流感减毒活疫苗效力试验检测新方法的建立

目的建立基于NP蛋白检测的流感减毒活疫苗病毒滴度检测方法,并进行初步应用。方法对病毒接种方式、流感病毒感染MDCK细胞培养时间、一抗和酶标二抗的稀释度、封闭液等ELISA反应条件进行筛选优化。采用建立的方法检测3批流感减毒活疫苗原液和1批疫苗成品,并与鸡胚培养法检测结果进行比较。结果选择直接接种法作为接种方式,病毒感染细胞后培养48 h进行NP蛋白表达的检测; ELISA检测条件一抗稀释度为1∶4 000,酶标二抗稀释度为1∶4 000,2%牛血清白蛋白封闭2 h,检测结果 P/N值最大。用建立的方法检测流感减毒活疫苗原液及疫苗成品效力,与鸡胚培养法检测结果差异无统计学意义(P0.05),具有较好的一致性。结论建立了基于NP蛋白检测的流感减毒活疫苗效力试验方法,可用于生产过程中流感减毒活疫苗原液和成品的检定。     

预防呼吸道及肠道病毒性疾病的活疫苗

<正> 与灭活疫苗和亚单位疫苗相比,活疫苗的显著优点是能诱导更有效的局部粘膜免疫力及更持续的免疫反应能力。而活疫苗的最大缺点则是遗传不稳定使得减毒特性丧失的可能性。目前,应用以下5种策略使这种可能性减小到最小程度,同时又不致于影响减毒与免疫原性之间的平衡,这种平衡是所有活疫苗株所必需具备的。 一、用琴纳(Tennel)法研制活疫苗株 这种方法是用与人病毒抗原相关的动物病毒株作为疫苗,免疫接种于人体以预防人病毒引起的疾病。通常,在其自然宿主上适应力很好的动物病毒接种于人体后,它们的繁殖能力不是太强,因此对人来说是减毒的。目前,人们将传统的琴纳法与现代病毒遗传学、分子生物学及免疫学技术相结合,研制     

轮状病毒灭活疫苗和减毒活疫苗序贯免疫的体液免疫应答效果

目的:评价采用轮状病毒灭活疫苗进行初始免疫,减毒活疫苗进行加强免疫的序贯免疫方案的体液免疫应答效果。方法:将实验小鼠随机分为4组(口服疫苗组、序贯疫苗组、口服对照组及序贯对照组),按相应方案免疫后,ELISA检测血清轮状病毒特异性IgG和IgA、肠道轮状病毒特异性IgA;微量中和实验检测血清病毒特异性中和抗体;同时采用ELISA分析口服活疫苗后病毒排出情况。结果:与对照组相比,序贯疫苗组小鼠产生的轮状病毒特异性血清IgG、IgA、中和抗体及肠道IgA水平显著升高。与口服疫苗组相比,序贯疫苗组的免疫方案诱发的轮状病毒特异性血清IgG、IgA、中和抗体水平显著升高,肠道IgA水平两组间没有显著差异。同时,与口服疫苗组相比,序贯疫苗组中轮状病毒灭活疫苗进行的初始免疫未影响第一次口服活疫苗后病毒的排出量和排出时间,但序贯疫苗组第二次口服活疫苗后病毒的排出量迅速减少,排毒时间快速缩短,与口服疫苗组第三次服苗后病毒的排出量和排出时间相似。结论: 轮状病毒灭活疫苗和减毒活疫苗序贯免疫可有效诱发小鼠全身和黏膜局部的体液免疫应答,该方案将有可能成为轮状病毒疫苗临床应用的候选方案。     

麻疹减毒活疫苗检定用Vero细胞代次对病毒滴度稳定性试验的影响

目的了解麻疹减毒活疫苗检定用Vero细胞代次的稳定性范围。方法用DMEM培养液将Vero细胞137代连续传至173代,检测其细胞活力,并观察Vero细胞的形态,接种麻疹减毒活疫苗参考品进行病毒滴定及热稳定性试验。结果 170代以下Vero细胞形态良好,细胞活力在85%以上,麻疹减毒活疫苗病毒滴度热稳定性试验的结果:137~169代Vero细胞活力及原麻疹减毒活疫苗病毒滴度热稳定性试验无统计学差异(P>0.05),170代以上代次的Vero细胞活力及原麻疹减毒活疫苗热稳定性试验有明显统计学差异(P<0.01)。结论 169代以下Vero细胞为检定用麻疹减毒活疫苗最佳细胞代次。     

腮腺炎病毒抗血清的制备

以制备适用于疫苗生产检定中病毒鉴别试验和外源因子检查的高效价腮腺炎病毒抗血清为目的。用腮腺炎病毒接种SPF鸡胚尿囊腔,培养收取病毒尿液免疫SPF鸡,采集抗血清。腮腺炎病毒接种Vero细胞,培养病毒抗原经PEG沉淀,超速离心法纯化后免疫家兔采集抗血清。比较两种免疫方法所得病毒抗血清效价。结果显示SPF鸡抗腮腺炎病毒血清中和抗体GMT为1:1716,兔抗腮腺炎病毒血清中和抗体GMT为1:732。两种动物抗血清均适用于疫苗生产相关检定。免疫SPF鸡制备的病毒抗血清无特定病原及抗体污染,是毒种外源因子检测和疫苗鉴别试验的理想试剂。免疫SPF鸡制备病毒抗血清的程序简单,结果易于验证,有利于生物试剂标准化。     

北京株水痘疫苗生产工艺的建立

目的:建立北京株水痘疫苗生产工艺,采用此生产工艺生产水痘减毒活疫苗。方法:采用细胞工厂培养2BS细胞,感染北京株水痘-带状疱疹病毒工作种子批,同时感染Oka株水痘-带状疱疹病毒工作种子批作对照,经洗涤、离心、收获、原液合并、冻干制备水痘减毒活疫苗,并进行各项检定。结果:对照两种不同毒株生产的水痘减毒活疫苗无明显差异,且各项检测指标均符合要求。结论:根据结果显示,可使用此毒株生产工艺大规模生产北京株水痘疫苗,与Oka株生产水痘疫苗无差异。如果用此毒株生产水痘疫苗供应市场,将会打破Oka株水痘疫苗国内市场的垄断。     

BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗结果的影响分析

目的分析BHK_(21)细胞对蚀斑法检测乙型脑炎减毒活疫苗系统的影响,降低检测系统误差。方法比较BHK_(21)细胞以不同频次传代培养时的细胞生长状态,以及以不同接种浓度进行疫苗病毒滴度检测时,对检测系统稳定性的影响。结果 BHK_(21)细胞培养4 d传代,其形态良好、边缘光滑、胞质透光性好、细胞分散均匀、细胞活率达到95%以上;BHK_(21)以105细胞/m L浓度接种时,乙型脑炎减毒活疫苗及其病毒滴度参考品的变异系数最小,分别为0.66%和0.64%。结论 BHK_(21)细胞培养4 d传代、以105个/m L浓度接种时用蚀斑法检测病毒滴度系统最稳定,可供乙型脑炎减毒活疫苗滴度检测参考。     

高效价腮腺炎病毒抗血清的制备及检定

目的制备高效价腮腺炎病毒抗血清,用于麻腮、麻腮风、麻腮风水痘联合疫苗的病毒滴定及鉴别试验等指标的检定。方法将腮腺炎病毒ME株接种于SPF鸡胚尿囊腔中,优化病毒制备工艺条件,收获高滴度病毒原液,制备免疫抗原;采用皮下多点注射法免疫SPF豚鼠,制备抗血清并对其进行中和效价、中和能力以及特异性干扰试验的检定。结果 ME株腮腺炎病毒以102倍稀释接种鸡胚尿囊腔,培养5 d经-20℃预冷1 h后,收获的尿囊液病毒滴度最高;以其免疫豚鼠所制备的抗血清平均中和效价达1∶3 276,高于鸡抗腮腺炎病毒血清;当豚鼠抗腮腺炎病毒血清稀释度为1∶320时,可完全中和1 000 CCID50/m L的腮腺炎病毒;豚鼠抗腮腺炎病毒血清对Vero细胞、RK-13细胞及2BS细胞的生长,均未见干扰及细胞毒性作用;豚鼠抗腮腺炎病毒血清对异种病毒(麻疹病毒、风疹病毒和水痘病毒)滴度的干扰试验显示,各病毒滴度其试验组与对照组的差值均0.50 lg CCID50/m L,表明豚鼠抗腮腺炎病毒血清对麻疹病毒、风疹病毒和水痘病毒的滴度均无干扰;豚鼠抗腮腺炎病毒血清及鸡抗腮腺炎病毒血清对麻腮风水痘联合疫苗各病毒滴度均无干扰,且两种抗血清之间差异无统计学意义(P0.05)。结论采用优化后的病毒制备工艺条件及免疫方法,可获得较高效价的抗腮腺炎病毒血清,经检定和验证,均符合含腮腺炎成分疫苗检定抗血清使用要求。     

立谷热基因工程疫苗的研究

<正>近几年来有关立谷热(RVF)人用灭活疫苗和减毒活疫苗的研究均取得了明显的进展。但灭活疫苗存在疫苗产量低和需多次免疫及有副反应等缺点,而减毒活疫苗短期内尚难实际应用,故有关RVF的基因工程疫苗研究具有重要意义。随着RVF病毒核酸分子生物学研究的日益完善,有关RVF的基因工程疫苗的研究工作也取得了不少进展。不少学者分别在细菌、哺乳动物细胞和昆虫细胞等中表达了RVF病毒糖蛋白(G_1和G_2)基因(M基因)的     

TaqMan实时荧光定量RT-PCR检测甲型肝炎减毒活疫苗病毒含量的研究

目的：建立一种快速定量检测甲型肝炎减毒活疫苗病毒含量的实时荧光定量RT-PCR方法。方法对Gen-Bank中登陆的甲型肝炎减毒活疫苗株（ L-A-1）和其他甲型肝炎病毒基因组全序列比较分析,根据其高度保守的5′端非编码区设计针对甲型肝炎减毒活疫苗株特异性引物与探针,对荧光定量RT-PCR反应条件进行优化,检测该方法的特异性和灵敏性,并对甲型肝炎减毒活疫苗病毒含量进行定量检测。结果该方法对甲型肝炎减毒活疫苗株高度特异,扩增片段为207 bp,不与其他肠道病毒发生非特异性反应。在104 CCID50/管～10-1 CCID50/管之间有良好的扩增曲线,检测的灵敏度可达0．1CCID50～0．01CCID50,比普通RT-PCR高100倍。结论该方法具有快速、灵敏、特异、重复性好等优点,可应用于甲型肝炎减毒活疫苗生产过程中病毒含量滴度测定及指导疫苗成品的配制。     

Influenza H5 hemagglutinin DNA primes the antibody response elicited by the live attenuated influenza A/Vietnam/1203/2004 vaccine in ferrets

Priming immunization plays a key role in protecting individuals or populations to influenza viruses that are novel to humans. To identify the most promising vaccine priming strategy, we have evaluated different prime-boost regimens using inactivated, DNA and live attenuated vaccines in ferrets. Live attenuated influenza A/Vietnam/1203/2004 (H5N1) candidate vaccine (LAIV, VN04 ca) primed ferrets efficiently while inactivated H5N1 vaccine could not prime the immune response in seronegative ferrets unless an adjuvant was used. However, the H5 HA DNA vaccine alone was as successful as an adjuvanted inactivated VN04 vaccine in priming the immune response to VN04 ca virus. The serum antibody titers of ferrets primed with H5 HA DNA followed by intranasal vaccination of VN04 ca virus were comparable to that induced by two doses of VN04 ca virus. Both LAIV-LAIV and DNA-LAIV vaccine regimens could induce antibody responses that cross-neutralized antigenically distinct H5N1 virus isolates including A/HongKong/213/2003 (HK03) and prevented nasal infection of HK03 vaccine virus. Thus, H5 HA DNA vaccination may offer an alternative option for pandemic preparedness.     

Development and Pre-Clinical Evaluation of Two LAIV Strains against Potentially Pandemic H2N2 Influenza Virus

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.     

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.     

Induction of interferon in man by vaccines.

The purpose of this study was to extend the spectrum of vaccines with interferon-inducing potential in man. The vaccines selected for study were the commercially available attenuated poliomyelitis vaccine type 2 (Sabin strain) and the new live attenuated influenza A/England/42/72 (H3N2) vaccine ("Alice" strain). Five subjects, two of whom had low or undetectable polio type 2 neutralizing antibody levels were given the type 2 vaccine (10-4.7 TCID50) in the standard manner orally. Even though the two individuals with low titers experienced a fourfold or greater antibody rise and one of them shed the virus in his stool, neither they nor the remaining three volunteers developed detectable levels of interferon in their sera obtained at very closely spaced intervals from day 0 to day 25 following immunization. Fifteen subjects were given approximately 10-7.5 TCID50 of influenza A/England/42/72 (H3N2) by nasal drops. Specimens consisting of sera and nasal washings were obtained at closely timed intervals for 23 days, starting with day 3 following immunization. Interferon could be detected in three of nine (33.3%) subjects who had fourfold or greater HI antibody rises. No interferon was detected in nasal washings, however. It is concluded that poliomyelitis is not a good interferon inducers in man. Live attenuated influenza vaccine does induce an interferon response in subjects with low initial serum antibody titers. This response is at best modest. The latter finding also suggests that the attenuation of the Alice strain of influenza A vaccine is not dependent on its interferon inducing potential.     

不同代次牛肾原代细胞培养轮状病毒的比较研究

口服轮状病毒活疫苗(LLR株)生产用细胞基质为新生小牛肾原代细胞。原始的初代细胞(P0)产量小,一对牛肾平均生产7瓶细胞。将原始的初代细胞传代可使细胞产量显著增加,传代后(P2代)细胞产量可由7瓶增加为96~112瓶,细胞核型检查传至P5代的细胞染色体数目与初代细胞一致。细胞培养物均一性提高。P0代与P2代细胞病毒培养物滴度分别在6.2±1.5和6.5±0.5lgCCID50/ml,使用P2代细胞培养病毒,产量增加10~15倍。提高了疫苗生产的可控性和质量,生产规模显著放大,经济效益明显。     

Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.     

重组黄热病毒E蛋白结构域Ⅲ作为亚单位疫苗的抗原性和免疫原性

目的：表达纯化黄热病毒（YFV）囊膜蛋白（E蛋白）结构域Ⅲ,研究其作为亚单位疫苗预防YFV、日本脑炎病毒（JEV）感染的可能。方法：扩增YFVE蛋白结构域Ⅲ（YFDⅢ）的cDNA片段333bp,将其连接到原核表达载体pET-32a（＋）中,构建原核表达载体pET-YFDⅢ,转化感受态大肠杆菌Rosetta（DE3）,IPTG诱导表达重组YFDⅢ;用纯化的YFDⅢ免疫新西兰兔和BALB/c鼠,检测相关抗体滴度。结果：在大肠杆菌中可溶性表达了YFDⅢ融合蛋白,表达量约占菌体蛋白的50%;Western印迹及ELISA分析表明,纯化的YFDⅢ具有良好的抗原性和免疫原性;利用纯化的YFDⅢ免疫新西兰兔,获得了高达1∶4×105滴度的抗YFV抗体和1∶2×104滴度的抗JEV抗体;利用纯化的YFDⅢ免疫BALB/c鼠,获得了1∶7×104滴度的抗YFV抗体和1∶2×103滴度的抗JEV抗体。结论：重组YFDⅢ有较好的免疫原性,具有开发成亚单位疫苗的潜能。     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus

Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.