Construction of a highly efficient CRISPR/Cas9-mediated duck enteritis virus-based vaccine against H5N1 avian influenza virus and duck Tembusu virus infection

Figure 1. Schematic illustration of the novel candidate trivalent vaccine C-KCE-HA/PrM-E development. (A) Full-length of the attenuated commercial DEV vaccine strain (C-KCE). (B) Two portions of the genome C-KCE expanded to show the UL27, UL26, US7, and US8, and the gene junction regions are depicted. (C) The organization of transfer fragments Left arm-RFP-Right arm and Left arm-GFP-Right arm. (D) The recombinant C-KCE-RFP/GFP was generated using CRISPR/Cas9 system. (E) The organization of transfer fragments Left arm-HA-Right arm and Left arm-PrM-E-Right arm. (F) Following the CRISPR/Cas9 system mediated recombination, the recombinant C-KCE-HA/PrM-E was generated.

Figure 2. Characterization of the recombinant C-KCE-HA/PrM-E. (A) Verification of HA and PrM-E insertion in C-KCE by PCR. The marker used was DL2000plus. Lane 1 is the DL2000plus marker. The products in lane 2, 3, 4, and 5 was amplified from samples with primer pairs JDPrM-E-F/JDPrM-E-R, and lane 6 and 7 are negative C-KCE and the H2O control. The products in lane 8, 9, 10, and 11 was amplified from samples with primer pairs JDHA-F/JDHA-R, and lane 12 and 13 are negative C-KCE and the H2O control. Full-length gel is presented in the Supplementary Figure 1. (B) Multiplestep growth curves of C-KCE-HA/PrM-E and C-KCE in CEF cells. (C) Detection of HA, PrM, and E proteins expressions in C-KCE-HA/PrM-E-infected CEF cells by western blot. The precursor PrM-E indicated by the red arrowhead. (D) Confirmation of the expression of HA and E protein in C-KCE-HA/PrM-E-infected CEF cells by immunofluorescence. CEF cells infected with C-KCE or mock-infected CEF cells were used as controls. Full-length blots are included in the Supplementary 2.

Figure 3. Conferred protection from immunization of ducks with C-KCE-HA/PrM-E against virulent DEV challenge. Ducks were inoculated subcutaneously with 105 PFU of C-KCE-HA/PrM-E, 105 PFU of C-KCE, or PBS (n = 10 per group) as a control, then intramuscularly challenged with 100-fold duck lethal dose (DLD)50 HB/10 at 2 weeks (A), 4 weeks (B) post-vaccination (pv), respectively. Ducks were examined daily for 2 weeks after challenge.

Figure 4. C-KCE-HA/PrM-E viruses prime humoral responses. Ducks were vaccinated with PBS, 105 PFU of C-KCE or C-KCE-HA/PrM-E (n = 10 per group) subcutaneously. Sera were obtained at the indicated time points to detect the neutralization antibodies against XN/07 and df2. (A) XN/07-neutralizing antibodies were measured by neutralization assay on MDCK cells. (B) df2-neutralizing antibodies were determined by neutralization assay on BHK-21 cells. The dashed line shows the detection limit for a positive response.

Figure 5. Serum IFN-γ and IL-4 cytokine levels in ducks. Ducks were vaccinated with PBS, 105 PFU of C-KCE or C-KCE-HA/PrM-E (n = 3 per group) subcutaneously. On the day of final immunization (day 14), the serum levels of Th1-type cytokine (IFN-γ) and Th2-type cytokine (IL-4) in ducks were determined by ELISA. Data are shown as the mean ± SD. **P < 0.01, ***P < 0.001.

Figure 6. Peripheral blood T-lymphocyte proliferation assay. Ducks were vaccinated with PBS, 105 PFU of C-KCE or C-KCE-HA/PrM-E (n = 3 per group) subcutaneously. On the day of final immunization (day 14), duck peripheral blood lymphocytes were evaluated by MTT assay. Data are shown as the mean ± SD. Statistically significant differences (P < 0.01) are indicated by **(compared with C-KCE or PBS group). ConA, Concanavalin A.

Figure 7. C-KCE-HA/PrM-E protection against HPAIV H5N1 challenge. Ducks were inoculated subcutaneously with 105 PFU of C-KCE-HA/PrM-E, 105 PFU of C-KCE, or PBS (n = 10 per group) as a control, following which they were intramuscularly challenged with 100-fold duck lethal dose (DLD)50 XN/07 at 2 weeks (A) or 4 weeks (B) pv, respectively. The ducks were observed daily for 2 weeks following influenza virus challenge.