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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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Characterization of the recombinant PRRSV expressing the GM-CSF gene in MARC-145 cells. (A) Detection of the GM-CSF gene insertion in the recombinant virus. F was product of the parental virus; the numbers show the passages of the recombinant viruses; M was Marker DL2000. The recombinant viruses used in B, C and D were passaged in MARC-145 cells for 20 passages. (B) Detection of GM-CSF protein expression in recombinant virus-infected MARC-145 cells by IFA. Original Magnification 200×. (C) Growth kinetics comparison between rHuN4-GM-CSF and the parental virus in MARC-145 cells. The infection was done as mentioned above. The cell supernatants were harvested at the indicated time points and titrated in MARC-145 cells. (D) Plaque assays for the parental virus and rHuN4-GM-CSF. MARC-145 cells infected with viruses were stained with crystal violet at 4 days post infection.
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Fig4: Characterization of the recombinant PRRSV expressing the GM-CSF gene in MARC-145 cells. (A) Detection of the GM-CSF gene insertion in the recombinant virus. F was product of the parental virus; the numbers show the passages of the recombinant viruses; M was Marker DL2000. The recombinant viruses used in B, C and D were passaged in MARC-145 cells for 20 passages. (B) Detection of GM-CSF protein expression in recombinant virus-infected MARC-145 cells by IFA. Original Magnification 200×. (C) Growth kinetics comparison between rHuN4-GM-CSF and the parental virus in MARC-145 cells. The infection was done as mentioned above. The cell supernatants were harvested at the indicated time points and titrated in MARC-145 cells. (D) Plaque assays for the parental virus and rHuN4-GM-CSF. MARC-145 cells infected with viruses were stained with crystal violet at 4 days post infection.

Mentions: To investigate whether the inserted gene could be stably maintained in the recombinant virus, we serially passaged the virus on MARC-145 cells to 20 times. The GM-CSF gene encoded by the recombinant PRRSV was detected by PCR amplification (Figure 4A) and confirmed by Sanger sequencing (data not shown). Consistent with the previous IFA data, the expression of GM-CSF by MARC-145 cells infected with the recombinant virus could be detected by IFA after 20 passages (Figure 4B).Figure 4


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)

Characterization of the recombinant PRRSV expressing the GM-CSF gene in MARC-145 cells. (A) Detection of the GM-CSF gene insertion in the recombinant virus. F was product of the parental virus; the numbers show the passages of the recombinant viruses; M was Marker DL2000. The recombinant viruses used in B, C and D were passaged in MARC-145 cells for 20 passages. (B) Detection of GM-CSF protein expression in recombinant virus-infected MARC-145 cells by IFA. Original Magnification 200×. (C) Growth kinetics comparison between rHuN4-GM-CSF and the parental virus in MARC-145 cells. The infection was done as mentioned above. The cell supernatants were harvested at the indicated time points and titrated in MARC-145 cells. (D) Plaque assays for the parental virus and rHuN4-GM-CSF. MARC-145 cells infected with viruses were stained with crystal violet at 4 days post infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Characterization of the recombinant PRRSV expressing the GM-CSF gene in MARC-145 cells. (A) Detection of the GM-CSF gene insertion in the recombinant virus. F was product of the parental virus; the numbers show the passages of the recombinant viruses; M was Marker DL2000. The recombinant viruses used in B, C and D were passaged in MARC-145 cells for 20 passages. (B) Detection of GM-CSF protein expression in recombinant virus-infected MARC-145 cells by IFA. Original Magnification 200×. (C) Growth kinetics comparison between rHuN4-GM-CSF and the parental virus in MARC-145 cells. The infection was done as mentioned above. The cell supernatants were harvested at the indicated time points and titrated in MARC-145 cells. (D) Plaque assays for the parental virus and rHuN4-GM-CSF. MARC-145 cells infected with viruses were stained with crystal violet at 4 days post infection.
Mentions: To investigate whether the inserted gene could be stably maintained in the recombinant virus, we serially passaged the virus on MARC-145 cells to 20 times. The GM-CSF gene encoded by the recombinant PRRSV was detected by PCR amplification (Figure 4A) and confirmed by Sanger sequencing (data not shown). Consistent with the previous IFA data, the expression of GM-CSF by MARC-145 cells infected with the recombinant virus could be detected by IFA after 20 passages (Figure 4B).Figure 4

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