Two Epitopes Shared by Taenia crassiceps andTaenia solium Confer Protection against Murine T. crassiceps Cysticercosis along with a Prominent T1 Response

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^α-tert-butyloxycarbonyl derivatives ofl-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 C₁₈reversed-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 ofT. 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 withl-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 × 10⁵cells 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 β-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, orTcAg 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-γ) (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. crassicepscysticerci. 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. soliumtapeworm 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.