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.

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8 + T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env-and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4 ؉ T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.Adenoviruses have been a focus of interest as vaccine vectors for more than a decade and have been tested in various preclinical and clinical studies for vaccination against viral and bacterial infections (reviewed in reference 38). This interest is based on the ability of adenoviral vectors to induce high antibody titers and robust cytotoxic T-lymphocyte (CTL) responses and on the high immunogenicity of the vector, which might have an adjuvant effect on vaccination (17). Adenoviral vectors have also been extensively evaluated for immunization against HIV (reviewed in reference 1), where they were used either alone or in combination with plasmid DNA or protein in prime-boost immunizations. However, vaccination with adenoviral vectors against HIV showed no effectiveness in a large phase IIb study (4), but it is conceivable that the observed lack of effectiveness was due to the choice of vaccine antigen rather than the vector itself, as the vaccine relied exclusively on the induction of CTL responses, and the outcome was unexpected given previous results from studies in nonhuman primates (33,42). The findings of the phase IIb study brought about a shift of focus from the CTL response to a more balanced immune response, including neutralizing antibodies, that is now expected to be necessary for protection from HIV infection.Apart from adenoviral vectors that encode vaccine antigens, there have also been approaches to modify adenoviral capsid proteins to include antigenic epitopes. These were mostly inserted into external loops of the hexon protein (5,22,25,26,43), which is the main component of the adenovirus capsid, but also other components of the capsid, such as fiber, protein IX, and penton base, have been evaluated (22). These studies showed that incorporation of single epitopes into capsid proteins of adenovirus leads ...

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8 ؉ and CD4 ؉ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven. H epatitis delta virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes the most severe hepatitis in humans.Nearly all patients develop chronic HDV infection that has a high probability of progressing to liver cirrhosis and hepatocellular carcinoma (1, 2). Worldwide, approximately 15 million patients are affected with HDV. About 8% of HBV surface antigen (HBsAg)-positive patients in several European countries have tested positive for antibodies against HDV (2). Therapeutic options for HBV/HDV carriers are limited. Only in about 25% of the patients does alpha interferon therapy result in sustained viral clearance (3).HBV carriers are at risk of being superinfected with HDV. Therefore, a vaccine protecting HBV carriers from HDV superinfection would be eligible. A main obstacle for the design of a vaccine against HDV infection is the fact that antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HDV particle. The HDV protein/RNA complex is covered by the envelope protein of HBV (HBsAg). Therefore, classical vaccines which induce neutralizing antibodies cannot be expected to prevent HDV infection.Immunizations with nucleoproteins of, e.g., influenza A virus-, HBV-, or woodchuck hepatitis virus (WHV) induced virus-specific T cells and were able to suppress replication, e.g., by cytokine secretion. In a second step, these virus-specific T cells are able to eliminate infected cells by their cytolytic activity and thus prevent the spread of the virus (4-7). T cell vaccines may not provide sterile immunity, because they do not induce neutralizing antibodies. However, T cell vaccines may stop infection via the cellular immune response at a very early phase of infection. Most conventional vaccines for humans induce sufficient amounts of neutralizing antibodies which prevent infection. ...

BackgroundInert nanoparticles are attracting attention as carriers for protein-based vaccines. Here we evaluate the immunogenicity of the model antigen ovalbumin delivered on polystyrene particles and directly compare particulate delivery with adenovirus-based immunization.FindingsMice were vaccinated with soluble ovalbumin, ovalbumin-coated polystyrene particles of different sizes, or an adenovirus-based expression-display vector that encodes and displays a pIX-ovalbumin fusion protein. Antibody responses were clearly higher when ovalbumin was administered on polystyrene particles compared to soluble protein administration, regardless of the particle size. Compared to adenovirus-based immunization, antibody levels were lower if an equivalent amount of protein was delivered, and no cellular immune response was detectable.ConclusionsWe demonstrate in a side-by-side comparison that inert nanoparticles allow for the reduction of the administered antigen amount compared to immunization with soluble protein and induce strongly enhanced antibody responses, but responses are lower compared to adenovirus-based immunization.

BackgroundType 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.ResultsMice 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.ConclusionsOur 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.

. After antigen uptake and activation, DCs mature and migrate to lymphoid tissues, where they present antigen-derived peptides on major histocompatibility complex type II (MHC-II) molecules and provide stimulatory signals for antigen-specific T cells. Because of the important role of DCs in the induction of protective immunity, DC targeting of antigens has been a much-pursued strategy in the development of genetic and protein-based vaccines. For this, vaccine antigens were fused to antibodies or ligands of DC surface molecules and delivered directly as a protein vaccine or through encoding DNA in a genetic plasmid-or viral vectorbased vaccine regimen (4,33,37,43,44). A different approach is the coexpression of chemoattractant molecules by a genetic vaccine to recruit APCs to the site of vaccine delivery. This approach has been studied in immunotherapy of tumors (16,19,34,46) and also for vaccination against virus infections (5, 13, 26, 47), but it has not yet been tested in a retrovirus challenge model.In this vaccination study we sought to increase the presence of DCs at the site of vaccine delivery. For this, we coadministered adenovirus vectors encoding different chemokines along with viral antigens. Chemokines are a group of proinflammatory proteins of 6 to 14 kDa that act as ligands of G-protein-coupled receptors (reviewed in reference 31) expressed on leukocytes. Chemokines induce the migration of these cells and play an important role in both homeostasis and inflammation. For these different processes, some chemokines are expressed continuously in certain tissues, while others are only expressed in response to inflammatory stimuli. Depending on the expression of their respective receptors, chemokines can stimulate multiple cell types. In the present study we studied the effects of the chemokines CCL3, CCL20, CCL21, and CXCL14 on immune responses induced by an adenovirus-based vaccine. All four tested chemokines, while acting on differing ranges of target cells, are known to be chemoattractants for DCs (reviewed in reference 48).We analyzed the adjuvant effect of chemokines for retroviral immunity using an HIV vaccination mouse model and the Friend retrovirus (FV) model. FV is an immunosuppressive retroviral complex, consisting of the apathogenic Friend murine leukemia virus (F-MuLV) and the replication-defective but pathogenic spleen focus-forming virus, that causes splenomegaly and lethal erythroleukemia in susceptible mice (15). In contrast to the vaccination against HIV proteins, the FV model allows for challenging immunized mice with a pathogenic retrovirus. The FV infection model has offered valuable insights into the role of particular cell types in the immune response to a retroviral infection and into the basic requirements for immune protection. Using attenuated F-MuLV helper virus, it was demonstrated that complete protection from lethal FV challenge requires both humoral and cellular responses, comprising antibodies and CD4 ϩ and CD8 ϩ T cells (10). Although the correlates of immune protection ...

No abstract