Protection against Asiatic Taenia solium Induced by a Recombinant 45W-4B Protein

Pigs and eggs.Fifteen 50-day-old healthy pigs were purchased from a local area without occurrence of cysticercosis, in which anticysticercosis antibodies were not detected by enzyme-linked immunosorbent assay (ELISA). The experimental protocol was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

An adult worm recovered from a patient with taeniasis was completely scissored to release T. solium eggs. After being washed three times in saline, the released eggs were recovered by centrifugation and counted with a McMaster egg counter under a microscope. Viable and mature eggs were judged mainly by their morphology, such as structural integrity and visible hooks.

High expression and purification of the recombinant 45W-4B protein.The recombinant plasmid with removal of encoding signal peptide bp 51 at the 5′ terminus and encoding hydrophobic amino acid bp 57 at the 3′ terminus, designated as pGEX-45W-4B, was constructed previously in our lab. The cloning and expression of the recombinant 45W-4B protein were conducted as previously described (11). Briefly, the positive recombinant Escherichia coli BL21 cells were inoculated into 2× YT medium and induced with a final concentration of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 37°C for 5 h. Afterwards, the cells were collected by centrifugation, washed in phosphate-buffered saline (PBS; pH 7.2) several times, resuspended in cell lysis buffer containing 0.1 mg/ml lysozyme, and lysed using five freeze-thaw cycles. The fusion protein was purified using glutathione Sepharose 4B (Amersham) according to the protocols described before (17). The purity level and concentration of the purified protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a biophotometer (Eppendorf).

Preparation of crude antigens from fresh metacestodes.Crude extracts were prepared from T. solium cysticerci strictly according to the procedures as Cai et al. reported previously (2).

Immunization and experimental infection of pigs.In trial 1, 15 pigs were separated into three groups of five animals each. Vaccination of pigs with recombinant 45W-4B or crude antigens was performed prior to an experimental challenge infection with T. solium eggs. Immunizations were given intramuscularly in the neck. Pigs in group A were immunized with PBS used as the control. Group B was immunized with 1,200 μg of crude antigens extracted from T. solium metacestodes, and group C was immunized with 200 μg of purified 45W-4B. Twenty-one days later, a booster was conducted at the same site. Each pig was experimentally infected with 25,000 mature viable T. solium eggs 7 days after the booster. Control and immunized pigs were slaughtered 93 days after challenge. Naked-eye inspection was swiftly performed as soon as the experimental pigs were humanely slaughtered. Pig organs and tissues, including muscles, brains, and tongues, were sliced into small pieces with knives and scissors. Afterwards, dissected cysticerci were collected and counted.

In order to further evaluate the efficiency of the recombinant 45W-4B protein as an anticysticercosis vaccine, the vaccination trial in pigs was repeated as described for trial 1. As mentioned above, pigs in group A were immunized with PBS used as the control, and those in group B were immunized with 200 μg of purified 45W-4B. In the groups with immunization of recombinant 45W-4B or crude antigen in the two trials, an adjuvant, ISA 206 (Seppic, France), a kind of water-in-oil-in-water adjuvant, was utilized as an immune enhancer. The significance of the reduction induced by vaccination was determined using the Mann-Whitney test.

Detection of specific antibody in growing pigs by ELISA.Serum samples were collected and separated from each pig in the first vaccine trial 15, 40, 55, 75, 105, and 117 days postimmunization (PI). Specific antibody levels were detected by ELISA by the protocols described previously (17). In ELISA, the purified recombinant 45W-4B was used as a coating antigen. The antibody levels were compared using an unpaired t test.

Identification of the recombinant 45W-4B.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that the recombinant 45W-4B protein with a molecular mass of 40 kDa was mainly in the supernatant of the lysate. Using affinity chromatography, the highly purified target protein was obtained and its final concentration reached 3.2 mg/ml.

Anti-45W-4B specific antibody in the experimental pigs in trial 1.Compared with pigs in group A, pigs immunized with the recombinant 45W-4B vaccine obviously showed increases in the level of anti-45W-4B specific antibody 15 days PI (Table 1). Moreover, the antibody level of all the immunized pigs in group C reached a summit 30 days after the booster and lasted for long time, up to at least 3 months.

Protection against T. solium egg challenge.In trial 1, vaccination with the recombinant 45W-4B antigen significantly reduced the number of viable cysticerci recovered (P < 0.01), giving a 97.0% reduction, 1.1% higher than the 95.9% reduction in the pigs with immunization of crude extracts of metacestodes (Table 2). No significant differences were observed between the group immunized with 45W-4B and that immunized with crude antigens. There was only one pig completely protected against experimental infection with T. solium eggs, each, in groups B and C.

In the repeated vaccination, the reduction induced by the 45W-4B vaccine was up to 98.4%, slightly higher than that in group C in trial 1, and was obviously significant (P < 0.01) in comparison with the control. No viable cysticerci were found in three of five pigs immunized with the recombinant protein.