This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10⁶ (±4.34%) Da and 2.40×10⁶ (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×10⁶ (±2.94%) Da and 2.88×10⁶ (±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×10⁶ (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all < 1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R² of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.
Animals ; Antibodies, Viral ; Capsid Proteins ; Chromatography, Gel ; Circoviridae Infections/prevention & control* ; Circovirus ; Lasers ; Swine ; Vaccines, Inactivated ; Vaccines, Virus-Like Particle ; Viral Vaccines

Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of postweaning multisystemic wasting syndrome (PMWS). Co-infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) can result in severe economic losses to the swine industry. In this study, we constructed the recombinant adenovirus rAd-Cap-GP5 expressing Cap of PCV2 and GP5 of PRRSV. And the expression of Cap and GP5 protein in the HEK-293 cells inoculated with rAd-Cap-GP5 were confirmed by immunoperoxidase monolayer assay (IPMA), indirect immunofluorescence assay (IFA) and Western blotting, respectively. The immunogenicity of recombinant adenoviruses rAd-Cap-GP5 was examined in mice by vaccination with the recombinant adenovirus. The results showed that the mice could produce anti-PCV2 and PRRSV antibodies detected by indirect ELISA and virus neutralization assay. It indicated that rAd-Cap-GP5 could provide humoral immunity responses in mice. The recombinant adenovirus rAd-Cap-GP5 might be an attractive candidate vaccine for preventing the disease associated with PCV2 and PRRSV infection.
Adenoviridae ; genetics ; metabolism ; Animals ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; prevention & control ; virology ; Circovirus ; genetics ; metabolism ; Immunization ; Mice ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology