Dissecting the divergent effects of interferon-alpha on immune cells: Time to rethink combination therapy in chronic hepatitis B?

Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis BJournal of HepatologyVol. 58Issue 2PreviewA better understanding of the immunomodulatory effects of PegIFNα therapy could allow more rational optimisation of future therapeutic approaches in chronic HBV infection. In this study, we evaluated dynamic changes in the innate and adaptive arms of the immune system induced by PegIFNα. Full-Text PDF Chronic infection with the hepatitis B virus (HBV) continues to be a health burden of global impact. In spite of the availability of an effective vaccine for longer than two decades, about 360 million people are chronically infected worldwide and more than one million die each year due to HBV-associated liver pathologies. Since HBV does not cause direct cytopathic effects under normal infection conditions, liver injury is mostly caused by repeated attempts of the host's immune responses to control the infection. Over the years, such attempts set the stage for the development of severe liver pathologies, such as liver cirrhosis and hepatocellular carcinoma. Both virological and host factors appear to be involved in HBV persistence. While the ability of the HBV genome to form a stable minichromosome, the so-called covalently closed circular DNA (cccDNA), within the hepatocyte nuclei enables the virus to persist in infected hepatocytes [[1]Levrero M. Pollicino T. Petersen J. Belloni L. Raimondo G. Dandri M. Control of cccDNA function in hepatitis B virus infection.J Hepatol. 2009; 51: 581-592Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar], the establishment of a complex network of virus-host interactions may permit the virus to circumvent the activation of the host antiviral responses. In this regard, various studies have shown the potential capacity of HBV to suppress innate immunity [2Visvanathan K. Skinner N.A. Thompson A.J. Riordan S.M. Sozzi V. Edwards R. et al.Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.Hepatology. 2007; 45: 102-110Crossref PubMed Scopus (280) Google Scholar, 3Op den Brouw ML. Binda RS. van Roosmalen MH. Protzer U. Janssen HL. van der Molen RG. et al.Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus.Immunology. 2009; 126: 280-289Crossref PubMed Scopus (177) Google Scholar, 4Lang T. Lo C. Skinner N. Locarnini S. Visvanathan K. Mansell A. The hepatitis B e antigen (HBeAg) targets and suppresses activation of the toll-like receptor signaling pathway.J Hepatol. 2011; 55: 762-769Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 5Lutgehetmann M. Bornscheuer T. Volz T. Allweiss L. Bockmann J.H. Pollok J.M. et al.Hepatitis B virus limits response of human hepatocytes to interferon-alpha in chimeric mice.Gastroenterology. 2011; PubMed Google Scholar] and high levels of viral antigens (HBeAg and HBsAg) have been linked to T-cell exhaustion in chronically HBV-infected individuals [6Das A. Hoare M. Davies N. Lopes A.R. Dunn C. Kennedy P.T. et al.Functional skewing of the global CD8 T cell population in chronic hepatitis B virus infection.J Exp Med. 2008; 205: 2111-2124Crossref PubMed Scopus (191) Google Scholar, 7Boni C. Fisicaro P. Valdatta C. Amadei B. Di Vincenzo P. Giuberti T. et al.Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection.J Virol. 2007; 81: 4215-4225Crossref PubMed Scopus (701) Google Scholar]. Current treatment options involve the use of nucleos(t)ide analogs (NUCs), which efficiently block the HBV replication pathway by acting as inhibitors of the viral polymerase, and pegylated IFN-α (PegIFNα) which has been shown to have both immunomodulatory and direct antiviral effects. Since the therapeutic concepts available do not enable cccDNA eradication, the current major aim of HBV medical treatment is to prevent the progression of liver disease by suppressing HBV replication [[8]EASL EASL clinical practice guidelines: management of chronic hepatitis B.J Hepatol. 2009; 50: 227-242Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar]. Because NUCs do not target cccDNA transcription [1Levrero M. Pollicino T. Petersen J. Belloni L. Raimondo G. Dandri M. Control of cccDNA function in hepatitis B virus infection.J Hepatol. 2009; 51: 581-592Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 9Nguyen T. Locarnini S. Hepatitis: monitoring drug therapy for hepatitis B–a global challenge?.Nat Rev Gastroenterol Hepatol. 2009; 6: 565-567Crossref PubMed Scopus (16) Google Scholar], they have only modest effects on the production of circulating viral antigens (HBsAg, HBeAg). As a consequence, monotherapy with NUCs can be a life commitment, during which immunological control is rarely achieved. In contrast, reduction of serum HBsAg is more marked in patients treated with PegIFNα [[10]Brunetto M.R. Moriconi F. Bonino F. Lau G.K. Farci P. Yurdaydin C. et al.Hepatitis B virus surface antigen levels: a guide to sustained response to peginterferon alfa-2a in HBeAg-negative chronic hepatitis B.Hepatology. 2009; 49: 1141-1150Crossref PubMed Scopus (401) Google Scholar]. Since HBsAg loss and seroconversion represent the closest outcome to clinical cure, IFN-α treatment remains a valuable anti-HBV therapeutic approach. Yet, only a minority of HBV patients responds to IFN-α therapy and the mechanisms that drive viral antigen decline and sustained virological responses in IFN-α-treated patients are not entirely understood. IFN-α has been shown to induce a complex network of intracellular signaling on the infected hepatocytes. While studies in HBV-transgenic mice have reported its capacity to accelerate pgRNA degradation and core particle decay [11Wieland S.F. Guidotti L.G. Chisari F.V. Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice.J Virol. 2000; 74: 4165-4173Crossref PubMed Scopus (211) Google Scholar, 12Wieland S.F. Eustaquio A. Whitten-Bauer C. Boyd B. Chisari F.V. Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids.Proc Natl Acad Sci USA. 2005; 102: 9913-9917Crossref PubMed Scopus (146) Google Scholar, 13Uprichard S.L. Wieland S.F. Althage A. Chisari F.V. Transcriptional and posttranscriptional control of hepatitis B virus gene expression.Proc Natl Acad Sci USA. 2003; 100: 1310-1315Crossref PubMed Scopus (73) Google Scholar, 14Xu C. Guo H. Pan X.B. Mao R. Yu W. Xu X. et al.Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus.J Virol. 2010; 84: 9332-9340Crossref PubMed Scopus (98) Google Scholar], recent studies performed in vitro and in HBV-infected humanized mice showed that IFN-α can reduce the levels of both pregenomic and subgenomic HBV-RNAs by inducing epigenetic modifications of the histones bound to the cccDNA minichromosome [[15]Belloni L. Allweiss L. Guerrieri F. Pediconi N. Volz T. Pollicino T. et al.IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.J Clin Invest. 2012; 122: 529-537Crossref PubMed Scopus (440) Google Scholar]. Thus, it is conceivable that IFN-α directly contributes to the decline of viral antigen amounts (HBeAg, HBsAg) by targeting cccDNA transcription. On the other hand, the effectiveness of the immune status is known to be essential to achieve control of HBV infection and treatment outcome may be mostly triggered by the immune modulatory effects of PegIFNα on the innate and adaptive immune responses. Spontaneous resolution of viral infections typically requires combined innate and adaptive immune responses, since the main effectors of adaptive immunity are only activated following viral recognition by the innate immune system, which represents a first line defense against pathogens [[16]Maini M.K. Schurich A. The molecular basis of the failed immune response in chronic HBV: therapeutic implications.J Hepatol. 2010; 52: 616-619Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar]. In HBV infection, however, studies in chimpanzees [[17]Wieland S. Thimme R. Purcell R.H. Chisari F.V. Genomic analysis of the host response to hepatitis B virus infection.Proc Natl Acad Sci USA. 2004; 101: 6669-6674Crossref PubMed Scopus (551) Google Scholar] and humans [[18]Dunn C. Peppa D. Khanna P. Nebbia G. Jones M. Brendish N. et al.Temporal analysis of early immune responses in patients with acute hepatitis B virus infection.Gastroenterology. 2009; 137: 1289-1300Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar] have suggested that acute infection can be successfully controlled in the absence of a detectable IFN-α response. Remarkably, the expression of IFN-stimulated genes (ISGs) was found to remain unchanged in the liver of chimpanzees during the early phases of HBV infection and spread, while a clear induction of IFN-regulated genes and a large number of CD8+ T effector cells was detected during viral clearance, emphasizing the importance of the adaptive immune response in terminating the infection [[17]Wieland S. Thimme R. Purcell R.H. Chisari F.V. Genomic analysis of the host response to hepatitis B virus infection.Proc Natl Acad Sci USA. 2004; 101: 6669-6674Crossref PubMed Scopus (551) Google Scholar]. Accordingly, the depletion of CD8+ T cells resulted in loss of control in acute HBV infection [[19]Thimme R. Wieland S. Steiger C. Ghrayeb J. Reimann K.A. Purcell R.H. et al.CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection.J Virol. 2003; 77: 68-76Crossref PubMed Scopus (754) Google Scholar]. In chronic infection, however, distinct virus-mediated mechanisms may contribute to the limited effectiveness of the antiviral response and functional alterations of both innate and adaptive responses have been reported [[20]Dandri M. Locarnini S. New insight in the pathobiology of hepatitis B virus infection.Gut. 2012; 61: i6-17Crossref PubMed Scopus (199) Google Scholar]. Among the mechanisms that contribute to these functional alterations are the expression of inhibitory receptors on the surface of HBV-specific CD8+ T cells, such as PD-1 or CTLA-4, the suppressive action of the immunosuppressive cytokine IL-10 on NK cells and T cells, or the action of regulatory T cells [16Maini M.K. Schurich A. The molecular basis of the failed immune response in chronic HBV: therapeutic implications.J Hepatol. 2010; 52: 616-619Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Billerbeck E. Bottler T. Thimme R. Regulatory T cells in viral hepatitis.World J Gastroenterol. 2007; 13: 4858-4864PubMed Google Scholar]. Importantly, very little information is currently available about the effects of PegIFNα on the possible restoration of innate and adaptive immunity in chronic HBV-infection. In this issue, Micco et al. [[22]Micco L. Peppa D. Loggi E. Schurich A. Jefferson L. Cursaro C. et al.Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B.J Hepatol. 2013; 58: 225-233Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar] address this important issue and provide important novel insights into the immunomodulatory action of IFN-α. Indeed, one important finding of their study is that treatment with PegIFNα leads to a significant induction of the cytokine IL-15 and an expansion of natural killer (CD56bright NK) cells. Noteworthy, the expansion of functional CD56bright NK cells correlated with the peak of virological responses, suggesting a direct involvement of NK cells to the IFN-α-mediated antiviral effects. The receptor involved in the regulation of apoptosis/necrosis, TRAIL, may play an important role in this setting, since patients with a strong increase in NK cell TRAIL expression showed significantly greater reductions in viral load and a trend to greater reductions in HBsAg compared to patients without an increase in TRAIL expression. Although PegIFNα-administration may have also directly contributed to the decline of viral loads by suppressing viral transcription, it is worth noting that a similar association between the IFN-mediated TRAIL induction and therapy response has been reported in HCV infection [[23]Stegmann K.A. Bjorkstrom N.K. Veber H. Ciesek S. Riese P. Wiegand J. et al.Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection.Gastroenterology. 2010; 138: 1885-1897Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar]. Thus, the upregulation of TRAIL is most likely directly mediated by IFN-α. This hypothesis would be also in line with a previous study performed by the group of Mala Maini showing that IFN-α can upregulate NK cell TRAIL expression 10–30-fold in vitro [[24]Dunn C. Brunetto M. Reynolds G. Christophides T. Kennedy P.T. Lampertico P. et al.Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage.J Exp Med. 2007; 204: 667-680Crossref PubMed Scopus (344) Google Scholar]. Treatment with PegIFNα also led to an increased IFN-γ production of NK cells. This is an important finding, since previous studies have shown a selective impairment of NK cells obtained from chronically HBV infected patients to produce IFN-γ [25Oliviero B. Varchetta S. Paudice E. Michelone G. Zaramella M. Mavilio D. et al.Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections.Gastroenterology. 2009; 137 (1160 e1151–1157): 1151-1160Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 26Tjwa E.T. van Oord G.W. Hegmans J.P. Janssen H.L. Woltman A.M. Viral load reduction improves activation and function of natural killer cells in patients with chronic hepatitis B.J Hepatol. 2011; 54: 209-218Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 27Peppa D. Micco L. Javaid A. Kennedy P.T. Schurich A. Dunn C. et al.Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.PLoS Pathog. 2010; 6: e1001227Crossref PubMed Scopus (198) Google Scholar]. However, in contrast to their finding regarding TRAIL, Micco et al. did not observe a relationship between IFN-γ responsiveness and virological outcome in the small study cohort analyzed, possibly hinting towards a dominance of cytolytic TRAIL mediated effector functions [[22]Micco L. Peppa D. Loggi E. Schurich A. Jefferson L. Cursaro C. et al.Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B.J Hepatol. 2013; 58: 225-233Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar]. This is further supported by the finding of Dunn et al., that NK cells contribute to liver inflammation by inducing TRAIL-mediated death of the hepatocytes [[24]Dunn C. Brunetto M. Reynolds G. Christophides T. Kennedy P.T. Lampertico P. et al.Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage.J Exp Med. 2007; 204: 667-680Crossref PubMed Scopus (344) Google Scholar]. However, in that study, NK-mediated killing of hepatocytes was not completely blocked by TRAIL antibodies, suggesting that NK cells may also use additional ligands to mediate liver damage and viral control. The mechanisms responsible for the PegIFNα mediated restoration of NK cell effector functions are not fully understood. However, they are most likely not due to secondary effects, such as viremia reduction or a decrease in the levels of the immunosuppressive cytokine IL-10, which has been shown to suppress IFN-γ production by NK cells in chronic HBV infection [[27]Peppa D. Micco L. Javaid A. Kennedy P.T. Schurich A. Dunn C. et al.Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.PLoS Pathog. 2010; 6: e1001227Crossref PubMed Scopus (198) Google Scholar]. Indeed, Peppa et al. did not find a functional NK cell recovery after HBV suppression and Micco et al., did not find a decrease in the levels of IL-10 during PegIFNα therapy. Interestingly, however, PegIFNα treatment did lead to an increase in the expression of the activatory receptor NKp46. This is an important finding, since previous HCV infection studies have linked upregulation of NKp46 to an increased antiviral activity, that was mediated by cytolytic and non-cytolytic effector functions, as well as to an increased antifibrotic activity, that was possibly due to the higher cytotoxicity of NKp46high cells against human hepatic stellate cells [28Kramer B. Korner C. Kebschull M. Glassner A. Eisenhardt M. Nischalke H.D. et al.Natural killer p46 (High) expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis.Hepatology. 2012; 56: 1201-1213Crossref PubMed Scopus (108) Google Scholar, 29Golden-Mason L. Cox A.L. Randall J.A. Cheng L. Rosen H.R. Increased natural killer cell cytotoxicity and NKp30 expression protects against hepatitis C virus infection in high-risk individuals and inhibits replication in vitro.Hepatology. 2010; 52: 1581-1589Crossref PubMed Scopus (89) Google Scholar, 30Heeg M. Thimme R. Natural killer cells and hepatitis C: natural killer p46 expression linked to antiviral and antifibrotic activity.Hepatology. 2012; 56: 1197-1200Crossref PubMed Scopus (13) Google Scholar]. In contrast to the significant PegIFNα mediated functional augmentation of NK cells, quite different effects were observed on T cells. Indeed, PegIFNα therapy led to a striking reduction of CD8+ T cells, an effect that was most pronounced on predominantly end-stage effector cells compared to naïve and central memory subsets. Even more importantly, HBV-specific CD8+ T cells remained at low frequency and no restoration of their effector functions was observed. In line with this finding, the expression levels of inhibitory markers, such as PD-1 or CTLA-4, were not affected by IFN-α therapy. These results are in agreement with previous studies that have analyzed the immunomodulatory effects of IFN-α on T cells. For example, Penna et al. have recently reported that PegIFNα does not improve early peripheral blood HBV-specific T cell responses in HBeAg negative chronic hepatitis [[31]Penna A. Laccabue D. Libri I. Giuberti T. Schivazappa S. Alfieri A. et al.Peginterferon-alpha does not improve early peripheral blood HBV-specific T-cell responses in HBeAg-negative chronic hepatitis.J Hepatol. 2012; 56: 1239-1246Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar]. Most likely, IFN-α suppresses T cell responses by its strong antiproliferative effect. However, it is also possible that indirect effects, such an NK cell mediated inhibition of T cells, may contribute to the IFN mediated inhibition of T-cell responses. Whatever the explanation, the elegant results obtained by Micco et al. clearly demonstrate a differential boosting of innate and adaptive antiviral immune responses during PegIFNα treatment [[22]Micco L. Peppa D. Loggi E. Schurich A. Jefferson L. Cursaro C. et al.Differential boosting of innate and adaptive antiviral responses during pegylated-interferon-alpha therapy of chronic hepatitis B.J Hepatol. 2013; 58: 225-233Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar], whereby hinting at the limited capacities of this treatment regimen to restore, at least on short term, HBV-specifc T-cell functions. It is intriguing to note that HBV-specific CD8+ T-cell functions could be restored, at least in part, after long-term treatment with NUCs. In this regard, Boni et al. recently reported that HBV-specific T cells isolated from NUC-treated patients that had achieved complete control of infection displayed efficient responses after in vitro expansion [[32]Boni C. Laccabue D. Lampertico P. Giuberti T. Vigano M. Schivazappa S. et al.Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.Gastroenterology. 2012; 143: e969Google Scholar]. Although these cells appeared still dysfunctional ex vivo, they were comparable to those of patients who spontaneously resolved HBV infection and were significantly stronger compared to those of untreated chronically infected patients in their proliferative capacity in vitro. Thus, in contrast to lymphocytes isolated from IFN-treated patients, efficient suppression of HBV infection achieved after long-term treatment with NUCs may also favor some restoration of HBV-specific T-cell reactivity [[32]Boni C. Laccabue D. Lampertico P. Giuberti T. Vigano M. Schivazappa S. et al.Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.Gastroenterology. 2012; 143: e969Google Scholar]. In contrast, NUC therapy was not shown to lead to a functional recovery of NK cells [[27]Peppa D. Micco L. Javaid A. Kennedy P.T. Schurich A. Dunn C. et al.Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.PLoS Pathog. 2010; 6: e1001227Crossref PubMed Scopus (198) Google Scholar]. In sum, two very important conclusions can be drawn from these studies. First, PegIFNα and NUCs have differential effects on the innate and adaptive immune responses, and second, both drugs have shown some capabilities to restore impaired immune functions in chronic HBV infection. These results may have important clinical implications since they provide the rationale for a re-evaluation of combination therapy with PegIFNα and NUCs in chronic HBV infection. Indeed, it should be the goal of combination therapy to restore both arms of the immune system to improve the possibility of complete virus control. Combination therapy approaches that were evaluated in the past did not show improvement of post-therapy response rates [33Marcellin P. Lau G.K. Bonino F. Farci P. Hadziyannis S. Jin R. et al.Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B.N Engl J Med. 2004; 351: 1206-1217Crossref PubMed Scopus (1053) Google Scholar, 34Janssen H.L. van Zonneveld M. Senturk H. Zeuzem S. Akarca U.S. Cakaloglu Y. et al.Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial.Lancet. 2005; 365: 123-129Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar]. It should be noted, however, that less potent NUCs were used in those studies and both therapies were started at the same time. The time schedule of therapy might indeed be a critical issue in this scenario, since NUC therapy typically requires several weeks until HBV replication is completely suppressed and months before the HBsAg levels begin to decrease. This is in sharp contrast to the treatment with PegIFNα, which can induce a much more rapid HBsAg decline; presumably by inducing both suppression of cccDNA transcription [[15]Belloni L. Allweiss L. Guerrieri F. Pediconi N. Volz T. Pollicino T. et al.IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.J Clin Invest. 2012; 122: 529-537Crossref PubMed Scopus (440) Google Scholar] and enhancement of TRAIL-mediated cytotoxic effector functions against the infected hepatocytes [[24]Dunn C. Brunetto M. Reynolds G. Christophides T. Kennedy P.T. Lampertico P. et al.Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage.J Exp Med. 2007; 204: 667-680Crossref PubMed Scopus (344) Google Scholar]. Furthermore, combination therapy based on the use of PegIFNα and polymerase inhibitors has already revealed its stronger capacity in reducing the amounts of HBcAg-positive hepatocytes and cccDNA loads, compared to monotherapy [35Wursthorn K. Lutgehetmann M. Dandri M. Volz T. Buggisch P. Zollner B. et al.Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B.Hepatology. 2006; 44: 675-684Crossref PubMed Scopus (376) Google Scholar, 36Lutgehetmann M. Volzt T. Quaas A. Zankel M. Fischer C. Dandri M. et al.Sequential combination therapy leads to biochemical and histological improvement despite low ongoing intrahepatic hepatitis B virus replication.Antivir Ther. 2008; 13: 57-66PubMed Google Scholar]. Combining these clinical observations with the novel immunological findings discussed above, it is tempting to speculate that a late add-on therapy of PegIFNα to an ongoing NUC administration might be most beneficial. According to this scenario, NUC therapy would first lead to strong suppression of viremia, thereby assisting restoration of HBV-specific CD8+ T cells and, subsequently, PegIFNα will be added to accelerate the decline of circulating and intrahepatic viral antigens [[37]Allweiss L. Lütgehetmann M. Volz T. Bornscheuer T. Lohse A.W. Petersen J. et al.Pegylated-interferon-alpha alone or in combination with entecavir restores ISG responsiveness and reduces intrahepatic viral loads and antigenemia in hepatitis B virus infected humanized mice.J Hepatol. 2012; 56: S18-S19Abstract Full Text PDF Google Scholar] and to allow expansion of NK cells (Fig. 1). Importantly, the proof-of-principle of this approach has previously been reported by Kittner et al., showing HBsAg seroconversion in a subgroup of patients where PegIFNα was added late to a current NUC therapy [[38]Kittner J.M. Sprinzl M.F. Grambihler A. Weinmann A. Schattenberg J.M. Galle P.R. et al.Adding pegylated interferon to a current nucleos(t)ide therapy leads to HBsAg seroconversion in a subgroup of patients with chronic hepatitis B.J Clin Virol. 2012; 54: 93-95Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. IFN-mediated enhancement of NK cell activity may also augment cell injury and compensatory hepatocyte proliferation which, in turn, may increase the fraction of cccDNA-free hepatocytes and promote cccDNA destabilization [[39]Lutgehetmann M. Volz T. Kopke A. Broja T. Tigges E. Lohse A.W. et al.In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice.Hepatology. 2010; 52: 16-24Crossref PubMed Scopus (66) Google Scholar]. Finally, the use of drugs able to prevent (re)infection of the hepatocytes may be also envisaged [[40]Petersen J. Dandri M. Mier W. Lutgehetmann M. Volz T. von Weizsacker F. et al.Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein.Nat Biotechnol. 2008; 26: 335-341Crossref PubMed Scopus (325) Google Scholar]. Clearly, IFN-based combination therapy concepts need to be further investigated in larger, prospective studies, ideally together with a comprehensive analysis of both arms of the immune system. It is also conceivable that additional strategies will be needed to achieve efficient restoration of the adaptive immunity in chronically HBV infected patients [[41]Bertoletti A. Ferrari C. Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection.Gut. 2011; PubMed Google Scholar]. For example, a more potent restoration of HBV specific T-cell functions may be achieved by the additional blockade of inhibitory receptors [42Schurich A. Khanna P. Lopes A.R. Han K.J. Peppa D. Micco L. et al.Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-prone CD8 T cells in persistent hepatitis B virus infection.Hepatology. 2011; 53: 1494-1503Crossref PubMed Scopus (237) Google Scholar, 43Fisicaro P. Valdatta C. Massari M. Loggi E. Ravanetti L. Urbani S. et al.Combined blockade of programmed death-1 and activation of CD137 increase responses of human liver T cells against HB, V but not HCV.Gastroenterology. 2012; PubMed Google Scholar]. Also of particular interest are new approaches based on T-cell receptor gene transfer [44Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2093) Google Scholar, 45Protzer U. Abken H. Can engineered "designer" T cells outsmart chronic hepatitis B?.Hepat Res Treat. 2010; 2010: 901216PubMed Google Scholar] or the application of chimeric antigen receptors [[46]Bohne F. Chmielewski M. Ebert G. Wiegmann K. Kurschner T. Schulze A. et al.T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.Gastroenterology. 2008; 134: 239-247Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar]. Nonetheless, numerous studies have shown that IFN response is achieved only in a minority of treated patients. Although the existence of distinct host polymorphisms able to predict virological response to IFN-based therapy has not yet been elucidated [[32]Boni C. Laccabue D. Lampertico P. Giuberti T. Vigano M. Schivazappa S. et al.Restored function of HBV-specific T cells after long-term effective therapy with nucleos(t)ide analogues.Gastroenterology. 2012; 143: e969Google Scholar], further investigation of the molecular mechanisms involved in IFN response in chronically HBV infected patients will be indispensable, while the development of therapeutic strategies aiming at inducing stronger suppression of both HBV replication and antigen productivity is warranted. The novel insights into divergent immunomodulatory effects of IFN are an important step in refining treatment strategies with the ultimate goal to completely eradicate HBV. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.