Risk of HBV reactivation in HCC patients undergoing combination therapy of PD-1 inhibitors and angiogenesis inhibitors in the antiviral era.

BACKGROUND: Although routine antiviral therapy has been implemented in HCC patients, the risk of HBV reactivation (HBVr) remains with the use of programmed cell death-1(PD-1) blockade-based combination immunotherapy and the relevant risk factors are also unclear. Therefore, we aimed to identify the incidence and risk factors of HBVr in HCC patients undergoing combination therapy of PD-1 inhibitors and angiogenesis inhibitors and concurrent first-line antivirals. METHODS: We included a total of 218 HBV-related HCC patients with first-line antivirals who received PD-1 inhibitors alone or together with angiogenesis inhibitors. According to the anti-tumor therapy modalities, patients were divided into PD-1 inhibitors monotherapy group (anti-PD-1 group) and combination therapy group (anti-PD-1 plus angiogenesis inhibitors group). The primary study endpoint was the incidence of HBVr. RESULTS: HBVr occurred in 16 (7.3%) of the 218 patients, 2 cases were found in the anti-PD-1 group and the remaining 14 cases were in the combination group. The Cox proportional hazard model identified 2 independent risk factors for HBVr: combination therapy (hazard ratio [HR], 4.608, 95%CI 1.010-21.016, P = 0.048) and hepatitis B e antigen (HBeAg) positive (HR, 3.695, 95%CI 1.246-10.957, P = 0.018). Based on the above results, we developed a simple risk-scoring system and found that the high-risk group (score = 2) developed HBVr more frequently than the low-risk group (score = 0) (Odds ratio [OR], 17.000, 95%CI 1.946-148.526, P = 0.01). The area under the ROC curve (AUC-ROC) was 7.06 (95%CI 0.581-0.831, P = 0.006). CONCLUSION: HBeAg-positive patients receiving combination therapy have a 17-fold higher risk of HBVr than HBeAg-negative patients with PD-1 inhibitors monotherapy.

MDSC-targeted liposomal all-trans retinoic acid suppresses mMdscs and improves immunotherapy in HBV infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are evolving as a prominent determinant in cancer occurrence and development and are functionally found to suppress T cells in cancer. Not much research is done regarding its involvement in viral infections. This research was designed to investigate the role of MDSCs in hepatitis B virus (HBV) infection and how targeting these cells with our novel all-trans retinoic acid encapsulated liposomal formulation could improve immunotherapy in C57BL/6 mice. METHODS: Ten micrograms (10 µg) of plasmid adeno-associated virus (pAAV/HBV 1.2, genotype A) was injected hydrodynamically via the tail vein of C57BL/6 mice. An all-trans retinoic acid encapsulated liposomal formulation (L-ATRA) with sustained release properties was used in combination with tenofovir disoproxil fumarate (TDF), a nucleotide analog reverse transcriptase inhibitor (nRTI) to treat the HBV infection. The L-ATRA formulation was given at a dose of 5 mg/kg intravenously (IV) twice a week. The TDF was given orally at 30 mg/kg daily. RESULTS: Our results revealed that L-ATRA suppresses MDSCs in HBV infected mice and enhanced T-cell proliferation in vitro. In vivo studies showed higher and improved immunotherapeutic effect in mice that received L-ATRA and TDF concurrently in comparison with the groups that received monotherapy. Lower HBV DNA copies, lower concentrations of HBsAg and HBeAg, lower levels of ALT and AST and less liver damage were seen in the mice that received the combination therapy of L-ATRA + TDF. CONCLUSIONS: In effect, targeting MDSCs with the combination of L-ATRA and TDF effectively reduced mMDSC and improved immunotherapy in the HBV infected mice. Targeting MDSCs could provide a breakthrough in the fight against hepatitis B virus infection.

Design and Synthesis of (3-Phenylisoxazol-5-yl)methanimine Derivatives as Hepatitis B Virus Inhibitors.

Series of (3-phenylisoxazol-5-yl)methanimine derivatives were synthesized, and evaluated for anti-hepatitis B virus (HBV) activity in vitro. Half of them more effectively inhibited HBsAg than 3TC, and more favor to inhibit secretion of HBeAg than to HBsAg. Part of the compounds with significant inhibition on HBeAg were also effectively inhibit replication of HBV DNA. Compound (E)-3-(4-fluorophenyl)-5-((2-phenylhydrazineylidene)methyl)isoxazole inhibited excellently HBeAg with IC50 in 0.65 µM (3TC(Lamivudine) in 189.90 µM), inhibited HBV DNA in 20.52 µM (3TC in 26.23 µM). Structures of compounds were determined by NMR and HRMS methods, and chlorination on phenyl ring of phenylisoxazol-5-yl was confirmed by X-ray diffraction analysis, and the structure-activity relationships (SARs) of the derivatives was discussed. This work provided a new class of potent non-nucleoside anti-HBV agents.

A Semi-mechanistic Model to Characterize the Long-Term Dynamics of Hepatitis B Virus Markers During Treatment With Lamivudine and Pegylated Interferon.

Antiviral treatments against hepatitis B virus (HBV) suppress viral replication but do not eradicate the virus, and need therefore to be taken lifelong to avoid relapse. Mathematical models can be useful to support the development of curative anti-HBV agents; however, they mostly focus on short-term HBV DNA data and neglect the complex host-pathogen interaction. This work aimed to characterize the effect of treatment with lamivudine and/or pegylated interferon (Peg-IFN) in 1,300 patients (hepatitis B envelope antigen (HBeAg)-positive and HBeAg-negative) treated for 1 year. A mathematical model was developed incorporating two populations of infected cells, namely I 1 , with a high transcriptional activity, that progressively evolve into I 2 , at a rate δ tr , representing cells with integrated HBV DNA that have a lower transcriptional activity. Parameters of the model were estimated in patients treated with lamivudine or Peg-IFN alone (N = 894), and the model was then validated in patients treated with lamivudine plus Peg-IFN (N = 436) to predict the virological response after a year of combination treatment. Lamivudine had a larger effect in blocking viral production than Peg-IFN (99.4-99.9% vs. 91.8-95.1%); however, Peg-IFN had a significant immunomodulatory effect, leading to an enhancement of the loss rates of I 1 (×1.7 in HBeAg-positive patients), I 2 (> ×7 irrespective of HBeAg status), and δ tr (×4.6 and ×2.0 in HBeAg-positive and HBeAg-negative patients, respectively). Using this model, we were able to describe the synergy of the different effects occurring during treatment with combination and predicted an effect of 99.99% on blocking viral production. This framework can therefore support the optimization of combination therapy with new anti-HBV agents.

Long-term follow-up of treatment-naïve HBeAg-negative patients with chronic hepatitis B.

BACKGROUND/AIMS: We aimed to explore long-term results of oral antiviral agents in treatment-naïve "HBeAg negative chronic hepatitis B (CHB)" and determine the factors affecting the complete virological response. METHOD: Patients with HBeAg-negative CHB who used oral antiviral agents for at least 3 years were evaluated retrospectively. RESULTS: A total of 173 patients were recorded. The mean duration of treatment was 62.2 ± 28.9 months. Complete virological responses (CVR) were 82.8% (n = 53/64) in tenofovir disoproxil fumarate (TDF), 84.4% (n = 49/58) in lamivudine (LAM), 83.9% (n = 26/31) in entecavir (ETV), 95% in telbivudine (LdT) (n = 19/20) (p = 0.290). Multivariate analysis revealed age ≤ 40 (p = 0.012, 95%CI = 1.38-13.76, OR = 4.36) and baseline HBV DNA value (p = 0.003, 95%CI = 1.23-2.63, OR = 1.78) as independent factors for CVR. Virological breakthrough was detected in 29 (50%) patients on LAM therapy, two (6.4%) patients on ETV therapy, and two (10%) patients on LdT therapy. Treatment was switched to another antiviral agent due to osteoporosis in four patients in the TDF group, muscle pain in nine patients in the LDT group, and headache in one patient in the ETV group. Hepatocelluler cancer was detected in five patients. HBsAg seroclearance developed in two patients. Anti-HBs seroconversion was not detected. CONCLUSION: CVR was achieved at similar rates with all four antiviral agents, while younger age (≤ 40) and low baseline viral load were the main factors for virological response. However, drug resistance and virological breakthrough in the LAM group and side effects in the LdT group were detected during the long-term follow-up. Moreover, HBsAg seroclearance was achieved at very low rates with oral antiviral agents.

Effect of Hepatocyte Targeting Nanopreparation Syringopicroside on Duck Hepatitis B Virus and Evaluation of Its Safety.

Syringopicroside is a kind of iridoid monomer compound isolated from Syringa oblata exhibiting a potent effect against hepatitis B virus (HBV). The therapeutic effect and safety of syringopicroside-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (SYR-NP) were studied on the model of HBV-infected ducklings and on cultured HepG2.2.15 cells. HBV DNA in ducklings was assessed by fluorescence quantitative PCR. In HepG2.2.15 cells, the content of HBsAg and HBeAg were assayed. Acute toxicity of SYR-NP was studied in ICR mice in 12 h and 7 days after SYR-NP administration. The serum levels of HBV DNA in ducklings treated with SYR-NP in a high dose was significantly lower than in the control. In HepG2.2.15 cells treated with different doses of SYR-NP, the concentrations of HBsAg and HBeAg were significantly below the control. Acute toxicity test showed high safety of SYR-NP. Thus, SYR-NP can inhibit replication of HBV DNA and protect the liver tissue.

Inhibition of duck hepatitis B virus infection of liver cells by combined treatment with viral e antigen and carbohydrates.

The e antigen (eAg) of duck hepatitis B virus (DHBV) is a glycosylated secretory protein with a currently unknown function. We concentrated this antigen from the supernatants of persistently infected primary duck liver cell cultures by ammonium sulphate precipitation, adsorption chromatography over concanavalin A Sepharose, preparative isoelectric focusing and molecular sieve chromatography. The combined treatment of duck liver cells with DHBV eAg (DHBe) concentrate and alpha-methyl-d-mannopyranoside strongly inhibited DHBV replication at de novo infection. When DHBe was added to non-infected primary duck liver cells, it was found to be associated with liver sinusoidal endothelial cells. This binding could be inhibited by the addition of alpha-methyl-d-mannopyranoside and other sugar molecules. The inhibitory effect of DHBe on infection could play a role in maintaining viral persistence.

HBV-specific lymphoproliferative and cytokine responses in patients with chronic hepatitis B.

BACKGROUND/AIMS: Hepatitis B virus specific T cell responses are crucial for viral elimination but their nature is not fully understood. METHODS: We studied the regulation of proliferation and cytokine production after antigenic stimulation in peripheral blood mononuclear cells from chronically HBV-infected patients and subjects with natural immunity after recovery from an acute infection. Proliferation and production of interferon-gamma, IL-10 and tumor necrosis factor-alpha were determined after stimulation with HBcAg, HBeAg or HBsAg in the absence or presence of IL-12 or neutralizing antibodies to IL-12, interferon-gamma, IL-4, IL-10 or tumor necrosis factor-alpha. RESULTS: Upon stimulation with HBcAg or HBeAg, peripheral blood mononuclear cells from chronic hepatitis B virus patients displayed a clear class-II restricted proliferative response (SI greater than 2.5). Both interferon-gamma (less than 50 IU/ml) and IL-10 levels up to 600 pg/ml were detected. Proliferative or cytokine responses to HBsAg were very weak or absent. Addition of IL-12 to HBeAg-stimulated cultures increased the production of interferon-gamma to more than 200 IU/ml in all patients and slightly increased the production of IL-10. Neutralization of IL-10 increased the HBeAg-induced interferon-gamma production but had no effect on tumor necrosis factor-alpha production. Addition of anti-IL-4 or anti-tumor necrosis factor-alpha had no significant influence on proliferation or cytokine release. Importantly, in both chronic hepatitis B virus patients and naturally immune subjects, IL-12 induced proliferative and interferon-gamma responses in peripheral blood mononuclear cells stimulated with HBsAg. CONCLUSIONS: Our data indicate that peripheral blood mononuclear cells from chronic hepatitis B virus patients proliferate and produce interferon-gamma and IL-10 upon HBeAg but not upon HBsAg stimulation. IL-12 augments the HBeAg-induced responses and, additionally, provokes proliferation and interferon-gamma production in HBsAg-stimulated cultures.

[Hepatitis B virus antigen-induced specific TCR V beta gene subfamily amplifications and their diversity].

The human immune system confronts various antigens, then the recognitive structures of T lymphocytes must be diverse. In this study, we analyzed the TCR V beta 1-20 gene repertoire by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood lymphocytes (PBLs) from individuals vaccinated with recombinant hepatitis B vaccine and PBLs from healthy donors stimulated by supernatants of the HepG2 2215 cell line transfected by HBV-DNA. The specific amplifications of V beta 6, 14, V beta 6, 15 were observed in PBLs after vaccination and stimulation by HepG2 2215 cell line respectively. The results suggest that the V beta segments may be the specific recognitive units for these antigens and involved in restriction and cytotoxicity.

Effects of hepatitis B virus antigens on interferon-gamma production of peripheral blood mononuclear cells from hepatitis B virus carriers and healthy individuals.

BACKGROUND: Although the precise mechanisms of persistent infection by hepatitis B virus are not yet known, several lines of studies suggest that hepatitis B e antigen in sera might modulate the immune response of the host. The aim of this study was to clarify the effects of hepatitis B virus antigen on cytokine production of lymphocytes. METHODS: We studied the effects of recombinant hepatitis B core antigen and surface antigen on interferon-gamma production of phytohemagglutinin-stimulated peripheral blood mononuclear cells from hepatitis B virus carriers and healthy individuals. Hepatitis B core antigen used in this study shared the antigenic site responsible for hepatitis B core and e antigen. RESULTS: Although pre-incubation of peripheral blood mononuclear cells with hepatitis B core/e antigen followed by stimulation with phytohemagglutinin significantly reduced the production of interferon-gamma, pre-incubation with hepatitis B surface antigen did not affect them. CONCLUSIONS: These results indicate that hepatitis B core/e antigen has the ability to inhibit interferon-gamma production of lectin-stimulated peripheral blood mononuclear cells in vitro, suggesting that secretion of hepatitis B e antigen into sera might be how hepatitis B virus escapes the immuno-surveillance system of the host.