Viral envelope-specific antibodies in chronic hepatitis B virus infection

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ScienceDirect Viral envelope-specific antibodies in chronic hepatitis B virus infection Davide Corti1, Fabio Benigni1 and Daniel Shouval2 While the cellular immune response associated with acute and chronic HBV infection has been thoroughly studied, the B cell response in chronic hepatitis B and the role of antibodies raised against the HBV envelope antigens in controlling and prevention of infection requires further investigation. The detection of anti-HBs antibodies is considered as one of the biomarkers for functional cure of chronic hepatitis B virus infection, as well as for protective immunity. Indeed, vaccineinduced neutralizing anti-HBs antibodies have been shown to protect against HBV challenge. Yet, the therapeutic potential of viral envelope-specific antibodies and the mechanism involved in protection and prevention of cell-to-cell transmission warrants additional investigative efforts. In this review, we will provide a critical overview of the available preclinical and clinical literature supporting the putative role of active and passive vaccination and neutralizing envelope-specific antibodies for therapeutic intervention in combination regimens intended to cure persistent HBV infection. Addresses 1 Humabs BioMed SA, A Subsidiary of Vir Biotechnology, 6500 Bellinzona, Switzerland 2 Liver Unit, Institute for Gastroenterology and Hepatology, HadassahHebrew University Hospital, P.O. Box 12000, 91120 Jerusalem, Israel Corresponding authors: Corti, Davide ([email protected]), Shouval, Daniel ([email protected])

Current Opinion in Virology 2018, 30:48–57 This review comes from a themed issue on Antiviral strategies Edited by Antonio Bertoletti and Stephan Urban

https://doi.org/10.1016/j.coviro.2018.04.002 1879-6257/ã 2018 Elsevier B.V. All rights reserved.

associated with development of chronic infection concentrated on the role and failure of the cellular immune response in promoting HBV clearance. By contrast, information on the humoral immune response against HBV has mainly focused on development of HBV diagnostic tools. The present review is intended to provide an overview on the humoral immune response and B cell activation in acute and chronic-HBV infection and on the potential role of antibodies to the envelope proteins in preventing, containing and controlling HBV infection.

HBV neutralizing antibodies and beyond ‘Naturally’-occurring polyclonal antibodies generated by HBV infection are directed towards a variety of viral nucleocapsid and envelope antigens, including HBcAg [2,3], HBeAg [4–6], the viral polymerase, the X, as well as the L (large), M (middle) and S (small) HBsAg forms. Amongst these antibodies, only a small fraction is endowed with neutralizing activity and only two classes of HBV neutralizing antibodies have been described. The first class comprises antibodies targeting specific sites in the antigenic loop of HBsAg and capable of neutralizing viral entry, blocking the interaction with the pre-receptor heparan sulphate proteoglycan (HSPG) [7]. During chronic HBV infection these anti-HBs antibodies are depleted by the large amounts of circulating spherical and filamentous subviral particles. An additional antiviral mechanism for antibodies directed to the antigenic loop of HBsAg (but possibly occurring also for PreS1 antibodies) was suggested to involve endocytosis and the consequent intracellular blocking of HBV and HBsAg subviral particles release from infected hepatocytes [8,9]. The second class comprises antibodies targeting the receptor binding ‘site’ of the PreS1 domain. These antibodies block the interaction of virions with the sodium taurocholate co-transporting polypeptide (NTCP) receptor on hepatocytes [10,11], preventing the de novo infection of hepatocytes. Such antibodies may also interact with the L form of HBsAg displayed on filamentous, but not spherical, subviral envelope particles.

Introduction Our understanding of the molecular and immunologic pathophysiology of acute and chronic HBV infection has expanded significantly during the past 5 decades since the discovery of the HBV envelope ‘Australia antigen’ by Blumberg et al. [1]. Infection with the hepatitis B virus (HBV) leads to cellular and humoral immune responses, which determine the natural history of infection in the individual patient. Most studies on the immune response following acute HBV infection and the risk factors Current Opinion in Virology 2018, 30:48–57

In addition to their capacity to block viral entry via Fab recognition of virions, which is also the mechanism of action of the potent NCTP blocker Myrcludex B, neutralizing antibodies can act in vivo through a variety of Fcdependent additional mechanisms. These include killing and/or phagocytosis of infected cells, as well as clearance of viral and subviral particles, as already reported for other viruses such as influenza A virus and HIV-1 [12,13]. Early experiments with a human hepatoma cell line containing www.sciencedirect.com

Viral envelope-specific antibodies Corti, Benigni and Shouval 49

integrated HBV-DNA and expressing HBsAg on the cell surface, revealed that target cell lysis may be possible through a complement or antibody dependent cellular cytotoxicity (ADCC) in the presence of murine monoclonal anti-HBs antibodies (mAbs) [14]. Interestingly, long-term exposure of such a cell line to mAbs against distinct epitopes on HBV, led to viral breakthrough, most probably because of emergence of escape mutant (s). However, short-term treatment of HCC tumor bearing athymic mice expressing HBsAg with these anti-HBs mAbs led to tumor growth suppression [15]. Scarce information exist regarding the impact of anti-HBs antibodies on the replication of HBV. Two recent studies using mAbs targeting the ‘a’ determinant of the antigenic loop (mAb E6F6; [16]) and the NCTP-binding site of preS1 (2H5-A14; [17]) have shown that their ability to reduce HBV DNA and HBsAg levels in mouse models of established HBV infection was abolished when these antibodies were engineered in the Fc (D265A/N297A, dubbed DANA) to prevent binding to FcgRs. In addition, the preS1 mAb 2H5-A14, but not its DANA variant, reduced significantly the levels of covalently closed circular (ccc) DNA in HBV infected mice. The data presented in these studies suggest that the mAb effector mechanisms observed were mediated via elimination of viral and subviral particles, as well as infected hepatocytes by effector cells, such as Kuppfer cells. The role of 2H5A14 mAb mediated killing of infected hepatocytes by natural killer (NK) cells (i.e. ADCC) was shown in vitro, while its relevance in vivo was not investigated due to limitations of the mouse model used. The role of antibody-mediated complement killing for mAb 2H5-A14 was excluded using in vitro experiments, but its relevance in controlling the infection with therapeutic mAbs warrants additional studies. Of note, anti-HBcAg/HBcAg immune complexes were shown to be pathogenic and to lead to massive liver injury [18], similarly to what observed in severe human cases of influenza A virus infection [19]. Besides promoting HBsAg clearance and blocking viral spread [16,17], an additional potential advantage of anti-HBs antibody-based therapy relies on the possibility to generate or re-activate effective T cell responses via the formation of immune-complexes (IC) [20,21]. Antigen-antibody ICs may enhance the capture of HBsAg by immature phagocytic cells, triggering an effective crosspresentation and stimulating a revived cell-mediated immune response [20]. Indeed, the use of E6F6 mAb enhanced virus-specific CD8+ T-cell responses in HBVtolerant mice [16]. In a similar mouse model, the removal of circulating HBsAg by a neutralizing anti-HBs mAb (Ab-H) was reported to reduce tolerance and to re-establish B cell and CD4+ T cell responses to subsequent HBV vaccination with recombinant HBsAg, ultimately leading to anti-HBs seroconversion [22]. This approach termed ‘passive-active immunization’ may enhance the www.sciencedirect.com

resolution of persistent HBV infection by preventing new rounds of intrahepatic infection, which might eventually lead to the reduction of the cccDNA pool. The concept that an antibody response to HBsAg during the course of HBV disease might contribute to HBV clearance is further supported by the simultaneous detection of HBsAg and anti-HBs antibodies in some HBV carriers [23]. It has been suggested that such antibodies represent an atypical serological profile which is associated with the emergence of viral mutants. As an example, the introduction of novel N-glycosylation sites in the antigenic loop [24] alters the ‘a’ determinant antigenic structure and reduces the ability of anti-HBs antibodies to bind to the mutated HBsAg forms [25]. Overall these observations suggest that anti-HBs antibodies may mediate a long-lasting clearance phase that could favor the selection of escaping viral quasispecies. In this context, in a treatment-naı¨ve cohort of genotype A chronic hepatitis B (CHB) patients treated with tenofovir disoproxil fumarate (TDF), the detection of antibodies targeting both loop 1 and loop 2 of the HBsAg correlated with an outcome of HBsAg loss and seroconversion. The investigators proposed that the presence of these antibodies may represent a potential viral biomarker of HBsAg clearance (defined as ‘clearance profile’) [26]. In a more recent study, the detection of the ‘clearance profile’ was found to be predictive of HBsAg decline in patients receiving the RNAi ARC520 therapy [27]. Overall, the above observations highlight the potential of expanding the options for a functional cure of CHB by also including envelope-specific neutralizing antibodies.

B cell response in chronic hepatitis B patients HBsAg is suggested to play a role in the exhaustion of antigen-specific T cells in CHB [28]. A hallmark of HBV infection is the massive production and release of HBsAg subviral particles in quantities that largely exceed the number of infectious virions. This feature has been suggested to represent a viral strategy to evade both cellular and antibody-mediated responses in CHB patients. In addition, the suppression of antiviral T cell responses in CHB might also be dependent on the production of the immune-modulatory cytokine IL10 by regulatory B cells (Bregs) [29–31]. In this context, modulation of Bregs may represent a valuable and innovative therapeutic approach for CHB [32]. The importance of the B cell response in CHB is highlighted by the clinical observation that B cell depletion induced by anti-CD20 or anti-CD52 antibody therapies for lymphoma can lead to HBV reactivation both in inactive HBsAg carriers as well as in subjects who resolved HBV infections [33]. Of note, the risk of HBV reactivation is lower in patients with detectable anti-HBs Current Opinion in Virology 2018, 30:48–57

50 Antiviral strategies

antibodies [34]. The risk of HBV re-activation in these individuals is dependent on both viral and host factors [35], including HBV viral DNA load, serum HBV markers, virus genotype and mutations, as well as chemotherapy with corticosteroids or with other immune-suppressive agents [33]. The composition and functionality of memory B cells is altered in different chronic settings of infection, such as hepatitis C (HCV), HIV-1 [36,37], malaria, and tuberculosis, suggesting a link between disease and a defective B cell response. This is manifested in expansion of a phenotypically unique memory B cell population (defined as atypical, exhausted or tissue-like), characterized by low expression of the cell surface markers CD21 and CD27, high expression of inhibitory receptors, such as FcRL4 and FcRL5 [38–41,42], and up-regulation of T-bet transcription factor [43]. This peculiar population of B cells is considered dysfunctional, even though some evidence suggests that, at least for malaria, atypical B cells can proliferate and produce neutralizing antibodies [39]. Previous reports suggested that using highly sensitive immunoassays anti-HBs antibodies are found in CHB patients, but these are mainly detected as complexed to HBsAg [44]. However, recent data show that in CHB patients memory B cells exhibit a defective proliferation phenotype [45]. In other studies [46–48], the phenotype of B cells was shown to change along the different phases of CHB, culminating in a ‘hyper-activation’ status. Xu and co-authors [46] showed that in CHB patients the in vitro stimulation of B cells did not lead to the production of anti-HBs antibodies, as measured by ELISPOT; however, this defect was not linked to a specific memory B cell phenotype. Importantly, the impairment of B cell functions in CHB patients might also be related to the presence of defective T follicular helper cells [49,50]. Overall these findings suggest a critical role for B cell responses in controlling the subclinical infection in CHB patients and that memory HBsAg-specific B cells are functionally defective.

Limitations and pitfalls of HBV serological diagnostics The detection of antibodies directed to HBcAg, HBeAg and HBsAg is used as a diagnostic tool to distinguish between different clinical phases of acute and chronic HBV infection. The available immuno-assays to measure anti-HBs antibodies are based on the small S HBsAg and detect antibodies regardless if generated following ‘natural’ acute HBV infection, vaccination or seroconversion secondary to CHB resolution. Traditionally, detectable anti-HBs antibodies in the absence of HBsAg are considered the hallmark for ‘functional cure’ of CHB infection, although it was recently suggested that sustained HBsAg loss may be sufficient [51]. Furthermore, Current Opinion in Virology 2018, 30:48–57

currently used anti-HBs diagnostic assays may be suboptimal, since antibodies complexed with circulating HBsAg may be missed [52–55]. Thus the predictive power of detectable anti-HBs antibodies as biomarker of remission of infection is blurred in some chronic carriers by the concomitant detection of both HBsAg and anti-HBs antibodies, as discussed above [56,57]. Likewise, the assays for detection of HBsAg are also impaired by the formation of ICs by anti-HBs antibodies, such as in occult HBV infection, where HBsAg is not measured in the presence of HBV-DNA [58]. In this context, novel more sensitive HBsAg detection assays were recently developed, based on the disruption of HBV particles, leading to a dissociation of HBsAg from ICs and its denaturation, and displaying envelope epitopes into linear forms [59]. This approach was coupled with the use of capture and detection by mAbs targeting regions in HBsAg that are not readily accessible in the native form of the antigen, such as epitopes in the internal loop and in a structural region external to the determinant ‘a’. Another limitation of current HBsAg diagnostics is represented by the possibility that HBsAg mutants (including those observed in patients with integrated HBV-DNA into the HCC genomes) can escape from detection (depending on the fine specificity of the capture and detection antibodies used in the assay) and persist in HBV-infected individuals after the diagnostically defined ‘loss’ of HBsAg. Finally, most commercially available anti-HBs assays are not designed to detect or quantify anti-PreS1 or antiPreS2 antibodies, mainly due to the lack of international standards for quantitation of Pre-S1 and Pre-S2 antigens. It has been shown that Pre-S1 antigen levels in CHB correlate with the degree of viral load [60]. Furthermore, in newborns immunized with a PreS1/PreS2/S vaccine, the highest anti-HBs levels were measured in newborns exhibiting reactivity towards PreS1, but lacked antiPreS2 reactivity [61].

Role of envelope-specific antibodies in HBV prophylaxis and therapy Traditionally, with a few exceptions, polyclonal antibodies have had a time-limited, short-term use as therapeutic agents for chronic infectious diseases [62]. In recent decades, technological advances have had an impact on the control of persistent HBV infection [63]. Hepatitis B immunoglobulins for pre-exposure and postexposure prophylaxis

Polyclonal hepatitis B immunoglobulins (HBIG) are prepared using pooled plasma from healthy human donors who recovered from hepatitis B or were immunized against HBV with high concentration of anti-HBs antibodies. HBIG has been successfully used for several decades for post-exposure prophylaxis (PEP) against HBV infection in neonates born to HBsAg carrier www.sciencedirect.com

Viral envelope-specific antibodies Corti, Benigni and Shouval 51

mothers, as well as in HBV infected liver transplant patients [63,64]. In the liver transplant setting, HBIG is administered during the anhepatic phase of surgery, and then repeatedly thereafter (in combination with a nucleoside analogue) for maintenance of anti-HBs titers 100 mIU/ml. Concern has been raised that some infants born to HBsAg carrier mothers or liver transplant patients treated with HBIG, will develop vaccine escape mutants (VEM). Such VEMs have been identified in Italy, Singapore and China carrying different mutations, of which the most common is the G145R [65,66]. Although one of these mutants has been shown to cause HBV infection in experimentally infected chimpanzees, immunization with yeast derived HBV vaccines containing the adw HBsAg subtype protected primates against challenge with the G145R virus [67]. After a follow-up of 20 years, the overall impact of such VEMs seems to be low with little, if any, implications on public health prevention strategies [68]. The mode by which HBIGs interacts with circulating HBV and infected hepatocytes is not fully understood. Putative mechanisms include: (1) Anti-HBs antibodies in HBIG recognize the conformational ‘a’ determinant and/ or second loop epitopes leading to neutralization of circulating virions and HBsAg-containing particles [63,69]. (2) Blocking of binding to the NTCP receptor and prevention of de novo infection of hepatocytes [70] and (3) endocytosis of the anti-HBs antibodies in hepatocytes via FcRn leading to intracellular neutralization [8]. Monoclonal antibodies to HBsAg for pre-exposure and post-exposure prophylaxis

The high cost of HBIG, the difficulty in obtaining high antibody titer plasma for HBIG production, and the variable batch-to-batch concentration of the purified polyclonal end-product, led to a search for alternative strategies for pre-exposure and post-exposure prophylaxis. In theory, the use of mAbs directed against HBsAg would provide an advantage over HBIG. Early studies with different murine and humanized mAbs against HBsAg or Pre-S1 revealed that pre-incubation of HBV particles with one or a combination of two such mAbs neutralized the virus and prevented acute HBV infection for a period of up to one year in HBV infected chimpanzees [71]. A combination of two human mAbs (17.1.41 and 19.79.5, renamed Hepex-B) targeting distinct sites on the ‘a’ determinant was developed up to phase 1 by XTL Biopharmaceuticals and Cubist Pharmaceuticals [76]. These two mAbs exhibited a 500–2000-fold higher specific activity to HBsAg as compared to HBIG. Regretfully, the clinical development of these mAbs was halted in 2006 because of strategic decisions related to manufacturing and regulatory issues. More recently, a single human mAb (HB-C7A, renamed GC1102) is under development by Green Cross Pharma as a substitute to HBIG. This mAb was shown to prevent HBV infection of www.sciencedirect.com

chimpanzees up to 1 year after viral challenge [72] and is currently tested in a phase 2/3 clinical trial in South Korea to prevent infection after liver transplant. Clinical use of mAbs to HBsAg for therapy

Limited information is available on use of mAbs against HBV envelope proteins in CHB patients. Overall experience suggests that anti-HBs mAbs can neutralize circulating envelope protein(s). However, rebound and relapse are frequent following withdrawal of mAb therapy [76]. The use of anti-HBs mAbs in humans was pioneered in two hypo-gammaglobulinaemic HBV chronic patients by Lever and colleagues using a murine anti-HBs mAb (RFHBs1) that did not reduce HBsAg, but decreased HBeAg and DNA levels [73]. Subsequently, van Nunen and colleagues tested in CHB patients the efficacy of the anti-HBs human mAb tuvirumab, alone or in combination with alpha interferon [74]. Albeit long term clearance of HBsAg in serum was not observed, a reduction of HBsAg levels by at least 50% was observed in all patients. Of note, in three patients receiving the combination therapy with alpha interferon, circulating HBsAg became undetectable. Hepex-B mAbs were tested therapeutically in a mouse HBV trimera model and in HBsAg+ chimpanzees leading to a significant and transient drop in viral load and HBsAg, which rapidly returned to baseline levels upon cessation of treatment [75]. These observations were confirmed in a phase 1 clinical trial in CHB patients treated by a single or multiple doses of Hepex-B mAbs. This treatment led to suppression of circulating HBV-DNA and HBsAg in a dose dependent manner [76]. Using a mathematical model based on the kinetic profiles and decline of HBsAg and HBV-DNA levels, it was suggested that the mode of action of Hepex-B mAbs included blocking of HBsAg secretion and release from infected cells, as well as acceleration of HBV clearance [9]. Finally, GC1102/HB-C7A mAb, currently under development as a potential replacement for HBIGs, was recently tested therapeutically in CHB patients carrying low levels of HBsAg (i.e. 1000 IU/ml HBsAg, NCT02569372). The results of this study are not disclosed yet. Even though mAbs were not administered in molar excess over the concentration of HBsAg, the intravenous injection of monoclonal or polyclonal anti-HBs antibodies in HBsAg positive mice, chimpanzees and human subjects has so far been well tolerated without serious sequelae of renal or rheumatic immune complex disease. Similarly, no adverse events were reported in CHB patients receiving high doses of HBIG [77,78]. In this regard, it is advised that a molar excess of the anti-HBs antibodies should be administered to guarantee sustained Current Opinion in Virology 2018, 30:48–57

52 Antiviral strategies

Table 1 Selected list of PreS1 and HBsAg-specific monoclonal antibodies. Origin

POC (P/T) b

aa 20–26

Hu

Mice (P/T)

KR127/359 4 mAbs BX-182 G10 18/7 HBsAg mAbs Tuvirumab/OST-577 17.1.41/19.79.5; Hepex-B RFHBs1 HBC7a/GC1102

aa 37–45 N/A aa 6–10 N/A aa 31–34

Mu a Hu Mu Hu Mu

Chimp. (P) N/A Chimp. (P) N/A N/A

‘a’ det. 2 epitopes on ‘a’ det. ‘a’ det. (124–137) Conformational ‘a’ det.

Hu Hu Mu Hu

Human Human Human Human

E6F6 ADRI-2F3 c-4G4

linear epitope (‘sA’) Conformational ‘a’ det. N/A

Mu Hu Hu

Mice (T) N/A N/A

MAb ID PreS1 mAbs 2H5-A14

a b

Epitope

(T) (T) (T) (P/T)

Comment

Status

Block binding to NCTP; broadly reactive

Ph. 1/2a Ph1 in chronic patients Small study in 2 patients Ph2b for liver transplant/Ph2 in chronic patients POC in mice In vitro In vitro

Ref.

Precl.

[17]

Precl. Precl. Precl. Precl. Precl.

[94,95] [96] [97] [98] [99]

Halted Halted Halted Ongoing

[74,100] [75,76] [73] [72,101]

Precl. N/A N/A

[69,16] [102] [103]

Further humanized; N/A, data not available. P, prophylaxis; T, therapy; aa, amino acids; Hu, human; Mu, murine.

levels of uncomplexed antibodies to enable effective neutralization of circulating HBV to prevent viral intrahepatic spread. In this context, HBV entry into infectionnaı¨ve liver cells is now recognized to be important for the maintenance of the cccDNA intrahepatic pool [79]. Taken together, these findings suggest that mAbs targeting viral envelope epitopes might have a potential for combination therapies in CHB patients. The development of either anti-HBs mAbs with superior properties or

targeting other regions on the envelope protein, such as PreS1, should be further investigated. For instance, the murine E6F6 mAb was recently humanized and engineered to extend its half-life (YTE mutation in the Fc). However, the YTE mutation, while prolonging the in vivo persistence of the mAb, also resulted in a significantly lower clearance of HBsAg in cynomolgus macaques [80]. This unfavorable feature could be compensated by additional engineering of E6F6 mAb using the sweeping antibody technology [81].

Table 2 Monovalent hepatitis B vaccines, worldwide.h Type Plasma-derived, SHBs

Recombinant, yeast derived

Recombinant, mammalian-cell derived

Name (manufacturer) i

Envelope antigen

Hepatavax-BR (Merck & Co., USA) Hevac BR (Pasteur) KGCR (Korea Green Cross) RECOMBIVAX HBR (Merck & Co.) Engerix BR (SmithKline, Belgium) GP 943 (Takeda Chem, Japan) d Heplisav BTM (Dynavax) g GS-4774 (GlobeImmune/Gilead) Gen Hevac B (Pasteur, France) b Bio-Hep-B/Sci-B-Vac, (Biotechnology General, Israel)e,a AG-3 (Hepagene, Hepacare) (Medeva, UK; Evans, UK)e,f

SHBs SHBsAg, (MHB) SHBs a SHBs SHBs a SHBs,a MHBs SHBs HBsAg, HBcAg, HBx SHBs, MHBs SHBs, MHBs, LHBs

HBsAg, 5–40 mg/dose HBsAg, 5–20 mg/dose

SHBs, MHBs, LHBs

HBsAg/Pre-S2/Pre-S1, 10–20 mg/dose

Remarks

Ref.

HBsAg, 2.5–10 mg/dose HBsAg, 10–20 mg/dose HBsAg, 10 mg/dose HBsAg, 20 mg/dose g Heat-inactivated S. cerevisiae HBsAg/Pre-S2, mg/dose disc. HBsAg/Pre-S2/Pre-S1, 2.5–10 mg/dose

[104] [85]

a

SHBs-p24. Contain non-glycosylated and glycosylated p24, gp33, gp36. c Contain non-glycosylated and glycosylated p24, gp33, gp36, p39, gp42. d Licensed in Japan. e Licensed in Israel. f Licensed in Western Europe. g Contains the CPG adjuvant, 3000 mg/dose. h Reproduced with modifications and permission from Ref. [84]. i All HBV vaccines contain an aluminum hydroxide adjuvant except for HeplisavBTM. Additional yeast derived vaccines are produced in Japan, Korea, Cuba and Germany (Hansenula polymorpha). SHBs, small HBsAg; MHBs, medium HBsAg; LHBs, large HBsAg. b

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Viral envelope-specific antibodies Corti, Benigni and Shouval 53

Another example for a combined strategy included injection of a neutralizing anti-HBs mAb (mAb-H) into tolerant HBV carrier mice (AAV-HBV mice), followed by vaccination with recombinant HBsAg vaccine. This led to the production of protective anti-HBs antibodies and HBsAg loss, without inducing liver injury [22]. A similar approach was reported in liver transplant patients with HBV infection in Hong-Kong treated with lamivudine and monthly injections of a preS1/preS2/S HBV vaccine leading to active anti-HBs seroconversion of 50% [82]. Finally, monthly treatment of 8 lamivudine-treated CHB patients with HBIG followed by HBV vaccination, led to >1 log reduction of HBsAg levels in 50% of patients and in 25% to the discontinuation of antiviral therapy due to sustained HBsAg loss [78] (Table 1). Hepatitis B vaccines for pre-exposure prophylaxis

Vaccines against hepatitis B have been available for almost three decades [83,84]. Highly immunogenic, first generation, plasma-derived hepatitis B vaccines containing purified HBsAg from HBV carriers, were already developed in the late 1970s in the US and France. In the mid 1980s, 2nd generation recombinant hepatitis B vaccines coding for the

non-glycosylated form of small HBsAg were produced in yeasts and are currently used for universal vaccination of newborns and adults in >180 countries worldwide. Several attempts have been made to improve the immunogenicity of 2nd generation HBV vaccines using experimental adjuvants instead of alum. Adjuvants such as MPL, MF59 and CpG have enhanced vaccine immunogenicity, albeit associated with increased risk of local reactions at injection site. Third generation HBV vaccines were further developed in mammalian cells to also include glycosylated Pre-S2 or PreS1 and Pre-S2 regions [84–87] (Table 2). Anti-HBs antibodies are the most useful correlate of vaccine induced protection against HBV infection. By convention, an antiHBs titer of 10 mIU/ml obtained following immunization or after recovery from hepatitis B is considered protective against HBV challenge. Yet, the humoral immune response following vaccination differs depending on vaccine type and adjuvant. Thus, vaccinees of PreS/S vaccines have seroconverted much faster and with a significantly higher quantitative anti-HBs response as compared to subjects immunized with alum adjuvanted recombinant HBsAg [85]. This enhanced immunogenicity was linked to the presence of PreS epitopes, as well as to glycosylation of the envelope

Figure 1

SVP OTHER THERAPEUTIC APPROACHES FcgRs HBV

Inhibitors of viral trancription and translation cccDNA targeting Nucleocapsid assembly and packaging inhibitors Viral polymerase inhibitors Therapeutic vaccines Innate immunity modulators Checkpoint modulators Entry inhibitors Interferons

Phagocyte

NCTP HSPG

Entry

Hepatocyte

Increased viral antigen clearance Reduced immune exhaustion cccDNA Viral replication

FUNCTIONAL CURE

Viral proteins RNA Effector cell

Current Opinion in Virology

Mechanisms of action of envelope-specific mAbs. Schematic representation of the various interacting mechanisms for envelope-specific mAbs, mediating viral entry blockade, antigen clearance, and antibody-dependent antiviral effector actions, in the context of the different HBV therapeutic approaches available. SVP, subviral particles.

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54 Antiviral strategies

proteins. Protection against HBV has been documented for over 3 decades post successful immunization [88]. Once established through vaccination, immunity against HBV is usually maintained for the lifetime, even if anti-HBs levels become undetectable. Yet, immunization with HBV vaccines does not guarantee sterilizing immunity against HBV, since ‘silent’ anti-HBc seroconversion following exposure to HBV has been repeatedly documented in vaccinees and especially in those with waning anti-HBs levels post vaccination [89,90]. Furthermore, chimpanzees and humans vaccinated with recombinant HBsAg exposed to HBV can develop humoral and cellular immunity to the vaccine unrelated HBcAg and viral polymerase, providing indirect evidence that vaccine-induced immunity is not always sterilizing [91]. Immunization with yeast-derived HBV vaccines is associated with a non-responder rate of 5–10% in children or even higher in adults. Vaccination failure rates increase with age, obesity, smoking, male gender and systemic disease, as well as under conditions of immune suppression. Non-response to conventional vaccines is also genetically controlled through dominant response gene(s). The more immunogenic 3rd generation HBV vaccines comprising PreS/S epitopes have reduced the number of non-responders to conventional vaccination [84,92,93].

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Open questions and future directions

10. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H et al.: Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. elife 2012, 1:e00049.

A better understanding of the B cell phenotype and defects in CHB patients in the different phases of the disease is highly warranted. More information is needed regarding the epitope specificity and the evolution of the anti-HBs antibody response in patients undergoing HBsAg seroconversion. This added knowledge might lead to improved definitions of humoral correlates of disease control, possibly guiding the development of effective therapeutic strategies beyond the functional cure of CHB. Several strategies have and are being explored to enhance a functional cure and possibly even eradication. These include one or a combination of various approaches, such as: HBV receptor entry inhibitors, HBV-RNA interference, capsid inhibitors, HBsAg synthesis and release inhibitors, cccDNA-targeting drugs, immune modulation through checkpoint inhibitors or effector function enhancers, as well as new generation highly immunogenic therapeutic vaccines. Based on their multiple direct and indirect antiviral mechanisms of action, HBV-envelope-specific mAbs may be considered in combination regimens with one or more of the new anti-viral strategies mentioned above (Figure 1).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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