Ophélie Godon

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.

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.

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.

IgG subclasses determine pathways of anaphylaxis in mice

Background: Animal models have demonstrated that allergen‐specific IgG confers sensitivity to systemic anaphylaxis that relies on IgG Fc receptors (Fc&ggr;Rs). Mouse IgG2a and IgG2b bind activating Fc&ggr;RI, Fc&ggr;RIII, and Fc&ggr;RIV and inhibitory Fc&ggr;RIIB; mouse IgG1 binds only Fc&ggr;RIII and Fc&ggr;RIIB. Although these interactions are of strikingly different affinities, these 3 IgG subclasses have been shown to enable induction of systemic anaphylaxis. Objective: We sought to determine which pathways control the induction of IgG1‐, IgG2a‐, and IgG2b‐dependent passive systemic anaphylaxis. Methods: Mice were sensitized with IgG1, IgG2a, or IgG2b anti‐trinitrophenyl mAbs and challenged with trinitrophenyl‐BSA intravenously to induce systemic anaphylaxis that was monitored by using rectal temperature. Anaphylaxis was evaluated in mice deficient for Fc&ggr;Rs injected with mediator antagonists or in which basophils, monocytes/macrophages, or neutrophils had been depleted. Fc&ggr;R expression was evaluated on these cells before and after anaphylaxis. Results: Activating Fc&ggr;RIII is the receptor primarily responsible for all 3 models of anaphylaxis, and subsequent downregulation of this receptor was observed. These models differentially relied on histamine release and the contribution of mast cells, basophils, macrophages, and neutrophils. Strikingly, basophil contribution and histamine predominance in mice with IgG1‐ and IgG2b‐induced anaphylaxis correlated with the ability of inhibitory Fc&ggr;RIIB to negatively regulate these models of anaphylaxis. Conclusion: We propose that the differential expression of inhibitory Fc&ggr;RIIB on myeloid cells and its differential binding of IgG subclasses controls the contributions of mast cells, basophils, neutrophils, and macrophages to IgG subclass–dependent anaphylaxis. Collectively, our results unravel novel complexities in the involvement and regulation of cell populations in IgG‐dependent reactions in vivo.

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.

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.

Platelets expressing IgG receptor FcγRIIA/CD32A determine the severity of experimental anaphylaxis

Platelet-derived serotonin contributes to FcγRIIA/CD32A-induced IgG-dependent anaphylaxis. Potent platelets Anaphylaxis results from inappropriate immune responses to allergens. Human platelets express the IgG receptor FcγRIIA/CD32A and release inflammatory mediators in response to their engagement, but their contribution to anaphylaxis is not well understood. Beutier et al. developed mouse models that express either human FcγRIIA/CD32A alone or the full human IgG receptor complexity to understand the role of platelets in anaphylaxis. Anaphylaxis induced a marked decrease in platelet levels, but preventive platelet depletion reduced anaphylaxis severity. A clinical study of patients with drug-induced anaphylaxis revealed that a severe reaction was likewise associated with fewer circulating platelets. Activated platelets released serotonin, which contributed to anaphylaxis severity. These results reveal a critical role for platelets in IgG-mediated anaphylaxis. Platelets are key regulators of vascular integrity; however, their role in anaphylaxis, a life-threatening systemic allergic reaction characterized by the loss of vascular integrity and vascular leakage, remains unknown. Anaphylaxis is a consequence of inappropriate cellular responses triggered by antibodies to generally harmless antigens, resulting in a massive mediator release and rapidly occurring organ dysfunction. Human platelets express receptors for immunoglobulin G (IgG) antibodies and can release potent mediators, yet their contribution to anaphylaxis has not been previously addressed in mouse models, probably because mice do not express IgG receptors on platelets. We investigated the contribution of platelets to IgG-dependent anaphylaxis in human IgG receptor–expressing mouse models and a cohort of patients suffering from drug-induced anaphylaxis. Platelet counts dropped immediately and markedly upon anaphylaxis induction only when they expressed the human IgG receptor FcγRIIA/CD32A. Platelet depletion attenuated anaphylaxis, whereas thrombocythemia substantially worsened its severity. FcγRIIA-expressing platelets were directly activated by IgG immune complexes in vivo and were sufficient to restore susceptibility to anaphylaxis in resistant mice. Serotonin released by activated platelets contributed to anaphylaxis severity. Data from a cohort of patients suffering from drug-induced anaphylaxis indicated that platelet activation was associated with anaphylaxis severity and was accompanied by a reduction in circulating platelet numbers. Our findings identify platelets as critical players in IgG-dependent anaphylaxis and provide a rationale for the design of platelet-targeting strategies to attenuate the severity of anaphylactic reactions.

Evidence that neutrophils do not promote Echis carinatus venom-induced tissue destruction

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Innate Immune Basis for Rift Valley Fever Susceptibility in Mouse Models

Rift Valley fever virus (RVFV) leads to varied clinical manifestations in animals and in humans that range from moderate fever to fatal illness, suggesting that host immune responses are important determinants of the disease severity. We investigated the immune basis for the extreme susceptibility of MBT/Pas mice that die with mild to acute hepatitis by day 3 post-infection compared to more resistant BALB/cByJ mice that survive up to a week longer. Lower levels of neutrophils observed in the bone marrow and blood of infected MBT/Pas mice are unlikely to be causative of increased RVFV susceptibility as constitutive neutropenia in specific mutant mice did not change survival outcome. However, whereas MBT/Pas mice mounted an earlier inflammatory response accompanied by higher amounts of interferon (IFN)-α in the serum compared to BALB/cByJ mice, they failed to prevent high viral antigen load. Several immunological alterations were uncovered in infected MBT/Pas mice compared to BALB/cByJ mice, including low levels of leukocytes that expressed type I IFN receptor subunit 1 (IFNAR1) in the blood, spleen and liver, delayed leukocyte activation and decreased percentage of IFN-γ-producing leukocytes in the blood. These observations are consistent with the complex mode of inheritance of RVFV susceptibility in genetic studies.

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.