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.

Fig. 1

Schematic representation for the construction of rGP5 and rGP5-fljB. rGP5 fragment with the deletion of its signal peptide (green) and transmembrane regions (light grey) was amplified by overlap-PCR, and inserted into the BamHI and EcoRI digested expression vector pColdI or pGEX-6p-1 to create pCold-rGP5 or pGEX-6p-1-rGP5 respectively. Flagellin fljB gene (light blue) was amplified from the genomic DNA of attenuated Salmonella Typhimurium SL7207 strain and cloned into the EcoRI and SalI sites of pCold-rGP5, resulting in a recombinant plasmid pCold-rGP5-fljB. “L” represents the linker sequence GGGGS

Fig. 2

SDS-PAGE analysis of recombinant bacteria of BL21 (DE3)(pCold-rGP5) (a), BL21(DE3)(pGEX-6p-1-rGP5) (b) and BL21 (DE3)(pCold-rGP5-fljB) (c). (a) Lanes: M, molecular weight markers; 1, product of BL21(DE3)(pCold) induced by IPTG; 2, Lysate supernatant of bacteria bearing His-rGP5 induced by IPTG; 3, Inclusion bodies of bacteria bearing His-rGP5 induced by IPTG; 4, product of bacteria bearing His-rGP5 not induced. (b) Lanes: M, molecular weight markers; 1, product of BL21(DE3)(pGEX-6p-1) induced by IPTG; 2, Lysate supernatant of bacteria bearing GST-rGP5 induced by IPTG; 3, Inclusion bodies of bacteria bearing GST-rGP5 induced by IPTG. (c) Lanes: M, molecular weight markers; 1, product of BL21(DE3)(pCold) induced by IPTG; 2, product of bacteria bearing rGP5-FljB not induced; 3, Lysate supernatant of bacteria bearing rGP5-FljB induced by IPTG; 4, Inclusion bodies of bacteria bearing rGP5-FljB induced by IPTG

Fig. 3

SDS-PAGE analysis of purified His-rGP5 and rGP5-FljB proteins. The fused His-rGP5 (a) and rGP5-FljB (b) were purified via Ni-NTA affinity columns. Lanes: M, molecular weight markers; 1 and 2, purified His-rGP5 or rGP5-FljB proteins

Fig. 4

Western blotting analysis of His-rGP5 and rGP5-FljB proteins. (a) Analysis of His-rGP5 and rGP5-FljB with an anti-PRRSV polyclonal antibody. Lanes: M, molecular weight markers; 1, 1 × SDS-loading buffer; 2, purified His-rGP5 protein; 3, purified rGP5-FljB protein. (b) Analysis of rGP5-FljB with an anti-FljB polyclonal antibody. Lanes: M, molecular weight markers; 1, Lysate supernatant of BL21(DE3)(pCold-rGP5-FljB) induced by IPTG; 2, Inclusion bodies of BL21(DE3)(pCold-rGP5-FljB) induced by IPTG; 3, product of BL21(DE3)(pCold) induced by IPTG

Fig. 5

TLR5-specific activity of recombinant fusion proteins. The TLR5-specific activity of recombinant flagellin fusion protein was examined on the HEK293-mTLR5 cell line expressing mouse TLR5. Cells were treated with endotoxin-free recombinant proteins rGP5 or rGP5-FljB at the concentration of 10 and 100 ng/ml for 5 h. For positive controls, HEK293-mTLR5 cells were treated with the TLR5 agonist Flagellin. Supernatants were collected and expression levels of IL-8 were then evaluated by ELISA. Error bars indicate standard deviations of the means. Statistical significance was determined at p < 0.01 (**) or p < 0.001 (***)

Fig. 6

Immunization schedule and GP5-specific IgG antibody titers in serum. (a) C3H/HeJ mice were randomly divided into five groups (6 mice per group) and immunized intraperitoneally either with rGP5, rGP5-FljB, rGP5 + R848, rGP5 + aluminium adjuvant, or PBS, respectively. These mice were immunized three times on days 0, 14, and 28 at a dose of 50 μg rGP5, 50 μg rGP5-FljB, 10 μg R848 or isochoric aluminium adjuvant in 200 μL. Blood was collected from eye sockets on days 26, 40, 52 and 64 for analysis of anti-GP5 IgG titers by ELISA. (b) GP5-specific IgG antibody titers in serum at day 12 after the boost and third immunization. (c) IgG1 and IgG2a levels in serum at day 12 after the third immunization. Data reflects the mean ± SD by using the Student t test at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***)

Fig. 7

PRRSV-specific IgG antibody titers in serum at day 12 after the third immunization. C3H/HeJ mice were randomly divided into five groups (6 mice per group) and immunized intraperitoneally either with rGP5, rGP5-FljB, rGP5 + R848, rGP5 + aluminium adjuvant, or PBS, respectively. These mice were immunized three times on days 0, 14, and 28 at a dose of 50 μg rGP5, 50 μg rGP5-FljB, 10 μg R848 or isochoric aluminium adjuvant in 200 μL. Blood was collected from eye sockets on days 40 for analysis of anti-PRRSV IgG titers by ELISA. Data reflects the mean ± SD by using the Student t test at p < 0.01 (**) or p < 0.001 (***)

Fig. 8

The longevity of GP5-specific serum IgG titers. Mice were bled for 36 days at 12 days intervals after 3rd immunization, and serum IgG levels were determined by indirect ELISA. The ELISA plates were coated with GST-taged GP5 antigen. The time course of GP5-specific antibodies induced by rGP5 alone, rGP5-FljB or rGP5 + R848 was analyzed. Data reflects the mean ± SD by using the Student t test at p < 0.05 (*), p < 0.01 (**) or p < 0.001 (***)