Development Of An Inactivated Vaccine For Severe Fever With Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified bunyavirus in China in2010. Major symptoms of SFTS patients include high fever, thrombocytopenia, leukocytopenia, gastrointestinal symptoms, and lymphadenopathy. SFTS cases have been identified in at least13provinces in China, such as Henan, Hubei, Shandong, Jiangsu, Anhui, Liaoning, and the average case fatality rate of SFTS is approximately10%. There is currently no effective vaccine available. Therefore, developing a safe and effective preventive vaccine became urgently needed to cope with the outbreaks of SFTS.According to epidemiological distribution of SFTS in China,7SFTSV vaccine candidate strains isolated from the patients of different provinces were comparatively analyzed. The7strains were cultured on Vero cells and purified by3rounds plaque-to-plaque assay, and the virus titers of all strains reached higher than108TCID50/ml. The7SFTSV strains were inoculated and amplified in Vero cells, then the supernatant of cell culture was harvested and inactivated by p-propiolactone and purified by low-speed centrifugation, filtration, ultrafiltration, ultracentrifugation with20%sucrose cusion, virus particles with high purity and concentration were obtained. After determining the optimal experimental animal immunization procedure, these seven purified SFTSV strains were formulated into vaccines with aluminum adjuvant separately, and each strain immunized two rabbits using the same dose and immunization procedure. Rabbit serum were collected on0,14,28,42days, and cross neutralization test were performed by micro-neutralization test(MN) and plaque reduction neutralization test(PRNT) respectively. The antiserum of HB29strain showed the best immunogenicity and cross-protection ability. Its neutralizing antibody geometric mean titers against7SFTSV strains were1159(PRNT) and538(MN). The antiserum of AH12strain was with the neutralizing antibody geometric mean titers of861(PRNT) and302(MN). Genetic stability analysis showed that genomic homologies of each passage of7SFTSV strains remain at100%after10passages in Vero cells. Therefore, HB29and AH12are more suitable as vaccine strains.The preparation conditions of inactivated vaccine against SFTSV were explored. Firstly, optimal proportion of virus versus cells in inoculation was determined. Secondly, infected at MOI=0.01, SFTSV was cultured in Vero cells with four different media, serum-free DMEM, serum-free M199, DMEM containing2%FBS and DMEM containing0.5%human serum albumin. And the growth cures of SFTSV in the four media were analyzed, and serum-free DMEM was selected for vaccine production. Then, the inactivation effects on SFSTV with β-propiolactone and formalin at different concentrations were compared. The results showed that β-propiolactone at1:2000or1:4000concentrations inactivated virus well, and keep the protective antigenicity of virus. Finally, we conducted a preliminary exploration on purification of SFTSV using gel filter chromatography.Since SFTSV is a new virus, some experimental methods used in the vaccine development are needed to be established. We developed an assay for titration of SFTSV based on detection of nucleoprotein by double antibody sandwich ELISA. This method was compared with plaque assay and immunofluorescence assay, and shows the high correlations of these three methods. An identification test for SFTSV vaccine strain was also established. A neutralization test was performed using diluted virus and a fixed serum dilution.13monoclonal antibodies against SFTSV glycoproteins were prepared and characterized, and based on which a double antibody sandwich ELISA was developed to detect the SFTSV glycoproteins.