重组乙型肝炎疫苗(CHO细胞)生产工艺病毒灭活验证

目的:验证重组乙型肝炎疫苗(CHO细胞)生产工艺灭活病毒能力,评价疫苗安全性。方法:将乙型肝炎疫苗原液按1∶9比例加入指示病毒(麻疹病毒、VSV病毒),混匀后加入甲醛溶液,使甲醛终浓度达到1/2000和1/4000,放置于37℃,分别于0h、24h、48h、72h取样,用亚硫酸氢钠中和,贮存于-70℃待检或立即检测病毒滴度,对病毒检测阴性的样品盲传3代,进一步检测。结果:疫苗原液加入1/2000和1/4000的甲醛溶液,37℃作用24h、48h、72h,麻疹病毒滴度下降4.0个Log值,VSV病毒滴度下降5.0个Log值,且细胞盲传三代,均未出现细胞病变。结论:乙型肝炎疫苗原液在1/2000和1/4000甲醛浓度37℃72h作用下,均能有效灭活麻疹病毒和VSV病毒。     

低pH孵放法对马破伤风免疫球蛋白F(ab′)_2病毒灭活效果的验证

目的采用低pH孵放法对马破伤风免疫球蛋白F(ab′)_2样品中辛德毕斯病毒(sindbis virus, SINV)、水疱性口炎病毒(vesicular stomatitis virus, VSV)、脑心肌炎病毒(encephalomyocarditis virus, EMCV)的灭活效果进行验证。方法以SINV、VSV、EMCV作为指示病毒,将3批马破伤风免疫球蛋白F(ab′)_2的pH调至4.1±0.1,每批12瓶(3 mL/瓶),分别加入333μL指示病毒,25℃放置21 d灭活病毒,同时设置中性对照和阳性对照,并于0 d、1 d、3 d、7 d、14 d、21 d取样测定剩余病毒滴度,验证低pH孵放法的病毒灭活效果。结果马破伤风免疫球蛋白F(ab′)_2样品经低pH孵放处理21 d后,SINV、VSV和EMCV等3种指示病毒残余病毒滴度均≤0.5 lgTCID_(50)/0.1 mL,灭活效果分别达到5.50～5.83、5.70～6.00和6.00～6.17 lgTCID_(50)/0.1 mL。结论采用低pH孵放法处理马破伤风免疫球蛋白F(ab′)_2中SINV、VSV、EMCV,病毒滴度下降值均>4 lgTCID_(50)/0.1 mL,灭活效果较好。     

利用鸭乙型肝炎病毒感染模型评价血液成分中病毒的灭活效果

目的 利用鸭乙型肝炎病毒(DHBV)感染动物模型,评价亚甲蓝光化学病毒灭活方法对血液成分中DNA病毒的灭活效果。方法 将超离纯化的DHBV分别加入人血浆或人红细胞,经亚甲蓝光化学灭活病毒,将含不同基因组拷贝数DHBV的血浆成分经静脉感染1 d龄雏鸭。采用放射性核素核酸杂交法对血清中DHBV DNA进行检测,计算病毒灭活处理前、后人血浆及人红细胞中DHBV的半数感染计量(ID50)。结果 结果显示加入DHBV的血浆在未经灭活处理前对1 d龄雏鸭的ID50值为103.33,而经病毒灭活处理后ID50值为1010拷贝,灭活处理可使病毒感染性滴度下降达6个Log;加入DHBV的红细胞灭活前ID50值为103.35,经灭活处理后ID50值为108.35拷贝,灭活处理使病毒感染性滴度下降5个Log。结论 利用DHBV感染动物模型,可以检测到少量病毒在自然感染宿主体内的感染性,可用于评判血液成分中病毒灭活方法的效果,亚甲蓝光化学处理对血浆中DNA病毒的灭活效果较好于对红细胞中DNA病毒的灭活作用。     

发热伴血小板减少综合征病毒灭活病毒的纯化及免疫原性初步研究

为了解纯化后灭活发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的免疫原性,本研究通过超滤浓缩和分子筛层析相结合的方法对灭活的SFTSV进行纯化,采用Western blot和SDS-PAGE对灭活病毒的纯化样品进行分析和鉴定,采用双抗夹心ELISA方法对其中糖蛋白(Glycoprotein,GP)和核蛋白(Nucleoprotein,NP)的抗原含量进行检测。浓缩纯化的SFTSV灭活病毒,经电镜观察并通过BCA法测定其总蛋白浓度。然后,将纯化的灭活SFTSV按两种不同的免疫程序免疫新西兰兔,评价免疫原性和比较免疫效果。结果显示,SFTSV灭活并超滤浓缩后,分子筛层析可观察到两个洗脱峰,其中之一为灭活病毒颗粒,SDS-PAGE、Western blot和ELISA法分析均可检测到GP和NP抗原,而另一洗脱峰主要成分则为NP。浓缩后的纯化病毒纯度达90%以上,总蛋白浓度约为1.1mg/mL,且具有典型的布尼亚病毒电镜形态。纯化的SFTSV灭活病毒免疫实验动物后,可在血清中检出高滴度病毒特异性IgG和中和抗体,但抗体滴度受免疫程序的影响,0d、14d和28d天三针程序免疫动物的效果明显优于0d、7d和28d免疫程序组。本研究显示了灭活SFTSV培养液中除完整病毒颗粒外,还含有大量游离的NP,经分子筛层析纯化可获得高纯度灭活病毒,同时明确了纯化的SFTSV灭活病毒具有良好的免疫原性,可诱导产生高滴度中和抗体和病毒特异IgG抗体,可为灭活病毒疫苗的进一步研究提供重要的线索。     

低pH孵放、干热处理冻干静脉注射人免疫球蛋白的实验研究

主要介绍冻干静脉注射人免疫球蛋白 (IVIG)分别通过 60℃ 72h,80℃ 72h ,低pH孵放 2 1d处理后 ,IVIG的各项理化及生物活性的变化 ,以及IVIG经过低pH孵放的灭活病毒情况。实验结果表明 ,处理后的IVIG其IgG各组份相对含量、抗补体活性 (ACA)、前激肽释放酶激活剂 (PKA)、白喉抗体、抗 HBs等结果 ,除ACA ,PKA略有下降 ,其他指标无明显变化 ,各项指标均符合 2 0 0 0版《中国生物制品规程》 ;加热后没有新抗原产生。低pH孵放 2 1d通过加指示病毒的实验 ,病毒灭活效果达到 7.0 0logTCID50 / 0 .1mL以上 ,灭活病毒有效。     

S/D灭活血浆内脂包膜病毒及病毒灭活血浆的研究

研究磷酸三丁酯(TNBP)／Triton X-100对血浆内脂包膜病毒的灭活效果。用VSV病毒和Sindbis病毒作指示病毒,加入血浆后再加磷酸三丁酯／Triton X-100,观察病毒的滴度变化及对血浆蛋白的影响。结果发现终浓度各为1％的磷酸三丁酯／Triton X-100在60min内可以灭活血浆内的两种指示病毒,而血浆蛋白的组成和功能变化很小。经层折、超滤后血浆内磷酸三丁酯和Triton X-100的残余量分别低于5μg/ml,表明S／D处理血浆的安全性和治疗作用都很好,其制剂冰冻血浆或冻干血浆可用于临床治疗凝血因子缺乏症,或用作血容量扩张剂。     

干热法对人血浆血管性假血友病因子冻干品中病毒灭活效果的观察

目的探讨干热灭活法(dry heat treatment)对血管性假血友病因子(von willebrand factor,v WF)中猪细小病毒(porcine parvovirus,PPV)和脑心肌炎病毒(encephalomyocarditis virus,EMCV)的灭活效果。方法以PPV或EMCV作为灭活指示病毒与v WF混合,并分别在(80±1)℃4、8、12、24、36、48、72 h,(90±1)℃4、12、24、36、48 h以及(99±1)℃5、15、30 min进行干热灭活处理。将在干热灭活前以及干热灭活后不同温度时间段的v WF样品接种ST细胞(swine testis)或Vero细胞培养,观察细胞病变(cytopathic effect,CPE),用Reed-Muench法计算残余PPV和EMCV的病毒滴度。对无CPE的样品进行盲传3代培养,验证其灭活效果,并检测干热处理前后的v WF活性。结果v WF中EMCV滴度在(80±1)℃、(90±1)℃以及(99±1)℃灭活条件下,均下降超过4.0 lg TCID_(50)/0.1 m L,v WF中PPV的滴度下降也均超过4.0 lg TCID_(50)/0.1m L。干热法灭活后v WF的活性下降20%,质量稳定。结论干热法可以灭活v WF中的PPV以及EMCV,且对样品的活性影响在允许范围之内。     

板层人工角膜辐照法病毒灭活验证方法的建立及灭活效果验证

目的建立板层人工角膜钴-60照射病毒灭活验证方法,用该方法对脂包膜病毒灭活效果进行验证。方法以伪狂犬病毒(pseudorabies virus,PRV)为指示病毒,将干燥的板层人工角膜浸泡在高滴度病毒液中,在2~8℃浸泡5 h,经剪碎、研磨等步骤后4℃放置12 h,确立角膜吸附和释放病毒的条件。之后,将吸附病毒并呈干燥状态的角膜以0、5、10、15、20、25 k Gy辐照剂量进行钴-60辐照,以确立的条件释放病毒并进行滴定,考察灭活效果。结果板层角膜在2~8℃浸泡5 h可吸附足量病毒;病毒滴度可达到4 lg值以上,满足病毒灭活验证的要求。样品经25 k Gy剂量钴-60照射后灭活PRV为2.75~3.25 lg TCID50/100μL,灭活后的样品在敏感细胞上盲传3代均未出现细胞病变。结论成功建立了板层人工角膜病毒灭活验证的方法,并验证采用钴-60辐照法对PRV有较好的灭活效果。     

流感病毒对福尔马林敏感性的研究

采用三种流感病毒株：A1/京防/1/86、A3/济防/15/90、A3/武汉/359/95,进行敏感性试验。用不浓度的福尔马林灭活病毒,并在不同间隔时间检测血凝交价及灭活效果。结果显示,三种毒株经福尔马林灭活后,血凝滴度均有下降,病毒灭活时间也随着福尔马林浓度的降低而延长,说明相同亚型不同株别及不同亚型的流感病毒,对福尔马林的敏感程度均有差异。该试验对今后流感病毒的研究及其疫苗生产均有重要意义。     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

Immunologic status in Hodgkin patients: correlation with Epstein-Barr virus titers]

1. In Hodgkin's disease patient's immunological in vitro and in vivo parameters are of prognostic importance. 2. Skin test reactivity correlates to peripheral T-lymphocyte counts and Con A induced lymphoblastogenesis. 3. Con A is the most sensitive in vitro indicator for detecting latent immunodeficiency. 4. Hodgkin patients in long term remission after tumor reductive therapy exhibit a qualitative and quantitative lymphocyte defect. 5. In Hodgkin patients Herpes virus related antibody titers are elevated against Epstein Barr virus (EBV). The elevation coincides with a decreased T-cell number and function. Antibodies against other Herpes viruses (HSV, CMV, VZV) are in the normal range, when tested by the complement fixation method.     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.

Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI grew to lower titers in melanoma cells than did parental VZV. While VZV deleted for gI replicated in other human cells, the mutant virus replicated to very low titers in primary guinea pig and monkey cells and did not replicate in Vero cells. When compared with the parental virus, rescued viruses, in which the gI deletion was restored with a wild-type allele, showed a similarly sized gE and comparable growth patterns in melanoma and Vero cells. VZV deleted for gI entered Vero cells; however, viral DNA synthesis was impaired in these cells. The VZV gI mutant was slightly impaired for adsorption to human cells. Thus, VZV gI is required for replication of the virus in Vero cells, for efficient replication of the virus in nonhuman cells, and for normal processing of gE.     

Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes

For the manufacturing of recombinant protein therapeutics produced from mammalian cell culture, demonstrating the capacity of the purification process to effectively clear infectious viruses is a regulatory requirement. At least two process steps, using different mechanisms of virus removal and/or inactivation, should be validated in support of the regulatory approval process. For example, exposure of the product stream to low pH, detergents or solvent/detergent combinations is commonly incorporated in protein purification processes for the inactivation of lipid‐enveloped viruses. However, some proteins have limited stability at low pH or in the presence of the detergents, and alternative techniques for achieving the inactivation of enveloped viruses would be beneficial. We present here an alternative and novel approach for the rapid inactivation of enveloped viruses using pH‐neutral buffer solutions containing arginine. The implementation of this approach in a monoclonal antibody or Fc‐fusion protein purification process is described and illustrated with several different therapeutic proteins. The use of the neutral pH arginine solution was able to effectively inactivate two enveloped model viruses, with no measurable effect on the product quality of the investigated proteins. Thus, the use of pH‐neutral arginine containing buffer solutions provides an alternative means of virus inactivation where other forms of virus inactivation, such as low pH and/or solvent/detergent treatments are not possible or undesirable due to protein stability limitations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:108–112, 2014     

S/D法灭活血液制剂中脂包膜病毒效果验证的研究

选用不同核酸的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证S/D法处理对纤维蛋白原、凝血酶原复合物、凝血因子Ⅷ、静注丙种球蛋白、免疫血浆等血液制剂的病毒灭活效果。结果该法对所有被处理的血液制剂中的PRV及VSV灭活能力分别为≥3.38~5.88和≥3.50~4.75logTCID50/0.1ml,表明S/D法对两种病毒核酸类型的脂包膜病毒有良好的灭活效果。     

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.