Production And Evaluation Of Virus-like Particles(VLPs) Based On Capsid Protein Of Porcine Circovirus Type 2

Porcine circovirus type 2(PCV2) is the main and primary causative agent of post-weaning mμLtisystemic wasting syndrome(PMWS) in swine, and is also closely associated with many other diseases such as porcine dermatitis and nephropathy syndrome(PDNS), congenital tremor(CT), porcine respiratory disease complex(PRDC), etc. It affects pigs from 5 to 18 weeks of age, and the disease is characterized by emaciation, decreased weight gain, dyspnea, jaundice, diarrhea, dermatitis, enlarged lymph nodes, and viremia. PCV2 infections distribute worldwide and pose significant economic impact on swine production. Prevention and control of PCV2 mainly relies on vaccination with commercial PCV2 vaccines, including inactive, chimeric, and subunit vaccines. All these vaccines have shown promising efficacy in reducing PCV2 infection in swine, but they are not suitable for the widespread use in China because of high cost. Hence it is necessary for China to develop a low-cost, safe and efficacious PCV2 subunit vaccine.Capsid(Cap) protein is the major immunogenic protein of PCV2 and has the ability to self-assemble into virus-like particles(VLPs) in vitro. In this study, the recombinant Cap protein was produced as soluble form by high cell density fermentation(HCDC) of E.coli followed by,, purification of by cation-exchange chromatography and assembled into VLPs of PCV2. This study provides the foundation for developing novel subunit VLP vaccines of PCV2.Firstly, we optimize the fermentation conditions of high cell density cultivation for expressing the Cap protein from E.coli BL21(DE3) and established a relatively stable 10 L fermenter expression system that results in the harvest of about 93.9±1.9 g/L bacteria pellet. The pellet was then suspended in Tris-HCl(p H 8.0), and disrupted under-5°C using a high-pressure homogenizer. Upon centrifugation, the supernatant was mixed an 30% volume of saturated ammonium sulfate to remove bacterial proteins, and then clarified supernatant was purified through cation-exchange chromatography(IEC SP FF column). More than 40 mg/L of Cap protein with purity over 85% was obtained. Large production of soluble Cap protein is basic for the development PCV2 subunit vaccine.Secondly, we obtained the optimal conditions for the assembly of Cap protein into VLPs and developed a double antibody sandwich ELISA to evaluate the stability of Cap protein. The resμLts showed that the bioactivity of Cap protein remained stable when stored under 25°C, and temperature shifts affect to a great deal the bioactivity of Cap protein. The addition of protein stabilizers, glycine or glycerol, helps remain the stability of Cap protein.Lastly, VLPs based on Cap protein were prepared for vaccinating BALB/c mice and piglets. Immunological parameters and production performance of the immunized animals were measured to evaluate the efficacy of PCV2 VLP subunit vaccine. For immunization of BALB/c mice, VLPs were emμLsified respectively with adjuvants IMS1313, carbomer and 50 V mineral oil. The administrations of 0.9% Na Cl and a commercial PCV2 vaccine(Boehringer Ingelvac Circo FLEX®) were used as mock and positive controls. We find that mineral oil-emμLsified VLPs induces the same antibody level as that of positive controls, and the antibody maintained at high level since the second week. Aqueous adjuvant-emμLsified VLPs gave a relatively lower performance. 50 V mineral oil was then selected to be used as adjuvant for the immunization of piglets. 0.9% Na Cl and a commercial PCV2 vaccine(Boehringer Ingelvac Circo FLEX®) were also used as mock and positive controls. The resμLts between two successive field vaccination trials were discrepant. The weight gain from the mock-vaccinated piglets were significantly higher than VLP and positive control-immunized piglets in the first vaccination trial. However, in the second trial we obtained a reverse conclusion that indicates showed high weight gains in VLP and positive control-immunized piglets than from the mock-vaccinated piglets.Hence, we developed a high cell density cμLtivation system for expressing the Cap protein from E.coli BL21(DE3). Large quantity of soluble Cap protein with purity over 85% was obtained. The stability of Cap protein was evaluated using a double antibody sandwich ELISA. Animal vaccination trials revealed that the immunological parameters and production performance of immunization with 50 V mineral oil-emμLsified VLPs were comparable to those of a commercial PCV2 vaccine. Thus the VLPs based on Cap protein of PCV2 produced in this study has the potential to be applied as a safe and efficacious PCV2 subunit vaccine with low cost.