title: A porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidate based on the fusion protein of PRRSV glycoprotein 5 and the Toll-like Receptor-5 agonist Salmonella Typhimurium FljB.
abstract: BACKGROUND
  Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by PRRS virus (PRRSV). Glycoprotein 5 (GP5) from PRRSV is a key inducer of neutralizing antibodies. Flagellin, a toll-like receptor 5 (TLR-5) agonist, is an effective inducer of innate immune responses. This study presents a novel PRRSV vaccine candidate based on the adjuvant effect of Salmonella Typhimurium FljB fused with PRRSV GP5.
  
  
  RESULTS
  A truncated rGP5 gene lacking the signal peptide and transmembrane sequences was amplified and inserted into prokaryotic expression vectors, pColdI or pGEX-6p-1. Salmonella Typhimurium flagellin fljB was amplified and inserted into the plasmid pCold-rGP5, generating recombinant plasmid pCold-rGP5-fljB. Histidine (His)-tagged rGP5 and fusion protein rGP5-FljB were induced with isopropyl-β-d-thiogalactoside, verified by SDS-PAGE and western blotting, and purified via Ni-NTA affinity columns. The TLR-5-specific bioactivity of fusion protein rGP5-FljB was determined by detecting the expression levels of the cytokine IL-8 in HEK293-mTLR5 cells by sandwich ELISA. The purified endotoxin-free proteins were administered intraperitoneally in a C3H/HeJ mouse model. The results show that immunization with the fusion protein rGP5-FljB induced a significantly enhanced GP5-specific and PRRSV-specific IgG response that persisted for almost 5 weeks. Co-administration of the rGP5 with R848 or Alum also yielded a higher IgG response than administration of rGP5 alone. The IgG1/IgG2a ratio in the rGP5-FljB immunization group was significantly higher (9-fold) than that in the rGP5 alone group and was equivalent to the response in the rGP5 + Alum immunization group, suggesting a strong Th2 immune response was induced by the fusion protein.
  
  
  CONCLUSIONS
  Purified fusion protein rGP5-FljB is capable of activating the innate immune response, as demonstrated by the results of our TLR-5-specific bioactivity assay, and FljB has adjuvant activity, as shown by the results from our administration of rGP5-FljB in a mouse model. Our findings confirm that FljB could serve as an excellent adjuvant for the production of GP5-specific and PRRSV-specific IgG antibodies as part of an induction of a robust humoral immune response.
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. xiongdan_yzu@163.com.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. xiongdan_yzu@163.com.
authors.forename: Dan
authors.initials: D
authors.lastname: Xiong
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. songli_yzu@163.com.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. songli_yzu@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Song
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. 1215693697@qq.com.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. 1215693697@qq.com.
authors.forename: Xianyue
authors.initials: X
authors.lastname: Zhai
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. gszhong@yzu.edu.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. gszhong@yzu.edu.cn.
authors.forename: Shizhong
authors.initials: S
authors.lastname: Geng
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. zmpan@yzu.edu.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. zmpan@yzu.edu.cn.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Pan
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu, 225009, China. jiao@yzu.edu.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009, China. jiao@yzu.edu.cn.
authors.forename: Xinan
authors.initials: X
authors.lastname: Jiao
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D005408:Flagellin / Q000737:chemistry* / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G; D016209:Interleukin-8 / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D019318:Porcine Reproductive and Respiratory Syndrome / Q000517:prevention & control* / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000276:immunology*; D011994:Recombinant Proteins; D013552:Swine; D051198:Toll-Like Receptor 5 / Q000819:agonists*; D014759:Viral Envelope Proteins / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: C094427:FljB protein, Salmonella; D007074:Immunoglobulin G; D016209:Interleukin-8; D011994:Recombinant Proteins; D051198:Toll-Like Receptor 5; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C114789:glycoprotein 5, PRRSV; D005408:Flagellin