Vero细胞无血清培养技术的研究与应用

Vero细胞是世界卫生组织和我国生物制品规程认可的疫苗生产细胞系。随着对疫苗质量和安全性要求的不断提高,用无血清培养基取代含血清培养基培养Vero细胞已成为病毒疫苗生产的一个重要发展趋势。Vero细胞无血清培养的技术关键是研发或选择能支持细胞以贴附培养方式生长的无血清培养基。微载体培养是贴附依赖性细胞系规模化培养和病毒疫苗生产的有效技术途径。我们对Vero细胞无血清培养基的研发、Vero细胞无血清培养及病毒疫苗生产工艺做了讨论,对该领域存在的问题和发展策略进行了展望。     

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。     

Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.     

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...     

Expression of anti-HVEM single-chain antibody on tumor cells induces tumor-specific immunity with long-term memory

Genetic engineering of tumor cells to express immune-stimulatory molecules, including cytokines and co-stimulatory ligands, is a promising approach to generate highly efficient cancer vaccines. The co-signaling molecule, LIGHT, is particularly well suited for use in vaccine development as it delivers a potent co-stimulatory signal through the Herpes virus entry mediator (HVEM) receptor on T cells and facilitates tumor-specific T cell immunity. However, because LIGHT binds two additional receptors, lymphotoxin β receptor and Decoy receptor 3, there are significant concerns that tumor-associated LIGHT results in both unexpected adverse events and interference with the ability of the vaccine to enhance antitumor immunity. In order to overcome these problems, we generated tumor cells expressing the single-chain variable fragment (scFv) of anti-HVEM agonistic mAb on the cell surface. Tumor cells expressing anti-HVEM scFv induce a potent proliferation and cytokine production of co-cultured T cells. Inoculation of anti-HVEM scFv-expressing tumor results in a spontaneous tumor regression in CD4+ and CD8+ T cell-dependent fashion, associated with the induction of tumor-specific long-term memory. Stimulation of HVEM and 4-1BB co-stimulatory signals by anti-HVEM scFv-expressing tumor vaccine combined with anti-4-1BB mAb shows synergistic effects which achieve regression of pre-established tumor and T cell memory specific to parental tumor. Taken in concert, our data suggest that genetic engineering of tumor cells to selectively potentiate the HVEM signaling pathway is a promising antitumor vaccine therapy.     

Plasmid-DNA lipid and polymeric nanovaccines: a new strategic in vaccines development

Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid establishment of facilities for the development of new vaccines are stimulating significate changes in the vaccine industry, which is gradually moving towards cell culture production. One approach is the third generation of vaccines, which are based on the use of plasmid DNA (pDNA) containing transgenes that encode an antigen capable of mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Plasmid DNA vaccination has distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. The effectiveness of pDNA vaccines against viruses, bacteria, parasites and cancer cells has been demonstrated in preclinical and clinical assays. Furthermore, currently there are a few veterinary pDNA vaccines in the market. The application of a simple formulation of naked pDNA as a vaccine is attractive, but a low transfection efficiency is often obtained. The use of nanoparticles to increase transfection efficiency is an approach that has been tested clinically. This review provides a summary of vaccine production, advances and major challenges associated with pDNA lipid and polymeric nanovaccines applications.     

A new generation of HIV vaccines

WHO estimates that currently there are 40 million individuals living with HIV and there are 16000 new infections daily, worldwide. The best strategy to control the AIDS epidemic would be the development of an effective vaccine. New strategies for vaccine development have gained momentum over the past decade, some of which show greater promise in macaque models than did earlier protein-subunit or recombinant-canarypox strategies. These new vaccines include DNA vaccines and live viral vectors, and have been based on the generation of high levels of antiviral T cells. These vaccines do not prevent infection, but rather control virus replication with a rapid expansion and then contraction of antiviral T cells in response to the challenge infection. These recent vaccine successes in macaques raise hope that a vaccine can be developed that will successfully limit both the development of AIDS and viral transmission.     

High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7

Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines.     

Process intensification of EB66® cell cultivations leads to high-yield yellow fever and Zika virus production

A live-attenuated, human vaccine against mosquito-borne yellow fever virus has been available since the 1930s. The vaccine provides long-lasting immunity and consistent mass vaccination campaigns counter viral spread. However, traditional egg-based vaccine manufacturing requires about 12 months and vaccine supplies are chronically close to shortages. In particular, for urban outbreaks, vaccine demand can be covered rarely by global stockpiling. Thus, there is an urgent need for an improved vaccine production platform, ideally transferable to other flaviviruses including Zika virus. Here, we present a proof-of-concept study regarding cell culture-based yellow fever virus 17D (YFV) and wild-type Zika virus (ZIKV) production using duck embryo-derived EB66® cells. Based on comprehensive studies in shake flasks, 1-L bioreactor systems were operated with scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion systems for process intensification. EB66® cells grew in chemically defined medium to cell concentrations of 1.6 × 10⁸ cells/mL. Infection studies with EB66®-adapted virus led to maximum YFV titers of 7.3 × 10⁸ PFU/mL, which corresponds to about 10 million vaccine doses for the bioreactor harvest. For ZIKV, titers of 1.0 × 10¹⁰ PFU/mL were achieved. Processes were automated successfully using a capacitance probe to control perfusion rates based on on-line measured cell concentrations. The use of cryo-bags for direct inoculation of production bioreactors facilitates pre-culture preparation contributing to improved process robustness. In conclusion, this platform is a powerful option for next generation cell culture-based flavivirus vaccine manufacturing.

     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.     

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV‐positive tumour cells

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.     

Evaluation of tumorigenic potential of high yielding cloned MDCK cells for live-attenuated influenza vaccine using in vitro growth characteristics,metastatic gene expression and in vivo nude mice model

Several mammalian cell lines, including Madin–Darby canine kidney (MDCK) cells have been approved by regulators for manufacturing of human vaccines. A new MDCK 9B9-1E4 cloned cell line has been created which is capable of producing live attenuated influenza vaccine (LAIV) with high yield. This cell line was shown to be non tumorigenic in eight week old adult athymic nude mouse model. This property is desirable for vaccine production and is unique to this cell line and is not known to be shared by other MDCK cell lines that are currently used for vaccine production. This significant difference in tumorigenic phenotype required further characterization of this cell line to ensure its safety for use in vaccine production. This is particularly important for LAIV production where it is not possible to incorporate a virus inactivation and/or removal step during manufacturing. Characterization of this cell line included extensive adventitious agent testing, tumorigenicity and oncogenicity assessment studies. Here, we describe the development of tumorigenic MDCK cell lines for use as positive controls and in vitro methods to aid in the evaluation of the tumorigenicity of MDCK 9B9-1E4 cloned cells. Tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK 9B9-1E4 cloned cells. In this study we demonstrate the lack of tumorigenic potential of the MDCK 9B9-1E4 cloned cell line in adult athymic nude mice model.     

Vaccine process technology

The evolution of vaccines (e.g., live attenuated, recombinant) and vaccine production methods (e.g., in ovo, cell culture) are intimately tied to each other. As vaccine technology has advanced, the methods to produce the vaccine have advanced and new vaccine opportunities have been created. These technologies will continue to evolve as we strive for safer and more immunogenic vaccines and as our understanding of biology improves. The evolution of vaccine process technology has occurred in parallel to the remarkable growth in the development of therapeutic proteins as products; therefore, recent vaccine innovations can leverage the progress made in the broader biotechnology industry. Numerous important legacy vaccines are still in use today despite their traditional manufacturing processes, with further development focusing on improving stability (e.g., novel excipients) and updating formulation (e.g., combination vaccines) and delivery methods (e.g., skin patches). Modern vaccine development is currently exploiting a wide array of novel technologies to create safer and more efficacious vaccines including: viral vectors produced in animal cells, virus-like particles produced in yeast or insect cells, polysaccharide conjugation to carrier proteins, DNA plasmids produced in E. coli, and therapeutic cancer vaccines created by in vitro activation of patient leukocytes. Purification advances (e.g., membrane adsorption, precipitation) are increasing efficiency, while innovative analytical methods (e.g., microsphere-based multiplex assays, RNA microarrays) are improving process understanding. Novel adjuvants such as monophosphoryl lipid A, which acts on antigen presenting cell toll-like receptors, are expanding the previously conservative list of widely accepted vaccine adjuvants. As in other areas of biotechnology, process characterization by sophisticated analysis is critical not only to improve yields, but also to determine the final product quality. From a regulatory perspective, Quality by Design (QbD) and Process Analytical Technology (PAT) are important initiatives that can be applied effectively to many types of vaccine processes. Universal demand for vaccines requires that a manufacturer plan to supply tens and sometimes hundreds of millions of doses per year at low cost. To enable broader use, there is intense interest in improving temperature stability to allow for excursions from a rigid cold chain supply, especially at the point of vaccination. Finally, there is progress in novel routes of delivery to move away from the traditional intramuscular injection by syringe approach.     

The North East Water polynya (Greenland Sea)

Physical observations of the North East Water (NEW) polynya, located near the north-eastern corner of Greenland, are presented. Data were collected in June 1991 by RV Polarstern. An idea is put forward to explain how the NEW is generated. A northward coastal current interacts with a persistent shelf ice barrier under which water can flow but that retains ice floes and therefore protects the NEW area from ice advection. Since in summer, the combination of currents, barrier and air-sea heat balance gives rise to a polynya. The distribution of upper water column vertical stability in the NEW is also influenced by its generation process. Surface melt water is retained by the shelf ice barrier, causing neutral vertical stability in its lee. Sea ice melting and land runoff then act as two distinct sources of vertical stability enabling the development of plankton blooms, especially in the northern part of the NEW.     

Host-range restriction of vaccinia virus E3L deletion mutant can be overcome in vitro, but not in vivo, by expression of the influenza virus NS1 protein

During the last decades, research focused on vaccinia virus (VACV) pathogenesis has been intensified prompted by its potential beneficial application as a vector for vaccine development and anti-cancer therapies, but also due to the fear of its potential use as a bio-terrorism threat. Recombinant viruses lacking a type I interferon (IFN) antagonist are attenuated and hence good vaccine candidates. However, vaccine virus growth requires production in IFN-deficient systems, and thus viral IFN antagonists that are active in vitro, yet not in vivo, are of great value. The VACV E3 and influenza virus NS1 proteins are distinct double-stranded RNA-binding proteins that play an important role in pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. Based on the functional similarities between E3 and NS1, we investigated the ability of NS1 to replace the biological functions of E3 of VACV in both in vitro and in vivo systems. For this, we generated a VACV recombinant virus lacking the E3L gene, yet expressing NS1 (VVΔE3L/NS1). Our study revealed that NS1 can functionally replace E3 in cultured cells, rescuing the protein synthesis blockade, and preventing apoptosis and RNA breakdown. In contrast, in vivo the VVΔE3L/NS1 virus was highly attenuated after intranasal inoculation, as it was unable to spread to the lungs and other organs. These results indicate that there are commonalities but also functional differences in the roles of NS1 and E3 as inhibitors of the innate antiviral response, which could potentially be utilized for vaccine production purposes in the future.     

Bacterially Produced Recombinant Influenza Vaccines Based on Virus-Like Particles

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein''s neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.     

Effect of modifiers of natural biological response on the functional activity of macrophages (oncological aspect)

Induction of cytotoxicity by biological response modifiers (BRMs) is only one aspect of macrophage activation. After the use of BRMs there were other changes in the functional activity of cells and in particular their increased production or secretion of a number of growth factors. Thus, activation of macrophage antitumor activity induced by BCG vaccine was transitory while activation of growth factor production was more stable in time which finally led to increased proliferation of tumor cells. Combined use of cyclophosphamide and BCG vaccine significantly increased not only the toxicity induced by BCG vaccine but also their liberation of the growth factors. Such macrophages lost their ability to control the growth of a small number of the tumor cells cultivated in their presence. Development of ways for directed activation of macrophages aimed at elimination of the tumor cells which survived the chemotherapy should include evaluation of the combined effect of various BRMs and chemotherapeutics on both antitumor and protumor activity i. e. ability to produce the factors stimulating the tumor growth.     

犬肾细胞MDCK无血清贴壁及单细胞悬浮培养

旨在挖掘用于鉴定金黄色葡萄球菌的高特异性靶点及其PCR检测引物。采用C++语言编程,以金黄色葡萄球菌Staphylococcus aureus MRSA 252基因组编码序列为对象,对2 656个可编码区进行分析,获得特异性靶点序列,并设计PCR扩增引物。对包括葡萄球菌属11个种及其他细菌属在内的共计137株细菌验证引物特异性,筛选获得9个DNA序列,并设计了4对引物。经验证2对引物的特异性较好,其中引物SA3的基因组DNA检测限为13.7 fg/μL,菌体检测限为9.25×102 CFU/mL。结果验证     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

疫苗制备中外源病毒检测方法

疫苗安全性是公众及药品监管机构非常关注的问题。由于疫苗生产原材料如组织、细胞基质、血清、胰蛋白酶等来源于动物,因此疫苗产品存在外源病毒污染的风险。外源病毒对人有潜在危害,实施系统性的检测和采取严格的预防措施可以有效降低外源性因子污染的风险。几十年来,某些经典的检测法已经成为广谱筛查外源病毒污染的标准方法,但其存在一定的局限性。随着大规模基因并行测序、微阵列等分子生物学检测新方法的建立,疫苗外源因子检测方法日趋多样化,增加了检测疫苗外源因子污染种类和概率。