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.

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate

“…Inflammatory cytokines are key regulators of the animal immune system in regulating innate and adaptive immune responses ( 69 ). IFN-γ is the main effector of cellular immunity, which can activate CD8+ T lymphocytes and macrophages to kill foreign pathogens or infected cells ( 25 ). Most commercial vaccines focused only on humoral responses, including antigen-specific or neutralizing antibody responses; however, failed to induce strong cellular immunity.…”

“…We named this strain PCV2 SC2020. We designed primers tCap -F1 and tCap -R1 to amplify the truncated tCap gene according to the research by Chen ( 25 ). The PCR product was purified and cloned into pUCm-T using T Vector Rapid Cloning Kit (Sangon Biotech, Shanghai, China).…”

“…To determine whether the serum antibodies induced by recombinant spores could effectively neutralize the virus, we constructed an in vitro infection model for virus neutralization test.PK15 cells were cultured in a 96-well cell culture plate, when the confluence rate reached about 50%, performed microneutralization tests according to the method of Chen ( 25 ). Briefly, the serum samples (50 μL) collected on 42nd day were heat inactivated at 56°C for 30 min and two-fold serial diluted from 2 -1 to 2 -8 in DMEM medium.…”

“…Titers were determined as the reciprocal of the final serum dilution with a 70% or greater reduction in fluorescence under fluorescence microscopy. Finally, the serum neutralizing antibody titers were calculated by the Reed-Muench method ( 25 ).…”

Fusion Expression and Immune Effect of PCV2 Cap Protein Tandem Multiantigen Epitopes with CD154/GM-CSF

“…Therefore, the Cap protein is an important target in the design of a novel PCV2 vaccine using genetic engineering technology. Studies have shown that cytokines and multiple epitopes can enhance the immune effect of vaccines [12,13]. CD154 is a kind of II type transmembrane protein.…”