Construction, Expression And Immunological Activity Study Of A Novel HBV Therapeutic Vaccine-HBcAg And PreS1 Epitope Fusion Protein

Hepatitis B virus infection is one of severe infection diseases whichthreaten public health, however, there are not any effective therapeutic tools.It is estimated that there are 400 million carries of HBV according to WHO.Among these patients, about 5-10% of adults and 90% of children becomechronic HBV infection, leading to hepatic cirrhosis, liver failure andhepatocellular carcinoma. Currently, the two approved drugs for chronichepatitis are alpha-interferon and lamivudine, but their long-term effects arenot satisfied. Interferon-α achieves a short-term outcomes of around20–30% loss of HBeAg. The efficacy is even lower in Chinese patients,who are immunotolerant to HBV because of acquisition of the diseaseduring early childhood. Lamivudine, nucleoside analogues, is able toreduce HBV DNA to undetectable levels. However, cessation of treatmentusually leads to a rapid relapse of disease, and long-term treatment oftenresults in the selection of lamivudine resistant viral variants due to mutationof YMDD motif of the HBV polymerase gene. These outcomes emphasizethe need for novel therapeutic approaches. Specific immunotherapeuticstrategies have been proposed as possible alternatives to the use of IFN orantiviral drugs to break the non-responsiveness of T-cell immunity inchronically infected patients. Among these, specific vaccine therapies withrecombinant protein vaccines and gene vaccine and peptide vaccine havebeen studied with animal models or in human clinical trials.HBcAg is a subviral particle which consists of 183-185 amino acidsdepending on the virus subtypes, and can self-assemble into a virus-likeparticles(VLPs) structure containing 180 or 240 copies of monomer.HBcAg particles have been expressed successfully in E. coli, yeast cellsand insect cells, and the particles are similar in appearance and antigenicproperties to the native particles from patients. Currently, thephysico-chemical properties of HBcAg particles have been investigatedwith the following conclusions: (i) HBcAg consists of two domains:assembly domain (amino acids 1-149) and arginine-rich domain (aminoacids 150-183). The latter can non-specifically bind nucleic acids, to primespecific Th1 immunity by recombinant HBcAg based on its arginine-richregion and nucleotides bound to it;(ii) the major immunodominantregion(MIR) has been identified as the surface-exposed tip of the spikes(between amino acids 78-82) on the surface of the HBV core particles, thehybrid core proteins which generated by insertion of different sequenceswithin this region, can still assemble HBcAg chimeric particles;(iii) Thestrong immunogenicity of the HBcAg has been explained for its dualbehavior as a T-cell dependent and independent antigen, which is related toa potent B-cell activation to work efficiently as primary antigen presentingcells;(iv) HBcAg is a excellent carrier molecule because of its virus-likeparticle (VLP) structure, and its immunogenic properties can transfer evento foreign segments presented on the core particles.The preS1, as a subunit of HBsAg, has been used to develop the thirdgeneration HBV vaccine. It has been confirmed that the preS region play animportant role in the interaction between HBV and hepatocytes, and areceptor binding site was found within the preS1 sequence between aminoacids 21-47 which carries B and T cell recognition sites. The anti-serum toa synthetic peptide preS1 (21-47) could neutralize the virus by blockingvirus attachment to cells, and antibodies to a synthetic peptide mimickingpreS1 (21-47) could protect chimpanzees from HBV infection.In the present study, we constructed a prokaryotic expression vectorpBTcs1 which was created by inserting preS1 sequence into the e1 loopsequence of HBcAg. After expressed in E.coli HB101, the fusion proteinBTcs1 was purified by sucrose density gradient ultracentrifugation, and itsantigenicity and immunogenicity were analyzed. Our results indicated thatBTcs1 could react with specific anti-preS1 antibodies, and it could also selfassemble to form virus-like particles (VLPs). After immunized in Balb/cmice, BTcs1 could induce moderate anti-HBc and strong anti-preS1immune response, and a strong T cell proliferation response was detectable.The analysis of cytokine secretion and IgG isotype showed that the immuneresponse was mainly Th1 type. These results indicate that BTcs1 may bedeveloped a novel therapeutic and prophylactic vaccine against HBVinfection. The project includes three parts as following.Part Ⅰ Construction and identification of the prokaryoticexpression vector pBTc and pBTcs11. Construction of the prokaryotic expression vectorpBTc and pBTcs1The expression plasmid pBTc and pBTcs1 was constructed by usingDNA recombinant technology. In brief, HBV core gene coding for aminoacids 1-183 was amplified by polymerase chain reaction (PCR) and a leadersequence was ligated into the N-terminus of HBcAg. A NheⅠ restrictionenzyme site was produced in the e1 loop of HBV core sequence bysite-directed mutagenesis. The preS1 gene was also amplified by PCR andtwo restriction enzyme sites NheⅠ were introduced into the terminus ofpreS1 sequence respectively. After digested with NheⅠ, the preS1 fragmentwas cloned into HBV core sequence. To generate the expression vectorpBTcs1, the full-length fragment including preS1 and HBV core gene wascloned into pKK233.2 between NcoⅠ/PstⅠ.2. DNA analysis of the prokaryotic expression vector pBTcand pBTcs1Firstly, the plasmid pBTc and pBTcs1 were digested by NcoⅠ/PstⅠand NheⅠ, then the DNA sequence was analyzed by direct sequencing.The results of restriction enzyme and direct sequencing were consistentwith the expected sequence.3. Protein expression of the prokaryotic expression vectorpBTc and pBTcs1Protein expression was identified by Western blot, the specific antibodywas mouse anti-preS1 mAb or G.pig anti-HBc polyAb. The resultsindicated that clones of pBTc and pBTcs1 could probe with correspondingantibody specifically. This indicated that fusion gene could express inE.coli.Part Ⅱ Expression, purification and identification of fusionprotein BTcs11. Expression and purification of fusion protein BTcs1After identified, 2-3 pBTcs1 colonies were inoculated into 1L LBmedium. When OD600 was 4, the cells were harvested by centrifugation andthe pellets were lyzed by freeze thawing and lysis solution. The supernatantcontaining fusion protein BTcs1 was purified by sucrose density gradientultracentrifugation, and the positive fraction were collected for furtherdialysis. Result of DOT-BLOT indicated that the distribution of BTcs1 wasmainly in 30-50% sucrose.2. Identification of fusion protein BTcs1The purity of BTcs1 was analyzed by SDS-PAGE and SEC, resultsindicated that the purity of BTcs1 was beyond 95%. The antigenicity ofBTcs1 was analyzed by Western blot and result indicated that BTcs1 couldprobe with mouse anti-preS1 mAb or G.pig anti-HBc polyAb specifically.The particles formation was analyzed by electron microscope and resultindicated that BTcs1 could self assemble VLPs.Part Ⅲ Immunological activity analysis of fusion proteinBTcs11. Antibodies production and isotype analysisThe specific antibodies titer was tested by indirect ELISA. Afterimmunized in Balb/c mice, BTcs1 could induce high titers anti-preS1 andlow titers anti-HBc. On day 42 of immunization, the titers of anti-preS1 andanti-HBc were 12800 and 2400, respectively. The major isotype ofanti-HBc and anti-preS1 was IgG2a. This indicated that BTcs1 couldinduce Th1 type immune responses in Balb/c mice.2. Splenocyte proliferation assaysSplenocyte proliferation response was measured by [3H] thymidineincorporation methods, and the proliferation ability was expressed asstimulation index (SI). Results indicated that BTcs1 could inducesplenocyte proliferation at lower antigen dose. The degree of proliferationwas depended on antigen dose. The proliferation response was inducedmainly by T cell epitope of HBcAg.3. Analysis of Th1/Th2 type cytokines expressionSplenocytes harvested from immunized mice were stimulated withcorresponding antigen in vitro for 3 days. Then, 0.5 ml of supernatant wascollected and cytokines were analyzed by a mouse Th1/Th2 Cytokine CBAkit. Results indicated that BTcs1 could induce mainly Th1 type cytokinesexpression, especially the level of γ-IFN was significantly higher than thoseof control group (p<0.01).In this study, we modified the HBcAg gene using DNA recombinanttechnology, and preS1 epitope gene was inserted into modified HBcAggene for a prokaryotic expression vector pBTcs1. After transfected intoE.coli, fusion protein BTcs1 was expressed and purified by sucrose densitygradient ultracentrifugation. Then the immunological activity of BTcs1 wasanalyzed in Balb/c mice. Our results indicated that BTcs1 could induce hightiters protective antibodies and mainly Th1 type immune responses inBalb/c mice, indicate that BTcs1 may be developed for a novel HBVvaccine with both prophylactic and therapeutic functions. However, manyproblems still need to be solved. There is a long way to go before practicaluse in clinic.