Preparation Of NDV Antitumor Vaccine For Human Use

Treatment of tumors with the virus had a long history.100 years ago,some researchers discovered by chance that the tumor regressed after being treated by virus vaccine.From then on,researches on anti-tumor virus vaccine have made significant progess.Current studies have been developed from animal experiments, pre-clinical research,Ⅰ,Ⅱ,Ⅲstages of clinical trial to the formal approval of virus anti-tumor drugs.Many oncolytic viruses have been found and studied for clinical applications. Current researches focus on Newcastle disease virus(NDV),recombinant Newcastle disease virus,anti-tumor vaccine of Newcastle desease virus lyses solution,Reovirus, Adenovirus,adenovirus vector with altered tropism,adenovirus vector stably expressed in proliferation cell,conditionally replicating adenoviruses(CR-Ad), animal adenovirus vector,Herpes simplex virus(HSV-1),influenza virus, recombinant influenza virus,influenza virus genome with inserted exogenous genes, virogene depletion of Influenza virus,Influenza virus carrier,lentivirus vector using gene therapy,epstein-barr virus(EBV),Autonomous parvovirus,chicken anemia virus(CAV),targeted therapy through cell carrier of viral vector etc...The NDV is the cause pathogen which causes epidemic disease(Newcastle disease,ND) in birds Newcastle.Epidemic Newcastle disease was first discovered in Indonesia's Batavia in 1926.The second case was found in British Newcastle at the same year,so it was named epidemic Newcastle disease.NDV is a single chain RNA virus in poultry.Its RNA length is 15 kilobases and belong to a paramyxovirus typeⅠvirus.It can cause acute infectious disease of poultry,but not infect human.The NDV used for human tumor treatment began in the 1960s.In 1970s～1980s, it was widely used as interferon induced agent.Over the past 20 years,experimental and clinical studies showed that NDV can selectively kill a variety of human cancer cells,such as malignant melanoma,lung,liver,pancreatic cancer,breast cancer, cervical cancer,Ovarian cancer,malignant glioma,lymphoma,leukemia,stomach, kidney,bladder cancer,nasopharyngeal and larynx cancer,et al.In vitro and In vivo studies showed that roles of NDV on anti-tumor cells have distinctive features:1.NDV selectivity kill tumor cell,while without killing the normal cells.2.The NDV can propagate to release in the human tumor cell.But the tumors are not dissipated with the inactivated NDV treatment.3.The effects of the NDV anti-tumor function rely on its potency,dosage dependent(NDV amount) and interactions between tumor cells.There are three kinds of perspectives on the mechanisms of NDV anti-tumor effects:1.NDV directly induces tumor cell apoptosis;2.NDV helps the host to produce cytokine;3.The proteins of NDV combine with part or whole cell membranes,which repress tumor cells metabolism and enhance the immunogenicity of the tumor cell.This mechanism is associated with Oncolysis.The NDV vaccine is widely used in poultry to prevent Newcastle disease(ND).It is derived from the lysis tissue of infectious chicken embryos,which contain a large number of antigens of chicken source.Thus it is not suitable for human use.The predecessor of candidate NDV anti-tumor vaccine used in this project is harvested from urine cyst cavity of chick embryo.Because of its good effects in novel application in human cancer therapy,it was slected as a key technology project by Science and Technology Commission of China in the '80s and '90s.It was listed as a "August 5" major projects by Chinese Academy of Science and as a major drug project by the Chinese Ministry of Health.A variety of normative experiments were conducted in animals and in clinical trials of advanced cancer patients.Some hospitals are using it as a reagent for advanced cancer treatment volunteers.The main advantages of the NDV are simple preparation without special equipment,high NDV validity and identified anti-tumor effects.However,its chick origin resulted in limited production and poor quality control for the large application,especially not suitable for the modern pharmaceutical industry standardization and scale production.This project focuses on the improvement of the old vaccine production technology and innovation of the NDV vaccine preparation process with cell culture technology.We successfully use primary chick embryo cell culture system in preparation of NDV anti-tumor vaccine.In the new vaccine development process, we are faced with some problems:First,how to ensure the safety of the new vaccine ingredients in human use? Second problem is how to increase the activity and potency.In order to ensure the vaccine composition's safety in human use,this study explored the feasibility to reduce the residual bovine serum in the new vaccine.Due to changing the process from chick embryo allantoic cavity harvest to primary chick embryo cell culture,usage of calf serum,which will trigger the body's allergic reaction,is the most important difference compared to the original vaccine. Therefore,in the new vaccine development process,we focused on maintaining and improving NDV titer while reducing the residual bovine serum.So that the new vaccine achieve the requirements of national's human biological products standards. Through continuous testing and comparison,we have adopted in 1.using a small number of high-quality calf serum(2%) before addition of NDV to the cells and washing single cell layer with serum-free medium after the confluence of the cell to reduce the content calf serum.2.replacing calf serum to human albumin in addition of NDV.The residual of bovine serum protein contents is lower than 50 ng/ml (reverse indirect hemagglutination assay) after using of these methods,which reach the quality requirements of biological products in China.We also performed guinea pig allergy experiment with the new vaccine containing residual bovine serum but not human albumin.Experiment result is negative.Those results showed that the remnants of bovine serum in the new vaccine will not lead to human allergic problems compared with the old vaccine.The project was also committed to enhancing the activity of new vaccines and potency.The old vaccine has relatively high titers NDV Hemagglutinin because of the use of live chicken embryo culture.The anti-tumor activity is higher too.How to use the new technology to yield high NDV titer was the pursuit of our exploration. We improved cell culture NDV output titer and biological activity through increased drug amount,the appropriate drug inoculation,the right time to seed and harvest,4℃incubation overnight to release NDV,improved protective components,control the temperature of vacuum freeze-drying,and so on.By adopting these measures,the potency of new vaccines was improved to achieve the requirements.The process will be further modified to get even better results.We test whether the new anti-tumor NDV vaccine technology meet the relevant requirements not only by ourselves but also by professional organizations.The new NDV vaccine was confirmed to pass the test of efficacy,toxicity and safety. Thus,we developed a new vaccine for the treatment of multiple tumors.Summary,we explore a new set of production process in NDV anti-tumor vaccine by using cell culture technology.The production of the candidate strains for vaccine was suitable for the standardization of the product and quality control.It was good for the application in the modern pharmaceutical industrialized production. And the vaccine was effective,no-toxic and safe for human use.In addition,we test the anti-cancer and apoptosis induction roles of this newly developed vaccine on oral squamous cell carcinoma,which was not reported in the studies of anti-tumor NDV vaccine.The oral malignant tumor is clinical frequently encountered disease,mainly referring to the oral cancer in oral epithelium including tongue carcinoma,buccal carcinoma,gingival carcinoma,carcinoma of floor of mouth and carcinoma of hard palate,respectively.The oral squamous cell carcinoma is the most common oral cancer.It is about 90%.The anatomy of the oral cavity is complex.It is close to other important organs.Surgical dissection resulted in organ defects and functional disturbance,which often affect patients' quality of life.But the oral tumor is superficial and easy for NDV direct injection or partially applied.It has more broad prospects in the clinical application.In order to observe the anti-cancer of apoptosis induction effects of this newly developed vaccine,we chose oral squamous cell carcinoma(OSCC) cell lines,which include GNM and TSCCa,for our preliminary exploration.MTT assay,cell morphology in addition of NDV,apoptosis cell rate,and the measurement absorb a light value were performed to comprehensive assessment the vaccine candidate strains for anti-tumor NDV. Result:The NDV present obvious killing effect(P＜0.001) and can induce GNM and the TSCCa cell Apoptosis(P＜0.05) to the GNM and the TSCCa in vitro.The expression of survivin was detected by RT-PCR and western blot.The cell apoptosis, alteration of every phase of cell cycle and cell proliferation index(PI) were checked by flow cytometry(FCM).The function of killing by NDV infection in vitro was shown.NDV decreased the expression of survivin and induced apoptosis of TSCCa cells,reduced G2/M phase and S phase cells,meanwhile,proliferation index declined.Compared with negative control group,the difference was significant (P＜0.05).The NDV infection to TSCCa cells can inhibit the expression of survivin,affect the cell cycle and induce the apoptosis.