Influenza Inactive Virus Vaccine with the Fusion Peptide (rTα1-BP5) Enhances Protection Against Influenza Through Humoral and Cell-Mediated Immunity

Thymosin α1 (Tα1) and Bursopentin (BP5) are both immunopotentiators. To explore whether the thymosin α1-Bursopentin (rTα1-BP5) is an adjuvant or not, we cloned the gene of Tα1-BP5 and provided evidence that the gene of Tα1-BP5 in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. To evaluate the immune adjuvant properties of rTα1-BP5, chickens were immunized with rTα1-BP5 combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, Avian influenza virus (AIV)-neutralizing antibodies, levels of Th1-type cytokines (gamma interferon (IFN-γ)) and Th2-type cytokines (interleukin 4 (IL-4)), and lymphocyte proliferation responses were determined. We found that rTα1-BP5 enhanced HI antibody and antigen-specific immunoglobulin G (IgG) antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte. Furthermore, virus challenge experiments confirmed that rTα1-BP5 contributed to the inhibition replication of the virus (H9N2 AIV (A/chicken/ Jiangsu/NJ07/05) from chicken lungs. Altogether, these findings suggest that rTα1-BP5 is a novel adjuvant suitable for H9N2 avian influenza vaccine.


Introduction
Avian influenza virus (AIV) is an enveloped virus that belongs to the Orthomyxoviridae family and has an eight-segmented, single-stranded, negative-sense RNA genome.Among the proteins encoded by the genome, there are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [1].AIVs are classified into subtypes according to the combination of 16 HA and nine NA molecules.Among the many subtypes of AIV, H9N2 is thought to have originated from shorebirds and gulls, and has rapidly spread to become one of the most prevalent diseases in domestic poultry worldwide.It also causes serious economic loss in the poultry industry [2] (see Table 1).In domestic avian species in North America, H9N2 influenza viruses occur primarily in turkeys, occasionally in quail, and rarely if ever in chickens.The H9N2 virus subtype was first isolated from turkeys in 1966 [3], when the virus was associated with mild respiratory disease.
In Asia, long-term surveillance in live poultry markets in Hong Kong from 1975 to 1985 detected H9N2 influenza viruses in apparently healthy ducks but not in chickens [4].Since the early 1990s, H9N2 influenza viruses have become widespread in domestic chickens in Asia [5].Among the avian influenza A virus subtypes, H9N2 viruses have the potential to cause an influenza pandemic because they are widely prevalent in avian species in Asia and have demonstrated the ability to infect humans [6].In April 1999, two World Health Organization reference laboratories independently confirmed the isolation of avian influenza A (H9N2) viruses for the first time in humans [7].
The best protection against influenza virus infection remains effective vaccination [8].Inactivated vaccines have been undergoing clinical trials as pandemic vaccine candidates, and it has been shown that inactivated vaccines elicit strong humoral responses; however, it is commonly accepted that no adequate mucosal or cellular immunity is achieved [9].Adjuvants are able to improve the quantity and quality of innate immune responses by enhancing their speed and duration, and by inducing adequate adaptive immunity [10].To improve methods for influenza vaccine production, the current strategy of many investigators is to increase the efficacy of pandemic influenza vaccines by the addition of adjuvants to boost immune responses, such as aluminum salts, MF59, IC31 ® , and chitosan [11][12][13][14].
A defined peptide sequence able to stimulate specific immune cell subsets has the potential to act as an adjuvant for a variety of immunogens.The thymus is an important central immune organ for T-lymphocyte differentiation and maturation [15].It is capable of secreting many peptides with the functions of regulating the development of different phenotypic markers and lymphocyte [16].Thymosin alpha 1 (Tα1), an immunomodulatory peptide consisting of 28 amino acid residues, was isolated originally from calf thymus [17].As a biological response modifier (BRM), Tα1 has multiple biological activities in the immune system.It can promote specific lymphocyte functions, stimulate the production of lymphokines such as gamma interferon (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 2 (IL-2), macrophage migration inhibitory factor (MIF), and precursor stem cell into the CD4+/CD8+ T cells, increase Tcell proliferation, differentiation and maturation, and so on [18,19].Furthermore, it has the activities of antitumor and protection against oxidative damage [20].Consequently, Tα1 is widely used in clinic treating various diseases including immunodeficiency diseases, severe sepsis, and systemic infectious disorder [21].
The bursa of Fabricius (BF) is a primary humoral immune organ unique to birds and is the site of B-lymphocyte development and differentiation.The tripeptidebursin (LysHisGlyNH2) has been described as an endogenous B-cell stimulant or differentiation factor [22].BS and bursinlike peptide T-X-N-L-K-H-G significantly enhance the JEV subtype vaccine-induced immune response in immunized mice [23].Bursin-like epitope peptide (BLP) is one of bursin-like peptides and enhances immune responses in mice immunized with inactivated H9N2 avian influenza vaccine [24].Our previous study has been reported that Bursopentin (BP5) is a small peptide separated from BF, which amino acid sequence is CKDVY.We found that BP5 not only promotes T-cell and B-cell proliferation, enhances humoral immunity and cellular immunity but also balances Th1 and Th2 immune responses [25,26].
Although both Tα1 and BP5 have the potent adjuvant effects, this study designed and synthesized Tα1-BP5 fusion gene according to the preferential codons of Escherichia coli, fused with prokaryotic expression vector pET-32a, and then transferred into E. coli BL21 to induce its expression.Then, we tested whether rTα1-BP5 could enhance immune responses in chicken upon vaccination with H9N2 avian influenza whole-inactivated virus (WIV).Shanghai Science Peptide Biological Technology Co., Ltd.(Shanghai, China), and the purity was over 95%.

Chicken embryos, animals, and vaccines
Specific pathogen-free (SPF) Roman chicken and chicken embryos were obtained from the Henan Experimental Animal Research Center.Avian influenza virus A/Chicken/Jiangsu/ NJ08/05(H9N2) (10 7 TCID 50 /0.1 mL) was inoculated into the allantoic cavities of 10-day-old SPF chicken embryos; the embryos that died within 24 h were discarded, and the allantoic fluids were harvested from the infected embryos at 48 h postinfection and inactivated by treatment with 0.2% formalin.The inactivated virus was emulsified with mineral oil to make an oilformulated inactivated H9N2 AIV vaccine.One dose of the vaccine contained 10 7 TCID 50 /0.1 mL, which was equal to it before inactivation.Procedure and test of inactivated vaccine were described according to OIE Terrestrial Manual 2012 [29].

Gene cloning and expression of the recombinant fusion peptide Tα1-BP5
Gene of the recombinant fusion peptide thymosin α1-Bursopentin (Tα1-BP5) was designed according to the preferential codons of E. coli and amplified by splicing overlap extension polymerase chain reaction (SOE-PCR) method [30].Sequences of the primers used for the synthetic Tα1-BP5 are as follows: F1: 5′-CCG GAA TTC AGC GAC GCT GCT GTT GAC ACT AGC AGC GAA ATC ACT ACTA AAG ACT TG-3′; F2: 5′-GTT CGG GGT GCTG CCG CCG CCG CCG TTT TCA GCT TCT TCA ACA ACT TCT TTT TTT TCT TTC AAG TCT TTA GTA GT-3′; and F3: 5′-GGC GGC GGC GGC AGC TGC AAA AAT GTG TAT TAA GTC GAC TCG-3′, with EcoR I and SaI I site (underlined).The genes of Tα1 and BP5 were connected with the gene of the GGGGS linker to form the Tα1-BP5 fusion gene.And then, the amplified DNA fragment was digested by EcoR I and SaI I, and then ligated into the expression vector pET32a.
The ligation mix was transformed into competent DH5α cells and the single bacterial colony was selected by overnight growth on Luria broth (LB) agar plates containing 100-μg/mL ampicillin.The obtained recombinant plasmid pET32a-Tα1-BP5 was confirmed by restriction endonuclease digestion and DNA sequencing.The pET32a-Tα1-BP5 plasmid was transformed into E. coli BL21 (DE3) for inducing expression.The expression products were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).And the recombinant proteins were purified on a Ni-affinity chromatography column (Amersham Bioscience HiTrap chelating HP 5 mL × one column) following the manufacturer's instructions.

Activity testing of fusion peptide Tα1-BP5 (rTα1-BP5) in vitro
Thymus and spleens from 4 to 6-week-BALB/c mice with (20 ± 2) g were collected aseptically, put them at 200-mesh stainless screen mesh cells, and gently minced into single cell suspension with a syringe followed by adding Hank's solution.The red blood cells were removed by centrifugation at 500 rpm for 5 min.The supernatant was centrifuged at 500 rpm for 5 min.
The obtained pellet was washed with Hank's solution twice.The density of lymphocytes was adjusted to around 5 × 10 6 cells/mL using RPMI-1640 medium containing 10% FBS.ConA and PMA were added into thymic lymphocytes and splenic lymphocytes to make the concentra-tions reach 5 μg/mL and 300 ng/mL, respectively.The two kinds of solutions were subpackaged into a 96-well plate with 100 μL/well, respectively, and three parallel samples were set for each well.The plates were incubated in CO 2 incubator at 37°C for 6 h, followed by adding 100-μL/ well rTα1-BP5 (affinity chromatography purified through Ni column) with different concentrations (1.25, 2.5, 5.0, 10.0, and 20.0 μg/mL) and continued culturing for 72 h.Control groups (phosphate-buffered saline (PBS), 10.0 μg/mL thioredoxin, 10.0 μg/mL Tα1, and 10.0 μg/mL BP5) were used following the same procedures.MTT method was used to test the effect of rTα1-BP5 effect on thymic and splenic lymphocytes proliferation.Relative ratio of cell proliferation (%) = (experimental group OD 570 /control group OD 570 ) × 100% [31,32].

Detection of antibodies in serum
Chicken (n = 5 per group) sera were collected on 7 and 21 days after the first immunization.Serum antibody (HI and antigen-specific antibodies) titers were determined using standard HI microliter and enzyme-linked immunosorbent assay (ELISA) as described [25,33].Briefly, to detect the HI titers in chicken serum, sera were inactivated by incubation for 30 min at 56°C and serially diluted twofold in PBS, then transferred in duplicate to 96-well round-bottomed Steps Forwards in Diagnosing and Controlling Influenza plates.Standard avian influenza virus (A/Chicken/Shandong/6/96(H9N2)) antigen with four units was then added to each diluted serum samples in a volume of 50 μL, and followed by an equal volume of 0.5% chicken erythrocyte suspensions.The mixture was incubated for 1 h at room temperature before the results were read.The HI titers were defined as the highest serum dilution capable of preventing hemagglutination.
To evaluate the antigen-specific antibodies titers, ELISA plates were coated with 10-μg/mL recombinant influenza HA protein (expressed in E. coli BL21) and blocked with 1% bovine serum albumin (BSA) for 2 h at 37°C.Aliquots of diluted chicken sera were added to the plates, which were then incubated overnight, washed, and incubated with HRP-conjugated goat antichicken IgG.Finally, TMB was added and the reaction was stopped by the addition of 2N H 2 SO 4 and the absorbance was read at OD 450 .Each serum sample was repeated in quintuplicate.
The results were plotted as OD versus dilution (log scale).Titers at half maximal OD were determined by linear interpolation [34].

Cytokine assays
On 7 and 21 days after the first immunization, the serum levels of Th1-type cytokine (IFN-γ) in chickens were determined using commercial Chicken cytokines gamma interferon ELISA kits (Cusabio Biotech, MD, USA), whereas Th2-type cytokine (IL-4) was determined with another commercial Chicken cytokines interleukin 4 ELISA kits (Cusabio Biotech, USA).The procedure followed the manufacturer's instructions.

Lymphocyte proliferation response
To detect changes in cellular immunity, lymphocyte proliferation response was performed.Thymus and bursa of Fabricius were collected from immunized chickens at 7 and 21 days after the first immunization.The thymus and BF lymphocytes were isolated and maintained in 1640 medium supplemented with 10% FBS at 37°C with 5% CO 2 .The thymus lymphocytes (5 × 10 6 cells/mL) were seeded in a 96-well plate and incubated with 50 μL of ConA (40 μg/mL) at 40°C/ 5% CO 2 for 48 h, whereas the BF lymphocytes (5 × 10 6 cells/mL) were treated with 50 μL of PMA (1 μg/mL) in a 96-well plate at 40°C/5% CO 2 for 24 h.Then, the lymphocyte proliferation assay was performed using a standard MTT method as described previously [36,37].Then, the plate was incubated with 10 μL of 5 mg/mL MTT for 3 h.Finally, 100 μL of 10% (w/v) SDS in 0.01 M HCl was added into the plate and allowed to incubate for 2 h.A spectrophotometric measurement was taken at A 570 .

Virus challenge experiment
Two weeks after the second vaccination, chickens (n = 15 per group) were intranasally challenged with 2.5 × 10 6 TCID 50 avian influenza virus A/chicken/Jiangsu/JS-1/2002(H9N2) in 100 μL PBS.Five chickens per group were humanely sacrificed at 3, 5, and 7 days after virus challenge and the viral titers in their lungs assessed by plaque formation assays using MDCK cells as described [35].

Statistical analysis
Statistical analyses were performed using unpaired t-tests or one-way analysis of variance (ANOVA) F-statistics followed by GraphPad Prism 6 software.Data are presented as the mean ± standard deviation (SD).Turkey multiple comparison tests were used to assess differences among the five experimental groups, with differences being considered significant at P < 0.05 or P < 0.01.

Expression of the recombinant fusion peptide Tα1-BP5
The gene of Tα1-BP5 was amplified by SOE-PCR with the primers F1, F2, and F3.The PCR products were identified by electrophoresis, and then about 114bp strip was observed.The recombinant plasmid was extracted and identified with Hind III enzyme.The results showed that the recombinant plasmid was not digested by Hind III, indicating that the recombinant plasmid had deleted the Hind III restriction site (Figure 2A).Sequencing result showed that the gene of the recombinant fusion peptide Tα1-BP5 was inserted into pET32a vector, and it was consistent to the expected size (Table 3), which was suggested that the recombinant expression vector was constructed successfully, and it was named pET32a-Tα1-BP5.Then, pET32a-Tα1-BP5 was transferred into E. coli BL21 (DE3) for its expression, and the expressed products were detected by using SDS-PAGE.The result showed that Tα1-BP5 was expressed Steps Forwards in Diagnosing and Controlling Influenza and purified with the molecular weight of 31.4 kDa (Figure 2B), which is consistent with its predicted molecular weight.

Activity of rTα1-BP5 in vitro
The expressed product of TBP5 recombinant bacteria was affinity chromatography purified through protein Ni column and quantified through spectrophotometer.MTT method was used to test the effect of rTα1-BP5 on the proliferation of mouse thymic and splenic lymphocytes.
The results showed that all rTα1-BP5 with different concentrations (1.25, 2.5, 5.0, 10.0, and 20.0 μg/mL) could promote the proliferation of thymic and splenic lymphocytes compared to PBS group.rTα1-BP5 could stimulate thymic and splenic lymphocytes proliferation stronger than TP5 and BP5.The differences were significant (P < 0.05) on the concentrations of 5.0 and 20.0 μg/mL, and the differences were more significant (P < 0.01) on the concentrations of 10.0 μg/mL (Figure 3A and B).All these data demonstrated that rTα1-BP5 could promote the proliferation of mouse thymic T lymphocytes and splenic B lymphocytes.

rTα1-BP5 stimulates significant antigen-specific immune responses
To determine antigen-specific immune responses to immunization, chickens were immunized two times, then sera were taken on days 7 and 21 after the first immunization and detected for HI and anti-HA antibody titers.HI antibody titers of chickens immunized with inactivated vaccine, Tα1 combined with H9N2 WIV and BP5 combined with H9N2 WIV increased significantly compared with chickens immunized with the H9N2 WIV alone at days 7 and 21 (P < 0.05).However, HI antibody titers in chickens immunized with rTα1-BP5 combined with H9N2 WIV were significantly higher than in chicken immunized with BP5 combined with H9N2 WIV at days 7 and 21 (P < 0.05) (Figure 4A).Anti-HA IgG antibody was observed in immunized chickens on days 7 and 21 after the first immunization.rTα1-BP5 enhanced the secretion of IgG antibody on day 7 after the first immunization, and the effect was greater than that induced by H9N2 AIV vaccine, Tα1 combined with H9N2 WIV and BP5 combined with H9N2 WIV.On day 21 after the first immunization, BP5 significantly enhanced IgG antibody secretion levels compared with that induced by H9N2 vaccine, Tα1 combined with H9N2 WIV (P < 0.05), while the effect of rTα1-BP5 was the greatest than the other groups (P < 0.01) (Figure 4B).These results suggested that rTα1-BP5 stimulates significant antigen-specific immune responses.

rTα1-BP5 promoted the production of AIV-neutralizing antibody
To assess whether rTα1-BP5 can effectively enhance virus-neutralizing antibodies, chicken sera were collected on days 7 and 21 after the first immunization and the titers of AIV-neutralizing antibody were assessed.The result showed that the titers of neutralizing antibody of chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine were higher than that in chickens immunized with H9N2 WIV alone on day 7, while it was higher in chickens immunized with Tα1-BP5 plus H9N2 WIV than that of other groups.Consistent with this, AIV-neutralizing antibody titers of chicken injected with rTα1-BP5 plus H9N2 WIV were the highest on day 21 (Table 4).These results indicated that rTα1-BP5 significantly stimulates the production of AIV-neutralizing antibodies.a Chickens were vaccinated on days 0 (first boost) and 14 (second boost).Chicken sera (n = 5) were collected on days 7 and 21, and plaque-reducing neutralizing antibody titers were determined.The 50% plaque-reducing neutralizing titer (PRNT 50 ) was reported as the geometrical reciprocal of the serum dilution resulting in a 50% reduction in plaques.The data presented are means ± SD of results from five replicates.*, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
Table 4. Titers of plaque-reducing neutralizing antibody in groups of chicken.

rTα1-BP5 increases the production of both Th1-and Th2-type cytokines
We then examined the levels of Th1 (IFN-γ) and Th2 (IL-4) cytokines from immunized chickens.Compared with stimulation with H9N2 WIV alone, both IFN-γ and IL-4 secretion were remarkably increased after immunization with inactivated H9N2 AIV vaccine, Tα1 plus H9N2 WIV and BP5 plus H9N2 WIV at days 7 and 21, and the highest level of IFN-γ secretion was observed in the vaccination group with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 5A  and B).Taken together, the results suggested that rTα1-BP5 promoted the secretion of both Th1 and Th2 cytokines.Steps Forwards in Diagnosing and Controlling Influenza

rTα1-BP5 significantly enhances T-and B-lymphocyte proliferation
To investigate the effects of rTα1-BP5 on T-and B-lymphocyte proliferation, thymus and BF were collected from chickens immunized with rTα1-BP5 plus H9N2 WIV.T-lymphocyte proliferation responses of chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine were enhanced at 7 days compared with chickens immunized with H9N2 WIV alone (P < 0.05), whereas it was higher for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01).On day 21, T-lymphocyte proliferation responses of chickens immunized with Tα1 plus H9N2 WIV and H9N2 AIV vaccine were higher than chickens immunized with H9N2 WIV alone (P < 0.05), while it was highest for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 6A).Similarly, B-lymphocyte proliferation responses of chickens immunized with BP5 plus H9N2 WIV and H9N2 AIV vaccine were enhanced at 7 and 21 days compared with chickens immunized with H9N2 WIV alone (P < 0.05), whereas, it was highest for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 6B).The data indicated that rTα1-BP5 promoted T-and B-lymphocyte proliferative responses.

rTα1-BP5 significantly promotes immune protection against H9N2 AIV challenge
To evaluate whether rTα1-BP5 promotes immune protection against H9N2 AIV infection, viral titers in chicken lungs were evaluated at 3, 5, and 7 days after viral challenge by plaque formation assays.Chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine showed significant virus removal from the lungs at 3, 5, and 7 days after challenge compared with H9N2 WIV groups (P < 0.05), while the viral titers of lungs from chicken immunized with rTα1-BP5 plus H9N2 WIV were significantly lower than that in the H9N2 AIV vaccine group (P < 0.01).Moreover, chickens immunized with rTα1-BP5 plus H9N2 WIV had almost no detectable virus particles in the lungs 7 days after challenge (Figure 7A-C).The data indicated that rTα1-BP5 significantly promoted immune protection against H9N2 AIV challenge.

Discussion
In the event of an influenza pandemic, vaccination is one of the most effective ways of intervention in terms of reducing cost, disease, and even death.Appropriate adjuvant can enhance the immunogenicity of the vaccine and improve the immune responses [38,39].However, most of the adjuvants used in conjugation with antigen have unacceptable levels of side effects, only a few of them are used clinically [40].Thus, we need to find new and optimal adjuvant candidates for vaccine.In recent years, some small peptide immunostimulants were reported in use for vaccine adjuvants [41][42][43].Both Tα1 and BP5 are associated with immune regulation.Previous studies showed that both Tα1 and BP5 had high potential as an adjuvant for vaccines [26,44].
In this study, the fusion peptide of rTα1-BP5 was designed and synthesized, and to investigate it as an adjuvant for inducing immune responses in chickens upon vaccination with inactivated H9N2 avian influenza virus (WIV).An effective adjuvant should be able to enhance the levels of both humoral and cell-mediated immunity.To investigate the effect of rTα1-BP5 on humoral responses, chickens were immunized with H9N2 WIV combined with Tα1-BP5, and then titers of HI antibody, antigen-specific antibodies, and AIV-neutralizing antibodies were assessed.Then, we found that rTα1-BP5 significantly enhanced HI antibody and antigen-specific IgG antibodies titers, promoted the secretion of AIV-neutralizing antibodies, which suggested that rTα1-BP5 enhanced the levels of humoral immune responses in chickens when it was coimmunized with H9N2 WIV.
In addition to humoral immune responses, cellular immunity also plays an important role in fighting influenza virus infections [45].The levels of Th1-and Th2-type cytokines are important references to measure cellular immunity.And lymphocyte homeostasis is required for the maintenance of normal immune function [46].Th1-type cytokines mainly include IL-2, TNFα, and IFN-γ, whereas Th2-type cytokines include IL-4, IL-5, and IL-10 [47].Our study though analyzed the production of Th1 (IFN-γ)-and Th2 (IL-4)-type cytokines, and T-and B-lymphocytes proliferation in vaccinated chickens post immunization to evaluate the cell-mediated immunity.The results suggested that rTα1-BP5 promoted the secretion of both Th1 and Th2 cytokines and T-and B-lymphocyte proliferative responses.Overall, this study found that rTα1-BP5 not only enhanced the humoral immune responses but also promoted the cellmediated immune responses, and it had the potential to use as an adjuvant.
To further evaluate the influence of rTα1-BP5 as an adjuvant on the immunity protection provided by H9N2 AIV vaccine against AIV infection, chickens were intramuscularly challenged with H9N2 AIV (A/chicken/Jiangsu/JS-1/2002) on day 28 post immunization.After 3 days post challenge, the PBS group chickens that received the challenge virus were mildly depressed.No other clinical signs were observed in that group or any of the other groups, which is typical of low-pathogenicity AIV in chickens [48,49].At 7 days post challenge, only the PBS-challenged group had mild, grossly detectable lesions in both the respiratory and gastrointestinal tract.And we found that the viral titers of lungs from chicken immunized with rTα1-BP5 plus H9N2 WIV were significantly lower than all the other groups at 3 days.Chickens immunized with rTα1-BP5 plus H9N2 WIV had almost no detectable virus particles in the Steps Forwards in Diagnosing and Controlling Influenza lungs at 7 days after challenge.Our data indicated that rTα1-BP5 could effectively inhibit the replication of H9N2 AIV in chickens and promote virus clearance in the lungs of chickens.Thus, rTα1-BP5 had the potential to be used in vaccine formulations to provide improved protection against H9N2 AIV infection in poultry.
In summary, this study demonstrated that inactivated H9N2 AIV vaccine with Tα1-BP5 as an adjuvant enhanced strong immune responses at both humoral and cellular levels against AIV infection in chickens.These data may provide a novel insight to find new adjuvant in vaccines.

Figure 1 .
Figure 1.Experimental scheme of immunization, sample collection, and challenge.

Figure 3 .
Figure 3.The effects of rTα1-BP5 on the proliferation of thymic lymphocytes (A) or splenic lymphocytes (B) from immunized mice.The data presented are of five replicates.*, P < 0.05, compared with mice immunized with PBS, and **, P < 0.01, compared with mice immunized with BP5 or Tα1.

Figure 4 .
Figure 4. Effect of rTα1-BP5 to H9N2 AIV vaccination on antigen-specific HI titers and anti-HA IgG antibodies.Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the first immunization, and the serum HI titers (A) and IgG titers (B) were analyzed by HI assay and ELISA, respectively.The data presented are means ± SD of results from five replicates.*, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.

Figure 5 .
Figure 5.Effect of rTα1-BP5 to H9N2 AIV vaccination on cytokine production in chicken sera.Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the first immunization.Cytokine release was measured by using commercial chicken cytokines gamma interferon (IFN-γ) and interleukin 4 (IL-4) ELISA kits.The data presented are means ± SD of results from five replicates.*, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.

Figure 6 .
Figure 6.rTα1-BP5 significantly stimulates chicken T-and B-lymphocyte proliferation.Chickens were immunized two times, and chicken thymus and bursa of Fabricius were collected on days 7 and 21 after the first immunization.T-(A) and B (B)-lymphocyte proliferation assays were evaluated by MTT method.The data presented are means ± SD of results from five replicates.*, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.

Figure 7 .
Figure 7. AIV-viral titers of lung in chickens.Lung samples from individual chicken in each group (n = 5) were collected on days 3, 5, and 7 post challenge with 2.5 × 106 TCID 50 avian influenza virus (A/chicken/Jiangsu/JS-1/2002(H9N2)).Each lung sample was diluted to 1 mL with 1640 media.The titers are presented as pfu per mL.The data presented are means ± SD of results from five replicates.*, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.

Table 1 .
Abbreviations for full name.

Table 2 .
Animal groups and the experimental design.
a H9N2 WIV, inactivated H9N2 avian influenza whole-inactivated virus; H9N2 AIV vaccine, H9N2 avian influenza virus vaccine prepared with oil/water as an adjuvant.