Possibility of PD-1/PD-L1 Inhibitors for the Treatment of Patients with Chronic Hepatitis B Infection

Hepatitis B is a viral infection that affects around 292 million people worldwide. It can lead to serious complications such as cirrhosis and liver cancer [1]. After HBV infects host hepatocytes, it forms covalently closed circular DNA (cccDNA) in the infected hepatocytes, which serves as a template for its replication and is highly stable in the nucleus, resulting in chronic HBV infection [2, 3]. Thus, most experts now agree that absolute sterilizing therapy that eliminates all forms of HBV replication intermediates, including cccDNA, is very difficult. Functional cure of hepatitis B, also called clinical cure, is defined as undetectable hepatitis B surface antigen (HBsAg) and HBV-DNA after 6 months off therapy [4]. In this case, indicators of liver function are normal, but cccDNA is still present in hepatocytes. Importantly, HBV can still be activated if the body’s immunity is weakened [5]. HBsAg seroclearance has been shown to be associated with reduced risk of cirrhosis and even hepatocellular carcinoma (HCC) [6, 7]. Functional cure is likely to be the goal of the next wave of new HBV therapies.

Currently, available treatment for chronic hepatitis B (CHB) includes nucleos(t)ide analogs (NAs) and pegylated-interferon-alpha [8]. The site of NAs action is in the reverse transcription and positive and negative strand DNA synthesis stage. The mechanism of action of pegylated-interferon-alpha includes inhibition of virus replication in infected cells, inhibition of cell proliferation, and immunomodulatory effects. However, none of these mechanisms enables cccDNA clearance, and the antiviral course is long and prone to relapse after discontinuation [9]. Overall, NAs therapy does not usually achieve HBV eradication and rarely results in HBsAg loss [8, 10, 11]. The addition of one or a few new drugs to existing therapies might significantly improve response to therapy and functional cure rates [9]. This will reduce risk of antiviral drug resistance and the incidence of cirrhosis and HCC. To achieve durable clearance of circulating HBsAg and HBV DNA after treatment, functional cure targets therapeutic strategies for high viral and antigenic loads as well as inadequate host immune responses [12].

Exhausted T-cell (TEX) populations typically arise in CHB, and the interaction of programmed death-1 (PD-1) with its ligands limits T-cell activation and inhibits downstream antiviral signaling pathways through the T-cell receptor. HBV chronic infection leads to impaired surveillance of the immune system, which changes the liver immune microenvironment [13], and the emergence of immune tolerance against HBV occurs, which promotes the development of HCC. Blockade of PD-1 and its ligands by checkpoint inhibitors may help rejuvenate exhausted HBV-specific CD8⁺ T cells to their original immunosurveillance capacity, exert antiviral effects, and reverse the state of immune tolerance [14]. Therefore, the potential use of ICIs for enabling improved immune surveillance by enhancing/remodeling HBV-specific CD8⁺ T-cell responses might qualify as a component of a future antiviral functional therapy for achieving viral clearance.

Immunotherapy with ICIs has been increasingly used for the treatment of various cancers with rapid expansion of treatment indications, with objective response rates of 15–20% and good safety profiles [15]. Immunotherapy has become a double-edged sword in CHB patients – it may lead to hepatic adverse events or hepatitis reactivation, and it may also lead to a functional cure. A handful of case reports have demonstrated that HBV reactivation may occur in CHB patients or even in patients with resolved HBV infection during immunotherapy. However, the exact incidence rate and risk factors for HBV reactivation remain uncertain. This important clinical question should be addressed in a large cohort of patients in regions with a high prevalence of HBV infection.

HBV chronic infection is defined as persistence of HBsAg for 6 months or more after acute infection with HBV. After HBV infection, due to a small amount of HBV genome, cccDNA and integrated virus DNA may persist in the nucleus of hepatocytes, which will lead to persistence of HBV [16, 17]. A robust immune response is important to clear infections. HBV-specific CD8⁺ T cells either directly target infected cells for elimination via cytopathic mechanisms or suppress viral replication via noncytopathic cytokine (predominantly interferons) mediated pathways [18]. There is consensus that virus-specific CD8⁺ T cells most likely play a prominent role because they are able to clear the virus by noncytolytic and cytolytic effector functions as soon as viral antigens are presented by infected hepatocytes via major histocompatibility complex class I [19, 20]. Specific CD8⁺ T cells produced after acute viral infection are highly functional. In contrast, the T-cell response is relatively weak in patients with CHB infection. Effector CD8⁺ T cells are produced in the early stages of infection and gradually lose their function during chronic viral infection, a phenomenon known as exhaustion. The chronic lymphocytic choroid plexus meningitis virus infection mouse model described T-cell exhaustion for the first time [21]. It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Related studies have shown that in peripheral blood and liver tissue, chronic viral infection leads to enhanced expression of specific CD8⁺ T-cell immune checkpoint molecules such as PD-1, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and most notably PD-1 [22, 23]. It is also noted that the ligand for PD-1, PD-L1, is constitutively expressed on liver sinusoidal endothelial cells, Kupffer cells, and stellate cells [24, 25]. Thus, HBV-specific CD8⁺ T cells obtained from the liver show a higher degree of exhaustion than peripheral CD8⁺ T cells. Due to persistent antigen exposure, TEX loses robust effector functions, expresses multiple inhibitory receptors and is ultimately removed by apoptosis. Following an acutely resolved antigen encounter, PD-1 expression eventually declines. If these T cells are instead exposed to a persisting antigen for 2–4 weeks, T-cell exhaustion becomes established, and these cells do not recover normal memory differentiation simply by the removal of antigen exposure [26, 27]. Although this phenomenon prevents overreaction of the immune system leading to a short period of massive hepatocyte necrosis, continuous, high expression of inhibitory receptors is the main reason for the host's inability to eliminate the pathogen [28].

In the natural history of chronic virus infection, persistent viremia has been associated with upregulation of PD-1 expression on virus-specific CD8⁺ T cells [29‒32]. HBV chronic infection leads to impaired surveillance function of the immune system and an altered hepatic immune microenvironment. Immune tolerance is induced toward HBV and promotes hepatocarcinogenesis and development [13]. Relevant research indicates that PD-1 and Tim-3 may have sequential, differential, and cooperative effects on the development of cirrhosis and HCC in chronic HBV infection [33]. Hence, blocking PD-1 and/or its ligands by ICIs may help exhausted HBV-specific CD8⁺ T cells to regain their original immunosurveillance capacity and exert their antiviral effects. Therefore, harnessing ICIs to reshape the immune surveillance function of HBV-specific CD8⁺ T cells is a potential antiviral regimen and one of the research directions in the field of HBV antivirals. There are several ongoing clinical trials using PD-1/PD-L1 inhibitors to treat CHB (Table 1).

PD-1 is a cell surface receptor expressed on activated B and T lymphocytes and is an important immune checkpoint that is also expressed on natural killer cells, monocytes, and dendritic cells [34]. PD-1 is a member of the CD28 superfamily and binds two ligands (PD-L1 and PD-L2) that are widely expressed in the immune system and other cells in the body. PD-1 plays a critical role in maintaining immune tolerance and regulating the duration and intensity of the immune response by inhibiting T-cell activation and cytokine production through interaction with its two ligands, PD-L1 and PD-L2 [35, 36].

T cells adapt to persistent antigens during chronic viral infection by downregulating T-cell antigen receptor (TCR) responsiveness [37‒39]. After interaction with the PD-1 ligand, the immunoreceptor tyrosine-based switch pattern (ITSM) in the PD-1 cytoplasmic structural domain is phosphorylated and recruits the phosphatases SHP-1 and SHP-2. These phosphatases act on proximal signaling kinases of the TCR pathway to decrease TCR signaling, leading to reduced T-cell activation and cytokine production. As is shown in Figure 1, under sustained antigenic conditions, T cells may upregulate inhibitory receptors such as PD-1 to attenuate TCR signaling, leading to reduced T-cell activation and cytokine production [40‒42].

In the case of chronic infections, prolonged antigen exposure results in permanent expression of PD-1, which can limit immune-mediated clearance of pathogens [34]. Additionally, the extent of HBV-specific T-cell exhaustion and disease severity correlates with PD-1 expression in chronic viral infections [43, 44]. In such chronic viral infections, continuous presence of functional CD8⁺ T-cell is essential to keep the virus in check [45], and loss of CD8⁺ T-cell responses can lead to resurgence of the virus [24, 46]. Therefore, targeting these inhibitory receptors is one of the therapeutic strategies currently being explored, including PD-1/PD-L1 inhibitors.

Studies in chronic viral infection models have shown that the PD-1/PD-L1 pathway contributes to T-cell dysfunction and lack of viral control. Blocking PD-1 and PD-L1 interactions can reverse T-cell exhaustion and reinvigorate immune responses [22, 47‒50]. In various human chronic infections, including infection by HBV, high PD-1 levels are expressed by virus-specific T cells, and T-cell function improvement has been achieved in vitro by inhibiting the PD-1/PD-L1 interaction [30, 49, 51‒55]. The evidence provided by these articles not only shows the reduced effector functions and elevated PD-1 expression on TEX but also verifies that blockade of the PD-1/PD-L1 pathway might result in increased proliferation and IFN-γ and IL-2 production of CD8⁺ T cells [14, 22]. In a separate study of woodchuck hepatitis virus, Liu et al. [56] used PD-1 antibodies to block the PD-1/PD-L1 pathway in CD8⁺ T cells in vivo, leading to anti-woodchuck hepatitis virus antibody development and complete viral clearance in some woodchucks in combination with NA entecavir treatment and DNA vaccination.

To compare the different functions of CD8⁺ T cells after blocking inhibitory receptors such as PD-1, 2B4, Tim-3, CTLA-4, and BTLA, in vitro experiments involving CD8⁺ T cells isolated and compared from the peripheral blood of 98 patients with CHB showed that the functional molecules of the cells induced by PD-1 inhibitors lead to the most obvious functional recovery, suggesting that blocking PD-1/PD-L1 may play an important role in HBV antiviral treatment [57].

Blocking PD-1 can reactivate TEX and improve control of chronic infection and cancer. Although the therapy is promising, most cancer patients using PD-1 pathway inhibitors fail to achieve a lasting response, and progression eventually occurs in most [22, 58, 59]. Thus, blocking PD-1 may not promote long-term improvements in TEX and immunological memory development in TEX. In a mouse model, Pauken et al. [60] observed that this reactivation disappeared 8–11 weeks after treatment and that the number, proliferation, effector function, and inhibitory receptor expression of lymphocytic choroid plexus meningitis virus-specific CD8⁺ T cells in anti-PD-L1-treated mice were comparable to those in control-treated mice. In addition, although anti-PD-L1 treatment reduced the viral load immediately after treatment, the viral load after 4 months was similar to that of control group mice. However, Bénéchet et al. [61] questioned the antiviral ability of PD1 pathway blockers. Their study concluded that naïve CD8⁺ T cells undergoing intrahepatic priming can be rescued by IL-2 but not by anti-PD-L1. They treated Cor93 TN-injected MUP-core mice with anti-PD-L1 blocking antibodies, recombinant IL-2 coupled with anti-IL-2 antibodies (IL-2c), or a combination of both. IL-2c administration promoted expansion and differentiation of Cor93 T cells into IFN-γ-producing cytotoxic effector cells, whereas anti-PD-L1 treatment either failed to do so when given alone or did not show a synergistic effect when given in combination with IL-2c.

HBV reactivation is defined as a sudden and rapid increase in HBV-DNA level by at least 100-fold in patients with previously detectable HBV-DNA or reappearance of HBV-DNA viremia in individuals who did not have viremia before the initiation of immune-suppressive or biological modifier therapy or cancer chemotherapy [62]. HBV reactivation starts with viral replication, followed by liver injury that results from delayed immune reconstitution. The severity of liver injury varies greatly among individuals, ranging from a symptomatic rise in alanine transaminase levels to severe hepatitis or even liver failure [17]. HBV reactivation may be classified into two broad categories based on the baseline virologic profile: HBV reactivation in patients who are positive for HBsAg in the serum with or without detectable HBV-DNA viremia in the blood and reverse seroconversion [62].

Based on the ICIs mechanism of action, that is, activating the immune response, HBV reactivation is unlikely. However, a retrospective study reported that six of 114 (5.3%) cancer patients with chronic HBV infection experienced HBV reactivation at a median of 18 weeks from the onset of PD-1 or PD-L1 inhibitors [63]. There is even a case report of a patient with bronchial adenocarcinoma who developed HBV reactivation after a single durvalumab (anti-PD-L1) infusion and eventually died of multi-organ failure [64]. A total of 13 studies, including 2,561 patients, were included in the meta-analysis. This study reported a pooled incidence rate of HBV reactivation of 10.0% (95% CI, 4–18%), 1.0% (95% CI, 0–2%), and 0.0% (95% CI, 0–0%) in patients with chronic HBV infection without antiviral prophylaxis, chronic HBV infection with antiviral prophylaxis, and past HBV infection, respectively [65]. This was unexpected, and the mechanism of HBV reactivation induced by anti-PD-1/PD-L1 therapy is unclear. According to previous research, the possible reasons are as follows: first, PD-1 is an important immunosuppressive mediator that helps to prevent overwhelming liver damage. Therefore, blocking the PD-1/PD-L1 axis may lead to destruction of hepatocytes and release of the previously latent virus into circulation [20]. There is emerging evidence that excess inflammation can also promote viral replication. Immunological assays of CHB patients have associated high concentrations of PD-1-expressing cytotoxic T lymphocytes with a reduction in acute flares of hepatitis B, while a lower number of PD-1-expressing lymphocytes was associated with a higher number of acute flares [66]. Furthermore, PD-1 may suppress proliferation of T regulatory cells. Blockade of PD-1 may promote proliferation of T regulatory cells, which leads to increased immunosuppression and hence reactivation of HBV [67, 68]. Thus, the occurrence of HBV reactivation may appear as a paradoxical effect and suggests that ICIs treatments may disrupt the balance of chronic HBV infection and, in some conditions, lead to deterioration of liver function rather than to hypothetical improvement. More basic research is needed to reveal the underlying mechanisms of hepatitis virus reactivation due to anti-PD-1 therapy.

Disinhibition of T-cell function by ICIs can lead to a range of inflammatory side effects or immune-related adverse effects (irAEs) [69]. Blocking the PD-1/PD-L1 axis might damage the balance between the host immune system and viral control, which entails the risk of functional liver damage [70]. In addition, exhaustion of T cells might lead to poor response to ICIs. To evaluate the safety and efficacy of ICIs in patients with HBV/HCV infection and advanced-stage cancer, a systematic review identified 186 patients from 14 studies. A total of 22.0% (41/186) of patients with HBV/HCV experienced hepatic transaminase elevation (HTE) after ICIs therapy, and the incidence of grade 3 or 4 HTE was 10.8% (20/186). One patient experienced immune-mediated HTE and recovered with steroid use [71]. To reduce the incidence of irAEs, researchers are working on developing PD-1/PD-L1 inhibitors that target the liver, and a portion of the studies have yielded fairly promising results. At the 2023 Annual Meeting of the Asian Pacific Association for the Study of the Liver (APASL), researchers at Aligos Therapeutics, Inc. reported the discovery of an oral liver-targeted small molecule inhibitor of PD-L1 (ALG-093702). In mouse pharmacokinetic studies, ALG-093702 significantly higher concentrations in the liver than in other tissues (e.g., the lung, kidney, pancreas, skin, and colon) was found [72]. This suggests that the inhibitor is preferentially and significantly partitioned to the liver and has the potential to mitigate immune-related systemic toxicity. Meng et al. [73] designed an anti-PDL1-IFNα heterodimeric fusion protein, in which one arm was derived from an anti-PDL1 antibody and the other arm was IFN-α. The results in chronic HBV-carrying mice showed that anti-PDL1-IFNα heterodimers preferentially targeted the liver, leading to viral suppression and even functional cure [73]. However, given the strict trial enrollment criteria, patients with chronic HBV infection have long been considered ineligible for most clinical studies of ICIs due to the risk of disease reactivation, increased immune-related toxicities, and potential lack of efficacy. Therefore, it is unclear whether chronic viral infection will increase the incidence of irAEs caused by PD-1/PD-L1 inhibitors. The mechanisms and clinical outcomes related to these events require further elucidation.

Recent advances in ICIs and their remarkable success in oncology provide new hope that such strategies may be likewise promising for contributing to a functional cure for HBV. First, chronic viral infection leads to many inflammatory factors, stimulating T cells to maintain high expression of PD-1 and PD-L1. Therefore, the high expression of PD-1/PD-L1 is not an inducement for TEX but plays a maintenance role. Second, HBV-specific T cells in different infection stages have different phenotypes and functions. T cells in different infection stages have different responses to PD-1 receptor blockers, such as poor T-cell response in end-stage failure. Therefore, how to choose the right time to use PD-1/PD-L1 inhibitors remains a challenging question for the future. Third, anti-PD-1 alone is not sufficient to completely reverse the immune function damage in chronic HBV infection; combined with other immunotherapies, it may lead to an excessive immune response and liver cell damage. It is recommended that HBV-DNA positive patients receiving ICIs should be combined with viral load suppression therapies. Fourth, the effect of blocking PD-1 may not last for long. Long-term use may not effectively increase the function of T cells but may also increase side effects. In general, use of PD-1 inhibitors in cancer patients with CHB is safe, but PD-1 inhibitor treatment may break the immune balance. Compared with their potential role in treating chronic viral infection, PD-1 inhibitors may lead to HBV reactivation and require further research. Based on studies performed to date, ICIs may be at least one component of effective therapy for chronic HBV. However, the risks of such treatments are substantial, and caution must be exercised when considering use of these agents. Any of these drugs carries a substantial risk of adverse effects, including an increased risk of viral infection, autoimmune reaction, or exacerbation of autoimmune disease. Therefore, use of ICIs in chronic HBV infection will need to balance the possible clinical benefits and the risks associated with use of these therapies and will require close and careful monitoring.

The authors have no conflicts of interest to declare.

1. Ministry of Science and Technology of the People’s Republic of China, 13th Five-Year National Science and Technology Major Project, 2018ZX10715-013-003, Research on Intelligent Medical Application of Information Platform of Integrated Prevention and Control Demonstration Area Based on "Internet +", Research on Intelligent Medical Application of Information Platform of Integrated Prevention and Control Demonstration Area Based on "Internet +". Supported by the Fundamental Research Funds for the Central Universities. 2. Ministry of Science and Technology of the People’s Republic of China, 13th Five-Year National Key Research and Development Program of China, 2019YFC0840600 and 2019YFC0840609, Research on cloud platform and intelligent diagnosis and treatment technology for viral hepatitis diagnosis and treatment. 3. Zhejiang Natural Science Foundation (LQ21H030003), research on dual roles of platelet TNIK in hemostasis and hyperlipidemia-induced pathological thrombosis. 4. Supported by the Fundamental Research Funds for the Central Universities (226-2023-00050).

Q.X., T.Y., and M.S. designed the research, collected and analyzed the data, and wrote the manuscript.

Q.X. and Z.S. designed, supervised, and managed the research and checked the manuscript.

W.W. and M.S. participated in collecting the data and writing and modifying the draft of the manuscript.