunoglobulin were killed on the day of challenge, and the spleen cells from individual mice were stained and analyzed by FACScan (Becton Dickinson, Lincoln, N.J.). The depletion efficiencies were >97% for both CD4+ and CD8+ T cells in all experiments.

Statistical analysis. Difference in the levels of protection among groups were analyzed by Fisher's exact test. Difference in mean number of schizont counts in hepatocyte cultures in the presence of the test and control sera or purified antibodies were analyzed by independent sample t test (SPSS for Windows 8.0; SPSS Inc., Chicago, Ill.). Differences in parasitemia density among groups of mice in a passive immunization experiment were analyzed by repeated measure analysis of variances (SPSS for Windows 8.0). For all tests, P values of <= 0.05 were considered significant.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Epitope mapping. The results of epitope mapping by ELISA of NYLS2 and NYLS3 MAbs are summarized in Table 1. Among the 29 peptides tested, NYLS2 reacted with two peptides, GFSPEEMEAVASKFR (aa 136 to 150) and EMEAVASKFREVC (aa 141 to 153), with the highest reactivity against the GFSPEEMEAVASKFR peptide. These two peptides have in common the amino acid sequence EMEAVASKFR (aa 141 to 150), suggesting that this sequence may represent the minimal epitope for NYLS2 MAb. NYLS3 reacted with two peptides, SFPMNEESPLGFSPE (aa 126 to 140) and EESPLGFSPEEMEAV (aa 131 to 145), with the highest reactivity against SFPMNEESPLGFSPE. These two peptides have in common the sequence EESPLGFSPE (aa 131 to 140), which may represent the minimal epitope for NYLS3 MAb. Based on ELISA reactivity, peptides GFSPEEMEAVASKFR (aa 136 to 150), containing the NYLS2 epitope, and SFPMNEESPLGFSPE (aa 126 to 140), containing the NYLS3 epitope, were selected for further study.

                              
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  		TABLE 1.   Epitope mapping of NYLS2 and NYLS3 MAbs by ELISA against linear synthetic peptides

Active immunization with MAPs. We initially produced MAPs as immunogens because NYLS3 MAb has a significant inhibitory effect on the liver-stage parasite development in vitro and a modest effect in vivo (4), and we were interested in determining if polyclonal antibodies against the B-cell epitopes recognized by NYLS3 and NYLS2 MAbs would protect against sporozoite challenge. Mice immunized with three doses of MAP4(SFPMNEESPLGFSPE)3P2P30 produced high titers of antibodies to P. yoelii liver-stage parasites, with the IFAT titers ranging from 9,600 to 20,000 (CD1 > A/J = C57BL/6 > BALB/c), and high titers of antibodies against (SFPMNEESPLGFSPE)3 peptide, with the ELISA OD1.0 units ranging from 77,000 to 120,000 (A/J = CD1 > C57BL/6 > BALB/c (Table 2). Mice immunized with MAP4(GFSPEEMEAVASKFR)3P2P30 had antibody levels lower than the levels induced by MAP4(SFPMNEESPLGFSPE)3P2P30, with the IFAT titers ranging from 4,800 to 19,200 (CD1 > C57BL/6 > A/J > BALB/c) and ELISA OD1.0 units against (GFSPEEMEAVASKFR)3 ranging from 60,000 to 184,000 (C57BL/6 > CD1 > BALB/c > A/J) (Table 2). Although all four strains of mice studied produced high levels of antibodies to liver-stage parasites and parasite-derived peptides, only A/J and CD1 mice were protected against sporozoite challenge (Table 2). In the case of the inbred mice, this finding was similar to that observed with the SSP2 peptide (29). Protection did not correlate with antibody titers since C57BL/6 mice were not protected even though their antibody titers by ELISA and IFAT were comparable to those found in A/J and CD1 mice immunized with MAP4(SFPMNEESPLGFSPE)3P2P30. Furthermore, CD1 mice immunized with MAP4(GFSPEEMEAVASKFR)3P2P30 had IFAT and ELISA titers comparable to those of CD1 mice immunized with MAP4(SFPMNEESPLGFSPE)3P2P30 but had essentially no protection (14.3%), while mice immunized with MAP4(SFPMNEESPLGFSPE)3P2P30 had 62.5% protection.

                              
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  		TABLE 2.   Antibody levels and protection in mice immunized with three doses of 25 µg MAP4(SFPMNEESPLGFSPE)3P2P30 or MAP4(GFSPEEMEAVASKFR)3P2P30 in TiterMax and challenged with 100 P. yoelii sporozoites

Sera collected from all four strains of mice after each immunization with MAP vaccines were also analyzed by ELISA to determine the number of immunizations required for the induction of peak antibody levels. Sera collected from mice after the first immunization with MAP4(SFPMNEESPLGFSPE)3P2P30 had low to moderate levels of antibodies to (SFPMNEESPLGFSPE)3, with the ELISA OD1.0 units ranging from 3,000 to 72,000 (CD1 > BALB/c > C57BL/6 > A/J) (Fig. 2). After the second immunization, antibody levels were higher in CD1 mice (204,800 OD1.0 units) than in other strains of mice. We also observed that a third immunization did not boost antibody levels in these mice, indicating that two immunizations were optimal for induction of an antibody response. Similar results were obtained when sera from mice immunized with MAP4(GFSPEEMEAVASKFR)3P2P30 were tested against (GFSPEEMEAVASKFR)3 peptide (data not shown).


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  		FIG. 2.   Antibody levels in sera of A/J, C57BL/6, BALB/c, and CD1 mice immunized with MAP4(SFPMNEESPLGFSPE)3P2P30 in TiterMax. Control (cont.) sera were obtained from mice immunized with TiterMax alone. Serial dilutions of sera collected 3 weeks after the first and second immunizations and 10 days after the third immunization were analyzed by ELISA against (SFPMNEESPLGFSPE)3 as described in Materials and Methods. Data are shown as mean ± SD of the OD readings of quadruplicate assays.

To confirm that two doses of immunization were optimal for the induction of antibody response and to determine if protection was affected by the number of immunizations, group of 10 A/J mice (protected mouse strain) were immunized with one to three doses of MAP4(SFPMNEESPLGFSPE)3P2P30 at 3- and 6-week intervals. Sera collected 10 days after the last immunization (4 days before challenge with P. yoelii sporozoites) were assessed for antibodies. Mice immunized with a single dose of MAP had low antibody levels (IFAT titer = 200; ELISA OD1.0 units = 1,000) (Table 3). Mice immunized with two and three doses of MAP at 3-week intervals had 140- and 160-fold-higher levels of antiparasite antibodies, while the levels of antipeptide antibodies were 171- and 154-fold higher than with a single dose of vaccine, and mice immunized with two doses of MAP at 3-week intervals had higher levels of antipeptide antibodies than mice immunized with two doses of MAP at 6-week intervals. However, mice immunized with two or three doses of vaccine had similar antibody titers, confirming the results in the previous experiment (Fig. 2).

                              
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  		TABLE 3.   Antibody levels and protection in A/J mice immunized at different intervals with one to three doses of 25 µg of MAP4(SFPMNEESPLGFSPE)3P2P30 in TiterMax and challenged with 100 P. yoelii sporozoites

Mice immunized with a single dose of MAP4(SFPMNEESPLGFSPE)3P2P30 were not protected, but mice immunized with two and three doses of this MAP were protected (60 to 90%) (Table 3). The group that received two doses of MAP at 3-week intervals had the highest level of protection (90%).

Active immunization with linear peptides. Data presented in Table 2 demonstrated a genetic restriction of protection and a lack of an association between antibody levels and protection, which suggested that protection is not mediated by antibody but may be mediated by T cells. We have previously demonstrated that protection induced by the linear (NPNEPS)3, an SSP2 peptide, was comparable to that induced by MAP4(NPNEPS)3P2P30 and that protection was dependent on CD4+ T cells (29). To determine if this was the case with the HEP17 peptides, we immunized A/J mice at 3-week intervals with two or three doses of 25 µg of the linear synthetic peptide (SFPMNEESPLGFSPE)3 or (GFSPEEMEAVASKFR)3 in TiterMax. Sera collected from mice 10 days after the last immunization (4 days before challenge) were analyzed by IFAT against 44-h P. yoelii in vitro liver schizonts and by ELISA against the relevant peptides. Antibody levels were comparable in mice immunized with two and three doses of (SFPMNEESPLGFSPE)3 (Table 4, experiments 1 to 3). Similar to immunization with MAP vaccine (Table 3), the highest level of protection against sporozoite challenge that was induced by linear peptide (SFPMNEESPLGFSPE)3 was achieved after two immunizations (Table 4, experiment 2). There was no protection against challenge with P. yoelii-infected erythrocytes, indicating that the protection was directed against the infected hepatocytes (Table 4, experiment 1). Immunization with two doses of (GFSPEEMEAVASKFR)3 also induced high levels of antibody and protection (Table 4, experiment 4). These data demonstrated that the linear peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 were highly immunogenic and protective.

                              
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  		TABLE 4.   Antibody levels and protection in A/J mice immunized with 25 µg of linear peptides in TiterMax and challenged with 100 P. yoelii sporozoites or 200 P. yoelii-infected erythrocytes

To further evaluate whether the linear peptide containing putative epitopes of either the NYLS2 or NYLS3 MAb could induce protection, A/J mice were immunized with linear peptide SFPMNEESPLGFSPE or GFSPEEMEAVASKFR in TiterMax and challenged with 100 P. yoelii sporozoites. Data in Table 5 clearly demonstrate that both 15-aa peptides could induce protection in the absence of antibodies.

                              
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  		TABLE 5.   Protection in A/J mice immunized two times at 3-week intervals with linear 15-aa peptides in TiterMax and challenged with 100 P. yoelii sporozoites

Antibody isotypes. To further characterize the antibody responses, we conducted antibody subclass analysis. Immunization of mice with MAP4(SFPMNEESPLGFSPE)3P2P30 induced similar levels of IgG1, IgG2a, IgG2b, and IgG3 in A/J (protected), BALB/c (nonprotected), C57BL/6 (nonprotected), and CD1 (protected) mice (Fig. 3A, C, and D). The C57BL/6 (nonprotected) mice had lower levels of IgG2a than IgG1, IgG2b, and IgG3 (Fig. 3B). IgM antibodies were detected in all strains of mice at very low levels (Fig. 3). Similar results were noted following immunization of mice with MAP4(GFSPEEMEAVASKFR)3P2P30 (data not shown).


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  		FIG. 3.   Antibody isotypes in sera of A/J, C57BL/6, BALB/c, and CD1 mice immunized with MAP4(SFPMNEESPLGFSPE)3P2P30 in TiterMax. Sera collected from mice 10 days after the third immunization (prior to challenge) were analyzed by ELISA against (SFPMNEESPLGFSPE)3, using heavy-chain-specific goat anti-mouse immunoglobulins as detecting antibodies. Data are shown as mean ± SD of the OD readings of quadruplicate assays.

Immunization of A/J mice with linear peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 induced significantly higher levels of IgG2a than of IgG1, IgG2b, and IgG3, but IgM was undetectable (Fig. 4). In a subsequent experiment, immunization of three inbred mouse strains (A/J, C57BL/6, and BALB/cByJ) with these peptides in TiterMax protected only A/J mice against P. yoelii sporozoites challenge, and only A/J mice produced more IgG2a than IgG1 (data not shown).


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  		FIG. 4.   Antibody isotypes in sera of A/J mice immunized with linear synthetic peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 in TiterMax. Sera collected from mice 10 days after the second immunization (prior to challenge) were analyzed by ELISA against the relevant peptide, using heavy-chain-specific goat anti-mouse immunoglobulins as detecting antibodies. Data are shown as mean ± SD of the OD readings of quadruplicate assays.

Passive immunization. To further assess whether the polyclonal antibodies against the linear peptides could protect mice against sporozoite challenge, A/J mice were passively immunized with antibodies purified from sera of A/J mice immunized with (SFPMNEESPLGFSPE)3 or (GFSPEEMEAVASKFR)3 in TiterMax or from sera of TiterMax control mice. The recipient mice received P. yoelii sporozoites prior to antibody transfer. None of these mice were protected against sporozoite challenge (data not shown). Differences in parasitemia density among groups of mice were evaluated by using repeated measure analysis of variances. There was no difference in parasitemia density between groups of mice (data not shown).

ILSDA. Despite the genetic restriction of protection and the lack of association between antibody titers and protection, we could not exclude the possibility that antibodies that recognized parasite-infected hepatocytes play a role in the protection. Sera from immunized mice were analyzed by ILSDA to determine their in vitro inhibitory effect on liver-stage parasite development. Since HEP17 protein is not expressed at the sporozoite stage of the life cycle, immunization with HEP17 peptides did not induce antibodies against P. yoelii sporozoites (data not shown); therefore, the in vitro effect on sporozoite invasion of hepatocyte culture was not determined. Sera from MAP vaccine-immunized mice had low to moderate levels of inhibition (15 to 63%) compared to sera from TiterMax control mice (Table 6). The level of inhibition did not correlate with protection against sporozoite challenge.

                              
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  		TABLE 6.   Inhibition of liver-stage parasite development of sera from four strains of mice immunized with MAP vaccines in TiterMax

Sera from the protected A/J mice immunized with linear peptides (SFPMNEESPLGFSPE)3 and (GFSPEEMEAVASKFR)3 or the purified polyclonal antibodies had some inhibitory effect (31 to 56% inhibition) (Table 7). These inhibitory levels were lower than that produced by NYLS3 MAb (98% inhibition) (4), which recognizes SFPMNEESPLGFSPE. Nonetheless, these results support our contention that antibodies against HEP17 are not the main immune effector mechanism responsible for the sterile protection found after active immunization.

                              
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  		TABLE 7.   Inhibition of liver-stage parasite development of sera or purified antibodies from A/J mice immunized with linear synthetic peptides in TiterMax

Depletion. Since data indicated that protection is not mediated by antibodies, we then determined whether this protection required T cells and IFN-gamma . A/J mice were immunized with two doses of (SFPMNEESPLGFSPE)3 or (GFSPEEMEAVASKFR)3 in TiterMax, then treated with MAbs specific for CD4+ T cells, CD8+ T cells, or IFN-gamma , and challenged with P. yoelii sporozoites. Peptide-immunized untreated and TiterMax-immunized mice were included as positive and negative controls, respectively. Immunized untreated mice consistently had 100% portection. Treatment of (SFPMNEESPLGFSPE)3-immunized mice with rat immunoglobulin control or with a MAb specific for CD8+ T cells did not alter the level of protection. However, treatment with a MAb specific for CD4+ T cells significantly reduced the protection to the level found in mice immunized with adjuvant alone (10%), and treatment with anti-IFN-gamma MAb completely eliminated protection (Table 8, experiment 1). These findings indicate that protection induced by (SFPMNEESPLGFSPE)3 was dependent on CD4+ T cell and IFN-gamma but not on CD8+ T cells. Similar results were obtained for (GFSPEEMEAVASKFR)3-immunized mice (Table 8, experiment 2). Protection after CD4+ T-cell depletion was reduced to the same level as in mice immunized with adjuvant alone (30%), and anti-IFN-gamma MAb completely eliminated protection. Interestingly, in this experiment, CD8+ T-cell depletion was associated with a modest reduction in protection, but this reflects only 3 of 10 mice, and the difference was not significant (P = 0.21).

                              
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  		TABLE 8.   Depletion of CD4+ and CD8+ T cells from linear peptide-immunized A/J micea or treatment with anti-IFN-gamma eliminates protection


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

When sporozoites are inoculated by mosquitoes or by i.v. injection, they are extracellular in the circulation for less than 30 min before entering hepatocytes, the only host cell in the life cycle that consistently expresses major histocompatibility complex molecules on its surface. In the P. yoelii rodent model, the liver-stage cycle is approximately 48 h, and then the parasites emerge from the liver into the bloodstream and invade erythrocytes, initiating the erythrocytic-stage cycle of invasion, development, and rupture, responsible for the pathology and clinical manifestations of malaria.

Immunization of inbred A/J, C57BL/6, and BALB/c mice with linear synthetic peptides containing one and three copies of SFPMNEESPLGFSPE or GFSPEEMEAVASKFR protected A/J mice (Tables 4 and 5), but not C57BL/6 and BALB/c mice against parasite challenge (data not shown). Isotype analysis of sera from (SFPMNEESPLGFSPE)3- or (GFSPEEMEAVASKFR)3-immunized mice demonstrated that the protected A/J (Fig. 4) but not the nonprotected C57BL/6 and BALB/c mice (data not shown) produced higher levels of IgG2a than of IgG1 antibodies. These results suggest that protection induced in A/J mice by immunization with HEP17 linear synthetic peptides is associated with a Th1-type immune response and IFN-gamma production. It is possible that the nonprotected mice are not able to produce a sufficient quantity of IFN-gamma , which is absolutely required for this peptide-induced protection. The exact mechanism(s) underlying this protective immunity remain to be elucidated.

Antibodies in mice immunized with MAP vaccines or linear peptides have modest anti-infected hepatocyte activity (Table 7). Data demonstrate that polyclonal antibodies recognize and eliminate parasite-infected hepatocytes as previously reported for NYLS3 MAb (4). However, the level of inhibition noted in vitro (40 to 50%) was significantly less than that of the NYLS3 MAb (98%) (4). From experience, at least 90% activity in vitro is required before an in vivo protective effect is seen. The mechanism of antibody mediated inhibition of liver-stage parasite development remains to be delineated. Preliminary data indicated that HEP17 is exported from the parasitophorous vacuole into the cytoplasm of the infected hepatocyte and that the protective NYLS3 MAb, but not unrelated control MAb, enter the infected hepatocytes (data not shown), presumably by interacting with exported protein at the surface of the hepatocyte. However, this surface localization of HEP17 protein has not been established. In contrast, antibodies against sporozoites that confer complete protection either after passive transfer (3) or active immunization (30) inhibit parasite development in vitro by 92 to 99%.

The data presented herein clearly demonstrate that immunization of inbred A/J mice with a 45- or 15-aa linear, HEP17-derived synthetic peptide, (SFPMNEESPLGFSPE)3, (GFSPEEMEAVASKFR)3, SFPMNEESPLGFSPE, or GFSPEEMEAVASKFR, induces consistent sterile protective immunity against sporozoite challenge (Tables 4 and 5) that is dependent on CD4+ T cells and IFN-gamma (Table 8) but that is not dependent on antibodies (Table 5). The protective immunity must be directed against the infected hepatocyte because HEP17 is not expressed by sporozoites and antibodies against HEP17 do not recognize sporozoites (4), and immunization with HEP17-derived peptides did not protect against challenge with infected erythrocytes (Table 4).

Since protection induced by the peptide (SFPMNEESPLGFSPE)3 is completely dependent on CD4+ T cells and IFN-gamma , we hypothesize that HEP17 is processed within the infected hepatocytes and HEP17 peptides are then presented, in association with class II major histocompatibility complex (22) molecules on the cell surface, to CD4+ T cells. These T cells release IFN-gamma , which may then initiate a process leading to elimination of the infected hepatocytes. This may be due to induction of inducible nitric oxide synthase and nitric oxide production, as we have shown following immunization of mice with a HEP17 DNA vaccine (9), or by another mechanism that remains to be defined. Interestingly, immunization with another HEP17 peptide, (GFSPEEMEAVASKFR)3, induces protection that is dependent on CD8+ T cells, in addition to CD4+ T cells and IFN-gamma . Both cell types may produce IFN-gamma responsible for elimination of infected hepatocytes. Further work is necessary to clarify this hypothesis.

While the mechanism of protection has been defined at least in part, the characteristics of the protective peptides require further elucidation. The linear peptides described herein, and the SSP2 peptide previously reported (29), induce similar CD4+ T-cell-dependent protection. However, all these peptides also include B-cell epitopes recognized by MAbs derived by immunizing with either sporozoites (2) or infected hepatocytes (4), native parasite material.

The demonstration of CD4+ T-cell-mediated protection induced by these different linear peptides raises the possibility of developing a vaccine based on multiple linear CD4+ T-cell epitopes. We have recently proposed a method to accomplish this by using genomic sequence data to identify such epitopes (14). A vaccine based on these epitopes could then be constructed as a mixture of the peptides or large recombinant proteins administered with adjuvant or as DNA vaccines expressing all of the epitopes. Preliminary data obtained by mixing two HEP17 peptides (data not shown) suggests an additive effect, supporting this approach. Subsequent studies will be designed to validate this approach in the P. yoelii rodent model and extend this approach to the P. falciparum human malaria model.


    ACKNOWLEDGMENTS

We thank Martha Sedegah, Arnel Belmonte, and Rumeo Wallace for providing the P. yoelii-infected mosquitoes, Fred D. Finkelman for providing the anti-IFN-gamma MAb, and Dorina C. Maris for FACScan analysis.

This work was supported by Naval Medical Research Development and Command Work Units. 611102A.S13.00101.BFX.1431 and 612787A.870.00101.EFX.1432. The work was performed in part while D.L.D. held a National Research Council-Naval Medical Research Institute Research Associateship.


    FOOTNOTES

* Corresponding author. Mailing address: Malaria Program, Naval Medical Research Center, 12300 Washington Ave., Rockville, MD 20852. Phone: (301) 295-1177. Fax: (301) 295-6171. E-mail: charoenvity{at}nmripo.nmri.nnmc.navy.mil.

Editor:   J. M. Mansfield


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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Infection and Immunity, November 1999, p. 5604-5614, Vol. 67, No. 11
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Copyright © 1999, American Society for Microbiology. All rights reserved.


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