Woodchuck hepatitis virus core antigen-based DNA and protein vaccines induce qualitatively different immune responses that affect T cell recall responses and antiviral effects

AbstractT helper type 1 (Th1) immunity was considered to play a dominant role in viral clearance of hepadnaviral infection. However, pre-primed Th2 type responses were able to efficiently control hepadnaviral infection in animal models. We investigated how pre-primed Th1/2 responses control hepadnaviral replication using the newly established mouse models. DNA (pWHcIm, pCTLA-4-C) and protein vaccines based on the nucleocapsid protein (WHcAg) of woodchuck hepatitis virus (WHV) primed specific immune responses with distinct features. The pre-primed responses determined the characteristics of recall responses if challenged with a WHcAg-expressing adenoviral vector. Vaccination with pWHcIm and pCTLA4-C facilitated viral control in the hydrodynamic injection model and reduced WHV loads by about 3 and 2 logs in WHV-transgenic mice, respectively, despite of different kinetics of specific CD8+ T cell responses. Thus, pre-primed Th2-biased responses facilitate the development of CD8+ T cell responses in mice compared with naïve controls and thereby confer better viral control

Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

Here, we investigate the potential of CD70 co-expression during viral vector boost vaccination to improve an antigen-specific T cell response. To determine the chance of activating antigen-specific T cells by CD70, we used the HBV core antigen as a model antigen in a heterologous protein-prime, Modified Vaccinia virus Ankara (MVA) boost vaccination scheme. Both the HBV core and a CD70 expression cassette were co-expressed upon delivery by an MVA vector under the same promoter linked by a P2A site. To compare immunogenicity with and without CD70 co-expression, HBV-naïve, C57BL/6 (wt) mice and HBV-transgenic mice were prime-vaccinated using recombinant HBV core antigen followed by the MVA vector boost. Co-expression of CD70 increased the number of vaccine-induced HBV core-specific CD8 T cells by >2-fold and improved their effector functions in HBV-naïve mice. In vaccinated HBV1.3tg mice, the number and functionality of HBV core-specific CD8 T cells was slightly increased upon CD70 co-expression in low-viremic, but not in high-viremic animals. CD70 co-expression did not impact liver damage as indicated by ALT levels in the serum, but increased the number of vaccine-induced, proliferative T cell clusters in the liver. Overall, this study indicates that orchestrated co-expression of CD70 and a vaccine antigen may be an interesting and safe means of enhancing antigen-specific CD8 T cell responses using vector-based vaccines, although in our study it was not sufficient to break immune tolerance

Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

Here, we investigate the potential of CD70 co-expression during viral vector boost vaccination to improve an antigen-specific T cell response. To determine the chance of activating antigen-specific T cells by CD70, we used the HBV core antigen as a model antigen in a heterologous protein-prime, Modified Vaccinia virus Ankara (MVA) boost vaccination scheme. Both the HBV core and a CD70 expression cassette were co-expressed upon delivery by an MVA vector under the same promoter linked by a P2A site. To compare immunogenicity with and without CD70 co-expression, HBV-naïve, C57BL/6 (wt) mice and HBV-transgenic mice were prime-vaccinated using recombinant HBV core antigen followed by the MVA vector boost. Co-expression of CD70 increased the number of vaccine-induced HBV core-specific CD8 T cells by >2-fold and improved their effector functions in HBV-naïve mice. In vaccinated HBV1.3tg mice, the number and functionality of HBV core-specific CD8 T cells was slightly increased upon CD70 co-expression in low-viremic, but not in high-viremic animals. CD70 co-expression did not impact liver damage as indicated by ALT levels in the serum, but increased the number of vaccine-induced, proliferative T cell clusters in the liver. Overall, this study indicates that orchestrated co-expression of CD70 and a vaccine antigen may be an interesting and safe means of enhancing antigen-specific CD8 T cell responses using vector-based vaccines, although in our study it was not sufficient to break immune tolerance

The expression of PD-1 ligands and their involvement in regulation of T cell functions in acute and chronic woodchuck hepatitis virus infection.

BACKGROUND: The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV). METHODS: Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined. PRINCIPAL FINDINGS: Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks. CONCLUSIONS: Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection

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

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice.

In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction), compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies, and increased numbers and functionality of HBV-specific, CD8+ T-cells in mice with low, but not in mice with high levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia following administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells and HBV was eliminated

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

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