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

Abstract

Duck enteritis virus (DEV), duck tembusu virus (DTMUV), and highly pathogenic avian influenza virus (HPAIV) H5N1 are the most important viral pathogens in ducks, as they cause significant economic losses in the duck industry. Development of a novel vaccine simultaneously effective against these three viruses is the most economical method for reducing losses. In the present study, by utilizing a clustered regularly interspaced short palindromic repeats (CRISPR)/associated 9 (Cas9)-mediated gene editing strategy, we efficiently generated DEV recombinants (C-KCE-HA/PrM-E) that simultaneously encode the hemagglutinin (HA) gene of HPAIV H5N1 and pre-membrane proteins (PrM), as well as the envelope glycoprotein (E) gene of DTMUV, and its potential as a trivalent vaccine was also evaluated. Ducks immunized with C-KCE-HA/PrM-E enhanced both humoral and cell-mediated immune responses to H5N1 and DTMUV. Importantly, a single-dose of C-KCE-HA/PrM-E conferred solid protection against virulent H5N1, DTMUV, and DEV challenges. In conclusion, these results demonstrated for the first time that the CRISPR/Cas9 system can be applied for modification of the DEV genome rapidly and efficiently, and that recombinant C-KCE-HA/PrM-E can serve as a potential candidate trivalent vaccine to prevent H5N1, DTMUV, and DEV infections in ducks.

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.