TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice.

OBJECTIVE: To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). METHODS: TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. RESULTS: In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (i.e., capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. CONCLUSIONS: Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Plasmid vector-linked maturation of natural killer (NK) cells is coupled to antigen-dependent NK cell activation during DNA-based immunization in mice.

Plasmid DNA vaccines serve in a wide array of applications ranging from prophylactic vaccines to potential therapeutic tools against infectious diseases and cancer. In this study, we analyzed the mechanisms underlying the activation of natural killer (NK) cells and their potential role in adaptive immunity during DNA-based immunization against hepatitis B virus surface antigen in mice. We observed that the mature Mac-1(+) CD27(-) NK cell subset increased in the liver of mice early after DNA injection, whereas the number of the less mature Mac-1(+) CD27(+) NK cells in the liver and spleen was significantly reduced. This effect was attributed to bacterial sequences present in the plasmid backbone rather than to the encoded antigen and was not observed in immunized MyD88-deficient mice. The activation of NK cells by plasmid-DNA injection was associated with an increase in their effector functions that depended on the expressed antigen. Maturation of NK cells was abrogated in the absence of T cells, suggesting that cross talk exists between NK cells and antigen-specific T cells. Taken together, our data unravel the mechanics of plasmid vector-induced maturation of NK cells and plasmid-encoded antigen-dependent activation of NK cells required for a crucial role of NK cells in DNA vaccine-induced immunogenicity.

Therapeutic vaccines and immune-based therapies for the treatment of chronic hepatitis B: perspectives and challenges.

The treatment of chronic hepatitis B virus (HBV) infection has greatly improved over the last 10 years, but alternative treatments are still needed. Therapeutic vaccination is a promising new strategy for controlling chronic infection. However, this approach has not been as successful as initially anticipated for chronic hepatitis B. General impairment of the immune responses generated during persistent HBV infection, with exhausted T cells not responding correctly to therapeutic vaccination, is probably responsible for the poor clinical responses observed to date. Intensive research efforts are now focusing on increasing the efficacy of therapeutic vaccination without causing liver disease. Here we describe new approaches to use with therapeutic vaccination, in order to overcome the inhibitory mechanisms impairing immune responses. We also describe innovative strategies for generating functional immune responses and inducing sustained control of this persistent infection.

Changes to the natural killer cell repertoire after therapeutic hepatitis B DNA vaccination.

BACKGROUND: Improvements to the outcome of adaptive immune responses could be achieved by inducing specific natural killer (NK) cell subsets which can cooperate with dendritic cells to select efficient T cell responses. We previously reported the induction or reactivation of T cell responses in chronic hepatitis B patients vaccinated with a DNA encoding hepatitis B envelope proteins during a phase I clinical trial. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined changes in the peripheral NK cell populations occurring during this vaccine trial using flow cytometry analysis. Despite a constant number of NK cells in the periphery, a significant increase in the CD56(bright) population was observed after each vaccination and during the follow up. Among the 13 different NK cell markers studied by flow cytometry analysis, the expression of CD244 and NKG2D increased significantly in the CD56(bright) NK population. The ex vivo CD107a expression by CD56(bright) NK cells progressively increased in the vaccinated patients to reach levels that were significantly higher compared to chronically HBV-infected controls. Furthermore, modifications to the percentage of the CD56(bright) NK cell population were correlated with HBV-specific T cell responses detected by the ELISPOT assay. CONCLUSIONS/SIGNIFICANCE: These changes in the CD56(bright) population may suggest a NK helper effect on T cell adaptive responses. Activation of the innate and adaptive arms of the immune system by DNA immunization may be of particular importance to the efficacy of therapeutic interventions in a context of chronic infections. TRIAL REGISTRATION: ClinicalTrials.gov NCT00988767.

Hepatitis B virus as a gene delivery vector activating foreign antigenic T cell response that abrogates viral expression in mouse models.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is characterized by functionally impaired T cell responses. To ensure active immunotherapy, the immune response must be switched from exhausted T cells to functional effectors that can attain the liver and cure the viral infection. We thus designed a recombinant HBV (rHBV) containing a modified viral core gene that specifically delivers a foreign antigenic polyepitope to the liver. This recombinant virus could only be self-maintained in hepatocytes already infected by HBV through capsid complementation. A strong foreign epitope-specific T cell response was first primed in the periphery by way of DNA immunization in human leukocyte antigen (HLA)-A2/DR1 transgenic mice. After the hydrodynamic (hyd.) injection of rHBV, expression of the foreign antigenic polyepitope in hepatocytes attracted/reactivated a vigorous T cell response in situ. Most liver-infiltrating CD8(+) T cells proved to be functional effectors. Following DNA priming and hyd. injection, the rHBV-based expression of hepatitis B surface antigen (HBsAg) in mouse liver was almost completely inhibited without causing major liver injury. Studies in HBsAg/HLA-A2/DR1 transgenic mice further validated our approach. CONCLUSION: For the first time, HBV was used as a gene delivery vector, which strongly triggered functional T cell response and subsequently controlled the viral expression in the liver of surrogate mouse models for HBV infection. It might represent an innovative and promising strategy of active immunotherapy during HBV persistent infection. This concept could even be more generally extended to other chronic viral diseases.

Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients.

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.

Identification of novel HLA-DR1-restricted epitopes from the hepatitis B virus envelope protein in mice expressing HLA-DR1 and vaccinated human subjects.

Helper T lymphocytes that control CD8(+) T-cell and antibody responses are key elements for the resolution of infection by the hepatitis B virus and for the development of effective immunological memory after hepatitis B vaccination. We have used H-2 class II-deficient mice that express the human MHC class II molecule, HLA-DR1, to identify novel hepatitis B virus envelope-derived T helper epitopes. We confirmed the immunogenicity of a previously described HLA-DR1-restricted epitope, and identified three novel epitopes. CD4(+) T-cell immune responses against these epitopes were detected in peripheral blood mononuclear cells from HLA-DR1(+) individuals vaccinated against hepatitis B. We showed that subjects receiving the currently available hepatitis B vaccines do not develop cross-reactive T helper responses against one of the novel epitopes which are structurally variable between different hepatitis B virus subtypes. These findings highlight the need for developing vaccines against a wider range of viral subtypes, and establish humanized mice as a convenient tool for identifying new immunogenic epitopes from pathogens.

Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers.

Hepatitis B virus (HBV) is the major pathogen of chronic hepatitis and liver disease worldwide. Despite the availability of effective vaccines against hepatitis B for many years, over 370 million people remain persistently infected with HBV. Viral persistence is thought to be related to poor HBV-specific T-cell responses. Based on clinical data, the development of efficient methods capable of inducing strong T-cell responses is an important and primary step toward the development of immunotherapeutics against chronic HBV infection. We designed a phase I clinical trial in chronic HBV carriers to assess safety, tolerability and immunogenicity of a DNA vaccine expressing HBV small (S) and middle (preS2 +S) envelope proteins. After occurrence of lamivudine breakthrough, 10 HBeAg positive patients with chronic hepatitis B were followed longitudinally before, during and after DNA vaccine therapy. Immunizations were well tolerated and adverse physical events were mild and considered unrelated to the vaccine. Proliferative responses to hepatitis B surface antigen (HBsAg) were detected in two patients after DNA injections. Following three injections of vaccine, interferon (IFN)-gamma-producing T-cells specific for the preS2 or the S antigen were detectable in 50 and 100% of the patients, respectively. Each patient recognized at least one peptide within the envelope domain encoded by the vaccine. Anti-preS2 antibodies and seroconversion to anti-HBe were detected in two patients. This study shows evidences for the safety and immunological efficacy of HBV-DNA vaccination and demonstrates that DNA vaccination can restore or activate T-cell responses in chronic HBV carriers.

[Therapeutic vaccination against chronic hepatitis B virus infection]. / Traitement des infections chroniques dues au virus de l'hépatite B par vaccination thérapeutique.

Chronic liver disease and hepatocellular carcinoma associated with chronic hepatitis B virus (HBV) infection are among the most serious human health problems in highly endemic regions. Despite the existence for many years of effective vaccines against HBV, more than 370 million people remain persistently infected with HBV today. Currently available therapies fail to provide long-term control of viral replication in most patients. Viral persistence has been associated with a defect in the development of HBV-specific cell-mediated immunity. Strategies to boost or to broaden the weak virus-specific T-cell response of patients with chronic hepatitis B have been proposed as a means of terminating this persistent infection. The immunogenicity of HBV envelope- or capsid-based vaccines, new formulations for recombinant vaccines as well as DNA-based vaccines are currently under investigation in clinical trials. Although improvements are still required, vaccination would be the therapeutic procedure with the lowest cost and the potentially greatest benefit.

Therapeutic vaccination against chronic hepatitis B virus infection.

Chronic liver disease and hepatocellular carcinoma due to chronic hepatitis B virus (HBV) infection pose a major public health problem in highly endemic regions. Effective vaccines against HBV exist but more than 370 million people remain chronically infected with HBV For these patients there is a high risk to develop cirrhosis and hepatocellular carcinoma. Currently available therapies fail to control viral replication in the long term in most patients. Viral persistence has been associated with a defect in the development of HBV specific cellular immunity. Strategies to boost or to broaden the weak virus-specific T-cell response of patients with chronic hepatitis B have been proposed as a means of curing this persistent infection. HBV envelope- and nucleocapsid-based vaccines, new formulations for recombinant vaccines and DNA-based vaccines are currently being assessed in clinical trials. Improvements are clearly required, but vaccination is likely to be the cheapest and potentially most beneficial treatment.

Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers.

Despite the availability of effective hepatitis B vaccines for many years, over 370 million people remain persistently infected with hepatitis B virus (HBV). Viral persistence is thought to be related to poor HBV-specific T-cell responses. A phase I clinical trial was performed in chronic HBV carriers to investigate whether HBV DNA vaccination could restore T-cell responsiveness. Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV-specific T-cell responses were assessed by proliferation, ELISpot assays, and tetramer staining. Secondary end points included safety and the monitoring of HBV viraemia and serological markers. Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV-specific interferon gamma-secreting T cells were detectable before immunization, but the frequency of such responses was significantly increased by 3 DNA injections. Immunization was well tolerated. Serum HBV DNA levels decreased in 5 patients after 3 vaccine injections, and complete clearance was observed in 1 patient. In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective. We demonstrate that DNA vaccination can specifically but transiently activate T-cell responses in some chronic HBV carriers who do not respond to current antiviral therapies.