Immunomodulation and RNA interference alter hepatitis B virus–specific CD8 T‐cell recognition of infected HepG2‐NTCP

Kuipery, Adrian; Vasquez, Juan D. Sanchez; Mehrotra, Aman; Feld, Jordan J.; Janssen, Harry L.A.; Gehring, Adam J.

doi:10.1002/hep.32230

Cited by 7 publications

(8 citation statements)

References 27 publications

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“…[115,116] Additionally, both type I IFNs and IFNγ up-regulate antigen presentation, making HBV-infected hepatocytes better targets for CD8 T-cell-mediated attack. [117,118] TNFα that is produced after TLR stimulation has been shown to promote T-cell proliferation in liver in mouse models. [119] Therefore, the secondary benefits of innate immunomodulators may not be observed until they enter combination strategies.…”

Section: Innate Immunomodulatorsmentioning

confidence: 99%

Getting to HBV cure: The promising paths forward

et al. 2022

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Chronic HBV infection is a global public health burden estimated to impact nearly 300 million persons worldwide. Despite the advent of potent antiviral agents that effectively suppress viral replication, HBV cure remains difficult to achieve because of the persistence of covalently closed circular DNA (cccDNA), HBV‐DNA integration into the host genome, and impaired immune response. Indefinite treatment is necessary for most patients to maintain level of viral suppression. The success of direct‐acting antivirals (DAAs) for hepatitis C treatment has rejuvenated the search for a cure for chronic hepatitis B (CHB), though an HBV cure likely requires an additional layer: immunomodulators for restoration of robust immune responses. DAAs such as entry inhibitors, capsid assembly modulators, inhibitors of subviral particle release, cccDNA silencers, and RNA interference molecules have reached clinical development. Immunomodulators, namely innate immunomodulators (Toll‐like receptor agonists), therapeutic vaccines, checkpoint inhibitors, and monoclonal antibodies, are also progressing toward clinical development. The future of the HBV cure possibly lies in triple combination therapies with concerted action on replication inhibition, antigen reduction, and immune stimulation. Many obstacles remain, such as overcoming translational failures, choosing the right endpoint using the right biomarkers, and leveraging current treatments in combination regimens to enhance response rates. This review gives an overview of the current therapies for CHB, HBV biomarkers used to evaluate treatment response, and development of DAAs and immune‐targeting drugs and discusses the limitations and unanswered questions on the journey to an HBV cure.

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Section: Innate Immunomodulatorsmentioning

confidence: 99%

Getting to HBV cure: The promising paths forward

et al. 2022

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“…HepG2 and HepG-NTCP-A3 cells were cultured in Dulbeco's Modified Eagles Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Canada), L-glutamine, 100U penicillin/ streptomycin (Gibco Fisher Scientific, Canada), 1mM Sodium Pyruvate, 20mM HEPES, essential and non-essential amino acids (Corning, Manasas, VA, USA) and 5 µg plasmosin (Invivogen, Cedarlane, Canada) at 37 o C in a 5% humidified incubator as described [7]. HepG2-NTCP-A3 subclones were obtained by limiting dilution essentially as described [8].…”

Section: Generation Of Hepg2-ntcp Subclonesmentioning

confidence: 99%

“…For staining of NTCP expression, HepG2, HepG2-NTCP-A3 parental clone or its subclones B7, C2, D10, G4 and G7 were seeded into a 12-well plate and incubated with 200nM MyrB-Alexa-647 at 37 o C for 30 minutes as described [12,13]. Unbound peptide was removed by washing with 1xPBS, the cells were fixed with 4% paraformaldehyde (PFA) and imaged using EVOS FL-Auto 2 fluorescence microscope (Invitrogen, Canada) as described [7].…”

Section: Peptide Labelling and Staining Of Ntcp Expressing Cellsmentioning

confidence: 99%

“…HepG2-NTCP-A3 parental clone or its subclones were seeded at 500,000 cells per ml per well into a 12-well plate and infected with HepAD38-cell harvested HBV genotype D at 100, 200, 500 or 1000 Genome Equivalents (GEq)/ml in the presence of 2.5% dimethyl sulfoxide (DMSO; Sigma) and 4% or no polyethylene glycol (PEG 8000; Sigma) as described [7,[14][15][16]. The next day, the virus inoculum was removed, the cells were extensively washed with PBS and cultured in the presence of DMSO-2.5%.…”

Section: Hbv Infection and Hbv Dna Quantificationmentioning

confidence: 99%

“…After blocking, cells were incubated with rabbit polyclonal HBV anti-Core antibody C149 at 1:5000 dilution as described [21] as a primary and donkey anti-rabbit Alexa fluor-647 at 1:500 dilution (Thermo Scientific, Canada) as a secondary antibody. Nuclei were stained with DAPI and the cells were imaged using EVOS FL Auto 2 (Invitrogen, Canada) as described [7].…”

Section: Immunofluorescence For Hbv Core Proteinmentioning

confidence: 99%

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HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection

Zahoor¹,

Kuipery²,

Mosa³

et al. 2022

Preprint

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HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for accurate interpretation of in vitro data. However, variations in HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than 4-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

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The scientific basis of combination therapy for chronic hepatitis B functional cure

Lim

Baumert

Boni

et al. 2023

Nat Rev Gastroenterol Hepatol

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Functional cure of Chronic Hepatitis B(CHB) or HBsAg loss after 24 weeks off therapy is now the goal of treatment, but rarely achieved with current therapy.Understanding the HBV lifecycle and immunological defects that lead to persistence can identify targets for novel therapy. Broadly they fall into three categories, those reducing viral replication, those reducing antigen load and immunotherapies. These therapies are discussed in the context of early phase clinical trials hence represent insights from preliminary data. Profound viral suppression seems unlikely to achieve qHBsAg reduction or HBsAg loss. Combining nucleoside analogues(NA) and immunotherapy reduces qHBsAg levels and induces HBsAg loss in some patients, particularly those with lower qHBsAg levels. Even agents that are specifically designed to reduce viral antigen load may not be able to achieve sustained HBsAg loss when used alone. Thus there is a rationale for the use of combinations of reducing viral replication, antigen reduction and immunotherapy. Monitoring during therapy is important not just to predict HBsAg loss but also to understand mechanisms of HBsAg loss using viral and immunological biomarkers, and in selected cases intrahepatic sampling. We consider different paths to functional cure of CHB and the need to individualize therapy of this heterogenous infection until a therapeutic avenue for all CHB patients is available. Key Points• HBsAg loss after 6 months off therapy with undetectable HBV DNA is defined as functional cure, and is associated with improved clinical outcomes and is the optimal goal of Chronic Hepatitis B therapy.• Novel agents fall into three categories; those reducing viral replication, those reducing viral antigen load and immunotherapies. Combinations that lead to functional cure are being explored.• Profound viral suppression alone is unlikely to lead to functional cure or reduction in qHBsAg levels.• Combining replication inhibition with immunotherapy leads to some reduction in qHBsAg levels (<1 log) and HBsAg loss, usually in those with low baseline qHBsAg levels• Reducing viral antigen production reduces qHBsAg levels by up to 2 log, which may be sustained off therapy, and in combination with replication inhibitors or immunotherapy can achieve HBsAg loss in some instances, although not all were sustained.• There is likely to be more than one path to functional cure and finding the optimal one for each patient is the challenge.

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Immunomodulation and RNA interference alter hepatitis B virus–specific CD8 T‐cell recognition of infected HepG2‐NTCP

Cited by 7 publications

References 27 publications

Getting to HBV cure: The promising paths forward

Getting to HBV cure: The promising paths forward

HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection

The scientific basis of combination therapy for chronic hepatitis B functional cure

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