Development Of A Eukaryotic Expression Vector Derived From Semliki Forest Virus RNA Replicon And A Derivative RNA Vaccine Candidate Against Type 2 Porcine Circovirus Infections

Porcine circovirus (PCV), a closed circular single-stranded DNA virus, is a member of the genus of Circovirus within the family Circoviridae. There exist two genotypes, nonpathogenic PCV1 and pathogenic PCV2. PCV2 is considered to be the primary pathogen of postweaning multisystemic wasting syndrome (PMWS), and also closely associated with many newly emerging infectious diseases, such as porcine dermatitis and nephropathy syndrome (PDNS), proliferative and necrotizing pneumonia (PNP), porcine respiratory disease complex (PRDC) and congenital tremor (CT), etc. These diseases have caused great economic losses to pig industry in many countries. However, no vaccine is commercially available so far. So it is urgent to develop a safe and efficacious vaccine.PCV2 ORF2 encodes the sole structural protein Cap, capsid protein. It was shown that the recombinant Cap protein expressed by recombinant baculovirus self-assembled to form capsid-like particles, which was proved immunogenic. So Cap protein can be used as an immunogen mimicking viral particles to develop vaccines against PCV2 infections.Conventional DNA vaccines have some disadvantages. For example, they express low levels of heterologous proteins, and have the potential of integration into the host genome. Recently, replicative DNA expression vectors derived from RNA replicons (self-replicating RNAs) of some alphaviruses (e.g. Semliki Forest virus, SFV) have been developed and widely used to express foreign proteins and develop therapeutic and preventive vaccines against cancers and viral diseases. RNA replicons cause lysis of transfected cells eventually mediated by apoptosis. They cannot produce replication-competent infectious viruses, nor are they capable of integrating themselves into cell genome. Previous studies showed that lower dosage of replicative DNA vaccines was safer and more efficient than conventionalnon-replicative DNA vaccines.pSFVl vector is a useful eukaryotic expression vector derived from SFV RNA replicon. However, it needs in vitro transcription of RNA, which restricts its wide application. So it is necessary to modify the vector for convenient use. Two pairs of specific primers were designed to amplify the sequences spnning human cytomegalovirus immediate early promoter (PCMV) and SV40 polyadenylation signal (SV40 polyA) from plasmid pEGFP-Nl by PCR. The amplified PCMVIE and SV40 polyA were cloned into the unique Sphl and Spel sites, respectively, of pSFVl, creating a recombinant plasmid designated as pSFVJCS. Subsequently, enhanced green fluorescent protein (EGFP) gene was cloned into pSFVlCS, resulting in a recombinant plasmid named pSFVlCS-EGFP. BHK-21 or 293T cells transfected with pSFVlCS-EGFP were shown to express EGFP gene efficiently, indicating that the modified expression vector can express a foreign protein.Next, PCV Cap gene was chosen as a model to verify the feasibility of using pSFVlCS as a potential RNA vaccine vector. The Cap gene was amplified from the genomic DNA of PK-15 cells infected with PCV2 JXL strain by PCR using a pair of specific primers based on the genomic sequence of PCV2 JXL strain, then cloned as a BamHl fragment into the unique BamHL site of pSFVlCS, creating a recombinant plasmid designated as pSFVlCS-Cap. The resultant plasmid was then transfected into BHK-21 and 293T cells. The results showed that the Cap gene was expressed efficiently in pSFVlCS-Cap-transfected cells, as demonstrated by indirect immunofluorescence assay (IFA). BALB/c mice inoculated with 10 or 100 jug of pSFVlCS-Cap developed anti-Cap protein specific antibodies, as indicated by IFA as well as indirect ELISA based on recombinant baculovirus-produced recombinant Cap protein, indicating that pSFVlCS-Cap can induce specific immune response in mice.In conclusion, the recombinant plasmid pSFVlCS-Cap harboring PCV2 Cap gene can express Cap protein, and is able to induce specific antibodies in mice. This study demonstrates that the resulting expression vector can be used for expressing eukaryotic proteins and as a replicative genetic...