Orf virus DNA vaccines expressing ORFV 011 and ORFV 059 chimeric protein enhances immunogenicity

Background ORFV attenuated live vaccines have been the main prophylactic measure against contagious ecthyma in sheep and goats in the last decades, which play an important role in preventing the outbreak of the disease. However, the available vaccines do not induce lasting immunity in sheep and goats. On the other hand, variation in the terminal genome of Orf virus vaccine strains during cell culture adaptation may affect the efficacy of a vaccine. Currently, there are no more effective antiviral treatments available for contagious ecthyma. Results We constructed three eukaryotic expression vectors pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV011/ORFV059 and tested their immunogenicity in mouse model. High level expression of the recombinant proteins ORFV011, ORFV059 and ORFV011/ORFV059 was confirmed by western blotting analysis and indirect fluorescence antibody (IFA) tests. The ORFV-specific antibody titers and serum IgG1/IgG2a titers, the proliferation of lymphocytes and ORFV-specific cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α) were examined to evaluate the immune responses of the vaccinated mice. We found that mice inoculated with pcDNA3.1-ORFV 011/ORFV059 had significantly stronger immunological responses than those inoculated with pcDNA3.1-ORFV011, pcDNA3.1-ORFV059, or pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059. Compared to other vaccine plasmids immunized groups, pcDNA3.1-ORFV011/ORFV059 immunized group enhances immunogenicity. Conclusions We concluded that DNA vaccine pcDNA3.1-ORFV011/ORFV059 expressing ORFV011 and ORFV059 chemeric-proteins can significantly improve the potency of DNA vaccination and could be served as more effective and safe approach for new vaccines against ORFV.


Background
Orf virus (ORFV) is the prototype species of the Parapoxvirus genus, which causes contagious ecthyma in sheep and goats. The disease is also known as Orf, contagious pustular dermatitis, infectious labial dermatitis, scabby mouth, and sore mouth. The disease, which is distributed worldwide and endemic in most sheep and/ or goat-raising countries, is characterized by proliferative and self-limiting lesions around the muzzle and lips (scabby mouth) of infected animals, and sometimes also affects the gums and tongue, especially in young lambs [1,2]. The disease has a very high morbidity. Though mortality is low and usually does not exceed ten percent, mortality rates of up to 10% and 93% have been reported in lambs and kids [3][4][5]. The disease is frequently severe enough to create substantial welfare problems in flocks [6]. This, in turn, has an economic impact on sheep farmers due to the accompanying decreases in production.
In recent years, reports of severe Orf outbreaks in flocks have been gradually increased [7][8][9][10]. In addition, a mild form of the disease has been described in wild ruminants and in humans, in which is characterized by self-limiting, painful pustular lesions on the hands and fingers [11,12]. Several ORFV attenuated live vaccines have been used worldwide since 1981 and form the main prophylactic measure against contagious ecthyma in sheep and goats [13]. However, Conventional ORFV attenuated live vaccines are less effective at preventing the disease at present. It mainly due to the available vaccines do not induce lasting immunity in sheep and the rapid changes in the genomes of Orf virus vaccine strains during cell culture adaptation, particularly involving the ends of viral genome [14].
The host immune response to ORFV has been extensively studied, yet many aspects of the complex hostvirus interactions remain unclear. Several studies have demonstrated that the major envelop proteins of ORFV could induce a strong immune response [15,16]. As a major immunogenic protein, the ORFV011 protein can induce a strong antibody response by stimulating lymphocytes derived from draining lymph nodes [17]. In addition, the potential of the ORFV059 protein to act as an antigen in subunit vaccines against antigenically identical Orf viral strains has been indicated. Furthermore, it seems to be responsible for induction of neutralizing antibodies in the host, and plays an important role in the viral cycle [15,18]. Considering the immunogenicity of the ORFV 011 and ORFV059 proteins, it is possible that the chimeric expression of the ORFV011 and ORFV059 proteins could induce stronger immune responses.
In this study, we assembled DNA vaccine plasmids expressing the two major immunodominant proteins (ORFV011 and ORFV059) of the Orf virus, individually and simultaneously. The expression of the recombinant proteins in vitro was investigated by western blotting analysis and indirect fluorescence antibody (IFA) tests. The levels of protective humoral and cellular immune responses induced by the recombinant ORFV DNA vaccines were investigated in a mouse model.

Virus and cells
A newly identified fatal strain of Orf virus was isolated from scab specimens collected from skin lesions of a 6week-old small-tailed Han sheep affected with Orf virus in November 2008 in the Jilin province of China [9]. Primary ovine fetal turbinate (OFTu) cells were maintained in minimal essential medium (MEM) (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2 mM L-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 20 μg of nystatin/ml. The virus was propagated in OFTu cells. For virus harvest, cell culture supernatant from infected cells were collected when approximately 90% of the culture showed cytopathic effects (CPE). After three freeze-thaw cycles, the supernatant was then cleared at 500 × g for 10 min at 4°C and stored at -80°C. The virus was purified by sucrose gradient centrifugation. Infectivity titre was assayed by the plaque method in OFTu cell culture and calculated as plaque forming units (PFU).

Expression of recombinant proteins in vitro
OFTu cells were cultured in MEM containing 10% fetal bovine serum (FBS) in a 5% CO 2 incubator at 37°C.

Immunization
Large-scale preparations of DNA vaccine plasmids were obtained using alkaline lysis. Sixty 6-8-week-old female Balb/c mice (purchased from the Laboratory Animal Center of Jilin University in China) were used for experiments in accordance with the guidelines for animal experimentation of Jilin University. The animals were maintained under pathogen-free conditions, randomly divided into six groups (ten mice per group), and immunized by skin scarification [19] with one of the following formulations: (π) the A group was immunized with PBS, (θ) the B group with 100 μg pcDNA3.1(+) plasmids, (ρ) the C group with 100 μg pcDNA3.1-ORFV011 plasmids, (σ) the D group with 100 μg pcDNA3.1-ORFV059 plasmids, (τ) the E group with 50 μg pcDNA3.1-ORFV011 plasmids plus 50 μg pcDNA3.1-ORFV059 plasmids, and () the F group with 100 μg pcDNA3.1-ORFV011/ORFV059 plasmids. The mice were boosted in the same manner on days 14 and 28. Blood samples were collected from the tail artery at weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 post immunization. The sera were inactivated at 56°C for 30 min and stored at -80°C until assayed by ELISA (IgG at each time-points, IgG1/IgG2a isotype at 1 week post the final boost immunization, virus-neutralizing antibody at 2 weeks post immunization). On day 10 after the final boost immunization, 5 mice of each group were sacrificed by cervical dislocation, and their splenocytes were isolated for a lymphocyte proliferation assay and flow cytometry assay.

ORFV-specific antibody titer assay
ORFV-specific antibody responses were determined using an indirect ELISA, with the purified ORFV as the antigen. Briefly, Costar High Binding 96-well plates (eBioscience, San Diego, CA) were coated overnight at 4°C with 0.1 μg of purified ORFV diluted in 100 μl of 0.1 M NaHCO 3 buffer (pH 9.6). The plates were blocked with PBS/0.1% Tween-20/2% BSA (150 μl/well) for 1 h at 37°C. After washing three times with PBS/ 0.1% Tween-20, sera were added in a dilution of 1:20 in PBS/0.1% Tween-20/2% BSA. The plates were incubated for 90 min at 37°C, washed three times, and then incubated with 100 μl of horseradish-peroxidase (HRP)-conjugated goat anti-mouse IgG (Zhongshan Biotechnology Company, Beijing, China) at a dilution of 1:5000 in PBS/ 0.1% Tween-20/2% BSA for 1 h at 37°C. After three washes, the presence of IgG was detected with 100 μl of TMB according to the manufacturer's instructions. The reaction was stopped by adding 50 μl of 2 M H 2 SO 4 . The OD value was read at 450 nm. Results were expressed as an antibody endpoint titer, determined when the OD value is 3-fold greater than the background value obtained with a 1:20 dilution of serum from PBS-injected mice. The purified ORFV antigen was replaced with OFTu cell supernanant as a negative control.

Immunoglobulin isotyping ELISA
In order to determine the IgG1 and IgG2a subtypes in mice immunized with ORFV vaccine plasmids, purified ORFV was diluted to optimal concentrations in 0.1 M NaHCO3 buffer (pH 9.6), which was used as coating antigen. Costar High Binding 96-well plates (eBioscience, San Diego, CA) were coated with 0.1 μg of purified ORFV diluted in 100 μl of 0.1 M NaHCO3 buffer (pH 9.6). The plates were blocked with PBS/0.1% Tween-20/2% BSA (150 μl/well) for 1 h at 37°C. After washing three times with PBS/0.1% Tween-20, the sera were diluted at 1:20 in PBS/0.1% Tween-20. After a 1 h incubation, the plates were washed with PBS/0.1% Tween-20 and goat anti mouse IgG1, IgG2a (Sigma Chemical Co., SA Locus, Mo.) diluted to 1:1000 were used to detect IgG1 and IgG2a subtypes, respectively. After incubating for 60 min, the plates were washed with PBS/0.1% Tween-20, and horseradish-peroxidase (HRP)-conjugated rabbit anti-goat IgG (Zhongshan Biotechnology Company, Beijing, China) diluted to 1:4000 in PBS/0.1% Tween-20 was added to each well. The reaction was visualized after 1 h of incubation with TMB. The reaction was stopped by adding 50 μl of 2 M H 2 SO 4 . The OD value was read at 450 nm. Results were expressed as an antibody endpoint titer, determined when the OD value is 3-fold greater than the background value obtained with a 1:20 dilution of serum from PBS-injected mice.

Determination of virus-neutralizing titers
To perform the virus neutralization assay, serum samples were diluted 1:5 in MEM medium and heat-activated for 30 min at 56°C. This 1:5 starting dilution of inactivated sera was serially diluted two-fold in MEM medium in 96-well flat bottom plates and incubated with 0.1 μg of purified ORFV diluted in 50 μl MEM for 90 min at 37°C. All sera were run in triplicate. Subsequently, 100 μl of OFTu cell suspension were added to each well at a concentration of 45,000 cells/well. The plates were incubated for 5 days at 37°C in a 5% CO 2 incubator. The neutralization titer of a serum is the dilution at which 50% of the wells are protected against virus infection. The titer is expressed as ND50 and calculated using the Reed and Muench method.

Splenocyte preparations
Single-cell suspensions of spleens were prepared from the immunized mice at day 10 post the final boost immunization. Briefly, spleens from freshly killed mice were disrupted by using monofilament nylon filters, and the cells were collected and centrifuged at 500 × g for 20 min. The cell pellets were suspended in 10 ml RPMI 1640 and then centrifuged at 500 × g for 10 min at room temperature. The cells were resuspended in RPMI 1640 supplemented with 10% FBS at a concentration of 1 × 10 7 cells/ml before the lymphocyte proliferation assay and flow cytometry assay.

Cellular proliferation assay
Untreated splenocytes were cultured in RPMI 1640 medium with 10% fetal serum. The cells were suspended in complete RPMI 1640 to achieve the concentration of 1 × 10 6 splenocytes/well. The primary incubation was at 37°C in a 5% CO 2 incubator for 72 h, followed by addition of 10 μl MTT (5 mg/ml) per well and a secondary incubation at 37°C in a 5% CO 2 incubator for another 4 h. The microplates were centrifuged, the unreacted MTT was removed, then 100 μl dimethyl sulfoxide (DMSO) was added per well to solubilize formazan. The plates were then incubated for 10 min with shaking. The absorbance of the plate was read using a 570 nm filter within minutes following shaking. The treated cells were cultured under the same conditions, but with the addition of purified ORFV antigen (at a final concentration of 5 μg/ml) in complete RPMI 1640 medium. The mitogen, Con A was added at 5 μg/ml concentration in the positive control wells. The geometric means and standard deviations for triplicate sets of samples were calculated. Lymphocyte proliferation is expressed as a stimulation index (SI), which is defined as the mean of experimental data divided by the mean of the unstimulated control [20,21].

Flow cytometry assay
In order to determine the changes of specific T cell subsets in mice immunized with ORFV vaccine plasmids, the flow cytometry assay were performed. 1 × 10 6 splenocytes collected from the immunized mice at day 10 post the final boost immunization were suspended in PBS and stained with FITC-conjugated anti-mouse CD3, PE-Cy5-conjugated anti-mouse CD8, and PE-conjugated anti-mouse CD4 for 30 min on ice. After three washes with PBS, the cells were centrifuged at 500 × g for 10 min. The cells were suspended in 200 μl PBS. At least 10,000 cells were analyzed per sample. The stained cells were analyzed by a BD FACSAria flow cytometer, and the data were analyzed with Cellquest V3.3 software.

Statistical analysis
All data were analyzed using the statistical software program Systat 10 (SPSS). The distribution of data was determined using descriptive statistics. Data which were not normally distributed were transformed by ranking. Differences in ELISA titers were investigated using oneway analysis of variance (ANOVA) performed on the rank. The means of the rank-transformed variables were compared using Tukey's multiple comparison test. Values of * P < 0.05, ** P < 0.01 were considered statistically significant.  (Figure 2a and 2b).

Subsets of specific T lymphocyte cells
To examine whether or not immunized mice by skin scarification developed an induced T-cell response against ORFV, T lymphocyte cells from mice in groups (pcDNA3.1-ORFV011 group, pcDNA3.1-ORFV059 group, pcDNA3.1-ORFV 011/ORFV059 group, pcDNA3.1-ORFV 011 plus pcDNA3.1-ORFV group, pcDNA3.1(+) group and PBS group) were respectively enriched from the spleens 10 days after the final boost immunization and cultured in RPMI 1640 medium. As shown in Figure 7, the percentages of CD4 + and CD8 + cells in mice immunized with DNA vaccine plasmids increased significantly compared to mice immunized with the PBS and pcDNA3.1(+). The ratios of CD4 + /CD8 + cells in mice immunized with vaccine plasmids were higher than the control groups. These results further indicate that the DNA vaccine constructs can induce a T-cell response in mice and activate cell immunity response.

ORFV-specific cytokine production in the splenocytes culture supernatants
We also investigated the responsiveness of spleen cells from immunized mice against ORFV. We determined the levels of cytokines production (IFN-γ, TNF-α, IL-2, IL-4 and IL-6) in the supernatant of splenocytes stimulated with purified ORFV. Little levels of IFN-γ, TNF-α and IL-2 were observed in mice immunized with empty vector or PBS. The levels of IFN-γ, TNF-α and IL-2 produced by splenocytes immunized with ORFV DNA vaccine plasmids were significantly higher than that with pcDNA3.1 (+) or PBS (**P < 0.01). The levels of Th2 type cytokines (IL-4 and IL-6) increased slightly in ORFV DNA vaccine plasmids immunized mice compared with pcDNA3.1 (+) or PBS immunized mice, but the changes were not so dramatic (Figure 8). The results mentioned above indicated that an efficient antigen-specific T cell activation, most probably CD8 + T cells, was induced by ORFV DNA vaccine plasmids expressing the MHC class I epitope.

Discussion
The continuing occurrence of Orf in the ovine and caprine herds all over the world emphasizes the need to develop novel vaccines with improved properties compared to the currently used attenuated live vaccines. One promising approach is the use of viral DNA vaccines because they are safe and can induce both neutralizing antibodies and cellular immune responses while maintaining the advantages of subunit and traditional vaccines [22,23]. To our knowledge, the current study is the first report demonstrating the potential of an ORFV DNA vaccine to induce strong immune responses in mice.
ORFV 011 and ORFV 059 are two major immunodominant proteins which have been demonstrated to induce a strong humoral response against ORFV [17,24]. In our study, ORFV DNA vaccines can induced specific humoral and cellular responses in mice. The levels of ORFV-specific IgG antibodies were greatly enhanced following two boosts. In addition, the higher neutralizing antibodies were detected in pcDNA3.1-ORFV 059, pcDNA3.1-ORFV 011 plus pcDNA3.1-ORFV 059 or pcDNA3.1-ORFV 011/ORFV 059 groups. Our findings are supported by a previous study showing that ORFV 059 protein could induce the production of neutralizing antibodies [15]. The measurement of certain antibody isotypes correlates directly to type 1 or type 2 T cell responses [25]. For example, IgG2a in mice [26] is associated with in vivo production of IFN-γ and can be used as an indicator to measure the type 1 responses. Here, the IgG2a:IgG1 isotypes in immunized mice were assayed by ELISA. The levels of anti-ORFV IgG1 and IgG2a in DNA vaccine plasmids immunizations groups were higher than control groups. We further examined whether there was any difference in the type of T helper responses among these groups by using the IgG2a/IgG1 ratio as a surrogate marker. Interestingly, the ratios of IgG2a/IgG1 in mice receiving DNA vaccine plasmids immunizations were more than 1, indicating that DNA vaccine plasmid constructs induced predominantly a Th1-type immune response after the final boost immunization.
helper (CD4 + ) and CTL (CD8 + ) cells. CD4 + T cells can increase the number of memory cells. The memory cells respond rapidly when re-exposed to pathogens and thus play a vital role in protection against viral challenge [27,28]. After activation, naive antigen-specific CD8 + T cells are able to proliferate quickly and differentiate into potent effector cells capable of rapid cytokine production and cytolytic killing of target cells [29]. CD4 + lymphocytes are further subdivided into Th1 and Th2 on the basis of the type of released cytokines. The Th1 cell subset mainly includes cells secreting IL-2, TNF-α, and INF-γ. The main roles of these cytokines include enhancing killer cell cytotoxicity and cell-mediated immune response. The Th2 cell subset mainly includes cells producing IL-4, IL-6, and IL-10. The role of Th2 cytokines is to promote antibody production and mediate humoral immune response. In the study, we investigated the responsiveness of spleen cells from immunized mice against ORFV. The levels of cytokines production (IFNγ, TNF-α, IL-2, IL-4 and IL-6) in the supernatant of splenocytes stimulated with purified ORFV were determined by ELISA assay. The levels of IFN-γ, TNF-α and IL-2 produced by splenocytes immunized with ORFV DNA vaccine plasmids were significantly higher than that with pcDNA3.1 (+) or PBS (**P < 0.01). The result indicated that an efficient antigen-specific T cell activation, most probably CD8 + T cells, was induced by ORFV DNA vaccine plasmids expressing the MHC class I epitope.
To further address the T cell response, we examined the CD4 + and CD8 + splenocytes collected from immunized mice by flow cytometry analysis. The percentages of CD4 + and CD8 + cells in mice immunized with DNA vaccine plasmids increased significantly compared to mice immunized with the PBS and pcDNA3.1(+). The ratios of CD4 + /CD8 + cells in mice immunized with vaccine plasmids were higher than the control groups. These results further indicate that the DNA vaccine constructs can induce a T-cell response in mice and activate cell immunity response.
Taken together, these data indicate that in vaccinated mice, the ORFV011 and ORFV059 can be expressed and secreted, induce specific humoral and cellular immune responses by activate both B and T cells, and the chimeric expression of ORFV011 and ORFV059 could significantly improve the potency of DNA vaccination. We concluded that DNA vaccine pcDNA3.1-ORFV011/ ORFV059 expressing ORFV011 and ORFV059 chemeric-proteins can significantly improve the potency of DNA vaccination and could be served as more effective and safe approach for new vaccines against ORFV.
Further studies are being carried out in lambs to understand the similarities and differences in the response between mice and lambs. These studies will provide a basis for the development of an ORFV DNA vaccine ready for clinical trial.

Conclusions
We concluded that DNA vaccine pcDNA3.1-ORFV011/ ORFV059 expressing ORFV011 and ORFV059 chemeric-proteins can significantly improve the potency of DNA vaccination and could be served as more effective and safe approach for new vaccines against ORFV.