Abbreviations for full name.
Abstract
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 Th1- and 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.
Keywords
- thymosin α1 (Tα1)
- Bursopentin (BP5)
- fusion peptide
- avian influenza vaccine
- adjuvant
1. Introduction
Avian influenza virus (AIV) is an enveloped virus that belongs to the
Abbreviation | Full name |
---|---|
Tα1 | Thymosin α1 |
BP5 | Bursopentin |
rTα1-BP5 | Thymosin α1-Bursopentin |
WIV | Whole-inactivated virus |
AIV | Avian influenza virus |
HA | Hemagglutinin |
NA | Neuraminidase |
BRM | Biological response modifier |
MIF | Macrophage migration inhibitory factor |
BF | Bursa of Fabricius |
BLP | Bursin-like epitope peptide |
HI | Hemagglutination inhibition assay |
IFN-γ | Interferon-γ |
IL-4 | Interleukin-4 |
TNF-α | Tumor necrosis factor-α |
Th | T-helper type |
Th1 | T-helper type 1 |
Th2 | T-helper type 2 |
MDCK | Madin-Darby canine kidney |
FBS | Fetal bovine serum |
MTT | 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide |
IPTG | Isopropyl-β-d-thiogalactoside |
ConA | Concanavalin A |
PMA | Phorbol-12-myristate-13-acetate |
TMB | Tetramethyl benzidine |
HRP | Horseradish peroxidase |
SPF | Specific pathogen-free |
SOE-PCR | Splicing overlap extension PCR method |
TRX | Thioredoxin |
TCID50 | 50% tissue culture infective dose |
PBS | phosphate-buffered saline |
pfu | Plaque-forming unit |
OD | Optical density |
IgG | Immunoglobulin G |
ELISA | Enzyme-linked immunosorbent assay |
PRNT50 | 50% plaque-reducing neutralizing titer |
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 [11–14].
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 T-cell 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 bursin-like 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
2. Materials and methods
2.1. Plasmid, viruses, and reagents
pET-32a (+),
2.2. 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) (107 TCID50/0.1 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 oil-formulated inactivated H9N2 AIV vaccine. One dose of the vaccine contained 107 TCID50/0.1 mL, which was equal to it before inactivation. Procedure and test of inactivated vaccine were described according to OIE Terrestrial Manual 2012 [29].
2.3. 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
2.4. 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 × 106 cells/mL using RPMI-1640 medium containing 10% FBS. ConA and PMA were added into thymic lymphocytes and splenic lymphocytes to make the concentrations 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 CO2 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 OD570/control group OD570) × 100% [31, 32].
2.5. Immunization of chickens
All animal experiments were approved by the Henan University of Science and Technology Animal Care and Use Committee. Twenty-one-day-old SPF Roman chickens were randomly divided into six experimental groups of 25 chickens each and intramuscularly immunized two times on days 0 and 14 with (i) 100 μL PBS as a negative control, (ii) 100 μL H9N2 WIV (A/Chicken/Jiangsu/NJ08/05, 107 TCID50/0.1 mL), (iii) a mixture of 100 μL H9N2 WIV and Tα1 (50 μg), (iv) a mixture of 100 μL H9N2 WIV and BP5 (50 μg), (v) a mixture of 100 μL H9N2 WIV and rTα1-BP5 (50 μg), and (vi) 100 μL oil-formulated inactivated H9N2 AIV vaccine (A/Chicken/Jiangsu/NJ08/05, 107 TCID50/0.1 mL) as a positive control (Table 2).
Group | Vaccination on days 0 and 14a |
---|---|
1 | 100 μL PBS |
2 | 107 TCID50 H9N2 WIV |
3 | 107 TCID50 H9N2 WIV + 50 μg Tα1 |
4 | 107 TCID50 H9N2 WIV + 50 μg BP5 |
5 | 107 TCID50 H9N2 WIV + 50 μg rTα1-BP5 |
6 | 107 TCID50 H9N2 AIV vaccine |
The details of the animal experiment time points are shown in Figure 1.
2.6. Detection of antibodies in serum
Chicken (
To evaluate the antigen-specific antibodies titers, ELISA plates were coated with 10-µg/mL recombinant influenza HA protein (expressed in
2.7. Determination of AIV-neutralizing antibodies
Inactivated sera were incubated with 100 plaque-forming unit (pfu) of avian influenza virus (A/Chicken/Jiangsu/JS-1/2002(H9N2), and the titers of AIV-neutralizing antibodies determined as described [35].
2.8. 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.
2.9. 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% CO2. The thymus lymphocytes (5 × 106 cells/mL) were seeded in a 96-well plate and incubated with 50 μL of ConA (40 μg/mL) at 40°C/5% CO2 for 48 h, whereas the BF lymphocytes (5 × 106 cells/mL) were treated with 50 μL of PMA (1 μg/mL) in a 96-well plate at 40°C/5% CO2 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 A570.
2.10. Virus challenge experiment
Two weeks after the second vaccination, chickens (
2.11. Statistical analysis
Statistical analyses were performed using unpaired
3. Results
3.1. 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
Name | Amino acid sequence |
---|---|
Tα 1-BP5 |
Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly Gly Gly Gly Ser Cys Lys Asp Val Tyr |
3.2. 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 (
3.3. 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 (
3.4. 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.
Treatment | PRNT50a | |
---|---|---|
First boost | Second boost | |
PBS | – | – |
H9N2 WIV | 10 ± 0.38 | 19 ± 0.21 |
H9N2 WIV + Tα1 | 15 ± 0.30* | 22 ± 0.25* |
H9N2 WIV + BP5 | 18 ± 0.32* | 28 ± 0.43* |
H9N2 WIV + rTα1-BP5 | 22 ± 0.24** | 32 ± 0.19** |
Inactivated H9N2 AIV vaccine | 16 ± 0.15* | 25 ± 0.54** |
3.5. 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 (
3.6. 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 (
3.7. 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 (
4. 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 [41–43]. 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 co-immunized 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, TNF-α, 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 cell-mediated 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 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.
Acknowledgments
This work was supported by Grant no. 31101792 from the National Natural Science Foundation of China and no. 2012GGJS-077 from the Foundation for University Key Teacher by Higher Education of Henan Province.
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