Expression and characterization of PRRSV ORF5a protein using an alphavirus-derived replicon vector system

1. Mark Mogler1,2; Kurt Kamrud1,3; DL Hank Harris1,3,4 1Harrisvaccines Inc d/b/a Sirrah-Bios, Ames, Iowa; Department of 2VMPM, 3Animal Science, 4VDPAM, Iowa State University, Ames, Iowa Expression and characterization of PRRSV ORF5a protein using an alphavirus-derived replicon vector system Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) remains a very important disease of swine worldwide. Recent investigations in both PRRSV and equine arteritis virus have identified a previously unknown viral protein produced from the subgenomic mRNA5, designated ORF5a protein. In PRRSV, this protein consists of 51 amino acids, and possesses a conserved RQ-rich domain on the carboxyl terminus. The protein has been demonstrated in highly purified virus preparations, and has been successfully expressed in bacterial cells. Our group has been evaluating vaccine candidates by expressing various PRRSV structural proteins using an alphavirus-derived replicon vector system. This approach utilizes an expression vector derived from Venezuelan equine encephalitis virus (VEEV) strain TC-83. The desired protein can be expressed in eukaryotic cells and harvested for use as a vaccine antigen. Alternatively, the RNA replicon can be packaged into a VEEV capsid and envelope for use as a single-cycle viral vector, termed a replicon particle (RP) (Figure 1). Cloning and sequencing Native ORF5a sequence from PRRSV genomic RNA was amplified using primers “ORF5a-AscI-F” and “ORF5a-PacI-R” with OneStep RT-PCR (Qiagen) reagents. These primers (Table 1) include restriction sites to facilitate further cloning steps. The RT-PCR product was confirmed to have the correct-sized band by agarose gel electrophoresis, then subcloned into pCR2.1-TOPO and transformed into TOP-10 E. coli (Invitrogen). Colonies were screened for inserts and sequenced to confirm that native nucleotide sequence had been maintained (Table 2). Replicon construction The alphavirus-derived replicon plasmid (pVEK) contains TC-83 non-structural protein 1-4 genes, an IRES element derived from enterovirus 71, a multiple cloning site and selection markers. The replicon plasmid and TOPO-ORF5a plasmid were digested with AscI and PacI, and ORF5a DNA fragment was ligated into the multiple cloning site. Assembled replicon clones were screened for inserts and confirmed by sequencing. A replicon clone containing the ORF5a gene, termed “pVEK-ORF5a” was selected for further study. Peptide N12 Peptide C13 Peptide RQ12 Anti-peptide antibody purification Synthetic peptides (GenScript) corresponding to various amino acid residues of ORF5a protein (Figure 3) were used to purify antibodies from convalescent sera. Briefly, synthetic peptide was coupled to NHS-activated agarose (Thermo Pierce) and incubated with sera overnight at 4°C. After washing with PBS, the bound antibodies were eluted with 0.1M glycine·HCl (pH 3.0) and neutralized with 1M phosphate. Purified antibodies were used in Western blot to determine reactivity with ORF5a protein (Figure 4). Antibodies purified against the peptide “N12” reacted with recombinant ORF5a protein. Antibodies purified against peptide “RQ12” and peptide “C13” did not detect recombinant ORF5a protein. Self-replicating mRNA or “replicon” Helper mRNA TC-83structural genes 5’- Non-structural genes IRES ORF5a gene - 3’ -3’ Replicon RNA and helper RNA are introduced into cells by electroporation Replicon RNA only is introduced into cells by electroporation kDa Single-cycle vaccine particles, containing the replicon RNA expressing gene(s) of interest are harvested and used to formulate the vaccine. Protein encoded by the gene(s) of interest is expressed at high levels and harvested for use as the vaccine antigen. Figure 4. Western blot of recombinant ORF5a protein detected by either convalescent sera or anti-peptide purified antibodies. The convalescent sera is the same source as Figure 2. Subunit protein production replicon particle production process Expression analysis Replicon RNA was transcribed in vitro using linearized pVEK-ORF5a plasmid as template and RiboMax T7 transcription reagents (Promega). The purified replicon RNA was electroporated into Vero cells, which were seeded into a roller bottle and incubated overnight. Cells were collected, washed with PBS and lysed with RIPA buffer (Thermo). Cell lysates were analyzed for ORF5a protein expression by Western blot. Briefly, prepared cell lysates and control antigens were run on a 12% SDS-PAGE gel (Invitrogen). The proteins were then transferred to a PVDF membrane, blocked with 5% non-fat dry milk, then incubated with primary antibody at 4°C overnight. The next day the blots were washed and incubated with secondary antibody for 1 hour at room temp. Blots were then washed and developed with TMB substrate (KPL). The primary antibodies were swine origin (convalescent sera, described in Figure 2) and the secondary antibody was goat-anti-swine IgG-HRP (Fitzgerald). Figure 1. Process description of both subunit protein and replicon particles using the alphavirus-derived replicon vector system. RP production Replicon RNA (pVEK-ORF5a) and helper RNA (TC-83 glyoprotein and capsid) were co-electroporated into Vero cells (Figure 1). After overnight incubation, RP were harvested by affinity chromatography using cellufine sulfate (Chisso). Titration of RP was accomplished by IFA targeting the nsp2 of TC-83 (Figure 5). Aliquots of RP were frozen at -80°C prior to use. Results Construction of alphavirus vector The ORF5a gene from a Type 2 PRRSV strain was successfully cloned into an alphavirus replicon vector. The gene does not encode any marker epitopes, such as 6xHis. This construct can be used to produce either recombinant protein or replicon particles (Figure 1). Expression of ORF5a protein In Western blot analysis, recombinant ORF5a protein migrated and reacted similarly to ORF5a found in purified PRRSV preparations. The ORF5a protein migrates slowly compared to its predicted mass, confirming what has been described by other investigators. Both the native and recombinant ORF5a proteins were detected using sera taken from infected pigs (Figure 2). Anti-peptide antibody purification Antibodies from convalescent sera were purified using synthetic peptides. Antibodies purified using the peptide corresponding to the N-terminal twelve residues of ORF5a protein reacted with recombinant ORF5a protein on Western blot (Figure 3 & 4). Antibodies purified using two other peptides did not detect the antigen. Immunization of pigs with ORF5a replicon particles Young pig vaccination trials evaluating RP expressing ORF5a protein are ongoing. Figure 5. Immunofluorescent straining of RP-infected Vero cells using anti-nsp2 antibody (left). Roller bottle apparatus used in both recombinant protein and RP production (right). Young pig immunization Weaned pigs were obtained at three weeks of age from a PRRSV-negative source farm. Pigs were randomized into two groups. One group received RP expressing ORF5a protein, and the other group received a sham RP vaccine. This study is currently in progress. Sera from this study will be tested for anti-PRRSV activity using Western blot, ELISA, and virus neutralization assays. kDa Selected references Firth et al (2011). Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production. J Gen Virol 92: 1097-106. Johnson et al (2011). Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by a alternative ORF5 present in all arteriviruses. J Gen Virol 92:1107-16. Kamrud et al (2010) Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle. J Gen Virol 91:1723-7. Figure 2. Western blot of native and recombinant ORF5a protein. The primary antibody is convalescent sera from a pig hyperimmunized by two successive PRRSV challenges (strain HLV013). The authors would like to thank Jill Gander, Ryan Vander Veen, Kara Burrack, Kay Kimpston-Burkgren, Pam Whitson, and Ashley Baert.