Development Of Capsid Protein Virus-like Particles Of Porcine Circovirus Type 2 As Antigen To Study Its Immunogenicity

Porcine circovirus type 2 (PCV2) is closely associated with postweaning multisystemic wasting syndrome (PMWS) in swine. PMWS is characterized by emaciation, decreased weight gain, dyspnea, jaundice, diarrhea, dermatitis, enlarged lymph nodes, and viremia. It affects pigs 5 to 18 weeks of age, with 1%–10% mortality rates. PCV2 infection distributed worldwide and has had a significant economic impact on swine production. Commercial PCV2 vaccines, including inactive, chimeric, and subunit vaccines, have been licensed both domestically and overseas. All these vaccines have shown active efficacy in reducing PCV2 infection in swine; however, the PCV2 vaccines are very expensive (10–48 RMB for each dose) because of the high production costs of the vaccine antigen. Swine farms have to face very high vaccine costs, but the vaccine cannot be used in all commercial swine farms. The ORF2 of PCV2 encodes the major structural capsid (Cap) protein, which is also the major immunogenic protein, which has the ability to independently self-assemble and form virus-like particles (VLPs) in vitro. The small ubiquitin-like modifier (SUMO) fusion expression system is used to express the whole native Cap (nCap) protein successfully by making it highly soluble in Escherichia coli, which provides an exceptional opportunity for developing the PCV2 VLP vaccine.1. The ORF2 gene that encodes the Cap protein was cloned into a prokaryotic expression vector pSK with two tags of SUMO and 6×histidines, and transformed into JM109 competent cells; the positive plasmid was designated as p-SMK-Cap. The recombinant Cap protein, a highly soluble recombinant protein, was expressed in BL21-Condon Plus (DE3) competent cells, observed after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the recombinant Cap protein (45 kDa) was confirmed using western blot analysis.2. The fusion tag of recombinant protein was digested with SUMO protease. The recombinant Cap was purified using Ni²⁺affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole nCap protein self-assembled into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size of 23 kDa, a diameter of 17 nm, and a shape that resembled the authentic Cap.3. Mice were vaccinated with three types of antigens, including His-SUMO-Cap, nCap, and InvCap. The specific antibodies against Cap were monitored using an indirect enzyme-linked immunosorbent assay, and the cytokine expression levels of interleukin 4 (IL-4), IL-10, IL-12, and interferon?(IFN-?) from PBMC increased after vaccination with the three antigens. The T lymphocyte subsets (CD3⁺T, CD4⁺T, and CD8Porcine circovirus type 2 (PCV2) is closely associated with postweaning multisystemic wasting syndrome (PMWS) in swine. PMWS is characterized by emaciation, decreased weight gain, dyspnea, jaundice, diarrhea, dermatitis, enlarged lymph nodes, and viremia. It affects pigs 5 to 18 weeks of age, with 1%–10% mortality rates. PCV2 infection distributed worldwide and has had a significant economic impact on swine production. Commercial PCV2 vaccines, including inactive, chimeric, and subunit vaccines, have been licensed both domestically and overseas. All these vaccines have shown active efficacy in reducing PCV2 infection in swine; however, the PCV2 vaccines are very expensive (10–48 RMB for each dose) because of the high production costs of the vaccine antigen. Swine farms have to face very high vaccine costs, but the vaccine cannot be used in all commercial swine farms. The ORF2 of PCV2 encodes the major structural capsid (Cap) protein, which is also the major immunogenic protein, which has the ability to independently self-assemble and form virus-like particles (VLPs) in vitro. The small ubiquitin-like modifier (SUMO) fusion expression system is used to express the whole native Cap (nCap) protein successfully by making it highly soluble in Escherichia coli, which provides an exceptional opportunity for developing the PCV2 VLP vaccine. 1. The ORF2 gene that encodes the Cap protein was cloned into a prokaryotic expression vector pSK with two tags of SUMO and 6×histidines, and transformed into JM109 competent cells; the positive plasmid was designated as p-SMK-Cap. The recombinant Cap protein, a highly soluble recombinant protein, was expressed in BL21-Condon Plus (DE3) competent cells, observed after sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the recombinant Cap protein (45 kDa) was confirmed using western blot analysis. 2. The fusion tag of recombinant protein was digested with SUMO protease. The recombinant Cap was purified using Ni2⁺ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole nCap protein self-assembled into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size of 23 kDa, a diameter of 17 nm, and a shape that resembled the authentic Cap. 3. Mice were vaccinated with three types of antigens, including His-SUMO-Cap, nCap, and InvCap. The specific antibodies against Cap were monitored using an indirect enzyme-linked immunosorbent assay, and the cytokine expression levels of interleukin 4 (IL-4), IL-10, IL-12, and interferon?(IFN-?) from PBMC increased after vaccination with the three antigens. The T lymphocyte subsets (CD3⁺T, CD4⁺T, and CD8⁺T) were analyzed at 21 and 28 days after vaccination using flow cytometry. The CD3⁺ T cell counts varied significantly between the control and the experimental groups (p < 0.05), whereas the percentages of the CD3⁺, CD3⁺/CD4⁺, and CD3⁺/CD8⁺ cells were not statistically significant among the different Cap antigens. 4. The size of PCV2 Cap VLPs was determined using a Zetasizer Nano. The piglets were immunized with the nCap antigen, whereas the InvCap was used as the control antigen. The serum samples were T) were analyzed at 21 and 28 days after vaccination using flow cytometry. The CD3⁺ T cell counts varied significantly between the control and the experimental groups (p < 0.05), whereas the percentages of the CD3⁺, CD3⁺/CD4⁺, and CD3⁺/CD8⁺ cells were not statistically significant among the different Cap antigens.4. The size of PCV2 Cap VLPs was determined using a Zetasizer Nano. The piglets were immunized with the nCap antigen, whereas the InvCap was used as the control antigen. The serum samples were collected at 7-day intervals. The level of serum antibodies increased significantly after 7 days until 28 days. The cellular immune responses were evaluated using a lymphocyte proliferation assay and flow cytometry and were very significant. Higher levels of IL-4, IL-10, and IFN-?were elicited with the nCap antigen compared with the control piglets. Thus, the Cap VLP antigen induces specific cellular and humoral immune responses and has significant potential as a subunit vaccine against PCV2 infection.