Elsevier

Vaccine

Volume 25, Issue 49, 28 November 2007, Pages 8220-8227
Vaccine

Construction and immunogenicity of pseudotype baculovirus expressing GP5 and M protein of porcine reproductive and respiratory syndrome virus

https://doi.org/10.1016/j.vaccine.2007.09.069Get rights and content

Abstract

Baculovirus, which is extensively utilized as an excellent tool for production of recombinant protein in insect cells, has recently emerged as a novel and attractive gene delivery vehicle for mammalian cells. In the present study, a pseudotype baculovirus (with the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope) was used as vector to construct recombinant baculovirus coexpressing GP5 and M protein of porcine reproductive and respiratory syndrome virus (PRRSV), under the transcriptional control of two independent cytomegalovirus immediate early (CMV-IE) enhancer/promoters. The resultant recombinant baculovirus (BV-G-5m6) efficiently expressed PRRSV GP5 and M protein in mammalian cells. Intramuscular injection of BV-G-5m6 with various doses (1 × 108, 1 × 109, and 1 × 1010 PFU/mouse) induced the production of PRRSV-specific neutralizing antibodies and gamma interferon (IFN-γ) under dose-dependent pattern. Furthermore, BV-G-5m6 performed better immunogenicity, even at low dose (108 PFU), than DNA construct (pCI-5m6) encoding the same antigens, as demonstrated by significantly enhanced neutralizing antibodies and IFN-γ production. These results indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop new generation of vaccine against PRRSV infection.

Introduction

The baculovirus Autographa californica multiple Nucleopolyhedrovirus (AcMNPV) has traditionally been used as an excellent tool to overexpress recombinant proteins in insect cells. Its host specificity was originally thought to be restricted to cells derived from arthropods. However, in 1995, Hofmann et al. reported that recombinant baculovirus containing cytomegalovirus immediate-early (CMV-IE) promoter can drive the expression of a luciferase reporter gene in human hepatocytes [1]. Since this initial report, the baculovirus/mammalian expression system has been employed with different mammalian cell-active promoters to deliver genes into mammalian cells, and these studies also rapidly expanded the list of permissive mammalian cells that can be efficiently transduced by recombinant baculovirus [2]. Furthermore, it has been reported that a pseudotype baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope can extend the host range and increase the transduction efficiency in mammalian cells [3]. Thanks to highly efficient gene delivery, baculovirus has captured increasing interest as a novel vector for gene therapy and vaccine development [2], [4].

The use of baculovirus as tool for direct vaccination was initially described by Aoki et al., who found that a recombinant baculovirus expressing glycoprotein B (gB) of pseudorabies virus induces gB-specific antibodies in mice [5]. More recently, several groups have investigated the potential of pseudotype bacuolvirus as vaccine vector and demonstrated that direct vaccination with recombinant pseudotype baculovirus can induce high-level humoral and cell-mediated immunity against various antigens, such as influenza virus HA [6], plasmodium falciparum circumsporozoite protein [7], hepatitis C virus E2 glycoprotein [8]. Indeed, as a vaccine vector, baculovirus possesses several striking features in the following aspects: (1) It can accommodate a large foreign DNA insertions (>30 kb); (2) construction, manipulation, and production of recombinant baculovirus are relatively simpler and faster, and high titers (>1010 PFU/ml) can be easily reached; (3) it is non-cytotoxic and non-replicative in mammalian cells even at high multiplicity of infection (MOI); (4) there is no pre-existing antibodies against baculovious in animals that might interfere with administration of recombinant genes in the host [2], [4].

Porcine reproductive and respiratory syndrome (PRRS), characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs, is now considered one of the most important diseases in countries with intensive swine industries. The causative agent, PRRS virus (PRRSV), is a positive-strand RNA virus belonging to the family Arteriviridae[9]. The genome of PRRSV is approximately 15 kb in length and contains nine open reading frames (ORF1a/1b, ORF2a/2b, ORF3-7) [10], [11]. ORF5 and ORF6 encode two major membrane-associated proteins, the envelope glycoprotein GP5 and the non-glycosylated membrane protein M, respectively, which form disulfide-linked heterodimers (GP5/M) in the virus particle [12].

PRRSV infection poses a challenge to current vaccination strategies. Concerns of inadequate efficacy and lack of safety surround killed vaccines or modified live vaccines, respectively. Recently, several experimental vaccines by expressing the major immunogenic protein(s) of PRRSV, including DNA vaccines [13], [14], vectored vaccines (adenovirus [15], [16], pseudorabies virus [17], [18], and Mycobacterium bovis BCG [19]), have been developed. However, only limited protective immunity could be obtained with these experimental vaccines. Hence, new strategy should be developed for safer and more efficacious vaccines against PRRSV infection.

In this study, the baculovirus/mammalian expression system was used to express the two major antigens of PRRSV, GP5 and M protein. Mice inoculated with the recombinant baculovirus developed PRRSV-specific immune responses. Moreover, in comparison with DNA construct encoding the same antigens, immunization with recombinant baculovirus induced higher PRRSV-specific neutralizing antibodies and gamma interferon (IFN-γ). These results indicate that baculovirus-mediated gene delivery can be used as an alternative strategy to develop new generation of vaccine against PRRSV infection.

Section snippets

Virus and cells

The PRRSV strain YA1, which was isolated from the lungs of field pigs at the acute stage of PRRSV infection in the China and identified as a high-virulence North American type isolate [20], was used in this study. The PRRSV strain NVSL97–7895 (GenBank accession number AY545985), a highly pathogenic PRRSV isolate, was kindly provided by Dr. Chowdhury, College of Veterinary Medicine, Kansas State University. PRRS viruses were propagated and titered in MARC-145 cells. Pig kidney cells (PK-15) were

Construction and in vitro expression of recombinant baculovirus BV-G-5m6

Our previous studies have demonstrated that the pseudotype baculovirus BV-G-EGFP exhibited high transduction efficiency and high-level expression of reporter protein in mammalian cells [21]. To investigate whether the obtained baculovirus BV-G-5m6 has higher transduction efficiency and whether can express PRRSV GP5 and M protein in mammalian cells, PK-15 cells were transduced with BV-G-5m6 at an MOI of 100 and immunofluorescence assay was performed. As showed in Fig. 1, bright fluorescence

Discussion

Although baculovirus carrying an appropriate mammalian cell-active promoter exhibit high transduction efficiency and high-level expression of heterologous proteins in mammalian cells in vitro[1], it is easy to be inactivated in vivo by complement system of the host [24], which will limit its application for gene therapy and vaccine. Recent studies have demonstrated that a pseudotype baculovirus displaying the glycoprotein of vesicular stomatitis virus (VSV-G) on the envelope can extend the host

Acknowledgements

We thank Dr. G.Z. Tong and S.I. Chowdhury for generously providing the MAbs and virus. This work was supported by State 863 High Technology R&D Project of China (2006AA10A204), the National Basic Research Program (973) of China (2005CB523200), Key Technology R&D Programme (2007BAD86B02), New Century Excellent Talent Project (NCET-06-0662) and the National Natural Sciences Foundation of China (No.30571385, 30400322).

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