Immunological characterization of a multi-epitope vaccine against Echinococcus granulosus based on the extracellular structural domain of CTLA-4

Purpose The aim of this study was to provide potential for the development of an efficient multi-epitope vaccine against Echinococcus granulosus by exploring the immunological properties and immune efficacy of the recombinant antigen CTLA-4IgV-EgG1Y162-2(4) for the effective prevention of E. granulosus . Methods Target antigen sequences were retrieved from NCBI. Epitopes of CTLA-4IgV-EgG1Y162-2(4) were predicted using IEDB and SYFPEITHI, and physicochemical properties and tertiary structure were analyzed via ProtParam, ProtScale, DNASTAR, and SWISS-MODEL. The recombinant plasmid was constructed, expressed in Escherichia coli BL21(DE3), and purified by Ni-NTA affinity chromatography, with identity confirmed by SDS-PAGE and Western blotting. Murine dendritic cells (DCs) were cultured with the recombinant protein for 24 h and analyzed by flow cytometry for maturation and antigen binding. Balb/c mice were immunized with the recombinant protein and adjuvant; DC maturation was assessed by flow cytometry, splenocyte proliferation was evaluated by CCK-8, and serum IFN-γ, IL-4, and IgG were assessed by enzyme-linked immunosorbent assay (ELISA). Immunized mice were challenged with E. granulosus ; after 120 days, the cyst wet weight inhibition rate was calculated, and spleen DC maturation, Th1/Th2 profiles, and serum cytokines were analyzed. Results The amino acid sequence of CTLA-4IgV-EgG1Y162-2(4) was obtained, and epitope analysis revealed six T-cell and B-cell co-associated epitopes, with no epitope displacement caused by the CTLA-4 extracellular region or GSGGSG linker. Bioinformatics tools successfully predicted its physicochemical properties and spatial structure. The recombinant plasmid was constructed and expressed in BL21 cells. The purified protein was confirmed by His antibody and showed specific Western blot reactivity with CE-infected sera. CTLA-4IgV-EgG1Y162-2(4) promoted DC maturation both in vitro and in vivo , enhanced splenocyte proliferation, and elevated IFN-γ, IL-4, and IgG levels. In the challenge experiment, this group showed lower infection rates and cyst wet weights, and higher Th1 and lower Th2 responses, with favorable IFN-γ/IL-4 profiles. Conclusion This study substantiates that CTLA-4IgV-EgG1Y162-2(4) stands as a multi-epitope vaccine characterized by remarkable immunogenicity and antigenicity, thus furnishing an indispensable theoretical and empirical foundation for the management and treatment of cystic echinococcosis (CE).