The Development And Immunogenicity Studies Of Sudan Ebola Virus Like Particles

Ebola virus(EBOV)belongs to the family filoviruses,leading to lethal hemorrhagic fevers in humans and non-human primates with a case fatality rate of 50-90%,which poses a potential threat to public health.According to the WHO,there have been 28,616 confirmed and suspected cases of infection worldwide as of June 10,2016,with 1,1310 deaths.Sudan EVD broke out in 1976,1979,2000,2004,2011,2014,for an aggregate case-fatality rate up to 53.76%,but,as of now there are no approved specific drugs or vaccines to protect against the frequent outbreak of EVD.Accordingly,developing safe and effective filovirus vaccines remains a top priority.VLPs are highly ordered repetitive structures,which assembled by one or several proteins,aided by their natural ability of viral proteins bind naturally.VLPs were similar to the real virus in the structure and size.The granule structure is beneficial to antigen presenting cell uptake,which can stimulate powerful innate and adaptive immune responses,and VLP-based vaccines have been safely and effectively administered in humans.VLPs as emerging vaccine platform,has been widely used in human papillomvirus vaccine,hepatitis B virus vaccine and hepatitis E virus vaccine.Compared attenuated with inactivated,VLPs vaccine is more safe and effective in clinical application,and will not cause vector immunity problems associated with live carrier vaccine.Chapter one: Prokaryotic expression of Sudan Ebolavirus GP and the development of recombinant protein-based indirect ELISA for antibody detection.In this study a fragment of SUDV GP was amplified by PCR,and then cloned into prokaryotic expression vector pET30a(+).Expression was induced with IPTG.The expressed products were purified with His-Ni affinity chromatography according to the manufacturer's directions and identified using SDS-PAGE and Western Blotting.The indirect ELISA technique was established using the purified protein as a coating antigen,and optimized the reaction conditions.The results show that SUDV GP was expressed efficiently in the form of inclusion bodies.The condition for this ELISA was as follows:the antigen preparation was 2?g/pool,dilution of sera was 1:320,time for incubation of sera was 1.5 h,HRP-labeled goat anti-mouse IgG was 1:10 000 and the time for incubation of TMB was 7 min at room temperature.The ELISA systems based recombinant GP have good specificity with the coincidence rate of determination result and that of known serum samples was 100%.The reproducibility of ELISA was 1:40 960,and the CVs of test results in intra-and inter-assays were both less than 10%.SUDV GP fragment was expressed efficiently and the indirect ELISA for SUDV antibody was developed successfully,which is useful in the evaluation of vaccine,the monitoring of SUDV and the Development of kit.Chapter two: Production and identification of Sudan Ebola virus-like particles.In this study,we amplified the full length GP gene and VP40 gene by PCR,rBac-GP-GP and rBac-VP40-VP40 were generated in baculovirus system,recombinant baculovirus were determined by immunofluorescence assay based anti-GP polyclonal sera and anti-VP40 polyclonal sera,extraction and PCR identification of recombinant baculovirus genomes were in P4.The Titers of P4 were determined using a rapid titration kit.To generate SUDV VLPs,Sf9 cells were co-infected with r BV-GP-GP and rBV-VP40-VP40 with MOI=2.5:1.Culture supernatants were harvested and purification by a 10-30-50% discontinuous sucrose gradient,the bands between 30-50% sucrose were collected and re-suspended in endotoxin-free PBS.Purified SUDV VLPs were processed and examined by Western Blotting and transmission electron microscopy.The result of IFA using anti-GP polyclonal sera and anti-VP40 polyclonal sera demonstrated rBV-GP-GP and rBV-VP40-VP40 were successfully expressed in sf9 cells,genome PCR identification showed successfully amplified the specificity of the corresponding fragment size product.Electron microscopy results demonstrated that SUDV VLPs with the typical filamentous morphology are structurally similar to the native virus,which were approximately 80 nm in diameter and 800-1500 nm in length,Western blot were performed by anti-GP polyclonal sera and anti-VP40 polyclonal sera,and the result showed that both bands corresponding to VP40 and GP about 100 KD and 40 KD are present in the VLPs preparations product.These results demonstrated that rBV-GP-GP and rBV-VP40-VP40 were successfully expressed in Sf9 cells virus-like particles by the co-infection of SUDV GP and VP40 in baculovirus expression system.Chapter three: Immunogenicity of Sudan Ebola virus-like particles.Purified SUDV VLPs Mouse inoculated with purified SUDV VLPs and mixed polysaccharide, Alum,Freund,Prodavx,Montanide Gel 02 PR,Montanide IMS 1313 VG NST or Montanide ISA 201 VG adjuvant,and mice in control group were vaccinated with PBS alone.Treatment mice were vaccinated twice at 3-week intervals,levels of GP-specific antibodies were detected by indirect ELISA to assessment the best adjuvants for SUDV VLPs immune effect.Treatment mice were vaccinated intramuscularly with SUDV VLPs and mixed with best adjuvant twice at 3-week intervals,and control group were inoculated with PBS alone or adjuvant alone.In order to evaluated the cellular immune response,Inguinal lymph node and splenocytes samples were harvested from vaccinated mice at 7 days after the first immunization,and 1week after the second immunization,single-cell suspensions of Lymphocytes were isolated from the spleens to detect cytokine secretion by ELISpot and ELISA.To make an estimate of the humoral immune response of vaccination,we evaluated the neutralizing antibody responses in BABL/c mice by neutralization test using pseudotype virus following immunization with SUDV VLPs.The results show that after the second immunization,SUDV VLPs induced strong specific IgG antibodies responses against the SUDV GP fragments.The IgG titer in Montanide ISA 201 VG group was higher than other groups.The percent of activated B cell were significantly changed in the SUDV VLPs groups than that of in the PBS group and adjuvant group.With the stimulation of the SUDV GP antigen,both IFN-? and IL-4 secreted by splenocyte of SUDV VLPs vaccinated mice were significantly higher than that of adjuvant or PBS vaccinated mice.The levels of cytokines secreted from spleen cells were significant increased,such as IL-2,IFN-?,TNF-?,IL-4 and IL-10.SUDV VLPs could robust induce virus-neutralizing antibodies responses against HIV-based SUDV GP pseudovirion after two vaccinations.The above results show that SUDV VLPs has potential to be developed to a safe and effective vaccine of Sudan ebolavirus disease.These data demonstrated that SUDV VLPs have excellent humoral immunity and cellular immunity in mouse,provide candidate vaccine which can be effective in preventing SUDV infection.