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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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Related in: MedlinePlus

Expression of GM-CSF by rHuN4-GM-CSF. Cells were infected at a multiplicity of infection (MOI) of 1 with viruses and incubated for 24 hours before analysis. (A) The virus-infected MARC-145 cells were fixed and tested by IFA to determine the expression of PRRSV N protein and porcine GM-CSF. Original Magnification 200×. (B) Western blot detection of GM-CSF in the supernatant fraction (sup) or cell lysates (cell lys) of MARC-145 cells infected with viruses.
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Fig3: Expression of GM-CSF by rHuN4-GM-CSF. Cells were infected at a multiplicity of infection (MOI) of 1 with viruses and incubated for 24 hours before analysis. (A) The virus-infected MARC-145 cells were fixed and tested by IFA to determine the expression of PRRSV N protein and porcine GM-CSF. Original Magnification 200×. (B) Western blot detection of GM-CSF in the supernatant fraction (sup) or cell lysates (cell lys) of MARC-145 cells infected with viruses.

Mentions: We next investigated whether the inserted GM-CSF gene could be expressed in recombinant PRRSV-infected cells. As expected, mouse anti-GM-CSF monoclonal antibody (mAb) specifically detected the MARC-145 cells infected with rHuN4-GM-CSF in IFA (Figure 3A), but not cells infected with the HuN4-F112 control virus. As shown in Figure 3B, the expression of GM-CSF protein (22 kDa) was detected in the supernatant and cell lysates of MARC-145 cells that infected with the recombinant rHuN4-GM-CSF, but not the parental virus, by Western blotting using anti-porcine GM-CSF mAb.Figure 3


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)

Expression of GM-CSF by rHuN4-GM-CSF. Cells were infected at a multiplicity of infection (MOI) of 1 with viruses and incubated for 24 hours before analysis. (A) The virus-infected MARC-145 cells were fixed and tested by IFA to determine the expression of PRRSV N protein and porcine GM-CSF. Original Magnification 200×. (B) Western blot detection of GM-CSF in the supernatant fraction (sup) or cell lysates (cell lys) of MARC-145 cells infected with viruses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Expression of GM-CSF by rHuN4-GM-CSF. Cells were infected at a multiplicity of infection (MOI) of 1 with viruses and incubated for 24 hours before analysis. (A) The virus-infected MARC-145 cells were fixed and tested by IFA to determine the expression of PRRSV N protein and porcine GM-CSF. Original Magnification 200×. (B) Western blot detection of GM-CSF in the supernatant fraction (sup) or cell lysates (cell lys) of MARC-145 cells infected with viruses.
Mentions: We next investigated whether the inserted GM-CSF gene could be expressed in recombinant PRRSV-infected cells. As expected, mouse anti-GM-CSF monoclonal antibody (mAb) specifically detected the MARC-145 cells infected with rHuN4-GM-CSF in IFA (Figure 3A), but not cells infected with the HuN4-F112 control virus. As shown in Figure 3B, the expression of GM-CSF protein (22 kDa) was detected in the supernatant and cell lysates of MARC-145 cells that infected with the recombinant rHuN4-GM-CSF, but not the parental virus, by Western blotting using anti-porcine GM-CSF mAb.Figure 3

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