Design and development of a chimeric vaccine candidate against zoonotic hepatitis E and foot-and-mouth disease

Fig. 1. Protrusion index of key-epitope sites of HEV and FMDV antigens within the HEV-FMDV chimeric proteins as calculated by the Ellipro web server. This figure shows the protrusion index of the key-epitope sites: a in the HEV antigens alone and when they are attached to the N-terminal of the FMDV Seq 8 antigen; b in the HEV antigens alone and when they are attached to the C-terminal of the FMDV Seq 8 antigen; and c in the FMDV Seq 8 antigen when it is attached to the N-terminal or C-terminal of the different HEV antigens. Next, the total areas under the curves of the protrusion indexes were calculated for each protein using GraphPadPrizm5 Software (d–f) and are shown in d for the HEV antigens alone and when they are attached to either the N- or C-terminal of the FMDV Seq 8 antigen; and in e for the FMDV Seq 8 antigen when it is attached to either the N- or C-terminal of the HEV antigens. The normalized AUCs of the protrusion indexes of the key epitopes for both HEV and FMDV fragments in the different chimeric proteins are summarized in f

Fig. 2. Prediction of the flexibility of HEV-FMDV chimeric proteins. The different conformational models of Seq 8-P216 (a) and Seq 8-P222 (b) predicted by the CABS-flex server were superimposed and visualized by Pymol. The fluctuations of the protrusion index of the key epitope sites between the different conformations were calculated for both Seq 8-P216 (c) and Seq 8-P222 (d): the black circles represent the PI of the epitope sites in the input models; the lines represent the PI for the same residues in the 12 models predicted by the molecular dynamics simulation; the black arrows indicate the residues with a PI decrease in most of the 12 models compared to the input model. The overall exposure of HEV and FMDV epitopes in Seq 8-P216 and Seq 8-P222 chimeric proteins were computed for each model (e): the exposure is represented as the area under the curve of the protrusion index of the key-epitope sites (the method is detailed in the “Antigenicity analysis” section); circles and diamonds in red represent the overall antigenicity of the initial models; circles and diamonds in black represent the overall antigenicity of different models predicted in the molecular dynamics simulations

Fig. 3. Expression and purification of the HEV-FMDV chimeric proteins. a A 15% SDS-PAGE gel showing the overexpression of Seq 8, Seq 8-P166, Seq 8-P216 and Seq 8-P222 at the expected molecular weights of 9.4, 27.4, 33.4 and 33.9 kDa respectively; and no extra-bands are visible before the IPTG induction (negative control). M: molecular weight marker; C (−): negative control, before IPTG induction; C (+): induction of P222 expression as a positive control. b SDS-PAGE analysis of the solubility of the expressed proteins; P: pellet (insoluble fraction); S: supernatant (soluble fraction). c SDS-PAGE analysis of the purified FMDV antigen and HEV-FMDV chimeric proteins; M: molecular weight marker; CL: cell lysates after passing through the Ni–NTA agarose column; E1 and E2: elution 1 and elution 2 respectively. d Determination of the protein concentrations using the Bradford protein assay: the protein concentration was calculated in the diluted samples then multiplied by 5 (dilution factor) to determine the concentrations in the stock solutions

Fig. 4. Stability of FMDV and HEV-FMDV recombinant proteins stored at different temperatures and analyzed by SDS-PAGE after 2 weeks (a, b), 6 weeks (c, d) and 10 weeks (e). f Electron micrographs of Seq 8-P216 and Seq 8-P222 VLPs generated in E. coli; the VLPs were negatively stained with 2% uranyl acetate; bar: 100 nm

Fig. 5. Antigenicity of the HEV-FMDV chimeric proteins. a Western blot analysis using anti-HEV 5G5 monoclonal antibody (left), commercial porcine polyclonal antibodies used against FMD (middle) and serum of mice free of anti-HEV and anti-FMDV antibodies (right); M: molecular weight marker; lanes 1-4: Seq 8, Seq 8-P216, Seq 8-P222 and P222, respectively. b Assessment by indirect ELISA of the reactivity of the chimeric antigens against the anti-FMDV antibodies present in the sera of pigs infected with FMDV strains. c Assessment of non-specific reactivity of the chimeric antigens using mice sera free of anti-FMDV and anti-HEV antibodies. d Reactivity of the chimeric antigens against the HEV-neutralizing 5G5 monoclonal antibody

Fig. 6. Immunogenicity of the HEV-FMDV chimeric proteins. This figure shows the levels of the HEV- and FMDV-specific antibodies in the sera of the immunized mice at different post-immunization time points (the sera were diluted 1:25,600). The different groups were analyzed by two-way ANOVA followed by Tukey’s test. a Titers of HEV-specific antibodies; b Titers of FMDV-specific antibodies; *P < 0.05, **P < 0.01; ***P < 0.001; ns: no significance, P > 0.05; red arrows: first and second immunizations