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Construction and in vitro evaluation of a recombinant live attenuated PRRSV expressing GM-CSF.

Yu L, Zhou Y, Jiang Y, Tong W, Yang S, Gao F, Wang K, Li L, Xia T, Cheng Q, Tong G - Virol. J. (2014)

Bottom Line: Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens.A novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued.Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. yulingxue1984@126.com.

ABSTRACT

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be an important problem for the swine industry. Inactivated vaccines and modified-live virus vaccines are widely used in the field; however, the efficacy of these PRRSV vaccines is suboptimal due to poor immunogenicity. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens. The purpose of this study was to determine if GM-CSF could increase the efficiency of PRRSV vaccine.

Methods: The GM-CSF gene was inserted in the HuN4-F112 vaccine strain by overlap PCR. The expression of GM-CSF by the recombinant virus was confirmed with methods of indirect immunofluorescent assay (IFA) and Western blotting. The stability of recombinant virus was assessed by cDNA sequence and IFA after 20 passages. To detect the biological activity of GM-CSF expressed by the recombinant virus, bone marrow-derived dendritic cells (BMDCs) were isolated and co-cultured with the recombinant virus or parental virus and the surface phenotypes of BMDCs were examined by flow cytometric analysis. The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were evaluated by ProcartaPlexTM Multiplex Immunoassays and qRT-PCR.

Results: A novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued. The GM-CSF protein was stable expressed in recombinant virus-infected cells after 20 passages. Analysis of virus replication kinetics showed that the novel vaccine strain expressing GM-CSF had a similar replication rate as the parental virus. In vitro studies showed that infection of porcine BMDCs with rHuN4-GM-CSF resulted in increased surface expression of MHCI+, MHCII + and CD80/86+ that was dependent on virus expressed GM-CSF. The expression of representative cytokines was significantly up-regulated when BMDCs were incubated with the recombinant GM-CSF expressing virus.

Conclusions: Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.

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Cytokines secreted by BMDCs treated with rHuN4-GM-CSF. The expression of cytokines determined by ProcartaPlexTM Multiplex Immunoassays. BMDCs were incubated with either rHuN4-GM-CSF or the parental virus at MOI of 0.3 at 24, 48, 72 and 96 hpi. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. (A) IL-1β; (B) IL-12; (C) IL-4; (D) IFN-γ; (E) IFN-α. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.
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Fig6: Cytokines secreted by BMDCs treated with rHuN4-GM-CSF. The expression of cytokines determined by ProcartaPlexTM Multiplex Immunoassays. BMDCs were incubated with either rHuN4-GM-CSF or the parental virus at MOI of 0.3 at 24, 48, 72 and 96 hpi. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. (A) IL-1β; (B) IL-12; (C) IL-4; (D) IFN-γ; (E) IFN-α. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.

Mentions: The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were as evaluated by ProcartaPlex™ Multiplex Immunoassays. As shown in Figure 6A, B and D, a significantly higher level of IL-1β, IL-12 and IFN-γ were observed for rHuN4-GM-CSF infected BMDCs compared to that of cells infected with the parental virus from 24 to 96 hpi. Despite the low levels, we detected slightly higher expression of IL-4 and TNF-α in BMDC treated by rHuN4-GM-CSF than treated by the parental virus (Figure 6C and E).Figure 6


Construction and in vitro evaluation of a recombinant live attenuated PRRSV expressing GM-CSF.

Yu L, Zhou Y, Jiang Y, Tong W, Yang S, Gao F, Wang K, Li L, Xia T, Cheng Q, Tong G - Virol. J. (2014)

Cytokines secreted by BMDCs treated with rHuN4-GM-CSF. The expression of cytokines determined by ProcartaPlexTM Multiplex Immunoassays. BMDCs were incubated with either rHuN4-GM-CSF or the parental virus at MOI of 0.3 at 24, 48, 72 and 96 hpi. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. (A) IL-1β; (B) IL-12; (C) IL-4; (D) IFN-γ; (E) IFN-α. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4255968&req=5

Fig6: Cytokines secreted by BMDCs treated with rHuN4-GM-CSF. The expression of cytokines determined by ProcartaPlexTM Multiplex Immunoassays. BMDCs were incubated with either rHuN4-GM-CSF or the parental virus at MOI of 0.3 at 24, 48, 72 and 96 hpi. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. (A) IL-1β; (B) IL-12; (C) IL-4; (D) IFN-γ; (E) IFN-α. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.
Mentions: The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were as evaluated by ProcartaPlex™ Multiplex Immunoassays. As shown in Figure 6A, B and D, a significantly higher level of IL-1β, IL-12 and IFN-γ were observed for rHuN4-GM-CSF infected BMDCs compared to that of cells infected with the parental virus from 24 to 96 hpi. Despite the low levels, we detected slightly higher expression of IL-4 and TNF-α in BMDC treated by rHuN4-GM-CSF than treated by the parental virus (Figure 6C and E).Figure 6

Bottom Line: Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens.A novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued.Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai, 200241, China. yulingxue1984@126.com.

ABSTRACT

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be an important problem for the swine industry. Inactivated vaccines and modified-live virus vaccines are widely used in the field; however, the efficacy of these PRRSV vaccines is suboptimal due to poor immunogenicity. Granulocyte-macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens. The purpose of this study was to determine if GM-CSF could increase the efficiency of PRRSV vaccine.

Methods: The GM-CSF gene was inserted in the HuN4-F112 vaccine strain by overlap PCR. The expression of GM-CSF by the recombinant virus was confirmed with methods of indirect immunofluorescent assay (IFA) and Western blotting. The stability of recombinant virus was assessed by cDNA sequence and IFA after 20 passages. To detect the biological activity of GM-CSF expressed by the recombinant virus, bone marrow-derived dendritic cells (BMDCs) were isolated and co-cultured with the recombinant virus or parental virus and the surface phenotypes of BMDCs were examined by flow cytometric analysis. The cytokines secreted by BMDCs infected with PRRSV, or treated with LPS, GM-CSF or medium alone were evaluated by ProcartaPlexTM Multiplex Immunoassays and qRT-PCR.

Results: A novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued. The GM-CSF protein was stable expressed in recombinant virus-infected cells after 20 passages. Analysis of virus replication kinetics showed that the novel vaccine strain expressing GM-CSF had a similar replication rate as the parental virus. In vitro studies showed that infection of porcine BMDCs with rHuN4-GM-CSF resulted in increased surface expression of MHCI+, MHCII + and CD80/86+ that was dependent on virus expressed GM-CSF. The expression of representative cytokines was significantly up-regulated when BMDCs were incubated with the recombinant GM-CSF expressing virus.

Conclusions: Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.

Show MeSH
Related in: MedlinePlus