人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

目的 研发高效广谱的人高致病性禽流感病毒H5N1实验疫苗.方法 首先构建了含H5N1(安徽株)结构基因[血凝素(HA)、神经氨酸酶(NA)、基质蛋白M1与M2]的两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒(rTTV天坛株)疫苗,采用不同剂量(104 PFU或107PFU)或组合(疫苗单独或联合)方式于0、4周二次免疫BALB/c小鼠,初步比较分析抗原特异的体液(HA血凝抑制抗体、NA特异性抗体、中和抗体)与细胞免疫应答(IFN-γ ELISPOT)特点.结果 重组痘苗病毒疫苗可有效表达H5N1靶抗原;高剂量组的重组痘苗病毒疫苗可快速激发较强的针对各个抗原的抗体与针对血凝素与神经氨酸酶蛋白的细胞免疫应答,含血凝素蛋白的重组痘苗病毒疫苗亦可诱导明显的中和抗体;但各组重组痘苗病毒疫苗所激发的针对基质蛋白(M1,M2)的细胞免疫应答均较弱;两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒疫苗联合应用所激发的针对基质蛋白2(M2)的体液免疫应答明显强于单双顺反子(HAop/M2)疫苗单独应用.结论 本研究中制备的各组重组痘苗病毒疫苗可诱导多个抗原特异的体液与细胞免疫应答,该研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础.     

重组H9N2禽流感病毒的构建及其在不同细胞中的复制特性

目的利用反向遗传学技术构建具有感染性的H9N2亚型禽流感病毒,并揭示其在不同细胞中的复制特性。方法以A/Quail/Hong Kong/G1/97(H9N2)禽流感病毒基因组RNA为模板,用RT-PCR技术获得该病毒的8条完整基因片段,并分别将它们插入到p HW2000载体上,最终在293T细胞中包装产生重组H9N2病毒。并将重组的H9N2病毒感染不同细胞,观察病毒在不同细胞上的增殖状况来揭示该病毒在不同细胞上的复制特性。结果应用流感病毒8质粒反向遗传学技术,成功获得重组病毒,并揭示了该毒株在不同细胞(A549,MDCK,Vero)中的复制特性,发现A549人肺腺癌细胞更适宜H9N2病毒的正常复制。此外还分析总结了该毒株的与病毒毒力相关的重要位点特征。结论成功建立了H9N2禽流感病毒的反向遗传系统,该系统的建立为在分子水平研究H9N2病毒以及制备H9N2禽流感疫苗提供了依据。     

人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia ( Tiantan strain) virus ( rTTV ) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2), or bicistron expressing the neuraminidase(NA) and matrix protein 1 (M1). The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose ( 104 PFU, 107 PFU)using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell(SFC) with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.     

人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia ( Tiantan strain) virus ( rTTV ) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2), or bicistron expressing the neuraminidase(NA) and matrix protein 1 (M1). The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose ( 104 PFU, 107 PFU)using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell(SFC) with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.     

利用反向遗传学技术构建H5N9重组流感病毒株

目的 利用反向遗传学技术构建来源人感染禽流感病毒H5N1和H7N9 HA和NA基因的H5N9亚型禽流感病毒.方法 全基因合成A/Beijing/01/2003(H5N1)禽流感病毒HA基因片段和A/Zhejiang/DTID-ZJU10/2013(H7N9)禽流感病毒NA基因片段,插入到pHW2000载体,与携带有A/Puerto Rico/8/34(H1N1)的6个内部基因的pHW2000重组质粒一起转染293T和MDCK混合细胞,拯救H5N9重组病毒.结果 核酸测序、HA和NA基因转录和表达检测、细胞病变分析确定利用该反向遗传学系统可以成功拯救H5N9病毒.重组H5N9病毒在MDCK细胞上复制增殖能力低于相同方法拯救H1N1病毒.结论 利用反向遗传学技术成功构建一株H5N9重组病毒.     

基于鞭毛蛋白的H5亚型禽流感疫苗候选株的构建及鉴定

目的构建基于鞭毛蛋白的H5亚型禽流感疫苗候选株。方法以H5N1禽流感病毒血凝素(HA)基因和鼠伤寒沙门菌SL7202基因组为模板,通过重叠PCR扩增出HA1-2-fljB基因片段,将其插入原核表达载体pET32a(+),构建重组质粒pET32a-HA1-2-fljB,再将其转化表达菌E.coli BL21(DE3),IPTG诱导目的蛋白表达。SDS-PAGE和Western blot法鉴定分析融合蛋白HA1-2-fljB。通过刺激HEK293-TLR5细胞,检测融合蛋白的TLR5生物学活性。结果正确构建出重组质粒pET32aHA1-2-fljB,SDS-PAGE显示,融合蛋白HA1-2-fljB相对分子质量(Mr)约为100 000,Western blot法表明融合蛋白具有良好的免疫反应性。TLR5生物活性实验结果显示,与HA1-2蛋白刺激组相比,HA1-2-fljB诱导HEK293-TLR5细胞分泌了高水平的IL-8(P0.01)。结论成功表达出具有TLR5活性的融合蛋白HA1-2-fljB,为研究H5亚型禽流感新型疫苗奠定了基础。     

重组禽流感疫苗株rH5N3的生物学特性与免疫效果

目的评价重组禽流感疫苗株rH5N3的生物学特性和免疫效果。方法将rH5N3注射SPF鸡胚和SPF鸡，观察其致病性；rH5N3及其亲本毒株A／Goose／HLJ／QFY／04分别在MDCK细胞单层和SPF鸡胚中增值，研究其生长特性；rH5N3在SPF鸡胚中连续传代，测定第10代rH5N3的核酸序列，评价其遗传稳定性；制备油包水型灭活rH5N3疫苗接种SPF鸡，免疫后不同时间采血测HI抗体滴度；rH5N3灭活疫苗接种SPF鸡，免疫后3周采血测定HI抗体滴度，同时进行H5N1亚型强毒株攻击，观察其保护作用。结果rH5N3疫苗株对鸡和鸡胚均无致病性；其在鸡胚尿囊液和细胞培养上清液的HA效价极大提高，分别为1：2048和1：512；第10代rH5N3的核酸序列与其亲本毒株相同；灭活rH5N3疫苗免疫鸡后2周即可诱导产生高效价HI抗体，且免疫后18周依然保持较高水平；该疫苗对强毒株包括国内早期分离的A／Goose／Guangdon∥1196和近期分离的A／Goose／HLJ／QFY／04都能产生完全的保护作用，免疫鸡攻毒后不发病、不排毒、不死亡。结论rH5N3疫苗株具有安全、高产、稳定等特点，且具良好的免疫原性，免疫动物完全能够抵抗强毒株的致死性攻击。该疫苗株的成功研制为H5N1亚型高致病性禽流感的防控提供了新的技术保障。     

反向遗传学技术在负链RNA病毒研究中的建立及应用

反向遗传学技术作为一种新兴的分子生物学实验操作技术,在生命科学研究的诸多领域均有广泛应用,它通过构建RNA病毒的感染性分子克隆,在DNA分子水平上对其进行体外操作,从而达到研究病毒结构与功能的目的。本文就反向遗传学操作技术在负链RNA病毒研究中的建立及应用作一综述。     

痘苗病毒载体在冠状病毒反向遗传学中的应用

反向遗传学方法是研究RNA病毒的主要方法,痘苗病毒载体则是感染性克隆技术的主要媒介,目前这一技术已趋向成熟并扩大其应用领域及对象.本文对痘苗病毒载体的构建、在冠状病毒反向遗传学中的应用情况以及目前该领域的进展作一综述.     

痘苗病毒载体在冠状病毒反向遗传学中的应用

反向遗传学方法是研究RNA病毒的主要方法，痘苗病毒载体则是感染性克隆技术的主要媒介，目前这一技术已趋向成熟并扩大其应用领域及对象。本文对痘苗病毒载体的构建、在冠状病毒反向遗传学中的应用情况以及目前该领域的进展作一综述。     

禽流感重组禽痘病毒rFPV-HA-NA活载体疫苗的研究

目的：确定重组禽痘病毒rFPV-HA-NA疫苗株的最佳免疫剂量、免疫日龄、免疫产生时间及免疫持续期。方法：用重组禽痘病毒rFPV-HA-NA疫苗免疫SPF鸡，免疫后用HPAIVH5N1和H7N1AIV进行致死性攻击，观察疫苗免疫后的保护效果。结果：大约含100个PFU的重组病毒即能使机体获得对强毒攻击的100％保护；对1日龄SPF鸡进行免疫接种，4周后能100％抵抗HPAIV的致死性攻击；2周和3周龄的免疫鸡对免疫周1后的强毒攻击具有100％的保护力；重组病毒在免疫后10个月时，其诱导产生的血凝抑制（Haemagglutinin inhibition，HI）抗体仍保持在2-3log2水平，并可以提供100％抵抗病毒的致死性感染。结论：重组禽痘病毒rFPV-HA-NA疫苗是一种安全、高效的基因工程疫苗，它有望在不久的将来替代全病毒灭活疫苗用于高致病力禽流感的预防。     

用反向遗传技术产生冷适应致弱的重组A型人流感病毒的研究

目的 以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒.方法 对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变.PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体.该重组质粒与PR8进行"7 1"组合的病毒拯救,共转染COS-1细胞.结果 经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒.结论 利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础.     

Preparation of a standardized, efficacious agricultural H5N3 vaccine by reverse genetics

Options for the control of emerging and reemerging H5N1 influenza viruses include improvements in biosecurity and the use of inactivated vaccines. Commercially available H5N2 influenza vaccine prevents disease signs and reduces virus load but does not completely prevent virus shedding after challenge with H5N1 virus. By using reverse genetics, we prepared an H5N3 vaccine whose hemagglutinin is 99.6% homologous to that of A/CK/HK/86.3/02 (H5N1). We used the internal genes of A/PR/8/34 and the H5 of A/Goose/HK/437.4/99 (H5N1) after deletion of basic amino acids from its connecting peptide region. The resulting virus was not lethal to chicken embryos and grew to high HA titers in eggs, allowing preparation of HA protein-standardized vaccine in unconcentrated allantoic fluid. The N3 neuraminidase, derived from A/Duck/Germany/1215/73 (H2N3), permitted discrimination between vaccinated and naturally infected birds. The virus construct failed to replicate in quail and chickens. Similar to parental A/PR/8/34 (H1N1), it replicated in mice and ferrets and spread to the brains of mice; therefore, it should not be used as a live-attenuated vaccine. The H5N3 vaccine, at doses of 1.2 microg HA, induced HI antibodies in chickens and prevented death, signs of disease, and markedly reduced virus shedding after challenge with A/CK/HK/86.3/02 (H5N1) but did not provide sterilizing immunity. Thus, reverse genetics allows the inexpensive preparation of standardized, efficacious H5N3 poultry vaccines that may also reduce the reemergence of H5N1 genotypes.     

H5N1流感疫苗株在Vero细胞中的研究[英文]

目的产生在Vero(非洲绿猴肾)细胞中能高适应生长的重配H5N1流感病毒疫苗株,并对其生物学特性进行初步测定。方法修饰A/Puerto Rico/8/34(H1N1)(PR8)的NS基因为Vero细胞适应型,并合成NS基因片段,构建质粒pHW2000-NS;通过RT-PCR方法,扩增疫苗株A/Anhui/01/2005(H5N1)的HA、NA基因片段,构建质粒pHW2000-HA、pHW2000-NA;以PR8的其它5个内部基因作为骨架,按照1+2+5模式8质粒共转染Vero细胞拯救流感病毒,并对拯救病毒的生物学特性进行检测。结果由Vero拯救出具有高适应生长特性的H5N1亚型人禽流感病毒疫苗株,并检测了重配疫苗株的生物学特性。结论成功从Vero细胞拯救了具有Vero细胞高适应性生长特性的H5N1亚型人禽流感病毒疫苗株,为应用Vero细胞大规模生产流感疫苗奠定了基础。     

H5亚型禽流感灭活疫苗病毒基因的定量检测及其与HI抗体效价之间关系的分析

目的建立H5亚型禽流感病毒灭活疫苗中病毒核酸的特异、敏感、快速的定量检测方法，分析其与HI抗体效价之间的相关性。探索禽流感灭活疫苗效力体外检验的方法。方法选取H5亚型禽流感病毒（avian influenza virus，AIV）血凝素（hemagglutinin，HA）基因保守序列，使用Prinler Express 2．0软件设计了特异性引物和TaqMan MGB（minor groove binder）探针，利用实时荧光PCR技术来定量检测禽流感灭活疫苗，通过体外转录，制备含有选定检测序列的RNA标准品，绘制标准曲线。对50批H5亚型禽流感灭活疫苗进行了荧光定量PCR检测，测定了疫苗的HA基因拷贝数，同时测定了50批疫苗相应的HI抗体效价。进行了疫苗的HA基因拷贝数与HI抗体效价之间相关性的分析。结果该方法的灵敏度为10拷贝，反应，标准曲线的相关系数为0．998003，对H9亚型禽流感灭活疫苗和其他禽病疫苗无交叉反应，特异性好、重复性佳。疫苗的HA基因拷贝数与HI抗体效价不相关。结论荧光定量PCR方法为H5亚型禽流感病毒灭活疫苗检测提供了一种特异、敏感、快速的定量检测方法。     

Efficacy of inactivated vaccines against H5N1 avian influenza infection in ducks

The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.     

Protective efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse genetics

We generated a high-growth H5N1/PR8 virus by plasmid-based reverse genetics. The virulence associated multiple basic amino acids of the HA gene were removed, and the resulting virus is attenuated for chickens and chicken eggs. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When SPF chickens were inoculated with 0.3 ml of the vaccine, the hemagglutinin-inhibition (HI) antibody became detectable at 1 week post-vaccination (p.v.) and reached a peak of 10log2 at 6 weeks p.v. then slowly declined to 4log2 at 43 weeks p.v. Challenge studies performed at 2, 3 and 43 weeks p.v. indicated that all of the chickens were completely protected from disease signs and death. Ducks and geese were completely protected from highly pathogenic H5N1 virus challenge 3 weeks p.v. The duration of protective immunity in ducks and geese was investigated by detecting the HI antibody of the field vaccinated birds, and the results indicated that 3 doses of the vaccine inoculation in geese could induce a 34 weeks protection, while 2 doses induced more than 52 weeks protection in ducks. We first reported that an oil-emulsion inactivated vaccine derived from a high-growth H5N1 vaccine induced approximately 10 months of protective immunity in chickens and demonstrated that the oil-emulsion inactivated avian influenza vaccine is immunogenic for geese and ducks. These results provide useful information for the application of vaccines to the control of H5N1 avian influenza in poultry, including chickens and domestic waterfowl.