Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine

Abstract

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 10⁷ of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (10⁷) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4⁺ T cells and CD4⁺/CD8⁺ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Keywords: 4Cap(d41) vaccine; CD4+ T cells; Cap protein; IFN-γ; PCV2; baculovirus surface display vectors; cellular immune response; virus neutralization test.

Figure 1

DNA electrophoresis analysis of the PCV2-Cap (d41) gene PCR product and confirmation of the pBacSC-Cap (d41) plasmid. A Lane M represents DNA marker (Bio-100 bp DNA ladder); lanes 1-3 represent PCR amplified PCV2-Cap (d41) gene fragments, which have restriction enzyme cleavage positions (Lane 1: XhoI/PstI, lane 2: NotI/SalI and lane 3: XhoI/BsiWI). The expected fragment size is 579 bp in length. B The pBacSC-Cap (d41) vector was cleaved by restriction enzymes XhoI and PstI to confirm the successful gene recombination, which was in line with the expected fragment size of about 579 bp in length.

Figure 2

Schematic illustration of novel baculovirus surface display vectors carrying multiple expression cassettes. To increase the expression levels of the PCV2 Cap(d41) protein, four novel baculoviral vectors pBacSC, pBacDD-2, pBacDD-3, and pBacDD-4, which carry multiple expression cassettes were constructed. The Cap(d41)-gp64-SP-TM-CTD fragment was inserted into the respective baculoviral vectors and the resultant recombinant plasmids were named pBacSC-Cap(d41), pBacDD-2Cap(d41), pBacDD-3Cap(d41), and pBacDD-4Cap(d41) (A–D).

Figure 3

Confirmation of recombinant baculovirus surface vectors and expression levels of the Cap (d41) protein using the respective baculovirus surface vectors. A Four recombinant plasmids pBacDD-2Cap(d41), pGem-T-easy-DD-2Cap(d41), pBacDD-3Cap(d41), and pBacDD-4Cap(d41) were digested with the respective restriction enzymes for confirming the presence of the Cap(d41) gene of PCV2. B Images of BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41) recombinant baculovirus-infected Sf-9 cells at 3 days post-infection. All images were magnified at 200×. C Confirmation of the expression of Cap(d41)-gp64-TM-CTD protein in Sf-9 cells. The cells were infected with recombinant viruses BacCE, BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41), respectively, at an MOI of 10, harvested 3 days post infection, and subjected to western blot assay using anti-PCV2 sera (lanes 2–6). The Cap(d41) protein has two different fragments (Cap(d41)-gp64-SP-TM-CTD with a molecular weight of 33 kDa; Cap(d41)-gp64-TM-CTD with a molecular weight of 28 kDa. Sf-9 cells and BacCE were used as the negative control. Signals in all western blots were quantified using Image J software. The levels in the Cap(d41) were considered one-fold. β-actin was used as an internal control for normalization. The expression folds indicated below each lane were normalized against values for Cap(d41). Data represent the mean ± SD. A p value less than 0.05 was considered significant.

Figure 4

Optimum conditions for production of the PCV2 Cap(d41) protein. Effects of different infection times, MOI, and cell numbers for expressing the Cap(d41) protein were tested. A, B Cell numbers (10⁷) of Sf-9 cells were infected with MOI of 10 (A) or infected with different MOI (B) for 4 days. Cell numbers (10⁷) of Sf-9 cells were infected with different recombinant baculoviruses as indicated. The Sf-9 cell alone and CE recombinant baculoviruses were used as the negative control. 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. The results were calculated from the data shown in the upper portion of each panel. All data shown represent the mean ± SD calculated from three independent experiments. A student t-test was conducted for analysis and a value of P < 0.05 was considered statistically significant. C Expression levels of the PCV2 Cap(d41) protein in different cell numbers infected with recombinant baculovirus at an MOI of 10 for 4 days was examined. 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. The results were calculated from the data shown in the left portion of each panel. All data shown represent the mean ± SD calculated from three independent experiments. A student t-test was conducted for analysis and a value of P < 0.05 was considered statistically significant. D The immune effect of different doses of Cap(d41) protein and evaluation of the effective dose. Different cell numbers (10⁵, 10⁶ and 10⁷) of Sf-9 cells were infected with recombinant baculoviruses BacSC-Cap(d41) and BacDD-4Cap(d41), respectively, at an MOI of 10 for 3 days, respectively and cell lysates were collected to immunize mice. After two vaccinations, antibody titers were analyzed by ELISA. Data represent the mean ± SD. A p value less than 0.05 was considered significant.

Figure 5

Serum neutralization (SN) titers and the level of IFN-γ in mice immunized with various immunogens. A The SN titers in mice induced by 4Cap(d41) and commercial vaccines. 5 × 10⁷ Sf-9 cells were infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 10 for 3 days. Cell lysates were collected to prepare the 4Cap(d41) vaccine. Eight-week-old mice were vaccinated via intraperitoneal injection at weeks 0 and 2 with PBS, CE, 4Cap(d41), and commercial vaccines as formulated with complete and incomplete adjuvants. As negative controls, three mice were injected with PBS and CE (5 × 10⁷ BacCE-infected Sf-9 cell lysates). Each mouse received one booster shot in week 2, and blood samples were taken at week 5 for the SN titer assay. B The fluorescence results were converted into neutralizing antibody titers and presented as a data chart (B). Data represent the mean ± SD. C Analysis of IFN-γ by ELISA was performed using the IFN-γ ELISA Kit. The value of OD₄₀₅ nm was measured with an ELISA reader. Data represent the mean ± SD. A P value less than 0.05 was considered significant.

Figure 6

Serum neutralization (SN) titers in swine immunized with various immunogens. 2-month-old SPF pigs were immunized at the base of the ear by the intramuscular route with PBS, CE, 4Cap(d41) (80 μg), and commercial vaccines, respectively. Sera samples were collected at 14 and 28 days after primary immunization for virus neutralization tests. Data represent the mean ± SD. A P value less than 0.05 was considered significant

Figure 7

Lymphocyte proliferation assay and analysis of IFN-γ and IL-4 by ELISA. A Lymphocyte proliferation assay was done using PBMCs of swine as described in “Materials and methods” section. The optical density (OD) was determined at 490 nm, and the stimulation index (SI) was calculated as follows: SI = mean OD of PCV2 stimulated cells/mean OD of unstimulated cells. B Analysis of IFN-γ and IL-4 by ELISA was performed using the IFN-γ and IL-4 ELISA Kit. The value of OD₄₀₅ nm was measured with an ELISA reader. Data represent the mean ± SD. A P value less than 0.05 was considered significant.

Figure 8

Detection of anti-PCV2 titers by ELISA. 2-month-old SPF pigs were immunized at the base of the ear by the intramuscular route with CE, 4Cap(d41), and commercial vaccines, respectively. Serum samples were collected every week after the first immunization to determine the Cap-specific ELISA antibodies. In the eighth week after the first immunization, all vaccinated pigs were intranasally injected with 2 ml of PCV2 (10^5.7TCID₅₀/mL). Data represent the mean ± SD. An asterisk (*) shows a statistically significant difference between the indicated groups (P < 0.05).

Figure 9

Quantification of PCV2 viral loads in sera from the challenged pigs. 2-month-old SPF pigs were immunized at the base of the ear by the intramuscular route with CE, 4Cap(d41), and commercial vaccines, respectively. Serum samples were collected every week after the first immunization. In the eighth week after the first immunization, all vaccinated pigs were challenged via intranasal injection with 2 ml of PCV2 (10^5.7TCID50/mL). The 8^th week after the first immunization is designated as the 0^th week. PCV2 viral loads were detected by real-time quantitative PCR one week post challenge. Data are presented as mean ± SD. Viral loads were determined as the mean of the logarithmic DNA copy number per ml (log copies/ml). An asterisk (*) shows a statistically significant difference between the indicated groups (P < 0.05).