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

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1 Dissecting the divergent effects of interferon-alpha on immune cells: Time to rethink combination therapy in chronic hepatitis B? Robert Thimme 1,, Maura Dandri 2 1 Dept. Internal Medicine II, University Hospital, Freiburg, Germany; 2 Dept. Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany See Article, pages Chronic infection with the hepatitis B virus () 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 -associated liver pathologies. Since 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 persistence. While the ability of the genome to form a stable minichromosome, the so-called covalently closed circular DA (), within the hepatocyte nuclei enables the virus to persist in infected hepatocytes [1], 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 to suppress innate immunity [2 5] and high levels of viral antigens (HBeAg and HBsAg) have been linked to T-cell exhaustion in chronically -infected individuals [6,7]. Current treatment options involve the use of nucleos(t)ide analogs (UCs), which efficiently block the replication pathway by acting as inhibitors of the viral polymerase, and pegylated -a (Pega) which has been shown to have both immunomodulatory and direct antiviral effects. Since the therapeutic concepts available do not enable eradication, the current major aim of medical treatment is to prevent the progression of liver disease by suppressing replication [8]. Because UCs do not target transcription [1,9], they have only modest effects on the production of circulating viral antigens (HBsAg, HBeAg). As a consequence, monotherapy with UCs can be a life commitment, during which immunological control is rarely achieved. In contrast, reduction of serum HBsAg is more Keywords: Hepatitis B virus; Interferon; cells; + T cells. Received 7 ovember 2012; accepted 8 ovember 2012 q DOI of original article: Corresponding author. address: robert.thimme@uniklinik-freiburg.de (R. Thimme). marked in patients treated with Pega [10]. Since HBsAg loss and seroconversion represent the closest outcome to clinical cure, -a treatment remains a valuable anti- therapeutic approach. Yet, only a minority of patients responds to a therapy and the mechanisms that drive viral antigen decline and sustained virological responses in -a-treated patients are not entirely understood. -a has been shown to induce a complex network of intracellular signaling on the infected hepatocytes. While studies in -transgenic mice have reported its capacity to accelerate pgra degradation and core particle decay [11 14], recent studies performed in vitro and in -infected humanized mice showed that -a can reduce the levels of both pregenomic and subgenomic -RAs by inducing epigenetic modifications of the histones bound to the minichromosome [15]. Thus, it is conceivable that -a directly contributes to the decline of viral antigen amounts (HBeAg, HBsAg) by targeting transcription. On the other hand, the effectiveness of the immune status is known to be essential to achieve control of infection and treatment outcome may be mostly triggered by the immune modulatory effects of Pega 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]. In infection, however, studies in chimpanzees [17] and humans [18] have suggested that acute infection can be successfully controlled in the absence of a detectable -a response. Remarkably, the expression of -stimulated genes (ISGs) was found to remain unchanged in the liver of chimpanzees during the early phases of infection and spread, while a clear induction of -regulated genes and a large number of + T effector cells was detected during viral clearance, emphasizing the importance of the adaptive immune response in terminating the infection [17]. Accordingly, the depletion of + T cells resulted in loss of control in acute infection [19]. 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 Journal of Hepatology 2013 vol. 58 j

2 responses have been reported [20]. Among the mechanisms that contribute to these functional alterations are the expression of inhibitory receptors on the surface of -specific + T cells, such as PD-1 or, the suppressive action of the immunosuppressive cytokine IL-10 on cells and T cells, or the action of regulatory T cells [16,21]. Importantly, very little information is currently available about the effects of Pega on the possible restoration of innate and adaptive immunity in chronic infection. In this issue, Micco et al. [22] address this important issue and provide important novel insights into the immunomodulatory action of -a. Indeed, one important finding of their study is that treatment with Pega leads to a significant induction of the cytokine IL-15 and an expansion of natural killer (CD56 bright ) cells. oteworthy, the expansion of functional CD56 bright cells correlated with the peak of virological responses, suggesting a direct involvement of cells to the -a-mediated antiviral effects. The receptor involved in the regulation of apoptosis/necrosis,, may play an important role in this setting, since patients with a strong increase in cell expression showed significantly greater reductions in viral load and a trend to greater reductions in HBsAg compared to patients without an increase in expression. Although Pega-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 -mediated induction and therapy response has been reported in HCV infection [23]. Thus, the upregulation of is most likely directly mediated by -a. This hypothesis would be also in line with a previous study performed by the group of Mala Maini showing that -a can upregulate cell expression fold in vitro [24]. Treatment with Pega also led to an increased -c production of cells. This is an important finding, since previous studies have shown a selective impairment of cells obtained from chronically infected patients to produce -c [25 27]. However, in contrast to their finding regarding, Micco et al. did not observe a relationship between -c responsiveness and virological outcome in the small study cohort analyzed, possibly hinting towards a dominance of cytolytic mediated effector functions [22]. This is further supported by the finding of Dunn et al., that cells contribute to liver inflammation by inducing -mediated death of the hepatocytes [24]. However, in that study, -mediated killing of hepatocytes was not completely blocked by antibodies, suggesting that cells may also use additional ligands to mediate liver damage and viral control. The mechanisms responsible for the Pega mediated restoration of 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 -c production by cells in chronic infection [27]. Indeed, Peppa et al. did not find a functional cell recovery after suppression and Micco et al., did not find a decrease in the levels of IL-10 during Pega therapy. Interestingly, however, Pega treatment did lead to an increase in the expression of the activatory receptor p46. This is an important finding, since previous HCV infection studies have linked upregulation of p46 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 p46 high cells against human hepatic stellate cells [28 30]. In contrast to the significant Pega mediated functional augmentation of cells, quite different effects were observed on T cells. Indeed, Pega therapy led to a striking reduction of + T cells, an effect that was most pronounced on predominantly end-stage effector cells compared to naïve and central memory subsets. Even more importantly, -specific + 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, were not affected by -a therapy. These results are in agreement with previous studies that have analyzed the immunomodulatory effects of -a on T cells. For example, Penna et al. have recently reported that Pega does not improve early peripheral blood -specific T cell responses in HBeAg negative chronic hepatitis [31]. Most likely, -a suppresses T cell responses by its strong antiproliferative effect. However, it is also possible that indirect effects, such an cell mediated inhibition of T cells, may contribute to the 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 Pega treatment [22], whereby hinting at the limited capacities of this treatment regimen to restore, at least on short term, -specifc T-cell functions. It is intriguing to note that -specific + T-cell functions could be restored, at least in part, after long-term treatment with UCs. In this regard, Boni et al. recently reported that -specific T cells isolated from UC-treated patients that had achieved complete control of infection displayed efficient responses after in vitro expansion [32]. Although these cells appeared still dysfunctional ex vivo, they were comparable to those of patients who spontaneously resolved 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 -treated patients, efficient suppression of infection achieved after long-term treatment with UCs may also favor some restoration of -specific T-cell reactivity [32]. In contrast, UC therapy was not shown to lead to a functional recovery of cells [27]. In sum, two very important conclusions can be drawn from these studies. First, Pega and UCs 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 infection. These results may have important clinical implications since they provide the rationale for a re-evaluation of combination therapy with Pega and UCs in chronic 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 [33,34]. It should be noted, however, that less potent UCs 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 UC therapy typically requires several weeks until replication is completely suppressed and months before the HBsAg levels begin to decrease. This is in sharp contrast to the treatment with Pega, which can induce a much 206 Journal of Hepatology 2013 vol. 58 j

3 JOURAL OF HEPATOLOGY Chronic infection p46high UC therapy p46high F γ I F F UC + Peg therapy p46high p46high Peg therapy Restoration of immune response Fig. 1. Combination therapy may lead to restoration of both arms of the immune system, cells (top row) as well as + T cells (middle row), that are typically dysfunctional during chronic infection and only partially restored by either therapy. Effects on human hepatocytes are shown in the bottom row: after driven transcription of the viral RAs, the pregenomic RA (pgra) is encapsidated and reverse transcribed by the polymerase. Through Golgi and endoplasmatic reticulum the core particles acquire the envelope and are secreted. Transcription of the subgenomic -RAs leads to the production of the envelope proteins, which are needed for virion secretion and to produce the non-infectious subviral particles (SVPs). While UCs therapy efficiently suppresses replication, administration can also suppress transcription and hence viral antigen production. more rapid HBsAg decline; presumably by inducing both suppression of transcription [15] and enhancement of -mediated cytotoxic effector functions against the infected hepatocytes [24]. Furthermore, combination therapy based on the use of Pega and polymerase inhibitors has already revealed its stronger capacity in reducing the amounts of HBcAg-positive hepatocytes and loads, compared to monotherapy [35,36]. Combining these clinical observations with the novel immunological findings discussed above, it is tempting to speculate that a late add-on therapy of Pega to an ongoing UC administration might be most beneficial. According to this scenario, UC therapy would first lead to strong suppression of viremia, thereby assisting restoration of -specific + T cells and, subsequently, Pega will be added to accelerate the decline of circulating and intrahepatic viral antigens [37] and to allow expansion of 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 Pega was added late to a current UC therapy [38]. -mediated enhancement of cell activity may also augment cell injury and compensatory hepatocyte proliferation which, in turn, may increase the fraction of -free hepatocytes and promote destabilization [39]. Finally, the use of drugs able to prevent (re)infection of the hepatocytes may be also envisaged [40]. Clearly, -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 infected patients [41]. For example, a more potent restoration of specific T-cell functions may be achieved by the additional blockade of inhibitory receptors [42,43]. Also of particular interest are new approaches based on T-cell receptor gene transfer [44,45] or the application of chimeric antigen receptors [46]. onetheless, numerous studies have shown that response is achieved only in a minority of treated patients. Although the existence of distinct host polymorphisms able to predict virological response to -based therapy has not yet been elucidated [32], further investigation of the molecular mechanisms involved in response in chronically infected patients will be indispensable, while the development of therapeutic strategies aiming Journal of Hepatology 2013 vol. 58 j

4 at inducing stronger suppression of both replication and antigen productivity is warranted. The novel insights into divergent immunomodulatory effects of are an important step in refining treatment strategies with the ultimate goal to completely eradicate. Conflict of interest The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of function in hepatitis B virus infection. J Hepatol 2009;51: [2] Visvanathan K, Skinner A, Thompson AJ, Riordan SM, Sozzi V, Edwards R, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology 2007;45: [3] Op 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. 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