Study Of A Recombinant Adenovirus Vector Vaccine Expressing The E2 And â–³E2 Antigen Of Hcv

Objective:Infection with hepatitis C virus (HCV) is a worldwide problem that has affected approximately 170 to 200 million persons. So far, there is no specific strategy available to prevent HCV infection. Hence, a vaccine that prevents and treats chronic HCV infections will be an important public health contribution.The protein E2 of HCV is the most important antigen that induces the neutralizing antibody of HCV. However, this antigen could combine with the cell surface protein, CD81, which could result in abnormal activation and proliferation of B cells, and would inhibit humoral immunoresponse. Thus the natural E2 antigen might not be suitable target antigen for HCV vaccine. Nevertheless, studies suggested that mutations in the CD81 binding segments (481-491aa and 544-551aa) would not affect the normal spatial structure of E2 and the neutralizing epitope of this antigen also would not be lost. Therefore, it may be a possible way to introduce mutations in these fragments to optimize the E2 as a suitable target antigen in vaccine development.Replication-defective adenovirus has shown more advantages than other vector systems, such as infecting more types of cell, higher efficiency of gene transfer, stronger antigen expression and inducing a higher protective immune to target protein, raising expression of cytokines, and so on, all of which have made it a promising vector system for gene transfer.In this study, the adenovirus-based recombinant vaccines rAd/E2 and rAd/△E2 were constructed, and the immune effects of these vaccines were observed, which paved a new way to the development of HCV vaccine.Methods: (1) Amplifition and identification of target gene: The DNA fragment of E2 was amplified by PCR from the plasmid pEGFP-HCV/E1E2 and inserted into pGEM-T vector. The linking production was then transformed into competent E.coli DH5α.The plasmids were extracted and identified by PCR as well as restriction endonucleases digestion and sequencing. (2) Construction of the eukaryotic expressing plasmid pcDNA3.0/E2: The fragment of E2 was obtained from pGEM-T/E2 by proper endonucleases digestion and then inserted into pcDNA3.0 vector which was cut by the same enzymes to construct the eukaryotic expressing plasmid pcDNA3.0/E2. After transformation,the recombinant plasmid was verified by PCR and enzymes cutting. (3) Construction of pcDNA3.0/△E2: Three aa were selected to be mutated in the E2 protein of HCV, one of which located in aa regions 481-491(cysteine to phenylalanine); and the other two located in aa regions 544-551(proline to leucine or alanine respectively). Accordingly, two pairs of complementary mutagenic oligonucleotides that contained the desired mutations were well designed and synthesized, and then a PCR-based, site-directed mutagenesis were performed using pcDNA3.0/E2 as template. After one step amplifications was completed the products were digested by DpnI to remove the template plasmid so as to increase the mutagenesis efficiency. Then the digested productions were transformed into competent E.coli DH5α, which could repair the nicks of those linear productions automatically. As a result, two mutants were obtained, one of which was named pcDNA3.0/△E2①, and the other was called pcDNA3.0/△E2②. Likewise, the same method used the back pair of two complementary synthetic oligonucleotides as primers and plasmids pcDNA3.0/△E2①as template to synthesize the third production named pcDNA3.0/△E2③, which contains both of the mutated points of△E2①and△E2②. (4) Construction of the recombinant adenoviral transfer plasmid pAdTrack-CMV/E2 and pAdTrack-CMV/△E2: The segment of E2 was retrieved from pGEM-T/E2 by suitable restriction enzymes and the fragment of△E2 was cut from pcDNA3.0/△E2 by the same enzymes and then both were purified by agarose gel electrophoresis. The fragment obtained was respectively subcloned into the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the recombinant adenoviral transfer plasmid pAdTrack-CMV/E2 or pAdTrack-CMV/△E2. Competent E.coli DH5αwere transformed, and selected by Kanamycin. The plasmids were extracted and then verified by PCR and enzymes cutting. As a result, the recombinant adenoviral transfer plasmid pAdTrack-CMV/E2 and pAdTrack-CMV/△E2 were formed successfully. (5) Generation of recombinant adenoviral plasmid: The recombinant transfer plasmid pAdTrack-CMV/E2 and pAdTrack-CMV/△E2 were both linearized by Pme I and then co-transformed into E.coli strain BJ5183 respectively with pAdEasy-1 that is the viral DNA plasmid using two-steps method. The recombinant viral DNA plasmids were identified by the PacI restriction enzyme analysis. Once identified successfully, the recombinant adenoviral plasmids were transformed into competent E.coli DH5αfor large preparation of DNA, and the resultant DNA were purified by PEG. (6) Packaging and amplification of the recombinant adenovirus particle The recombinant adenoviral plasmids were linearized by PacI digestion and then transfected into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000, according to the manufacturer's guidelines, to produce viral particles. The expression of green fluorescent protein (GFP), which is the tagging protein of the recombined virus, was monitored dynamically to appraise the packaging result. The cells and supernatant were collected using frozen-thawing method, and as a result, the first generation of the recombinant virus was gotten. The recombinant virus was amplified for 2 o 4 passages in 293 cells, and the four passage virus were purified using the adenovirus purification kit. (7) Determination of viral titration: Cell-free virus stocks were diluted serially in serum-free medium at ten times, and then infected the 293 cells growing on the 96-wells plates confluented seventy to eighty percent. According to AdEasy vector system application manual, the viral titration was measured by plaque forming units (PFU) methods. (8) The recombinant virus rAd/E2 and rAd/△E2 were both treated with proteinase K, and then identified by PCR. Simultaneously, the expressing of target protein encoded by the recombinant adenovirus was detected by reverse transcription PCR, and at the same time, the expressing of protein E2 in the infected 293 cells was detected by Western blotting. (9) Amplifing and purifying the Ad packaged in the past year using the same method, and then testing the titrers of the virus. (10) Large preparation was performed to obtain the plasmids of pcDNA3/E2 from transformed E.coli DH5α, and then purified by PEG8000. (11) Animal essay: BALB/c mice aged 6-8 weeks were divided into four groups: Ad/E2 group were immunized with 4.0×107pfu/100μl of Ad/E2 for two times at an interval of two weeks; pcDNA3/E2 group, immunized with 100μg plasmids for three times at the interval of three weeks; Ad group, immunized two times at two weeks'interval; PBS group, immunized with 100μl PBS three times at three weeks'interval. All of the mice were inoculated with intramuscular injection. Three weeks after the last immunization, splenocytes of the mice in each group were stimulated by inactivated rAd/E2 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay.Results: (1) The clone vector pGEM-T/E2 was constructed successfully; endonuclease analysis and sequencing result showed that the inserted genes were the same as espected. (2) The eukaryotic expressing plasmid pcDNA3.0/E2 and the mutational plasmid of pcDNA3.0/△E2 were constructed prosperously. pcDNA3.0/△E2①, was mutated at aa regions 481-491, from cysteine to phenylalanine; In pcDNA3.0/△E2②, the two mutations were located in aa regions 544-551, mutated from proline to leucine or alanine; the last one was known as pcDNA3.0/△E2③, which contained the three mutations above-mentioned. (3) The recombinant adenoviral transfer plasmid pAdTrack-CMV/E2 and Track-CMV/△E2 was constructed successfully. (4) Recombinant adenoviral plasmid pAd/E2 and pAd/△E2 were generated successfully. Restriction cutting with PacI yielded a large fragment of approximate 30 kb, and a smaller fragment of 4.5 kb. The lengths of the fragments were the same as expected. (5) Recombinant adenovirus rAd/E2 and rAd/△E2 were packaged and amplified successfully. (6) The titers of recombinant adenovirus rAd/E2 and rAd/△E2 from passage 1 to passage 4 were respectively as follows: 2.2×104 pfu/ml, 4.0×107 pfu/ml, 1.0× 108pfu/ml, 7.5×108 pfu/ml; 2.5×104 pfu/ml, 1.0×107 pfu/ml, 2.65×108pfu/ml, 6.0×108pfu/ml; 3.0×104pfu/ml, 4.1×107pfu/ml, 3.7×108pfu/ml, 9.5×108pfu/ml; 1.8×104pfu/ml, 4.0×106pfu/ml, 6.7×107pfu/ml, 3.9×108pfu/ml. (7) The recombinant virus rAd/E2 and rAd/△E2, treated with proteinase K, were identified by PCR, all of them showed specific strand by agarose gel electrophoresis. The expressing of protein E2 in the 293 cells infected by the recombinant adenovirus was confirmed by reverse transcription PCR as well as Western blotting, and likewise the expressing of protein△E2 was certified by reverse transcription PCR too. (8) Splenocytes from the vaccinated mice showed different proliferative activity to different stimulants of inactivated rAd/E2 or ConA. After stimulated by ConA, which was positive control, the proliferative activities of all of the four groups were increased. While when stimulated by rAd/E2, there was no significant difference in the proliferative activity among groups. (9) Two kinds of target cells were used in the cytotoxic assay, one was the SP2/0 cells infected by rAd/E2, and the other was the SP2/0 transfected by the plasmid of pcDNA3.0/E2. When the first one was used as target cell, the specific lymphocytic CTL cytotoxic activity of Ad/E2 group, pcDNA3/E2 group as well as Ad group were efficiently enhanced, which was remarkably stronger than that in the mice injected with PBS. Meanwhile, both of the Ad/E2 group and Ad group showed significantly stronger specific lymphocytic CTL cytotoxic activity than the pcDNA3/E2 group. However, the difference between Ad/E2 group and Ad group was not significant (P>0.05). While the second ones were used as target cell, there was no difference in the specific lymphocytic CTL cytotoxic activity among all the four groups.Conclusion: In this study, AdEasy adenovirus system was used to construct and package the recombinant adenovirus-based vaccine rAd/E2 and rAd/△E2, which might pave a new way to the development of HCV vaccine.