Novel therapeutic approaches aim to achieve sustained viral suppression and restoration of anti-HBV immunity through a finite duration of therapy
[20][21]. They can be divided into two types: inhibition of alternative steps of viral replication (i.e., virus-directing agents) and direct enhancement of host immunity (immunomodulatory agents). The two approaches are closely intercalated, as HBV itself plays a major role in the immunopathogenesis of CHB. In neonatal infection, the HBV antigens behave as neo-self-antigens and elicit tolerance as opposed to activation of HBV-specific T cells, which will lead to clonal deletion or clonal downregulation of high-affinity T-cell clones that are specific to all structural and non-structural HBV antigens (HBs, HBe/HBc, Pol, and X)
[22]. Without effective antiviral functions, the virus persists in the body, and chronic exposure to the viral proteins results in impaired activation of toll-like receptor pathways
[23] and lymphocyte dysfunction
[24] through mechanisms including clonal anergy, clonal ignorance, T-regulatory activity, and immune checkpoints
[25]. In contrast to patients with self-limiting acute HBV infection, CHB patients do not have strong, multiple, and sustained cytotoxic T lymphocyte responses
[26][27]. Enhanced and sustained expressions of different types of inhibitory markers on CD8
+T cells have been reported, which include programmed cell-death protein-1 (PD-1), T-cell immunoglobulin and mucin-domain-containing 3 (TIM-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and CD244
[28][29]. In CHB, B lymphocytes, albeit with static numbers in the peripheral blood, are functionally deficient and exhibit atypical phenotypes (CD21
- CD27
-) with enhanced PD-1 expression, resulting in impaired production of neutralizing antibodies
[30]. HBsAg also enhances regulatory T-cell [Treg (CD4
+CD25
+FoxP3
+)] and myeloid-derived suppressor cells (MDSC) activity
[31][32]. These mechanisms (i.e., chronic viral antigen exposure and the resultant numerical and/or functional depletion of cells in both innate and adaptive immune systems) have been proposed to contribute to the HBV chronicity, i.e., persistence of HBV beyond 6 months without development of polyclonal HBV-specific lymphocytes
[2]. It is generally accepted that HBsAg seroclearance is the result of regaining immunologic control of HBV from the immune exhaustion exerted by the huge numbers of viral proteins. Therefore, with the help of virus-directing agents to reduce the viral proteins, the host immunity can be restored and, thus, in turn the virus can be controlled. On the other hand, immunomodulatory agents directly suppress viral replication via enhancement of cellular effector functions (). In the following sections, only agents that are already in the clinical phases of development will be discussed. As mentioned above, a functional cure is the desirable endpoint and should be achieved in ≥30% of patients for drugs tested in phase 3 trials
[11]. For early-phase development, i.e., phase 1 and 2 trials, no recommendations or guidance have been proposed but are usually substituted by achieving a pre-defined HBsAg level or magnitude of HBsAg decline. This is likely to be related to the fact that HBsAg seroclearance is preceded by a low HBsAg level
[33][34], and only agents that demonstrate potent effect in suppression of HBsAg in early-phase trials will show potential to achieve the desirable endpoint for phase 3 trials. Therefore, HBsAg decline is the most frequently assessed parameter for assessing the efficacy of anti-HBV drugs in the trials discussed below. In addition to exploring whether novel agents are able to achieve a higher rate of functional cure, it would be highly appreciable if novel agents could also have a more long-term effect on cccDNA reduction.
3.2. Enhancement of Host Immunity
As mentioned above, chronic viral antigen exposure weakens HBV-specific immunity and leads to virus-specific T-cell anergy, leading to immune exhaustion in the host. Novel therapies aiming to restore or enhance the host immunity are being actively developed ().
Toll-like receptors (TLRs) are pattern-recognition receptors (PRRs) that upon activation would stimulate various leukocytes in both the innate and adaptive system. GS-9620 (Vesatolimod) is a TLR7 agonist. In the phase 2 double-blind placebo-controlled trial, GS-9620 induced ≥2-fold expression of the interferon-stimulated gene (ISG15) especially in female subjects. However, no significant serum interferon (IFN) alpha expression or HBsAg decline was demonstrated
[70]. GS-9688 (Selgantolimod) is a TLR8 agonist and achieved modest decline in HBsAg when given in combination with NA in the phase 2 study. GS-9688 induced dose-dependent cytokine responses (IL-12p40, IL-1RA, IFNγ) and shifts in peripheral immune cell subsets
[71].
HBV-specific T-cell function can be restored by autoantibodies that block the inhibitory molecules. Nivolumab is a monoclonal antibody against PD-1 that is approved for treatment of various malignancies. When given at a reduced dose in combination with GS-4774 (a therapeutic vaccine; see below), nivolumab led to a modest reduction in HBsAg level (0.16 to 0.3 log reduction) at 12 weeks, and one patient achieved HBsAg seroclearance that was preceded by ALT flare and an increase in peripheral HBsAg-specific T cells
[72].
IMC-I109V is a novel immunotherapy. It is a soluble bi-specific T-cell-engaging fusion protein comprised of a soluble affinity-enhanced T-cell receptor fused to a humanized anti-CD3 single chain variable fragment. As a type of immune-mobilizing monoclonal T-cell receptor against virus (ImmTAV), IMC-I109V TCR recognizes HBsAg presented by specific human leucocyte antigen (HLA)-A*02:01 on the surface of infected hepatocytes. Upon engagement of TCR with HBsAg, the effector domain will bind to CD3 on any surrounding T cell of a different family, which is redirected to the complex, thereby engaging the global T-cell population and compensating for the defective HBV-specific CD8 cells in CHB. In vitro study has confirmed that ImmTAV-Env (ImmTAV molecules that are specific for HLA-A*02:01-restricted HBV epitopes derived from the viral envelope proteins) can redirect T cells from healthy and HBV-infected donors toward HCC cells containing integrated DNA. The induced cytokine release was suppressible by corticosteroid, and the redirected T-cells induced cytolysis of HBV-infected cells and antigen-positive HCC cells, leading to reduction of HBeAg and loss of cells expressing viral RNA
[73]. IMC-I109V is currently in phase 1/2 clinical trial, involving mainly non-cirrhotic and virally suppressed HBeAg-negative CHB patients. The reported frequencies of HLA-A*02:01 are highly variable among different ethnicities. For instance, its prevalence is 11–20% in Asians and 23–60% in Caucasians
[74]. Subsequently, a HLA non-restrictive approach (HLA-E) has been developed and this would allow drug target engagement in all patients
[75]. It is worth noting that adverse reactions related to infusion of IMC-I109V such as hypersensitivity, anaphylaxis, and cytokine release syndrome have been reported in treatment with tebentafusp, another novel bispecific TCR-anti-CD3 directed against gp100 for patients with advanced melanoma, which might require immunosuppressive therapy (e.g., corticosteroids) for treatment
[76]. Given the excellent safety profile of current antiviral treatment with NAs, it is crucial to establish the safety of T-cell therapy in CHB patients, which means achieving a safe and effective level of cytolysis as induced by the redirected T cells, which is therapeutically adequate to achieve HBsAg seroclearance but not to the level of excessive hepatocyte death, causing hepatic decompensation
[77].
Therapeutic vaccines for CHB make use of different vaccination doses and frequencies to stimulate an anti-HBV immune response. Different viral components have been used, including DNA or peptide vaccines, vector or cell-based vaccines, and combinations of core, X, and polymerase antigens in addition to HBsAg. GS-4774, a yeast-based vaccine that consists of highly immunogenic recombinant HBcAg, HBsAg, and HBx epitopes, led to HBsAg decline ≥0.5 logs at week 24 in 3 out of 50 CHB patients who received it every 4 weeks, and no patient experienced a functional cure
[78]. ABX-203 (HeberNasvac), a yeast-based vaccine that comprises HBsAg and HBcAg virus-like particles, was studied in phase 1–3 clinical trials. It led to HBsAg seroclearance in two out of six CHB patients who had prior PEG-IFN treatment and were given ABX-203 intranasally every 2 weeks for up to 5 years
[79]. TG-1050, an adenovirus 5-based vaccine that expresses HBV polymerase and domains of core and HBsAg, was shown to induce specific IFNγ-producing T cells and led to minor reductions of HBsAg and significant reductions in HBcrAg in NA-treated CHB patients
[80].
GC1102 is a recombinant monoclonal hepatitis B immunoglobulin (HBIg) with enhanced affinity to HBsAg. HBsAg seroclearance was achieved in 22.2% CHB patients whose baseline HBsAg were ≤1000 IU/mL after 7 weeks of treatment with GC1102
[81]. VIR-3434 is another recombinant HBIg that is currently being evaluated in a phase 1 clinical trial. Initial data showed that six out of eight patients achieved a mean reduction of 1.3 logs of HBsAg by day 8
[82]. It is worth noting that both trials of recombinant monoclonal antibodies recruit CHB patients with baseline HBsAg ≤ 1000 IU/mL. Similar to NAPs and STOPS, it is expected that reduction in liver and circulating viral antigen load using neutralizing antibodies will restore adaptive immunity
[65]. Historically, HBIG derived from HBsAg-vaccinated subjects could lead to emergence and/or selection of immune escape HBV mutants, particularly in the ‘a’ determinant loop, that enables viral persistence despite antibody titers. Cases of fulminant hepatic failure resulting from immune escape mutants have been reported in liver allograft recipients
[83][84][85][86]. This again highlights the importance of safety during drug development. The potential risk of recombinant HBIG-induced escape mutations and subsequent hepatic failure can be minimized by selecting patients with low viral load, e.g., low HBsAg titers as in the GC1102 trial, the use of next generation monoclonal antibodies that display improved broadly neutralizing potential against different HBV strains and escape mutants
[87], and the concomitant use of another antiviral agent, e.g., NAs to reduce the immune selection pressure.
SB9200 (Inarigrivir) is a dual agonist of retinoic acid inducible gene-1 and nucleotide-binding oligomerization domain that consists of host PRRs that activate the innate immune pathway. SB9200-induced HBsAg decline ≥ 0.5 logs from baseline in 22% patients
[88]. However, the developing company prematurely terminated the phase 2b trial after the occurrence of unexpected serious adverse events, including one patient death in the Phase 2b CATALYST trial of SB9200 in January 2020.
4. Combination Strategies
With the efficacy and safety data of the individual novel agents mentioned above, the approach of combining two or more novel agents to reduce viral antigen load, inhibit viral replication, and stimulate immunity is being actively explored.
The triple combination of RNAi (monthly injections for three doses of JNJ-3989) + CpAM (daily oral doses of JNJ-6379 for 85 days) + NA (daily oral doses beyond the end of CpAM dosing) led to mean 1.7 logs reduction in HBsAg on day 113 in 12 CHB patients. In addition, other viral products including HBV DNA, HBV RNA, and HBcrAg were profoundly suppressed. This combination therapy was in general well-tolerated with no serious or severe adverse events reported. Mild ALT flares were observed in five patients and were attributed to therapeutic flares [89].
A few more combination therapies are currently being evaluated in a phase 2 study that evaluates the safety and efficacy of multiple combination therapies (ClinicalTrials.gov identifier: NCT04225715). One of them is a similar example as the above with triple combination with RNAi (RG6346) + CpAM (RO7049389) + NA. Other examples include: triple combination of RNAi (RG6346) + TLR7 agonist (RO7020531) + NA, triple combination with RNAi (RG6346) + PEG-IFN + NUC, and triple combination with CpAM (RO7049389) + TLR agonist (RO7020531) + NA. The approach of sequential use of therapeutic vaccines after antigen knockdown by RNAi has been explored in mice models that showed encouraging results in suppression of viral burden and stimulation in the number of functional HBV-specific T cells and production of HBV-neutralizing antibodies [90]. Clinical studies using this approach are awaited.
Some agents will require combination with NAs, especially CpAMs, NAPs, and most immune modulatory agents, in contrast to RNAi. This is likely to be related to whether the antiviral effect of the individual agent leads to silencing of cccDNA transcriptional activity, which is the case for RNAi via post-transcriptional knockdown of HBV transcripts. Theoretically, sequential combinational therapy will result in rapid decline in HBsAg (by virus-directing agents) which is beneficial for subsequent immune stimulation therapy [91]. In transgenic mice models, GaINac-conjugated siRNA followed by therapeutic vaccine showed significantly stronger immune stimulatory effect in terms of development of polyfunctional, HBV-specific CD8+ T cells compared to mice given control RNAs with NA, and therapeutic vaccine [90]. Although many RNAi-based combination therapies are still tested in clinical trials in combination with NAs, it remains to be unveiled whether the viral suppressive effects of NA can be substituted by other virus-directing agents.
5. Conclusions
The currently preferred treatment endpoint for CHB is functional cure. Novel agents work on different steps of viral replication or the host’s immune system, in order to reduce viral burden, inhibit viral replication, and restore host immunity. The therapeutic effects of both virus-directed and host-immunity-directed agents are intercalated and are equally crucial for achieving a functional cure. Most agents currently in clinical phases of development demonstrated favorable results in suppression of viral proteins and genomic materials, with initially promising results of enhancing the functional cure of CHB. With an increasing number of novel agents entering clinical phases of development, it is expected that each agent should demonstrate favorable safety data, and long-term follow-up data in the subjects that participated in these trials will be an important consideration. Most trials aimed to achieve broad eligibility by recruiting CHB patients with variable duration of infection and viral activity. However, it is likely that specific subgroups of patients will benefit most from selected agents so that a finite duration of therapy (partial cure or functional cure if HBsAg seroclearance can be achieved) instead of lifelong NA treatment can become feasible. For all trials mentioned, no cirrhotic patients were included and this will need to be addressed when more safety data are available. Combination therapy with two or more novel agents theoretically leads to synergistic therapeutic effects. NA and/or PEG-IFN are still the backbone of these combination regimens. The best cocktail of therapies is being sought, and different regimens are likely to be needed for different patient populations with various viral factors (e.g., HBsAg levels, previous treatment exposure), and host factors (e.g., HLA allele, gender).