Roles Of The Adjuvant LNT On The Enhancement Of Antigen Specific Immune Responses To HCV/NS3 DNA Vaccination

Part one: Preparation and identification of a hepatitis C virus nonstructural protein 3(NS3) DNA vaccineObjective: To prepare , identify and quantitate a hepatitis C virus nonstructural protein 3(NS3) DNA vaccine.Methods: The competent HB101 E.coli was transformed with pcDNA3.1-HCV/NS3,after amplification and extraction,the amplified products was quantitatively analyzed and identified by the agarose gel electrophoresis.Result : The competent HB101 E.coli was successfully transformed with pcDNA3.1-HCV/NS3.Single bacterial clone largely grew on the plate containing ampicillin incubated at 37℃。After amplitication and extraction,The concentration of DNA vaccine was from 1.5μg/μl to 1.8μg/μl, ,and the DNA purity was nearly 1.8.It was confirmed that the amplified plasmid contained the part of HCV NS3 DNA fragment which was 1.8 Kb by the agarose gel electrophoresis.Conclusion: HCV/NS3 DNA vaccine is successfully prepared for immunization in our lab. It is laid a foundation for further research in the immunity of the HCV/NS3 DNA vaccine.Part two: The effect of the adjuvant- lentinan on the specific humoral immunity generated by HCV/NS3 DNA vaccineObjective: To investigate the immunization of lentinan combining with HCV NS3 DNA vaccine on the antigen specific humoral immune response.Methods: Balb/c mice were immuned twice at 10 days intervals with 100μg HCV/NS3 plasmid by direct intramuscular injection.The different dose of LNT were given intragastrically.The mice were bled from the tail and serum was prepared at 1,3 and 5 weeks after primary DNA immunization.The plates were coated with HCV/NS3 protein and the anti-HCV/NS3 antibody level in the serum was determined by ELISA. Also the cytokines as IL-4 and IFN-γin serum were detected by ELISA technique.Results: Each immunization group produced different levels of specific IgG Abs one week after the last immunization . Particularly, compared to the group immunized with pcDNANS3 alone, a significantly enhanced level of specific IgG Abs was obtained in the group immunized with pcDNANS3 plus 0.5mg/ml LNT as adjuvant(P<0.05). The groups combination with 1mg/ml or 0.1mg/ml LNT seemed to be not help in the improvement of humoral immune responses. Moreover, with the lasting of time, the level of specific IgG Abs was gradually increased in the groups of immunized with HCV/NS3 alone or plus 0.5mg/ml LNT. Compared to the group of DNA immunized alone, the secretion of IFN-γwas increased in the groups of LNT (1mg/ml, 0.5mg/ml, 0.1mg/ml) plus HCV/NS3 DNA vaccine. Furthermore, 0.5mg/ml LNT plus DNA could increase a higher lever of IFN-γin serum compared to DNA alone(P<0.01). However the production of IL-4 in serum is very low and there were no obviously differences between all the groups(P>0.05).Conclusion: Specific IgG Abs is detected by immunized BALB/c mice twice with HCV/NS3 plasmid. Middle dose of LNT (0.5mg/ml) combining with HCV/NS3 plasmid can induce strong humoral immunity compared with HCV/NS3 plasmid . IFN-γsecretion is indicative of a Th1-biased response, while IL-4 production is indicative of a Th2-biased response. This result suggest that the combination of pcDNANS3 with LNT preferentially induced a Th1-type respinse.Part three: The effect of the adjuvant- lentinan on the specific cell immunity generated by HCV/NS3 DNA vaccineObjective: To investigate the immunization of lentinan combining with HCV/NS3 DNA vaccine on the antigen specific cell immune response.Methods: Balb/c mice were given different dose of lentinan plus the same dose of NS3 plasmid (100μg/per mouse) in a prime and boost immunization strategy. 10 days after the second injection, spleens were taken, prepared to cell suspension and cultured with NS3 peptide as effector cells. In addition, NS3 cocultured P815 cells were used as target cells. NS3 specific T cell proliferation and cytotoxicity T lymphocyte reaction (CTL)were analyzed by MTT methods.Results: When co-administrated with LNT, the levels of specific T lymphocyte proliferation were obviously higher than using NS3 plasmid alone except the group combination with 1.0mg/ml LNT. It was showed that the high and middle dose of LNT plus NS3 plasmid induced stronger NS3 specific CTL reaction at the ratio of effector to target as 50:1 and 25:1,compared with the single use of NS3 plasmid(P<0.05). There is no difference in the CTL response between the group immunized with pcDNANS3 alone and the group immunized with pcDNANS3 plus 0.1mg/ml LNT. With the increase of the ratio of effector to targetor,the percentage of specific lysis was gradually increasing.Conclusion: Immunized BALB/c mice with the middle dose of lentinan (0.5mg/ml) combining with HCV NS3 plasmid can induce strong antigen specific CTL reaction and T cell proliferation. It is suggested that lentinan could be used as adjuvant for HCV NS3 vaccine to provoke antigen specific immune response.