Co-Expression of Chicken IL-2 and IL-7 Enhances the Immunogenicity and Protective Efficacy of a VP2-Expressing DNA Vaccine against IBDV in Chickens

Chicken infectious bursal disease (IBD) is still incompletely controlled worldwide. Although IBD virus (IBDV) VP2 DNA vaccine was considered a safe vaccine for IBD prevention, the immunogenicity by itself remains poor, resulting in the failure of effectively protecting chickens from infection. We and others demonstrated that chicken IL-2 (chIL-2) and chIL-7 have the capacity to enhance the immunogenicity of the VP2 DNA vaccine. However, whether chIL-2 and chIL-7 can mutually enhance the immunogenicity of VP2 DNA vaccine and thereby augment the latter’s protection efficacy remains unknown. By using chIL-2/chIL-7 bicistronic gene vector to co-immunize the chickens together with the VP2 DNA vaccine, we now show that chIL-2 and chIL-7 significantly increased IBDV VP2-specific antibody titers, T cell proliferation, and IFN-γ production, resulting in the ultimate enhancement of vaccine-induced protection efficacy relative to that of chIL-2 or chIL-7 gene vectors alone. These results suggest that chIL-2 and chIL-7 can mutually enhance VP2 DNA vaccine’s efficacy, thereby establishing a concrete foundation for future optimization of IBDV VP2 DNA vaccine to prevent/treat chicken IBD.


Introduction
Infectious bursal disease (IBD) caused by IBD virus (IBDV) is a highly contagious and immunosuppressive disease affecting the poultry industry worldwide. IBDV specifically infects developing B cells in the bursa of Fabricius of young chickens, causing the destruction of the antibody-producing B cell precursors, with consequent lymphoid depletion of B cells and bursa atrophy, culminating in immunosuppression, vaccination failure, and susceptibility to other microbial infections [1].
IBD has not been effectively controlled so far although vaccination programs have been extensively implemented worldwide using live attenuated or inactivated IBDV vaccines, outbreaks of IBD still occur [8]. The live attenuated vaccine often exhibits strong immunogenicity, but is often associated with the emergence of highly virulent and variant strains, probably due to selection pressure from the administration of the live attenuated IBDV vaccine [9]. In addition, the live attenuated vaccine has often caused chicken immunosuppression and sub-clinical infection [10]. The inactivated IBDV vaccine is comparably safe but often displays relatively weak immunogenicity and low protective efficacy. Therefore, it is imperative to develop a safe and effective IBDV vaccine. In recent years, IBDV VP2-based DNA vaccine has been extensively studied as a potential candidate vaccine for the development of effective IBDV vaccine [11,12] because VP2 DNA vaccine can induce humoral and cellular immune responses [12], including neutralizing antibodies [6]. However, neither the full-length nor the truncated VP2 vaccine fully protected chickens from IBDV infection.
Recently, some immunostimulatory cytokines are shown to be more effective biological adjuvants that enhance the immunogenicity of IBDV DNA vaccine. Chicken IL-2 [13], chIL-6 [14], chIL-18 [15], and chIFN-γ [16] have all been demonstrated to possess strong biological adjuvant activities that boost the efficacy of IBDV VP2 DNA vaccine. Our previous work showed that chIL-7 gene vector also had potent adjuvant activity and enhanced the immunogenicity of IBDV VP2 DNA vaccine [17]. Consistently, recombinant chIL-7 significantly boosted the immunogenicity of inactivated IBDV vaccine [18].
IL-2, -7, -9, -15, and -21 belong to the common γ-chain cytokine family [19] and have essential roles in the maintenance of immune homeostasis [20,21]. IL-2 drives the development and maturation of B cells, and also promotes T cell differentiation into antigen-specific effector and memory populations [21,22]. In contrast, IL-7 stimulates the differentiation of multipotent hematopoietic stem cells into lymphoid progenitor cells and proliferation of all cells in the lymphoid lineage (B cells, T cells, and natural killer (NK) cells) [23,24]. Our previous work showed that mouse IL-2 and IL-7 can mutually enhance the immunogenicity of ovalbumin (OVA) DNA vaccine [25]. However, whether their chicken counterparts behave similarly remains unclear. To bridge this knowledge gap, we used a bicistronic expression vector for synchronously expressing chIL-2 and chIL-7 as an adjuvant for IBDV VP2 DNA vaccine. We found that chIL-2 and chIL-7 can indeed mutually enhance the immunogenicity of IBDV VP2 DNA vaccine in IBD chickens, as shown by dramatically increased chicken humoral and cellular immune responses against the VP2 DNA vaccine and consequent augmented protective efficacy. Our results thus establish an essential basis for further improving the immune effect of the IBDV VP2 DNA vaccines.

Vaccine, Antibodies, and Proteins
Attenuated live IBDV vaccine was from Ringpu (Baoding, China). Mouse anti-His monoclonal antibody and goat anti-mouse IgG-AP antibody were from Abcam (Cambridge, MA, USA). Recombinant IBDV VP2 protein was prepared in our laboratory as previously described [17].

IBDV Propagation and Titer Determination
Propagation and titration of IBDV were performed as previously described [26]. Briefly, four-week-old SPF white leghorn chickens were inoculated with 10 4 median embryo infectious dose (EID 50 ) virulent IBDV (0.2 mL per bird) by the nasal and eye-drop routes. Infected bursae were harvested at 3 days post-inoculation, homogenized with PBS, frozen (−70 • C) and thawed three times. Homogenates were clarified by centrifugation at 1000 g for 10 min at 4 • C. The virus titers in supernatant were determined using SPF chicken embryos and expressed as EID 50 . Non-virulent IBDV, a cell-adapted virus, was propagated in DF-1 chicken embryonic fibroblast cells. Virus titers were determined by Reed-Muench method and expressed as the 50% tissue culture infective dose (TCID 50 ).

Animal Experiment
A total of 300 SPF white leghorn chickens (21-day old) were randomly divided into 10 groups, 30 in each group (Table 1), and then each group was further divided into three groups, namely, an antibody tracking test group (8 birds), cellular immune evaluation group (8 birds), and challenge group (14 birds). The first group of chickens were not immunized and served as a negative control group. Chickens in group 10 were immunized orally with attenuated IBDV as positive control. Chickens in groups 2-9 were intramusculally injected with different immunization vectors (see Table 1). The immunized chickens were then boosted with the same vector and dose twice, 7 days apart. Chickens in group 10 were boosted with the same IBDV vaccine at the same titer at 10 days after the first immunization. At 2 days before the first immunization and at 0, 14, 28, 42, and 56 days after the first immunization ( Figure 1), the blood samples were collected by the wing vein of the chicken from antibody tracking test group and the sera were isolated, and the titer of IBDV-specific antibodies were determined by enzyme-linked immunosorbent assay (ELISA). On the 35th day after immunization, chickens in the cellular immune evaluation group (8 birds) were euthanized, the splenic lymphocytes were aseptically separated by Ficoll density gradient centrifugation, cell proliferation index was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and IFN-γ and IL-4 expressions were determined by ELISA. At 35 d after immunization, chickens in the challenge group (14 birds) were challenged with 1 × 10 3 ELD 50 IBDV virulent strain (amplified using chicken embryo) by oral administration. Chicken mortality, bursa/body ratio (B/B ratio), bursa lesion score, and protective efficacy were evaluated by corresponding methods. proliferation index was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and IFN-γ and IL-4 expressions were determined by ELISA. At 35 d after immunization, chickens in the challenge group (14 birds) were challenged with 1 × 10 3 ELD50 IBDV virulent strain (amplified using chicken embryo) by oral administration. Chicken mortality, bursa/body ratio (B/B ratio), bursa lesion score, and protective efficacy were evaluated by corresponding methods.

Detection of Serum Antibody Titers
Antibody titers were determined by ELISA. Briefly, the 96-well plates were coated with 100 µL of VP2 protein (10 mg/mL) in coating buffer (0.05 M carbonate buffer, pH9.6) overnight at 4 • C and blocked with 3% non-fat milk in PBST. Then the plates were incubated with 100 µL of 2-fold serial diluted chicken sera at 4 • C for 1h. After washing with PBST, wells were incubated with 100 µL HRP-conjugated goat anti chicken IgG (Sigma, St. Louis, MO, USA) for 1 h at 37 • C. The freshly-prepared 3,3 ,5,5 -Tetramethylbenzidine (TMB) solution (100 µL) was added for color development at 37 • C for 1 h. 50 µL of 2 M H 2 SO 4 were then added to each well to stop the reaction and the values were read using a microplate reader at 450 nm.

Lymphocyte Proliferation Assay
MTT assay was used to measure lymphocyte proliferation [17]. Briefly, immunized chickens were euthanized at 35 days post-immunization and lymphocytes were isolated from spleens. Splenocytes were plated in 96-well plates at 2 × 10 6 cells/well in RPMI-1640 medium with 10% FBS, and stimulated, in vitro, with concanavalin (Con A, 5 mg/mL, Sigma) as a positive control, and specific antigen VP2 protein (5 mg/mL) at 37 • C for 72 h. 20 µL MTT (5 mg/mL) were added to each well and incubated for 4 h. Cells were collected and incubated with 150 µL dimethyl sulfoxide (DMSO) to dissolve intracellular MTT. Supernatant was then transferred to another 96-well plate and the OD 450 value was read in a microplate reader.

Detection of Cytokine Production
Spleen lymphocytes isolated from immunized chicken were stimulated with the VP2 protein as above. The IFN-γ and IL-4 levels in the culture medium were determined by ELISA using IFN-γ (SEA049Ga) and IL-4 (SEA077Ga) kits from Cloud-Clone Corp (Katy, TX, USA) and following the manufacturer's instructions.

Viral Challenge Study
At 35 d post-immunization, the chickens in the challenge subgroups (14 birds in each group) were orally challenged with 1 × 10 3 EID 50 virulent IBDV. The challenged chickens were observed clinically for 8 d and mortalities were recorded. Chickens and bursae were weighed and B/B ratios were calculated by (bursal weight/body weight) × 1000. Bursal lesion scores were evaluated based on the histopathological severity of bursae [11]. Protection efficacy was defined by the number of chickens with histopathological BF lesion scores of 0 and 1 divided by the number of chickens in the group.

Statistics
The significance of differences between experimental groups was evaluated by one-way analysis of variance (ANOVA) with Dunnett's post-comparison test for multiple groups and Student's t-test was used for a single comparison of the two groups, respectively.

Construction of chIL-2 and chIL-7 Expression Vectors
To construct chIL-2 and chIL-7 expression vectors, the chIL-2 (432bp) gene was first amplified from chicken spleen by RT-PCR and then inserted into pcDNA3.1A plasmid to generate a His-tagged and non-tagged chIL-2 eukaryotic expression vectors, pcDNA-chIL2/H and pcDNA-chIL2. To construct chIL-2 and chIL-7 bicistronic expression vector, the VP2 gene in pcDNA-VP2/H-IRES-chIL7/H and pcDNA-VP2-IRES-chIL7 plasmids were substituted by chIL-2 gene to generate His-tag-fused and no-fused chIL-2 and chIL-7 bicistronic expression vectors: pcDNA-chIL2/H-IRES-chIL7/H and pcDNA-chIL2-IRES-chIL7, respectively ( Figure 2). The chIL-7 gene vectors (His-tag fused and no-fused) were prepared previously in our laboratory [17]. The sequence of chIL-2 gene amplified in this study was identified to be consistent with that in GenBank (GenBank: AF000631.1) and the inserting sites of the genes in expression vectors were identified to be correct by restriction analysis and sequencing.

Viral Challenge Study
At 35 d post-immunization, the chickens in the challenge subgroups (14 birds in each group) were orally challenged with 1 × 10 3 EID50 virulent IBDV. The challenged chickens were observed clinically for 8 d and mortalities were recorded. Chickens and bursae were weighed and B/B ratios were calculated by (bursal weight/body weight) × 1000. Bursal lesion scores were evaluated based on the histopathological severity of bursae [11]. Protection efficacy was defined by the number of chickens with histopathological BF lesion scores of 0 and 1 divided by the number of chickens in the group.

Statistics
The significance of differences between experimental groups was evaluated by one-way analysis of variance (ANOVA) with Dunnett's post-comparison test for multiple groups and Student's t-test was used for a single comparison of the two groups, respectively.

Construction of chIL-2 and chIL-7 Expression Vectors
To construct chIL-2 and chIL-7 expression vectors, the chIL-2 (432bp) gene was first amplified from chicken spleen by RT-PCR and then inserted into pcDNA3.1A plasmid to generate a His-tagged and non-tagged chIL-2 eukaryotic expression vectors, pcDNA-chIL2/H and pcDNA-chIL2. To construct chIL-2 and chIL-7 bicistronic expression vector, the VP2 gene in pcDNA-VP2/H-IRES-chIL7/H and pcDNA-VP2-IRES-chIL7 plasmids were substituted by chIL-2 gene to generate His-tag-fused and no-fused chIL-2 and chIL-7 bicistronic expression vectors: pcDNA-chIL2/H-IRES-chIL7/H and pcDNA-chIL2-IRES-chIL7, respectively ( Figure 2). The chIL-7 gene vectors (His-tag fused and no-fused) were prepared previously in our laboratory [17]. The sequence of chIL-2 gene amplified in this study was identified to be consistent with that in GenBank (GenBank: AF000631.1) and the inserting sites of the genes in expression vectors were identified to be correct by restriction analysis and sequencing.

In Vitro and In Vivo Expression of chIL-2 and chIL-7
To determine whether the chIL-2 and chIL-7 vectors can mediate chIL-2 and chIL-7 expressions in eukaryotic cells in a secretory manner, we first tested the vector-mediated chIL-2 and chIL-7 expression in vitro. The HEK293T cells were transfected with His-tag-fused chIL-2 (pcDNA-chIL2/H) and chIL-7 (pcDNA-chIL7/H) gene vectors and pcDNA-chIL2/H-IRES-chIL7/H (pcDNA3.1A empty vector was transfected under the same conditions as a negative control). The expressed chIL-2 and chIL-7 in the culture media were detected by immunoblotting using

In Vitro and In Vivo Expression of chIL-2 and chIL-7
To determine whether the chIL-2 and chIL-7 vectors can mediate chIL-2 and chIL-7 expressions in eukaryotic cells in a secretory manner, we first tested the vector-mediated chIL-2 and chIL-7 expression in vitro. The HEK293T cells were transfected with His-tag-fused chIL-2 (pcDNA-chIL2/H) and chIL-7 (pcDNA-chIL7/H) gene vectors and pcDNA-chIL2/H-IRES-chIL7/H (pcDNA3.1A empty vector was transfected under the same conditions as a negative control). The expressed chIL-2 and chIL-7 in the culture media were detected by immunoblotting using anti-His-tag antibody. As shown in Figure 3A, the specific protein band of chIL-2 (about 15 kDa) and chIL-7 (about 25 kDa) were detected in the culture anti-His-tag antibody. As shown in Figure 3A, the specific protein band of chIL-2 (about 15 kDa) and chIL-7 (about 25 kDa) were detected in the culture media from all three related vectors, indicating that the constructed vectors, either their individual or bicistronic vector, could mediate the corresponding gene expressions in a secretory manner in vitro.
To determine whether the chIL-2 and chIL-7 vectors can mediate their gene expressions in vivo, we used RT-PCR method to detect chIL-2 and chIL-7 expressions in vector-injected chicken muscle tissues at 2 d after immunization. Just like in vitro experiments, VP2, chIL-2, and chIL-7 expressions were detected in the muscle tissues ( Figure 3B-D), indicating that constructed expression vectors can mediate their corresponding gene expressions in vivo.

ChckenIL-2 and chIL-7 Genes Mutually Enhance Humoral Immune Response to IBDV VP2 DNA Vaccine in Immunized Chickens
To investigate whether chIL-2 and chIL-7 genes have the mutual enhancement on humoral immune response to the VP2 DNA vaccine in chickens, we co-immunized the SPF chickens with different plasmids (Table 1) and measured the antibody titers at the different time post immunization by ELISA ( Figure 4A). VP2-specific antibodies were detectable from day 14 and peaked on day 42 post-immunization, the titers were significantly increased in all immunized chickens. Importantly, the antibody titers in pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 co-immunized chickens were significantly higher than that of either pcDNA-VP2/pcDNA-chIL2 (p < 0.01) or pcDNA-VP2/pcDNA-chIL7 co-immunized chickens (p < 0.01) ( Figure 4A). Furthermore, the high-level neutralizing antibody against IBDV was also detected in co-immunized chickens with pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 plasmids ( Figure 4B). All above results indicate that both chIL-2 and chIL-7 enhance IBDV VP2 DNA vaccine immunogenicity, and co-administration of chIL-2/chIL-7 genes with IBDV VP2 DNA vaccine resulted in a significant increase in IBDV-specific To determine whether the chIL-2 and chIL-7 vectors can mediate their gene expressions in vivo, we used RT-PCR method to detect chIL-2 and chIL-7 expressions in vector-injected chicken muscle tissues at 2 d after immunization. Just like in vitro experiments, VP2, chIL-2, and chIL-7 expressions were detected in the muscle tissues ( Figure 3B-D), indicating that constructed expression vectors can mediate their corresponding gene expressions in vivo.

ChckenIL-2 and chIL-7 Genes Mutually Enhance Humoral Immune Response to IBDV VP2 DNA Vaccine in Immunized Chickens
To investigate whether chIL-2 and chIL-7 genes have the mutual enhancement on humoral immune response to the VP2 DNA vaccine in chickens, we co-immunized the SPF chickens with different plasmids (Table 1) and measured the antibody titers at the different time post immunization by ELISA ( Figure 4A). VP2-specific antibodies were detectable from day 14 and peaked on day 42 post-immunization, the titers were significantly increased in all immunized chickens. Importantly, the antibody titers in pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 co-immunized chickens were significantly higher than that of either pcDNA-VP2/pcDNA-chIL2 (p < 0.01) or pcDNA-VP2/pcDNA-chIL7 co-immunized chickens (p < 0.01) ( Figure 4A). Furthermore, the high-level neutralizing antibody against IBDV was also detected in co-immunized chickens with pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 plasmids ( Figure 4B). All above results indicate that both chIL-2 and chIL-7 enhance IBDV VP2 DNA vaccine immunogenicity, and co-administration of chIL-2/chIL-7 genes with IBDV VP2 DNA vaccine resulted in a significant increase in IBDV-specific antibody titers compared with separate administration, indicating that chIL-2 and chIL-7 possess mutual enhancement properties on chicken humoral immune response against IBDV.

Chicken IL-2 and chIL-7 Genes Mutually Enhance VP2 DNA Vaccine-Induced Cellular Immune Response in Immunized Chickens
To analyze whether chIL-2 and chIL-7 genes have the mutual enhancement on IBDV VP2 DNA vaccine-induced chicken cellular immune responses, the lymphocyte proliferation and cytokine production of immunized chickens were analyzed. Lymphocytes were isolated from spleen of the immunized chicken at 35 d post-immunization and stimulated with the VP2 protein (or Con A as a positive control). The cell proliferation was measured with MTT method and showed that the lymphocyte stimulation indexes (SI) of pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 co-immunized and IBDV vaccine-immunized chickens were significantly higher than those of pcDNA-VP2 immunized (p < 0.01), pcDNA-VP2/pcDNA-chIL2 co-immunized, and pcDNA-VP2/pcDNA-chIL7

Chicken IL-2 and chIL-7 Genes Mutually Enhance VP2 DNA Vaccine-Induced Cellular Immune Response in Immunized Chickens
To analyze whether chIL-2 and chIL-7 genes have the mutual enhancement on IBDV VP2 DNA vaccine-induced chicken cellular immune responses, the lymphocyte proliferation and cytokine production of immunized chickens were analyzed. Lymphocytes were isolated from spleen of the immunized chicken at 35 d post-immunization and stimulated with the VP2 protein (or Con A as a positive control). The cell proliferation was measured with MTT method and showed that the lymphocyte stimulation indexes (SI) of pcDNA-VP2/pcDNA-chIL2-IRES-chIL7 co-immunized and IBDV vaccine-immunized chickens were significantly higher than those of pcDNA-VP2 immunized (p < 0.01), pcDNA-VP2/pcDNA-chIL2 co-immunized, and pcDNA-VP2/pcDNA-chIL7 co-immunized chickens (p < 0.05) ( Figure 5A). These results indicate that chIL-2 and chIL-7 have mutual enhancement on VP2 Viruses 2019, 11, 476 9 of 17 DNA vaccine-induced lymphocyte proliferation. To further test their mutual enhancement on cellular immune responses, IFN-γ and IL-4 expressions in spleen lymphocytes from the different immunized chickens were measured by ELISA after stimulation with the VP2 protein in vitro. Results showed that the IFN-γ and IL-4 levels in culture medium of lymphocytes from chIL-2/chIL-7 gene co-immunized chickens significantly higher than those from chIL-2 or chIL-7 gene co-immunized chickens ( Figure 5B,C). Those above results indicate that chIL-2 and IL-7 have mutual enhancement on IBDV VP2 DNA vaccine-induced chicken cellular immune responses.
Viruses 2019, 11, x FOR PEER REVIEW 9 of 16 co-immunized chickens (p < 0.05) ( Figure 5A). These results indicate that chIL-2 and chIL-7 have mutual enhancement on VP2 DNA vaccine-induced lymphocyte proliferation. To further test their mutual enhancement on cellular immune responses, IFN-γ and IL-4 expressions in spleen lymphocytes from the different immunized chickens were measured by ELISA after stimulation with the VP2 protein in vitro. Results showed that the IFN-γ and IL-4 levels in culture medium of lymphocytes from chIL-2/chIL-7 gene co-immunized chickens significantly higher than those from chIL-2 or chIL-7 gene co-immunized chickens ( Figure 5B,C). Those above results indicate that chIL-2 and IL-7 have mutual enhancement on IBDV VP2 DNA vaccine-induced chicken cellular immune responses.

Discussion
Relative to IBDV VP2 protein, IBDV VP2 DNA vaccine is widely considered to be a more effective vaccine since the latter is more potent at stimulating humoral and cellular immune responses that inhibit pathogen infection. Consistent with this notion, plasmid-based VP2 gene vector showed~75% protective efficacy against virulent IBDV challenge [12]. Although the VP2 DNA vaccine has relatively high immunogenicity and an acceptable biosafety profile, it still falls short on conveying 100% protection from IBDV infection. Previous work has argued that both humoral and cellular immune responses may be needed for full protection. Several attempts that incorporate additional adjuvant(s) have been made to improve VP2 DNA vaccine's immunogenicity, including cytokines [15,17,18,28,30], heat shock protein (HSP) [31], CpG-DON [32], and β-defensin-1 [33]. By far, cytokines appear to be the mostly studied biological adjuvants aiming at boosting the efficacy of IBDV VP2 DNA vaccines. For example, chicken common γ-chain cytokine family members (IL-2, IL-7, IL-9, IL-15, and IL-21) [13,30] have been extensively tested for their adjuvanticity for IBDV VP2 DNA vaccine.
Similar to its mammalian counterparts, chicken IL-2 can induce spleen T cell proliferation, increase the activity of NK cells [34], and enhance the immunogenicity of IBDV VP2 DNA vaccine [13,30]. In contrast, IL-7 is shown to stimulate B cell and T cell differentiation, proliferation, maturation, and maintenance [23,24]. Due to its potent immunostimulating property, IL-7 was used to treat immunosuppression diseases [35,36]. Additionally, IL-7 can also enhance vaccine immunogenicity by functioning as an adjuvant [37,38]. Our previous work demonstrated that the canine IL-7 gene can enhance the immunogenicity of canine parvovirus VP2 DNA vaccine [39,40]. Whether chicken IL-7 (chIL-7) gene behaves similarly has remained to be elucidated. Recently, we cloned the chIL-7 and characterized its biological function, and found that chIL-7 could induce B cell and T cell activation [41]. We also found that chIL-7 gene vector and recombinant chIL-7 could enhance the immunogenicities of IBDV VP2 DNA vaccine or an inactivated IBDV vaccine, respectively [17,18].
To further enhance the immunogenicity and thereby increase the protective efficacy of IBDV VP2 DNA vaccine, we used chIL-2 and chIL-7 bicistronic gene vector to further improve immune efficacy of the VP2 DNA vaccine. As expected, chIL-2 and chIL-7 mutually enhanced the immunogenicity of IBDV VP2 DNA vaccine, and the chIL-2/chIL-7 bicistronic gene vector also significantly increased VP2 DNA vaccine-induced humoral and cellular immune responses relative to those of chIL-2 or chIL-7 vector alone. Protection from infection was increased from~80% in the chIL-2 or chIL-7 vector alone groups to 93% seen in the IL-2/IL-7 bicistronic vector group. These results shed light on the future optimization of DNA vaccine efficacy in the chicken IBD prevention and control.

Conclusions
In summary, in this study we have demonstrated that chIL-2 and chIL-7 can mutually enhance the immunogenicity of the IBDV VP2 DNA vaccine. The IL-2/IL-7 bicistronic vector significantly increases chickens' immune response elicited by the IBDV VP2 DNA vaccine, thereby resulting in significantly better protection against virulent IBDV challenge. This study opens up new ways (i.e., cytokines co-administration) to boost DNA vaccine for chicken IBD.