Preparation, Characterization And Preliminary Efficacy Evaluation Of Genetic Engineering Hand-Foot-and- Mouth Disease (EV71) Vaccine

Hand, foot and mouth disease(HFMD) is a global contagious disease, which has become one of the serious public health issues mainly occurring in the Asia-Pacific region of the developing country. In China, since its initial identifcation in Shanghai in 1981, outbreaks of HFMD have occurred throughout the country. Given its major incidence for C class infectious diseases, HFMD was known as the 21 st century“Polio”. The causative pathogens of HFMD are members of the Enterovirus genus of the Picornaviridae family. On the basis of HFMD epidemiological study, Enterovirus71(EV71) and Coxsackie A16(CVA16) are the most common etiological agent,which together accounted for more than 80% of all infections. Children under 5 years old are most susceptible, with the number of cases accounting for 90% of the total patients. Clinical trials have found that EV71 infection sometimes induce a variety of severe neurological complications and even death, while CVA16 infection showed a mild clinical syndromes. Currently, no clinically effective therapeutic drugs for patients of severve HFMD. Therefore, the development and vaccination of prophylactic vaccines, as one of the important ways to control HFMD occurred is of vital importance.In view of the HFMD has become a widespread public health concern in the mainland China, and the large outbreak and neurotropism of pathogen EV71, the EV71 pathogen has become a current research focus of HFMD. This study will make EV71 pathogen the main research object of genetic engineering vaccine of HFMD.Now, the inactivated vaccine and virus-like particles(VLPs) vaccine are as the main research form of HFMD. However, due to the continuous development of genetic engineering technique,VLPs vaccines with its unique natural advantages have been widely used. Moreover, the VLPs vaccines of hepatitis B virus(HBV) and human papilloma virus(HPV) have been available, which provides a theoretical basis for the research of the EV71 VLPs vaccine. Although there has been some recent reports of EV71 VLPs vaccine prepared respectively by yeast expression system(Pichia pastoris and Saccharomyces cerevisiae) and insect-baculovirus expression vector system(BEVS), the purificationmethods of these VLPs are all used laboratory methods(sucrose density gradient or Cs Cl density gradient ultracentrifugation).Because of its own limitations using this laboratory purification method, which is time-consuming, laborious and costly, this method is not good for vaccines of large-scale production or clinical research. For these reasons, chromatograhy purification is becoming an imperative key factor of the development of EV71 VLPs vaccine.On that basis, this study does a comprehensive, concrete and in-depth research on above issues. We first optimized the expression conditions of EV71 VLPs vaccine,and then established a new kind of mutlistep chromatograhy purification method,followed by characterizing and identifying the EV71 VLPs efficacy. This study aims to prepare EV71 VLPs genetic engineering vaccine that is in conformity with the related regulation stated in Pharmacopoeia, by which to solve the current EV71 VLPs vaccine problems existing in the development of purification of laboratory method.This research mainly comprises of the the following two parts:Part one: optimization of upstream expression conditions and establishment of downstream purification methods of EV71 VLPs vaccineFirstly, according to the HFMD epidemic epidemiological investigation in mainland China, here the EV71 C4 epidemic strain(EU703814.1) had been chosen,and the EV71 VLPs were prepared using by BEVS, and the conditions of insect cell culture and recombinant BEVS expression were also optimized in this study. Finally we successfully screened the optimal expression conditions of EV71 VLPs vaccine.Secondly, chromatography purification is an important means of preparing high quality EV71 VLPs vaccine. In this study, a new flowing-through mode chromatography(Capto TM Core700), a strong mix mode anion exchange chromatography(CaptoTMadhere) and a weak hydrophobic chromatography(CaptoTM butyl) were jointly used. Through these chromatography columns combined use, we establish a new multistep chromatograhy purification method to successfully purify the EV71 VLPs. Through the identification of EV71 VLPs purity, we conclude this new purification method is available to prepare EV71 VLPs vaccine.Part two: characterization and preliminary efficacy evaluation of EV71 VLPs vaccineFirstly, we used the immunofluorescence staining and confocal microscopy and the in situ immunogold staining to detect the cellular expression and localization of EV71 VLPs, which showed that EV71 VLPs purified in this study mainly exist in thecytoplasm. To identify the EV71 VLPs vaccine purified by this multistep chromatography whether is in conformity with the related regulation stated in Pharmacopoeia, such as the purity, host-cell impurities, morphology, structure,diameter, amino acid sequence, isoelectric point(PI), molecular weight, glycosylation site, and so on, which all have been tested.Secondly, the protein-protein interaction system, Octet Red96 system(Forte Bio),was used to detect the affinity between the antibody and antigen, and the good antigenicity of the EV71 VLPs vaccine was verified, which has high affinity with22A12 m Ab; The neutralizing antibody acquired byanimal immunization was evaluated by pseudovirus detection system established by our laboratory. Compared with the EV71 inactivated vaccine, EV71 VLPs vaccine has high neutralization antibody titer, which suggests EV71 VLPs is of good immunogenicity.Third, the antibody protection test in vivo is one of the most important indicators to evaluate the effectiveness of EV71 VLPs vaccine purified by this study method.This study found, through experiments such astransmission electron microscope(TEM), high-performance liquid chromatography(HPLC), amimal immunizationand suckling mice challenged with a lethal dose of homologous virus, the EV71 VLPs not only have good stability but also have 100% immune protection in suckling mice,which can prevent the EV71 homologous virus attacks.In conclusion, according to shake flask and wave bioreactor culture, we optimized the Sf9 cell culturing conditions and the expression condition of EV71 VLPs vaccine, which lay a foundation for the downstream purification of EV71 VLPs vaccine. The HFMD genetic engineering EV71 VLPs vaccine, which was produced from insect-baculovirus expression vector system and prepared by multistep chromatograhy purification, was successfully cultured, purified, and characterized.The result showed that the purity of the product can reach more than 95%, and the recovery rate is 31.52%, and there has good features of characterization and stability,and about 215 neutralization antibody titer can be induced after the immunization,which can have 100% immune protection against EV71 homologous virus attacks in suckling mice. Consequently, the multistep chromatography purification method established in this study is promising to be the theory basis of large-scale production of EV71 VLPs vaccine, and the EV71 VLPs vaccine prepared by this method also is hoped to be a kind of effective vaccine, HFMD genetic engineering vaccine.The innovation of this paper as bellows: We established a new type of multistep chromatographic purification process for the first time, and the the purity is more than95% EV71 VLPs vaccine was successfully achieved. We also first verify the post-translation modification of EV71 VLPs in Sf9 cells, of which the specific N-glycosylation modification site is located at VP1 N176 of EV71 VLPs, and analyze the multi-subunit EV71 VLPs primary structure for the first time. To locate the expression of this vaccine at the cellular level, to detect the morphology, structural,stability, and antigenic characterization, to evaluate the immunogenicity of EV71 VLPs vaccine in the animal level, and to verify the immune protection of EV71 VLPs vaccine by using the existing suckling mice model.