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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: 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.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-?-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.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.

SUBMITTER: Xiong D

PROVIDER: S-EPMC4489122 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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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.

Xiong Dan D Song Li L Zhai Xianyue X Geng Shizhong S Pan Zhiming Z Jiao Xinan X

BMC veterinary research 20150523

<h4>Background</h4>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 PR ...[more]

PMID: 26001608

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Project description:Porcine reproductive and respiratory syndrome (PRRS) is a serious viral disease affecting the global swine industry. Its causative agent, PRRS virus (PRRSV), is an enveloped virus, and therefore membrane fusion between its envelope and host cell target membrane is critical for viral infection. Though much research has focused on PRRSV infection, the detailed mechanisms involved in its membrane fusion remain to be elucidated. In the present study, we performed confocal microscopy in combination with a constitutively active (CA) or dominant negative (DN) mutant, specific inhibitors, and small interfering RNAs (siRNAs), as well as multiple other approaches, to explore PRRSV membrane fusion. We first observed that PRRSV membrane fusion occurred in Rab11-recycling endosomes during early infection using labeled virions and subcellular markers. We further demonstrated that low pH and cathepsin E in Rab11-recycling endosomes are critical for PRRSV membrane fusion. Moreover, PRRSV glycoprotein 5 (GP5) is identified as being cleaved by cathepsin E during this process. Taken together, our findings provide in-depth information regarding PRRSV pathogenesis, which support a novel basis for the development of antiviral drugs and vaccines.IMPORTANCE PRRS, caused by PRRSV, is an economically critical factor in pig farming worldwide. As PRRSV is a lipid membrane-wrapped virus, merging of the PRRSV envelope with the host cell membrane is indispensable for viral infection. However, there is a lack of knowledge on its membrane fusion. Here, we first explored when and where PRRSV membrane fusion occurs. Furthermore, we determined which host cell factors were involved in the process. Importantly, PRRSV GP5 is shown to be cleaved by cathepsin E during membrane fusion. Our work not only provides information on PRRSV membrane fusion for the first time but also deepens our understanding of the molecular mechanisms of PRRSV infection, which provides a foundation for future applications in the prevention and control of PRRS.

Project description:PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4+ T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of TLR3, 7, 8, and 9. Our results showed that the expression of TLR3, 7, and 9 were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (TNF-α, IL-6, IL-1β and IL-12). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of IL-10 expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.

Dataset Information

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.

Publications

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.

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