Dataset Information

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

ABSTRACT: 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 ?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-?, IL-12 and TNF-? 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.

SUBMITTER: Pirestani M

PROVIDER: S-EPMC4345088 | biostudies-literature | 2014 Oct-Dec

REPOSITORIES: biostudies-literature

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

Pirestani Majid M Dalimi Abdolhossein A Sarvi Shahabodin S Khoramabadi Nima N

Iranian journal of parasitology 20141001 4

<h4>Background</h4>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.<h4>Methods</h4>The recombinant plasmid pET32-a+ used for Eg95 expression was constructed with the EG95 gene of Echinococcus granulosus fused with the thioredoxin tag. This recom ...[more]

PMID: 25759730

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Project description:BACKGROUND:Cystic echinococcosis is a chronic disease caused by infection with the larvae of Echinococcus granulosus. The parasite's ability to establish persistent infection is partly due to its evolving immune evasion strategies. One strategy may involve the protective effect of arginase, which impedes the control of pathogens or tumors, whereas it remains largely unknown during E. granulosus infection. Here, we analyzed whether arginase was produced in peritoneal cells and assessed its role in immunosuppression in mice infected with protoscoleces of E. granulosus. METHODS:BALB/c mice injected with protoscoleces of E. granulosus were used to evaluate the expression of arginase (ARG) in mRNA and protein levels. The profiles of ARG-1 expression in peritoneal cells and CD3ζ expression in T cells from spleens were assessed at different time points (3, 6, 9 and 12 months post-infection) by flow cytometry. In vitro, peritoneal cells were co-cultured with purified T cells in a transwell system, and the levels of CD3ζ re-expression were compared by flow cytometry. Meanwhile, the changes of L-arginine and its related metabolites in serum were tested. RESULTS:Compared to the control group, the peritoneal cells from infected mice showed higher levels of ARG-1 mRNA and protein, unchanged ARG-2 and iNOS. Enhanced ARG-1 expression was present in SSClowCD11b+F4/80+, CD11b+CD11c+, CD11b+Gr-1+Ly-6C+Ly-6G-, CD11b+Gr-1+Ly-6C-Ly-6G+, CD11b+Gr-1+ and CD11b+Ly-6G+ cells. The proportion of cells and the proportion of ARG-1 expression in corresponding cells exhibited a rising trend along with the extension of infection time, except for fluctuations in SSClowCD11b+F4/80+ and CD11b+CD11c+ cells at 12 months post-infection, whereas the expression of CD3ζ chain in CD4+ and CD8+ T cells showed a descending trend. Purified T cells showed declined re-expression of CD3ζ when co-cultured with peritoneal cells from infected mice, and CD3ζ was regenerated by supplement of L-arginine or arginase inhibitor BEC, rather than NOS inhibitor L-NMMA or catalase. Meanwhile, the concentrations of L-arginine, L-citrulline and NO decreased, and those of L-ornithine and urea increased in serum post-infection. CONCLUSIONS:Our findings demonstrated that ARG-1 expression is enhanced in multiple myeloid cells from peritoneum and promotes immune evasion of E. granulosus in mice by inhibiting the expression of T cell receptor CD3ζ chain and antagonism against iNOS.

Dataset Information

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

Publications

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

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