Vaccine immunization is now one of the most effective ways to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. Impurity is one of the main factors affecting vaccine safety and efficacy. Here we present a novel innovative PRRSV purification approach based on surface display technology. First, a bifunctional protein PA-GRFT (protein anchor-griffithsin), the crucial factor in the purification process, was successfully produced in Escherichia coli yielding 80 mg/L of broth culture. Then PRRSV purification was performed by incubation of PA-GRFT with PRRSV and gram-positive enhancer matrix (GEM) particles, followed by centrifugation to collect virions loaded onto GEM particles. Our results showed that most of the bulk impurities had been removed, and PA-GRFT could capture PRRSV onto GEM particles. Our lactic acid bacteria-based purification method, which is promising as ease of operation, low cost and easy to scale-up, may represent a candidate method for the large-scale purification of this virus for vaccine production.
The porcine circovirus type 2 (PCV2) capsid (Cap) protein and porcine parvovirus (PPV) VP2 protein have been studied in vaccines to control postweaning multisystemic wasting syndrome (PMWS). Virus-like particle (VLP) vaccines are nonreplicative vectors that deliver epitopes and induce immune responses. However, most VLP vaccines are recombinant proteins expressed in eukaryotic systems and are expensive and complex. In this study, the full-length PCV2-Cap and PPV-VP2 proteins were expressed in Escherichia coli, which selfassembled into VLPs. The highly soluble proteins were purified using Ni-chelating affinity chromatography. The proteins self-assembled into VLPs of *20 nm (Cap VLP) and 25 nm (VP2 VLP) in diameter. The immunogenicities of Cap VLP and VP2 VLP were determined in piglets coinfected with PPV and PCV2 postimmunization. The results suggested that Cap VLP and VP2 VLP did not antagonize each other. The combined vaccine induced stronger humoral and cellular immune responses and provided the best protection against PPV and PCV2 coinfection. On a farm containing PMWS-infected pigs, the combined Cap VLP and VP2 VLP vaccine significantly improved piglet growth indices; the average daily weight gains were significantly higher than those of the Cap VLP vaccine and nonimmunized groups. Thus, Cap and VP2 protein expression in E. coli is feasible for large-scale VLP vaccine production. The combined vaccine may be a promising candidate vaccine for better preventing PMWS-associated diseases coinfected with PCV2 and PPV.
Aims: Purification of porcine circovirus type 2 (PCV2) using Gram-positive enhancer matrix (GEM) surface display technology and immunogenicity evaluation of the purified antigen. Methods and Results: A recombinant bifunctional protein containing a protein anchor domain and a 'virus anchor' domain was designed as a protein linker (PL) between PCV2 and GEM particles. By incubating with PL and GEM particles sequentially, PCV2 could be purified and enriched through a simple centrifugation process with GEM surface display technology. Our data showed that one unit (2Á5 9 10 9 particles) of GEM particles with 80 lg PL could purify 100 ml of PCV2-containing culture supernatant (viral titre: 10 6Á5 TCID 50 per ml À1 ) with a recovery rate up to 99Á6%. The impurity removal efficiency of this method, calculated according to decreased total protein content during purification, was approximately 98%. Furthermore, in vivo experimentation showed that piglets immunized with purified PCV2 could elicit strong immune responses to prevent against PCV2 infection. Conclusion: Porcine circovirus type 2 could be efficiently purified and enriched with GEM display technology via a crucial PL, and the purified PCV2 could elicit effective immune responses against PCV2 infection. Significance and Impact of the Study: The GEM-based purification method established here is cost-efficient and high-throughput, and may represent a promising large-scale purification method for PCV2 vaccine production.
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