PRRSV-specific Antibodies Research Articles

Preliminary Findings of a Previously Unrecognized Porcine Primary Immunodeficiency Disorder

Weaned pigs from a line bred for increased feed efficiency were enrolled in a study of the role of host genes in the response to infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Four of the pigs were euthanatized early in the study due to weight loss with illness and poor body condition; 2 pigs before PRRSV infection and the other 2 pigs approximately 2 weeks after virus inoculation. The 2 inoculated pigs failed to produce PRRSV-specific antibodies. Gross findings included pneumonia, absence of a detectable thymus, and small secondary lymphoid tissues. Histologically, lymph nodes, spleen, tonsils, and Peyer's patches were sparsely cellular with decreased to absent T and B lymphocytes.

Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1(©)-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with interferon α/γ was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1(©)-α-γ-GP35 could provide partially protective efficacy, which was similar to the pVAX1(©)-α-γ (expressing interferon α/γ). For long-lasting protection, pigs inoculated with pVAX1(©)-α-γ-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-γ, and IL-4, than those vaccinated with pVAX1(©)-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1(©)-α-γ-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1(©)-GP35 group. It indicated that pVAX1(©)-α-γ-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1(©)-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.

Mannan oligosaccharide increases serum concentrations of antibodies and inflammatory mediators in weanling pigs experimentally infected with porcine reproductive and respiratory syndrome virus1,2

ABSTRACTMannan-containing products are capable of modulating immune responses in animals. However, different products may have diverse immunomodulation. The experiment was conducted to examine effects of mannan oligosaccharide (Actigen; ACT) on growth performance and serum concentrations of antibodies and inflammatory mediators in weanling pigs (Sus scrofa) experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 32 PRRSV-negative pigs (3 wk old) were randomly assigned from within blocks to 1 of 4 treatments in a 2 by 2 factorial arrangement [2 types of diet: control (0%) and ACT addition (0.04%); and with and without PRRSV] in a randomized complete block design. Pigs were blocked by initial BW within sex. Ancestry was equalized across treatments. Pigs (8/treatment) were kept individually in each pen. After 2 wk of an 8-wk period of feeding the treatments, pigs received an intranasal inoculation of PRRSV or sham medium at 5 wk of age. Infection by PRRSV decreased ADG, ADFI, and G:F throughout the experiment (P < 0.01). Actigen did not affect ADG (P = 0.450), but decreased (P = 0.047) ADFI from 28 to 42 days postinoculation (DPI). During that time, ACT improved G:F in infected pigs but not in sham controls (interaction, P = 0.009). Dietary ACT did not affect viremia in infected pigs (P > 0.05), but increased PRRSV-specific antibody titer at 35 DPI (P = 0.042). Infection with PRRSV induced the febrile responses of pigs from 3 to 10 DPI (P < 0.001) with return to normal at 14 DPI. During the experimental period, the rectal temperature of pigs was found slightly elevated by ACT (P = 0.045). Infected pigs had greater serum concentrations of IL-1β, tumor necrosis factor (TNF)-α, IL-12, interferon (IFN)-γ, IL-10, and haptoglobin (Hp) than sham controls (P < 0.001). These results indicate that PRRSV stimulated secretion of cytokines involved in innate, T-helper 1, and T-regulatory immune responses. Actigen tended to decrease the serum TNF-α concentration regardless of PRRSV (P = 0.058). The ACT × PRRSV interaction was significant for IL-1β (P = 0.016), IL-12 (P = 0.026), and Hp (P = 0.047), suggesting that infected pigs fed ACT had greater serum concentrations of these mediators than those fed the control. The increases in IL-1β and IL-12 may favorably promote innate and T-cell immune functions in infected pigs fed ACT. Feeding ACT may be useful as ACT is related to increased PRRSV antibody titers and G:F in infected pigs at certain times during infection.

Development of a Fluorescent Microsphere Immunoassay for Detection of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus Using Oral Fluid Samples as an Alternative to Serum-Based Assays

For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies developed in response to porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, we developed a multiplexed fluorescent microsphere immunoassay (FMIA) for detection of PRRSV-specific antibodies in oral fluid and serum samples. Recombinant nucleocapsid protein (N) and nonstructural protein 7 (nsp7) from both PRRSV genotypes (type I and type II) were used as antigens and covalently coupled to Luminex fluorescent microspheres. Based on an evaluation of 488 oral fluid samples with known serostatus, the oral fluid-based FMIAs achieved >92% sensitivity and 91% specificity. For serum samples (n = 1,639), the FMIAs reached >98% sensitivity and 95% specificity. The assay was further employed to investigate the kinetics of the antibody response in infected pigs. In oral fluid, the N protein was more sensitive for the detection of early infection (7 and 14 days postinfection), but nsp7 detected a higher level and longer duration of antibody response (28 days postinfection). In serum, the antibodies specific to nsp7 and N proteins were detected as early as 7 days postinfection, and the responses lasted more than 202 days. This study provides a framework from which a more robust assay could be developed to profile the immune response to multiple PRRSV antigens in a single test. The development of oral fluid-based diagnostic tests will change the way we survey diseases in swine herds and improve our ability to cheaply and efficiently track PRRSV infections in both populations and individual animals.

Hemagglutinating virus of Japan envelope (HVJ-E) can enhance the immune responses of swine immunized with killed PRRSV vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically detrimental pig pathogen that causes significant losses for the pig industry. The immunostimulatory effects of hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy and the adjuvant efficacy of HVJ-E have been previously evaluated. The objective of this study was to investigate the adjuvant effects of HVJ-E on immunization with killed PRRSV vaccine, and to evaluate the protective effects of this immunization strategy against virulent PRRSV infection in piglets. Next, the PRRSV-specific antibody response, lymphocyte proliferation, PRRSV-specific IL-2, IL-10 and IFN-γ production, and the overall protection efficacy were evaluated to assess the immune responses of the piglets. The results showed that the piglets inoculated simultaneously with killed PRRSV vaccine and HVJ-E had a significantly stronger immune response than those inoculated with killed PRRSV vaccine alone. Our results suggest that HVJ-E could be employed as an effective adjuvant to enhance the humoral and cellular responses of piglets to PRRSV.

Plasmid containing CpG motifs enhances the efficacy of porcine reproductive and respiratory syndrome live attenuated vaccine

Porcine reproductive and respiratory syndrome (PRRS) is now among the most important swine diseases that affect the Chinese swine industry. Both killed and live attenuated vaccines are currently used against the disease, but neither of them could provide full protection after vaccination. In the present study, the adjuvanticity of a plasmid containing CpG motifs (pUC18-CpG) was introduced to enhance the efficacy of a commercial PRRS live attenuated vaccine. After vaccination, PRRSV-specific antibodies, PRRSV-specific cytokines, and clinical parameters were studied and compared between different vaccinated groups. During a following challenge study, co-administration of pUC18-CpG with the vaccine could confer higher protection rate. Our results have shown that co-administration of pUC18-CpG with the vaccine could elicit more potent adaptive immune response and provide better protection.

Multiplex Method for Simultaneous Serological Detection of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to the porcine respiratory disease complex (PRDC). Routine serological diagnosis and surveillance play an important role in the prevention of PRDC, as it is a leading cause of economic losses to the swine industry. We herein describe an advanced microsphere-based immunoassay that permits the simultaneous detection of antibodies to PCV2 and PRRSV, thereby reducing the time and effort involved in testing. Recombinant PRRSV nucleoprotein antigen and the PCV2 capsid antigen were coupled to fluorophore-dyed beads with distinct spectral addresses. Weekly serum samples from 72 pigs that were experimentally exposed to either PCV2, PRRSV, or both PCV2 and PRRSV were used to validate the microbead assay (MBA) in comparison with the "gold standard" enzyme-linked immunosorbent assays. The kinetics of the PCV2- and PRRSV-specific antibody responses measured by the microbead assay were comparable to those of the standard assays; Spearman's rank correlations were 0.72 (P < 0.001) for PRRSV and 0.80 (P < 0.001) for PCV2. Diagnostic sensitivity and specificity were determined using field sera whose positive or negative status was determined by the standard tests. The diagnostic sensitivity and specificity were both 98% for PCV2 and were 91% and 93%, respectively, for PRRSV (kappa coefficients, 0.85 and 0.67 for PCV2 and PRRSV, respectively). Multiplexing did not interfere with assay performance or diagnostic sensitivity. Therefore, the described study demonstrates proof of concept for the development of more versatile and economical microbead array-based multiplex serological test panels for veterinary use.

Mannan oligosaccharide improves immune responses and growth efficiency of nursery pigs experimentally infected with porcine reproductive and respiratory syndrome virus1,2

This study was conducted to determine whether the ingestion of mannan oligosaccharide (MOS, Bio-Mos) alters the immune response of nursery pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 64 pigs (3 wk old), free of PRRSV, were used in 2 separate but similar experiments conducted sequentially. Pigs were blocked by initial BW. Sex and ancestry were equalized across treatments. Pigs were randomly assigned from within blocks to 1 of 4 treatments in a 2 × 2 factorial arrangement [2 types of diet: control (0%) and MOS addition (0.2%); 2 levels of PRRSV: with and without]. There were 8 replicate chambers of 2 pigs each. After 2 wk of a 4-wk period of feeding the treatments, pigs were intranasally inoculated with PRRSV or a sterile medium at 5 wk of age. The PRRSV challenge decreased ADG, ADFI, and G:F throughout the experiment (P < 0.001). Feeding MOS improved G:F of the pigs during d 7 to 14 (P=0.041) postinfection (PI). Serum concentrations of tumor necrosis factor (TNF)-α, C-reactive protein, and haptoglobin were increased by PRRSV (P < 0.001). The MOS × PRRSV interaction was significant for TNF-α at d 14 PI (P=0.028), suggesting that infected pigs fed MOS had less TNF-α than those fed the control. Dietary MOS increased serum IL-10 at d 14 PI (P=0.036). Further, MOS-fed pigs had greater numbers of white blood cells (WBC) at d 3 (P=0.048) and 7 PI (P=0.042) and lymphocytes at d 7 PI (P=0.023) than control-fed pigs. In contrast, PRRSV decreased (P < 0.01) WBC numbers until d 14 PI. Dietary MOS appeared (P=0.060) to increase the neutrophils in PRRSV-infected pigs at d 3 PI, but no (P=0.202) MOS × PRRSV interaction was found. Infection with PRRSV increased rectal temperature (RT) of pigs at d 3 PI (P < 0.001) and continued to affect the infected pigs fed the control diet until d 14 PI. The MOS × PRRSV interaction for RT was found at d 7 (P < 0.01) and 10 (P=0.098) PI, indicating that the infected pigs fed MOS had a decreased RT compared with those fed the control. This could explain why feed efficiency was improved by MOS. No effect (P > 0.05) of treatments on viremia or PRRSV-specific antibody was observed. These results suggest that MOS is associated with rapidly increased numbers of WBC at the early stage of infection and alleviates PRRSV-induced effects on G:F and fever. The results also indicate that the reduced intensity of inflammation by MOS may be related to changes in inflammatory mediator levels at the end of the acute phase.

Intranasal delivery of whole cell lysate of Mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease to pork producers worldwide. Commercially, both live and killed PRRSV vaccines are available to control PRRS, but they are not always successful. Based on the results of mucosal immunization studies in other viral models, a good mucosal vaccine may be an effective way to elicit protective immunity to control PRRS outbreaks. In the present study, mucosal adjuvanticity of Mycobacterium tuberculosis whole cell lysate ( Mtb WCL) was evaluated in pigs administered a modified live PRRS virus vaccine (PRRS-MLV) intranasally. A Mtb WCL mediated increase in the frequency of NK cells, CD8 +and CD4 + T cells, and γδ T cells in pig lungs were detected. Importantly, an increased and early generation of PRRSV specific neutralizing antibodies were detected in PRRS-MLV+ Mtb WCL compared to pigs inoculated with vaccine alone. In addition, there was an increased secretion of Th1 cytokines (IFNγ and IL-12) that correlated with a reciprocal reduction in the production of immunosuppressive cytokines (IL-10 and TGFβ) as well as T-regulatory cells in pigs vaccinated with PRRS-MLV+ Mtb WCL. Further, a complete rescue in arginase levels in the lungs mediated through Mtb WCL was observed in pigs inoculated with PRRS-MLV. In conclusion, Mtb WCL may be a potent mucosal adjuvant for PRRS-MLV in order to potentiate the anti-PRRSV specific immune responses to control PRRS effectively.

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Despite great efforts of pig holders, veterinarians, researchers and vaccine developers, the virus still causes major production losses. It is clear that efficient and correct monitoring and rational development of vaccines are crucial in the combat against this pathogen. PRRSV-specific monoclonal antibodies (mAbs) are essential tools for both diagnostic and research purposes. This study describes the production of PRRSV GP3-, GP5- and N-specific hybridomas and an extensive characterization of the mAbs. The N-specific mAbs generated in this study appear to be useful tools for diagnostics, as they were found to react with genetically very different PRRSV isolates and may serve to discriminate between European and American type PRRSV isolates. These mAbs also allowed detection of the PRRSV N protein in both formalin-fixed, paraffin-embedded tissue sections and frozen tissue sections of PRRSV-infected lungs, further illustrating their diagnostic value. Different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested shows virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the “major neutralizing epitope” suggested for American type PRRSV. The current findings argue against an important role of the identified antigenic regions in direct antibody-mediated neutralization of European type PRRSV in vivo. However, it is also clear that findings concerning a specific PRRSV epitope cannot always be generalized, as the antigenic determinants and their biological properties may differ radically between different virus isolates.

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.

Stability of porcine reproductive and respiratory syndrome virus and antibody in swine oral fluid

Objective: To evaluate the stability of porcine reproductive and respiratory syndrome virus (PRRSV) and anti-PRRSV antibodies in oral fluid as a function of time and temperature. Materials and methods: A 4-L pool of swine oral fluid was collected from 16-week-old finisher pigs. To ensure uniform, quantifiable levels of virus and antibody over time, 4 mL of PRRSV isolate ISU-P containing 1 × 1012 RNA copies per mL and 10 mL of concentrated hyperimmune anti-PRRSV antibodies were added to the pool. The pool was then divided into three equal portions: no treatment, chlorhexidine digluconate at 0.01% by volume, and isothiazolinone at 3 ppm. Each treatment was tested in triplicate at each of five temperatures (-20°C, 4°C, 10°C, 20°C, and 30°C). Samples were removed at specific intervals (0, 12, 24, 48, 72, 144, 216, and 288 hours), stored at -80°C, and then assayed for PRRSV RNA; IgM, IgA, and IgG PRRSV-specific antibody; and culturable bacteria per mL. Results: The stabilities of anti-PRRSV antibody and detectable PRRSV were temperature-dependent, with antimicrobial treatment providing no improvements in stability at lower temperatures. Both virus and antibody were stable at ≤ 10°C over 12 days of storage. Implication: Conventional serum storage protocols (freezing or refrigeration at 4°C) preserves PRRSV and anti-PRRSV antibody in oral-fluid diagnostic samples without the use of preservatives.

Plasmid containing CpG oligodeoxynucleotides can augment the immune responses of pigs immunized with porcine reproductive and respiratory syndrome killed virus vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of porcine reproductive and respiratory syndrome (PRRS), causes serious health problems in swine, resulting in great economic losses to the swine industry worldwide. The porcine reproductive and respiratory syndrome (PRRS) killed virus (KV) vaccine has been widely used in China but has been linked with side effects in pigs and only partial protection. A few studies have demonstrated the immunostimulatory adjuvant effects of the synthetic CpG oligodeoxynucleotide (ODN) motifs in humans and animals. In this study, the adjuvant efficacy and the protective effects of recombinant plasmid containing CpG ODN, in combination with PRRS KV vaccine against PRRSV were evaluated. The concentration of PRRSV-specific antibodies and cytokines and the clinical parameters (body temperature, weight gain and behavior) on the challenge and post-challenge virological profiles (virus distribution in tissues and virus titers in serum at autopsy) were investigated to define the immune responses of pigs. Our findings indicated that the immune responses of the pigs immunized with plasmid containing CpG ODN and PRRS KV vaccine were stronger than those induced by PRRS KV vaccine or PBS alone. Furthermore, the protection ratios, PRRSV titers in serum samples and virus tissue distribution, all demonstrated that the plasmid containing CpG ODN could augment the immune responses and improve the protective effect of PRRSV vaccination.

Changes in Lymphocyte Subsets and Cytokines During European Porcine Reproductive and Respiratory Syndrome: Increased Expression of IL-12 and IL-10 and Proliferation of CD4−CD8high

The changes in peripheral blood mononuclear cells (PBMCs) have been studied in several reports in an attempt to determine the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, how these changes are evoked after PRRSV infection has not yet been clarified. The aim of this study was to analyze the changes seen in lymphocyte subsets and immunomodulatory cytokine expression in pigs after an acute experimental infection with a European PRRSV field isolate. Pigs were inoculated intramuscularly with PRRSV field isolate 2982. Samples from blood, medial retropharyngeal and tracheobronchial lymph nodes, and spleen were collected at different time points for flow cytometry studies and for cytokine expression by ELISA. CD21(+) cell counts increased in PBMCs and tracheobronchial lymph node cells from 17 to 24 dpi, coinciding with an increase in PRRSV-specific antibody titer in blood. CD3(+) T-cell counts increased mainly due to an enhancement of CD4(-)CD8(high) and CD4(+)CD8(+) T cells. CD4(-)CD8(low) T cells were decreased in all organs studied, whereas CD4(+)CD8(-) T cells decreased only in the spleen. The drop in viremia correlated with an enhancement of CD4(-)CD8(high) T cells, and with a higher expression of interleukin-10 (IL-10) and interleukin-12 p40 (IL-12 p40). No efficient interferon-gamma (IFN-gamma) response was detected during the acute phase of the infection, and the expression of interferon-alpha (IFN-alpha) was late and reached its maximum expression once the viremia decreased. These results point to IL-10 and IL-12 as cytokines that might play a significant role in the PRRSV immune response, as may CD4(-)CD8(high) T cells.

GM-CSF fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus increased the immune responses and protective efficacy against virulent PRRSV challenge

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. Current vaccination strategies only provide a limited protection against PRRSV infection. This study was aimed to construct the recombinant adenovirus co-expressing GP3 and GP5 of highly pathogenic PRRSV fused with swine granulocyte-macrophage colony stimulating factor (GM-CSF) (rAd-GF35), and to detect the immune response in mice and pigs. The results showed that the rAd-GF35 could induce significantly higher PRRSV-specific neutralizing antibodies than the recombinant adenovirus only expressing GP3 and GP5 (rAd-GP35). Moreover, the fusion of GM-CSF markedly increased the secretion of IFN-γ and IL-4 in PRRSV-stimulated mice lymphocytes culture and pigs sera. Following challenge with PRRSV, piglets inoculated with recombinant rAd-GF35 had lighter clinical signs, lower viremia and less gross lesion of lungs, as compared to that of rAd-GP35 immunized group. It demonstrated that GM-CSF fused with GP3 and GP5 of PRRSV could significantly enhance the humoral and cellular immune responses and provide protection against PRRSV challenge in pigs. The recombinant adenovirus rAd-GF35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infection.

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-γ 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.

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 immune responses of mice inoculated recombinant adenoviruses expressing GP5 by fusion with GP3 and/or GP4 of PRRS virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important causes of economic losses of the swine industry. PRRS virus (PRRSV) infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants expressing fusion protein GP3–GP5, GP4–GP5, or GP3–GP4–GP5 were developed as potential vaccine against PRRSV in a mouse model. Six groups of BALB/c mice (24 mice per group) were inoculated subcutaneously twice at 2-week intervals with above mentioned recombinants and other adenoviruses expressing single GP3, GP4, or GP5 protein. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post-boost-immunization. However, mice immunized with recombinant adenoviruses rAd-GP3–GP5, rAd-GP4–GP5, and rAd-GP3–GP4–GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP3, rAd-GP4 or rAd-GP5 alone. It was also found that mice immunized with rAd-GP3–GP5 and rAd-GP3–GP4–GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-GP3 and rAd-GP5. These findings suggested that the recombinant adenoviruses expressing fusion proteins GP3–GP5 or GP3–GP4–GP5 might be an attractive candidate vaccine for preventing PRRSV infection.

Development of an optical biosensor using gold nanoparticles and quantum dots for the detection of Porcine Reproductive and Respiratory Syndrome Virus

Current diagnostic methods to detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) have many disadvantages and are ill suited for on-site detection of the highly contagious virus, making effective disease eradication difficult. A novel PRRSV detection method is therefore proposed, which utilizes nanophotonic and optical biosensor technology. The biosensor consists of two labeled biomolecule architectures: a PRRSV-specific capture antibody labeled with a fluorescent dye bound to a protein that is labeled with a quantum dot and a PRRSV-specific capture antibody labeled with a fluorescent dye bound to a protein that is labeled with a gold nanoparticle. When exposed to PRRSV, a binding event between the virus particle and antibody takes place, resulting in a conformational change within the biosensor complex that increases the distance between the fluorescent molecules or the fluorescent/quencher molecules. The resulting change in the fluorescence spectrum of the sensor occurs due to a nonradiative signal transduction method, Fluorescence Resonance Energy Transfer (FRET), between the fluorescent dye and the quantum dot or gold nanoparticle. Results have demonstrated that both sensor architectures are able to rapidly and effectively detect PRRSV in solution with a detection limit of 3 particles/μl, illustrating that the optical biosensor is a feasible method for PRRSV detection.

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

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 × 10 8, 1 × 10 9, and 1 × 10 10 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 (10 8 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.