应用昆虫细胞表达预防性人乳头瘤病毒疫苗和应用填充床生物反应器表达H1N1流感病毒疫苗的研究
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摘要
动物细胞培养起始于上世纪60年代初，由于动物细胞体系十分适合表达有生物活性的生物大分子，具有重要医用价值的疫苗、基因工程药物、抗体药物、蛋白药物等生物制品都可应用动物细胞表达。按照动物细胞不同的培养方式可分为悬浮培养、贴壁培养。本论文分两部分研究内容，第一部分为应用WAVE反应器悬浮培养昆虫细胞表达人乳头瘤病毒预防性疫苗的研究；第二部分为应用一次性填充床生物反应器贴壁培养MDCK细胞表达H1N1流感病毒的研究。
一、人乳头瘤病毒预防性疫苗研究
人类多种癌症，特别是子宫颈癌经相关的分子流行病学资料证明与HPV有着密切的关系。HPV的型别众多，人们不断地在皮肤癌症、疣状表皮等病理组织中发现新的HPV型别。目前为止已知型别已经突破200种。
根据中国大陆地区流行病学调查结果，选用在大陆地区的第三大优势型别-HPV58型作为疫苗的组分之一，确定以HPV16、18、58、6、11型为主要疫苗型别的五价疫苗。期望此疫苗可以保护75%以上的由HPV16、18、58引起子宫颈癌，还可预防由HPV6、11引起的低度癌前病变和生殖器疣。
主要内容如下：
HPV各型VLP的构建表达：通过PCR合成HPV16型、18型、58型、6型、11型主要衣壳蛋白全长L1目的基因，根据核定位信号设计PCR引物扩增截短型HPV16型、18型、58型、6型、11型目的基因。测序正确后分别重组入杆状病毒表达系统穿梭质粒pFastBac1，通过基因移位作用将目的基因片段重组入Bacmid基因组，提取重组的Bacmid DNA，M13引物PCR鉴定正确。各亚型重组杆状病毒Bacmid-HPVL1转染Sf9细胞，大量感染昆虫细胞后，分别表达52-55kD大小的蛋白，经Western-Blot鉴定，与Camvir1单克隆抗体产生特异性反应，证明转染成功目的蛋白为HPVL1蛋白。应用蔗糖垫离心结合氯化铯密度梯度超速离心纯化HPV各亚型VLP，将各亚型VLP透析后的样品放置于透射电镜下观察。发现其形成的病毒样颗粒与野生型病毒的大小和形态相似，在氯化铯溶液中密度为1.27g/ml。
HPV各型VLP免疫原性研究：通过全长型（HPV16-505）和截短型（HPV16-483）假病毒中和抗体试验证明：去除HPV16L1C端核定位信号的截短型（HPV16-483）中和抗体滴度显著高于全长型（HPV16-505）。说明截短型所诱发的体液免疫应答水平显著高于全长型。通过上述研究结果可知，去除核定位信号的VLP可能更易于被B淋巴细胞识别，有利于产生高滴度的中和抗体。因此在中和抗体实验中，仅对五种亚型的截短型VLP进行研究。
HPV16型、18型、6型单价疫苗免疫分别免疫小鼠后，均可产生高滴度的型特异中和抗体。1618双价疫苗和16186三价疫苗，分别免疫小鼠后均可产生针对于相应型别高滴度的中和抗体。多价HPV疫苗联合免疫时，随着疫苗型别增多，各型别的中和抗体滴度均出现降低的趋势，即存在免疫干扰现象。
HPV VLP生产工艺初步研究：以HPV6L1为例，研究了昆虫细胞体系的VLP疫苗生产工艺研究。首先比较不同接毒量、感染密度、细胞收获时间对HPV6L1蛋白表达量的影响。对比不同原理生物反应器培养昆虫细胞的区别，发现WAVE生物反应器更适合悬浮培养昆虫细胞。为了考察不同裂解方法对sf9昆虫细胞裂解的影响，结果表明与超声法、化学裂解法相比，低渗裂解法更适合于昆虫细胞裂解。又比较不同孔径的中空纤维微滤膜对sf9昆虫细胞收获的影响，选择了GE公司面积为420cm2的三种孔径微滤膜（0.10,0.22,和0.45μm），结果表明，与0.10,0.22μm中空纤维微滤膜相比，孔径为0.45μm中空纤维微滤膜具有更大的平均膜孔径，滤膜阻力较小，有利于培养基的透过，浓缩过程中可以缓解引起的膜表面凝胶层，因此具有更快的处理速度，其平均透过通量可达90L/hm2。在此基础上结合细胞破碎相关的实验结果，设计应用中空纤维系统作为一个整合系统，实现细胞收获液的浓缩、洗滤、抽提、澄清步骤。此工艺避免了离心机，死端过滤等实验步骤，而且在抽提过程中，采用低渗的方法裂解昆虫细胞，释放目的蛋白，避免了裂解剂（Triton X-100）的加入和超声仪的使用，使整个操作都处于密闭和无菌状态，更适合于工业化生产。
在柱层析初级分离纯化阶段，选择DMAE弱阴离子层析纯化HPV6L1蛋白，与Q Sepharose XL强阴离子层析相比，DMAE弱阴离子层析纯化的HPV6L1蛋白纯度更高，基本达到初级分离纯化的要求。之后在柱层析精制分离纯化阶段尝试了应用Octyl Sepherose FF、Butyl-S Sepherose FF、Fractogel EMDTMAE、CM Sepherose FF等层析介质进行精纯工艺摸索。考虑到初级分离纯化阶段采用的是阴离子交换层析，不同阶段采用不同原理的层析介质纯化蛋白更有效。因此在精制分离纯化阶段，选择Octyl Sepherose FF疏水层析纯化HPV6L1蛋白。又摸索了类病毒颗粒体外组装工艺，经解组装、重组装步骤后，获得了均一稳定的病毒样颗粒。对于上述优化的放大纯化工艺所制备的HPV6L1VLPs，进行了SF9细胞宿主DNA残留量；SF9昆虫细胞宿主蛋白残留量；HPV VLP疫苗抗原含量；内毒素等检测。
综上所述，本研究利用杆状病毒-昆虫细胞表达系统表达了HPV各型VLP，应用假病毒中和抗体体系评价了各型VLP疫苗的免疫原性。初步建立了昆虫细胞体系的HPV6L1的生产工艺和质量研究体系。以上研究结果为研制适合中国大陆地区昆虫细胞体系人乳头瘤病毒预防性疫苗提供参考。
二、应用一次性填充床生物反应器表达MDCK细胞表达H1N1流感病毒
流行性感冒是由流感病毒引起的，呼吸系统常见的传染性疾病，流感的临床特征为鼻咽部的急性炎症。流感是一种发病率高，传染性强，并且与患者的年龄、性别无关的传染病。接种合适的流感疫苗是避免流感传染最有效的方法。相关人群的发病率和死亡率通过接种疫苗可以明显降低，同时也减少了流感病毒的继续感染的机会。
鸡胚生产流感疫苗已有多年历史，临床应用表明其具有较好的免疫效果和安全性。其缺点在于：需要提供足够的SPF级鸡蛋来生产流感疫苗，鸡胚的质量标准和方法学研究不易控制和标准化；同时可能存在鸡胚带有禽流感病毒的危险；并且大规模SPF级种蛋的供应难以应对流感大暴发。由于流感病毒能够在多种动物细胞内繁殖，因此可以应用哺乳动物细胞大规模生产流感疫苗来满足流感的大流行性以及新发流感的需求。MDCK细胞(MDCK Cell Lines)是在上世纪五十年代由Madin和Darby经Cocker Spaniel犬的肾脏组织分离后培养获得，MDCK细胞通常是以贴壁方式生长的上皮样细胞。由于其病毒感染效率高、增殖快，且不易变异，被公认为最适于流感病毒疫苗生产的细胞系。
MDCK细胞为贴壁细胞，可用各型生物反应器大规模培养，其中包括：微载体生物反应器，填充床反应器，中空纤维生物反应器。与传统的填充床反应器不同，安普生物反应器采用外循环式纸片灌注培养方式，其分为灌注袋、培养袋两部分。灌注袋中放置，细胞培养液经培养袋置于特制摇床中，通过激流反应补充氧气，再经蠕动泵泵进灌注袋提供给吸附在聚酯纸片载体的贴壁细胞。安普生物反应器在细胞培养过程中通过PID模式可以自动控制相关的细胞生长参数。所用的一次性聚酯纸片载体内部相互交织的三维结构可提供传统培养模式无法比拟的表面积，适合多种类型细胞吸附生长，细胞增殖速度快维持时间长，符合生物安全要求。同时细胞聚集可形成支撑保护，减少培养基流动时对细胞产生的剪切力。因为贴壁细胞可吸附在聚酯纸片载体的表面和内部结构里，避免了由于培养基的流动可能造成的损伤，不需要中空纤维等特殊的分离细胞装置，更换培养液更方便。因此采用安普生物反应器可实现在含有血清的培养基条件下培养MDCK细胞，待细胞长满后迅速更换为适合病毒繁殖的无血清培养基，从而获得高效价的流感病毒。而与传统生物反应器微载体悬浮培养MDCK细胞表达流感病毒相比，省去了多次洗涤更换培养基的步骤，并且降低了由于载体间的相互碰撞容易造成细胞损伤细胞损伤。
本研究首先将用鸡胚传代得来的H1N1毒株(A/New Caledonia/20/99)感染MDCK细胞（66代），并进行传代稳定性试验。随后应用安普微型反应器考察MDCK细胞数目与葡萄糖消耗速率（GCR）之间关系，实现通过监控葡萄糖消耗速率（GCR）掌握MDCK细胞数量的方法。同时在安普微型反应器上考察TPCK胰酶浓度、MOI、接种量对安普微型反应器上MDCK细胞生长和流感病毒产量的影响。通过以上研究，选择接种量为0.05和TPCK-胰酶浓度为2.5μg/ml作为AP20C生物反应器感染参数。在安普AP20C生物反应器进行MDCK细胞生长和流感病毒产量的放大实验，试验中流感病毒滴度最高为512HA units/100μl并且一直保持到96小时。而此时的TCID50为7.3×10~7/ml。为了考察生物反应器表达流感病毒的重复性，以同样的培养条件和相同感染参数又进行了两次实验。结果证实感染3天后，两次生物反应器的流感病毒滴度分别为7.8×10~7和6.7×10~7/ml，血凝值分别为768和512。与第二次生物反应器试验基本一致。此外，安普生物反应器所使用的灌注袋和培养袋经钴60辐照灭菌，一次性使用后即可抛弃，省略原有的灭菌步骤减少相关附属管道从而降低了生产成本，生物反应器无需进行清洗、消毒等工作，缩短生物反应器培养间隔周期，疫苗生产效率得到了明显提高。
综上所述，我们应用安普微型反应器通过监控葡萄糖消耗速率（GCR）来实现间接评估MDCK细胞数量的方法，并考察TPCK胰酶浓度、MOI、接种量对安普微型反应器上MDCK细胞生长和流感病毒产量的影响。应用上述优化的感染参数在一次性安普AP20C生物反应器进行MDCK细胞生长和H1N1流感病毒产量的放大实验。以上研究结果为研制MDCK细胞基质的流感疫苗奠定了基础。
The paper describes the HPV preventive vaccine research which based insectcell-baculovirus expression system. To develop new types influenza vaccine, thethesis also describes the application Disposable packed bed bioreactor expressingMDCK cells expressing the H1N1influenza virus research.
1. Study on Prophylactic Human Papillomavirus Vaccines
By IARC in a number of countries for the investigation of the HPV types thatcause invasive cervical cancer (ICC). The histological diagnosis of99.7%of the1000ICC cases found positive for HPV DNA. Forty HPV subtypes related tocervical cancer have been discovered.HPV16and18are two of the most prevalenttypes in cervical cancer worldwide.So far known HPV types already exceeded200.
In this paper, the HPV epidemiology survey in mainland China results as wellas the worldwide based on the choice of cervical cancer in women of mainlandChina's third-largest advantage type-HPV58type as a vaccine component, todetermine of HPV16,18,58,6,11is prophylactic human papillomavirus vaccinesvaccine type. Expectations pentavalent prophylactic human papillomavirus vaccineswe developed the face of the region can prevent75%of cervical cancer caused byHPV16,18,58, but also the prevention of the low-risk cancer caused by HPV6,11precancerous lesions and genital warts.
Using PCR technique, major capsid protein full length L1gene of HPV16, type18, type58, type6, type11were amplified, truncated HPV16type, type18, type58,type6,type11gene were also amplified by nuclear localization signal. The clonedgenes were subcloned into pFastBac-1.The recombinant pFastBac-1were used totransform DH10Bac.Infected insect cells expressed52-55kD protein aftertransfected, which identified by the Western-Blot, with Camvir-1monoclonalantibody specific reactions successfully. HPV subtypes VLP were purified bygradient ultracentrifugation, the various VLPs was placed in a transmission electronmicroscope. It found that the formation of the virus-like particles in a cesiumchloride solution, similar to the size and morphology of wild-type virus, the density is about1.27g/ml.
A successful HPV prophylactic vaccine is judged by whether induced highneutralizing antibody. Therefore, how to effectively and quickly detect a protectiveefficacy of neutralizing antibodies, become the key to the success of HPVprophylactic vaccines. Due to the human papillomavirus (HPV) has stricttissue-specific and species-specific, limited infection of human skin and mucosaltissue, is extremely difficult to cultivate in vitro tissue culture system. Currently,there have been a considerable number of neutralizing antibodies in a method forneutralizing antibody titers in the serum to be tested. However, these methods allhave some limitations: some complex method of operation, long life cycle.Somemethods only detect several HPV types; Some methods likely to cause false positiveresults; Some lower sensitivity; Some trials containing radioactive Isotope causeharm to the human and the environment.
According to the characteristics of antibody assay, we selected luciferase as areporter gene of the HPV-pseudovirus neutralizing antibodies.The advantage ofluciferase as a reporter gene as described above shown with a microplate reader withthe use of high throughput detection and can simultaneously detect the96samples.The traditional luciferase system requires cell lysate which were lysed to releaseluciferase. For high-throughput detection operation is somewhat cumbersome. Inorder to be suitable for high-throughput detection, using the Promega Bright-Glo Luciferase system, without the medium was removed or washed cells in this systembefore joining the detection reagent. The cells can be cultured with a96-well plateand testing, and very suitable for continuous operation in the automation system.
The neutralization assay was used to detect the neutralizing antibodies inducedby the full-length (HPV16-505) and the truncated (HPV16-483). We also found thatthe antibody titer of removal of HPV16L1C-terminal of the nuclear localizationsignal (HPV16-483) was significantly higher than that of full-length (HPV16-505).Description truncated L1of the humoral immune response induced significantlyhigher levels of full-length. Therefore, in the neutralizing antibody experiments, wehave select five truncated VLP to research.
HPV16,18,6type monovalent vaccine iwere immunized mice which alsoinduce high titers of type-specific antibody. The bivalent vaccine16/1816/18/6trivalent vaccine, were immunized mice could induce high titer specific neutralizingantibodies. Polyvalent HPV vaccines combined immunodeficiency, with the vaccinetype number indrease, various types of neutralizing antibody titers were decreased,immunological interference phenomenon.
We compared the difference of the different principles bioreactor cultures ofinsect cells and found that the WAVE Bioreactor more suitable for suspensionculture of insect cells. In order to investigate the impact of different lysis methods ofsf9insect cell, the results show that compared with the ultrasonic method, chemicallysis, hypotonic lysis method is more suitable for insect cell lysates.。 We alsocompare different pore size hollow fiber microfiltration membrane for sf9insectcells harvested, we selected GE420cm2microfiltration membranes (0.10,0.22, and0.45μm), the results show that, compared with the0.10and0.22μm hollow fibermicrofiltration membrane0.45μm membrane with a larger average membrane poresize which membrane resistance is small, favor the medium through the enrichmentprocess can alleviate the cause the film surface of the gel layer, and thus has a fasterprocessing speed, mean through flux of up to90L/hm2. In combination on this basis,we design application of the hollow fiber system as an integrated system, theconcentrated cell harvest, diafiltration, extraction and clarification step. This processavoids the centrifuge, and dead-end filtration experimental procedure, and in theextraction process, we use the hypotonic lysis of insect cells, the release of the targetprotein, and to avoid the lysis agent (Triton X-100) was added and ultrasonic, sothat the operation in a sealed and sterile state, and more suitable for industrialproduction. In the primary separation of purification stage, we choose DMAEchromatograph (the weak anion chromatography) to purify HPV6L1protein,compared to Q Sepharose XL (strong anion exchange chromatography), HPV6L1protein purity higher by the DMAE chromatography purified. In the polishseparation of purification stage, we select Octyl Sepherose FF, Butyl-S SepheroseFF, Butyl-S Sepherose FF, Fractogel EMD TMAE,CM Sepherose FF media. For the primary separation of purification stage is anion exchange chromatography,different principles of chromatography media is more effective in the differentpurified stages Therefore we choose Octyl Sepherose FF in the polish stage. And weexplored the virus-like particles in vitro assembly process after disassembly andreassembly steps to obtain homogeneous and stable virus-like particles. Finalpurified HPV L1protein was characterized to confirme to be free of host cellproteins, and endotoxins.
In summary, this study was expressed HPV VLP by insect cell-baculovirusexpression system which evaluated the immunogenicity of the VLP vaccine in theapplication of pseudovirus-based neutralization assay. Next, studied the productionprocess of HPV6L1in insect cell system, which was more reasonable and suitablefor scale-up production.
Production of H1N1Influenza Virus Vaccines using a Packed-bedBioreactor
Influenza is a major cause of hospitalization and lower respiratory illnesses,especially in children and elderlyindividuals. Vaccination is the primar y method forpreventing influenza and its complications. Vaccination can not only reduce themorbidity and mortality of high-risk groups, and also ease symptoms and reduce theincidence of complications, reducing the probability of further spread. During aninfluenza pandemic, antiviral drugs are necessary, but not a substitute for thevaccine, so vaccination is the prevention of influenza.
Embryo culture technology to produce influenza inactivated vaccine has been60years of history, clinical applications for many years that they have good immuneeffects and clinical safety, protection rates are generally75%to90%. However,there are some limitations: such as the dependence of chickens, chick embryoproduction of influenza vaccines need to consume a large amount of SPF grade eggs,difficult to control quality, the method is difficult to standardize; chick embryo withthe potential viral contamination; and large-scale supply of SPF grade eggs difficultto cope with the outbreak of influenza. The cell culture derived vaccine does notrequire extensive advance planning and can be produced rapidly on a large scale in the event of an emerging pandemic. The most prominent adherent mammalian celllines for influenza vaccine development is Madine Darby Canine Kidney (MDCK)cells, which have been extensively studied. The MDCK cell line was derived from akidney of an apparently normal adult female cocker spaniel, September,1958, byS.H. Madin and N.B. Darby. MDCK cells are particularly favored, because it canyield high quantities of influenza virus.
Compared to traditional packed bed bioreactor, the Amprotein CurrentPerfusion Bioreactor (APCB) based on a non-sparging O2transfer method is used asa dissolved O2generator to culture immobilized cells in a perfusion column filledwith a non-woven polymer fiber carrier. During the cell culture, mixed gas iscontinually passed through the headspace utilizing the sterilizing filters provided onthe bioreactor bag. The gentle shaking motion of the bioreactor bag provides abubble-free oxygenated culture medium which flows into an inlet of the perfusioncolumn by a peristaltic pump, passes through the paper carrier to which MDCK cellsare attached, and exits at an outlet of the perfusion column to the inlet of thebioreactor bag for recirculation through the system. The shaking rate and gas flowhave been optimized by the system controller to provide an oxygenated culturemedium for high-density cell culture in the perfusion column without excessivefoaming or shear damage. While using serum containing DMEM in microcarriercultivations for influenza production requires time-consuming steps for washing andmedium exchange. Compared with cell suspension perfusion culture using anexpensive hollow fiber column, perfusion culture in the ACPB is much moreaffordable and scalable. Although using serum-free medium can eliminate the needfor washing cells before infection, cell growth is generally not as robust as that inserum-containing medium.Compared with microcarrier cultures, usingserum-containing DMEM for the production of influenza vaccine in the ACPB hasdefinite advantages in terms of the reduced cost and need for cleaning/validation.
The H1N1strain (A/New Caledonia/20/99) which passaged by the chickembryo infected MDCK cells (66passages), and test the stability. The mini-bioreactor was used to study the relationship between cell density and glucoseconsumption rate (GCR) and to optimize the infection parameters of the influenzaH1N1virus (A/New Caledonia/20/99). Then the MDCK cell culture and virusinfection was maintained in a disposable perfusion bioreactor (Amprotein CurrentPerfusion Bioreactor) with Proportional-Integral-Derivative (PID) control of pH,dissolved O2(DO), agitation and temperature. During six days of culture, the totalcell number increased from2.0×109to3.2×1010. The maximum virus titers of768hemagglutinin (HA) units/100μL and7.8×10~7TCID50/mL was obtained3days afterinfection. These results demonstrated that using a disposable perfusion bioreactor forlarge-scale cultivation of MDCK cells, through the control of DO, pH and otherconditions, is a convenient, stable technology for the industrial-scale production ofinfluenza vaccines.
In the ACPB, the bioreactor bag and perfusion column are constructed withpre-sterilized plastic. This design eliminates the need for cleaning, sterilization andassociated validation and thus shortens the implementation time to conform to GoodManufacturing Practices (GMP). The gamma-radiation sterilized ACPB reduces therisk of contamination due to equipment malfunction or operator error typical oftraditional bioreactors such as stirred tanks, spinners and hollow-fiber systems. Oncea cultivation cycle is completed, the culture is harvested, and a new ACPB canimmediately replace the discarded one on the shaking platform, which will greatlyimprove the capacity of vaccine production.
一、人乳头瘤病毒预防性疫苗研究
人类多种癌症，特别是子宫颈癌经相关的分子流行病学资料证明与HPV有着密切的关系。HPV的型别众多，人们不断地在皮肤癌症、疣状表皮等病理组织中发现新的HPV型别。目前为止已知型别已经突破200种。
根据中国大陆地区流行病学调查结果，选用在大陆地区的第三大优势型别-HPV58型作为疫苗的组分之一，确定以HPV16、18、58、6、11型为主要疫苗型别的五价疫苗。期望此疫苗可以保护75%以上的由HPV16、18、58引起子宫颈癌，还可预防由HPV6、11引起的低度癌前病变和生殖器疣。
主要内容如下：
HPV各型VLP的构建表达：通过PCR合成HPV16型、18型、58型、6型、11型主要衣壳蛋白全长L1目的基因，根据核定位信号设计PCR引物扩增截短型HPV16型、18型、58型、6型、11型目的基因。测序正确后分别重组入杆状病毒表达系统穿梭质粒pFastBac1，通过基因移位作用将目的基因片段重组入Bacmid基因组，提取重组的Bacmid DNA，M13引物PCR鉴定正确。各亚型重组杆状病毒Bacmid-HPVL1转染Sf9细胞，大量感染昆虫细胞后，分别表达52-55kD大小的蛋白，经Western-Blot鉴定，与Camvir1单克隆抗体产生特异性反应，证明转染成功目的蛋白为HPVL1蛋白。应用蔗糖垫离心结合氯化铯密度梯度超速离心纯化HPV各亚型VLP，将各亚型VLP透析后的样品放置于透射电镜下观察。发现其形成的病毒样颗粒与野生型病毒的大小和形态相似，在氯化铯溶液中密度为1.27g/ml。
HPV各型VLP免疫原性研究：通过全长型（HPV16-505）和截短型（HPV16-483）假病毒中和抗体试验证明：去除HPV16L1C端核定位信号的截短型（HPV16-483）中和抗体滴度显著高于全长型（HPV16-505）。说明截短型所诱发的体液免疫应答水平显著高于全长型。通过上述研究结果可知，去除核定位信号的VLP可能更易于被B淋巴细胞识别，有利于产生高滴度的中和抗体。因此在中和抗体实验中，仅对五种亚型的截短型VLP进行研究。
HPV16型、18型、6型单价疫苗免疫分别免疫小鼠后，均可产生高滴度的型特异中和抗体。1618双价疫苗和16186三价疫苗，分别免疫小鼠后均可产生针对于相应型别高滴度的中和抗体。多价HPV疫苗联合免疫时，随着疫苗型别增多，各型别的中和抗体滴度均出现降低的趋势，即存在免疫干扰现象。
HPV VLP生产工艺初步研究：以HPV6L1为例，研究了昆虫细胞体系的VLP疫苗生产工艺研究。首先比较不同接毒量、感染密度、细胞收获时间对HPV6L1蛋白表达量的影响。对比不同原理生物反应器培养昆虫细胞的区别，发现WAVE生物反应器更适合悬浮培养昆虫细胞。为了考察不同裂解方法对sf9昆虫细胞裂解的影响，结果表明与超声法、化学裂解法相比，低渗裂解法更适合于昆虫细胞裂解。又比较不同孔径的中空纤维微滤膜对sf9昆虫细胞收获的影响，选择了GE公司面积为420cm2的三种孔径微滤膜（0.10,0.22,和0.45μm），结果表明，与0.10,0.22μm中空纤维微滤膜相比，孔径为0.45μm中空纤维微滤膜具有更大的平均膜孔径，滤膜阻力较小，有利于培养基的透过，浓缩过程中可以缓解引起的膜表面凝胶层，因此具有更快的处理速度，其平均透过通量可达90L/hm2。在此基础上结合细胞破碎相关的实验结果，设计应用中空纤维系统作为一个整合系统，实现细胞收获液的浓缩、洗滤、抽提、澄清步骤。此工艺避免了离心机，死端过滤等实验步骤，而且在抽提过程中，采用低渗的方法裂解昆虫细胞，释放目的蛋白，避免了裂解剂（Triton X-100）的加入和超声仪的使用，使整个操作都处于密闭和无菌状态，更适合于工业化生产。
在柱层析初级分离纯化阶段，选择DMAE弱阴离子层析纯化HPV6L1蛋白，与Q Sepharose XL强阴离子层析相比，DMAE弱阴离子层析纯化的HPV6L1蛋白纯度更高，基本达到初级分离纯化的要求。之后在柱层析精制分离纯化阶段尝试了应用Octyl Sepherose FF、Butyl-S Sepherose FF、Fractogel EMDTMAE、CM Sepherose FF等层析介质进行精纯工艺摸索。考虑到初级分离纯化阶段采用的是阴离子交换层析，不同阶段采用不同原理的层析介质纯化蛋白更有效。因此在精制分离纯化阶段，选择Octyl Sepherose FF疏水层析纯化HPV6L1蛋白。又摸索了类病毒颗粒体外组装工艺，经解组装、重组装步骤后，获得了均一稳定的病毒样颗粒。对于上述优化的放大纯化工艺所制备的HPV6L1VLPs，进行了SF9细胞宿主DNA残留量；SF9昆虫细胞宿主蛋白残留量；HPV VLP疫苗抗原含量；内毒素等检测。
综上所述，本研究利用杆状病毒-昆虫细胞表达系统表达了HPV各型VLP，应用假病毒中和抗体体系评价了各型VLP疫苗的免疫原性。初步建立了昆虫细胞体系的HPV6L1的生产工艺和质量研究体系。以上研究结果为研制适合中国大陆地区昆虫细胞体系人乳头瘤病毒预防性疫苗提供参考。
二、应用一次性填充床生物反应器表达MDCK细胞表达H1N1流感病毒
流行性感冒是由流感病毒引起的，呼吸系统常见的传染性疾病，流感的临床特征为鼻咽部的急性炎症。流感是一种发病率高，传染性强，并且与患者的年龄、性别无关的传染病。接种合适的流感疫苗是避免流感传染最有效的方法。相关人群的发病率和死亡率通过接种疫苗可以明显降低，同时也减少了流感病毒的继续感染的机会。
鸡胚生产流感疫苗已有多年历史，临床应用表明其具有较好的免疫效果和安全性。其缺点在于：需要提供足够的SPF级鸡蛋来生产流感疫苗，鸡胚的质量标准和方法学研究不易控制和标准化；同时可能存在鸡胚带有禽流感病毒的危险；并且大规模SPF级种蛋的供应难以应对流感大暴发。由于流感病毒能够在多种动物细胞内繁殖，因此可以应用哺乳动物细胞大规模生产流感疫苗来满足流感的大流行性以及新发流感的需求。MDCK细胞(MDCK Cell Lines)是在上世纪五十年代由Madin和Darby经Cocker Spaniel犬的肾脏组织分离后培养获得，MDCK细胞通常是以贴壁方式生长的上皮样细胞。由于其病毒感染效率高、增殖快，且不易变异，被公认为最适于流感病毒疫苗生产的细胞系。
MDCK细胞为贴壁细胞，可用各型生物反应器大规模培养，其中包括：微载体生物反应器，填充床反应器，中空纤维生物反应器。与传统的填充床反应器不同，安普生物反应器采用外循环式纸片灌注培养方式，其分为灌注袋、培养袋两部分。灌注袋中放置，细胞培养液经培养袋置于特制摇床中，通过激流反应补充氧气，再经蠕动泵泵进灌注袋提供给吸附在聚酯纸片载体的贴壁细胞。安普生物反应器在细胞培养过程中通过PID模式可以自动控制相关的细胞生长参数。所用的一次性聚酯纸片载体内部相互交织的三维结构可提供传统培养模式无法比拟的表面积，适合多种类型细胞吸附生长，细胞增殖速度快维持时间长，符合生物安全要求。同时细胞聚集可形成支撑保护，减少培养基流动时对细胞产生的剪切力。因为贴壁细胞可吸附在聚酯纸片载体的表面和内部结构里，避免了由于培养基的流动可能造成的损伤，不需要中空纤维等特殊的分离细胞装置，更换培养液更方便。因此采用安普生物反应器可实现在含有血清的培养基条件下培养MDCK细胞，待细胞长满后迅速更换为适合病毒繁殖的无血清培养基，从而获得高效价的流感病毒。而与传统生物反应器微载体悬浮培养MDCK细胞表达流感病毒相比，省去了多次洗涤更换培养基的步骤，并且降低了由于载体间的相互碰撞容易造成细胞损伤细胞损伤。
本研究首先将用鸡胚传代得来的H1N1毒株(A/New Caledonia/20/99)感染MDCK细胞（66代），并进行传代稳定性试验。随后应用安普微型反应器考察MDCK细胞数目与葡萄糖消耗速率（GCR）之间关系，实现通过监控葡萄糖消耗速率（GCR）掌握MDCK细胞数量的方法。同时在安普微型反应器上考察TPCK胰酶浓度、MOI、接种量对安普微型反应器上MDCK细胞生长和流感病毒产量的影响。通过以上研究，选择接种量为0.05和TPCK-胰酶浓度为2.5μg/ml作为AP20C生物反应器感染参数。在安普AP20C生物反应器进行MDCK细胞生长和流感病毒产量的放大实验，试验中流感病毒滴度最高为512HA units/100μl并且一直保持到96小时。而此时的TCID50为7.3×10~7/ml。为了考察生物反应器表达流感病毒的重复性，以同样的培养条件和相同感染参数又进行了两次实验。结果证实感染3天后，两次生物反应器的流感病毒滴度分别为7.8×10~7和6.7×10~7/ml，血凝值分别为768和512。与第二次生物反应器试验基本一致。此外，安普生物反应器所使用的灌注袋和培养袋经钴60辐照灭菌，一次性使用后即可抛弃，省略原有的灭菌步骤减少相关附属管道从而降低了生产成本，生物反应器无需进行清洗、消毒等工作，缩短生物反应器培养间隔周期，疫苗生产效率得到了明显提高。
综上所述，我们应用安普微型反应器通过监控葡萄糖消耗速率（GCR）来实现间接评估MDCK细胞数量的方法，并考察TPCK胰酶浓度、MOI、接种量对安普微型反应器上MDCK细胞生长和流感病毒产量的影响。应用上述优化的感染参数在一次性安普AP20C生物反应器进行MDCK细胞生长和H1N1流感病毒产量的放大实验。以上研究结果为研制MDCK细胞基质的流感疫苗奠定了基础。
The paper describes the HPV preventive vaccine research which based insectcell-baculovirus expression system. To develop new types influenza vaccine, thethesis also describes the application Disposable packed bed bioreactor expressingMDCK cells expressing the H1N1influenza virus research.
1. Study on Prophylactic Human Papillomavirus Vaccines
By IARC in a number of countries for the investigation of the HPV types thatcause invasive cervical cancer (ICC). The histological diagnosis of99.7%of the1000ICC cases found positive for HPV DNA. Forty HPV subtypes related tocervical cancer have been discovered.HPV16and18are two of the most prevalenttypes in cervical cancer worldwide.So far known HPV types already exceeded200.
In this paper, the HPV epidemiology survey in mainland China results as wellas the worldwide based on the choice of cervical cancer in women of mainlandChina's third-largest advantage type-HPV58type as a vaccine component, todetermine of HPV16,18,58,6,11is prophylactic human papillomavirus vaccinesvaccine type. Expectations pentavalent prophylactic human papillomavirus vaccineswe developed the face of the region can prevent75%of cervical cancer caused byHPV16,18,58, but also the prevention of the low-risk cancer caused by HPV6,11precancerous lesions and genital warts.
Using PCR technique, major capsid protein full length L1gene of HPV16, type18, type58, type6, type11were amplified, truncated HPV16type, type18, type58,type6,type11gene were also amplified by nuclear localization signal. The clonedgenes were subcloned into pFastBac-1.The recombinant pFastBac-1were used totransform DH10Bac.Infected insect cells expressed52-55kD protein aftertransfected, which identified by the Western-Blot, with Camvir-1monoclonalantibody specific reactions successfully. HPV subtypes VLP were purified bygradient ultracentrifugation, the various VLPs was placed in a transmission electronmicroscope. It found that the formation of the virus-like particles in a cesiumchloride solution, similar to the size and morphology of wild-type virus, the density is about1.27g/ml.
A successful HPV prophylactic vaccine is judged by whether induced highneutralizing antibody. Therefore, how to effectively and quickly detect a protectiveefficacy of neutralizing antibodies, become the key to the success of HPVprophylactic vaccines. Due to the human papillomavirus (HPV) has stricttissue-specific and species-specific, limited infection of human skin and mucosaltissue, is extremely difficult to cultivate in vitro tissue culture system. Currently,there have been a considerable number of neutralizing antibodies in a method forneutralizing antibody titers in the serum to be tested. However, these methods allhave some limitations: some complex method of operation, long life cycle.Somemethods only detect several HPV types; Some methods likely to cause false positiveresults; Some lower sensitivity; Some trials containing radioactive Isotope causeharm to the human and the environment.
According to the characteristics of antibody assay, we selected luciferase as areporter gene of the HPV-pseudovirus neutralizing antibodies.The advantage ofluciferase as a reporter gene as described above shown with a microplate reader withthe use of high throughput detection and can simultaneously detect the96samples.The traditional luciferase system requires cell lysate which were lysed to releaseluciferase. For high-throughput detection operation is somewhat cumbersome. Inorder to be suitable for high-throughput detection, using the Promega Bright-Glo Luciferase system, without the medium was removed or washed cells in this systembefore joining the detection reagent. The cells can be cultured with a96-well plateand testing, and very suitable for continuous operation in the automation system.
The neutralization assay was used to detect the neutralizing antibodies inducedby the full-length (HPV16-505) and the truncated (HPV16-483). We also found thatthe antibody titer of removal of HPV16L1C-terminal of the nuclear localizationsignal (HPV16-483) was significantly higher than that of full-length (HPV16-505).Description truncated L1of the humoral immune response induced significantlyhigher levels of full-length. Therefore, in the neutralizing antibody experiments, wehave select five truncated VLP to research.
HPV16,18,6type monovalent vaccine iwere immunized mice which alsoinduce high titers of type-specific antibody. The bivalent vaccine16/1816/18/6trivalent vaccine, were immunized mice could induce high titer specific neutralizingantibodies. Polyvalent HPV vaccines combined immunodeficiency, with the vaccinetype number indrease, various types of neutralizing antibody titers were decreased,immunological interference phenomenon.
We compared the difference of the different principles bioreactor cultures ofinsect cells and found that the WAVE Bioreactor more suitable for suspensionculture of insect cells. In order to investigate the impact of different lysis methods ofsf9insect cell, the results show that compared with the ultrasonic method, chemicallysis, hypotonic lysis method is more suitable for insect cell lysates.。 We alsocompare different pore size hollow fiber microfiltration membrane for sf9insectcells harvested, we selected GE420cm2microfiltration membranes (0.10,0.22, and0.45μm), the results show that, compared with the0.10and0.22μm hollow fibermicrofiltration membrane0.45μm membrane with a larger average membrane poresize which membrane resistance is small, favor the medium through the enrichmentprocess can alleviate the cause the film surface of the gel layer, and thus has a fasterprocessing speed, mean through flux of up to90L/hm2. In combination on this basis,we design application of the hollow fiber system as an integrated system, theconcentrated cell harvest, diafiltration, extraction and clarification step. This processavoids the centrifuge, and dead-end filtration experimental procedure, and in theextraction process, we use the hypotonic lysis of insect cells, the release of the targetprotein, and to avoid the lysis agent (Triton X-100) was added and ultrasonic, sothat the operation in a sealed and sterile state, and more suitable for industrialproduction. In the primary separation of purification stage, we choose DMAEchromatograph (the weak anion chromatography) to purify HPV6L1protein,compared to Q Sepharose XL (strong anion exchange chromatography), HPV6L1protein purity higher by the DMAE chromatography purified. In the polishseparation of purification stage, we select Octyl Sepherose FF, Butyl-S SepheroseFF, Butyl-S Sepherose FF, Fractogel EMD TMAE,CM Sepherose FF media. For the primary separation of purification stage is anion exchange chromatography,different principles of chromatography media is more effective in the differentpurified stages Therefore we choose Octyl Sepherose FF in the polish stage. And weexplored the virus-like particles in vitro assembly process after disassembly andreassembly steps to obtain homogeneous and stable virus-like particles. Finalpurified HPV L1protein was characterized to confirme to be free of host cellproteins, and endotoxins.
In summary, this study was expressed HPV VLP by insect cell-baculovirusexpression system which evaluated the immunogenicity of the VLP vaccine in theapplication of pseudovirus-based neutralization assay. Next, studied the productionprocess of HPV6L1in insect cell system, which was more reasonable and suitablefor scale-up production.
Production of H1N1Influenza Virus Vaccines using a Packed-bedBioreactor
Influenza is a major cause of hospitalization and lower respiratory illnesses,especially in children and elderlyindividuals. Vaccination is the primar y method forpreventing influenza and its complications. Vaccination can not only reduce themorbidity and mortality of high-risk groups, and also ease symptoms and reduce theincidence of complications, reducing the probability of further spread. During aninfluenza pandemic, antiviral drugs are necessary, but not a substitute for thevaccine, so vaccination is the prevention of influenza.
Embryo culture technology to produce influenza inactivated vaccine has been60years of history, clinical applications for many years that they have good immuneeffects and clinical safety, protection rates are generally75%to90%. However,there are some limitations: such as the dependence of chickens, chick embryoproduction of influenza vaccines need to consume a large amount of SPF grade eggs,difficult to control quality, the method is difficult to standardize; chick embryo withthe potential viral contamination; and large-scale supply of SPF grade eggs difficultto cope with the outbreak of influenza. The cell culture derived vaccine does notrequire extensive advance planning and can be produced rapidly on a large scale in the event of an emerging pandemic. The most prominent adherent mammalian celllines for influenza vaccine development is Madine Darby Canine Kidney (MDCK)cells, which have been extensively studied. The MDCK cell line was derived from akidney of an apparently normal adult female cocker spaniel, September,1958, byS.H. Madin and N.B. Darby. MDCK cells are particularly favored, because it canyield high quantities of influenza virus.
Compared to traditional packed bed bioreactor, the Amprotein CurrentPerfusion Bioreactor (APCB) based on a non-sparging O2transfer method is used asa dissolved O2generator to culture immobilized cells in a perfusion column filledwith a non-woven polymer fiber carrier. During the cell culture, mixed gas iscontinually passed through the headspace utilizing the sterilizing filters provided onthe bioreactor bag. The gentle shaking motion of the bioreactor bag provides abubble-free oxygenated culture medium which flows into an inlet of the perfusioncolumn by a peristaltic pump, passes through the paper carrier to which MDCK cellsare attached, and exits at an outlet of the perfusion column to the inlet of thebioreactor bag for recirculation through the system. The shaking rate and gas flowhave been optimized by the system controller to provide an oxygenated culturemedium for high-density cell culture in the perfusion column without excessivefoaming or shear damage. While using serum containing DMEM in microcarriercultivations for influenza production requires time-consuming steps for washing andmedium exchange. Compared with cell suspension perfusion culture using anexpensive hollow fiber column, perfusion culture in the ACPB is much moreaffordable and scalable. Although using serum-free medium can eliminate the needfor washing cells before infection, cell growth is generally not as robust as that inserum-containing medium.Compared with microcarrier cultures, usingserum-containing DMEM for the production of influenza vaccine in the ACPB hasdefinite advantages in terms of the reduced cost and need for cleaning/validation.
The H1N1strain (A/New Caledonia/20/99) which passaged by the chickembryo infected MDCK cells (66passages), and test the stability. The mini-bioreactor was used to study the relationship between cell density and glucoseconsumption rate (GCR) and to optimize the infection parameters of the influenzaH1N1virus (A/New Caledonia/20/99). Then the MDCK cell culture and virusinfection was maintained in a disposable perfusion bioreactor (Amprotein CurrentPerfusion Bioreactor) with Proportional-Integral-Derivative (PID) control of pH,dissolved O2(DO), agitation and temperature. During six days of culture, the totalcell number increased from2.0×109to3.2×1010. The maximum virus titers of768hemagglutinin (HA) units/100μL and7.8×10~7TCID50/mL was obtained3days afterinfection. These results demonstrated that using a disposable perfusion bioreactor forlarge-scale cultivation of MDCK cells, through the control of DO, pH and otherconditions, is a convenient, stable technology for the industrial-scale production ofinfluenza vaccines.
In the ACPB, the bioreactor bag and perfusion column are constructed withpre-sterilized plastic. This design eliminates the need for cleaning, sterilization andassociated validation and thus shortens the implementation time to conform to GoodManufacturing Practices (GMP). The gamma-radiation sterilized ACPB reduces therisk of contamination due to equipment malfunction or operator error typical oftraditional bioreactors such as stirred tanks, spinners and hollow-fiber systems. Oncea cultivation cycle is completed, the culture is harvested, and a new ACPB canimmediately replace the discarded one on the shaking platform, which will greatlyimprove the capacity of vaccine production.
引文
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