Immunological Characterization Of The Recombinant Adenoviral Vectors Carrying Chimiric Gene G1/G2-S0.7-HSP70C、Ub-G1/G2 -S0.7 Of Hantavirus

Hantaviruses are the etiologic agents for two acute febrile disorders of man: hemorrhagic fever with renal syndrome(HFRS) and hantavirus pulmonary syndrome(HPS).Until now, there is no WHO-approved hantavirus vaccine available. However at least three different inactivated vaccines, based on purified suckling-mouse brains, golden hamster kidney cells or Mongolian gerbil kidney cells, were developed in Korea and China, and which are being used only locally. One serious problem is that these vaccines have poor immunogenicity for eliciting neutralizing antibodies and cell-mediated immunity. It is important to develop more effective vaccines.GP plays an important role in stimulating neutralizing antibodies and keeping humans from HV infection, but its immunogenicity is so weak that the antibody ?titer elicited by GP is low.NP can provoke cell-mediated immunity and keep humans from being infected by HV. It also has been proved that NP contains neutralizing epitopes. NP play an important role in evoking cellular immune response. Previous work has proved that the 0.7 kb fragment of the S segment close to the N-terminus could produce the same immunological effect as the complete NP. our previous experiments indicated that the fusion proteins G1S0.7 and G2S0.7 could effectively elicit neutralizing antibodies and a cellular immune response. Then, we replaced the CMV promoter with CAG promoter to the adenoviral vector, and incorporated HSP70C and Ub genes into the recombinant adenoviral vectors to enhance the expression of the fusion protein.In this study, we will compare the expression level as well as immunized mice with recombinant adenovirus, and make initiatory animal protection tests.1. C57BL/6 mice were inoculated by intraperitoneal injection, HFRS inactivated vaccine, Adenovirus-Lac Z and normal mice were employed as controls. The results from ELISA showed that the specific antibodies titers of mice immunized with rAd-G1S0.7-pCAG、rAd-G1S0.7-pCAG-HSP70C and rAd-G2S0.7-pCAG-HSP70C were the highest, which can be 1:160; The results from microcell- cultured neutralization test, neutralization titers of mice immunized with rAd-G1S0.7-pCAG-HSP70C were the highest, which can be 1:40 higher than the vaccine control(1:20); The results from ELISPOT showed that:(1)The IFN-γof rAd-G1S0.7-pCAG、rAd-G1S0.7-pCAG–HSP70C and rAd-G2S0.7-pCAG-HSP70C were higher than the other experimental groups(P<0.05);(2)The IL-2 of rAd-G1S0.7-pCAG was higher than the other experimental groups(P<0.05);(3)The IL-4 of rAd-G1S0.7-pCAG-HSP70C and rAd-G2S0.7–pCAG–HSP70C were higher than the other experimental groups( P<0.05 ) ;(4)IL-10 did not change remarkably during all the groups(P>0.05);The results from Cell-mediated cytotoxicity assay showed that the specific cytotoxic effects of the rAd-G1S0.7-pCAG and rAd-G1S0.7–pCAG -HSP70C were stronger than the other experimental groups(P<0.05). 2. C57BL/6 mice were infected with hantavirus by intramuscular injection Then we detected the specific antigen from C57BL/6 mousses? Livers and spleens by ELISA after 3 days. THE results showed that rAd-G1S0.7-pCAG and rAd-G1S0.7-pCAG-HSP70C could give a stronger protection for C57BL/6 mouses?Livers and spleens than other experimental groups(P<0.05).To conclude, rAd-G1S0.7-pCAG、rAd-G1S0.7-pCAG-HSP70C and rAd- G2S0.7-pCAG-HSP70C can elicit a higher humeral-mediated immunity level; rAd-G1S0.7-pCAG、rAd-G2S0.7-pCAG、rAd-G1S0.7-pCAG-HSP70C and rAd- G2S0.7-pCAG-HSP70C can elicit a higher cell-mediated immunity; rAd-G1S0.7- pCAG and rAd-G1S0.7-pCAG-HSP70C can give a stronger protection for C57 BL/6 mouses?Livers and spleens. We will chose rAd-G1S0.7-pCAG、rAd-G2 S 0.7-pCAG、rAd-G1S0.7-pCAG-HSP70C and rAd-G2S0.7-pCAG-HSP70C to be the candidate vaccine and do long-term toxicity test、acute toxicity test and pharmacokinetics about them。...