A novel method for synthetic vaccine construction based on protein assembly

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

In vivo cytotoxicity assay. Naïve WT C57BL/6 mice were immunized subcutaneously with 30 pmol of aDEC205-Sc-OVA 8 -ED3 or Sc-OVA 8 -ED3, along with CpG1826 and Poly I:C as an adjuvant, as described above. Five days after the secondary vaccination, the immunized mice were injected intravenously with the peptide-pulsed target cells for in vivo killing. Briefly, congenic naïve splenocytes (2 3 10 7 cells/ml) were pulsed with 10 mg/ml OT1 peptide for 1.5 h at 37uC. After one wash in PBS, the cells were labeled with 5 mM CFSE (CFSE high ) (Invitrogen, Carlsbad, CA, USA) for 10 min, after which 20% FBS was immediately added to terminate the labeling reaction. The cells were then washed twice in PBS before use. Meanwhile, unpulsed splenocytes were labeled with 0.5 mM CFSE (CFSE low ) following the same procedure. A 151 mixture of CFSE high and CFSE low cells (2.5 3 10 6 cells from each population) was co-transferred to the immunized mice. Twenty hours later, the spleens were harvested, and single-cell suspensions were prepared and analyzed by flow cytometry. The percentage of specific killing was evaluated with the following formula: % specific killing 5 1 2 % survival, in which % survival 5 (the number of OT1-pulsed target cells remaining)/(the number of un-pulsed target cells remaining).