DNA immunization against very virulent infectious bursal disease virus with VP2-4-3 gene and chicken IL-6 gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2-4-3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin-6 (ChIL-6) genes were constructed and designated as pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 respectively. Several DNA vaccination experiments were performed: 1-week-old chickens were intramuscularly injected with only plasmid pcDNA3-VP2, pALTER-MAX-VP2-4-3 or mixture with pALTER-MAX-ChIL-6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 three times conferred protection for 90% of chickens. Enzyme-linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER-MAX-ChIL-6 were higher than those immunized simply with plasmid pcDNA3-VP2 or pALTER-MAX-VP2-4-3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT-PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2-4-3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL-6 plasmid significantly increased the protection after challenge with the very virulent strain.     

CpG寡核苷酸对IBDV VP2基因真核表达质粒免疫增效作用

以传染性法氏囊病病毒(IBDV)VP2蛋白基因表达质粒DNA为免疫原，以CpG的寡核苷酸(CpG-0DN)为免疫佐剂，肌肉注射于14日龄SPF鸡，1周后加强免疫1次，2次免疫后15d和21d分别测定血清ELISA抗体效价，并于免疫后21d用IBDV99儿强毒株攻毒和进行病理学观察。结果显示，(1)VP2基因重组质粒DNA与CpG共同免疫组的ELISA抗体水平明显高于VP2重组质粒免疫组；(2)IBD弱毒苗与VP2重组质粒免疫组抗体水平明显高于VP2重组质粒免疫组，且比VP2基因重组质粒DNA与CpG共同免疫组略高；(3)VP2基因重组质粒DNA与CpG共同免疫组及IBD弱毒苗与VP2重组质粒免疫组可明显降低IBDV强毒攻击后引起的急性发病率和死亡率。由此表明，CpG寡核苷酸对IBDV VP2蛋白基因真核表达质粒免疫具有明显增强作用，有很大的应用前景。     

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-��

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.     

鸡传染性支气管炎病毒S1基因免疫对鸡的保护作用

将鸡传染性支气管炎病毒肾型 Ｔ 株 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 连接于ｐｃ Ｄ Ｎ Ａ３ 的 Ｈｉｎｄ Ｉ Ｉ Ｉ与 Ｂａ ｍ Ｈ Ｉ位点之间构建含有 Ｃ Ｍ Ｖ 启动子及 Ｂ Ｇ Ｈｐｏｌｙ Ａ 信号序列的鸡传染性支气管炎病毒 Ｓ１ 蛋白真核表达质粒。实验证明, Ｓ Ｐ Ｆ 鸡肌注免疫后血清 Ｉｇ Ｇ 抗体逐渐升高,至第３５ 日龄左右达到高峰,攻毒后血清 Ｉｇ Ｇ 抗体先下降而后升高,血清 Ｉｇ Ｇ 抗体升高幅度不及 Ｉ Ｂ 油苗免疫组。质粒 Ｄ Ｎ Ａ 免疫鸡攻毒后有４０ ％ 的鸡可耐过强毒的攻击,说明 Ｓ１ 基因在体内获得了表达并使鸡产生了一定的免疫力。     

传染性法氏囊病超强病毒株VP2基因原核表达及其免疫原性检测

为检测传染性法氏囊病超强毒株(vvIBDV)重组VP2蛋白的免疫原性,本研究利用RT-PCR方法扩增vvIBDV的结构蛋白VP2基因,并将其克隆到表达载体pGEX-4T-3中,构建重组表达质粒pGEX-VP2。将其转化受体菌E.coli BL21(DE3)plysS,经IPTG诱导后,SDS-PAGE电泳和western blot分析表明,表达的重组蛋白约69 ku,并以包涵体形式存在。表达的重组蛋白经纯化后,免疫6周龄BALB/c小鼠制备免疫血清,ELISA分析表明制备的血清效价在1∶5 120以上,表明vvIBDV VP2具有良好的免疫原性,为建立vvIBDV的ELISA检测方法提供了试验依据。     

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

鸡传染性法氏囊病（IBD）是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒（HVT）活载体疫苗,本研究构建了表达鸡传染性法氏囊病病毒（IBDV）保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体,获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下,连接于入门质粒pENTR,构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应,构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞（CEF）,拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测,并绘制重组病毒体外生长曲线,分析其体外复制特性。结果表明,重组病毒rHVT-VP2能够稳定表达VP2蛋白,rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体,并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础,对IBD的防控具有重要意义。     

减毒沙门氏菌为载体传递DNA疫苗诱导对新城疫病毒的免疫保护

探讨了以减毒鼠伤寒沙门氏茵为栽体传递新城疫病毒DNA疫苗的安全性、免疫原性和可行性。将含新城疫病毒(NDV)F48E9株融合蛋白(F)基因的真核表达质粒pcDNA3-F的重组减毒鼠伤寒沙门氏菌ZJ111株(ZJ111／pcD-NA3一F菌株)，以10^8CFU进行首免，2周后二免，三免后4周攻击强毒株F48E9，观察其安全性和免疫原性，同时设只含空载体pcDNA3的ZJ111／pcDNA3菌株对照及口服PBS对照。结果表明：重组ZJ111／pcDNA3-F菌株具有良好的安全性。对强毒株攻击的保护率达64．7％。重组ZJ111／pcDNA3-F菌株不仅能诱导雏鸡产生NDVELISA抗体，而且诱导产生的法氏囊B淋巴细胞和胸腺T淋巴细胞增殖反应显著高于ZJ111／pcDNA3时照组。这些结果提示，减毒沙门氏菌为载体不仅可直接将NDVF基因呈递给鸡体细胞进行表达，产生抗NDV的体液免疫，而且还可诱导细胞免疫应答。     

Induction of protective immunity in chickens immunised with plasmid DNA encoding infectious bursal disease virus antigens

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). The vp2 gene of IBDV strains GP40 and D78, and the vp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA from E. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.     

Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana

Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens-mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.     

DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens

The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p < 0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.     

Soluble Expression,Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus

To obtain the capsid VP2 with high quality, VP2 gene of very virulent infectious bursal disease virus (vvIBDV) Gx was cloned and inserted into pCold-Ⅰ and the prokaryotic expression plasmid pCold-Ⅰ-GxVP2 was constructed. In engineering bacteria Transetta(DE3), the induction conditions of protein VP2 expression were optimized.With affinity chromatography and gel filtration, protein VP2 was purified. With the monoclonal antibody directed Western blotting, protein VP2 was identified. Using SPF chicken, immunocompetence of VP2 was evaluated. The results showed that the dissoluble protein VP2 was expressed successfully in Transetta(DE3) in cold-shock conditions; Protein VP2 was purified and the concentration was 542 μg/mL; The purified protein VP2 not only reacted with the monoclonal antibody against protein VP2, but also induced specific immune response in immunized chickens. In general, with 15℃ of cold-shock condition, 120 r/min of shaking culture, 1 mmol/L of IPTG,inducting for 24 h, soluble capsid VP2 of IBDV with immunocompetence was successfully expressed and purified.The preparation of highly purified, soluble capsid protein with functional activity laid the foundation for further researches on the pathogenic mechanism.     

传染性法氏囊病病毒超强毒株衣壳蛋白的可溶性表达、纯化与活性分析

为获得高质量的病毒衣壳蛋白VP2,本研究克隆了传染性法氏囊病病毒（infectious bursal disease virus,IBDV）超强毒株Gx的VP2基因,并亚克隆至载体pCold-Ⅰ,进而获得重组原核表达质粒pCold-Ⅰ-GxVP2,在Transetta（DE3）工程菌中优化条件进行IBDV衣壳蛋白VP2的可溶性表达,运用Ni-NTA亲和层析和凝胶过滤两种技术串联的方法进行蛋白纯化,运用单克隆抗体介导的Western blotting技术鉴定纯化蛋白的特异性,免疫SPF鸡鉴定纯化蛋白的免疫活性。结果显示,在冷休克条件下,IBDV衣壳蛋白VP2在Transetta（DE3）工程菌中实现了可溶性表达;通过纯化获得了高纯度的VP2,浓度为542 μg/mL;该蛋白不仅能与VP2单克隆抗体特异性反应,还能刺激鸡产生特异性免疫应答,具有良好的免疫活性。本研究在IPTG为1 mmol/L,15℃、120 r/min,诱导时间为24 h的条件下,实现了有免疫活性的IBDV衣壳蛋白VP2的可溶性表达和纯化。高纯度、可溶、具有功能活性的衣壳蛋白的制备,为深入开展IBDV致病机制研究奠定了基础。     

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 microg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.     

Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV

In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.     

传染性法氏囊病超强毒Gx株的致弱研究

本研究成功将鸡传染性法氏囊病超强毒vvIBDVGx株通过SPF鸡胚的快速培育及鸡胚成纤维细胞传代致弱，揭示了vvIBDV从超强毒力向弱毒力转化过程中，其主要结构蛋白VP2基因核苷酸及推导的氨基酸序列的变化规律。对不同代次细胞毒进行了序列分析。发现细胞毒在第7代以前，VP2基因序列没有改变。与欧洲标准超强毒氨基酸同源性达100%；细胞毒第8代，有个别核苷酸发生了改变。但没有影响氨基酸序列；细胞毒第9代是变化复杂的过渡代；10代毒VP2基因与欧洲标准弱毒Cu-1氨基酸序列同源性达97%；以后的细胞适应毒至20代。其VP2基因序列不再改变。致病性实验表明原代毒对4周龄SPF鸡致死率为64%。细胞毒第5代的致死率为60%，而20代毒对鸡无致病性。在鸡体内连续传代6代不返强。     

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.     

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.     

ADV分离强毒株全基因突变重组质粒的构建、表达及其免疫原性的分析

为研制水貂阿留申病核酸疫苗,应用重叠延伸PCR技术去除ADV VP2基因中编码428~446位氨基酸的核苷酸序列,与pc DNA3.1(~+)载体连接,构建全基因突变重组质粒pc DNA3.1-ADV-428,在此基础上,截去编码487~501位氨基酸的核苷酸序列,构建全基因突变重组质粒pc DNA3.1-ADV-428-487。将构建的重组质粒经肌肉注射免疫小鼠,应用间接ELISA法检测接种后14、28、42、56 d抗ADV抗体水平;流式细胞术检测接种后第42天小鼠脾细胞CD3~+、CD4~+和CD8~+T淋巴细胞亚群。结果显示,小鼠接种质粒后CD3~+、CD4~+和CD8~+T淋巴细胞亚群数量均明显增加,第42天抗ADV抗体水平达峰值。本试验通过对ADV全基因突变重组质粒的免疫原性进行分析,为水貂阿留申病核酸疫苗的研制提供了参考。