Yunbo Jiang

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses interferon-β production by interfering with the RIG-I signaling pathway

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s. Accumulating evidences have revealed that PRRSV infection fails to induce type I interferon (IFN-α/β), which are normally induced rapidly during virus replication in virus-infected cells. However, the potential mechanisms remain largely unclear. In this study, we showed that PRRSV infection activated the signal transduction components of NF-κB and AP-1, but not of interferon regulatory factor 3 (IRF3), an essential IFN-β transcription factor. Furthermore, PRRSV infection significantly blocked synthetic dsRNA-induced IFN-β production and IRF3 nuclear translocation. To better understand the upstream signaling events that suppress IRF3 activation, we further investigated the roles of individual components of the retinoic acid-inducible gene I (RIG-I)- and Toll-like receptor 3 (TLR3)-mediated signaling pathway for IFN-β production during PRRSV infection. We observed that PRRSV infection significantly inhibited dsRNA-induced IRF3 activation and IFN-β generation by inactivating IFN-β promoter stimulator 1 (IPS-1), an adaptor molecule of RIG-I. In contrast, PRRSV infection only partially reduced the activation of TIR domain-containing adaptor inducing IFN-β (TRIF), an adaptor molecule of TLR3. Our results suggest that PRRSV infection suppresses production of IFN-β primarily by interfering with the IPS-1 activation in the RIG-I signaling pathway.

Recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses.

Em2007, a porcine reproductive and respiratory syndrome virus (PRRSV) variant with a unique 68 aa deletion in Nsp2, was recently isolated in China. Phylogenetic and molecular evolutionary analyses indicated that Em2007 is a natural recombinant between a vaccine strain of PRRSV and circulating virus. We also tested its pathogenicity in piglets.

Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene

Since May 2006, a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV), which causes continuous high fever and a high proportion of deaths in vaccinated pigs of all ages, has emerged and prevailed in Mainland China. Huge efforts should be made towards the development of an efficient vaccine against the highly pathogenic PRRSV. Although the ORF5-encoded GP5 is the most important immunogenic protein, accumulating evidences have demonstrated that incomplete protection conferred by GP5-based vaccines. The inability to induce robust protective immunity has been postulated to be associated with the presence of a non-neutralizing decoy epitope and heavy glycosylation in close to its neutralizing epitope. In this study, a synthetic ORF5 gene (SynORF5) was engineered with the codon usage optimized for mammalian cell expression based on the native ORF5 gene of highly pathogenic PRRSV strain WUH3. Additional modifications, i.e., inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the non-neutralizing decoy epitope, and mutating four potential N-glycosylation sites (N30, N34, N35 and N51) were also included in the synthetic ORF5 gene. The immunogenicity of the SynORF5-encoded GP5 was evaluated by DNA vaccination in mice and piglets. Results showed that significantly enhanced GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-gamma level, as well as lymphocyte proliferation response, could be induced in mice and piglets immunized with DNA construct encoding the modified GP5 than those received DNA vaccine expressing the native GP5. The enhanced immunogenicity of the modified GP5 will be useful to facilitate the development of efficient vaccines against the highly pathogenic PRRSV in the future.

Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection.

Enhanced immunogenicity of the modified GP5 of porcine reproductive and respiratory syndrome virus.

The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing PRRSV GP5. More recent evidence has demonstrated a non-neutralizing decoy epitope upstream of the neutralizing epitope of GP5, which might prevent the development of a strong neutralizing antibody response against PRRSV. In the present study, we modified the ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy epitope to minimize or eliminate the decoy effect of the non-neutralizing epitope. The immunogenicity of the modified GP5 was further evaluated using DNA vaccination. The results showed that significantly enhanced neutralizing antibodies were elicited in mice immunized with the DNA construct expressing the modified GP5 compared with the native GP5. Slightly increased levels of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicate that the high immunogenicity of the modified GP5 might facilitate the development of improved PRRS vaccines in the future.

Molecular cloning and functional characterization of porcine IFN-β promoter stimulator 1 (IPS-1)

The IFN-beta promoter stimulator 1 (IPS-1), also known as MAVS/VISA/Cardif, is an adaptor molecule for the retinoic-acid-inducible protein I (RIG-I) or melanoma-differentiation-associated gene 5 (MDA5) that recognizes intracellular double-stranded RNA (dsRNA) and triggers a signal for producing type I IFN. In the present study, porcine IPS-1 cDNA was cloned, using RT-PCR coupled with rapid amplification of cDNA ends (RACE)-PCR, from porcine peripheral blood mononuclear cells. The open reading frame of porcine IPS-1 consists of 1575bp encoding 524 amino acids. The putative porcine IPS-1 protein contains a N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to mouse, rat, monkey, human and cattle counterparts, ranging from 59% to 79%. Semi-quantitative RT-PCR showed that porcine IPS-1 mRNA was widely expressed in different tissues. Porcine kidney (PK-15) cells transfected with a DNA construct encoding porcine IPS-1 produced type I IFN, and activated IRF3 and NF-kappaB. Deletion mutant analyses further revealed that both the CARD-like domain and transmembrane domain are essential for these functions. In addition, poly(I:C)-induced porcine IFN-beta promoter activation in PK-15 cells was significantly reduced by siRNA targeting IPS-1, indicating that IPS-1 is an important immunoregulator in the porcine innate immune system. The availability of porcine IPS-1 and establishment of its function in the type I IFN signaling pathway provides a useful molecule for defining its role during the course of pig infectious diseases.

Activation of NF-κB by nucleocapsid protein of the porcine reproductive and respiratory syndrome virus.

Nuclear factor kappa B (NF-κB) is a critical transcription factor in innate and adaptive immune response as well as cell proliferation and survival. Previous studies have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection activated NF-κB pathways through IκB degradation in MARC-145 cells and alveolar macrophages. To evaluate the mechanisms behind this, we investigated the role of PRRSV structural proteins in the regulation of NF-κB. In this study, we screened the structural proteins of PRRSV by NF-κB DNA-binding assay and luciferase activity assay and demonstrated that PRRSV nucleocapsid (N) protein could activate NF-κB in MARC-145 cells. Furthermore, we revealed that the region between aa 30 and 73 of N protein was essential for its function in the activation of NF-κB. These results presented here provide a basis for understanding molecular mechanism of PRRSV infection and inflammation response.

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice.

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 microg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.

A suicidal DNA vaccine co-expressing two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus antigens induce protective responses.

We constructed a suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing GP5m (a modified GP5) and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV). In mice immunization, specific immune responses were elicited by the suicidal DNA vaccine pSFV-ORF5m/ORF6. The immunogenicity and protective efficiency was then evaluated in piglets immunized with pSFV-ORF5m/ORF6 before virus challenge: PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses were developed. Post-PRRSV challenge, these immune responses were further boosted and partial protection was obtained.

Enhanced immunogenicity to food-and-mouth disease virus in mice vaccination with alphaviral replicon-based DNA vaccine expressing the capsid precursor polypeptide (P1)

Recently, alphavirus replicon-based DNA vaccines, also known as suicidal DNA vaccines, have emerged as an important strategy to enhance the potency of DNA vaccines. In this study, two different types of DNA vaccines encoding the capsid precursor polypeptide (P1) of foot-and-mouth disease virus (FMDV) were constructed and the immunogenicity were investigated and compared in mouse model. The first DNA vaccine, pcDP1, is a conventional plasmid DNA vaccine in which P1 was driven directly by a cytomegalovirus promoter. The second DNA vaccine, pSCAP1, is a Semliki Forest virus (SFV) replicon-based DNA vaccine encoding the same antigen. In vitro expression and characterization indicated that two vaccine vectors could correctly produce the P1 antigen. However, pSCAP1 could induce obvious apoptosis of the transfected cells. After immunization in BALB/c mice, the P1-specific ELISA antibodies, neutralizing antibodies, as well as lymphocyte proliferative responses induced by pSCAP1 were significantly higher than those obtained in mice immunized with pcDP1. Notably, mice immunized with the pSCAP1 had the determined ability of clearing virus in their sera after FMDV challenge. These results indicate that the SFV replicon-based DNA vaccine pSCAP1 are more effective than conventional DNA vaccine and it can be considered a promising approach for the development of a safety and efficacious vaccine against FMDV.