A novel method for synthetic vaccine construction based on protein assembly

Liu, Zhida; Zhou, Hang; Wang, Wenjun; Tan, Wenjie; Fu, Yang-Xin; Zhu, Mingzhao

Synthetic aDEC205-Sc-OVA 8 -ED3 vaccine generates enhanced cytotoxic T-cell response. We next tested whether the synthetic vaccine could generate an efficient cytotoxic T-cell response. This was measured by IFNc intracellular staining and in vivo specific killing assays. Naïve WT C57BL/6 mice were immunized twice with the aDEC205-Sc-OVA 8 -ED3 fusion protein or the Sc-OVA 8 -ED3 protein at a 7-day interval. CpG1826/Poly I:C was used as adjuvant. For the IFNc intracellular staining assay, 5 days after the last injection, splenocytes were isolated from the immunized mice and stimulated with 5 mg/ml OT1 peptide in a U-bottom 96-well plate for 6 h. The IFNc-producing CD8 T cells were then stained and analyzed by flow cytometry. The splenocytes from the aDEC205-Sc-OVA 8 -ED3-vaccinated mice showed a significantly higher percentage of IFNc-producing CD8 T cells compared with the splenocytes from Sc-OVA 8 -ED3-immunized mice (Figures 4a and 4b ). For the in vivo specific killing assay, 4 days after the secondary injection, target cells (an equal mixture of OVA 8 peptide-loaded, CFSE-high, labeled naïve splenocytes and non-loaded, CFSE-low, labeled naïve splenocytes) were transferred to the immunized mice. Next, 20 h later, the proportion of CFSE-labeled cells in the spleen was analyzed. A significantly higher specific killing rate was found in mice immunized with the aDEC205-Sc-OVA 8 -ED3 vaccine Synthetic aDEC205-Sc-OVA 8 -ED3 vaccine induces better antigenspecific antibody response. It has been shown that targeting antigens to DEC205 can induce strong antibody responses in the presence of adjuvants. We next tested whether synthetic aDEC205-Sc-OVA 8 -ED3 can elicit a better antibody response against the antigen TBEV ED3. Naïve WT C57BL/6 mice were immunized twice with 300 pmol aDEC205-Sc-OVA 8 -ED3 fusion protein or Sc-OVA 8 -ED3 protein, together with CpG1826/Poly I:C as an adjuvant, at a 14-day interval. Seven days after the last vaccination, the mice were bled, and anti-TBEV ED3 antibody was measured by ELISA. Compared with the Sc-OVA 8 -ED3 control vaccine, aDEC205-Sc-OVA 8 -ED3 elicited a significantly increased antibody response against TBEV ED3 ( Figure 5) . Thus, the synthetic DCtargeting vaccine is efficient in inducing both T-and B-cell responses.