Immune Responses To Plasmid DNA Encoding The Hepatitis C Virus Core Deletion Mutant In Mice

HCV infection is becoming a major problem of health worldwide. It is believed that HCV has infected an estimated 170 million people. There are more than 85% of HCV infected people will become chronic carriers after acute infection with the risk of developing cirrhosis and hepatocellular carcinomas. Up to now, only a minority of patients benefit from antiviral therapies. The development of a preventive ,and possibly even more, a therapeutic vaccine is thus highly desirable.HCV core protein has been studied as candidate of target of DNA vaccine for a long time. Data show that plasmid encoding HCV core protein could induce core-specific humoral and cellular immune responses. However, the immune responses are rather low when immunized with those encoding full-length core protein. Although multiple strategies have been used by many researchers, the core-specific immune responses still remains to be optimized. One of the reasons may be that the expression of core protein after DNA inoculation is not sufficient to produce enough antigen. Another reason may be that the core protein has inhibitory effects to immune responses. The aim of our study is to modify HCV core gene by deleting of certain peptides which seems to affect expression or immune recognition in order to enhancecore-specific immune responses. Methods and Results:1. HCV core protein cDNA was amplified by RT-PCR from the sera of 2 patients with chronic hepatitis C from Xi'an. The cDNA fragments were inserted into pGEM-Teasy vetor and identified by restriction enzyme and DNA sequencing . Phylogenetic analysis showed that the 4 HCV core cDNA clones were genotype 1b, named as HCV-CE1-2-1, HCV-CE1-2-2, HCV-CE1-3-3, HCV-CE1-3-4. The HCV-CE1-3-3 was used as the template of next PCR reaction.2. Four HCV core gene fragments, encoding full-length core(C573), core with 170-191aa deleted(C507), core with 60-80aa deleted(C510) and core with 170-191aa and 60-80 aa deleted(C444), were amplified by PCR and splicing overlap extension(SOE) PCR from HCV-CE1-3-3 core cDNA clone. Subsequently, they were cloned into pCI-neo to construct the recombinant eukaryotic expression vector respectively, named as pCI-C573n pCI-C507> pCI-C444 pCI-C510. The recombinant plasmids were then transfected into P815 cell line using Iipoefectamine2000 and detected transient expresssion by IFA and analyzed with confocal scanning. Semi-quantification of fluorescence signal showed pCI-C507(130.50 ± 11.86) > pCI-C444(109.29 + 9.11) > pCI-C510(84.05±7.05)>pCI-C573(44.29±8.08 ) and the level of the three mutant were significantly higher than that of pCI-C573(P<0.001). P815 cells were selected by G418 to establish cell line for stable expression and confirmed through IFA and Western blot.3. Twenty five BALB/c mice were randomly divided into 5 groups and immunized with three doses of recombinant plasmids constructed above respectively on weeks 0,2 and 6. Ten weeks after the first immunization, mice...