Rabies virus-like particles expressed in HEK293 cells

Rabies is an infectious viral disease with a mortality rate close to 100%. Currently, there is a need to generate cheaper and more immunogenic vaccines for the effective prevention of rabies, mostly in developing countries. Virus-like particles have been widely used in viral vaccine production due to their high immunogenicity and safety during the production process. Rabies virus glycoprotein is the major antigen to trigger a protective immune response and the only protein capable of generating virus neutralizing antibodies. In this study we describe the development of a recombinant stable cell line for the production of rabies virus-like particles (RV-VLPs) expressing the rabies virus glycoprotein by lentivirus-based transduction of HEK293 cells. Protein expression was analyzed by flow cytometry, fluorescence microscopy, western blot and ELISA. Particles were purified from culture supernatant and their size and morphology were studied. Furthermore, mice were immunized with RV-VLPs, formulated with adjuvant, and these particles were able to produce a specific antibody response, demonstrating that these virus-like particles present a promising rabies vaccine candidate.     

Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing

Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 10⁹ IVP/mL demonstrating the scale-up potential of the process.     

3-氯-1,2-丙二醇对人胚肾293细胞DNA损伤作用

目的 研究3-氯-1,2-丙二醇(3-MCPD)对人胚肾293(HEK293)细胞DNA损伤作用。方法 以HEK293细胞为靶细胞,3-MCPD设5个剂量组:0,1.25,2.5,5,10 mmol/L,应用单细胞凝胶电泳检测3-MCPD诱导的HEK293细胞DNA损伤;将3-MCPD设4个剂量组:0,2.5,5,10 mmol/L,以2,7-二氢二氯荧光素(DCFH)法测定HEK293细胞内活性氧(ROS)水平;将3-MCPD设4个剂量组:0,1.25,2.5,5 mmol/L,通过免疫组化法测定8-羟基脱氧鸟苷(8-OHdG)在HEK293细胞内的表达水平。结果 与对照组比较,1.25~10 mmol/L的染毒组细胞DNA链断裂,细胞形成彗星样脱尾,尾DNA%明显上升,且呈剂量依赖关系(P<0.05)。2.5~10 mmol/L的染毒组细胞内ROS水平表达升高,在10 mmol/L时明显升高(P<0.05)。1.25~5 mmol/L的染毒组细胞内8-OHdG表达水平上升,在2.5和5 mmol/L时8-OHdG表达明显增强(P<0.05)。结论 3-MCPD导致了HEK293细胞DNA损伤,可能是通过细胞内ROS升高及8-OHdG形成增加所造成的氧化性DNA损伤作用。     

ERK信号转导通路在CdCl2诱导HEK293细胞低剂量兴奋效应中的作用

目的 探讨ERK信号转导通路在CdCl2诱导HEK293细胞低剂量兴奋效应过程中的作用.方法 以0、0.0005、0.005、0.05、0.5、5、50和500μmol/L浓度的氯化镉(CdCl2)分别染毒HEK293细胞12h和24h;在染毒前分别用ERK1/2抑制剂PD98059(100μmol/L)、U0126(...     

短串联重复序列图谱分析法鉴定HEK293细胞传代的稳定性

目的 用短串联重复序列(STR)图谱20位点分析法对疫苗研发用HEK293细胞的传代稳定性进行研究.方法 对购自美国菌毒种保管中心(ATCC)的HEK293细胞进行传代,按中国药典三部要求建立主种子库(第40代),工作种子库(第43代)和生产终末细胞(第65代),并进行STR检测;按检测要求进行细胞扩增,DNA提取,PCR扩增及STR20位点分析.结果 STR图谱20位点分析法已全面覆盖美国ATCC 9位点和中检院16位点法,三级传代细胞检测结果与ATCC标准株29EcRShh(Adv5)匹配度100％,与中检院检测结果完全一致,无外源细胞的污染.结论 本实验室传代建库的HEK293细胞遗传稳定,无外源细胞污染,符合疫苗研发用要求.     

大鼠心脏kir2.2、kir2.3通道重组质粒构建及真核表达

目的 构建大鼠心脏kir2.2、kir2.3通道真核表达质粒,并鉴定其在人胚胎肾细胞（HEK293）中的表达。方法 提取大鼠心脏组织细胞RNA,逆转录扩增kir2.2、kir2.3通道编码基因,克隆至真核表达质粒pEGFP-N1中,构建重组质粒并转染HEK293细胞,应用全细胞膜片钳法定kir2.2、kir2.3通道电流。结果 重组质粒pEGFP-N1-kir2.2及pEGFP-N1-kir2.3 经双酶切和测序证实构建正确,并在HEK293细胞中成功表达,且记录到相应通道电流。结论 成功构建了大鼠心脏kir2.2、kir2.3通道真核表达质粒,并在HEK293细胞中成功表达。     

Influence of HEK293 metabolism on the production of viral vectors and vaccine

Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals including viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of recombinant proteins and a well-established system for the production of viral vectors. Understanding metabolic requirements for high productivity in HEK293 cells remains an important area of investigation. Many authors have presented approaches for increased productivity through optimization of cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed to improving feeding strategies by addition of exhausted or critical substrates and eventually removal of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for optimization through better understanding of the cellular metabolism under different operating conditions. This review will present both approaches and their successes with regards to improvement of viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating remaining issues to address and problems to resolve to maximize the productivity of viral vectors in HEK293 cells.     

Ebola epidemic in war-torn Democratic Republic of Congo, 2018: Acceptability and patient satisfaction of the recombinant Vesicular Stomatitis Virus – Zaire Ebolavirus Vaccine

Background

The current Ebola outbreak in Eastern Democratic Republic of the Congo (DRC) is the second largest in history and the first in which the recombinant Vesicular Stomatitis Virus – Zaire Ebolavirus (rVSV-ZEBOV) vaccine has been used at scale. We assessed side-effects, satisfaction, and attitudes toward the new vaccine.

Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform

Large quantities of antigens are required since protective antigens, such as classical swine fever virus (CSFV) E2 protein, are widely used in diagnostic reagents and subunit vaccines. Compared to clonal cell lines and transient gene expression, stable cell pools provide a potential alternative platform to rapidly produce large amounts of antigens. In this work, firstly, Human embryonic kidney 293 T (HEK293T) cell pools expressing E2 protein were developed by transduction of lentiviral vectors. On the one hand, the SP7 was selected from 7 well-performing signal peptides to remarkably increase the production of E2 protein. On the other hand, it was found that high MOI could improve the expression of E2 protein by increasing gene copy numbers. Moreover, the HEK293T cell pools were evaluated for stability by passages and batch cultures, demonstrating that the cell pools were stable for at least 90 days. And then, the performance of the cell pools in batch, fed-batch, and semi-perfusion was studied. Among them, the titer of E2 protein was up to 2 g/L in semi-perfusion, which is currently the highest to the authors’ knowledge. Finally, the aggregations and immunogenicity of the E2 protein were analyzed by SDS-PAGE and immunization of mice, respectively. There was no significant difference in aggregations and antibody titers of E2 protein in three culture methods. These results suggest that stable HEK293T cell pools are a promising and robust platform for rapid and efficient production of recombinant proteins.     

Characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles co-produced in HEK-293SF

One of the concerns associated with the use of influenza virus-like particles (VLPs) as vaccine candidate or delivery system is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out not only the viral antigenic proteins but also host proteins. In addition, the intrinsic nature of cells to produce membrane derived vesicles or extracellular vesicles (EVs), which have similar size to the VLPs, makes VLP purification process challenging. To further characterize these particles and identify proteins that are unique to each population, comparative proteomic analyses were completed to ultimately provide guidance for rational design of separation protocols. The VLPs were produced in suspension and serum free media by transient transfection of an inducible clone of a Human Embryonic Kidney (HEK-293SF) cells expressing HA and NA (H1N1/A/Puerto Rico/8/34), with a plasmid containing the gag gene of HIV-1 fused to GFP. EVs were produced independently from the non-transformed HEK-293SF cell line as a control for comparative studies. Both preparations were characterized for total nucleic acids and protein concentrations and extensively analyzed by nanoLC-MS/MS for their protein compositions. The proteomic analyses showed that aside from the recombinant VLP proteins, nucleolin was the most abundant host cell protein uniquely identified within VLPs (considering the MASCOT score value) while lactotransferrin and heat shock protein 90 were the most abundant proteins in EVs. Overall, this comparative study identifies potential target proteins as specific markers to guide VLP purification and discusses the biogenesis of enveloped particles released in HEK-293 cell suspension cultures emphasizing on the biological functions of host cell proteins identified.     

New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: Studies on growth,metabolism and virus propagation

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8 × 10⁶ cells/mL were obtained with doubling times of 23 h for AGE1.CR and 35 h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 log HA/100 μL for influenza virus, 3.2 × 10⁸ pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.     

Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate

Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate.     

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”.In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100 L scale for several biologics, including recombinant viruses.Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.     

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150–200 nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.     

镉致HEK293细胞凋亡中氧化应激作用

目的探讨镉在诱导人胚胎肾细胞（HEK293）凋亡中氧化应激的作用。方法采用流式细胞仪检测细胞凋亡率;采用流式细胞仪和激光共聚焦检测胞浆内的活性氧（ROS）水平,采用分光光度法检测细胞内丙二醛（MDA）、还原型谷胱甘肽（GSH）含量,蛋白印迹法检测Bcl-2蛋白、Caspase-3酶原表达量的变化。结果在一定浓度范围内随着镉浓度的升高,凋亡率升高。在60μmol/L氯化镉诱导细胞6h,凋亡率达到最高;细胞内ROS生成显著增多;MND含量明显增多（P〈0.01）,GSH含量大幅下降（P〈0.01）;Bcl-2表达量较镉处理3h时表达量有所下降;Cas-pase-3酶原蛋白表达下降。结论镉诱导HEK293细胞凋亡的机制与ROS上升及其导致的氧化损伤、Bcl-2蛋白表达下调和Caspase-3的激活有关。     

Production and antigenic properties of influenza virus from suspension MDCK-siat7e cells in a bench-scale bioreactor

In efforts to overcome limitations associated with egg-based influenza vaccines, mammalian cell substrates have gradually emerged as potential production platforms. Recently, a suspension Madin Darby canine kidney (MDCK) cell line for influenza virus production was created by expressing the human siat7e gene. To examine the broad susceptibility of this novel cell line, the scalability of the production process, and the antigenic stability of cell-derived progeny viruses, infection experiments using four current influenza vaccine strains (A/California/07/2009 X-179A H1N1, A/Brisbane/59/2007 IVR-148 H1N1, A/Uruguay/716/2007 X-175C H3N2, and B/Brisbane/60/2008) were performed. In small-scale experiments, this cell line was found to support high-titer replication of all four virus strains. Subsequently, production in a bench-scale bioreactor and the antigenic characteristics of progeny viruses were assessed. High titers of hemagglutinin (at least 1:512) were produced in a 2-L bench-scale bioreactor with all four strains. Immunoblot results demonstrated higher yields in the cells than those obtained in chicken embryonated eggs with three of the four tested strains. Progeny viruses collected after serial passages in this cell line exhibited minimal mutations in the HA-encoding gene. Hemagglutination inhibition (HAI) assays using ferret antiserum confirmed the antigenic stability. As a proof-of-concept this work demonstrates that by using a proper strategy, high yields of biologically active hemagglutinin can be produced from scalable cultures of suspension MDCK-siat7e cells.     

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF^® (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀ (HA units/100 μL) and 1.8 × 10¹⁰ virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.     

Endo G基因过表达对镉诱导HEK-293细胞凋亡影响

目的观察细胞凋亡因子核酸内切酶G(endonuclease G,Endo G)基因过表达在镉诱导人胚胎肾细胞HEK-293凋亡中的影响。方法从人肝癌细胞系(human hepatocarcinoma cells,HepG 2)中提取RNA,经逆转录获取互补DNA cDNA),通过聚合酶链反应(PCR)扩增Endo G基因,与pcDNA 3.0相连,构建pcDNA 3.0-Endo G真核表达载体,转染HEK-293细胞,并结合不同浓度氯化镉处理细胞;通过蛋白印迹实验(Western blot)验证基因过表达,吖啶橙/溴乙锭(AO/EB)双染法及流式细胞术(FCM)检测Endo G基因过表达对镉诱导HEK-293细胞凋亡的影响,噻唑蓝法(MTT)检测细胞存活率。结果成功构建pcDNA 3.0-EndoG真核表达载体,Endo G基因扩增片段大小约1 100 bp;该载体转染细胞后,Endo G成功过表达,使HEK-293细胞凋亡率>37%,且随氯化镉浓度增加,Endo G表达量先上升后下降。结论Endo G以诱导细胞早期凋亡和晚期凋亡为主要形式。     

Ethanol potentiates dopamine uptake and increases cell surface distribution of dopamine transporters expressed in SK-N-SH and HEK-293 cells.

Ethanol increases dopaminergic release in the reward and reinforcement areas of the brain. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT). In vitro electrophysiological and biochemical studies in Xenopus laevis oocytes have previously shown ethanol potentiates DAT function and increases transporter-binding sites. The potentiating effect of ethanol on the transporter is eliminated in Xenopus oocytes by the DAT mutation glycine 130 to threonine. However, ethanol's action on DAT functional regulation has yet to be examined in mammalian cell expression systems. To further understand the molecular mechanisms of ethanol's action on DAT, we determined the direct mechanistic action of short-term (相似文献   

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.