Evaluation of Immunogenicity of Novel Isoform of EG95 (EG95-5G1) From Echinococcus granulosus in BALB/C Mice

Background: Echinococcosis is a zoonotic parasitic disease of humans and various herbivorous domestic animals transmitted by the contact with domestic and wild carnivores, mainly dogs and foxes. The aim of this study is the production, purification and evaluation immunogenicity of new construction of EG95 protein. Methods: The recombinant plasmid pET32-a(+) used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The purification was performed under denaturing conditions in the presence of 8M urea by Ni-NTA column and dialysis. The purified recombinant proteins were confirmed with western blot analysis using polyclonal antiserum. To find out the immunogenicity of the purified protein, the BALB/c mice (10 mice/group) were immunized by injecting 20 mu g rEG95 protein formulated in Freund's and alum adjuvant. Results: Immunization of mice with rEG95 using CFA/IFA and alum adjuvant generated high level of total antibody. In proliferation assay, the lymphocytes were able to mount a strong proliferative response with related production of IFN-gamma, IL-12 and TNF-alpha but with low secretion of either IL-4 or IL-10. The humoral and cellular immune responses against rEG95 suggested a mixed Th1/Th2 response with high intensity toward Th1. Conclusion: Our findings suggest that new construct of rEG95 formulated with CFA/IFA and alum adjuvant elicited strong cellular and humoral responses supporting further development of this vaccine candidate.