Immunological Characterization Of The Modified Recombinant Adenoviral Vectors Carrying Chemiric G1S0.7 Or G2S0.7 Of Hantavirus

Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaviruses (HTV), and there is no specific treatment right now. There are a few inactivated vaccines licensed for use in China, but their prophylactic effect has prompted mixed reviews. One clear problem is the vaccines'poor immunogenicity for eliciting neutralizing antibodies and cell-mediated immunity. It is clearly indicates a need for developing more effective vaccines.It is well demonstrated that GP is a main candidate protein for the HTV genetically engineered vaccine: It plays an important role in stimulating neutralizing antibodies and in protecting humans and animals from HTV infection; however, its immunogenicity is weak, the antibody elicited by GP is produced later, and the titer is low. Among the structural proteins, NP has the strongest immunogenicity and it can elicit a high titer and a long-lasting antibody response. It is disputed that NP contains neutralizing epitopes, but the fact that NP can protect animals from HTV infection is totally confirmed. Besides, NP contains antigenic sites associated with cytotoxic T-lymphocyte (CTL) responses. Thererfore, NP plays an important role in evoking cellular immune response against HTV infection. Previous work has demonstrated that the antigenic sites of NP are mainly distributed at the 0.7 kb fragment of the S segment, which is close to the N-terminus. Mice immunized with the truncated protein expressed in prokaryotic vector could produce the same immunological effect as immunized with the complete NP. Further experiments indicated that mice immunized with the fusion proteins G1S0.7 (G1 of the M segment and a 0.7 kb fragment of the S segment) and G2S0.7 (G2 of the M segment and a 0.7 kb fragment of the S segment) effectively elicit specific anti-NP, anti-GP and neutralizing antibodies and a cellular immune response. And the immune response produced by the recombinant adenovirus containing the chemiric genes was more efficient than other systems. But the expreesion level of the fused proteins was not satisfied, and the cell-mediated immunity of mice which were immuned with the expressed products was still low. Efficient strategies are need for the problems.In this study, we chose the human adenoviral type 5 replication-incompetent systems to express the fusion proteins G1S0.7 and G2S0.7. To improve the expression levels, we made several modifications to the adenoviral vector pShuttle, including replacing the CMV promoter (Human cytomegalovirus early enhancer/promoter) with CAG promoter (hybrid of Human cytomegalovirus early enhancer and chickenβ-actin promoter). We also incorporated WPRE (Woodchuck Hepatitis Virus post-transcriptional regulatory element) alone, or in conjunction with, the new promoter CAG. After packaging the recombinant adenoviruses and infecting HEK 293 cells, we identified the fusion proteins and compared the expression level as well as immunized mice with recombinant adenoviruses, a series of immunological assays were taken and the immunological characteristics were studied.1. By designing and synthesizing the CAG promoter, the classic CMV promoter of the adenoviral vectors pShuttle containing chimeric genes G1S0.7 or G2S0.7 were replaced, naming G1S0.7-pCAG and G2S0.7-pCAG; Designed and synthesized the WPRE, incorporated at the 3′UTR of pShuttle carrying chimeric genes G1S0.7 or G2S0.7, naming G1S0.7-WPRE and G2S0.7-WPRE; By both replacing the promoter and incorporating WPRE, the reconstructed vectors containing chimeric genes G1S0.7 or G2S0.7 were modified, naming G1S0.7-pCAG-WPRE and G2S0.7-pCAG-WPRE, respectively.2. Reconstructed pShuttle vestors containing chimeric genes, and the pAdeno-X DNA were all digested with PI-SceⅠand I-CeuⅠ, and the ligation products were transformated into E.coli JM109. Clones were identified by XhoⅠdigestion, PCR amplified and PI-SceⅠand I-CeuⅠdouble digestion. After transfected HEK 293 cells, the recombinant Adenoviruses were packaged, naming rAd-G1S0.7-pCAG, rAd-G2S0.7-pCAG, rAd-G1S0.7-WPRE, rAd-G2S0.7-WPRE, rAd-G1S0.7-pCAG-WPRE and rAd-G2S0.7-pCAG-WPRE.3. By purifing and determinating the viral titers, immunofluorescence assays and Western blot were employed to identify the expression products, the unmodified recombinant adenoviruses rAd-G1S0.7 and rAd-G2S0.7 were used as controls. A positive fluorescence could be observed in 293 cells 48 hrs after infection in IFA. The most intense fluorescence for the target proteins were all observed in vector with the CAG promoter groups. Western blot results indicated that the reconstructed vectors with a CAG promoter and carrying the chemiric genes G1S0.7 or G2S0.7, expressed 2.1-fold and 2.3-fold more protein than the unmodified vectors, respectively.4. C57BL/6 mice were inoculated by intraperitoneal injection, HFRS inactivated vaccine, Adenovirus-Lac Z and normal mice were employed as controls. ELISA, microcell-cultured neutralization test, ELISPOT and CTL assay were performed for the immunological characteristics of recombinant adenoviruses. The results showed that among all the experimental groups the titers of mice immunized with rAd-G1S0.7-pCAG were the highest, which were 1: 80 and 1: 320, respectively. In the microcell-cultured neutralization test, a low titer of neutralization antibodies were observed in all experimental groups (1: 10~1: 40), of which the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the vaccine control (1: 10~1: 20). The levels of cytokines stimulated by splenocytes were quite different: the INF-γand IL-2 of rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were higher than the other experimental groups and vaccine control (P<0.05), while TNF-αand IL-10 did not change remarkedly during all the groups. Cell-mediated cytotoxicity assay results indicated that all the recombinant adenoviruses induced specific cytotoxic effects on target cells, which was enhanced in accord with the rise of E/T ration. Among all the experimental groups the specific cytotoxic effects of the rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG were stronger than the other experimental groups and vaccine control at the E/T ration of 100: 1 and 50: 1(P<0.05).By replacing the promoter, we obtained 2 recombinant adenviruese rAd-G1S0.7-pCAG and rAd-G2S0.7-pCAG, which could express the fusion proteins in a relative high level. Animal experiments showed that these recombinant adenviruses induced a stronger humoral immunoresponse and cellullar immunologic response, especially the cellullar immunologic response, which were stronger than the vaccine control. Next step, we hope to obtain more recombinant adenoviruses which could induce effective cell-mediated immunity by inserting the adjuvant such as ubiquitin and HSP70.