Maryline Bourgine

Anti-HBV DNA vaccination does not prevent relapse after discontinuation of analogues in the treatment of chronic hepatitis B: a randomised trial—ANRS HB02 VAC-ADN

Objective The antiviral efficacy of nucleos(t)ide analogues whose main limitation is relapse after discontinuation requires long-term therapy. To overcome the risk of relapse and virological breakthrough during long-term therapy, we performed a phase I/II, open, prospective, multicentre trial using a HBV envelope-expressing DNA vaccine. Design 70 patients treated effectively with nucleos(t)ide analogues for a median of 3 years (HBV DNA <12 IU/mL for at least 12 months) were randomised into two groups: one received five intramuscular injections of vaccine (weeks 0, 8, 16, 40 and 44) and one did not receive the vaccine. Analogues were stopped after an additional 48 weeks of treatment in patients who maintained HBV DNA <12 IU/mL with no clinical progression and monthly HBV DNA for 6 months. The primary endpoint was defined as viral reactivation at week 72 (HBV DNA >120 IU/mL) or impossibility of stopping treatment at week 48. Results Reactivation occurred in 97% of each group after a median 28 days without liver failure but with an HBV DNA <2000 IU/mL in 33%; 99% of adverse reactions were mild to moderate. Immune responses were evaluated by enzyme-linked immunosorbent spot and proliferation assays: there was no difference in the percentage of patients with interferon-γ secreting cells and a specific T-cell proliferation to HBcAg but not to HBsAg after reactivation in each group. Conclusions Although it is fairly well tolerated, the HBV DNA vaccine does not decrease the risk of relapse in HBV-treated patients or the rate of virological breakthrough, and does not restore the anti-HBV immune response despite effective viral suppression by analogues. Trial registration number NCT00536627.

Adeno-Associated Virus-Mediated Gene Transfer Leads to Persistent Hepatitis B Virus Replication in Mice Expressing HLA-A2 and HLA-DR1 Molecules

ABSTRACT Hepatitis B virus (HBV) persistence may be due to impaired HBV-specific immune responses being unable to eliminate efficiently or cure infected hepatocytes. The immune mechanisms that lead to HBV persistence have not been completely identified, and no appropriate animal model is available for such studies. Therefore, we established a chronic HBV infection model in a mouse strain with human leukocyte antigen A2/DR1 (HLA-A2/DR1) transgenes and an H-2 class I/class II knockout. The liver of these mice was transduced with adeno-associated virus serotype 2/8 (AAV2/8) carrying a replication-competent HBV DNA genome. In all AAV2/8-transduced mice, hepatitis B virus surface antigen, hepatitis B virus e antigen, and HBV DNA persisted in serum for at least 1 year. Viral replication intermediates and transcripts were detected in the livers of the AAV-injected mice. The hepatitis B core antigen was expressed in 60% of hepatocytes. No significant inflammation was observed in the liver. This was linked to a higher number of regulatory T cells in liver than in controls and a defect in HBV-specific functional T-cell responses. Despite the substantial tolerance resulting from expression of HBV antigens in hepatocytes, we succeeded in priming functional HBV-specific T-cell responses in peripheral tissues, which subsequently reached the liver. This AAV2/8-HBV-transduced HLA-A2/DR1 murine model recapitulates virological and immunological characteristics of chronic HBV infection, and it could be useful for the development of new treatments and immune-based therapies or therapeutic vaccines for chronic HBV infections.

Immunological and Antiviral Responses After Therapeutic DNA Immunization in Chronic Hepatitis B Patients Efficiently Treated by Analogues

A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ-producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ-producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4+ T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ-producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ-producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4(+) T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.

Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection

Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus–cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy.

Optimization of immune responses induced by therapeutic vaccination with cross-reactive antigens in a humanized hepatitis B surface antigen transgenic mouse model

The absence of relevant animal models of chronic hepatitis B virus (HBV) infection has hampered the evaluation and development of therapeutic HBV vaccines. In this study, we generated a novel transgenic mouse lineage that expresses human class I and II HLA molecules and the hepatitis B surface antigen (HBsAg). HBsAg and hepatitis B core antigen (HBcAg) administered as plasmid DNAs and recombinant proteins, either alone or in combination, were evaluated as therapeutic vaccine candidates in this mouse model. Our results emphasize the importance of the route of administration in breaking HBsAg tolerance. Although immunizing the transgenic mice with DNA encoding homologous HBsAg was sufficient to induce CD8+ T-cell responses, HBsAg from a heterologous subtype was required to induce a CD4+ T-cell response. Importantly, only prime-boost immunization protocols that combined plasmid DNA injection followed by protein injection induced the production of antibodies against the HBsAg expressed by the transgenic mice.

Adenoviral Delivery of Recombinant Hepatitis B Virus Expressing Foreign Antigenic Epitopes for Immunotherapy of Persistent Viral Infection

ABSTRACT We previously reported a proof-of-concept study for curing chronic hepatitis B virus (HBV) infection using a foreign-antigen recombinant HBV (rHBV) as a gene therapy vector. Targeted elimination of wild-type HBV (wtHBV)-infected cells could be achieved by functionally activating an in situ T-cell response against the foreign antigen. However, as chronic HBV infection spreads to all hepatocytes, specific targeting of virus-infected cells is thought to be less critical. It is also feared that rHBV may not induce active immunization in a setting resembling natural infection. For this immunotherapeutic approach to be practically viable, in the present study, we used a recombinant adenovirus (rAd) vector for rHBV delivery. The rAd vector allowed efficient transduction of wtHBV-producing HepG2 cells, with transferred rHBV undergoing dominant viral replication. Progeny rHBV virions proved to be infectious, as demonstrated in primary tupaia hepatocytes. These results greatly expanded the antiviral capacity of the replication-defective rAd/rHBV in wtHBV-infected liver tissue. With prior priming in the periphery, transduction with rAd/rHBV attracted a substantial influx of the foreign-antigen-specific T-effector cells into the liver. Despite the fully activated T-cell response, active expression of rHBV was observed for a prolonged time, which is essential for rHBV to achieve sustained expansion. In a mouse model of HBV persistence established by infection with a recombinant adeno-associated virus carrying the wtHBV genome, rAd/rHBV-based immunotherapy elicited a foreign-antigen-specific T-cell response that triggered effective viral clearance and subsequent seroconversion to HBV. It therefore represents an efficient strategy to overcome immune tolerance, thereby eliminating chronic HBV infection. IMPORTANCE Adenovirus-delivered rHBV activated a foreign-antigen-specific T-cell response that abrogated HBV persistence in a mouse model. Our study provides further evidence of the potential of foreign-antigen-based immunotherapy for the treatment of chronic HBV infection.

Human Hematopoietic Reconstitution and HLA-Restricted Responses in Nonpermissive Alymphoid Mice

We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.

Recognition of core-derived epitopes from a novel HBV-targeted immunotherapeutic by T-cells from patients infected by different viral genotypes.

Hepatitis B virus (HBV) infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Current therapies based on nucleos(t)ide analogs or pegylated-interferon-α lead to control of viral replication in most patients but rarely achieve cure. A potential strategy to control chronic hepatitis B is to restore or induce functional anti-HBV T-cell immune responses using HBV-specific immunotherapeutics. However, viral diversity is a challenge to the development of this class of products as HBV genotypes display a sequence diversity of up to 8%. We have developed a novel HBV-targeted immunotherapeutic, TG1050, based on a non-replicative Adenovirus vector encoding a unique and large fusion protein composed of multiple antigenic regions derived from a HBV genotype D sequence. Using peripheral blood mononuclear cells from 23 patients chronically infected by five distinct genotypes (gt A, B, C, D and E) and various sets of peptides encompassing conserved versus divergent regions of HBV core we have measured ability of TG1050 genotype D core-derived peptides to be recognized by T-cells from patients infected by various genotypes. Overall, PBMCs from 78% of genotype B or C- and 100% genotype A or E-infected patients lead to detection of HBV core-specific T-cells recognizing genotype D antigenic domains located both in conserved and variable regions. This proof-of-concept study supports the clinical development of TG1050 in large patient populations independently of infecting genotypes.

Identification of a small molecule that primes the type I interferon response to cytosolic DNA

The type I interferon response plays a pivotal role in host defense against infectious agents and tumors, and promising therapeutic approaches rely on small molecules designed to boost this system. To identify such compounds, we developed a high-throughput screening assay based on HEK-293 cells expressing luciferase under the control of Interferon-Stimulated Response Elements (ISRE). An original library of 10,000 synthetic compounds was screened, and we identified a series of 1H-benzimidazole-4-carboxamide compounds inducing the ISRE promoter sequence, specific cellular Interferon-Stimulated Genes (ISGs), and the phosphorylation of Interferon Regulatory Factor (IRF) 3. ISRE induction by ChX710, a prototypical member of this chemical series, was dependent on the adaptor MAVS and IRF1, but was IRF3 independent. Although it was unable to trigger type I IFN secretion per se, ChX710 efficiently primed cellular response to transfected plasmid DNA as assessed by potent synergistic effects on IFN-β secretion and ISG expression levels. This cellular response was dependent on STING, a key adaptor involved in the sensing of cytosolic DNA and immune activation by various pathogens, stress signals and tumorigenesis. Our results demonstrate that cellular response to cytosolic DNA can be boosted with a small molecule, and potential applications in antimicrobial and cancer therapies are discussed.

Nasal route favors the induction of CD4 + T cell responses in the liver of HBV-carrier mice immunized with a recombinant hepatitis B surface- and core-based therapeutic vaccine

&NA; Immunization routes and number of doses remain largely unexplored in therapeutic vaccination. The aim of the present work is to evaluate their impact on immune responses in naïve and hepatitis B virus (HBV)‐carrier mouse models following immunization with a non‐adjuvanted recombinant vaccine comprising the hepatitis B surface (HBsAg) and core (HBcAg) antigens. Mice were immunized either by intranasal (i.n.), subcutaneous (s.c.) or simultaneous (i.n. + s.c.) routes. Humoral immunity was detected in all the animal models with the induction of a potent antibody (Ab) response against HBcAg, which was stronger than the anti‐HBs response. In the HBV‐carrier mouse model, the anti‐HBs response was predominantly subtype‐specific and preferentially induced by the i.n. route. However, the Ab titers were not sufficient to clear the high concentration of HBsAg present in the sera of these mice. The i.n. route was the most efficacious at inducing cellular immune responses, in particular CD4+ T cells. In naïve mice, cellular responses in spleen were strong and mainly due to CD4+ T cells whereas the CD8+ T‐cell response was low. In HBV‐carrier mice, high frequencies of HBs‐specific CD4+ T cells secreting interferon (IFN)‐&ggr;, interleukin (IL)‐2 and tumor necrosis factor (TNF)‐&agr; were found in liver only after i.n. immunization. Increased frequencies of CD4+ T cells expressing the integrin CD49a in liver suggest a role of nasal route in the cellular homing process. Multiple dose schedules appear to be a prerequisite for protein‐based immunization in order to overcome immunotolerance in HBV‐carrier mice. These findings provide new avenues for further preclinical and clinical development.