Ribonuclease h (rNaseH) Inhibitors

RNaseH is essential for HBV replication and is therefore a potential target for anti-HBV therapy [reviewed in^[36]]. Recent low-throughput screening of compound classes with anti-Human Immunodeficiency Virus RNaseH activity led to identification of HBV RNaseH inhibitors that are now beginning to be tested in animal models of HBV infection. The future role of RNaseH inhibitors in gaining a better control of the virus is still to be defined.

Entry Inhibitors

In 2012, Yan and colleagues have identified the bile acid pump sodium taurocholate cotransporting polypeptide (NTCP) as the entry receptor of both HBV and hepatitis D virus (HDV).^[37,38] The synthetic lipopeptide Myrcludex B competes with the viral envelop pre-S1 motif for NTCP binding thereby blocking HBV entry.^[39,40] This could potentially be used to prevent infection post-exposure, after liver transplantation in recipients with CHB and in neonates of HBV positive mothers. Its use in the context of CHB as a therapeutic module is under debate; in theory, entry inhibition could supplement other antivirals by inhibition of de novoinfection of naive hepatocytes. This may provide the proper time window for the infected hepatocytes to be eliminated following their accelerated turn-over (induced by immune modulation), thereby "clearing" the liver from HBV.^[41,42] A phase 2a clinical study with once daily S.C Myrcludex B showed >1log reduction in serum HBV DNA among 75% of patients. The drug was safe although higher dosage was associated with a clinically insignificant elevation in serum bile acid levels.^[43] Albeit, HBsAg level was merely affected following 24w of treatment, therefore, further studies with higher dosage, prolonged treatment or combination therapies are needed.

Modulation of the Immune System

HBV persistence and the maintenance of its cccDNA mainly result from an insufficient immune response against the virus.^[44] An efficient control of viral infection requires a concerted action of both innate and adaptive immune responses. Previous studies suggest that HBV is a stealth virus that only weakly induces the innate immune response, thereby escaping recognition.^[45,46] Conjointly, CHB is associated with a weak or even absent virus-specific T-cell reactivity, which is largely attributed to a T-cell exhaustion state, characterized by poor cytotoxic activity, impaired cytokine production and expression of inhibitory receptors [reviewed in^[47]]. Thus, recruitment of the innate immune response against HBV infection and restoring the antiviral function of exhausted T cells are coveted fields of research [reviewed in^[48]].

Targeting the Innate Immune System

TLR7 Agonists. The innate immune response is activated by the host surveillance system via pathogen recognition receptors (PRRs) such as toll-like receptors (TLRs). TLR-7 chiefly recognizes viral single-stranded RNAs. Upon TLR-7 binding, a cascade of IFN and other cytokines/chemokines production is induced, stimulating both natural killer (NK) cells and cytotoxic T lymphocytes, hence simultaneously activating the innate and adaptive immune responses.^[49]

In a recent study performed in a woodchuck model of CHB, short duration of treatment with the TLR7 agonist GS-9620, resulted in a robust and sustained antiviral response including reductions in hepatic woodchuck hepatitis virus (WHV) DNA, WHV RNA as well as in WHV cccDNA and WHV surface antigen (WHsAg).^[50] A subset of the animals sero-converted to become positive for WHs antibodies (WHsAb). Moreover, animals achieving viral load decline exhibited a mark reduction in HCC incidence (only 10% compared to over 70% among the placebo group).

Two phase Ib double blind studies with GS-9620 were conducted.^[51] Oral administration of GS-9620 was found to be safe and well tolerated, but no change in HBsAg or HBV DNA levels was observed after a short course of therapy. Of note, in the vast majority of patients, no detectable serum IFNα level was measured. Further studies using higher dosages and a longer duration of therapy will hopefully yield better results. A phase II study comparing the combination of GS-9620 and tenofovir vs. tenofovir alone is under way.

STING Agonists. The stimulator of IFN genes (STING) is an intracellular DNA sensor that is activated by cyclic GMP-AMP Synthase (cGAS) in response to cytosolic DNA.^[52] DMXAA (Vadimezan or ASA404) is a STING agonist that produces a type I IFN-dominant cytokine response, which efficiently suppresses HBV replication in cultured murine hepatocytes and in a murine model of HBV infection.^[53]DMXAA induces a more potent antiviral activity against HBV as compared to TLR agonists, as well as a favourable cytokine response with less pro-inflammatory characteristics, possibly resulting in less inflammation and tissue damage. Pending further in vivo studies, DMXAA is hypothesized to suppress HBV replication, viral transcription and possibly promoting cccDNA decay. This may result in attenuation of T-cell exhaustion thereby restoring the HBV-specific T-cell response. As DMXAA was initially developed as a vascular disrupting agent with antitumor activity, it has already been evaluated in phase II clinical trials for treating various cancers, with considerable safety and efficacy information available.^[54,55]

Targeting the Adaptive Immune System.Specific T- and B-cell responses to HBV remain crucial for elimination of the virus and its cccDNA. The adaptive immune response may be targeted in various ways; the two leading approaches include stimulation via therapeutic vaccinations and blockade of immune inhibitory pathways thought to be responsible for T-cell exhaustion.

Therapeutic Vaccines. Numerous studies using therapeutic vaccinations against chronic HBV have taken place, using diverse antigens or DNA fragments as target molecules.

Vaccination with a recombinant HBsAg, commonly used as a prophylactic vaccine, has been tested as a means to promote antiviral immune response among chronically infected patients. As a staggering viral load may lead to an insufficient immune response,^[56–59]pre-treatment with NUCs prior to administering a therapeutic vaccine is frequently performed. Once the viral load is decreased, antigenic presentation of viral particles could potentially evoke a profound T-cell response thus breaking immune tolerance. Despite this, a randomized control trial of HBsAg-based vaccine co-administered with NUCs therapy did not show any superiority in comparison to NUCs mono-therapy.^[60] Likewise, a randomized, open-label dose-ascending study showed the safety and immunogenicity of GS-4774, a recombinant, heat-killed, yeast-based vaccine engineered to express HBV-specific antigens among healthy individuals.^[61] However, in a recent multicenter study performed in CHB patients who were virally suppressed with NUCs, there was no advantage to the addition of GS-4774 in terms of HBsAg suppression or loss.^[62]

It is important to keep in mind that although the rational for combining NUCs with therapeutic vaccines is obvious, it is conceivable that the effect of innovative therapies may largely differ between patients on long-term NUCs therapy and treatment naïve patients.

DNA vaccination represents another approach to restore antiviral immune response by promoting antiviral CD8 T cell as well as CD4 T cell and antibody responses. A phase I study performed in HBV patients following lamivudine breakthrough showed that a DNA vaccine containing the small and middle regions of the HBV envelop was safe and also elicited an immunological response in a high proportion of patients.^[63] However, a recent open prospective phase I/II study in patients who had been treated with NUCs for a median of 3 years and who had undetectable DNA for at least a year, showed that five IM injections of surface based DNA vaccine did not prevent HBV reactivation after NUCs discontinuation and did not restore anti-HBV immune response.^[64]

In summary, although therapeutic vaccinations show great success in vitro and in animal models, thus far clinical trials display disappointing results.

PD-1/PD-L1 Inhibitors. CD8 T cells express an inhibitory receptor designated programmed cell death-1 (PD-1) which functions as a negative regulator of the immune system^[65]and is thought to play a critical role in T-cell exhaustion.^[66]Ex vivo studies have shown that the level of CD8+ T-cell response inversely correlates with the level of HBV viremia, that HBV specific CD8+ T cells express high levels of PD-1 and that interfering with the interaction between PD-1 and its natural ligand, programmed cell death ligand-1 (PDL-1) results in improvement of the HBV-specific T-cell function.^[67] Interestingly, antiviral therapy with NUCs reduces the expression of PD-1 immensely, thereby enhancing immunity against HBV.^[68] This finding gave rise to trials combining NUCs with PD-1 inhibitors and therapeutic vaccinations, so far yielding encouraging results. More recently, a study performed in a WHV model has shown that combining therapeutic vaccination and PD-L1 inhibitor upon suppression of viral replication with NUCs, resulted in enhanced HBV specific T-cell activation and a much more robust and sustained suppression of both, WHV DNA and WHsAg levels.^[69] Of note, restoration of cytotoxic T-cell response bears a potential concern of massive cell destruction and therefore safety issues must be addressed before implementation of the anti PD-1 therapy in clinical trials.

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