HSP70 fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus enhanced the immune responses and protective efficacy against virulent PRRSV challenge in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in pig industry all over the world. Current vaccination strategies provide only a limited protection. In this study recombinant adenoviruses expressing GP3/GP5 of highly pathogenic PRRSV and heat shock protein 70 (HSP70) gene of Heamophilus parasuis were constructed, and the immune responses and protective efficacy against homologous challenge were examined in pigs. The results showed that all animals vaccinated with rAd-GP35 (co-expressing GP3-GP5), rAd-HS35 and rAd-HSA35 (co-expressing GP3-GP5 fused with HSP70 using different linkers), developed specific anti-PRRSV ELISA antibody and neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that of rAd-GP35. Moreover, the fusion of HSP70 markedly induced both IFN-γ and IL-4 in pigs’ sera. Following challenge with PRRSV, pigs inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less pathological lesion of lungs, as compared to those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that of rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could induce enhanced immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infections.     

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.     

Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing GP5/GP3 of porcine reproductive and respiratory syndrome virus and swine IL-18

Two recombinant fowlpox viruses (rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3) containing the ORF5/ORF3 cDNAs of PRRSV (strain Chang Chun) and IL-18 of swine were constructed and evaluated for theirs abilities to induce humoral and cellular responses in piglets. In addition, their abilities to protect piglets against homologous virus challenge were examined. All piglets were given booster vaccinations at 21 days after the initial inoculation, and all piglets were challenged at 60 after the initial inoculation. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody and neutralizing antibody, as well as T-lymphocyte proliferation response. To evaluate the cellular immune function, IFN-gamma production in pigs serum and T-lymphocytes (CD4 and CD8 T cells) in peripheral blood were examined. Following challenge with a pathogenic strain of PRRSV (strain Chang Chun), piglets inoculated with recombinant fowlpox virus (rFPV) showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection. The results demonstrated the potential use of a fowlpox virus-based recombinant vaccine in the control and prevention of PRRSV infections.     

Efficacy of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: Clinical protection and cell-mediated immunity

The purpose of this study was to assess clinical protection in pigs vaccinated with a commercially available attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Porcilis^® PRRS) and then naturally exposed under field conditions to a heterologous (Italian cluster) strain of virulent PRRSV. A total of 30, 4-week-old pigs seronegative for PRRSV were allocated to 1 of 3 groups (IM, ID, and C groups). At 5 weeks of age, pigs of groups IM (n = 10 pigs) and ID (n = 10 pigs) were vaccinated intramuscularly and intradermally, respectively, with modified live PRRSV-1 vaccine (Porcilis^® PRRS). Pigs of group C (n = 10 pigs) were kept as non-vaccinated controls. At post-vaccination (PV) days 0, 7, 14, 28, and 45, blood samples were collected for detection of vaccine virus (PCR) and antibody response (ELISA), identification of changes in lymphocyte subpopulations by cytometry, and IFN-γ PRRSV-specific secreting cells (SC) by ELISpot. At PV day 45, pigs of A, B, and C groups were moved to a site 3 conventional finishing herd with a history of respiratory disease caused by PRRSV and the most common bacteria to be exposed to a natural challenge. The PRRSV field strain, belonging to the Italian cluster of the PRRSV-1, demonstrated a 84% identity with the vaccine virus (DV strain) at ORF5 sequencing. At 0 (exposure day = 45 days PV), 4, 7, 11, 14, 19, 21, 28, and 34 days post-exposure (PE) blood samples were collected for detection and titration of PRRSV and antibody, as well as for lymphocyte and IFN-γ measurement as described above. Throughout the post-exposure period, all pigs were observed daily for clinical signs. The overall clinical signs were reduced by 68 and 72%, respectively in the intramuscularly and intradermally vaccinated pigs compared to controls. Respiratory signs were reduced by 72 and 80%, respectively in the IM and ID groups. Clinical protection was associated with marked activation of cell-mediated immune response. The highest levels of specific IFN-γ production at 21–34 days PE were concomitant and associated to changes in natural killer (NK) cells, γ/δ T, and cytotoxic T lymphocytes in the blood. In our field study, evidences of EU attenuated vaccine-induced clinical protection against natural exposure to a genetically diverse (84% homology) PRRSV-1 isolate (Italian cluster) was demonstrated by the statistically significant reduction in clinical signs in terms of incidence, duration and severity and by a more efficient cell-mediated immune response in the vaccinated pigs as compared to the unvaccinated controls.     

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.     

Impact of genetic diversity of European-type porcine reproductive and respiratory syndrome virus strains on vaccine efficacy

The aim of this study was to find out how efficiently pigs that are vaccinated with an attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine based on a virus from the Lelystad cluster are protected against a European wild-type strain from the same or another genetic cluster. Two experiments were performed. In each experiment, 5-week-old PRRSV-seronegative pigs were vaccinated intramuscularly with 10(4.5) TCID50 of a commercial vaccine based on a European virus strain from the Lelystad cluster. Non-vaccinated pigs were included as controls. At 5, 9, 15, 20, 28, 35 and 42 days post vaccination (PV), broncho-alveolar lavage (BAL) fluids and blood were collected to determine vaccine virus quantities. Forty-nine days PV, pigs were challenged intranasally with 10(6.0) TCID50 of a European wild-type strain, belonging either to the Lelystad cluster (98% nucleotide identity in ORF5 with vaccine strain) (experiment A) or to an Italian cluster (84% nucleotide identity in ORF5 with vaccine strain) (experiment B). At 5, 9, 15, 20 and 27 days post challenge (PC), BAL fluids and blood were collected to determine virus quantities. Vaccine virus was first detected in BAL fluids and blood at 5 days PV and reached highest quantities between 9 and 15 days PV. One pig was positive in its BAL fluid until 42 days PV. After challenge, virus was isolated from BAL fluids and blood of all non-vaccinated control pigs. All vaccinated pigs challenged with the Lelystad strain remained negative for virus, while virus was present in BAL fluids and blood of all vaccinated pigs after challenge with the Italian strain. Mean virus titres of the vaccinated pigs challenged with the Italian strain were significantly lower than those of the non-vaccinated control pigs (P <0.05) at 9, 15 and 20 days PC. Thus, the genetic diversity within European-type PRRSV may affect the efficacy of the current European-type vaccines.     

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP(5) glycoprotein and improves immune response in challenged pigs

Pigs exposed to GP(5) protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP(5) than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response.     

A porcine reproductive and respiratory syndrome virus candidate vaccine based on the synthetic attenuated virus engineering approach is attenuated and effective in protecting against homologous virus challenge

Current porcine reproductive and respiratory syndrome virus (PRRSV) vaccines sometimes fail to provide adequate immunity to protect pigs from PRRSV-induced disease. This may be due to antigenic differences among PRRSV strains. Rapid production of attenuated farm-specific homologous vaccines is a feasible alternative to commercial vaccines. In this study, attenuation and efficacy of a codon-pair de-optimized candidate vaccine generated by synthetic attenuated virus engineering approach (SAVE5) were tested in a conventional growing pig model. Forty pigs were vaccinated intranasally or intramuscularly with SAVE5 at day 0 (D0). The remaining 28 pigs were sham-vaccinated with saline. At D42, 30 vaccinated and 19 sham-vaccinated pigs were challenged with the homologous PRRSV strain VR2385. The experiment was terminated at D54. The SAVE5 virus was effectively attenuated as evidenced by a low magnitude of SAVE5 viremia for 1–5 consecutive weeks in 35.9% (14/39) of the vaccinated pigs, lack of detectable nasal SAVE5 shedding and failure to transmit the vaccine virus from pig to pig. By D42, all vaccinated pigs with detectable SAVE5 viremia also had detectable anti-PRRSV IgG. Anti-IgG positive vaccinated pigs were protected from subsequent VR2385 challenge as evidenced by lack of VR2385 viremia and nasal shedding, significantly reduced macroscopic and microscopic lung lesions and significantly reduced amount of PRRSV antigen in lungs compared to the non-vaccinated VR2385-challenged positive control pigs. The nasal vaccination route appeared to be more effective in inducing protective immunity in a larger number of pigs compared to the intramuscular route. Vaccinated pigs without detectable SAVE5 viremia did not seroconvert and were fully susceptible to VR2385 challenge. Under the study conditions, the SAVE approach was successful in attenuating PRRSV strain VR2385 and protected against homologous virus challenge. Virus dosage likely needs to be adjusted to induce replication and protection in a higher percentage of vaccinated pigs.     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

In vivo targeting of porcine reproductive and respiratory syndrome virus antigen through porcine DC-SIGN to dendritic cells elicits antigen-specific CD4T cell immunity in pigs

Immunogenicity of protein subunit vaccines may be dramatically improved by targeting them through antibodies specific to c-type lectin receptors (CLRs) of dendritic cells in mice, cattle, and primates. This novel vaccine development approach has not yet been explored in pigs or other species largely due to the lack of key reagents. In this study, we demonstrate that porcine reproductive and respiratory syndrome virus (PRRSV) antigen was targeted efficiently to dendritic cells through antibodies specific to a porcine CLR molecule DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) in pigs. A recombinant PRRSV antigen (shGP45M) was constructed by fusing secretory-competent subunits of GP4, GP5 and M proteins derived from genetically-shuffled strains of PRRSV. In vaccinated pigs, when the PRRSV shGP45M antigen was delivered through a recombinant mouse-porcine chimeric antibody specific to the porcine DC-SIGN (pDC-SIGN) neck domain, porcine dendritic cells rapidly internalized them in vitro and induced higher numbers of antigen-specific interferon-γ producing CD4T cells compared to the pigs receiving non-targeted PRRSV shGP45M antigen. The pDC-SIGN targeting of recombinant antigen subunits may serve as an alternative or complementary strategy to existing vaccines to improve protective immunity against PRRSV by inducing efficient T cell responses.     

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection.     

Protection and immune response in pigs intradermally vaccinated against porcine reproductive and respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) field strain

The purpose of this study was to assess the immune response in pigs intradermally vaccinated with a commercially available attenuated porcine reproductive and respiratory virus (PRRSV) vaccine (Porcilis PRRS) and subsequently exposed to a heterologous (Italian cluster) field strain of virulent PRRSV. A total of 18, 4-week-old pigs seronegative for PRRSV were allocated to 1 of 3 groups (groups A, B, and C). At 5 weeks of age, pigs of groups A (n=6 pigs) and B (n=6 pigs) were vaccinated intramuscularly and intradermally, respectively, with Porcilis PRRS. The more conventional intramuscular route of vaccination was included for comparative purposes with the intradermal route of vaccination (performed with the I.D.A.L. vaccinator). Pigs of group C (n=6 pigs) were kept as nonvaccinated controls. At post-vaccination (PV) days 7, 14, 21, 28, and 35, blood samples were collected for detection of vaccine virus (PCR) and antibodies (ELISA), and for changes in PBMC (flow cytometry). At PV day 35, pigs of all groups were each exposed (challenged) intranasally to a heterologous field strain (78% ORF5 sequence homology between vaccine and field virus) belonging to the Italian cluster of the European genotype of PRRSV. At post-challenge (PC) days 0, 3, 7, 10, 13, and 17, blood samples were collected for detection and quantitation of virus and antibodies, and for changes in PBMC as described above for blood samples collected PV. Throughout the experiment all pigs were observed daily for clinical signs. At PC days 7 and 17, two pigs and four pigs, respectively, of each group were euthanized and examined for macroscopic lesions. Following vaccination some pigs of groups A and B had a detectable viremia that in two pigs (one pig of group A and one pig of group B) lasted until PV day 28. However, all pigs (groups A, B, and C) remained clinically normal. All vaccinated pigs developed a serological response (ELISA) to PRRSV. Presumptive evidence for vaccine-induced protective immunity against the heterologous challenge strain was provided by finding that viremia following challenge was generally less (incidence) and significantly less (titers) in vaccinated pigs than in nonvaccinated pigs. No differences were apparent between pigs vaccinated intramuscularly and those vaccinated intradermally. The absence of virulent-virus-induced clinical signs and macroscopic lesions in nonvaccinated as well as in vaccinated pigs precluded a more definitive evaluation of the magnitude of protective immunity provided by vaccination or by the route of vaccination. Some likely treatment-associated changes in lymphocyte subpopulations were observed among the three treatment groups. These changes and their potential relationship to protective immunity are discussed.     

Immune response of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus

Pigs were immunised with recombinant BCG (rBCG) expressing a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) (rBCGGP5) and M protein (rBCGM) of porcine reproductive and respiratory syndrome virus (PRRSV). At 30 days post-inoculation (dpi), pigs inoculated with rBCGGP5 and rBCGM developed a specific humoral immune response against the viral proteins, as detected by commercial ELISA and Western blot tests, and at 60 dpi, three out of five animals developed neutralizing antibodies with titers ranging from 1:4 to 1:8. At 67 dpi, an IFN-gamma response against BCG antigens, but not against the viral proteins, was detected by ELISPOT in inoculated pigs. Following challenge with a pathogenic strain of PRRSV, pigs inoculated with rBCG showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection.     

Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: Assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination

A Luminex (Luminex Corp., Austin, TX) multiplex swine cytokine assay was developed to measure 8 cytokines simultaneously in pig serum for use in assessment of vaccine candidates. The fluorescent microsphere immunoassay (FMIA) was tested on archived sera in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This FMIA simultaneously detects innate (IL-1β, IL-8, IFN-α, TNF-α, IL-12), regulatory (IL-10), Th1 (IFN-γ) and Th2 (IL-4) cytokines. These proteins were measured to evaluate serum cytokine levels associated with vaccination strategies that provided for different levels of protective immunity against PRRSV. Pigs were vaccinated with a modified-live virus (MLV) vaccine and subsequently challenged with a non-identical PRRSV isolate (93% identity in the glycoprotein (GP5) gene). Protection (as defined by no serum viremia) was observed in the MLV vaccinated pigs after PRRSV challenge but not those vaccinated with killed virus vaccine with adjuvant (KV/ADJ) (99% identity in the GP5 gene to the challenge strain) or non-vaccinates. Significantly elevated levels of IL-12 were observed in the KV/ADJ group compared to MLV vaccinated and control groups. However, this significant increase in serum IL-12 did not correlate with protection against PRRSV viremia. Additional studies using this assay to measure the local cytokine tissue responses may help in defining a protective cytokine response and would be useful for the targeted design of efficacious vaccines, not only for PRRSV, but also for other swine pathogens.     

CD40 ligand expressed in adenovirus can improve the immunogenicity of the GP3 and GP5 of porcine reproductive and respiratory syndrome virus in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused heavy economic losses in swine industry worldwide. Current vaccination strategies only provide a limited protective efficacy, thus immune modulators are being considered to enhance the effectiveness of PRRSV vaccines. In this study, the recombinant adenoviruses expressing porcine CD40 ligand (CD40L) and GP3/GP5 of PRRSV were constructed and the immune responses were examined in pigs. The results showed that rAd-CD40L-GP35 (co-expressing CD40L and GP3-GP5) or rAd-GP35 (expressing GP3-GP5) plus rAd-CD40L (expressing CD40L) could provide significant higher specific anti-PRRSV ELISA antibody and neutralizing antibody. And the levels of proliferative responses of peripheral blood mononuclear cells (PBMC), IFN-γ and IL-4 were markedly increased in rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 groups than those in rAd-GP35 group. Following homologous challenge with Chinese isolate of the North-American genotype of PRRSV, pigs inoculated with recombinant rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 showed lighter clinical signs and lower viremia, as compared to those in rAd-GP35 group. It indicated that porcine CD40L could effectively increase humoral and cell-mediated immune responses of GP3 and GP5 of PRRSV. Porcine CD40L might be used as an attractive adjuvant or immunotargeting strategies to enhance the PRRSV subunit vaccine responses in swine.     

Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat in swine industry and causes heavy economic losses worldwide. Currently, the available vaccines are the inactivated and attenuated virus vaccines, but the use of PRRSV in their production raises the issue of safety. We developed a chimeric virus-like particles (VLPs) vaccine candidate for PRRSV protection. The chimeric VLPs was composed of M1 protein from H1N1 influenza virus and a fusion protein, denoted as NA/GP5, containing the cytoplasmic and transmembrane domains of H1N1 virus NA protein and PRRSV GP5 protein. Vaccination of BALB/c mice with 10 μg of chimeirc VLPs by intramuscular immunization stimulated antibody responses to GP5 protein, and induced cellular immune response. The data suggested that the chimeric VLP vaccine candidate may provide a new strategy for further development of vaccines against PRRSV infection.     

Construction and immunogenicity of DNA vaccines encoding fusion protein of murine complement C3d-p28 and GP5 gene of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. The commercial vaccines only provide a limited protection against PRRSV infection. At present, DNA vaccine is the focus on the new vaccines. The gene fragment (p28) coding for the molecular adjuvants complement protein C3d (mC3d) from BALB/c mouse was cloned and expressed as a fusion protein for its application in the vaccine study of mice. Three potential vaccines construct units were engineered to contain two, four and six copies of mC3d-p28 coding gene linked to the GP5 gene of PRRSV and one vaccine expressing GP5 alone (pcDNA3.1-GP5) was constructed. Subsequently, the vaccines’ abilities to elicit the humoral and cellular immune responses were investigated in mice. These results showed that significantly enhanced GP5-specific ELISA antibody, GP5-specific neutralizing antibody, IFN-γ level, and IL-4 level, could be induced in mice immunized with DNA construct units encoding the pcDNA3.1-C3d-p28.n-GP5 than those received DNA vaccine expressing GP5 alone (pcDNA3.1-GP5). Analysis of the immunogenicity of different repeats of mC3d-p28 revealed that mC3d-p28 had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of mC3d-p28 may be necessary for efficient enhancement of antigen specific immune responses. This approach may provide a new strategy for the development of efficient vaccines against the PRRSV for pigs in the future.     

Virological kinetics and immunological responses to a porcine reproductive and respiratory syndrome virus infection of pigs at different ages

The objective of this study was to measure the effect of two variables (pig age and virus strain) on selected responses (clinical signs, viraemia, virus excretion and seroconversion) of pigs following exposure to porcine reproductive and respiratory syndrome (PRRS) virus. Therefore, young (6 till 8 weeks old) and old (6 months old) pigs were infected with 3 different PRRSV strains, i.e. LV ter Huurne (LVTH), LV4.2.1 and SDSU#73. Regardless of the strain used for exposure, young pigs were more susceptible to infection as shown by a higher number of viraemic and virus excreting pigs. Strain differences were also evident. LV ter Huurne induced virus excretion in a higher number of pigs and with a higher virus titre, whereas SDSU#73 induced most severe clinical signs. LV4.2.1 induced viraemia and virus excretion in a low number of pigs. The kinetics of the antibody response differed per virus strain. The results presented here are useful in developing a less expensive standardised infection model, consisting of young pigs intranasally infected with a virulent PRRSV strain, to study the efficacy of new vaccine strains.     

Novel Nsp2 deletion based on molecular epidemiology and evolution of porcine reproductive and respiratory syndrome virus in Shandong Province from 2013 to 2014

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease affecting swine worldwide. In this study, a total of 385 samples were collected from Shandong pig farms during 2013 and 2014, when pigs were not inoculated with any vaccine. Results indicated that, out of 385 samples, 47 (12.21%) were PRRSV-RNA-positive. The gene sequence analysis of 12 ORF5, 12 ORF7, and 8 Nsp2 of these samples was used to determine the molecular epidemiology of PRRSV in different parts of China’s Shandong Province. The phylogenetic tree based on these 3 genes indicated that the Chinese PRRSV strains could be divided into five subgroups and two large groups. The 8 study strains were clustered into subgroup IV, another 4 strains into subgroup I. The first 8 strains shared considerable homology with VR-2332 in ORF5 (96–97.5%), the other 4 strains shared considerable homology with JXA1 (94–98%). Phylogenetic tree of GP5 showed that the eight isolates formed a tightly novel clustered branch, subgroup V, which resembled but differed from isolate VR-2332. When examined using Nsp2 alone, the first 8 strains showed considerable homology with a U.S. vaccine strain, Ingelvac MLV (89.6–98.4%). One novel pattern of deletion was observed in Nsp2. The genetic diversity of genotype 2 PRRSV tended to vary in the field. The emergence of novel variants will probably be the next significant branch of PRRSV study.     

Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.