Maryline Mancini-Bourgine

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.

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 (ie, 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.

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.

Hepatitis B Virus Splice-Generated Protein Induces T-Cell Responses in HLA-Transgenic Mice and Hepatitis B Virus-Infected Patients

ABSTRACT 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.

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

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. (HEPATOLOGY 2009.)

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

ABSTRACT 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.

O031 : TG1050, a novel immunotherapeutic to treat chronic hepatitis B, can control HBsAg and provoke HBsAg seroconversion in HBV-persistent mouse models

Methods: We infected mice with a hepatotropic virus and challenged these mice by injecting TNF. Using knockout mice, as well as biochemical and multiparametric histological methods we analyzed the influence of different pattern recognition receptors. Additionally, we isolated mitochondria from virus-infected mice and performed metabolomic investigations to analyze the role of mitochondria in virus recognition as well as their role in apoptosis execution. Results: We demonstrate that viral infection in hepatocytes is recognized even in the absence of classical pattern recognition receptor signaling. Rather, viral infection of hepatocytes modulates metabolic processes, leads to increased levels of the pro-apoptotic BCL-2 family members Bax and Bad that cause damage to mitochondria. This leads to a downregulation of the anti-apoptotic protein XIAP (X-chromosome linked inhibiting of apoptosis protein). Downregulation of XIAP facilitates TNF-induced activation of the initiator caspase 8 that together with increased mitochondrial sensitivity to activated caspase 8 is responsible for TNF-induced cell death in virus-infected hepatocytes. Conclusions: We have identified a novel immune-sensing mechanisms in virus-infected hepatocytes that links effector molecules generated by virus-specific T cells to cell-autonomous induction of death selectively in virus-infected hepatocytes.