HBV can be transmitted through infected bodily fluids such as blood and semen, which can be caused immune-mediated liver disease (7). HBV does not directly damage cells. The inflammation and necrosis of liver tissue are mainly due to the host’s recognition of invading antigens and the activation of its own immune system, which targets and destroys infected hepatocytes. Liver injury caused by excessive immune activation can further contribute to liver fibrotic disease and HCC during chronic HBV infection (18). HBV is highly effective in invading recognition by the innate immune system owing to its unique replication strategies, such as the use of capped and polyglandulated transcripts similar to host-derived mRNAs or the restriction of RNA/DNA genomes produced by replication to nucleocapsid particles in the cytoplasm (19). The HBV genotypes can be classified based on their genome sequences from A to J with many subtypes (20). The pathogenicity, virulence, clinical outcome, and response of HBV to type I IFN treatment are associated with its genotype. HBV DNA levels and hepatitis B E antigen (HBeAg) seroconversion rates were lower in patients infected with HBV genotypes C or D than those with HBV genotypes A or B (21). HBeAg seroconversion rate refers to patients who no longer express HBeAg and produce anti-HBeAg antibodies (22). HBV infection can be divided into the following four stages: immune tolerance, HBeAg positive immune active, HBeAg negative immune inactive (CHB loss or low replication), and HBeAg negative immune active (23). Serological markers should be discovered to determine the disease stage. General serum markers can diagnose CHB and help in distinguishing between acute and chronic infections. Common serological tests can detect HBV surface antigen (HBS), HBeAg, HBV surface antibody (anti-HBS), hepatitis B core antibody (anti-HBC), HBV envelope antibody (anti-HBE), and HBV DNA (24).

During the HBV life cycle, HBV DNA is transformed into a highly stable double-stranded circular DNA structure called cccDNA, which is an important stage in the nucleus of the liver cells. During this stage, cccDNA is integrated into the host genome as a template for viral RNA transcription, and cccDNA hides in the nuclei of the liver cell nuclei and serves as a template for viral replication (25) ( Figure 1 ). HBV infectious virions are enveloped nucleocapsids that selectively enter hepatocytes and deliver incomplete circular DNA genomes, which initiates multiple viral replication processes (26). The circulating virions are initially attached to heparan sulfate proteoglycans (HSPG) (27), then viral surface proteins facilitate their entry into host hepatocytes. The preS1 domain is a crucial structure for mediating large surface proteins (28). HBV can enter the hepatocytes, which is co-mediated by surface molecules called sodium taurocholate cotransport polypeptides (NTCPs) (29). After the entry of the virus, the HBV nucleocapsid containing relaxed circular DNA (rcDNA) is delivered into the nucleus, where host enzymes transform the viral genome into cccDNA (30). Human RNA polymerase II mediates cccDNA transcription to produce pregenomic RNA (pgRNA). PgRNAs are mRNAs of core proteins and polymerases that serve as templates for HBV DNA replication (31). PgRNA is reverse transcribed to form incomplete rcDNA, wherein the HBV capsid is coated with HBsAg to become mature virus particles (32). Capsid-containing rcDNAs are transported back to the nucleus to increase the cccDNA content or enter multivesicular bodies. They come into contact with viral envelope proteins and exit hepatocytes to circulate in the blood as infectious virions (33).

Establishing animal HBV infection models is important for elucidating the mechanism underlying the immune response to HBV infection, which leads to hepatitis and the progression of liver injury and repair. Establishing relevant animal models has facilitated the development of methods to control chronic HBV infection and the study of ISG regulatory pathways. Mice have good immune system characteristics and are easy to handle. However, they cannot naturally be infected with HBV. Therefore, many studies have established various HBV infection models in transgenic mice using gene editing and humanized liver technologies (34). Past studies have found that sterile alpha motif domain-containing 4A (SAMD4A) is an important anti-HBV ISG by overexpressing or knocking down ISGs in HBV transgenic mice (35). Besides, interferon alpha-inducible protein 27 (IFI27) as ISG can inhibit HBV gene expression and DNA replication in mouse models (36). Other studies have shown that the steady-state level of HBV DNA in ubiquitin specific peptidase 18 (USP18) (UBP43) deficient mice is significantly reduced (37). Moreover, some studies have used the human liver chimeric mouse model and shown that HBV/HDV infection significantly induced ISG expression (38). Chimpanzees are the only immunocompetent animals that are naturally susceptible to HBV, and they are the main animal model for studying HBV infection (39). However, their HBV-related studies were limited because of ethical issues. Other animals, such as woodchucks, are naturally infected with hepatitis viruses similar to HBV (40). Woodchucks can be infected with woodchucks hepatitis virus (WHV), and ducks can be infected with duck hepatitis virus (41). These viruses have characteristics similar to HBV infection in humans. Some studies have investigated the changes in ISG expression after HBV infection using custom woodchuck microarray platforms (42). Moreover, another HBV-like virus, woolly monkey HBV (WMHBV), can infect its natural host, woolly monkeys, and was investigated for antiviral therapies for HBV infection (43). Other smaller non-human primate models are also being developed, such as tupaias, cynomolgus monkeys, and rhesus monkeys. The development of these animal models is crucial for studying HBV infection (44) ( Table 1 ). HBV has a high species specificity. However, recent advances in transgenic mice, humanized mice, and strategies to make macaques more susceptible to HBV infection are gradually improving our ability to study HBV in a more suitable in vivo environment (47).

The prophylactic vaccine for HBV is adopted in all developed countries. It is a common and crucial measure for preventing and controlling HBV (48). However, this vaccine does not affect patients with prolonged infections. Currently, treatment for these patients is limited to immunomodulators, including many direct-acting antivirals (DAAs), known as third-generation nuclear analogs (NUCs), such as entecavir, tenofovir, and tenofovir alanine or regular and pegylated type I IFNs (7). Induction of IFN expression occurs in response to viral or bacterial infection. With the development of recombinant IFNs, IFNs have been increasingly applied in HBV treatment, and have become a more popular treatment option (49).

Systematic reviews and meta-analyses of the role of conventional IFN-α in patients with HBeAg-positive CHB have found that it can improve their biological, serological, and virological responses. Treatment with higher doses of IFN-α and a longer duration of continuous administration can have a better therapeutic effect; however, it can also lead to side effects and increased treatment costs (68). IFN-α is presently the first choice of antiviral therapy for children with CHB older than one year, whereas PEG-IFN-α-2a is the recommended treatment for children with CHB older than three years. The results showed that antiviral monotherapy with IFNα-2B or PEG-IFNα-2a was well tolerated and effective in CHB children compared with adults with higher HBeAg seroconversion rates and HBsAg clearance rates (69). Many studies have shown that standard IFN-α has a specific role in anti-HBV infection; however, pure IFN-α is not commonly given as a therapy in clinical trials (70). Some studies have shown that IFN-α treatment is ineffective in most patients with HBV infection possibly because HBV prevents the induction of IFN-α signaling and interferes with ISG transcription in hepatocytes by inhibiting STAT1 nuclear translocation, which results in a low IFN-α therapeutic effectiveness (71). Overall, its antiviral effects in patients with CHB are modest for unknown reasons but may include inadequate delivery to the infected liver, tolerance of infected hepatocytes to IFN-α signaling, or other mechanisms (72).

Clinical results showed that PEG-IFN-α-2b was effective in treating HBeAg-positive CHB (73). In addition, PEG-IFN-α monotherapy was effective in 298 Chinese inactive HBV carriers, with good tolerability and safety (74). Using PEG-IFN-α in treating HBeAg-positive patients with CHB could inhibit viral production to some extent in 10%–40% of patients, and the HBeAg serum conversion rate of patients was about 25%–30%. Loss of HBsAg expression was observed in approximately 5% of patients six months after treatment discontinuation (75). Treatment regimens with PEG-IFN-α should be determined based on host-related factors and viral predictive markers, such as age, alanine transaminase (ALT) levels, viral load, and HBV genotype (76). Moreover, hepatitis B core-related antigen and HBsAb levels at the end of treatment can help determine the curative effect of PEG-IFN-α-based treatment in patients with CHB (77).

Combining IFN-α and PEG-IFN-α with other drugs is currently an attractive approach. The co-administration of ribavirin and IFN-α may be effective in treating viremic anti-HBE-positive patients with CHB who have not responded well to previous IFN treatment (78). Another clinical trial showed that sequential combination therapy with lamivudine and IFN-α induced a sustained virological response, including HBS seroconversion, in patients with CHB who were unresponsive to IFN-α alone. This observation suggests that this treatment regimen needs to be further evaluated in clinical trials (79). NVR3-778 is one of the core protein allosteric modulators (CpAMs), which has been shown to reactivate the host innate immune response by inducing the expression of ISGs (80, 81). Clinical studies have shown that combining PEG-IFN-α and NVR3-778 exerts a good antiviral effect in vivo (82). In addition, combining entecavir or tenofovir with PEG-IFN-α can reduce HBsAg levels consistently (83). Additional treatment with PEG-IFN-α results in higher serological response rates than monotherapy and may facilitate NAs discontinuation (84).

Furthermore, current regimens that may be of more interest include combining IFNs with traditional Chinese medicine (TCM) (85). Many studies have reported that TCM and related active compounds extracted from TCM have a potential anti-HBV activity, including Salvia miltiorrhiza, Astragalus, Oxymatrine, Artemisinin, and Vogoning. TCM preparations have better safety than IFN-α regarding dose-dependent side effects and drug resistance and are potential candidates for anti-HBV therapies (86). TCM preparations combined with IFNs considerably decreased serum HBeAg, increased serum HBV DNA clearance rates, and improved serum ALT normalization compared with IFNs alone (87). Moreover, a polysaccharide from Radix isatidis (Isatis indigotica Fortune) can exert an antiviral effect by activating the IFN-α-dependent JAK/STAT signaling pathway and increasing anti-HBV protein levels (88). Despite many IFN-related clinical trials, stronger evidence and more detailed experiments are needed to evaluate the safety and efficacy of combination therapy. In addition, more studies are needed to develop more convenient and effective IFN-α-based HBV treatment strategies.

All type I IFNs, including IFN-α and IFN-β, are regulated by the IFN-α/β receptor (IFNAR) complex, which contains two subunits, IFNAR1 and IFNAR2 (91). However, type I IFN binding to IFNAR can induce ISG expression and activate the JAK/STAT signaling pathway (92). The heterotrimeric ISG factor 3 (ISGF3) transcription factor complex comprises phosphorylated STAT1/STAT2 and interferon regulatory factor 9, and type I IFNs can activate ISGF3 expression via the JAK/STAT signaling pathway (14). Activated ISGF3 binds to ISG upstream promoter regions in the nucleus in response to IFN stimulation ( Figure 2 ). Furthermore, studies have shown that the increased interaction between STAT1 methylation and STAT1- protein inhibitor of activated STAT-1 is involved in IFN-α HBV antagonism, and the antiviral effect of IFN-α can be enhanced by increasing the expression of methylated STAT1 and S-adenosyl methionine (93). In addition, the unbiased high-throughput RNA interference technology was used to screen cells that showed HBV inhibition after IFN-α treatment. Among 711 epigenetic modifiers, SET domain containing 2-mediated K525 STAT1 methylation is an important antiviral signaling mechanism (94). Activating the JAK signaling pathway further induces alternative signaling pathways such as mitogen-activated kinase-like protein, phosphatidylinositol 3-kinase, and nuclear factor Kappa-light chain enhancer of activated B cells (NF-κB), amplifying the strength and magnitude of type I IFN signaling (49). Though previous studies considered ISGs as IFN-induced protein-coding mRNAs, recent studies have shown that IFNs also mediate changes in the expression of many non-coding RNAs, including long non-coding RNAs and miRNAs (95).

The ISG gene pool is complex and large. Next-generation RNA sequencing studies have shown that IFNs regulate ~10% of all human genes. Moreover, studies comprehensively examining ISG expression in transcriptomes of different animals identified 62 core ISGs (96). Furthermore, several antiviral ISGs with critical roles have been discovered by identifying genes that are aberrantly expressed during viral infection inhibition. Among them, anti-myxovirus protein (MX)1 is the first classical effector molecule found to inhibit virus entry, primarily by preventing early-stage viral replication (97). In addition, interferon-induced transmembrane protein 3 (IFITM3) prevented the membrane fusion process of virus entry into cells via the endocytic pathway (98). Protein kinase R (PKR) and zinc antiviral protein are typical ISGs that inhibit viral protein production (99, 100). Therefore, different ISGs can block the HBV life cycle via corresponding pathways and play an essential role in regulating IFN-induced immune response and antiviral processes.

IFN-α is an antiviral drug with a limited treatment course. It acts on important biological processes including HBV replication and transcription by enhancing immune cell function, increasing cytokine levels, inducing ISG expression, and activating multi-antiviral proteins via the IFN signaling pathway, thereby playing a dual role in immune and antiviral regulation (10). Various ISGs exert anti-HBV effects in the host via different mechanisms ( Table 2 ). Host cells infected with viruses can immediately recognize their pathogen-associated molecular pattern, promoting the viability of B cells activated by transcription factors IFN regulator 3 or 7 and NF-κB. This process initiates the expression of the genes of type I IFNs and proinflammatory cytokines, inducing downstream ISGs to establish an antiviral host cell environment with antiviral effects (97).

IFN-α-induced ISG MX2 reduces HBV cccDNA expression by inhibiting viral RNA synthesis, an important anti-HBV function. MX2 represents a novel HBV inhibitor with therapeutic potential (101). APOBEC3G is an IFN-α-induced cytosine deaminase that deaminates cytosine to uracil in single-stranded DNA replication, inhibiting the coding and replication ability of HBV (110). In a study, cell-based assays were performed to screen 285 human ISGs to check their anti-HBV activity, finding SAMD4A to be an important anti-HBV ISG and a strong repressor of HBV replication. It can be used in IFN-HBV treatment. SAMD4A/B expression was associated with human HBV sensitivity (35). In addition, IFN-α-inducible protein 6 (IFI6) inhibited HBV replication in cell and mouse model by reducing the expression of the gene of HBV enhancer II and core promoter (EnhII/Cp); thus, increasing IFI6 expression may be a potential therapeutic approach for inhibiting HBV infection (102).

Another study showed that the SPRY domain of tripartite motif containing 14 (TRIM14) interacted with the C-terminus of the HBV X protein (HBx) and might block HBV replication by inhibiting the formation of the structural maintenance of chromosome protein (SMC)-HBx- DNA damage-binding protein 1 (DDB1) complex (103). Other studies have shown that the IFN-interleukin (IL)-27-TRIM25 signaling pathway is induced by type I IFNs and inhibits HBV replication, identifying the ISG TRIM25 as a potential therapeutic target for HBV infection (104). In addition, TRIM5γ and TRIM31 were identified as key genes interacting with HBx that promote its degradation among the 145 ISGs examined, identifying them as potential therapeutic strategies for IFN-resistant patients with HBV infection (111). ISG20 is a 3′-5′ exonuclease that binds and degrades HBV transcripts (105). ISG20 is primarily induced by IFN-β, reducing HBV gene expression and inhibiting HBV enhancer activity by binding to EnhII/Cp regions (112). Moreover, m6A reader protein YTH domain family 2 (YTHDF2) regulates ISG20 expression by selectively recognizing and processing N6-methyladenosine (m6A)-modified HBV transcripts for degradation (105).

Moreover, studies have shown that IFN-α treatment significantly decreases microRNA-122 (miR-122) expression in hepatocytes, targeting ISG 5′-nucleotidase, cytosolic III (NT5C3), an inhibitor of miR-122 expression, and potentially inhibiting IFN-α function in HBV treatment (106). Other studies have shown that hepatocyte-specific miR-122 expression positively correlates with adenosine deaminase acting on RNA gene (ADAR1) expression. Exogenous miR-122 reduces HBV RNA and DNA, and p53 is also involved in the ADAR1-mediated reduction of HBV RNA (113). In addition, studies have shown that IFN-α attenuates mitochondrial signaling protein (MAVS) by RNA editing, which is mediated by ADAR1 antiviral therapy. These results indicate that combining MAVS with IFN-α has potential clinical applications in the studies on HBV infection (107).

ISG stimulator of interferon response the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) interactor (STING) is an important DNA-mediated regulator regulating the natural immune response of the body and a potential therapeutic target in HBV infection (114). Studies have shown that activation of STING signaling pathway can effectively reduce the severity of liver injury in chronic HBV mouse models, which may be a promising approach to prevent HBV virus proliferation and HBV-related liver fibrosis (115, 116). Furthermore, IFN-α reduces HBV cccDNA content by regulating the general control non-repressed 5 protein-mediated succinylation of histone H3K79 in HBV-infected human liver-chimeric mice. Therefore, IFN-α can inhibit HBV transcription at the epigenetic level (117). Indoleamine 2, 3-dioxygenase (IDO) is an ISG that can effectively reduce intracellular HBV DNA levels and the main IFN-γ regulatory gene in hepatocytes to produce an anti-HBV response (108). Moreover, the downstream signaling pathway of IFN-λ was identified by a proteomic method. IFITM3, 5′-3′ exoribonuclease 2 (XRN2), and 5’-nucleotidase, cytosolic IIIA (NT5C3A) expression were upregulated, and ISG transcription was activated to inhibit HBV replication (118).

In addition, ISG expression as a predictor of clinical efficacy is also an attractive strategy. Single nucleotide polymorphisms (SNPs) in the 2′,5′-oligoadenylate synthetase gene (OAS) in patients play a major role in predicting the efficacy of IFN treatment against CHB (119). Additionally, SNPs in IL28B and OAS were correlated with the clinical efficacy of IFN therapy in children with CHB, suggesting that they might be a new important consideration in treating CHB with IFNs (120).

According to recent studies, ISGs may also be involved in the mechanism by which HBV antagonizes IFNs and inhibits IFN efficacy. Studies have shown that IFN-α treatment activates STAT1 nuclear translocation and ISG expression. Therefore, HBV inhibits STAT1 nuclear translocation and interferes with ISG transcription in hepatocytes, blocking IFN-α signaling and causing a poor treatment response (71). In addition, HBV has molecular mechanisms that promote resistance to IFN therapy. HBV infection increases HBV polymerase levels and inhibits ISG induction, resulting in the poor antiviral efficacy of IFN-α in HBV mouse model (121). In addition, HBV precore protein P22 can reduce ISG expression and IFN-stimulated response element activity and inhibit IFN-α signaling by blocking the JAK/STAT signaling pathway and STAT nuclear translocation (122). Spliceosome-associated factor 1 can reduce the antiviral activity of IFN-α by attenuating JAK/STAT signaling and reducing the expression of ISGs such as MX, OAS, and PKR in HepG2 cells (123).

Moreover, IL-6 expression impaired the efficiency of IFN-α-mediated HBV suppression in hepatocytes by upregulating the suppressor of the cytokine signaling 3 genes (SOCS3). Therefore, SOCS3 downregulation can improve the antiviral activity of IFNs in HBV-replicating hepatocytes to a certain extent, representing a novel therapeutic strategy that may effectively target HBV infection (124). Other studies have shown that HBV can promote miR-146a transcription, inhibiting STAT1 and leading to IFN resistance. Therefore, this mechanism represents a promising research target for recovering the effects of IFN-α in HBV treatment (125). Moreover, the homologous to the E6-AP carboxyl terminus and RLD domain containing E3 ubiquitin-protein ligase 5-mediated modification of HBx by ISG15 increased HBV replication, resulting in HBV resistance to IFN-α therapy (126). Understanding the interaction between HBV and ISGs and ISG regulation by HBV to produce IFN antagonism will be helpful for further anti-HBV research ( Figure 3 ).