T-cell engager antibodies enable T cells to control HBV infection and to target HBsAg-positive hepatoma in mice.

BACKGROUND & AIMS: Current antiviral therapies control but rarely eliminate HBV, leaving chronic HBV carriers at risk of developing hepatocellular carcinoma (HCC). Lacking or dysfunctional virus-specific adaptive immunity prevents control of HBV and allows the virus to persist. Restoring antiviral T-cell immunity could lead to HBV elimination and cure of chronically infected patients. METHODS: We constructed bispecific T-cell engager antibodies that are designed to induce antiviral immunity through simultaneous binding of HBV envelope proteins (HBVenv) on infected hepatocytes and CD3 or CD28 on T cells. T-cell engager antibodies were employed in co-cultures with healthy donor lymphocytes and HBV-infected target cells. Activation of the T-cell response was determined by detection of pro-inflammatory cytokines, effector function (by cytotoxicity) and antiviral effects. To study in vivo efficacy, immune-deficient mice were transplanted with HBVenv-positive and -negative hepatoma cells. RESULTS: The 2 T-cell engager antibodies synergistically activated T cells to become polyfunctional effectors that in turn elicited potent antiviral effects by killing infected cells and in addition controlled HBV via non-cytolytic, cytokine-mediated antiviral mechanisms. In vivo in mice, the antibodies attracted T cells specifically to the tumors expressing HBVenv resulting in T-cell activation, tumor infiltration and reduction of tumor burden. CONCLUSION: This study demonstrates that the administration of HBVenv-targeting T-cell engager antibodies facilitates a robust T-cell redirection towards HBV-positive target cells and provides a feasible and promising approach for the treatment of chronic viral hepatitis and HBV-associated HCC. LAY SUMMARY: T-cell engager antibodies are an interesting, novel therapeutic tool to restore immunity in patients with chronic hepatitis B. As bispecific antibodies, they bind envelope proteins on the surface of the hepatitis B virus (HBV) and CD3 or CD28 on T cells. This way, they induce a potent antiviral and cytotoxic T-cell response that leads to the elimination of HBV-positive cells. These bispecific T-cell engager antibodies are exciting therapeutic candidates for chronic hepatitis B and HBV-associated hepatocellular carcinoma.

Interferon-induced degradation of the persistent hepatitis B virus cccDNA form depends on ISG20.

Hepatitis B virus (HBV) persists by depositing a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that cannot be targeted by available antivirals. Interferons can diminish HBV cccDNA via APOBEC3-mediated deamination. Here, we show that overexpression of APOBEC3A alone is not sufficient to reduce HBV cccDNA that requires additional treatment of cells with interferon indicating involvement of an interferon-stimulated gene (ISG) in cccDNA degradation. Transcriptome analyses identify ISG20 as the only type I and II interferon-induced, nuclear protein with annotated nuclease activity. ISG20 localizes to nucleoli of interferon-stimulated hepatocytes and is enriched on deoxyuridine-containing single-stranded DNA that mimics transcriptionally active, APOBEC3A-deaminated HBV DNA. ISG20 expression is detected in human livers in acute, self-limiting but not in chronic hepatitis B. ISG20 depletion mitigates the interferon-induced loss of cccDNA, and co-expression with APOBEC3A is sufficient to diminish cccDNA. In conclusion, non-cytolytic HBV cccDNA decline requires the concerted action of a deaminase and a nuclease. Our findings highlight that ISGs may cooperate in their antiviral activity that may be explored for therapeutic targeting.

Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels.

BACKGROUND & AIMS: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. METHODS: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. RESULTS: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5-12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. CONCLUSIONS: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. LAY SUMMARY: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis.

BACKGROUND & AIMS: Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. METHODS: We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). RESULTS: Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. CONCLUSIONS: IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-ß receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-ß receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases-for example, by lymphotoxin-ß receptor activation-allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

p38 MAPK/PP2Acα/TTP pathway on the connection of TNF-α and caspases activation on hydroquinone-induced apoptosis.

This study investigated tumor necrosis factor-α (TNF-α)-mediated death pathway contribution to hydroquinone (HQ) cytotoxicity in human leukemia U937 cells. HQ-induced apoptosis of human leukemia U937 cells was characterized by the increase in mitochondrial membrane depolarization, procaspase-8 degradation and tBid production. Downregulation of Fas-associated death domain protein (FADD) blocked HQ-induced procaspase-8 degradation and rescued the viability of HQ-treated cells, suggesting the involvement of a death receptor-mediated pathway in HQ-induced cell death. HQ induced increased TNF-α mRNA stability led to TNF-α protein expression upregulation, whereas HQ suppressed TNF-α-mediated NFκB pathway activation. HQ elicited protein phosphatase 2A catalytic subunit α (PP2Acα) upregulation via p38 mitogen-activated protein kinase (MAPK)-mediated CREB/c-Jun/ATF-2 phosphorylation, and PP2Acα upregulation was found to promote tristetraprolin (TTP) degradation. Suppression of p38 MAPK activation and protein phosphatase 2A (PP2A) activity abrogated TNF-α upregulation and procaspase degradation in HQ-treated cells. Overexpression of TTP suppressed HQ-induced TNF-α upregulation and restored the viability of HQ-treated cells. Moreover, TTP overexpression increased TNF-α mRNA decay in HQ-treated cells. Taken together, our data indicate that HQ elicits TNF-α upregulation via p38 MAPK/PP2A-mediated TTP downregulation, and suggest that the TNF-α-mediated death pathway is involved in HQ-induced U937 cell death. The same pathway was also proven to be involved in the HQ-induced death of human leukemia HL-60 and Jurkat cells.

Structure-function studies on inhibitory activity of Bungarus multicinctus protease inhibitor-like protein on matrix metalloprotease-2, and invasion and migration of human neuroblastoma SK-N-SH cells.

In view of the findings that several Kunitz-type protein inhibitors suppress tumor invasion and metastasis, the aim of the present study is to explore whether Bungarus multicinctus protease inhibitor-like protein-2 (PILP-2) and PILP-3 exhibit anti-tumor activity. Although approximately 28% of amino acid substitutions occurred between PILP-2 and PILP-3, molecular modeling suggested that PILP-2 and PILP-3 shared similar folded structures. Unlike PILP-2, PILP-3 showed a notable activity in abolishing migration and invasion of human neuroblastoma SK-N-SH cells. The ability of PILP-3 to inhibit matrix metalloprotease-2 (MMP-2) activity was higher than that of PILP-2. Pull-down assay revealed protein-protein interaction between PILP-3 and MMP-2. In contrast to mutation on N-terminal region, replacement of amino acids at C-terminus attenuated notably the ability of PILP-3 to inhibit cell invasion, cell migration and MMP-2 activity as well as the binding capability of PILP-3 with MMP-2. Molecular docking showed that N-terminal region of PILP-2 and PILP-3 fitted into the cleft around the active site of MMP-2 catalytic domain. In contrast to that of PILP-2, C-terminal region of PILP-3 was suggested to be in close contact with catalytic domain of MMP-2. Collectively, our data indicate that PILP-3 is a MMP-2 inhibitor and shows an activity in inhibiting migration and invasion of neuroblastoma, and suggest that intact C-terminus is crucial to the activities of PILP-3.

Genetic organization of Bungarus multicinctus protease inhibitor-like proteins.

The structural organization of the genes encoding Bungarus multicinctus protease inhibitor-like proteins (PILPs), PILP-1, PILP-2 and PILP-3, are reported in this study. Unlike PILP-2 and PILP-3, recombinant PILP-1 exhibited inhibitory activity on trypsin. PILP genes and B chain genes shared identical organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 chain and B2 chain genes were absent in that of PILP genes. Noticeably, intronic insertion in the second intron of B chain genes appeared in the promoter region of PILP-1 gene, but not in that of PILP-2 and PILP-3 genes. Comparative analyses of PILP genes and B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that PILP genes and B chain genes originate from a common ancestor, and that accelerated evolution may diversify PILP and B chain genes as that proposed for snake venom phospholipase A(2), neurotoxin and cardiotoxin genes.