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. 2024 Jul 16;15:1429909. doi: 10.3389/fimmu.2024.1429909

Figure 1.

Figure 1

Characterization and comparison of EBOV GP vaccines. (A) Schematic of the Th GP-FL vaccine against EBOV. The Th vaccine consist of the N-terminal human Ig-kappa signal sequence (hIgKss), the BCG T helper epitopes (P25 and P10) which are flanked by the cathepsin B cleavage site (TVGL), GP1 which includes the mucin-like domain (MLD), the furin cleavage site, GP2, and the C-terminal hexahistadine tag (6xHis). GP1 and GP2 are held together by a disulfide bond. For the WT GP-FL version of the vaccine, the BCG T helper epitopes (P25 and P10) flanked by the cathepsin B cleavage sites are absent. The MLD deleted versions of these vaccines were also constructed (Th GPΔM and WT GPΔM). (B) SDS-PAGE under reducing conditions of purified EBOV GPs as shown by Coomassie gel staining and Western blotting with anti-His antibody. The GP1 and GP2 fragments, which are normally held together by disulfide bonds, were separately resolved under the reducing and denaturing conditions of the SDS-PAGE analysis. The expected size for GP2, which consists of the C terminal region of the EBOV GP after the MLD is 25 KDa. The expected size for the GP1 precursor is 120 KDa and 60 KDa for the full length and the MLD deleted version of the EBOV GP, respectively. The GP0 fragment in the full-length versions of the GP constructs was detected as two or more bands of ~120-145 KDa, consistent with glycosylation and disordered structure of the MLD. Purity of isolated GPs was ≥ 90% based on Coomassie blue staining of the gels, and protein yields were determined by BCA protein assay (WT GP-ΔM: 2060 μg/ml, WT GP-FL: 220 μg/ml, Th GP-ΔM: 2972 μg/ml, Th GP-FL: 80 μg/ml). (C) ELISA with antibody ADI-15878 specific for EBOV GP conformational epitope was used to probe purified EBOV GPs. The ovalbumin version of the Th vaccine (Th OVA) served as a negative control to show the specificity of ADI-15878 antibody against EBOV GP. (D) Processing and presentation of BCG Th epitopes was shown by incubating purified EBOV GPs for 16 hours with dendritic cells and in the presence of a CD4+ T cell hybridomas specific for P25 of Ag85B (left) or P10 of TB9.8 (right). Supernatants were analyzed by sandwich ELISA for IL-2 (indicated as absorbance (Abs) values for conversion of the assay substrate. Multiple columns were analyzed by Kruskal-Wallis one-way ANOVA, followed by Dunn’s multiple comparison test; (***p < 0.001, **p < 0.01, *p < 0.05).