Studies On The Antigenic Epitopes And Localization Of TSOL18 Of Taenia Solium On Taenia Solium Oncospheres

Taeniid cestodes infect humans and livestock, causing considerable morbidity and mortality, as well as economic loss. Substantial progress has been made toward the development of recombinant vaccines against cysticercosis in livestock animals.Cysticercosis is an important zoonosis caused by Cysticercus cellulosae, which infect human and pigs. It is a major public health and a potential threat to human beings in most areas of China, especially in minority regions, and it causes economic loss. Vaccines are primary ways to prevent cysticercosis. TSOL18 is a specifically expressed antigen at T. solium oncosphere stage and secreted in intermediate host infected by oncosphere at early stage. The immune serum of TSOL18 can kill oncospheres in vitro and recent studies demonstrated that most of the TSOL18-vaccinated pigs are protected against experimental infection. Thus, TSOL18 is one of the important potential antigens of cysticercosis vaccine. Mapping of TSOL18 antigenic epitopes is critical for vaccine design or diagnosis based on TSOL18 protein.Recombinant Pichia pastoris cells were constructed previously in our laboratory. Recombinant TSOL18 antigen was produced in a 5-liter bioreactor and induced using methanol about 72 h. The amount of TSOL18 secreted by P. pastoris was up to 2.00 g/L, accounting for more than 80% proteins in total supernatant, which was determined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified by Sephadex G-100 gel chromatography with about 90% of purity, which can fully meet the requirements of antigen to produce monoclonal antibody.The purified TSOL18 antigen was used to immunize Balb/C mice with Ferunds' complete and incomplete adjuvant. After three times'immunization and the booster injection via intraperitoneal administration, cell fusion of immunized speen cell and SP2/0 was performed with 50% PEG-1500. Twelve hybridoma cell lines were obtained stably secreting TSOL18 MAbs after being screened with TSOL18 antigen. The antibodies were identified using mouse monoclonal antibody isotyping kit as the IgG1,IgG2a,IgG2b and IgM class with light chain ofκtype. Two different recognized epitopes were identified by ELISA adding test. Specificity and titer of the MAbs was determined using indirect ELISA, and their titers were 102 and 106 in the ascites, respectively. The two of monoclonal antibodies interacted with the TSOL18 specifically. The stability and chromosome type of the hybridoma cells was performed, which proved that the monoclonal antibodies were suitable for the conventional experiment.Taenia solium eggs were hatched in a 10% sodium hypochlorite solution and viewed under a microscope. When the embryophoric blocks were disaggregating from the majority of the eggs, water was added. Taenia solium oncospheres were activated by incubation in an artificial intestinal fluid (AIF) solution of 0.5% pancreatin,0.2% sodium carbonate (anhydrous) and 1% pig bile. The activated oncospheres were centrifuged and resuspended in fresh RPMI-1640 three times. The hatching rate of T. solium eggs was approximately 95%, and the activation rate of the oncospheres approximately was 72%. Activated oncospheres were motile. The effect of the MAb on oncospheres was tested by in vitro oncospheres-killing assay. Sera from pigs vaccinated with the recombinant T. solium oncosphere protein TSOL18 killed 73% of developing T. solium larvae in vitro, and the MAbs against TSOL18 killed 52%, which was measured by 1% methylene blue. Results proved that the TSOL18 MAbs have some killing effect on T. solium oncospheres.MAb were used to define the B cell epitopes of TSOL18 with phage-displayed random dodecapeptide library (Ph.D.-12), and fifty-nine of the positive phage clones were sequenced and analyzed. The predominant mimotopes were ETTKLQRFQAML (L1) found in 83%, followed by DHTXF in 15%(L2:DHTLFAASHNHR, DHTLFSTGHSHG, and DHTFMQRYHTHQ). Comparison of the peptide sequences with native TSOL18 protein sequence using Clustal W software showed that they did not completely match, suggesting that the ETTKLQRFQAML and DHTXF sequences should be conformational epitopes. Detection of immunoreactivity of the two clones demonstrated that they were positively recognized by MAb. The binding specificity of phage clones L1 and 12 to the purified MAb was also measured and confirmed by competitive ELISA. A significant inhibition in phage L1 or L2 was obtained. The purified MAb was observed to bind with phage L1 or L2, and their affinity to TSOL18 protein reduced under the existence of the phage L1 or L2.To evaluate the immune responses of the selected mimotopes, phage clones L1 and L2 were chosen to immunize Balb/C mice using intraperitoneal administration. Serum samples were collected and the sera raised by clones L1 or L2 reacted specifically with the TSOL18 protein. The purified phages of L1 and L2 were reacted with sera of TSOL18 immunized pigs using Enzyme-linked immunosorbent assay and the binding sensitivity of individual clones with swine cysticercosis sera was 85% and 79%, respectively. These phage mimotopes may have potential for further use as diagnostic reagents and immunogens against porcine cysticercosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with interest antibody.Immunogold labelling was used to determine the localisation of the host-protective antigen TSOL18 in T. solium oncospheres. Specific immunogold staining was evident for TSOL18 in the hooks, cytoplasm and the secretory granules of bilateral glandular cells. The specificity of the labeling was evident from the lack of reactivity seen using control sera.