Recombination Of Mini-circle Hsv Amplicon Expressing IFN-γ In The Epithelium And Evaluation Of Its Antiviral Effect Against HSV-2

Background:Herpes simplex virus infects human bodies through broken skin or mucous membranes, and cause infection at different locus, such as corneal herpes, herpes simplex viral meningitis and encephalitis, genital herpes etc., even cause blindness or death, and lurk in the body for life with a periodic asymptomatic or symptomatic recurrence and shed among humans. Its infection rate in adult is 60%~95%. Currently, the clinical treatment to infection of herpes simplex virus, such as chemical nucleoside analogues(acyclovir) can not efficiently inhibit replication of HSV viruses. It is meaningful to expore effective anti-viral methods. Studies found that good anti-HSV effect with little no-target effects was got by IFN-γ expressed by conventional HSV amplicon vectors with bacterial sequence. It has found that the bacterial sequence could inhibit the expression of transgenes. Several studies have confirmed that mini-circle DNA free of bacterial sequence can mediate long-term and stable expression of foreign genes. Production of mini-circle amplicon in Escherichia coli mediated by integrase ФC31 was labor-intensive and time-consuming. our previous studies found that Cre-loxp recombinantion in mammalian cells have efficiently increase the production of mini-circle HSV amplicons. Objective:In this study, one adenovirus vector with opimized expression cassette of transgene and another adenovirus vector Adv-Cre expressing recombinase Cre were inoculated into the genital tract of mouse model infected with HSV-2 to see if mini-circle HSV amplicons could be produced in genital membrane, and the effect of IFN-γexpressed by mini-circle HSV against HSV-2 was evaluated. Methods:1. Recombination in the cultured cells: Two kind of adenovirus vectors expressing different reporter genes Ds Red or Lac Z, Adv-loxp-DOPCp-loxp and Adv-loxp-LOPCp-loxp with the promoter and coding sequence of reporter genes placing at both ends of HSV viral genes Ori S and Pac, were constructed by LR recombination. Adv-cre and Adv-loxp-DOPCp-loxp or Adv-loxp-LOPCp-loxp co-infected 2-2 cells, then the production of mini-circle amplicons MC-OPD or MC-OPL were checked by observing red fluorescence or in situ β-gal staining and PCR.2. Recombination in the genital tract: Adv-cre and Adv-loxp-DOPCp-loxp or Adv-loxp-LOPCp-loxp were inoculated into the genital tract of mouse model with acute HSV-2 infection. The in vivo formation of pseudovirus of MC HSV amplicons, MC-OPD or MC-OPL, were examined by collecting vaginal lavage fluid to perform virus culture and PCR(Group MC-OPD) or PCR and in situ β-gal staining of paraffin section of genital tract(Group MC-OPL).3. Anti-HSV-2 effects of m IFN-γ expressed by mini-circle amplicons: according to the same methods, Adv-loxp-IRDOPCp-loxp with two expression cassettes(expressing m IFN-γ and Ds Red) was constructed. Production of MC-OPIRD were evaluated by observing red fluorescence and m IFN-γ by ELISA kit in vitro. Its anti-viral effects were analysed by comparing survival rates of mice. Results:1. Recombination in the cultured cells: 2-2 cells co-infected by Adv-Cre and Adv-loxp-DOPCp-loxp or Adv-loxp-LOPCp-loxp expressed a wide range of red fluorescence, and bands of interest can be amplified through PCR. Recombinant MC-HSV amplicons were packaged by replication-defective HSV-1-del ICP27 virus to get a virus mixture with recombinant MC-HSV amplicon pseudovirus and HSV-1-del ICP27 virus.2. Recombination in the genital tract: Mice model with acute infection in genital tract were testfied by observing symptoms and HE staining of paraffin section of genital tract. Adv-Cre and Adv-loxp-DOPCp-loxp or Adv-loxp-LOPCp-loxp were inoculated into the genital tract of successful mouse modle. Bands of interest could be amplified by PCR using vaginal lavage as template. The production of MC-OPD or MC-OPL pseudovirus were testfied by viral cluture or in situ β-gal staining.3. Anti-HSV-2 effects of m IFN-γ expressed by mini-circle amplicon: MC-OPIRD HSV amplicon pseudovirus was produced by co-infection of Adv-Cre and Adv-loxp-IRDOPCp-loxp and packaged by HSV-1-del ICP27 virus in 2-2 cells, and verified by observing red fluorescence and testing m IFN-γ by ELISA kits. The results showed that the expression levels of m IFN-γ mediated by MC-OPIRD amplicon pseudovirus were significantly higher compared with control group(p<0.01). Survival analysis showed that the survival rate in the experimental group was higher than control group(p<0.01). Conclusions:Mini-circle HSV amplicon produced by Cre-loxp recombination mediated by adenoviral vectors could be formed in the epithelial cells of mouse genital tract and packaged by HSV-2 existed in the mucosal infections. IFN- γ expressed by mini-circle amplicon pseudotyped virus could inhibit replication of HSV-2 during productive infection.