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。     

国内外关于人用疫苗病毒安全性检测的研究

人用疫苗产品安全性检测的重点是确定生产基质及原辅料有无潜在病毒污染。按照2010版中国药典(以下简称CP)病毒外源因子检查,并与欧洲药典7.0版(以下简称EP7.0)和美国药典(以下简称USP34/NF29)进行比较,发现2010版CP在细胞基质病毒外源因子检测要求上比2005版CP有较大的提高,并对检测结果有效性的控制进一步加强。USP34/NF29规定检测的病毒种类繁多,并且根据种属来源的特异性及病毒的亲嗜性,列出了敏感指示细胞系,而2010版CP对上述部分内容未作规定或欠详细表述。因此在今后药典的标准提高方面,应结合国内外相关病毒外源因子检测的实际情况,加强疫苗安全性;以EP7.0或USP34/NF29标准为依据进行药品研发及药品审评工作的人员应注意其中的差别,勿混淆套用。     

弱毒疫苗中鸡传染性贫血病毒低剂量污染对SPF鸡体重和疫苗免疫抗体的影响

为了观察使用污染有低剂量鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)弱毒疫苗后对鸡免疫和体重的影响,本研究通过人工模拟试验观察了CIAV低剂量污染对SPF鸡体重以及对NDV疫苗抗体产生的影响。结果显示使用每羽份污染10个EID50CIAV和5个EID50CIAV两种剂量的NDV疫苗后均造成了SPF鸡体重的下降,与使用未污染疫苗组相比差异显著;并且使用污染两种剂量CIAV的疫苗后NDV抗体水平与使用无污染组相比也降低了,差异显著。使用污染两种剂量CIAV的NDV疫苗后第2周开始均检测到一定比例的CIAV抗体阳性,而通过核酸检测在使用污染疫苗后第1周就检测到很高比例的CIAV核酸阳性。研究结果不仅展示了弱毒疫苗中CIAV污染对SPF鸡生产性能和免疫机能的影响,也提示我们在通过SPF鸡检查法检测外源病毒污染时增加对病毒核酸检测有助于节省检测时间和提高检出率。     

狂犬病毒在Vero细胞上的适应传代研究

我国目前用于预防狂犬病的疫苗主要是地鼠肾细胞狂犬病疫苗,由于其所用毒种是脑毒种,不仅容易导致外源因子污染,还会将脑组织成分带入疫苗。为了排除鼠脑毒种带来的外源因子污染,应用Vero细胞做基质生产液体毒种,可以得到理想的病毒滴度,而且免疫原性也较好。1材料与方法1．1材料1．1．1病毒株CTN－1V5由中国药品生物制品检定所提供。1．1．2细胞及其培养Vero细胞来源于ATCC,用常规方法培养。1．2方法1．2．1毒种的传代方法取CTN－1V5干燥毒种稀释后与Vero细胞混种于培养液中,用199培养液,33℃培养168h收获病毒液,如此连续…     

Vero细胞狂犬疫苗研究和生产进展

自ＷＨＯ推荐Ｖｅｒｏ细胞用于疫苗生产后,国内外相继进行以Ｖｅｒｏ细胞为基质的人用狂犬疫苗的研究和应用,Ｖｅｒｏ细胞以繁殖快,质量可高效控制,不污染外源因子,对不同毒株有广泛的适应性,且可在细胞反应器中进行工业化生产,病毒滴度高、成本低、疫苗质量好,有较大的发展前景。     

用CaG株毒种制备精制狂犬病疫苗的研究

通过对狂犬病毒aG株在金黄地鼠肾细胞上传代培养,获得一种新型狂犬病毒毒株,即地鼠肾细胞适应株(CaG株)[1].由于该毒种具有生产方法简单,成本低廉,且外源因子污染机率小等优点,因此试用该毒种生产精制狂犬病疫苗.在相同条件下,分别用豚鼠脑毒种和细胞毒种各生产3批病毒原液,经相同纯化工艺制备成精制狂犬病疫苗.经初步检定用细胞毒种制备的疫苗安全性良好,疫苗免疫效价与豚鼠脑毒种疫苗无明显差异.     

基于甲病毒的RNA复制子疫苗

RNA复制子疫苗利用源自病毒能够自主复制的RNA,结构蛋白基因由外源抗原基因取代,保留了非结构蛋白基因,非结构蛋白可控制载体RNA在胞浆中高水平复制和外源基因的高水平表达。RNA复制子疫苗克服了传统疫苗和普通DNA疫苗存在的缺点,具有免疫效果显著、安全性好、应用范围广等优点,具有很好的应用前景。用于RNA复制子疫苗的载体主要源自甲病毒,本文以甲病毒载体为例,简要阐明RNA复制子疫苗的基本原理和特点,并对其应用作一综述。     

RNA复制子疫苗研究进展

最近兴起的RNA复制子疫苗,利用源自病毒的能够自主复制的RNA,其结构蛋白基因由外源抗原基因取代,保留了非结构蛋白(RNA复制酶)基因。RNA复制酶可使RNA载体在细胞质中高水平复制,并实现外源抗原基因的高水平表达,可同时诱导细胞免疫和体液免疫应答。大量双链RNA可诱导被感染细胞凋亡,宿主细胞的凋亡有利于免疫系统识别外源抗原。RNA复制子疫苗克服了传统疫苗和普通DNA疫苗存在的缺点,具有抗原表达效率高、安全性好、应用范围广等优点,因而被视为一种发展前景很好的疫苗形式。目前已对一些疾病模型基于复制子的治疗性和预防性疫苗进行了研究(涉及的对象包括病毒、肿瘤以及细菌毒素等),并对某些不足之处进行了改进。     

活载体疫苗的研究进展

免疫学与生物工程等领域取得的巨大进步,为发展基因工程疫苗提供了更多的理论和技术支持。活载体疫苗(Live Vector Vaccine,LVV),是以细菌或病毒作为外源抗原和治疗因子的载体系统,是目前最具发展潜力的基因工程疫苗之一。不同疫苗载体、抗原呈递方式和对宿主细胞免疫系统启动机制等研究的深入,开拓了活载体疫苗的实用价值。对国际上相关研究予以重点综述,以期为深入开展该领域研究提供依据。     

用于疫苗生产的人二倍体细胞研究进展*

人二倍体细胞(human diploid cells, HDCs)作为制备疫苗的重要培养细胞基质备受人们的关注。由于人二倍体细胞与人类基因组相同、无外源因子、对多种病毒易感性、无潜在致瘤性、所制备的人二倍体疫苗(human diploid cell vaccine, HDCV)具有良好的免疫原性和安全性,适合于疫苗的工业化生产。当前,人群中使用的灭活疫苗、减毒疫苗或亚单位疫苗等均依赖于原代细胞、传代细胞和人二倍体细胞,其中用于疫苗制备的人二倍体细胞主要有WI-38、MRC-5、2BS和KMB-17等细胞系。然而,人二倍体细胞为有限性细胞,细胞的来源和培养技术等存在某些缺陷,进而影响其应用。综述了用于疫苗生产的人二倍体细胞及其疫苗制备技术的研究进展,并分析了存在的问题及改进策略。     

Proposed algorithm to investigate latent and occult viruses in vaccine cell substrates by chemical induction

The recent urgency to develop new vaccines for emerging and re-emerging diseases, such as pandemic influenza, has necessitated the use of cell substrates not previously used in the manufacture of licensed vaccines. A major safety concern in the use of novel cell substrates is the presence of potential adventitious agents, such as latent and occult viruses, that may not be detected by currently used conventional assays. In cases where the novel cell substrate is known to be tumorigenic, there are additional safety issues related to tumorigenicity of intact cells and oncogenicity of residual cellular DNA. We have developed a strategy for evaluating vaccine cell substrates for the presence of latent/occult viruses, including endogenous retroviruses, latent RNA viruses and oncogenic DNA viruses, by optimizing conditions for chemical induction of viruses and using a combination of broad and specific assays to enable detection of known and novel viruses.     

Detection of mutants in polio vaccine viruses using pooled antipoliovirus monoclonal antibodies.

We prepared six monoclonal antibodies (mAbs) for type 1 polioviruses, and analysed their neutralizing specificities for use in safety tests in oral poliomyelitis vaccine (OPV) production. Pools of two or more individual mAbs showed high neutralizing activity against high-titre (approximately 10(7) CCID (50)/25 microl) of Sabin type 1 virus. It was demonstrated that the pooled mAbs can be utilized effectively in detection tests of adventitious viruses, which are among the safety tests in OPV production. Moreover, some pooled mAbs were shown to be capable of detecting very small amounts of type 1 virulent viruses and mutants in high-titre Sabin type 1 virus suspensions. Neutralizing antibody titres of these pooled mAbs decreased with increasing numbers of mutants containing neurovirulent activity in high-titre Sabin type 1 viruses which were repeatedly passaged in culture. It is expected that these pooled mAbs will contribute greatly to safety tests for OPV production.     

植物口服疫苗的研究进展

植物口服疫苗是通过转基因植物生产,通过口服的方式预防疾病的生物制品.作为一种新型疫苗,其研究开始于三十几年前.由于植物口服疫苗可以最大程度地降低传统疫苗的潜在风险,在疫苗生产中具有优势,因此拥有良好的商业生产前景.植物疫苗价格低廉,生产过程安全,可产生与注射疫苗相似效价效果,无论是在控制养殖业抗生素滥用的情况下作为替代...     

Vaccine and adjuvant design for emerging viruses: mutations, deletions, segments and signaling

Vaccination is currently the most effective strategy to medically control viral diseases. However, developing vaccines is a long and expensive process, and traditional methods, such as attenuating wild-type viruses by serial passage, may not be suitable for all viruses and may lead to vaccine safety considerations, particularly in the case of the vaccination of particular patient groups, such as the immunocompromised and the elderly. In particular, developing vaccines against emerging viral pathogens adds a further level of complexity, as they may only be administered to small groups of people or only in response to a specific event or threat, limiting our ability to study and evaluate responses. In this commentary, we discuss how novel techniques may be used to engineer a new generation of vaccine candidates as we move toward a more targeted vaccine design strategy, driven by our understanding of the mechanisms of viral pathogenesis, attenuation and the signaling events which are required to develop a lasting, protective immunity. We will also briefly discuss the potential future role of vaccine adjuvants, which could be used to bridge the gap between vaccine safety, and lasting immunity from a single vaccination.     

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu^®); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10³⁴. Residual MDCK-DNA is ≤10 ng per dose and the ß-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.     

Risk perception,risk management and safety assessment: What can governments do to increase public confidence in their vaccine system?

For decades vaccine program managers and governments have devoted many resources to addressing public vaccine concerns, vaccine risk perception, risk management and safety assessment. Despite ever growing evidence that vaccines are safe and effective, public concerns continue. Education and evidence based scientific messages have not ended concerns. How can governments and programs more effectively address the public’s vaccine concerns and increase confidence in the vaccine safety system? Vaccination hesitation has been attributed to concerns about vaccine safety, perceptions of high vaccine risks and low disease risk and consequences. Even when the public believes vaccines are important for protection many still have concerns about vaccine safety. This overview explores how heuristics affect public perception of vaccines and vaccine safety, how the public finds and uses vaccine information, and then proposes strategies for changes in the approach to vaccine safety communications. Facts and evidence confirming the safety of vaccines are not enough. Vaccine beliefs and behaviours must be shaped. This will require a shift in the what, when, how and why of vaccine risk and benefit communication content and practice. A change to a behavioural change strategy such as the WHO COMBI program that has been applied to disease eradication efforts is suggested.     

Enteric Viruses of Humans and Animals in Aquatic Environments: Health Risks, Detection, and Potential Water Quality Assessment Tools

Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools.     

Enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools.

Waterborne enteric viruses threaten both human and animal health. These pathogens are host specific and cause a wide range of diseases and symptoms in humans or other animals. While considerable research has documented the risk of enteric viruses to human health from contact with contaminated water, the current bacterial indicator-based methods for evaluation of water quality are often ineffectual proxies for pathogenic viruses. Additionally, relatively little work has specifically investigated the risk of waterborne viruses to animal health, and this risk currently is not addressed by routine water quality assessments. Nonetheless, because of their host specificity, enteric viruses can fulfill a unique role both for assessing health risks and as measures of contamination source in a watershed, yet the use of animal, as well as human, host-specific viruses in determining sources of fecal pollution has received little attention. With improved molecular detection assays, viruses from key host groups can be targeted directly using PCR amplification or hybridization with a high level of sensitivity and specificity. A multispecies viral analysis would provide needed information for controlling pollution by source, determining human health risks based on assessments of human virus loading and exposure, and determining potential risks to production animal health and could indicate the potential for the presence of other zoonotic pathogens. While there is a need to better understand the prevalence and environmental distribution of nonhuman enteric viruses, the development of improved methods for specific and sensitive detection will facilitate the use of these microbes for library-independent source tracking and water quality assessment tools.     

Multiplex PCR for rapid detection of minute virus of mice,bovine parvovirus,and bovine herpesvirus during the manufacture of cell culture-derived biopharmaceuticals

Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals since numerous adventitious viruses get contaminated during the manufacturing process. In particular, Chinese hamster ovary (CHO) cells are highly susceptible to minute virus of mice (MVM), bovine parvovirus (BPV), and bovine herpesvirus (BHV). Therefore, viral detection during CHO cell culturing is necessary to ensure the safety of biopharmaceuticals against viruses. In this study, a multiplex PCR assay was developed and subsequently evaluated for its effectiveness to simultaneously detect MVM, BPV and BHV during the manufacture of cell culture-derived biopharmaceuticals. Specific primers for MVM, BPV, and BHV were selected, and a multiplex PCR was optimized. The sensitivity of the assay was 6.49 × 10¹ TCID₅₀/mL for MVM, 7.23 × 10² TCID₅₀/mL for BPV, and 5.80 × 10¹ TCID₅₀/mL for BHV. The multiplex PCR assay was very specific to MVM, BPV, and BHV and was subsequently applied to the validation of CHO cells artificially infected with each virus. It could detect each viral DNA from CHO cells as well as culture supernatants. Therefore, we concluded that the multiplex PCR assay is invaluable for detecting adventitious viruses during the manufacture of cell culture-derived biopharmaceuticals.