Experimental Study On HCV Epitope Vaccine Using HBsAg As Carrier

Hepatitis C,also named as viral hepatitis C,is a viral hepatitis caused by infection with the hepatitis C virus(HCV).HCV is a blood borne virus that is mainly transmitted through blood transfusions,acupuncture,and drug use.HCV infection is prevalent globally.According to statistics from the World Health Organization(WHO),the global infection rate of HCV is about 3%,with an estimated 180 million people infected with HCV and approximately 35000 new cases of hepatitis C each year.Persistent infection with HCV can lead to liver fibrosis and cirrhosis,and some patients may develop into hepatocellular carcinoma(HCC),with decompensated liver function and even death.In the next 20 years,the mortality rate of patients caused by diseases related to HCV infection,such as liver failure and hepatocellular carcinoma,will continue to increase,posing great harm to patients’health and life.Therefore,HCV infection has become a major issue that seriously affects society and public health at this stage.At present,although some encouraging results have been obtained in research related to HCV infection,there is an urgent need for the development of new and efficient hepatitis C vaccines as the spread of HCV has not been fully controlled and there is no universal hepatitis C vaccine available.With the deepening of research on HCV epitopes,epitope vaccines are highly likely to be a feasible strategy for the development of HCV vaccines.Therefore,in this study,the feasibility of using HBsAg as a vaccine carrier was explored,and two highly conserved T epitopes of HCV were selected.HBsAg was used as the carrier for this epitope to prepare recombinant proteins HBsAg-T1 and HBsAg-T2.Furthermore,the immunogenicity of the recombinant mixed protein HBsAg-T1+HBsAg-T2 was analyzed,providing a preliminary experimental basis for the development of epitope vaccines for anti HCV treatment.1.Feasibility study on HBsAg as a vaccine carrierAt the 778-786 nucleotide site of the hepatitis B virus(HBV)surface antigen(HBsAg)gene S(Gen Bank registration number AF280817),a Spe I restriction enzyme digestion site was added by PCR method,and a recombinant eukaryotic expression plasmid pc DNA-S-RFP was constructed based on this.Subsequently,the plasmid was transfected into Hela cells and the expression of red fluorescent protein(RFP)was continuously observed using a fluorescence inverted microscope,Hela cells were found to emit red fluorescence and RFP was expressed normally,confirming the feasibility of HBsAg as a vaccine immune vector.2.Preparation of HCV recombinant epitope vaccineTwo T cell epitopes T1(35-44aa)and T2(131-140aa)of the HCV core protein and NS3 protein were artificially synthesized and recombined into the 778-786nucleotide site of the HBV S gene.The recombinant prokaryotic expression plasmids p Trc-S-T1 and p Trc-S-T2 were successfully constructed.The plasmid was transformed into competent Escherichia coli,and the virus particle like recombinant proteins HBsAg-T1 and HBsAg-T2 were extracted and purified by alkali lysis and sucrose density gradient centrifugation.3.Immunogenicity Study of HCV Recombinant Epitope VaccineUsing HBsAg protein as a control,ICR mice were immunized with recombinant mixed protein HBsAg-T1+HBsAg-T2 by subcutaneous injection into the abdomen.Samples were taken at the appropriate time to analyze the T cell subpopulations in spleen lymphocytes,tumor suppression,cytokines in spleen lymphocytes and serum,as well as the proliferative activity of non-specific T lymphocytes in spleen lymphocytes.At the same time,the peripheral blood lymphocytes of patients with acute HCV infection were isolated,and HBsAg was used as the control.The cells were stimulated with HBsAg-T1+HBsAg-T2,and the cytotoxic T lymphocyte(CTL)activity was detected by cytotoxicity test.The results confirmed that compared with the control group,the number of tumor formation and tumor size in mice immunized with mixed protein were significantly reduced.The results of flow cytometry showed that after three immunizations,the levels of CD8~+T cells in splenic lymphocytes of the HBsAg-T1+HBsAg-T2 group were higher than those of the IFN in lymphocytes of the control group and HBsAg-T1+HBsAg-T2 immunized mice-γHigher than HBsAg immunized mice,while IL-4 levels are lower.The ELISA test results showed that the serum IL-10 levels in the HBsAg-T1+HBsAg-T2 immunized group were significantly lower than those in the HBsAg immunized group(P<0.01).The MTT test results confirmed that the proliferation activity of spleen cells in the HBsAg-T1+HBsAg-T2group and HBsAg immune group was significantly higher than that in the Con A stimulated control group.Compared with the HBsAg group,HBsAg-T1+HBsAg-T2has a stronger ability to promote non-specific T cell proliferation in mice,and the upregulation of cellular immune response is more significant(P<0.01).The CTL activity analysis results also showed that when the target to effect ratio was 10:1,the CTL killing rate of the HBsAg-T1+HBsAg-T2 immune group was(77.42%±3.81)%,twice that of the HBsAg immune group(33.86±2.21)%,and nine times that of the control group(8.47±0.15)%(P<0.01).Therefore,this study confirms that HBsAg can be used as a carrier for epitope vaccines,and the prepared HBsAg-T1+HBsAg-T2 recombinant mixed protein can induce strong cellular immune responses,making it a potential therapeutic vaccine for HCV and worthy of further research.