Limits...
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.

Show MeSH

Related in: MedlinePlus

Maturation and activation of BMDCs stimulated by rHuN4-GM-CSF. BMHCs as DC precursors were cultured with or without GM-CSF. The cells were infected with viruses. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. Maturation of BMDCs treated by GM-CSF and then inoculated virus at 48 hpi (A). Activation of BMDCs were cultured without GM-CSF, but directly infected with viruses at 48 hpi (B) and 72 hpi (C). All data are the means from three independent experiments with cells from different donors. 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
getmorefigures.php?uid=PMC4255968&req=5

Fig5: Maturation and activation of BMDCs stimulated by rHuN4-GM-CSF. BMHCs as DC precursors were cultured with or without GM-CSF. The cells were infected with viruses. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. Maturation of BMDCs treated by GM-CSF and then inoculated virus at 48 hpi (A). Activation of BMDCs were cultured without GM-CSF, but directly infected with viruses at 48 hpi (B) and 72 hpi (C). All data are the means from three independent experiments with cells from different donors. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.

Mentions: To test the biological activity of GM-CSF expressed by the recombinant virus, BMDCs were isolated from porcine bone marrow, and co-cultured with the recombinant virus or parental virus. BMDCs treated with lipopolysaccharide (LPS) were included as a positive control. BMDCs were infected with PRRSV at MOI of 0.03 and the surface phenotypes of these cells were examined by flow cytometric analysis at 48 hpi. As shown in Figure 5A, infection with rHuN4-GM-CSF promoted better maturation and/or activation of BMDCs than infection with the parental virus, when they were pretreated with GM-CSF, as shown by significantly more MHC I+/CD 80/86+ and MHC II+/CD 80/86+ double positive cells.Figure 5


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)

Maturation and activation of BMDCs stimulated by rHuN4-GM-CSF. BMHCs as DC precursors were cultured with or without GM-CSF. The cells were infected with viruses. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. Maturation of BMDCs treated by GM-CSF and then inoculated virus at 48 hpi (A). Activation of BMDCs were cultured without GM-CSF, but directly infected with viruses at 48 hpi (B) and 72 hpi (C). All data are the means from three independent experiments with cells from different donors. 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

Fig5: Maturation and activation of BMDCs stimulated by rHuN4-GM-CSF. BMHCs as DC precursors were cultured with or without GM-CSF. The cells were infected with viruses. LPS and GM-CSF were used as a positive control, and the medium from untreated cells (Mock) served as a negative control. Maturation of BMDCs treated by GM-CSF and then inoculated virus at 48 hpi (A). Activation of BMDCs were cultured without GM-CSF, but directly infected with viruses at 48 hpi (B) and 72 hpi (C). All data are the means from three independent experiments with cells from different donors. The horizontal lines represent the geometric mean for each group, and statistical analysis was performed. *: P < 0.05; **: P < 0.01.
Mentions: To test the biological activity of GM-CSF expressed by the recombinant virus, BMDCs were isolated from porcine bone marrow, and co-cultured with the recombinant virus or parental virus. BMDCs treated with lipopolysaccharide (LPS) were included as a positive control. BMDCs were infected with PRRSV at MOI of 0.03 and the surface phenotypes of these cells were examined by flow cytometric analysis at 48 hpi. As shown in Figure 5A, infection with rHuN4-GM-CSF promoted better maturation and/or activation of BMDCs than infection with the parental virus, when they were pretreated with GM-CSF, as shown by significantly more MHC I+/CD 80/86+ and MHC II+/CD 80/86+ double positive cells.Figure 5

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