INTRODUCTION

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Taenia solium cysticercosis is a common parasitic disease of the central nervous system of humans in several countries in Latin America, Africa, and Asia, where it represents a major health and economic problem (2, 28). The life cycle of this parasite includes a larval phase (cysticercus) that affects both pigs and humans after the ingestion of T. solium eggs. The parasite's life cycle is completed when humans consume improperly cooked cysticercotic pork and the adult intestinal tapeworm develops and, in turn, produces millions of eggs that are shed in human feces. In regions of endemic infection, transmission is clearly related to prevailing low standards of personal hygiene and environmental sanitation control (i.e., open air fecalism) in areas where rustic rearing of pigs is practiced by the rural population (pigs roaming about freely in search of edibles and/or deliberately fed with human feces [11]). Regrettably, control of transmission by general improvement of the social, economical, and educational status in developing countries or by proper and strict meat inspection programs is not within reach in the near future. However, since the pig is an indispensable intermediate host, transmission could be hindered by lowering the prevalence of pig cysticercosis through vaccination. Development of an effective vaccine to be used in pigs is being pursued by a number of scientists, with promising results (9, 15-17).

Because of the high costs of experimentation in pigs, murine cysticercosis caused by Taenia crassiceps has been used to test and select promising antigens before they are tested in pigs (13, 21). Thus, it has been shown that total T. crassiceps antigens can cross-protect pigs against T. solium cysticerosis. However, the effects of vaccination with whole-antigen extracts were strongly dose dependent; besides, some antigens were found to be protective while others led to facilitation of the infection (22). Such complications with the use of whole-antigen extracts led us to redirect our research to the identification of individual protective antigens (14, 26). Using recombinant DNA technology, several vaccine candidates were identified in murine T. crassiceps cysticercosis with crude lysates of the respective clones as the immunogen (13, 14). One of them, KETc7, which has a protective capacity confirmed by DNA immunization (1, 20), includes at least one protective epitope of 17 amino acids (GK1). GK1 is also expressed in T. solium oncospheres (25), the parasite's developmental stage most vulnerable to immunological attack (19). Two additional protective clones, KETc1 and KETc12 (14), were also identified. Herein we report the protective capacity against T. crassiceps murine cysticerosis of the peptides deduced from these last two clones. Furthermore, we describe the localization of the peptides in each parasite stage of T. solium and T. crassiceps, the immune response they elicit in immunized micewhere T1 is most prominentand propose them as additional components for a synthetic vaccine to be tested in pigs in an attempt to block T. solium transmission.

	MATERIALS AND METHODS

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Peptides. Two T. crassiceps-derived peptides (14) that are shared by T. solium (24), KETc1 [APMSTPSATSVR(G)] and KETc12 [GNLLLSCL(G)], were synthesized by stepwise solid-phase synthesis with N-tert-butyloxycarbonyl derivatives of L-amino acids on phenyl-acetamidomethyl resin (Sigma Chemical Co., St. Louis, Mo.). The peptides were 95% pure as judged by high-pressure liquid chromatography in an analytical C₁₈ reversed-phase column (3.9 by 150 mm; Delta Pak [Waters]). The correct amino acid sequence of each peptide was confirmed by protein sequencing on a pulsed-liquid-phase protein sequencer (Applied Biosystems) at the National Institute of Cardiology, Mexico City.

Mice. BALB/cAnN mice, previously characterized as susceptible to cysticercosis (3), were used in vaccine trials. The original murine stock was purchased from M. Bevan (University of Washington) and then bred and kept in our animal facilities by the "single-line breeding" system for more than 30 generations. All mice used were males that were 5 to 7 weeks of age at the beginning of the experiments. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animals Resources, National Research Council, Washington, D.C.

Immunization of mice and serum collection. Groups of six to nine BALB/cAnN mice were subcutaneously immunized with two doses of 10 µg of each individual peptide in saponin (Sigma Chemical Co.) per mouse at a concentration of 100 µg/mouse as described elsewhere (25). This dose was determined as optimal in collateral experiments (data not shown). Ten days later, the mice were given a booster with the same immunizing dose of the same peptide in the same adjuvant as used before. Immune sera were obtained from each individual mouse before and after each immunization and stored at 70°C until individually tested for the presence of specific antibodies.

Parasites and cysticercal antigens. The ORF strain of T. crassiceps (4) has been maintained by serial passage in BALB/cAnN female mice for 15 years in our animal facilities. Cysticerci for infection were harvested from the peritoneal cavity of mice 1 to 3 months after inoculation of 10 nonbudding small cysticerci (2 to 3 mm in diameter) per animal. The soluble antigens were recovered from similar cysticerci by a previously described procedure (18). Whole T. solium cysticerci were dissected from skeletal muscle of highly infected pork carcasses 2 to 4 h after slaughter in an abattoir in Zacatepec, Morelos, Mexico; embedded in optimun-cutting-temperature compound (Miles, Inc.), and frozen at 70°C until used in immunofluorescence assays (see below). Segments from T. solium tapeworm and eggs were obtained from the feces of an infected man in Puebla, Mexico. The tapeworm was recovered after treatment with a single oral dose (2 g) of niclosamide (Yomesan; kindly supplied by Bayer). After being washed in saline plus antibiotics (100 U of penicillin per ml plus 100 µg of streptomycin per ml), several gravid proglottids were separated for immunofluorescence assays.

ELISA for antibody measurements. T. crassiceps whole soluble antigens (TcAg) were obtained as previously described (18) and used as the source of antigens in an enzyme-linked immunosorbent assay (ELISA) to measure the antibody response induced by peptide immunization by using a procedure described elsewhere (25).

Proliferation assay. Spleen cells from control and KETc1- and KETc12-immunized mice were harvested 15 days after the second immunization and cultured in RPMI 1640 medium supplemented with L-glutamine (0.2 mM), nonessential amino acids (0.01 mM), penicillin (100 U/ml), streptomycin (100 µg/ml) and fetal bovine serum FBS (10%). The cells were cultured with the appropriate concentration of concanavalin A (ConA) (5 µg/ml), KETc1 (50 µg/ml), KETc12 (10 µg/ml), or TcAg (10 µg/ml) and incubated at 37°C in a 5% CO₂ humidified atmosphere in flat-bottomed microtiter plates at a cell concentration of 2 × 10⁵ cells per 200 µl of final volume. Then 10⁵ peritoneal cells recovered from the same mice were added to each well in a volume of 50 µl. Peritoneal cells were obtained by ex vivo lavage with 5 ml of RPMI 1640 medium. After 72 h, the cultured cells were pulsed (1 µCi per well) for a further 18 h with [methyl-³H]thymidine (Amersham Life Science, Little Chalfont, United Kingdom). Then all the cells were harvested and the amount of incorporated label was measured by counting in a 1205 -plate spectrometer (Wallac).

Spleen cell phenotype analysis. After 3 days of in vitro culture with medium, TcAg, or peptides, splenocytes were harvested and CD8 and CD4 expression was determined by staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (Pharmingen, San Diego, Calif.) and phycoerythrin-conjugated anti-CD4 (Pharmingen), respectively, by a previously reported procedure (25). Parallel samples of the cells were stained with the corresponding isotype control to account for nonspecific staining of the cells. Briefly, the cells were washed with phosphate-buffered saline (PBS) containing 10% gamma globulin-depleted FBS plus 0.02% NaN₃ and incubated with the indicated antibodies at 4°C for 30 min. After being washed, the splenocytes were resuspended in cold 1% formaldehyde in isotonic solution and analyzed with a FACScan instrument (Becton Dickinson, Palo Alto, Calif.). The results are expressed as a percentage of positive cells.

Cytokine measurements. For detection of intracellular cytokines, spleen cells were treated with medium, KETc1, KETc12, or TcAg and cultured for 60 h. To inhibit cytokine secretion, brefeldin A (2 µM) was added to the cell cultures 10 h before the assay. At harvest, the cells were centrifuged at 500 × g for 10 min and washed twice in ice-cold PBS containing 10% gamma globulin-depleted FBS plus 0.02% NaN₃. CD3 and interleukin (IL) expression were determined by two-color fluorescence-activated cell sorting (FACS) as previously described (25). Briefly, the cells were stained with biotin anti-CD3 (Pharmingen) and then streptavidin-FITC (Sigma) was added. Intracellular cytokines were assayed by using a cytoStain TM kit (Pharmingen) to fix and permeabilize the cells. To stain intracellular cytokines, fixed and permeabilized cells were incubated with phycoerythrin-conjugated monoclonal rat anti-IL-2, anti-IL-4, anti-IL-10, or anti-gamma interferon (INF-) (all from Pharmingen). Parallel samples of the cells were stained with isotype control to account for nonspecific cell staining. Then 10⁵ cells were analyzed with a CD3⁺ lymphocyte gate as defined by light scatter in a FACScan instrument. The results are expressed as a percentage of positive cells.

Experimental challenge. Metacestodes used in challenge infections were harvested from the peritoneal cavity of BALB/cAnN female mice carrying the ORF strain of T. crassiceps cysticerci. Ten small (diameter, ca. 2 mm), nonbudding larvae were suspended in 0.5 M NaCl-0.01 M sodium phosphate buffer (pH 7.2) and intraperitoneally injected into each challenged mouse using a 27-gauge needle (this procedure disrupts the cysticerci upon entry, but the fragments reorganize into cystic structures in a matter of a few days [24]). Mice were killed 30 days after infection, and the cysts found inside the peritoneal cavity were counted. In this form of infection, the parasites do not migrate to another location in the host. The variation in individual parasite intensities within groups of vaccinated and control mice was attributed to differences in the infectivity of each parasite inoculum. In consequence, each experiment measuring levels of immunity by parasite intensity always included a group of nonimmunized mice to assess the infectivity of each inoculum. Thus, the effects of immunization measured in each experiment were contrasted with the control group.

Immunolocalization of KETc1 and KETc12 protein. T. crassiceps cysticerci and T. solium specimens (cysticerci and gravid proglottids) were placed on ice in a 50-ml conical plastic-bottom centrifuge tube containing ice-cold PBS. All tissues were treated to prepare slides as previously reported (20). The slides were rehydrated and blocked with 1% bovine serum albumin (BSA) in PBS plus 0.1% Triton X-100 (pH 7.2) (PAT) for 1 h. A second blocking in cysticercus-infected tissue sections was performed with sheep anti-mouse IgG (whole antibody; Amersham) diluted 1:100 in PBS plus 0.1% BSA, and then the samples were incubated for 1 h at 4°C. Slides of T. solium tapeworm and eggs were incubated 1 h at 4°C with horse serum diluted 1:100 in PBS plus 0.1% BSA as a second blocking agent. The solutions were removed, and the slides were overlaid with the appropriate sera from noninfected (negative control), infected (positive control), or anti-KETc1- or anti-KETc12-immunized mice diluted 1:10,000 in PBS plus 0.1% BSA, incubated overnight at 4°C, and then washed twice in PBS (pH 7.2). Finally, sections were incubated with FITC-labeled goat anti-mouse immunoglobulin G (Zymed) diluted 1:50 for 1 h at room temperature. The slides were washed twice and mounted with aqueous mounting solution (Zymed). Preparations were observed with an epifluorescence microscope Olympus BH2-RFCA.

Statistical analysis. Statistical comparison of individual parasite intensities between groups was performed by the Kruskal-Wallis nonparametric analysis of variance ANOVA test because many mice contained zero parasites in the immunized groups and because parasite intensity is a discontinuous variable (i.e., 0, 1, 2, ... n parasites). Data were considered statistically significant at P < 0.05. A Student-Newman-Keuls multiple-comparison test was used to measure the statistical significance between the immune response elicited in vaccinated and control mice.

	RESULTS

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Protective effect of peptide immunization against T. crassiceps cysticercosis. The effect of peptide immunization on the number of cysticerci recovered from mice immunized with KETc1 and KETc12 or adjuvant alone (controls) is shown in Table 1: 66.7, 75.3 or 100% protection was induced using KETc1 as immunogen, and 52.7, 73.4, or 88.1% protection was induced using KETc12 as immunogen. Some mice were completely protected (no parasites) by immunization with either KETc1 or KETc12.

Antibody response induced by KETc1 and KETc12 immunization. To test for the presence of a B-cell epitope(s) within the two peptides, the levels of induced anti-KETc1 and anti-KETc12 specific antibodies were assessed. T. crassiceps cysticercal antigens (Fig. 1) as well as each of the peptides were used as antigens in ELISAs (data not shown). Figure 1 shows low but detectable levels of serum antibodies in both KETc1- and KETc12-immunized mice.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Antibody levels determined by ELISA in individual control (C) and immunized (I) mice against TcAg. The mean level of antibodies was significantly higher in immunized mice than in controls. O.D., optical density.

Immunolocalization of KETc1 and KETc12 in the parasite. Pooled sera with the highest antibody levels induced by KETc1 and KETc12 immunizations were used to immunolocalize the native antigen in both T. crassiceps and T. solium (Fig. 2 and 3). KETc1 and KETc12 were expressed in the tegument of T. crassiceps cysticerci, albeit with different distributions. KETc1 was restricted to the tegument of both cysticerci (Fig. 2E and F), while in T. solium it was found in the most external part of the tegument and also in the cuticular folds of the spiral canal (Fig. 2F). KETc12 (Fig. 2G and H) was very abundant in both metacestodes. Nevertheless, the T. crassiceps tegument showed an intensely positive wall surface and parenchyma, especially around the calcareous corpuscules (Fig. 2G). KETc12 was also detected in the oncosphere of the egg as numerous points (Fig. 3G), in contrast to KETc1, which was almost negative. Both epitopes were present in tapeworm tissue: KETc1 was very abundant on the most external side of the tegument (Fig. 3F), and KETc12 was distributed along the tegument's depth (Fig. 3H). When sera from infected mice were used, all structures were fluorescent (Fig. 2C and D and 3C and D). The specificity of these antibody reactions was demonstrated by the lack of reactivity of normal mouse serum with the used tissues (Fig. 2A and B and 3A and B).

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Immunofluorescent staining of T. crassiceps (A, C, E, and G) and T. solium (B, D, F, and H) cysticerci. Sections of 6 µm were processed and incubated with pooled sera from noninfected mice (A and B), T. crassiceps-infected mice (C and D), and KETc1-immunized (E and F) and KETc12-immunized (G and H) mice. The tegument (t) and the parenchyma (p) are evident in both cysticerci (C and D). In T. crassiceps cysticerci (E), KETc1 antigen shows a protruding and intensely positive signal in the tegument, while in T. solium cysticerci (F) it is clearly evident in the cuticular folds of the spiral canal (cf). KETc12 is quite abundant in both metacestodes; it is evident in the tegument and in the parenchyma of T. crassiceps (G) as well as in the tegument, parenchyma, and flame cells (arrows) of T. solium (H). Bar, 40 µm.

View larger version (65K): [in this window] [in a new window]   FIG. 3.   Immunofluorescence staining of the T. solium oncosphere (A, C, E, and G) and proglottid tegument (B, D, F, and H). Sections of 6 µm were processed and incubated with pooled sera from noninfected mice (A and B), T. crassiceps-infected mice (C and D), and KETc1-immunized (E and F) and KETc12-immunized (G and H) mice. It is evident that the oncosphere (o) and the distal cytoplasm region (dc) (C and D, respectively) stain positively. Some structures of the perinuclear cytoplasm region (pe), like the protoplasmic extensions of the tegumental cells, are also apparently positive. KETc1 antigen is almost negative in the oncosphere and appears as little positive spots (arrows); in contrast, in adult tissue (F) it is quite evident in the distal cytoplasm region of the tegument. The KETc12 antigen is only slightly present in the oncosphere (o) but is quite conspicuous in the distal cytoplasm region and in the perinuclear cytoplasm region of the adult tissue. Bar, 40 µm.

Assessment of T-cell epitopes on the KETc1 and KETc12 peptides. The proliferative response of spleen cells from mice immunized with KETc1 or KETc12 or saponin alone is reported in Table 2. Spleen cells from mice injected in vivo with free peptides or saponin were stimulated in vitro with the corresponding peptide, TcAg, or ConA in previously determined optimal concentrations. Table 2 shows that in vitro stimulation with KETc1 or KETc12, as well as with cysticercal antigens, induced a significantly greater proliferative response in cells from immunized mice than in those from control mice. Cells from mice injected with saponin (controls) showed no proliferative response above background levels.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Flow cytometer analysis of spleen cells from KETc1- and KETc12-immunized mice with or without in vitro stimulation with the respective peptide or antigen (TcAg) R1 denotes the region of proliferating cells in the SSC/FSC plot (side-scatter/forward-scatter plot), and the number below indicates the percentage of cells in this region. CD4+ and CD8+ cell percentage expression was determined in the defined R1 gate.

	DISCUSSION

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Our results show that KETc1 and KETc12 induce protection against experimental murine T. crassiceps cysticercosis and that both are B- and T-cell epitopes. The protective capacity and the immunity induced by these two peptides closely resemble those induced by GK1, a previously reported protective epitope also shared by T. crassiceps and T. solium (25). It should be noted that there are two forms of expressing protection; i.e., reduction in parasite intensity and proportion of totally parasite-ridden mice. Because T. crassiceps cysticerci multiply asexually in the peritoneal cavity of infected mice, the reduction in parasite intensity is highly dependent on the time of assessment after infection; the effects of vaccination tending to disappear in late infections. Sterile immunity is attained only if the initial inoculum is totally destroyed by the immune system response. These and previous results are in accordance with the notion that if a single T. crassiceps cysticercus evades or survives the initial immune attack of the murine host, it will multiply and eventually reach very high parasite intensities indeed (3). This initial immune attack would appear must successful if a strong T1 response is induced, as we have shown here and others have shown previously (23). In older infections, when massive parasite intensities (>10³) are achieved, T2 responses predominate and perhaps downregulate T1 (23). These two peptides would appear to touch off the protective T1 response more efficiently than the T2 response.

The immunologic assays performed in our experiments indicate the immune mechanisms involved in infection control. It has been repeatedly stated that protection induced by vaccination against T. crassiceps murine cysticercosis is T1 related whereas antibodies and other T2 molecules are less effective (1). In this study, results point in the same direction: while antibodies are erratically and weakly induced by both KETc1 and KETc12, IL-2 induction is noticeably increased 5.7- to 10.1-fold in immunized mice relative to control mice. The same is true for IFN-, the characteristic inflammatory cytokine, which activates macrophages in the vicinity of the parasite and triggers their well-known damaging effects (25). In addition to the preponderance of the inflammatory interleukins IFN- and IL-2, the low profiles of IL-4 and IL-10, which inhibit the proliferation of the T2 responses, could well explain the low levels of antibodies elicited by both peptides. Protective immunity in the context of a T1 response has also been related to innate resistance conditions (23, 27). Despite the low levels of specific antibodies induced by both peptides, their possible protective role cannot be excluded. This is of particular relevance considering the recent finding of the capacity of anti-GK1 antibodies to block T. solium cysticercus conversion to tapeworms (5). The fact that more than 50% of human neurocysticercosis patients make antibodies against KETc1 and KETc12 (8) strengthens our interest in these two epitopes.

Based on the different anatomic distribution of KETc1 and KETc12 in T. solium cysticerci and in oncospheres, which also differ from that of GK1, it would appear that all three peptides should be included in a vaccine against pig cysticercosis to maximize the number of targets. In spite of the risk associated with extrapolating from T. crassiceps and mice to T. solium and pigs, optimism about vaccine development prevails because of the many examples of effective immunity induced against different cestodes in diverse hosts. Also, the extensive similarities among cestode infections in terms of their natural history, pathology, and antigenic composition all point to possibly similar effects of vaccination (6, 7, 10). In fact, different sources of protective antigens have been successfully used as vaccines against porcine cysticercosis, one using recombinant antigen from Taenia ovis (12) and the other using antigen from T. crassiceps itself (13).

In the hope of increasing the efficiency of vaccination, it is advisable that KETc1 and KETc12 plus GK1, all of which induce high levels of protection in the murine model of cysticercosis and are present at all stages of T. solium development, be considered as candidates for inclusion in a mixed polyepitopic synthetic vaccine to be used against T. solium cysticercosis in pigs.