Hepatocyte-intrinsic innate immunity in hepatitis B virus infection: A focused review

Abstract

Chronic hepatitis B virus (HBV) infection remains a major health burden worldwide. To establish a persistence infection, HBV needs to evade both adaptive and innate immune surveillance. Multiple mechanisms for adaptive immunity evasion have been established, but how HBV evades the innate surveillance is less clear. There are three types of host cells involving in the innate immune responses against HBV infection: Hepatocytes, hepatic nonparenchymal cells and conventional innate immune cells. Among these, hepatocytes are the only target cells that are susceptible to HBV infection and the only confirmed site where HBV replication takes place. This review focuses on the hepatocyte-intrinsic innate immunity; one of the earliest host defense responses. After entering hepatocytes, the viral components can be sensed by the cellular pattern recognition receptors. This triggers downstream antiviral responses capable of inhibiting viral replication and even degrading the viral DNA genome directly or indirectly. However, HBV has evolved a variety of sophisticated strategies to evade intracellular immune defense, resulting in the establishment of infection. Here, we provide insights into the mechanisms of the intrinsic innate immune response of hepatocytes and how HBV escapes these defense mechanisms. Hopefully, this will lay the foundation for the development of novel anti-HBV therapies.

Key Words: Hepatitis B virus; Innate immunity; Immune evasion; Pathogen recognition receptors; Pathogen-associated molecular patterns

Core Tip: Despite the advancements in hepatitis B virus (HBV) immunology, the mechanism by which hepatocytes exert their innate immune responses against HBV infection remains poorly understood. This article focused on the complex interplay between hepatocytes and the innate immune response during HBV infection and summarized the recent findings about how HBV evading the innate response to promoter viral persistence. These insights may be helpful for developing novel anti-HBV immune-based therapies.

INTRODUCTION

Although there have been effective prophylactic vaccines for tens of years, chronic hepatitis B (CHB) virus (HBV) infection remains a major public health problem[1]. The World Health Organization estimated that, in 2022, about 296 million people had CHB worldwide, and 1.1 million people died of hepatitis-B-related complications, especially liver cirrhosis and hepatocellular carcinoma[2].

HBV is a small, enveloped DNA virus with unique replication mechanisms characterized by a protein-primed reverse transcription process that relates to but differs from retroviral replication[3,4]. Upon entering the host hepatocytes, the virus releases its partially double-strand, relaxed circular DNA (rcDNA) genome into the nucleus, where the rcDNA is converted to covalently closed circular DNA (cccDNA), which acts as the transcription template for all the viral RNAs, including the pregenomic RNA (pgRNA) and subgenomic mRNAs encoding seven viral antigens: DNA polymerase (P protein); core protein (HBc); precore protein (or HBe); X protein (HBx); and three coterminal surface proteins (large, middle and small HBs). The viral replication begins with the encapsidation of pgRNA into the progeny capsid, where the pgRNA is reverse transcribed by the co-packaged P protein to new rcDNA genomes[3,5]. The majority of newly formed rcDNA-containing-capsids are enveloped and released from the cell as progeny virions, but some mature capsids can be recycled back to the nucleus, where their rcDNA is released and reconverted to cccDNAs to replenish the cccDNA pool[6-9].

It is believed that adaptive immunity is largely responsible for HBV clearance upon infection[10]. The robust HBV-specific cytotoxic CD8⁺ T cells response directly contributes to the spontaneous resolution of infection[11]; while CD4⁺ T cells facilitate the activation, proliferation and maintenance of CD8⁺ T cells[12]. In addition, the neutralizing antibody response can prevent HBV from entering hepatocytes. In most acutely infected people, the cellular and humoral immune responses can clear the infectious virions from the body and induce life-long immunity[13,14]. Unfortunately, for a minority of infected people, the virus persists; probably due to the weak or impaired immune responses[15]. Multiple mechanisms for adaptive immunity evasion have been established. For example, high viral load and long-term exposure to excessive circulating HBs and HBe leads to T cell exhaustion in CHB patients[16].

However, if and how HBV induces an innate immune response remains controversial. HBV has been designated as a stealth virus that does not activate innate antiviral responses[17]. Nevertheless, the current prevailing view is that the innate immune system can sense HBV infection but the virus has evolved efficient strategies to antagonize the antiviral activity exerted by innate immunity[18-21]. Accumulating evidence supports that the innate immune system is also important to control HBV infection, by which it limits viral entry, replication and packaging, and instructs the adaptive immune system[22]. For instance, HBV replication in humans is generally undetectable until about 1 month after infection, suggesting cell-intrinsic innate immunity may inhibit early virus replication[23,24].

The dynamic interaction between virus and host immune defenses determines the pathogenesis of HBV[25]. While viral clearance is association with T-cell-mediated cytolytic elimination of infected hepatocytes, emerging evidence underscores the critical role of noncytolytic control mechanisms driven by innate immune components. Pattern recognition receptors (PRRs) detect HBV-associated molecular patterns, triggering antiviral cytokine cascades (e.g., type I/III interferons; IFNs) and priming adaptive immunity[26]. These responses suppress viral replication without widespread hepatocyte destruction. However, HBV uses immune evasion procedures, such as inhibiting PRR signaling and subverting IFN-stimulated gene (ISG) expression, which contribute to viral persistence and progression to chronic hepatitis.

Harnessing the host’s intrinsic antiviral defenses–through targeted immunomodulation (e.g., PRR agonists and cytokine therapies) or broad-spectrum strategies (e.g., checkpoint inhibitors) – holds promise for achieving durable, sterilizing cure by restoring immune control over residual cccDNA reservoirs[27]. A deeper understanding of innate immune dysregulation in chronic HBV infection, particularly the interplay between viral evasion and host defense signaling, is essential for translating mechanistic insights into curative regimens[28].

There are three types of host cells involved in anti-HBV innate immunity: Hepatocytes; hepatic nonparenchymal cells, such as liver sinusoidal endothelial cells and Kupffer cells; and conventional innate immune cells, such as dendritic cells and natural killer cells[29]. Hepatocytes, which are the parenchyma cells in the liver, are the target of HBV infection and the hepatocyte-intrinsic innate immunity provides the first line of defense against viral invasion[19]. This review focuses on summarizing how HBV triggers and escapes the hepatocyte-intrinsic innate immune response.

SENSING HBV BY PPRs EXPRESSED IN HEPATOCYTES

During infection, the innate immune system senses the pathogen-associated molecular patterns (PAMPs) of viruses via diverse host PRRs, including toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and DNA receptors like cyclic GMP-AMP synthase (cGAS) and the absent in melanoma 2-like receptors (ALRs)[26]. The PRRs recruit their distinct adaptor molecules, triggering the downstream antiviral signaling cascade, inducing IFNs and other inflammatory cytokines.

The human TLRs, comprising 10 members, localize either on the cell surface or in intracellular vesicles[29]. TLR2 locates to the cellular membrane and can recognize a wide range of PAMPs, including viral glycoproteins and bacteria cell wall components such as lipoteichoic acid, peptidoglycan and porins[30]. It was demonstrated in an in vitro study that primary human hepatocytes (PHHs) can sense HBV particles via TLR2, leading to the activation of anti-HBV innate immune response[28]. Further evidence has suggested that the TLR2 signaling pathway is activated by HBc; the glycoprotein at the center of HBV particles[31]. TLR3 is located on endosomal membranes and is responsible for recognition of viral double-stranded RNA[32]. Based on the results from PH5CH8 cell line, Li et al[33] concluded that poly (I-C), a double-strand RNA analog, can activate TLR3-dependent antiviral signaling pathways in hepatocytes. TLR3 Ligands inhibit HBV replication via activation of innate immunity in PHHs[34].

RLRs, including RIG-I and melanoma differentiation-associated protein 5 (MDA-5), are the major innate sensors for viral RNAs within cytosol[35]. Several studies have suggested that different RLR members recognize the viral RNAs derived from different HBV genotypes[20]. Sato et al[36] found that PHHs can sense HBV genotype A, B and C infection via RIG-I-mediated recognition of the 5’-epsilon (ε) stem-loop structure in HBV pgRNA, leading to the production of type III IFNs; while Lu et al[37] reported that HBV genotype D is sensed by MDA-5, but not RIG-I, based on the results from hepatoma cell line Huh-7 transfected with a HBV replicon plasmid.

NLRs, another class of intracellular sensors for PAMPs, comprise 22 members in humans; among which, NLR family pyrin domain-containing 3 (NLRP3), located in hepatocytes and other cell types, is the most well-studied inflammasome sensors[38]. Xie et al[39] reported that HBx can activate the NLRP3 inflammasome in human hepatocyte cell line HL7702 under oxidative stress. Based on the analysis of liver biopsies from patients with CHB, NLRP3 inflammasomes are activated in the liver[40]. HBx plays key roles in NLRP3-associated liver inflammation by inducing the release of reactive oxygen species[40-42].

As a typical DNA virus, at least two types of HBV dsDNA (namely rcDNA and cccDNA) may be recognized by intracellular DNA sensors[19]. Several in vitro studies have illustrated that these HBV dsDNA, either as the whole genomes or DNA fragments, can activate anti-HBV innate immunity via the cGAS-STING pathway[43-46]. Recently, IFI16, a member of the ALRs, has been identified as a unique innate sensor in hepatocyte nuclei for HBV cccDNA[47]. Binding of IFI16 to cccDNA not only leads to activation of antiviral innate immune response but also performs direct epigenetic inhibition of cccDNA transcription by targeting an IFN-stimulated response element present in cccDNA[47].

EVASION OF HEPATOCYTE-INTRINSIC INNATE IMMUNITY

Passive avoidance strategies

In sharp contrast to the strong IFN response to hepatitis C virus infection, the lack of detectable hepatic expression of immune-related genes, especially IFN genes and ISGs, in chimpanzees with acute HBV infection leads to the hypothesis that HBV may passively avoid innate immunity[17,48]. After entering host hepatocytes, the viral rcDNA genome, rather than exposed to intracellular DNA sensor, is encapsulated within the capsid, which protects rcDNA from innate recognition[21]. Next, the nucleocapsid-encapsulated rcDNA is transported to the cell nuclei and converted to cccDNA[49]. In hepatocyte nuclei, the cccDNA that serves as the viral original replication template is organized as a minichromosome and mimics the host chromosomes structurally. This appears to be a key strategy to avoid the sensing of cccDNA[21]. Viral RNAs are major targets for innate sensor recognition, but all the subgenomic HBV RNAs transcribed from cccDNA are similar to host mRNAs by their 5’-cap and 3’-poly (A) tail, thus they are ignored by RNA sensors[21]. Among all the viral RNA transcripts, only pgRNA contains an unusual ε stem-loop structure that is prone to recognition[21,50]. However, the pgRNA is rapidly encapsidated by HBc, sequestering it from cytosolic PRRs.

Active escape strategies

HBV may actively suppress the hepatocyte-intrinsic innate immune response by multiple mechanisms, including downregulating PPRs and their adaptor proteins, interfering with inflammatory signaling pathways, as well as altering epigenetic modification[51,52] (Figure 1). During chronic infection, HBV suppresses expression of innate sensors, leading to an impaired innate response and IFN production[53]. Analysis of liver biopsies from a large cohort of CHB patients has shown that many innate immunity-related genes, including ISGs that encode TLRs/adaptor proteins, type I/III IFN and other proinflammatory chemokines/cytokines are significantly downregulated[54]. By comparing the cytokine expression profiles among Huh-7, HepG2 and HepG2.2.15 cells, detectable interleukin (IL)-10 expression was found only in HepG2.2.15 cells, suggesting that HBV can induce hepatocytes to secrete anti-inflammatory cytokines (e.g., IL-10) and create an immunoinhibitory microenvironment[55]. In a more recent study, HBV was shown to actively evade innate immunity by suppressing the expression of cGAS and its effector genes in PHHs, NTCP-overexpressing hepatoma cell line (HepG2-NTCP) and humanized liver chimeric mice. To counteract the RIG-I-mediated innate defense induced by viral pgRNA, HBV can induce Parkin-dependent recruitment of the linear ubiquitin assembly complex to disrupt mitochondrial antiviral signaling protein (MAVS; the cytoplasmic adaptor protein for RLRs) signalosome and attenuate IRF3 activation, leading to inhibition of IFN-β synthesis[56]. Using an HBV-expressing cell model (HepG2.2.15), Hou et al[57] reported that HBV infection can suppress the IFN-induced antiviral response by disrupting the Janus kinase–signal transducer and activator of transcription 1 signaling pathway via upregulation of miR-146a transcription in hepatocytes. Zhou et al[58] demonstrated that HBV can suppress RLR signaling via activation of glycolysis. Specifically, HBV-induced lactate directly binds to MAVS and prevents its aggregation and mitochondrial localization, consequently sequestering MAVS from RIG-I.

Figure 1 Overview of the anti-hepatitis B virus hepatic innate immunity in hepatocytes. The viral components of hepatitis B virus (HBV) can be recognized by diverse pattern recognition receptors in hepatocytes, for example: HBV core protein in nucleocapsid can be recognized by Toll-like receptor (TLR)-2 Located on cell membranes (a); the epsilon stem-loop structure in HBV pregenomic RNA can be recognized either by RIG-I-like receptors (RLRs); RIG-I and MDA5 in cytosol or by TLR3 Located on endosome membrane (b); the HBV double-strand DNAs can be recognized by DNA sensors like cGAS or IFI16 (c). The recognition triggers the downstream antiviral innate response. HBV have evolved efficient strategies to counteract the innate response, including: (1) Downregulation of TLRs, damping the recognition of HBV infection; (2) Induce the production of IL-10 that can impair natural killer and T cell function; (3) Epigenetic downregulation of interferons (IFNs) and IFN-stimulating genes expression by recruiting the histone-methyltransferase EZH2 onto targeted gene promoters; (4) HBe binds to TRAM and Mal to disrupt the TLR2 signaling pathway; (5) The interaction of viral antigens (HBx, HBs and HBe) with mitochondrial antiviral signaling protein disrupts RLRs signaling pathway; (6) HBV P protein binds to DEAD-box RNA helicase 3 and block its interaction with TANK-binding kinase 1/IκB kinase epsilon, subsequently inhibiting IFN-β production; (7) Inactive STING with viral antigens (e.g. HBV P protein) to disrupt DNA sensors pathway; and (8) HBx relieves the histone methyltransferase SET domain bifurcated histone lysine methyltransferase 1-associated transcriptional silencing of covalently closed circular DNA, thereby promoting viral replication.

HBV proteins, including P protein, HBc, HBe, HBs and HBx, may contribute to inhibiting innate immune response by diverse mechanisms[18,19,59]. Lei et al[60] reported that IFN-α resistance is association with lower levels of IL-1β in CHB patients, while in vitro experiments indicated that HBV P protein is responsible for suppression of inflammasome activation and IL-1β production. To counteract the TLRs/RLRs pathways sensing viral dsRNA, HBV P protein can directly bind to DEAD-box RNA helicase 3 (DDX3) and block its interaction with TANK-binding kinase 1 (TBK1)/IκB kinase epsilon (IKKε) complex, subsequently inhibiting IFN regulatory factor 3 activation and IFN-β production[61,62]. P protein can suppress nuclear factor (NF)-κB signaling by inhibiting activity of IKKs via interaction with heat shock protein 90β[63,64]. Besides interfering with RNA-sensing pathway, HBV P protein can block the innate DNA-sensing pathways, which results in suppression of IFN-β synthesis by direct interaction with the stimulator of interferon genes (STING) and subsequent disruption of its K63-linked ubiquitination[65]. HBc was shown to inhibit the expression of IFN-induced GTP-binding protein MxA at transcriptional level by directly interacting with the promoter region of MxA, thus contributing to impairment of the antiviral effect exerted by IFN[66]. At an early time after infection, HBc from incoming HBV can quickly recruit the histone-methyltransferase EZH2 onto targeted gene promoters, resulting in the epigenetic downregulation of IFNs and ISG expression in hepatocytes[18,67].

HBe and HBs are the two secreted viral proteins involved in multiple immune evasion processes[18]. They have been reported to inhibit IFN-β synthesis by suppressing MAVS expression and interrupting the binding of MAVS to RIG-I[53]. They are able to antagonize the activation of TLR pathways in murine hepatocytes and hepatic nonparenchymal cells[18,68]. Mechanistically, HBe, but not HBc, can interact with the specific Toll/IL-1 receptor (TIR)-containing proteins TRAM and Mal, thus disrupting the homotypic TIR: TIR interaction that is critical for TLR-mediated signaling, finally suppressing the TLR- and TIR-induced activation of NF-κB and IFN-β[69]. Using hepatocyte cell models infected with HBV and liver samples from CHB patients, Wang et al[70] revealed that HBe can suppress NF-κB signaling by interact with NF-κB essential modulator (NEMO). Because NEMO is a regulatory subunit for IKK that controls NF-κB signaling, the binding of HBe to NEMO inhibits its TRAF6-dependent, K63-linked ubiquitination, and thereby downregulates NF-κB activity. HBs can interfere with NF-κB pathway by interacting with the TAK1–TAB2 complex[71]. Specifically, when bound to TAK1 and TAB2, HBs suppresses ubiquitination of the TAK1–TAB2 complex and blocks TAK1 phosphorylation, thereby inhibiting the NF-κB signaling pathway.

As a multifunctional viral regulator of cellular signal transduction and transcription pathways, HBx can negatively regulate the innate response through diverse strategies. To abolish extracellular IFN-α-mediated signal transduction, HBx can downregulate IFN-α receptor expression in hepatocytes[72]. To antagonize the intracellular RNA-sensing pathway, HBx can interfere with RIG-I/MAVS signaling by ubiquitination of MAVS[73,74]. Surprisingly, HBx can also function as a deubiquitinating enzyme to cleave K63-linked polyubiquitin chains from many critical proteins involving in the signaling cascades of IFN-I induction, including IRF3, IRF7, RIG-I, RIG-I 2CARD, TRAF3 and IKKi, thus blocking subsequent antiviral signal transduction[75]. In addition, HBx suppresses the transcription of TRIM22 (a mediator of IFN-induced antiviral response) by inducing a single CpG (+71) methylation in its 5′-UTR, demonstrating an epigenetic regulation-related mechanism of IFN resistance[76]. HBx can also act as an epigenetic modulator for the chromatin structure of HBV cccDNA minichromosome. For example, it can relieve the transcriptional silencing of cccDNA mediated by SET domain bifurcated histone lysine methyltransferase 1, a major histone methyltransferase responsible for methylation of H3 on lysine 9, consequently promoting viral replication[52,77,78].

Recently, the N6 methyladenosine (m⁶A) epitranscriptomic modification of viral pgRNA and host PTEN (phosphatase and tensin homolog; a nIRF-3 nuclear translocation) mRNA was identified a novel strategy to antagonize the innate response[79,80]. As discussed earlier, the 5’-ε stem-loop structure of HBV pgRNA is a target for RIG-I recognition. However, the m⁶A modification on 5’-ε of pgRNA recruits the m⁶A reader proteins YTHDF2 and YTHDF3, thus sequestering HBV pgRNA from cytosolic RIG-I[80]. As for PTEN mRNA, its m⁶A modifications induced by HBV infection destabilize the transcript with a corresponding decrease in PTEN protein levels, leading to inhibition of IRF-3 nuclear import and subsequent IFN synthesis[79]. Specifically, HBx was identified as a key modulator for regulating the HBV-induced m⁶A modification of pgRNA and PTEN mRNA. During HBV infection, HBx recruits the m6A methyltransferases (METTL3 and 14) onto HBV cccDNA and PTEN chromosomal loci, and achieves cotranscriptional m⁶A modification of their corresponding RNA transcripts; namely, HBV pgRNA and PTEN mRNA, respectively[80,81].

NOVEL ANTI-HBV AGENTS TARGETING INNATE IMMUNE COMPONENTS

IFN-α, the only approved anti-HBV immunotherapeutic, can occasionally result in functional cure of CHB in some patients, but it suffers severe systemic side effects as well as poor response rate[82]. This has necessitated development of novel anti-HBV immunotherapies with minimal side effects and increased therapeutic effects. The latest clinical progress in anti-HBV immunotherapeutic referring to innate immunity is summarized (Table 1).

Table 1 Current anti-hepatitis B virus candidate therapeutics based on innate immunity.

Drug name	Target	Sponsor	Phase	Ref.
Vesatolimod	TLR7 agonist	Gilead	II	[87]
Ruzotolimod	TLR7 agonist	Roche	II	[88]
JNJ-64794964 (JNJ-4964/AL-034/TQ-A3334)	TLR7 agonist	Chia Tai Tianqing/Janssen	II	[95]
Selgantolimod	TLR8 agonist	Gilead	II	[89,90]
Cavrotolimod	TLR9 agonist	Bluejay	I	[96]
Inarigivir	RIG-I agonist	Spring bank/Gilead	II (terminated)	[91]
Recombinant IL2	IL2	University of Science and Technology of China	Investigator-Initiated Trial	[85]

TLR: Toll-like receptor; RIG-I: Retinoic acid-inducible gene I; IL2: Interleukin-2.

Apart from IFNs, the efficacy and safety of several other cytokines, including IL-12 and IL-2, have been evaluated in CHB patients. As early as 2000–2001, two phase I/II clinical studies have demonstrated that treatment of CHB with IL-12 is safe and tolerable, and appears to be active against HBV infection in terms of leading to HBV DNA and HBeAg clearance in some patients, but it had no advantage in comparison to currently available treatments[83,84]. A recent clinical study reported that the sequential administration of recombinant IL-2 is effective in nonresponder patients after IFN-α therapy by restoring HBV-specific CD8⁺ T-cell responses, suggesting the CD8 cis-targeted sequential IL-2 therapy may be a promising treatment for refractory CHB[85,86].

TLR and RIG-I agonists represent a novel promising immunotherapeutic strategy against chronic HBV infection. TLR agonists such as vesatolimod (formerly GS-9620; TLR7 agonist), ruzotolimod (formerly RO7020531 or RG-7854; TLR7 agonist) and selgantolimod (formerly GS-9688; TLR8 agonist) are well tolerated and can lead to serological changes associated with progression to durable functional cure in a small number of CHB patients[87-90]. The RIG-I agonist inarigivir (formerly SB9200) stimulates prolonged IFN-α/β secretion and broad ISGs activation, with clinical data suggesting superior activity for reduction of HBsAg compared to nucleos(t)ide analogs such as entecavir[91]. However, the clinical development of inarigivir was terminated due to safety concerns[92].

FUTURE PERSPECTIVES

Research into the immune escape mechanisms of HBV has potential for developing therapies that disrupt viral persistence. Future research should prioritize targeting the stealth strategies of HBV. Novel PRR agonists or inhibitors of HBV-encoded immune antagonists (e.g., HBx or HBs) could restore innate sensing and amplify antiviral responses.

A deeper understanding of host–virus coevolution is also essential. The ability of HBV to epigenetically silence cccDNA and mimic host chromatin modifications highlights the need for therapies that reverse viral epigenetic camouflage, such as histone deacetylase inhibitors or other transcriptional activators to expose viral DNA to immune detection. The unraveling of heterogeneity in innate immune evasion across infected hepatocytes, by using cutting-edge technologies such as single-cell multiomics, will help to guide personalized therapies.

CONCLUSION

It is well established that the activation of PRR signaling and promotion of IFN synthesis play important roles in the control of HBV infection. However, to maintain persistent infection, HBV has evolved multiple sophisticated mechanisms to evade the antiviral innate immune response. The comprehensive understanding of the interactions between HBV and innate immune system is essential for developing curative treatment strategies against HBV. Novel immunotherapeutics such as TLR/RIG-I agonists have demonstrated impressive antiviral efficacy in clinical trials, but the challenges such as immune hyperactivation risks and inefficient HBsAg loss highlight the need for combination therapies to achieve sustained functional cure. For example, a recent clinical trial has reported that the combination of TLR7 agonist (ruzotolimod) or pegylated IFNα with salnesiran, an siRNA targeting the conserved region of S gene in HBV genome, can achieve high rates of HBsAg seroconversion in large cohorts of CHB patients[93-96].

The limitations in the field include the lack of physiologically relevant models that recreate the immune evasion of HBV in human hepatocytes within a functional immune microenvironment. Current in vitro systems (e.g., NTCP-reconstituted HepG2 cells) poorly model innate signaling complexity, while animal models (e.g., HBV-infected humanized mice) fail to mirror the immune exhaustion of chronic infection. With the construction of reliable experimental models, the interactions between HBV and host innate immunity will be better understood, thereby providing novel insights into anti-HBV therapies.

ACKNOWLEDGEMENTS

We would like to thank Dr. Guochuang Chen for helpful and constructive comments during the preparation of the Figures and Tables.