Introduction

Porcine reproductive and respiratory syndrome (PRRS) is an emerged viral disease and causes significant economic losses to the swine industry today [1]. The etiological agent is porcine reproductive and respiratory syndrome virus (PRRSV), which is a small, enveloped single-stranded, positive-sense RNA virus. It is a member of the genus Arterivirus, family Arteriviridae, order Nidovirales [2]. PRRSV contains the genome of a single-stranded positive-sense RNA of approximately 15 kb in size, and the genome codes for two large non-structural polyproteins (PP1a and PP1a/1b) in the 5′-terminal 12 kb region, and eight structural proteins in the 3′-terminal 3 kb region: GP2 (glycoprotein 2), E (small envelope), GP3, GP4, GP5, GP5a, M (membrane), and N (nucleocapsid) proteins in order [3–5]. GP5a is a small membrane protein newly identified and encoded in the internal ORF within ORF5 with unknown function [4, 5].

Among the structural proteins of PRRSV, GP3, GP4, and GP5 are associated with the development of neutralizing antibodies and protection [6, 7]. GP3 plays an important role in clearing the virus infection and provides protection in piglets against PRRSV infection in the absence of noticeable neutralizing antibody response [8]. GP5 can induce higher titers of neutralizing antibody, and three B cell epitopes in GP5 have been identified using monoclonal antibodies [9].

PRRS has become one of the most economically important diseases to the global pork industry [10]. Especially, a highly pathogenic PRRSV (HP-PRRSV) appeared in China in spring 2006 and caused heavy economic losses [11–13]. Currently, attenuated live vaccine could protect piglets from lethal challenge and might be a candidate vaccine against the HP-PRRSV [14], but there is a possibility that the attenuated virus returned to high virulence [15]. Thus, DNA vaccine is developed as one of the most promising alternatives to conventional vaccines. Immunization with plasmid DNA is able to elicit both cell-mediated and humoral immune responses [16, 17] against antigens derived from numerous viral, bacterial and parasitic pathogens. Recently, although the use of a DNA vaccine for PRRSV has been reported, only partial protection was obtained [18]. Co-delivery of immunomodulators is being considered as an approach to enhance the effectiveness of PRRSV DNA vaccines.

Interferon alpha (IFNα) belongs to type I interferons and is one of the first lines of host cell defense against viral infection [19]. Recombinant porcine IFNα could inhibit the growth of PRRSV in alveolar macrophage cultures [20]. In addition to its antiviral activity, IFNα can function as an adjuvant when co-administered with a DNA vaccine [21]. Type II IFN (IFNγ) is a multifunctional cytokine produced by T-helper 1 (Th1) and natural killer (NK) cells. The antiviral activity of IFNγ against PRRSV has been demonstrated in porcine macrophages [22]. IFNγ was also evaluated as an effective adjuvant for DNA vaccine constructs encoding for HIV and SIV [23].

Although the signal pathways elicited by each type of IFN differ, the combination of type I and type II IFNs can synergistically induce gene expression [24]. Coactivation of the IFN signaling pathways produce an increased effect in blocking the replication of a number of viruses, including herpes simplex virus [25], vaccinia virus [26], hepatitis C virus [27], and mouse hepatitis virus [28]. In our research group, we first found that a combination of IFNα and IFNγ could act synergistically to block PRRSV replication both in vitro and in vivo. The combined anti-PRRSV activity of IFNα and IFNγ was significantly higher than IFNα or IFNγ individually (unpublished data).

In this study, we first constructed a DNA vaccine pVAX1©-α-γ-GP35 co-expressing GP3 and GP5 of PRRSV with interferon α/γ, then evaluated the immediate and long-lasting protective efficacy in pigs challenged with HP-PRRSV.

Materials and methods

Viruses and cells

HP-PRRSV strain SD-JN was kept in our laboratory and described elsewhere [29]. Vesicular stomatitis virus (VSV) was gifted by Dr. Bing Huang (Institute of Poultry Science, Shandong Academy of Agricultural Sciences, Jinan, China). MARC-145 cells were used for propagation and titration of HP-PRRSV SD-JN strain. The infected cell lysate (F8 passage) was clarified, titrated, diluted to 1 × 105 TCID50/ml, and stored at −20 °C to be used for animal challenge. The SD-JN PRRSV whole virion antigen was purified and quantitated by optical density (OD) measurement as described previously [7, 30, 39] and used for indirect enzyme-linked immunosorbent assay (iELISA), T lymphocyte proliferation, and cytokine assays. HEK-293A cells (ATCC CRL1573) were used for transfection of plasmids. All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM l-glutamine, 100 U penicillin/ml, and 100 μg streptomycin/ml.

RT-PCR for amplification of mature peptide of porcine IFNα and IFNγ gene

Based on porcine IFNα (GenBank accession no. X57191) and IFNγ (GenBank accession no. DQ839398) gene sequence, two pairs of PCR primers named, IFNα-Fwd/IFNα-Rev and IFNγ-Fwd/IFNγ-Rev, were designed (Table 1). Porcine spleen cells from 8-week-old Yorkshire swines were isolated by mechanical disruption and filtration through a 75-μm cell filter followed by hypotonic lysis of erythrocytes, and then stimulated with ConA (10 μg/ml; Sigma-Aldrich, St. Louis, MO) for 24 h in vitro. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. The cDNA was synthesized using oligo d(T)12–18 primer. Then, the active mature peptide of porcine IFNα or IFNγ gene was amplified from the cDNA. The amplification was performed in a 50-μl reaction mixture containing 1.5 mM MgCl2, 1 × PCR buffer, 0.2 mM of each dNTP, 20 pmol of each primer, 1.5 U of TaqDNA polymerase (Invitrogen, Carlsbad, CA), and 2 μl of cDNA. The reaction was run in a thermocycler (DNA Engine, PTC-0200; Bio-Rad Laboratories, Hercules, CA) with the following program: denaturation at 94 °C for 5 min, 30 cycles composed of denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s and extension at 72 °C for 1 min, and was ended with a final extension step of 10 min at 72 °C. Then, the PCR product of porcine IFNα or IFNγ gene was individually cloned into pMD18-T vector (TaKaRa Biotechnology Co. Ltd., Dalian, China) according to the manufacturer’s protocol. The positive plasmids were named pMT-IFNα and pMT-IFNγ, respectively.

Table 1 Primer sequences for amplification of porcine IFNα and IFNγ gene from porcine spleen cells and GP3/5 genes from PRRSV SD-JN strain
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Amplification of GP3 and GP5 gene of PRRSV SD-JN

To amplify GP3 and GP5 genes from PRRSV SD-JN strain, the primers GP3-Fwd and GP3-Rev for GP3 gene, and the primers GP5-Fwd and GP5-Rev for GP5 gene, were designed based on the GP3 (GenBank accession no. JQ627636) and GP5 (GenBank accession no. FJ422123) sequence of PRRSV SD-JN isolate as shown in Table 1.

The viral RNA was extracted from PRRSV SD-JN isolate using TRIzol reagent as per the instruction of manufacture. Reverse transcription was performed at 55 °C for 60 min with 12 μl total RNA, 1 μl SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA), 1 μl oligo(dT)12–18, 1 μl 0.1 M DTT, 4 μl 5 × First-Strand buffer, and 1 μl 10 mM dNTPs. The PCR amplification was performed as above.

Construction of the expression plasmids: pVAX1©-α-γ, pVAX1©-GP35, and pVAX1©-α-γ-GP35

The plasmid pVAX1©-α-γ co-expressing IFNα and IFNγ was created by overlapping extension PCR [31] and standard recombinant DNA procedures. Primer IFNα-2A-Rev was designed based on 2A of foot-and-mouth disease virus (FMDV)-type A12 strain 119 (GenBank accession no. M10975) which encodes the 17 amino acids self-cleaving 2A protease (NFDLLKLAGDVESNPGP) as reported [32] (Table 1). IFNα-2A gene containing 2A gene of FMDV was amplified from plasmid pMT-IFNα using primers IFNα-Fwd and IFNα-2A-Rev. IFNγ-2A gene was also amplified from plasmid pMT-IFNγ using primers IFNγ-2A-Fwd and IFNγ-Rev. Then, the purified PCR products of IFNα-2A and IFNγ-2A gene were used as templates, and overlapping extension PCR was conducted with primers IFNα-Fwd and IFNγ-Rev. Finally, the gene of IFNα-2A-IFNγ was inserted into the position between the KpnI and XhoI sites in pVAX1© vector (Invitrogen, Carlsbad, CA) to produce pVAX1©-α-γ (Fig. 1).

Fig. 1
figure 1

Schematic construction of recombinant plasmids. The IFNα, IFNγ, and GP3–GP5 gene were inserted into pVAX1© vector. The linkers between these genes were five glycine gene or 2A gene of FMDV

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When constructing the pVAX1©-GP35 expressing GP3–GP5 fusion protein, to avoid the interference resulting from the direct ligation between GP3 and GP5, a linker of five glycine residues was inserted between the GP3 gene and GP5 gene so that GP3 and GP5 protein could maintain its individual form as described elsewhere [33] (Fig. 1). pVAX1©-GP35 was constructed in the same way using overlapping extension PCR with primers GP3-Fwd and GP5-Rev as above (Table 1).

In order to generate pVAX1©-α-γ-GP35 expressing IFNα, IFNγ, and GP3–GP5, IFNα-2A-IFNγ gene or GP3–GP5 fusion gene was individually amplified from plasmid pVAX1©-α-γ or pVAX1©-GP35 using primer pair IFNα-Fwd/IFNαγ-2A-Rev or GP35-2A-Fwd/GP5-Rev (Table 1). 2A gene of FMDV was also introduced between IFNα-IFNγ gene and GP3–GP5 fusion gene by overlapping extension PCR. Finally, IFNα-2A-IFNγ-2A-GP3–GP5 gene was amplified with primers IFNα-Fwd and GP5-Rev and then cloned into pVAX1© vector using KpnI and XhoI sites (Fig. 1).

All the expression plasmids were sequenced to confirm the correct tandem in frame insertion of individual gene.

Identification of IFNα, IFNγ, GP3, and GP5 expressed in the constructs

DNA transfection of eukaryotic expression plasmids pVAX1©-α-γ, pVAX1©-GP35, pVAX1©-α-γ-GP35, or empty vector pVAX1© was carried out using Lipofectamine™ 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). HEK-293A cells were seeded in 35-mm diameter dishes and grown to 70 % confluency. Solutions of 5.0 μl Lipofectamine™ 2000 and 2.0 μg individual plasmid were diluted in 1.0 ml Opti-MEM and added to HEK-293A cells monolayers. After 6 h of incubation, the transfection mixtures were removed and replaced with DMEM supplemented with 10 % FBS. Cells were transfected with individual plasmid in duplicate. At 24-h post-transfection, cells of one dish were washed with PBS and lysed in the buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 1 % NP-40, and 1 mM PMSF] for western blot analysis. Cells of the other dish were frozen and thawed three times for detecting the antiviral activity of IFN.

In order to compare the expression levels of pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35, empty vector pVAX1© (2.0 μg), pVAX1©-α-γ (2.0 μg), pVAX1©-GP35 (2.0 μg), pVAX1©-α-γ (2.0 μg) + pVAX1©-GP35 (2.0 μg), or pVAX1©-α-γ-GP35 (2.0 μg) was transfected into HEK-293A cells as above. Cell lysates were subjected to western blot analysis.

Western blot assay

Western blots were used to evaluate protein's expression by pVAX1©-α-γ, pVAX1©-GP35, and pVAX1©-α-γ-GP35. Cell lysates were centrifuged at 13,400×g for 20 min in a microcentrifuge (Eppendorf 5415R). Supernatants were collected and boiled in SDS-PAGE loading buffer [60 mM Tris-HCl (PH 6.8), 2 % SDS, 0.1 % bromphenol blue, 25 % glycerol, 5 % β-mercaptoethanol] for 5 min, followed by 10 % SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5 % non-fat dry milk in TBST [10 mM Tris-HCl (PH 8.0), 150 mM NaCl, and 0.1 % Tween 20] for 4 h at room temperature and then incubated with PRRSV-specific antiserum of pigs (1:100 diluted in TBST), mouse anti-IFNα, or anti-IFNγ serum (prepared by immunizing mice with purified mature peptide of porcine IFNα and IFNγ expressed by pET-32a (+) vector in E. coli BL21, kept in our laboratory, 1:100 diluted in TBST) overnight at 4 °C. After washing five times for 10 min each with TBST, the membranes were incubated with horseradish peroxidase-conjugated Staphylococcal Protein A (SPA-HRP, Boshide, Wuhan, China) or HRP-conjugated goat anti-mouse IgG (Boshide, Wuhan, China) for 1 h, washed five times for 10 min each with TBST, and developed using Supersignal® West Pico Chemiluminescent Substrate according to the manufacturer’s suggestions (Pierce, Rockford, IL).

In order to compare the expression levels of pVAX1©-α-γ + pVAX1©-GP35 and pVAX1©-α-γ-GP35, the cell lysates of empty vector, pVAX1©, pVAX1©-α-γ, pVAX1©-GP35, pVAX1©-α-γ + pVAX1©-GP35, or pVAX1©-α-γ-GP35, were also subjected to western blot analysis as above. The primary antibodies were mixtures of PRRSV-specific antiserum of pigs, mouse anti-IFNα, and anti-IFNγ serum, while the secondary antibodies were mixtures of SPA-HRP and HRP-conjugated goat anti-mouse IgG. Blot of β-actin was used as loading control.

Antiviral activity of IFN expressed by pVAX1©-α-γ and pVAX1©-α-γ-GP35

Cells transfected with individual plasmid were frozen and thawed three times and then centrifuged at 13,400×g for 10 min in a microcentrifuge (Eppendorf 5415R). Supernatants were harvested and assayed for anti-VSV and anti-PRRSV activity. Serial dilutions of supernatant fluids were transferred to preformed monolayers of MARC-145 cells and incubated at 37 °C with 5 % CO2 for 24 h and removed. Then, the cells were infected with 100 TCID50 of VSV or PRRSV SD-JN strain and incubated at 37 °C with 5 % CO2 for 48–72 h. Cytopathic effects (CPE) was used to determine the end-point titers that were calculated as the reciprocal of the highest supernatant fluids dilution to neutralize 100 TCID50 of VSV or PRRSV SD-JN strain in 50 % of the wells.

Preparation of DNA plasmids

All plasmids for DNA immunizations were grown in E. coli DH5α strain (Invitrogen, Carlsbad, CA), and large-scale preparation of the plasmid DNA was carried out by alkaline lysis using Qiagen EndoFree Plasmid-Giga kits (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions.

Vaccination of pigs with the constructs

Sixty 21-day-old crossbred (Landrace × local stock) pigs were obtained from a local farm. All pigs were tested to be free from PRRSV, porcine circovirus 2 (PCV-2), porcine parvovirus (PPV), pseudorabies virus (PRV), and Actinobacillus pleuropneumoniae (APP) infections and proven to be seronegative for PRRS by ELISA and serum neutralization (SN) assay. The animals were then randomly divided into 12 groups with five pigs each group, numbered, and housed in separate rooms. Six groups were used in Experiment 1, and the other six groups were used in Experiment 2.

Experiment 1 (immediate protection experiment)

Groups 1.1–1.4 were inoculated intramuscularly with pVAX1©-α-γ (500 μg), pVAX1©-GP35 (500 μg), pVAX1©-α-γ (500 μg) + pVAX1©-GP35 (500 μg), and pVAX1©-α-γ-GP35 (500 μg) in 1 ml PBS, respectively. Groups 1.5 and 1.6 were negative controls injected with 500 μg of pVAX1© vector in 1 ml PBS and 1 ml PBS, respectively. The plasmid DNA or PBS were injected in the cervical region muscles. At 2 days post inoculation (dpi), all pigs were challenged intranasally with 5 × 104 TCID50 PRRSV SD-JN strain, following which the animals were monitored daily for 14 days.

The severity of the clinical signs was evaluated daily after challenge as reported [34]. Observations included behavior, respiration, and cough. Scores for each of three individual observations ranged from 1 to 4. The overall score for clinical condition was determined by sum of daily observations of behavior, respiration, and cough. For example, a clinically normal animal would be given a total score of 3 (i.e., behavior = 1, respiration = 1, and cough = 1), an animal with maximum clinical illness would be given a total score of 9 (i.e., behavior = 3, respiration = 3, and cough = 3) and a dead animal would be given a total score of 12 (i.e., behavior = 4, respiration = 4, and cough = 4). Sequential blood samples were collected from all animals at 0, 3, 7, and 14 days post challenge (dpc) for detection of PRRSV. At the end of experiment, all pigs were humanely euthanized and the gross lesions of lungs were evaluated at necropsy. Lungs were evaluated by the percentage of lesions noted per lobe, following which, using a standard scoring system, an overall level of gross lung pathology was determined [35].

Experiment 2 (long-lasting protection experiment)

Groups 2.1–2.4 were inoculated intramuscularly with pVAX1©-α-γ (500 μg), pVAX1©-GP35 (500 μg), pVAX1©-α-γ (500 μg) + pVAX1©-GP35 (500 μg), or pVAX1©-α-γ-GP35 (500 μg) in 1 ml PBS. Groups 2.5 and 2.6 were negative controls injected with 500 μg of pVAX1© vector in 1 ml PBS and 1 ml PBS, respectively. The plasmid DNA or PBS was injected in the cervical region muscles, and the immunization was boosted 28 days later. The sera were collected from each pig at 28, 42, and 56 dpi to detect antibody to PRRSV using iELISA and SN assay. At 42 and 56 dpi, the heparinized blood was used to isolate peripheral blood mononuclear cells (PBMCs) for T-lymphocyte proliferation assay. Meanwhile, the supernatants of the lymphocytes stimulated with purified SD-JN PRRSV antigen at 42 dpi were obtained to detect the levels of Th1-type cytokine of IFN-γ and Th2-type cytokine of IL-4. At 56 dpi, all groups were challenged intranasally with 1 × 105 TCID50 PRRSV SD-JN strain and monitored daily for 14 days. The severity of the clinical signs was evaluated, and blood samples were collected at 0, 3, 7, and 14 dpc for PRRSV detection as above. The experiments were terminated on 14 dpc, and the animals were humanely euthanized. The gross lesions of lungs were also evaluated as above.

iELISA

The purified SD-JN PRRSV antigen was used as iELISA antigen and coated in 96-well plates at the concentration of 1.0 μg/ml. The plates were blocked with 0.15 % BSA in PBS. The sera of pigs were diluted 1:2 serially in PBS-T (PBS containing 0.5 % Tween80) and added into the plates. After incubation for 60 min at 37 °C, the wells were washed three times and incubated with SPA-HRP (Boshide, Wuhan, China) for 60 min at 37 °C. The plates were incubated with substrate solution O-phenilendiamine (OPD) at 37 °C for 15 min, and the reaction was stopped by adding 2 M H2SO4 solution in each well. The OD was 490 nm as recorded by an ELISA reader. Meanwhile, the PRRSV negative sera of pigs were used as negative control. The results were expressed as the ratio of OD490 nm produced by the serum samples compared with negative control serum. Sera, giving a ratio value higher than 2.1, were considered to be positive sera. The titers were expressed as the highest dilution of antibody producing 2.1 as ratio value.

Serum neutralization assays

Serum neutralization (SN) assays were performed as previously described [30]. All samples of sera from pigs were heat inactivated (56 °C, 30 min) and 1:2 serially diluted. Then, the serial dilutions of serum were mixed with equal volumes of 200 TCID50 SD-JN PRRSV. After incubation at 37 °C for 1 h, the mixtures were transferred to MARC-145 monolayers in 96-well tissue culture plates. Then, the plates were incubated and observed daily for up to 5 days for the appearance of CPE. Meanwhile, the PRRSV positive and negative sera of pigs were used as positive and negative controls, respectively. CPE was used to determine the end-point titers that were calculated as the reciprocal of the last serum dilution to neutralize 100 TCID50 of PRRSV in 50 % of the wells.

T lymphocyte proliferation assay

Lymphocytes were isolated from heparinized blood of pigs with lymphocyte separation medium (Boshide, Wuhan, China), suspended to 5 × 106/ml with RPMI complete medium (RPMI 1640 containing 10 % FCS, 2 mM l-glutamine, 50 μM β-mercaptoethanol, 200 U/ml penicillin, 200 μg/ml streptomycin, and 100 U/ml of mycostatin) and seeded in 96-well flat-bottom plates at the rare of 100 μl per well. Each cell sample was plated in triplicate. The culture was stimulated with purified SD-JN PRRSV antigen at a final concentration of 10 μg/ml or unstimulated, respectively. Meanwhile, phytohemagglutinin (PHA) (10 μg/ml) was used as a positive control. After incubation for 45 h at 37 °C with 5 % CO2, the proliferation responses were detected by a standard MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) method. T lymphocyte proliferation was expressed as stimulation index (SI), which is the ratio of OD570 nm of stimulated well to that of unstimulated one.

Cytokine assay

Subsets of Th cells can be distinguished by their pattern of cytokine expression. Th1 cells produce IFN-γ, IL-2, and lymphotoxin, and Th2 cells produce IL-4, IL-5, IL-10, and IL-13. To distinguish the subsets, PBMC (5 × 106/ml, 100 μl per well) isolated from the blood of pigs were stimulated with purified SD-JN PRRSV antigen at the final concentration of 10 μg/ml. After 72 h, the cells were centrifuged, and the supernatant was collected to examine the levels of the Th1-type cytokine of IFN-γ and Th2-type cytokine of IL-4 using commercially available porcine IFN-γ/IL-4 ELISA kits [sensitivity: 1 pg/ml, Adlitteram Diagnostic Laboratories (ADL, USA)] according to the manufacture’s instructions [7, 39].

Real-time PCR

Real-time PCR was carried out as described previously [7]. Total RNA was extracted from 300 μl serum collected at 0, 3, 7, and 14 dpc using TRIzol reagent. cDNA was synthesized using oligo d(T)12-18 primer and was performed as mentioned above. SYBR Green real-time PCR was performed to evaluate PRRSV RNA level, using the sequence of sense primer: 5′-AATAACAACGGCAAGCAGCAG-3′ and antisense primer: 5′-CCTCTGGACTGGTTT TGT TGG-3′. The cDNA was used as the template. The reaction was performed at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 61 °C for 1 min using the ABI 7300 detection system. For quantification, cDNA of SD-JN PRRSV were tenfold serially diluted and were used to generate the standard curve. The real-time PCR method was very sensitive and could detect even 0.01 TCID50 of PRRSV in the serum. PRRSV RNA quantity of samples was determined by linear extrapolation of the Ct value plotted against the standard curve.

Statistical analysis

Data were compared and the differences were determined by One-way repeated measurement ANOVA and least significance difference (LSD). A P value <0.05 was considered statistically significant [7].

Results

Construction of the expression plasmids: pVAX1©-α-γ, pVAX1©-GP35, and pVAX1©-α-γ-GP35

As shown in Fig. 1, The eukaryotic expression vectors individually encoding α-γ, GP35, and α-γ-GP35 were constructed, and DNA sequencing confirmed that the nucleotide sequence of the insert genes in recombinant plasmids had the same sequence as designed, as well as in the proper open reading frame.

Expression analyses of pVAX1©-α-γ, pVAX1©-GP35, and pVAX1©-α-γ-GP35 in HEK-293A cells

The expressions of the foreign proteins were examined by western blot with PRRSV-specific antiserum of pigs, mouse anti-IFNα, or anti-IFNγ serum. As shown in Fig. 2a, the specific band of ~65–70 kDa, being consistent with the predicted size of GP35, was clearly observed in cell lysates of pVAX1©-GP35 and pVAX1©-α-γ-GP35 as visualized with PRRSV-specific antiserum of pigs, whereas no band was found in cell lysates of pVAX1©-α-γ or empty vector pVAX1© plasmid. In addition, HEK-293A cells transfected with pVAX1©-α-γ or pVAX1©-α-γ-GP35 produced the specific band of IFNα (19 kDa) or IFNγ (17 kDa), respectively (Fig. 2b, c).

Fig. 2
figure 2

Western blot analysis of total lysates of HEK-293A cells transfected with pVAX1© empty vector (lane 1), pVAX1©-α-γ (lane 2), pVAX1©-GP35 (lane 3), pVAX1©-α-γ-GP35 (lane 4), and pVAX1©-α-γ + pVAX1©-GP35 (lane 5), respectively, using anti-PRRSV serum of pigs a, mouse anti-IFNα serum b, mouse anti-IFNγ serum c or mixtures of PRRSV-specific antiserum of pigs, mouse anti-IFNα, and anti-IFNγ serum d. Blot of β-actin was used as loading control

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To compare the expression levels of pVAX1©-α-γ + pVAX1©-GP35 and pVAX1©-α-γ-GP35, the cell lysates were analyzed by western blot using the mixtures of PRRSV-specific antiserum of pigs, mouse anti-IFNα, and anti-IFNγ serum. As shown in Fig. 2d, similar expression level of IFNα, IFNγ, or GP35 could readily be detectable by the mixtures of three antibodies from cell lysates of pVAX1©-α-γ + pVAX1©-GP35 and pVAX1©-α-γ-GP35.

To further detect the expressions of the IFNα or IFNγ, the antiviral activity of IFN was examined. MARC-145 cells were incubated with serial dilutions of supernatants from the HEK-293A cells transfected with individual plasmid, and then infected with 100 TCID50 of VSV or PRRSV SD-JN strain. As shown in Table 2, the anti-VSV and anti-PRRSV activities of pVAX1©-α-γ were very similar to those of pVAX1©-α-γ-GP35, but those of pVAX1© or pVAX1©-GP35 had no detectable activity. The anti-PRRSV activities of pVAX1©-α-γ and pVAX1©-α-γ-GP35 were lower than anti-VSV activity as VSV was more sensitive than PRRSV to IFN.

Table 2 Antiviral activity of IFN in the supernatants from the HEK-293A cells transfected with individual plasmid
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Body temperature change, clinical signs, and lung lesions after challenge (Experiment 1)

After challenge with HP-PRRSV, all pigs in pVAX1© and PBS control groups and pVAX1©-GP35 group had high fever (≥40.5 °C) and displayed a range of clinical signs, including inappetence, lethargy, skin cyanopathy, dyspnoea, coughing, and shivering. However, pigs vaccinated with pVAX1©-α-γ, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 only had low fever or a little fluctuation of rectal temperatures during 14 dpc (Fig. 3). The scores of clinical signs of pigs in pVAX1©-α-γ, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 groups were significantly lower than those in the other groups (P < 0.05), but no significant difference was observed among these groups (P > 0.05) (Table 3).

Fig. 3
figure 3

Rectal temperature of the pigs after challenge with HP-PRRSV isolate SD-JN in immediate protection experiment. The values were expressed as mean ± standard error

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Table 3 The scores of clinical signs of the pigs after challenge and lung lesions were recorded at 14 dpc in immediate protection experiment and long-lasting protection experiment
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At 14 dpc, all pigs were euthanized, and the scores of lung lesions were evaluated. The results showed that all pigs from pVAX1©-GP35, pVAX1©, and PBS groups had diffuse tan consolidation of the lungs. However, pigs in pVAX1©-α-γ, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 groups had very mild lung lesions, which was significantly lower than those of pVAX1©-GP35, pVAX1©, and PBS groups as shown in Table 3 (P < 0.05).

Viremia after challenge (Experiment 1)

At 0, 3, 7, and 14 dpc, the blood samples of the pigs were collected, and PRRSV RNA in the serum was monitored by real-time PCR. At 7 and 14 dpc, pigs inoculated with pVAX1©-α-γ, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 showed a significantly lower PRRSV RNA in blood than those with pVAX1©-GP35, pVAX1©, and PBS (P < 0.05) (Fig. 4).

Fig. 4
figure 4

Viremia of pigs inoculated with individual plasmid or PBS and challenged with HP-PRRSV isolate SD-JN in immediate protection experiment. PRRSV RNA concentrations of sera were collected at 0, 3, 7, and 14 dpc and detected by SYBR Green real-time PCR. The relative PRRSV RNA levels of samples were determined by linear extrapolation of the Ct value plotted against the standard curve. Data are shown as mean ± standard error for five pigs per group

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Humoral immune responses (Experiment 2)

The immunogenicity of the constructs was further investigated in pigs. The sera taken at 28, 42, and 56 dpi were used to detect the PRRSV-specific antibody level. As shown in Fig. 5, anti-PRRSV antibody in pigs vaccinated with pVAX1©-GP35, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 could be detected by ELISA at 28 dpi, and markedly increased after the booster. At 42 and 56 dpi, the levels of IgG from the groups of pVAX1©-α-γ + pVAX1©-GP35 and pVAX1©-α-γ-GP35 were significantly higher than that from the group of pVAX1©-GP35 (P < 0.05). There was no significant difference between the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 (P > 0.05). No PRRSV-specific antibody was detected in the groups of pVAX1©-α-γ, pVAX1©, or PBS.

Fig. 5
figure 5

iELISA analysis of sera from pigs immunized with individual plasmid or PBS. Serum samples (n = 5) were collected at various time-points and antibodies to purified SD-JN PRRSV antigen were detected using iELISA. Data are shown as mean ± standard error

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Serum samples were further evaluated the ability to neutralize PRRSV in vitro by SN assays. The results indicated that the levels of neutralizing antibodies in pVAX1©-α-γ-GP35, and pVAX1©-α-γ + pVAX1©-GP35 groups were also markedly higher than of pVAX1©-GP35 group at 42 and 56 dpi (Fig. 6). No neutralizing antibodies against PRRSV could be detected in pigs immunized with pVAX1©-α-γ, pVAX1©, or PBS. It indicated that pVAX1©-α-γ-GP35 could induce similar antibody level with pVAX1©-α-γ + pVAX1©-GP35 and enhance the humoral immune responses of GP3–GP5.

Fig. 6
figure 6

Neutralizing antibody responses in pigs immunized with individual plasmid or PBS. Serum samples (n = 5) were collected and detected by serum neutralization (SN) assays at various time-points. The titers of neutralizing antibodies were expressed as the reciprocal of the last serum dilution to neutralize 100 TCID50 of PRRSV in 50 % of the wells. Data are shown as mean ± standard error

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T lymphocyte proliferation responses (Experiment 2)

At 42 and 56 dpi, PBMCs were isolated, and the PRRSV-specific lymphocyte proliferation responses were detected. The results showed that pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 could induce significant higher levels of the proliferation at 42 and 56 dpi, compared with pVAX1©-GP35 (P < 0.05) (Fig. 7). However, there was no significant difference between the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 (P > 0.05). This result revealed that pVAX1©-α-γ-GP35 improved the cell-mediated immune response of the GP3–GP5 in pigs.

Fig. 7
figure 7

Lymphocyte-proliferative responses in pigs immunized with individual plasmid or PBS. Splenocytes samples (n = 5) were collected at days 42 and 56 dpi and were stimulated with purified SD-JN PRRSV antigen (10 μg/ml) in triplicate. After 45 h of stimulation, MTT was added, and the proliferation responses were detected by a standard MTT method. The PHA control sample showed a SI of 6–8. Data are shown as mean ± standard error

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Th1-type and Th2-type cytokine responses detected by cytokine ELISA kits (Experiment 2)

Cytokines play a dominant role in modulating immune responses against infection or in the effectiveness of vaccination. To monitor the expressions of cytokine, the supernatant of the lymphocytes stimulated with purified SD-JN PRRSV antigen were obtained to detect the levels of IFN-γ and IL-4 at 42 dpi using commercial ELISA kits. The results showed that the levels of IFN-γ and IL-4 from the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 were significantly higher than those from the group that received pVAX1©-GP35 (P < 0.05) (Fig. 8). The results demonstrated that pVAX1©-α-γ-GP35 could potentiate both Th1-type and Th2-type cytokine responses.

Fig. 8
figure 8

The concentrations (pg/ml) of Th1-type cytokine of IFN-γ and Th2-type cytokine of IL-4 in the supernatants. PBMC (5 × 106/ml, and 100 μl per well) were isolated from the blood of pigs at 42 dpi and stimulated with purified SD-JN PRRSV antigen (10 μg/ml). After 72 h, the supernatant fluids were collected to examine the levels of the Th1-type cytokine of IFN-γ and Th2-type cytokine of IL-4 using commercially available pig cytokine ELISA kits. Data are shown as mean ± standard error

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Body temperature change, clinical signs, and lung lesions after challenge (Experiment 2)

After challenge with HP-PRRSV, all pigs in pVAX1©-α-γ, pVAX1©, and PBS control groups had high fever (≥40.5 °C) and displayed a range of clinical signs as observed in the control groups in immediate protection experiment. Similar but light clinical signs were observed in group of pVAX1©-GP35. Pigs vaccinated with pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 only had very low fever or very little fluctuation of rectal temperatures during the 14 dpc (Fig. 9). Pigs vaccinated with pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 showed almost no clinical signs with a total clinical signs score of 3.4–3.5, which was significantly lower than the other groups (P < 0.001) (Table 3).

Fig. 9
figure 9

Rectal temperatures of the pigs after challenge with HP-PRRSV isolate SD-JN in long-lasting protection experiment. The values are expressed as mean ± standard error

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At 14 dpc, all pigs were euthanized, and the scores of lung lesions were evaluated. The results showed that all pigs from pVAX1©-α-γ, pVAX1© and PBS group had diffuse tan consolidation of the lungs. Pigs inoculated with pVAX1©-GP35 also showed light lung lesions. However, pigs in pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 groups had almost no lung lesions—significantly lower than those of pVAX1©-GP35 group as shown in Table 3 (P < 0.001).

Viremia after challenge (Experiment 2)

At 0, 3, 7, and 14 dpc, the blood samples of the pigs were collected, and PRRSV RNAs in the serum were detected by real-time PCR. At 7 and 14 dpc, pigs inoculated with pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 showed a significantly lower viremia in blood than that with pVAX1©-α-γ, pVAX1©, PBS (P < 0.001), and pVAX1©-GP35 (P < 0.05). Meanwhile, pigs inoculated with pVAX1©-α-γ, pVAX1©, and PBS had the highest viremia at both 7 and 14 dpc. In contrast, pVAX1©-GP35 group showed a little lower viremia (P < 0.05) (Fig. 10).

Fig. 10
figure 10

Viremia of pigs inoculated with individual plasmid or PBS and challenged with HP-PRRSV isolate SD-JN in long-lasting protection experiment. PRRSV RNA concentrations of sera were collected at 0, 3, 7, and 14 dpc and detected by SYBR Green real-time PCR. The relative PRRSV RNA levels of samples were determined by linear extrapolation of the Ct value plotted against the standard curve. Data are shown as mean ± standard error for five pigs per group

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Discussion

Despite the tremendous efforts invested in controlling PRRSV infections, the virus continues to plague the swine industry and damage pig production worldwide. Lack of safe and effective vaccine is the major barrier to control this disease. PRRSV genetic-engineered vaccines have recently been reported, including PRV or DNA vaccine expressing GP5 and M [36, 37], recombinant adenovirus or fowlpox virus co-expressing GP3 and GP5 [7, 30, 38, 39]. In order to increase the efficiency of the vaccine, the immune-modulating effects of cytokines, such as HSP70 [7], IL-18 [38] and GM-CSF [39], were evaluated as new adjuvants in PRRS vaccination strategies. In order to use the immunoadjuvant function and the synergistic anti-PRRSV effects of IFNα and IFNγ, IFNα/γ and GP3/5 were designed to be co-expressed in one eukaryotic expression vector. Our data clearly demonstrated that the pVAX1©-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with IFN α/γ, could effectively increase PRRSV-specific immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs.

Fusion protein strategy had been used in PRRS vaccine design and showed enhanced immune responses [30]. In this study, a linker of five glycine residues was inserted between GP3 and GP5 of PRRSV, and GP3–GP5 were expressed in fusion form (pVAX1©-GP35 and pVAX1©-α-γ-GP35, Fig. 2a). Recently, the 2A sequence of FMDV, which encoded a self-cleavage protease, was used to enable the expression of two proteins from one cistron [40]. Here, to maintain the individual antiviral effect of IFNα or IFN-γ, 2A linker was introduced between IFNα and IFN-γ (pVAX1©-α-γ and pVAX1©-α-γ-GP35) or between IFN-γ and GP35 (pVAX1©-α-γ-GP35). IFNα and IFN-γ were expressed correctly and showed the anti-VSV and anti-PRRSV activities (Fig. 2b, c; Table 2). Furthermore, the introduction of the 2A as a linker in pVAX1©-α-γ-GP35 resulted in a natural IFNα or IFN-γ in vivo, which may facilitate the adjuvant function [40].

Neutralizing antibodies play an important role in clearing PRRSV and preventing infection [41, 42]. It is known that GP3 and GP5 proteins have neutralizing epitopes [7, 9]. In this study, the animal experiment results indicated that pVAX1©-GP35 could induce neutralizing antibodies against PRRSV, but the titer was lower. In contrast, pigs vaccinated with pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 group had significantly higher neutralizing antibodies (Fig. 6). It indicated that the combination of IFN-α and IFN-γ could enhance the humoral immune responses of PRRSV GP35 fusion protein.

Cell-mediated immunity (CMI) is also extremely important in PRRSV infection [39]. As T lymphocyte proliferation response is generally related to CMI, data from the T cell proliferation assays indicated that the PRRSV-specific T lymphocyte proliferation responses from the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 were significantly higher than the group that received pVAX1©-GP35 (P < 0.05) (Fig. 7). It demonstrated that the combination of IFN-α and IFN-γ could enhance the cell-mediated immune responses of PRRSV GP35 fusion protein.

To assess the profile of cytokines after vaccination, the levels of the Th1-type cytokine of IFN-γ and Th2-type cytokine of IL-4 in the supernatants of PBMCs stimulated with purified PRRSV antigen were detected by commercial cytokine ELISA kits. The results showed that the levels of IFN-γ and IL-4 from the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 were significantly higher than those from the group that received pVAX1©-GP35 (P < 0.05) (Fig. 8). It confirmed that the combination of IFN-α and IFN-γ could facilitate both Th1-type and Th2-type cytokine responses.

To confirm the protective immune responses induced by pVAX1©-α-γ-GP35, the pigs were challenged with HP-PRRSV strain SD-JN. Temperature, viremia, clinical signs, and lung lesions were examined to evaluate the protective efficiency. It was shown that the younger pigs had exhibited much greater susceptibility to PRRSV than the older pigs [43]. Based on this report, 5 × 104 TCID50 challenge dose of PRRSV SD-JN strain was used in immediate protection experiment (23-day-old pigs), while in long-lasting protection experiment, the challenge dose of PRRSV SD-JN strain was 1 × 105 TCID50 (77-day-old pigs). Compared with the older pigs of pVAX1© or PBS control group in long-lasting protection experiment, although only half challenge dose was used, the younger pigs of pVAX1© or PBS control group in immediate protection experiment showed a little more intense viremia (Figs. 4, 10), and more severe clinical signs and lung lesions (Table 3). The result was consistent with the previous report [43].

In immediate protection experiment, the results showed that pVAX1©-α-γ-GP35 could provide similar protective efficiency with pVAX1©-α-γ, indicating that IFN-α and IFN-γ were natural folding and displayed the anti-PRRSV activity. However, pVAX1©-GP35 was not effectively working because the protective efficiency was based on the anti-PRRSV activity of IFN-α and IFN-γ. Although PRRSV infection was not fully prevented after homogenous challenge, pVAX1©-α-γ, pVAX1©-α-γ + pVAX1©-GP35, and pVAX1©-α-γ-GP35 produced significantly higher protective efficiency than the other groups (Figs. 3, 4; Table 3). In Experiment 2 (long-lasting protection experiment), the humoral immune responses, the PRRSV-specific lymphocyte proliferation responses, and the levels of IFN-γ and IL-4 of pigs in pVAX1©-GP35 group were better than those of the pVAX1©-α-γ-, pVAX1©- and PBS-inoculated groups (Figs. 5, 6, 7 and 8); these data could support the hypothesis that pVAX1©-GP35 was able to play its role. In long-lasting protection experiment, the results showed that all pigs in the pVAX1©-α-γ-, pVAX1© -, and PBS-inoculated groups showed severe clinical signs. The rectal temperatures rose the day after challenge in pVAX1©-α-γ-, pVAX1©-, and PBS-inoculated pigs, and peaked during 4–8 dpc, then decreased to normal level gradually, which result was coincident with the viremia detected by real-time PCR (Figs. 9, 10). However, pigs inoculated with pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 showed almost no clinical signs and almost no lung lesions, as compared with the other groups (P < 0.001) (Table 3). It indicated that the enhanced PRRSV-specific humoral and CMI of pVAX1©-α-γ-GP35 could provide the better protection against homogenous challenge. In the current research, as the combination of IFNα and IFNγ can act synergistically, we did not contain the group of pVAX1©-α (expressing IFNα) + pVAX1©-GP35 or pVAX1©-γ (expressing IFNγ) + pVAX1©-GP35, which would be taken up in the future studies. Based on the research of our lab, the HP-PRRSV strain is the dominant strain now in China, and only the HP-PRRSV strain was used in challenge experiment. Experiment on the heterologous challenge will also be performed in the future.

In both Experiments 1 and 2, it seemed that the effects of pVAX1©-α-γ-GP35 were always slightly better than that of pVAX1©-α-γ + pVAX1©-GP35. However, the statistical analysis showed that there was no significant difference between the groups of pVAX1©-α-γ-GP35 and pVAX1©-α-γ + pVAX1©-GP35 (P > 0.05) (Figs. 3, 4, 5, 6, 7, 8, 9, 10; Table 3). The DNA vaccine pVAX1©-α-γ-GP35 could provide similar protective efficiency with pVAX1©-α-γ + pVAX1©-GP35 in immediate and long-lasting protection. In contrast to pVAX1©-α-γ + pVAX1©-GP35, it was only needed to purify one kind of plasmid to obtain pVAX1©-α-γ-GP35, which was more economical and practical. In addition, the anti-PRRSV activity of pVAX1©-α-γ-GP35 could be used in immediate protection, and the enhanced humoral and cell-mediated immune responses of pVAX1©-α-γ-GP35 could be developed in long-lasting protection. After vaccination with pVAX1©-α-γ-GP35, pigs could partially resist PRRSV infection. When PRRSV-specific neutralizing antibodies and CMI were induced, the pigs could be better protected. Hence, it could be used as single therapeutic and prevention agent for HP-PRRSV in pig farms in China. To our knowledge, this study was the first to demonstrate that swine IFNα and IFNγ could be combined together to enhance the immune responses of GP3–GP5 of PRRSV. The DNA vaccine of pVAX1©-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections in pigs.