An experimental chimeric hepatitis E virus vaccine elicits both local and systemic immune responses

Figures

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  Figure 1. Schematic diagram of O2P2 protein, IBLP027 particle preparation and mice immunization. (A) pENHAcO2P2 is the expression plasmid used to generate the fusion protein. IBLP obtained by heat and acid treatment were mixed to obtained the LysM5O2P2-IBLP complexes for immunization of mice following either an oral or a combined schedule with subcutaneous + oral immunization of BALB/c mice. (B) The SDS-PAGE gel stained with Coomassie shows the IBLPs alone, and the complexes loaded with two different concentrations of LysM5O2P2, 5 and 40 μg, respectively.

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  Figure 2. Immunization protocol: mice were divided into three groups (5 mice each) and subjected to two immunization schemes: three oral doses or one subcutaneous dose followed by two oral doses. Group 1 (IBLP027): control group receiving only IBLP027. Group 2 (LysM5O2P2): immunized with the chimeric protein LysM5O2P2 without adjuvant. Group 3 (IBLP027-LysM5O2P2): immunized with LysM5O2P2 displayed on the surface of IBLP027.

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  Figure 3. Evaluation of the humoral immune response in mice following the oral immunization schedule. Mice were immunized orally with either IBLP027 (control group), the chimeric protein alone (LysM5O2P2) or the IBLP027-LysM5O2P2 complexes. (A) Specific anti-ORF2 IgA was determined by ELISA in gut fluids obtained from mice at day 35. (B) Specific anti-ORF2 IgG was determined by ELISA in sera obtained from mice after 14 (t1), 21 (t2) or 35 (t3) days after the 1st immunization. The cut-off value was set as the mean absorbance ±3 SD from a panel of negative sera (t0). ANOVA was followed by Tukey’s test (for pairwise comparisons of the means) to evaluate the differences between the groups. **p < 0.01, and ****p < 0.0001.

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  Figure 4. Evaluation of the humoral immune response in mice following the combined immunization schedule. Mice were immunized with a first subcutaneous dose, followed by two nasal boosters with either IBLP027 (control group), the chimeric protein alone (LysM5O2P2) or the IBLP027-LysM5O2P2 complexes. (A) Specific anti-ORF2 IgA was determined by ELISA in gut fluids obtained from mice at day 35. (B) Specific anti-ORF2 IgG was determined by ELISA in sera obtained from mice after 14 (t1), 21 (t2) or 35 (t3) days after the 1st immunization. The cut-off value was set as the mean absorbance ±3 SD from a panel of negative sera (t0). ANOVA was followed by Tukey’s test (for pairwise comparisons of the means) to evaluate the differences between the groups. ***p < 0.001, and ****p < 0.0001.

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  Figure 5. Evaluation of the cellular immune response in mice following the combined immunization schedule. Mice were immunized with a first subcutaneous dose, followed by two nasal boosters with either IBLP027 (control group), the chimeric protein alone (LysM5O2P2) or the IBLP027-LysM5O2P2 complexes. Peyer’s patches (A) or spleens (B) were taken at day 35. Cultured immune cells were challenged ex vivo with HEV ORF2 and the concentration of TNF-α, IFN-γ, IL-4 and IL-17 were determined in cell culture supernatants after 24 h of antigen stimulation. ANOVA was followed by Tukey’s test (for pairwise comparisons of the means) to evaluate the differences between the groups. ***p < 0.001, and ****p < 0.0001.

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  Figure 6. Resistance of the IBLP027-LysM5O2P2 complexes to artificial gastrointestinal fluids. After incubating the complexes in the respective buffers that simulate saliva (lines 3–4) for 5 min, gastric fluid for 30 min (lines 5–6) and pancreatic fluid for 90 min (lines 7–8), the complexes were suspended in Lämmli buffer and run in SDS-PAGE. The complexes without treatment served as control (lines 1–2).