Combination of DNA prime--adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis B in the woodchuck model.

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8+ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8+ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4+ and CD8+ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients

Cytotoxic T-cell responses detected in chronically WHV-infected woodchucks treated with the combination therapy.

<p>Summary of WHcAg-specific (A) and WHsAg-specific (B) degranulation responses evaluated by CD107a assay at representative time points of the experiment. PBMCs were expanded <i>in vitro</i> for 3 days with WHcAg-derived epitope c96-110 or WHsAg-derived epitope s220-234. The background value was calculated as a mean of all values detected for negative controls in all woodchucks at all time points. Presented values show the percentage of CD107a⁺ CD3⁺ CD4⁻ T-cells in the CD3⁺ CD4⁻ T-cell population. The black arrow represents beginning of the immunization regimen at week 8. (C–D) The kinetics of CD107a⁺ degranulation responses in WHV chronic carriers at all monitored time points of therapy. Chronically WHV-infected woodchucks were treated with ETV for 23 weeks. Four of them received subsequently 9 intramuscular immunizations with DNA plasmids, expressing WHcAg and WHsAg (blue arrows), Ad5WHc (red arrows), and Ad35WHc (green arrows). Two animals (number 61791 and 61795) were treated only with ETV and served as controls. The positive CTL responses are marked with “+” sign.</p

Determination of WHV infection markers in immunized and only entecavir-treated chronic WHV carrier woodchucks.

<p>Six chronically WHV-infected woodchucks were treated with ETV for 23 weeks. Four of them (61792, 61793, 61786 and 61789) received subsequently 9 intramuscular immunizations with DNA plasmids, expressing WHcAg and WHsAg (blue arrows), Ad5WHc (red arrows), and Ad35WHc (green arrows) (A). Two animals (number 61791 and 61795) were treated only with ETV and served as controls (B). Summarized comparison of WHV load (C) and WHsAg levels (D) detected in woodchucks receiving immunizations and only ETV-treated controls at representative time points of the therapy. The black arrow represents beginning of the immunization regimen at week 8. The viral DNA was extracted from woodchuck sera and the viral loads were quantified per ml of serum, using real-time PCR analysis. Serum WHsAg concentration was determined by electroimmunodiffusion using rabbit anti-WHs serum. WHsAg-specific antibodies were (anti-WHs) detected in woodchuck sera using protein G coupled to peroxidase. The GOT levels in woodchuck sera were quantified using the standard diagnostic methods. The GOT value above 50 IU/l was considered elevated (GE – genome equivalents; n.d. – not done; † - dead).</p

Cellular immune response induced by DNA prime – AdV boost immunization in WHV transgenic mice.

<p>Mice were primed two times by immunization with the pCGWHc plasmid. Four weeks later boosting immunization with Ad5WHc, Ad35WHc, or pCGWHc was performed. The group of mice immunized twice with pCGWHc in combination with Ad5WHc was boosted 4 weeks later for a second time with Ad35WHc. Mice immunized with ‘empty’ pCG and boosted with Ad5 expressing GFP served as controls. (A–B) Representative and summarised frequencies of WHcAg-specific CD8⁺ T-cells detected <i>ex vivo</i> in the population of splenic lymphocytes. Antigen-specific cells were detected using DimerX H2-Db fusion protein loaded with H2-Db-restricted CD8⁺ epitope c13-21. (C,E) Representative and summarised WHcAg-specific IFNγ⁺ CD8⁺ T-cell responses detected in the splenocytes expanded <i>in vitro</i> for 7 days with CD8⁺ T-cell epitope c13-21. (D,F) Representative and summarised WHcAg-specific IFNγ⁺ CD4⁺ T-cell responses in the splenocytes expanded <i>in vitro</i> for 7 days with CD4⁺ T-cell epitope c131-145. The bars represent the mean value obtained for each group of mice including SEM. Asterisks mark the statistically significant difference (<b>*</b><0.05;<b>**</b><0.005; <b>***</b><0.0005; <i>ns</i> – not significant).</p

Quantification of the WHV loads before and after the immunization trials in WHV transgenic mice.

<p>The viral load was evaluated by quantitative real-time PCR with the detection limit of 10³ genome equivalents (GE) per milliliter serum. For analysis DNA samples obtained from the serum of mice were used. The single pair of dots connected with the line represents values obtained from one mouse before (time point of cardiotoxin pretreatment) and after the immunization trials (week 2 after the last immunization). The correlations of the WHV loads for 4 immunization groups of mice are presented: (A) control group primed with ‘empty’ pCG and boosted with Ad5 expressing GFP, (B) group immunized only with DNA vaccine – pCGWHc, (C) group primed with pCGWHc and boosted with Ad5WHc, and (D) group primed with pCGWHc and boosted twice with Ad5WHc and Ad35WHc. The statistical analysis was performed using the Wilcoxon signed rank test (<b>*</b><0.05; <b>***</b><0.0005; <i>ns</i> – not significant).</p

Detection of WHV-specific antibodies induced by DNA prime – AdV boost immunization in WHV transgenic mice.

<p>Mice were primed two times by immunization with the pCGWHc plasmid. Four weeks later boosting immunization with Ad5WHc, Ad35WHc, or pCGWHc was performed. The group of mice immunized twice with pCGWHc in combination with Ad5WHc was boosted 4 weeks later for a second time with Ad35WHc. Mice immunized with ‘empty’ pCG and boosted with Ad5 expressing GFP served as controls. WHcAg-specific IgG (A), IgG_2a (B), IgG₁ (C) or WHsAg-specific IgG (D) antibodies were detected in sera collected two weeks after the last immunization (serum dilution 1∶500). Asterisks mark the significant difference (*<0.05; **<0.005, ***<0.0005; <i>ns</i> – not significant).</p

WHV-specific lymphoproliferative responses detected in chronically WHV-infected woodchucks treated with the combination therapy.

<p>(A) Immunization schedule. Six chronically WHV-infected woodchucks (number: 61786, 61789, 61791, 61792, 61793 and 61795) were treated with entecavir for 23 weeks. The drug was administered for 12 weeks in a dose of 1.4 mg ETV per week. From week 8 to 23 of the therapy, subcutaneous injections of 1 mg ETV were performed twice a week. At week 7, four of the six ETV-treated animals (number: 61786, 61789, 61792, and 61793) were pretreated with cardiotoxin (black arrow) and one week later the animals received in total 9 intramuscular subsequent immunizations with 0,5 mg of pCGWHc together with 0,5 mg of pWHsIm (time points of immunization marked by the blue arrows at weeks: 8, 10, 12, 25 and 27), 1×10¹⁰ PFU of Ad5WHc together with 0,5 mg of pWHsIm (red arrows at weeks 14 and 19) or 1×10¹⁰ PFU of Ad35WHc together with 0,5 mg of pWHsIm (green arrows at weeks 16 and 22). Two animals (number 61791 and 61795) were treated only with ETV and served as controls. WHcAg-specific (B) and WHsAg-specific (C) lymphoproliferative responses in vaccinated woodchucks. The PBMCs were stimulated with panel of 10 WHcAg-specific and 16 WHsAg-specific peptides in triplicates. After 5 days of culture, cells were pulsed with ²[³H]adenine for 16 h and the incorporation of ²[³H]adenine was measured. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003391#s2" target="_blank">Results</a> for triplicate cultures are presented as a mean stimulation index (SI). A SI≥3.0 was considered significant.</p

Determination of WHV replication in the livers of chronically WHV-infected woodchucks.

<p>Southern blot analysis was performed on the DNA obtained from the liver samples collected post-mortem or by liver biopsy from chronically WHV-infected woodchucks at given time points (W – week). Woodchucks number 61786, 61789, 61792 and 61793 were treated with combination therapy. Woodchucks 61791 and 61795 were treated only with ETV and served as controls. Total amount of 10 µg of isolated DNA was electrophoresed into agarose gel and then transferred onto nylon membrane. WHV replicative intermediates were detected by hybridization with [³²P] labelled full-length WHV strain 8 genome as a probe. The arrows indicate the relaxed circular WHV DNA (RC DNA; 3,0 kb) and single-stranded WHV DNA (ssDNA; approximately 1,5 kb).</p

Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

Background Type I interferons (IFNs) exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV) or HIV. Results Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4+ T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4+ T cell responses were enhanced by IFNα subtypes. Conclusions Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4+ T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines