Expression And Immunogenicity Of Fc Tagged GP5Glycoproteins Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is caused by a single-strandedpositive-sense RNA virus, PRRSV. The disease has brought huge economic loss to the pigindustry because of the widespread emergence and high pathogenicity of the viruses, andcurrent vaccines can not provide long-lasting effective protection to the animals against theinfection. GP5is a major glycosylated envelope protein, which plays an important role in theprocess of virus assembly and invasion, and the induction of immune responses to PRRSV.As a main inducer of neutralizing antibodies, GP5has become a leading target in the designof new vaccines against PRRSV. Previous studies have shown that the native GP5glycoprotein is poorly immunogenic and not able to induce robust protective responses,probably due to the presence of shielding N-linked glycans and an immunodomiantnon-neutralizing decoy epitope in close to its neutralizing epitope. In addition, the signalpeptide and three transmembrane domains can affect the expression level of GP5protein inmammalian and prokaryotic expression systems. Therefore, for the development of subunitvaccines targeting GP5, it is necessary to optimize the protein to improve its immunogenicityand the induction of neutralizing antibodies.To reconstruct the GP5protein, a highly pathogenic PRRSV strain was used as thetemplate. The GP5gene was truncated by deleting the signal peptide and transmembranedomains, and the extracellular and intracellular domains were linked with three repeats ofGGGGS amino acid sequences, then fused with a human IgG Fc tag to make the protein ofGP5-Fc. As the neutralizing epitopes locate in the ectodomain of GP5, another fusion proteinGP5N-Fc which containing only the ectodomain of GP5was also constructed. In this study,these two Fc tagged GP5proteins were produced using baculovirus/insect cell expressionsystem, and the truncated GP5protein was also produced using prokaryotic expressionsystem. The purified GP5, GP5-Fc and GP5N-Fc proteins were used to immunize mice, andserum samples were collected for further analyses. PRRSV specific antibodies were detectedby ELISA and Western-blot. The results showed that both GP5-Fc and GP5N-Fc elicitedsignificant serum responses to the functional GP5on PRRSV virons. Although thebacterium-derived GP5could elicit high level of GP5-specific antibodies, the titer of the antibodies to the virus particles was significantly lower than GP5-Fc and similar to theGP5N-Fc antiserum. We found in our research that GP5N-Fc failed in inducing significanttiter of neutralizing antibodies, despite the fact that the neutralizing epitopes only located inthe ectodomain. In contrast, GP5-Fc was shown as an effective inducer of neutralizingantibodies. It is speculated that the conformation of the ectodomain plays a critical role in thepresentation of the neutralizing epitopes for the inducing of neutralizing antibodies, and thepresence of the endodomain may benefit the foding of the ectodomain to form a properconformation. Meanwhile, the results suggested that the fusion protein GP5-Fc was a betterimmunogen.In summary, by the analysis of immunogenicity of the GP5, GP5-Fc and GP5N-Fcproteins, which come from different expression systems, we confirmed that thebacterium-derived GP5protein could induce neutralizing antibodies. More importantly, inthis study, we constructed a fusion protein GP5-Fc, which is not only well immunogenic, butalso an effective inducer of neutralizing antibodies in mice. This designed immunogen haspotential as a candidate for the future development of new generation vaccine against PRRSVinfection.