Development of Polycistronic Baculovirus Surface Display Vectors to Simultaneously Express Viral Proteins of Porcine Reproductive and Respiratory Syndrome and Analysis of Their Immunogenicity in Swine

Abstract

To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in E. coli. In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 10⁵ of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines.

Keywords: IFN-γ; IL-4; baculovirus surface display vectors; neutralizing antibody; porcine reproductive and respiratory syndrome virus.

Figure 1

PCR amplification of the sequences encoding the ectodomain of GP2, GP4, and mtGP5 of PRRSV and schematic illustration of novel baculovirus surface display vectors. (A,B) DNA electrophoresis analysis of PCR products of the truncated GP2, GP4, and mtGP5 genes of PRSSV. Lane M represents DNA marker (Bio 100 bp DNA ladder). (C,D) pBacDD-2GP2-2GP4 and pBacDD-4mtGP5 constructs were digested with SalI/BamHI and XhoI/KpnI, respectively, to confirm the correctness of the recombinant fragments. (E,F) Schematic illustration of baculovirus surface display vectors carrying multiple expression cassettes. The gp64 TM-CTD and the gp64 signal peptide sequences of baculovirus localized in the vectors are shown. The developed baculovirus surface display vectors pBacDD-2GP2-2GP4 (E) and pBacDD-4mtGP5 (F), which carry multiple expression cassettes were shown.

Figure 2

Construction of genetic recombinant baculoviruses BacSC-mtGP5, BacDD-2GP2-2GP4, and BacDD-4mtGP5. (A) Images of BacSC-mtGP5, BacDD-2GP2-2GP4, and BacDD-4mtGP5 baculovirus-infected SF-9 cells at 3 days post infection. Phase contrast images (upper panel) and fluorescence images (lower panel). All images were magnified at 200×. (B) Sf-9 insect cells were infected with the recombinant baculoviruses BacSC-mtGP5 and BacDD-4mtGP5, respectively, with an MOI of 20, harvested 3 days post infection, and subjected to Western blot using anti-His monoclonal antibody and PRRSV polyclonal antibodies, respectively. The ectodomain of His tagged mtGP5-gp64TM-CTD (10 kDa) fusion protein was detected. No proteins were detectable in the negative controls (BacCE and Sf-9 cell alone). The expected size of the His and baculovirus gp64TM-CTD fusion protein is approximately 5 KDa. Signals in Western blots were quantified using Image J software and shown in the right panel. (C) Anchoring of the ectodomain of His-tagged mtGP5-gp64TM-CTD fusion protein on the plasma membrane of Sf-9 cells as revealed by confocal microscopy. (D) Sf-9 cells were infected with the recombinant baculovirus BacDD-2GP2-2GP4 at an MOI of 20, harvested 3 days post infection, and then subjected to Western blot using anti-His monoclonal antibody and PRRSV polyclonal antibodies, respectively. BacCE and Sf-9 cells alone were used as negative controls. The expected sizes of His6-tagged GP2-gp64TM-CTD and His6-tagged GP4-gp64TM-CTD fusion proteins were approximately 23 kDa and 16 kDa, respectively. The expected sizes of the expressed fusion proteins are indicated on the right-hand side of each panel.

Figure 3

Optimum conditions for production of the truncated proteins of PRRSV. Effects of different infection times, MOI, and cell numbers for expressing the ectodomain of His-tagged GP2-gp64TM-CTD and His-tagged GP4-gp64TM-CTD fusion proteins of PRRSV were tested. (A,C) Sf-9 cells were infected with the genetic recombinant baculovirus BacDD-2GP2-2GP4 at different MOIs, infection times, and cell numbers. The Sf-9 cell alone and BacCE were used as negative controls. The expressed fusion proteins were probed using anti-His monoclonal antibodies. Signals in all Western blots were quantified using Image J software. β-actin was used as an internal control for normalization. Relative abundance (%) are shown. (B,D) The results were calculated from the data shown in panels A and C. All data shown represent the mean± SE calculated from three independent experiments.

Figure 4

Effects of different concentrations of FBS on co-expression of the truncated fusion proteins of PRRSV. (A) Sf-9 cells were infected with genetic recombinant baculovirus BacD4D-GP2-GP3-2GP4 at an MOI of 20 supplemented with different FBS concentrations. The Sf-9 cell alone and BacCE were used as negative controls. The expressed proteins were probed using anti-His monoclonal antibodies. Signals in all Western blots were quantified using Image J software. β-actin was used as an internal control for normalization. Relative abundance (%) are shown. (B) The results were calculated from the data shown in the left panel. All data shown represent the mean± SE calculated from three independent experiments.

Figure 5

Coomassie blue-stained SDS-PAGE of E. coli BL21(DE3) expressing the PRRSV proteins. The PRRSV proteins were expressed as shown in panels A–E. (A) The ectodomain of TrxA-His-GP2, (B) TrxA-His-GP4, (D) TrxA-His-mtGP5 (supernatant), and (E) TrxA-His-mtGP5 (pellet). E.coli BL21 (DE3) containing the indicated constructs were induced for different time points with IPTG at a final concentration of 0.4 mM in culture medium. The pET32a (empty vector) was used as the negative control. The expressed proteins were marked by red frame. (C,F) The expressed TrxA-His-GP2, TrxA-His-GP4, and TrxA-His-mtGP5 fusion proteins were probed using anti-His monoclonal antibodies or PRRSV polyclonal antibodies, respectively. The expected size of the expressed fusion proteins is indicated on the right-hand side of each panel. The expected size of the TrxA-His fusion protein is about 17 KDa.

Figure 6

Detection of ELISA titers, SN titers, and the levels of IL-4 and IFN-γ in 5-week-old SPF pigs immunized with various immunogens. (A) Detection of anti-PRRSV proteins (GP2, GP4, and mtGP5) titers by the developed ELISA. The 2-month-old SPF pigs were immunized at the base of the ear by the intramuscular route with BacCE (negative control) and the developed subunit vaccine (prepared from BacD4D-2GP2-2GP4- and BacD4D-4mtGP5-infected cells, 5x10⁷ cells), respectively. Serum samples were collected every week after the first immunization to determine the GP2, GP4, and mtGP5 protein-specific ELISA antibodies. (B) SN titers in SPF pigs immunized with BaCE (negative control), subunit vaccine, and commercial MLV vaccine (positive control), respectively. Sera were from 11 weeks post vaccination. (C) Analysis of IL-4 and IFN-γ by ELISA was performed using IL-4 and IFN-γ ELISA kits. SPF pigs immunized with the developed subunit vaccine and BacCE (negative control), respectively. The levels of IL-4 and IFN-γ in the 4th and 11th weeks after the first and second immunizations were analyzed. The value of OD₄₀₅ nm was measured with an ELISA reader. All data shown represent the mean ± SE calculated from three independent experiments.

Figure 7

PCR amplification of the ectodomain sequences of GP3 and M genes of PRRSV and schematic illustration of baculovirus surface display vectors carrying multiple expression cassettes. (A,B) DNA electrophoresis analysis of PCR products of the ectodomain of GP3 and M genes of PRRSV. (C,D) The resultant pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs digested with the respective restriction enzymes were analyzed by electrophoresis in a 1.2% agarose gel. (E,F) Schematic illustration of baculovirus surface display vectors pBacDD-GP2-GP3-2GP4 (E) and pBacDD-2mtGP5-2M (F), which carry multiple expression cassettes are shown. The baculovirus gp64TM-CTD and the signal peptide sequence of gp64 localized in the vectors are shown. The ectodomain of GP2, GP3, GP4, mtGP5, and M genes of PRRSV was inserted between the gp64 signal peptide and gp64TM-CTD of the surface display vectors under the control of the p10 or Pph promoter. (G) Sf-9 cells were transfected with the developed vectors to create recombinant baculoviruses BacDD-GP2-GP3-2GP4 and BacDD-2mtGP5-2M, respectively. Images of BacDD-GP2-GP3-2GP4 and BacDD-2mtGP5-2M recombinant baculovirus-infected Sf-9 cells at 3 days post infection are shown. All images were magnified at 200×. (H,I) To confirm that the target protein is successfully expressed in insect cells, the genetic recombinant baculoviruses BacDD-GP2-GP3-2GP4 and BacDD-2mtGP5-2M, and BacCE (empty vector) were used to infect insect cells at an MOI of 20 for three days, and the baculovirus-transfected Sf-9 cells and Sf-9 alone were collected for Western blot assays using PRRSV polyclonal antibodies and His monoclonal antibodies, respectively.