浙江省肾综合征出血热灭活疫苗的研究

目的　通过对分离到的汉坦病毒 (HV)免疫原性及病毒滴度的选育 ,选择出疫苗毒株及筛选出不破坏病毒血凝抗原的灭活剂和疫苗基质细胞 ,提高疫苗的规模化生产技术 ,生产出副反应轻 ,免疫原性和现场防病效果好的各种剂型的疫苗 ,满足人群免疫预防的需要。方法　分离到的HV株 ,应用多种方法进行免疫原性和动物保护试验选育 ;疫苗灭活剂筛选通过与甲醛灭活效果比较 ,观察对血凝素抗原的影响 ,以此作为判断指标 ;疫苗基质细胞建立主要通过观察病毒在各种细胞上的增殖滴度、基质细胞的贴壁时间及收液次数等方法 ,选择基质细胞。结果　从 45株病毒株中成功选育出Z10 (汉滩型 )和Z3 7(汉城型 )疫苗病毒株 ,两株疫苗免疫原性好、病毒滴度高 ;成功选择出 β 丙内酯作为疫苗灭活剂 ,它不破坏病毒的血凝素抗原 ,接种反应轻 ;建立的长爪沙鼠肾原代细胞作为疫苗基质 ,不仅病毒滴度高 ,而且贴壁效果好 ,可以做到多次收液的目的。结论　我们研制的疫苗无论是单价的Ⅰ型、Ⅱ型疫苗 ,还是双价 (Ⅰ型和Ⅱ型混合 )疫苗 ,接种人体后副反应轻 ,免疫效果好 ,现场防病效果也很理想 ,是目前控制肾综合征出血热流行最有效和经济的手段和措施 ,值得大力推广。     

VERO细胞狂犬病疫苗纯化工艺中样品浓度对纯化效果的影响

目的　研究浓缩 Vero细胞狂犬病疫苗蛋白浓度对纯化效果的影响。　方法　用超滤浓缩法对狂犬病疫苗进行不同倍数的超滤浓缩 ,将浓缩后不同倍数的样品分别用凝胶柱层析法进行纯化。　结果　样品浓度在 4 0 mg/ ml下时 ,狂犬病毒收集液中杂蛋白去除率可达 99%以上 ,小牛血清含量在 2 5～ 37.5 ng/ ml之间 ;浓度超过 4 0 mg/ ml时 ,随着样品浓度的增加 ,狂犬病毒收集液中杂蛋白的去除率逐渐下降 ,且有病毒丢失。　结论　采用凝胶柱层析法纯化狂犬病疫苗 ,蛋白浓度应在 4 0 mg/ ml之下     

β-丙内酯对狂犬病病毒的灭活效果

狂犬病是一种由狂犬病病毒（RV）感染引起的高度致死性脑脊髓炎,一旦发病,病死率几乎高达100％,预防接种狂犬病疫苗是控制狂犬病的最有效方法。目前,人用狂犬病疫苗均为灭活疫苗,其灭活剂主要有β-丙内酯（BPL）和甲醛1。虽然BPL早已应用于狂犬病疫苗生产,但未见其对狂犬病毒的灭活曲线的研究报道。我们对不同浓度BPL灭活RV的效果、灭活曲线等进行研究,现将结果报道如下。     

亚甲蓝光化学法病毒灭活血浆的制备应用探讨

目的探讨亚甲蓝光化学法(MB-P)病毒灭活前后新鲜冰冻血浆中凝血因子Ⅷ(FⅧ)、纤维蛋白原(Fib)和总蛋白(TP)、亚甲蓝浓度及血浆容量的变化,为临床应用病毒灭活血浆提供依据。方法随机抽取2015年7月~12月(每月10份)60份新鲜血浆,分成两组,一组进行病毒灭活,一组不灭活,灭活前后留样,制成新鲜冰冻血浆,48小时后融化,分别对病毒灭活前后血浆中的凝血因子Ⅷ(FⅧ)、纤维蛋白原(Fib)和总蛋白(TP)的含量进行测定,并检测病毒灭活前后血浆中亚甲蓝浓度和称取病毒灭活过滤MB前后血浆的重量,同时计算有效成分的回收率。结果经病毒灭活后新鲜冰冻血浆中凝血因子Ⅷ(FⅧ)、纤维蛋白原(Fib)和总蛋白(TP)的含量及亚甲蓝浓度、血浆重量明显降低,各项指标均有显著变化,P<0.01,差异均有统计学意义,凝血因子Ⅷ(FⅧ)、纤维蛋白原(Fib)和总蛋白(TP)回收率分别为84.81%、82.46%和93.76%,亚甲蓝的清除率可达83.93%,血浆回收率为93.97%。结论经MB-P病毒灭活血浆能提高血浆输注的安全性,同时,血浆中的有效成分及容量也有一定程度的损失,应加强从采血到血浆病毒灭活各环节的冷链及时间控制,严格按操作规程操作,保证原料血浆的有效成分处于较高水平。     

不同灭活方法对Vero细胞狂犬病疫苗效力的影响

传统的人用浓缩狂犬病疫苗是采用福尔马林进行灭活 ,而国际上新型疫苗早已采用 β -丙内酯做为灭活剂〔1〕。为了摒弃旧的生产工艺 ,提高我国生物制品产品质量 ,使之尽早与国际接轨 ,我们对两种灭活方法进行了试验研究。现报告如下。材料与方法　 (1)病毒原液制备 :将Ｖｅｒｏ细胞与ＣＴＮ - 1株狂犬病毒混合接种于 10立升转瓶 ,培养一定时间后 ,收获病毒液。 (2 )超滤浓缩与纯化 :收获的病毒经 0 45 μｍ滤膜去除细胞残片等物质 ,再以 10万分子量超滤膜进行浓缩 ,浓缩后的病毒液先加入特异蛋白作用一定时间 ,再经Ｓｅｐｈａｒｏｓｅ -ＦＦ…     

β-丙内酯在Vero细胞HFRS疫苗中的应用

目的 探讨β-丙内酯灭活双价Vero细胞肾综合征出血热纯化疫苗的最佳条件。方法 采用不同终浓度的β-丙内酯和不同的灭活时间对疫苗进行灭活，用细胞培养法进行病毒增殖试验，以确定灭活效果。应用高效液相色谱法分析β-丙内酯的最佳水解条件和3批双价Vero细胞出血热纯化灭活疫苗β-丙内酯残留虽。检测3批双价纯比灭活疫苗的免疫效果。结果 疫苗经终浓度为1：2000和1：4000的β-丙内酯作用24h，均可完全灭活病毒；疫苗中的β-丙内酯在37℃水浴下，随着水解时间的延长，其含量逐渐下降，水解2h后已无β-丙内酯检出；3批经β-丙内酯灭活的双价Vero细胞出血热纯化疫苗均未检出β-丙内酯残留，在家兔体内诱导产生针对汉滩型和汉城型病毒的中和抗体效价均达到1：20。结论 肾综合征出血热纯化疫苗经终浓度为1：4000β-丙内酪4℃灭活24h，然后再于37℃水浴中水解2h，既可达到完全灭活病毒且无β-丙内酯残留的目的；采用此方法灭活的双价Vero细胞肾综合征出血热纯化疫苗保持良好的免疫原性。     

双价纯化肾综合征出血热疫苗临床观察

用沙鼠肾原代细胞培养肾综合征出血热 (ＨＦＲＳ)Ｚ10(Ⅰ型 )和Ｚ37(Ⅱ型 )病毒 ,经 β丙内脂灭活 ,已研制成Ⅰ型、Ⅱ型和双价 (Ⅰ型加Ⅱ型 ) 3种ＨＦＲＳ疫苗 ,均已取得新药证书和生产文号 ,并形成规模生产 ,经 10 0 0余万现场人群接种 ,取得了很好的预防效果[1 ] 。为使产品的质量进一步提高 ,达到副反应更轻 ,接种针次减少的目的 ,进行了纯化疫苗的研究。材料与方法1　疫苗制造　利用Ｚ10型 (Ⅰ型 )和Ｚ37(Ⅱ型 )毒种 ,分别接种沙鼠肾原代细胞 ,经培养和多次收液 (每次收液病毒滴度ＴＣＩＤ50 达 10 6 0 /ｍｌ以上 )后合并 ,用 10万分子…     

甲醛超声雾化消毒器对脊髓灰质炎病毒灭活效果的实验观察

目的 检测甲醛超声雾化消毒器对脊髓灰质炎病毒灭活效果.方法 通过物表消毒模拟现场试验,检测甲醛超声雾化消毒器对污染于玻璃载体表面的脊髓灰质炎病毒灭活效果.结果 在试验条件下,当空气中甲醛初始浓度达到737 mg/m3时,薰蒸60 min,能够完全灭活污染于载体上的脊髓灰质炎病毒.结论 甲醛超声雾化消毒器对脊髓灰质炎病毒具有可靠的灭活效果.     

深圳市公共场所空气中甲醛污染状况的调查

目的　了解公共场所室内空气中甲醛污染状况。方法　用甲醛分析仪对深圳市 2 6 4间公共场所室内空气甲醛含量进行测定。结果　公共场所甲醛的总超标率为 18 83%。装修后开业第1～ 3个月公共场所室内空气中甲醛浓度的超标率为 4 4 97% ,1年以后的超标率为 7 2 5 % (P <0 0 1)。使用中央空调的公共场所甲醛超标率 (2 3 84 % )高于使用普通空调的场所 (11 2 7% ) (P <0 0 1)。结论　新装饰开业的公共场所室内空气中甲醛污染较严重。应加强室内通风换气及装饰材料的卫生。     

狂犬疫苗灭活后β-丙内酯水解的最佳pH值的选择

我国狂犬疫苗灭活剂是甲醛溶液。甲醛为致癌物,残留的游离甲醛随疫苗注入人体后产生刺激性反应。而β－丙内酯（ＢＰＬ）在国外已广泛用于各种疫苗的灭活,它是一种杂环类化合物（Ｃ３Ｈ４Ｏ２）,沸点１５５℃,常温下呈无色粘稠状液体,对病毒有很强的灭活作用。ＢＰＬ虽是致癌物,但极易水解,形成人体脂肪代谢产生β－羟基丙酸〔１〕,对人体无害。在不同ｐＨ值下,ＢＰＬ水解情况是不同的。我们将ＢＰＬ分别加到ｐＨ值为７－２、７－４、７－６、７－８、８－０疫苗中,并在３７℃水浴中水解２小时,用毛细管气相色谱法检测其含量变化…     

A fast and efficient purification platform for cell-based influenza viruses by flow-through chromatography

Since newly emerging influenza viruses with pandemic potentials occurred in recent years, the demand for producing pandemic influenza vaccines for human use is high. For the development of a quick and efficient vaccine production, we proposed an efficient purification platform from the harvest to the purified bulk for the cell-based influenza vaccine production. This platform based on flow-through chromatography and filtration steps and the process only involves a few purification steps, including depth filtration, inactivation by formaldehyde, microfiltration, ultrafiltration, anion-exchange and ligand-core chromatography and sterile filtration. In addition, in the proposed chromatography steps, no virus capture steps were employed, and the purification results were not affected by the virus strain variation, host cells and culturing systems. The results from different virus strains which produced by Vero or MDCK cells in different culturing systems also obtained 33–46% HA recovery yields by this platform. The overall removal rates of the protein and DNA concentration in the purified bulk were over 96.1% and 99.7%, respectively. The low residual cellular DNA concentrations were obtained ranged from 30 to 130 pg per human dose (15 µg/dose). All influenza H5N1 purified bulks met the regulatory requirements for human vaccine use.     

Development and qualification of the parallel line model for the estimation of human influenza haemagglutinin content using the single radial immunodiffusion assay

Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay.     

Optimization and qualification of a quantitative reversed-phase HPLC method for hemagglutinin in influenza preparations and its comparative evaluation with biochemical assays

A previously described reversed-phase HPLC (RP-HPLC) method based on fast separations on a non-porous silica stationary phase [1] was optimized and qualified for the quantitative determination of hemagglutinin (HA) in influenza vaccine preparations. Optimization of the gradient elution conditions led to improved separation of the HA1 subunit from other vaccine constituents. The sensitivity of the method was significantly increased by using native fluorescence detection, resulting in an approximately 10-fold increase as compared to UV-vis detection. This enabled the elimination of the concentration step described in the original method and allowed direct analysis of vaccine preparations. The method was qualified for linearity, range, limit of detection, limit of quantitation and precision. Overall, it was found to be linear over the range of 2.5-100 μg HA/mL for all subtypes examined. This range covered 50-150% of the concentration found for individual strains in seasonal influenza vaccines and in the pandemic H1N1 vaccine. The limit of detection and limit of quantitation for each subtype were found to be suitable for the method's intended purpose and compared well to values found by the single radial immunodiffusion (SRID). The repeatability of the method gave RSD values below 5% for both retention time and peak areas. As expected for intermediate precision, larger RSD values for peak area were obtained but were below 10% and deemed acceptable.The RP-HPLC results were compared to Western blot analysis using a HA universal antibody for a set of 15 monovalent A/California H1N1 preparations and showed good correlation. Similarly, the quantitative nature of the RP-HPLC method was assessed in relation to the SRID assay currently used for the determination of the HA content in bulk antigen and final vaccine preparations. Thus, for a series of 23 monovalent A/Brisbane/59/2007 H1N1 bulks, ranging between 12.7 and 15.9 μg HA/mL by SRID, the RP-HPLC values were found to be in very good agreement, ranging between 11.9 and 14.1 μg HA/mL (n = 5) for five determinations carried out on 5 different days.During the 2009-10 H1N1 influenza pandemic the quantitative RP-HPLC method was used alongside several other test methods for the analysis of pandemic H1N1 vaccine preparations that included bulk antigen and final vaccines. The HA content of vaccines formulated at 15 or 30 μg/mL was measured by RP-HPLC and SRID and results showed that the HA content determined by RP-HPLC correlated well to that determined by SRID and to values determined by Western blot. Overall, the results provided further evidence of the usefulness of RP-HPLC for the detection and quantitation of the HA content once a reference standard has been established.     

The stability and immunogenicity of inactivated MDCK cell-derived influenza H7N9 viruses

In recent years, cell-based influenza vaccines have gained a great interest over the egg-based vaccines. Several inactivated H7N9 vaccines have been evaluated in clinical trials, including whole-virion vaccines, split vaccines and subunit vaccines. Recently, we developed a new suspension MDCK (sMDCK) cell line for influenza viruses production. However, the properties of purified antigen from sMDCK cells remain unclear. In this study, the stability of influenza H7N9 vaccine bulk derived from sMDCK cells was investigated, and the data were compared with the vaccine antigen derived from our characterized adhesion MDCK (aMDCK) cells in serum-free medium. The influenza H7N9 bulks derived from sMDCK and aMDCK cells were stored at 2–8 °C for different periods of time, and a number of parameters selected to monitor the H7N9 vaccine antigen stability were evaluated at each interval (1, 3 and 12 months). The monitored parameters included virus morphology, hemagglutinin (HA) activity, HA concentration, antigenicity, and immunogenicity. The sMDCK-derived H7N9 bulk showed similar morphology to that of the aMDCK-derived H7N9 bulk, and there were no obvious changes after the extended storage periods. Furthermore, the HA titer, HA concentration, and antigenicity of sMDCK-derived H7N9 bulk were stable after 28 months of storage. Finally, the results of hemagglutination inhibition and neutralization tests showed that sMDCK- and aMDCK-derived H7N9 vaccines had comparable immunogenicity. These results indicated that sMDCK-derived H7N9 bulk has good stability compared to that of aMDCK-derived H7N9 bulk. Thus, the newly developed suspension MDCK cell line shows a great alternative for manufacturing cell-based influenza vaccines.     

Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry

Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1 μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.     

Antigenic stability of H1N1 pandemic vaccines correlates with vaccine strain

In 2009 a novel H1N1 influenza virus emerged and spread rapidly. Soon after vaccine lots were released, however, the shelf life was revised downward due to an unexpected decrease in HA potency. In this study, we found differences in both stability and antigenic content of two monovalent H1N1/2009 vaccine preparations. These appear to have arisen due to differences in the A/California/7/2009-like influenza strain used to prepare vaccine.     

2000—2007年佛山市甲1亚型（H1N1）流感病毒监测情况及其HA1基因的变异

目的分析2000—2007年佛山市甲1亚型（H1N1）毒株流行情况及其HA1基因变异情况。方法用MDCK细胞分离流感病毒，采用血球凝集抑制试验进行型别鉴定，选取每年2～4株甲1亚型毒株的细胞培养物提取病毒核酸，进行HA1基因的逆转录，对其核苷酸和氨基酸序列及亲缘关系进行分析。结果病毒分离结果显示2000—2007年间甲1亚型流感病毒在佛山市人群中的流行是间断性的，只在2000、2001、2005、2006年4个年份分离到甲1亚型流感毒株。毒株的HAl区氨基酸序列与流感疫苗推荐株A／NewCMedonia／20／99和A／Solomon Islands／3／2006相比，同源性分别为97．2％～99．7％和97．2％-98．5％。氨基酸残基平均替换数随年份的推移而增加，只有2006年的毒株在抗原决定簇发生了替换。结论与疫苗株比较，佛山市的甲1亚型流感毒株抗原性已逐渐发生了改变，应密切注意疫苗对毒株的免疫效果。     

A bivalent influenza VLP vaccine confers complete inhibition of virus replication in lungs

The conventional egg-grown influenza vaccines are trivalent. To test the feasibility of using multivalent influenza virus-like particles (VLPs) as an alternative influenza vaccine, we developed cell-derived influenza VLPs containing the hemagglutinin (HA) of the H1 subtype virus A/PR/8/34 or the H3 subtype virus A/Aichi/2/68 (X31). Mice immunized intramuscularly with bivalent influenza VLPs containing H1 and H3 HAs induced neutralizing activities against the homologous and closely related H1N1 strains A/PR/8/34 and A/WSN/33 as well as the H3N2 strains A/Aichi/2/68 (X31) and A/Hong Kong/68, but not the A/Philippines/2/82 strain isolated 14 years later. HA sequence and structure analysis indicated that antigenic distance could be a major factor in predicting cross-protection by VLP vaccines. The bivalent influenza VLP vaccine demonstrated advantages in broadening the protective immunity after lethal challenge infections when compared to a monovalent influenza VLP vaccine. High levels of the inflammatory cytokine IL-6 were observed in na?ve or unprotected immunized mice but not in protected mice upon lethal challenge. These results indicate that multivalent influenza VLP vaccines can be an effective antigen for developing safe and alternative vaccine to control the spread of influenza viruses.     

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.