Construction Of The Recombinant Adenovirus Expressing The CVB3 VP1 Protein With A Signal Peptide Of Human IL-2 And Study On Its Immunological Effects In Mice

Objective: Coxsackievirus Group B Type 3(CVB3) is a major pathogen of human viral myocarditis, which is the leading cause of neonate sudden death as well. There is no virus-specific preventive procedure against CVB3 infection available today.Gene vaccine affords an opportunity to pave a new way to prevent CVB3 infection. Although different vaccines constructed with pcDNA3 vector could induce the production of antibody, the titers of antibody induced were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy to improve immunogencity of the DNA vaccine so as to induce stronger CVB3-specific immune responses and enhance the protective efficacy.One of the key points of gene therapy is the exploitation of a transfer vector with high efficiency of gene transfer and target specificity. In the past years, a recombinant, replication-defective adenovirus system has been used as a transfer vector for experimental models of gene therapy. The fact that adenovirus could infect most types of cells with no requirement for cell division, transfer and express gene in a higher efficiency, and therefore robust the protective effect of the recombinant adenovirus-based vaccine, has made it a promising system for human gene therapy in vivo.VP1 is the major capsid protein of CVB3, which can induce both humoral and cellular immune responses. It had been reported that DNA vaccine expressing VP1 alone could induce the production of antibody, but the titers of antibody were usually low. The probable reason might be that VP1 protein could be expressed by the transfected cells but released from these cells and thus can not elicit antibody response effectively.Signal peptides may help proteins transport out of cells, so adding signal peptide gene to VP1 cDNA can probably promote the secretion of VP1 protein by the transfected cells. Human IL-2(hIL2) signal peptide is a quite hydrophobic sequence consisted of 20 amino acids. In the early study, we have constructed a secretable VP1 eukaryotic expressing plasmid pcDNA3/sVP1, which was constructed by fusing DNA sequence of the 11 N-terminal amino acid residues of mature hIL-2 with CVB3 VP1 and could express VP1 secretively. It has been shown that the gene vaccine could protect mice against CVB3 challenge.In this study, we constructed an adenovirus-based CVB3 vaccine Ad/sVP1, which could express sVP1 in 293 cells and evaluated the immune protective effect of the vaccine in mice.Methods: (1) The DNA fragment of sVP1 was amplified by PCR from the plasmid pcDNA3 encoding sVP1 protein. Gene fragments were inserted into pGEM-T vector respectively. Competent E.coli DH5αwere transformed and selected by Ampicillin, the recombinants were verified by electrophoresis analyses and gene sequencing. (2) The gene fragments were purified from agarose gel, and cloned into the transfer vector pAdTrack-CMV cut by the same endonucleases to construct the transfer plasmids AdTrack-CMV/sVP1. The transformed bacteria were selected by Kanamycin, the recombined plasmids extracted were verified by endonuclease analyses. (3) The recombinant plasmids were linearized with Pme I and co-transformed into E. coli strain BJ5183 respectively together with pAdEasy-1, the viral DNA plasmid. Recombinants were selected with kanamycin. Once achieved and verified, the recombinant adenoviral plasmids were transformed to competent E. coli DH5αfor greater yields and then purified by PEG. (4) Screening and amplification of recombinant adenoviral particles Linearized by PacI digestion, the recombinant adenoviral plasmids were transferred into HEK 293 cells (human embryo kidney 293 derived cell line) using LipofectamineTM2000 according to the manufacturer's guidelines, to produce viral particles, followed by screening the expression of green fluorescent protein(GFP). The recombinant viruses further amplified for 2 to 3 passages to get higher titers of the virus. (5) Cell lysate and supernatant of the cell culture were analyzed by Western blotting for the expression of the protein 48h post infection. (6) Viral particle titration Cell-free virus stocks were diluted serially in serum-free medium at ten times, and were titrated by blue forming units (BFU) methods according to the manual of Ad-Easy vector system application. (7)BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) twice on 0,16 day with 1×10⁷ pfu/ml of Ad/VP1,Ad/sVP1 and PBS respectively. Fourteen days after every injection, sera of each group were collected and the titers of CVB3-specific neutralizing antibodies and specific VP1 IgG were measured. Three weeks after the second immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, the other eight mice were challenged with 4LD₅₀ of CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD₅₀ CVB3 and sacrificed seven days later to evaluate the titers of blood viruses.Result: (1)The clone vector pGEM-T/sVP1 was constructed successfully; endonuclease analyses and sequencing result showed that the inserted genes were the same as reported.(2)Recombinant adenoviral transfer plasmid AdTrack-CMV/sVP1 was constructed successfully.(3) Recombinant adenoviral plasmid pAd/sVP1 was generated successfully. Restriction digest with Pac I yielded a large fragment of approximately 30 kb, and a smaller fragment of either 3.0 kb or 4.5 kb. (4) Recombinant adenoviruses Ad/sVP1 was generated successfully. The infected cells were lysed by freeze/thaw cycles, and supernatant was used to reinfect fresh HEK 293 cells.(5) The cell culture medium was screened for VP1 expression by Western blotting.(6)The titers of recombinant adenoviruses Ad/VP1 and Ad/sVP1 after passage 4 were as follows: 3.0×10⁹ pfu/ml,5.0×10⁸ pfu/ml。(7)When mice were immunized with the vaccines, the antibody titers increased time-dependently with the time of inoculation. The mean titers of neutralizing antibody after every immunization in Ad/VP1 group were 1:8.41,1:31.77, respectively, and that in Ad/sVP1 group were 1:8.91,1:44.87, while it was always lower than 1:5 in PBS group. More specifically, mice with Ad/sVP1 elicited the highest level of neutralizing antibody after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.05).(8)The titers of specific VP1 IgG after each inoculation were 1:56.10,1:168.27 in Ad/VP1 group, and 1:66.68,1:503.50 in Ad/sVP1 group. The mice immunized with Ad/sVP1 elicited the strongest specific VP1 IgG after the second immunization. When compared with that of Ad/VP1, the difference was significant (P<0.01).(9) Splenocytes from vaccinated mice showed different proliferative activity to different stimulants of CVB3 or ConA. When stimulated by CVB3, the difference of the three groups was not significant. While, when stimulated by ConA, the proliferative activity of mice of Ad/sVP1 group was higher than those of the other two groups (p<0.05).(10) The adenovirus-based CVB3 vaccines Ad/VP1 and Ad/sVP1 enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that that of the mice immunized with PBS (p<0.05).(11) After 3LD₅₀ of CVB3 challenge, the virus titers of blood in Ad/sVP1 group were only 2.58±0.25, significantly lower than that of Ad/VP1 group (3.41±0.21). (12) Protection of mice from death after lethal CVB3 (4LD₅₀) challenge was augmented to 58.33% with Ad/sVP1, while mice with Ad/VP1 was only 41.67% and no one survived in PBS group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons.Conclusion: (1)Adenovirus-based CVB3 vaccine Ad/sVP1 was constructed successfully. And the expression of VP1 was verified by Western blotting.(2)After administration of Recombinant adenovirus vaccine Ad/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times, the virus titers of blood were much lower, and protection of mice from death after lethal CVB3 (4LD₅₀) challenge was augmented to 58.33%.