Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Immunology > Immunobiology > The biology of the immune molecules ( immunochemistry )

Studies on Prophylactic Human Papillomavirus Vaccines Using Insect Cell and Production of H1N1Influenza Virus Vaccine Using a Packed-bed Bioreactor

Author SunBo
Tutor KongWei; JiangChunLai
School Jilin University
Course Microbiology
Keywords human papillomavirus cervical cancer pseudoviruses neutralizing antibodyassay baculovirus insect cells chromatography influenza virus packed bedbioreactor
CLC R392.11
Type PhD thesis
Year 2012
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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 VaccinesBy 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-bedBioreactorInfluenza 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.

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