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

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.

On the stability of sequences inserted into viral genomes

“…In another attempt, granulocyte–macrophage colony-stimulating factor (GM-CSF) was inserted in a PRRSV vaccine strain. The inserted gene was stably expressed upon serial passaging in vitro , and the presence of GM-CSF led to increased surface expression of MHCI+, MHCII+, and CD80/86+ (Yu et al. 2014).…”

Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs

“…GM-CSF is a multi-functional immune modulator, playing an important role in the differentiation of the progenitor cells into DCs in vitro and in vivo [ 44 ] and it has been employed as an adjuvant to enhance the immunogenicity in many viral vaccines [ 39 , 45 - 52 ]. In our studies, it was found that expression of GM-CSF by RABV could result in the recruitment and activation of more DCs than the parent virus in the murine [ 38 , 39 ] as well as in the canine model via parenteral as well as oral routes.…”

Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4

“…As a potential remedy to this problem, foreign gene was inserted between PRRSV ORF1b and ORF2a, along with a copy of TRS. This approach had particular advantage of minimizing the effects of expression and post-translational modification of viral proteins [ 33 ], and a series of recombinant PRRS viruses have been constructed using this approach [ 33 – 37 ]. In this study, the pIL-4 gene was inserted between N gene and 3′-UTR of a live attenuated PRRSV infectious clone using a similar strategy.…”