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 can bind to DEC205 1 DCs both in vitro and in vivo. To test the DC-targeting ability of the synthetic aDEC205-Sc-OVA 8 -ED3 fusion protein, splenocytes isolated from naïve WT C57BL/6 mice were incubated with the fusion protein or an isotype-control protein. After staining with a fluorescent secondary antibody, the cells were analyzed by flow cytometry. As shown in Figure 3a , conventional DCs were gated as MHC class II high and CD11c high cells, and the CD8a 1 DC and CD8a 2 DC subsets were then each further gated and analyzed. As shown in Figure 3b (top panel), the aDEC205 fusion protein preferentially bound to CD8a 1 DCs, rather than to CD8a 2 DCs or other immune cells (data not shown), which is consistent with the and assembly. The cDNA encoding the single chain of aDEC205 was genetically fused to SpyTag at the C-terminus, and the antigen containing the OT1 epitope and TBEV ED3 was fused to the C-terminus of SpyCatcherDN. Once mixed, an amide bond efficiently forms between SpyTag and SpyCatcher. (b) Synthetic vaccine production and purification. Purified aDEC205-SpyTag was mixed with SpyCatcherDN-OVA 8 -TBEV ED3 at a molar ratio of 151.5 at 4uC for 2 h. The assembled aDEC205-Sc-OVA 8 -ED3 adduct was purified by Protein A chromatography. Both the efficiency of the reaction and the purified adduct can be analyzed by SDS-PAGE. specific expression of DEC205 on the former subset of DCs. To further test its targeting ability in vivo, naïve WT mice were immunized with the aDEC205 fusion protein, together with CpG/ Poly I:C. Next, 24 h after immunization, draining lymph nodes (DLNs) were isolated and digested to form single-cell suspensions. aDEC205 fusion protein-bound cells were visualized with fluorescent secondary antibody staining followed by flow cytometry. Preferential targeting to CD8a 1 DCs was confirmed (Figure 3b , bottom panel). Thus, the SpyTag/SpyCatcher system allows assembly of SpyCatcher fusion proteins with aDEC205-SpyTag, without influencing its DC-targeting ability.