ABSTRACT
BACKGROUND: Porcine cirovirus type 1 (PCV1) and type 2 (PCV2) are circulating in Chinese pig herds and the infected pigs develop antibodies to both viruses. Current commercial available ELISA kits cannot differentiate PCV2-specific antibodies from the mixtures of PCV1 and PCV2 antibodies in PCV1/2-infected or PCV2-vaccinated pigs. Therefore, the need for developing PCV2-specific ELISA methods is urgent to evaluate PCV2 antibody level in exclusion of PCV1 antibody interference after PCV2 vaccination. RESULTS: Virus-like particles (VLPs) of PCV2 based on the recombinant Cap protein were expressed in Escherichia coli. A competing ELISA was established by using the VLPs as coating antigen and a PCV2-specific monoclonal antibody as the competing antibody. The competing ELISA was compared with the results obtained by using an immunoperoxidase monolayer assay on 160 serum samples. The sensitivity and specificity of this competing ELISA were determined as 96.5 and 96.0 %, at 2 standard deviation from the mean or 91.8 and 100 % at 3 standard deviations from the mean. Next, a serological survey of 1297 vaccinated serum samples collected from commercial pig herds in Beijing, Hunan and Henan provinces in China was conducted. The results showed that 85.9 % of sera having positive PCV2 antibodies. CONCLUSIONS: The competing ELISA we developed in this study was both sensitive and specific to PCV2 and was suitable for large-scale PCV2 antibody monitoring in exclusion of PCV1 antibody interference after PCV2 vaccination.
Subject(s)
Antibodies, Viral/blood , Circoviridae Infections/veterinary , Circovirus/immunology , Enzyme-Linked Immunosorbent Assay/veterinary , Swine Diseases/virology , Animals , Antibodies, Monoclonal , Antibodies, Viral/isolation & purification , Antigens, Viral , Capsid Proteins/immunology , Circoviridae Infections/blood , Circoviridae Infections/immunology , Circoviridae Infections/virology , Circovirus/classification , Enzyme-Linked Immunosorbent Assay/methods , Hot Temperature , Reproducibility of Results , Sensitivity and Specificity , Specific Pathogen-Free Organisms , Swine , Swine Diseases/blood , Swine Diseases/immunologyABSTRACT
Female BALB/c mice were immunized with a commercial PCV2 vaccine, and a monoclonal antibody (MAb) designated as 3H11 was achieved by hybridoma techniques. The MAb specifically reacted with Cap protein of PCV2, which has been identified by western blot. Immunoperoxidase monolayer assay results showed that 3H11 did not cross-react with PCV1-infected cells. Therefore, this work suggested that 3H11 could be a useful tool as a specific diagnostic reagent for PCV2 research.
Subject(s)
Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Capsid Proteins/immunology , Circovirus/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/immunology , Circovirus/pathogenicity , Epitope Mapping , Female , Mice , Mice, Inbred BALB C , SwineABSTRACT
BACKGROUND: Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. RESULTS: In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. CONCLUSIONS: Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.
Subject(s)
Cattle Diseases/prevention & control , Cattle Diseases/virology , Foot-and-Mouth Disease/prevention & control , Foot-and-Mouth Disease/virology , Vaccines, Virus-Like Particle/biosynthesis , Animals , Batch Cell Culture Techniques/methods , Cattle , Escherichia coli/genetics , Protein Engineering/methods , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/therapeutic useABSTRACT
We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.
