Preparation And Immunity Efficacy Of Recombinant Ebola VLP Vaccine

Ebola virus disease, which is caused by infection with the Ebola viruses, it has as an endemic infectious disease sporadically occuring in Central Africa in 1976. Especially species Zaire ebolavirus the mortality rates as high as 90%.Although ebola virus disease is considered a potential public health threat, there is no licensed drug or vaccine is currently. Complete protection in cynomolgus macaques against Ebola virus challenge was achieved with the VSV-based vaccine, recombinant r Ad5-based vaccine and so on. While these vaccines have been tested in phase I clinical trials, it is not getted any vaccine commercially available until now.In particular, a number of studies have shown that expression of viral structural proteins in cells leads to the assembly and release of particles that resemble virions in size and morphology, and these particles are designated virus-like particles(VLP).The features of VLP also stimulated great interest for their application in vaccine development and highly promising results have been getted.The world health organization has classified EBOV as level 4 virus the most hazards to human beings. Use the real virus to detect neutralizing antibody levels, undoubtedly hampered the study of the world’s most experiments, which to some extent, explain the reasons why the vaccine has not been licensed. The Pseudotyped virus infected host system can simulate the process of Ebola virus, but it lost its pathogenicity, it can be operated at a low security level laboratories. These research has provided a guarantee to evaluate Ebola-related research.The clinical phase of the Ebola vaccine used traditional needle injection immunization currently. To find innovative ways on immunization, it may become an important breakthrough in the development of Ebola vaccine, and when the analysis of the skin as an ideal site for the immune many advantages, and in combination with needle-free technology,the future will become the ideal vaccine inoculation mode, will also improve existing Ebola vaccine developed some drawbacks.Objectives: we used Bac-to-Bac system to express Zaire(EBOV-Z)-VLP candidate vaccine,and intradermally and intrmuscally immunized 6-8 week female Balb/c mice, established a screening model against Ebola virus to evaluate the immunogenicity and protective efficay of candidate vaccine.Methods :The sequences were getted from Gene Bank and analysised. We used complete recombinant Bacmid-VP40-GP to transfected insect cells, and express the recombinant ZEBOLA-VLP. Sucrose density gradient centrifugation were used to purify the recombinant vaccine. Western blot and SDS-PAGE results showed that recombinant vaccines target proteins were stably expressed. And electron microsocy observation showed recombinant ZEBOLA-VLP vaccine has the particle characteristics. After intradermally and intrmuscally immunized 6-8 weeks female BALB/c mice we used pseudotyped virus system and ELISA assay to detect the level of serum neutralizing antibody levels and specific Ig G to evaluate the immune response.Results:We were successfully constructed p Fast Bac DUAL-VP40-GP plasmid and highly expressed in insect cells by 30% and 60% sucrose gradient centrifugation plus S600 column chromatography to obtain a 95% pure recombinant ZEBOLA-VLP candidate vaccine antigens.ZEBOLA-VLP candidate vaccine antigens prepared by SDS-PAGE, Western blot have identified the protein, the virus particle electron microscopy structure of uniform size, HLPC determination of purity of 95%; by microscopic observation and false virus titer determination,fake viral packaging success, can be used in the next step to assay the antibody titer; Animal experiments confirmed, the immune recombinant ZEBOLA-VLP vaccine, each dose group could produce specific antibodies, and the intradermal needle-free injection of 10μg was optimal dose, which stimulates the production of the specific Ig G antibody titers were significantly higher than other groups, each group were measured fake virus neutralizingantibodies.Conclusion:1.We firstly developed the high-purity recombinant ZEBOLA-GP + VP40 VLP vaccines for the prevention of the current outbreak of the Zaire strain of Ebola epidemic and providing a new type of ebola vaccine.2. With the help of th HIV virus and slow for the current outbreak of the Zaire strain of Ebola epidemic successfully prepared high titer, good stability, high security recombinant Ebola fake viral, for the development of vaccine efficacy evaluations provide experimental materials.3, Restructuring ZEBOLA GP + VP40-VLP vaccine has a good safety and efficacy, mice vaccinated with ZVLP produce EBOV-specific antibodies, including neutralizing antibodies,with the total serum antibody levels associated with vaccine dosage. All in all, recombinant ZEBOLA-VLP candidate vaccines may be promising Ebola candidate vaccines in the future.