Toward Curative Immunomodulation Strategies for Chronic Hepatitis B

Subscriber access provided by TULANE UNIVERSITY

Review

Towards curative immunomodulation strategies for chronic HBV infection Wei Zhu, Hongyan Liu, and Xiaoyong Zhang ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

Towards curative immunomodulation strategies for chronic HBV infection Wei Zhu, Hongyan Liu, Xiaoyong Zhang*

State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of Viral Hepatitis Research, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838, North Guangzhou Avenue, Guangzhou, 510515, China

*Correspondence author, mailing address: Xiaoyong Zhang, MD, PhD Hepatology Unit and Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, No. 1838, North Guangzhou Avenue, Guangzhou 510515, China Phone: +86 20 62787830 E-mail: [email protected]

ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chronic hepatitis B virus (HBV) infection remains a major cause of morbidity and mortality worldwide. HBV surface antigen loss is considered a functional cure, and is an ideal goal for antiviral therapy. However, current treatment regimens, including nucleos(t)ide analogues or interferons monotherapy and combination therapy, rarely achieve this goal in chronic hepatitis B patients. NAs, as well as many direct antiviral drugs in ongoing development, are able to inhibit HBV replication and gene expression, but it is hard to achieve immune control and prevent recurrence after therapy cessation. Host immunity, especially HBV-specific T cell response, is proven to play a critical role in control or clearance of HBV infection. Considering HBV chronically infected patients display varying degrees of dysfunction regarding their immune system, novel approaches to enhancing antiviral immune responses are thus necessary in order to combine with current antiviral agents. In this review, we focus on the role of innate and adaptive immune responses in HBV immunopathogenesis, and discuss attractive strategies or drugs that aim to activate or rebuild antiviral immunity to achieve the goal for HBV functional cure.

Keywords: Hepatitis B virus; chronic hepatitis B; innate immunity; adaptive immunity; functional cure


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The hepatitis B virus (HBV) belongs to the Hepadnaviridae family of small, enveloped, primarily hepatotropic viruses with a partially double-stranded DNA genome approximately 3.2 kb in size.1 HBV infection poses a significant international public health burden. It was estimated that 240 million people get chronically infected globally.2 There are two major modalities for HBV transmission: horizontally via percutaneous or mucosal contact with infected bodily fluids or vertically from HBVinfected mothers to neonates.3-4 Vaccine prophylaxis is the most effective strategy to reduce HBV transmission. In patients with chronic hepatitis B (CHB), standardized antiviral therapy prevent the progression to end-stage liver diseases, such as hepatic failure, liver cirrhosis or hepatocellular carcinoma (HCC).5 Current approved antiviral agents for HBV, including interferons (IFN) and nucleos(t)ide analogues (NAs), inhibit HBV replication effectively and reduce transmission, but neither of them eliminate the virus.6 Generally, HBV surface antigen (HBsAg) loss is considered as “functional cure” which means undetectable HBV DNA in the serum after treatment cessation. Actually, HBsAg loss occur in only a minority of CHB patients received antivirals therapy. Therefore, it is still necessary to develop novel curative strategies for therapeutic intervention on chronic HBV infection. The host immune system plays a critical role in clinical outcomes of HBV infection. Interaction between the host immune system and the virus are correlative with clinical and virological parameters during the natural course of HBV infection. Although many features of acute immune response against HBV infection have been reported before, our understanding of the immunopathogenesis of CHB is still ongoing. In this review, we will focus on the current advances in both innate and adaptive immune responses to HBV infection and recently developed immunotherapeutic strategies for the treatment of chronic HBV infection. Acute self-limited HBV infection Acute HBV infection, leads to a robust T cell and B cell response specific for different viral antigens. Viremia starts to decrease shortly after antiviral T cells become detectable in the blood (approximately 8–10 weeks after HBV infection) and increased infiltration of CD8+ T cells in liver 7 Antiviral function of HBV-specific T cells appears



earlier than the initiation of severe liver damage, because the decreased in viremia precedes the peak value of serum liver enzymes in acute HBV infection.7 These early non-cytopathically antiviral effects are mediated by cytokines such as interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα).8-9 Additionally, cytopathically antiviral effects are mediated by granzyme and perforin after recognizing HBV positive hepatocytes by viral-specific CD8+ T cells.7 In chimpanzee and mouse models, humoral immune responses are also detectable during an acute HBV infection. The seroconversion of HBV e antigen (HBeAg) and HBsAg to anti-HBe and anti-HBs is used as biomarkers of recovery.10 During an acute HBV infection, anti-HBs detection is associated with disease resolution and virus control.11 Detection of anti-HBs, which occurs only late at infection, is later than detection of HBV-specific T cells.12 Although HBV DNA and HBsAg levels change significantly in acute HBV resolution, it is proved that the stable numbers of circulating HBsAg-specific B cells during the symptomatic phase of acute HBV infection. And HBsAg-specific B cells share the similar impaired function in both acute HBV and CHB patients.13 Chronic HBV infection The risk of developing chronic HBV infection is inversely proportional to age at time of infection. CHB develops in 90% of children infected before the age of one, 20–50% of children infected from the age of one to the age of five, and 5% of persons infected after the age of five.14 Studies on the immune response to early childhood infection help understanding the developing of CHB. Resent data revealed the immune tolerance in utero exposure to HBV based on trained immunity by comparing the cord blood of neonates from HBV-infected and uninfected mothers.15 Neonatal woodchuck hepatitis virus (WHV) infection was induced and characterized by increasing viral load and chronicity and temporal lacking of the acute intrahepatic effector mediated by IFN-γ and TNF-α leads to the neonatal WHV infection.16 The mouse HBV model showed the same age-dependent immune responses to HBV. Administration of naïve cell infusion into adult HBV transgenic mice primes adaptive immune response and achieves seroconversion from HBsAg to anti-HBs, however cell infusion treatment in young transgenic mice does not produce sufficient adaptive immune response. In addition,


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


young mice have lower hepatic chemokine (C-X-C motif) ligand 13 which is essential for induction of virus-specific B cells.17 This differential outcome has been attributed to age-dependent expression of costimulatory molecules on hepatic antigen-presenting cells18 and defective function of T-follicular helper cells, which are essential for induction of virus-specific T cells and B cells.19 The clinical process of HBV infection varies among subjects with different host factors, viral factors, and host-viral interactions. The natural course of chronic HBV infection is sub-divided into HBeAg-positive chronic infection, HBeAg-positive chronic hepatitis, HBeAg-negative chronic infection, HBeAg-negative chronic hepatitis20 (Table 1). Some infected subjects may enter a HBeAg-negative hepatitis phase, and even develop liver cirrhosis or HCC.21 Only a small minority of CHB subjects may achieve seroconversion from HBsAg to anti-HBs, and enter a phase which resembles functional cure. The HBeAg-positive chronic infection phase includes compromised HBV-specific T cell,22-23 normal levels of serum liver enzymes, and high levels of serum HBV DNA. The HBeAg-positive chronic hepatitis phase includes more severe liver disease and fluctuations in HBV DNA titers and serum liver enzymes levels.24 The HBeAg-negative chronic infection phase of low viral replication, with seroconversion from HBeAg to anti-HBe, reduced inflammation, and decreased HBV DNA level. However, delayed HBeAg seroconversion with persistently high viremia after forty years old suggests a higher risk of developing liver cirrhosis and liver cancer.25-26 Role of innate immune system in chronic HBV infection In general, the innate immune system constitutes the first line of host defense against infection. Most viruses activate the innate immune system by pathogen-associated molecular patterns (PAMPs).27 However, whether HBV can trigger the components of innate immunity remains controversial. It is reported that HBV does not activate innate immune as a stealth virus.28 HBV does not induce a hepatic IFN-stimulated genes (ISGs) response in a chimpanzee model.29 Meanwhile, WHV does not induce known ISGs in acute or chronical infection woodchuck models.30 No induction of IFNs was observed in the serum of acute HBV infected subjects by detection of serum cytokines.31 Proinflammatory cytokines were low or undetectable within the first 30 days of HBV



infection, and the kinetics of cytokines were also delayed compared with hepatitis C virus and human immunodeficiency virus infection.32 Recently, a study showed that no hepatic IFNs and ISGs induction was detected in chronically HBV infected patients who had different disease course, serum HBV DNA level and HBV genotypes. The innate immune signaling in liver is intact.33 Some reports, however, have claimed that HBV is sensed by the innate immune system, but it suppresses the activation of innate immune system.34 These studies employing HepaRG cells overexpressing HBV,35 HepG2 cells overexpressing other molecules36-37 or high dose of WHV38 as the model, suggest the interaction between HBV and innate immunity. However, more studies need to be performed in more natural HBV infection systems. NK cells have shown anti-HBV activity in acute infections of woodchucks and humans.38 In addition, NK cells regulate the antiviral T cell response in CHB and mediate a rapid contact-dependent killing of HBV-specific T cells.39 However, it was reported that NK cells are defective in cytokines production and non-cytopathic antiviral effects.40-41 The NKT cells in human liver are mostly composed by MR1restricted mucosal-associated invariant T (MAIT) cells instead of the classical CD1drestricted NKT cells which are abundant in mice.42 However, there are no data presented to suggest that HBV activates MAIT cells until now. Role of adaptive immune responses in chronic HBV infection Virus-specific CD4+ T lymphocytes are required for optimal induction of both efficient B cell and CD8+ T cell responses, so they are essential in controlling viral infections. HBV-specific CD4+ T cells appear in the blood 7-10 weeks after infection, in parallel with the emergence of HBV-specific CD8+ T cells and antibodies.43 In an acute selflimited HBV infection, Th1-polarized CD4+ T cells in peripheral blood contribute to successful control of HBV infection and liver injury.44 However, hepatic HBV-specific CD4+ T cells secrete IL4 and IL5 in addition to Th1-type cytokines, showing a distinct functional profile compared with those from the blood in chronic HBV infection.45 In addition, CD4+ CD25+ regulatory T cells serve as a negative regulator which suppresses the activation, proliferation, and IFN-γ production of both CD4+ and CD8+ T cells in chronic HBV infection.46-47


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HBV-specific CD8+ T lymphocyte is one of the keys to achieve successful control of HBV infection and liver injury. CD8+ T cells control HBV infection in a cytolytic and non-cytolytic manner.48-49 The peak of HBV-specific CD8+ T cell level (6-8 weeks after infection) is usually accompanied by the peak of liver injury50 and the powerful activation of bystander CD8+ T cells, accompanied by the peak of HBV-specific CD8+ T cells level, may also contributes to liver injury.51 Chronic HBV infection is characterized by an T cell failure (Figure 1), which exhibit impaired cytokine production, defective cytotoxic activity and sustained expression of multiple inhibitory receptors. Persistent stimulation or high level antigen stimulation of T cell receptor (TCR) can lead to T cell exhaustion and up-regulation of many inhibitory checkpoint molecules, including programmed death-1 (PD1), 2B4, cytotoxic T-lymphocyteassociated protein 4 (CTLA4), T cell immunoglobulin mucin 3 (Tim3), lymphocyteactivation gene 3 (Lag3), T cell Ig and ITIM domain (TIGIT), B- and T-lymphocyte attenuator (BTLA), CD160, CD162, the death receptor 5 and the pro-apoptotic mediator, bcl-2 interacting mediator cell death (BIM)in T cells.52-55 The level and duration of antigen stimulation correlate with the severity of dysfunction during chronic infection and typically, the higher the number of inhibitory receptors co-expressed by exhausted T cells, the more severe the level of T cell exhaustion.53 In the inhibitory checkpoint molecules, over 90% of hepatic HBV-specific CD8+ T cells present PD1 and 2B4.56-57 CD8+ or CD4+ T cells recognize HBV-infected hepatocytes through the major histocompatibility complex (MHC)-peptide complex on the cell surface.58 The downregulation of MHC molecule induced by HBV infection may also lead to CHB. In a WHV infection model, the expression of MHC class I molecules on hepatocytes and lymphoid cells was downregulated,59 and the same phenomenon was observed in humans.60-61 However there are also evidences suggesting chronic HBV infection does not down regulate MHC class I.62 Moreover, mutation of MHC-binding residues may reduce peptide-MHC binding scores, providing a plausible mechanism for HBV immune escape and development of CHB.63 Core mutants in patients with chronic genotype A or D reduce peptide-MHC affinity providing another possible mechanism



for HBV immune escape.63 In addition, some CD8+ T cell epitopes act as TCR antagonists to impair function of HBV-specific T cells and lead to HBV immune escape. For example, certain HBV core antigen64 and HBsAg65 bind the TCR and specifically inhibit the CD8+ T cell response. Further efforts should be made to understand immune escape and the interaction between host and virus. B cells-mediated humoral immunity is increasingly recognized to play an important role in control of HBV infection. Antibodies targeting the preS1 domain (amino acids 2–48) or the HBs antigenic loop (amino acids 104–163) can block HBV infection.66-67 Neutralizing antibodies targeting HBsAg administration not only reduced HBV spread but also suppressed HBsAg load and promoted HBV T cell responses in a mice model.68 Antibodies targeting HBV are thought to be T cell-dependent,69 however anti-HBsproducing B cells are compromised in the blood CHB patients compared with acute hepatitis patients,70-71 because of defective T helper follicular cells in CHB patients.72 HBsAg-specific and global B cells had an accumulation of CD21–CD27– atypical memory B cells with high expression of inhibitory receptors, interestingly, the function of HBsAg-specific B cells are reversible by B cell-maturing cytokines and PD-1 blockade.13, 73 In addition to neutralizing HBV and reducing HBV dissemination, HBVspecific antibodies may induce antibody dependent cell mediated cytotoxicity or complement- dependent cytotoxicity such as that found in HIV infection.74 Furthermore, B cells can act as antigen presenting cells to stimulate helper T cells.69 Immunomodulation strategies for therapeutic approaches against CHB By producing kinds of cytokines, HBV-specific T cells and antibodies, the host immune system controls and eliminates HBV in most adult patients without treatment. Therefor treatments targeting immune system lead to the possibility of a cure of HBV. The current advances in treatments for CHB are summarized (Figure 2). In addition, immunomodulation therapies for chronic HBV infection in clinical trials are summarized (Table 2). IFNs have been approved for HBV treatment, so it will not be discussed in this section. Toll-like receptors (TLRs) agonists The TLRs are a class of proteins representing a particularly important group of pattern-


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


recognition receptors and play a key role in the innate immune system.31 Kinds of TLR ligands stimulation suppresses HBV replication and gene expression in vitro and in vivo.75 Intravenous injection of ligands for TLR3, TLR4, TLR5, TLR7 or TLR9 inhibited HBV replication non-cytopathically by type I IFN induction in HBV transgenic mice model.76 TLR2 or TLR4 ligands were able to inhibit HBV and WHV replication through IFN-independent pathways in vitro.77-78 Recently, a TLR7 selective agonist GS-9620 for CHB therapy was reported.79 GS-9620 showed no direct antiviral activity in HBV-infected primary human hepatocytes (PHH). But conditioned media from human PBMCs treated by GS-9620 induced a prolonged reduction in HBV DNA, RNA and antigen levels in PHH and HepaRG cells in a type I IFN-dependent manner.80 Oral administration of GS-9620 reduced serum and liver HBV DNA and HBsAg, increased serum IFNα, and the expression of ISGs in the blood and liver in HBV infected chimpanzees.81 Clinical usage of GS-9620 showed good safety and a dosedependent ISG15 mRNA induction in patients with CHB, however, no clinically significant changes in HBsAg or HBV DNA levels were observed.82 TLR8 is mainly expressed in myeloid immune cells and its agonist is the stronger activator of innate immunity in the liver.83 Most recently, GS-9688, a selective small molecule agonist of TLR8 was also developed by Gilead company.84 GS-9688 reduced serum WHV DNA and surface antigen in chronical WHV infection.85 GS-9688 is currently in a Phase 1 clinical trial. Retinoic acid inducible gene I (RIG-I) agonist RIG-I typically recognizes short (

Toward Curative Immunomodulation Strategies for Chronic Hepatitis B

Recommend Documents