Towards curative therapy of chronic viral hepatitis

Übersicht

Towards curative therapy of chronic viral hepatitis Kurative Therapien der chronischen Virushepatitis

Authors Ralf Bartenschlager1, 2, 3, Stephan Urban1, 2*, Ulrike Protzer4, 5, 6*

Key words viral hepatitis, hepatitis virus, hepatitis B, hepatitis D, hepatitis C, curative therapy Schlüsselwörter Virushepatitis, Hepatitis Virus, Hepatitis B, Hepatitis D, Hepatitis C, Kurative Therapie received 04.10.2018 accepted 18.12.2018

Tuberkulose. Diese Zahlen belegen eindrücklich, dass mehr Anstrengungen unternommen werden müssen, um bessere Therapien, die idealerweise kurativ sind, zu entwickeln und auf breiter Basis verfügbar zu machen, so wie es beispielsweise bei der Therapie der HIV-Infektion erreicht wurde. Ein wichtiger Schritt in diese Richtung war die Entwicklung von kurativen Therapien der Hepatitis-C-Virus-Infektion. Im Fall des Hepatitis-D-Virus wurden neue Wirkstoffe entwickelt, mit deren Zulassung wir schon sehr bald rechnen können. Bei der chronischen Hepatitis B hinkt die Entwicklung hinterher, aber motiviert durch die Therapieerfolge in der chronischen Hepatitis C und der Verfügbarkeit neuer Infektionssysteme wurde das Interesse der pharmazeutischen Industrie an einer kurativen Therapie der Hepatitis B geweckt. Zahlreiche Wirkstoffkandidaten befinden sich zurzeit in präklinischer und klinischer Entwicklung und man darf hoffen, dass diese die Klinik erreichen und zumindest bei einem Teil der Patienten kurativ wirken. In dieser Übersicht fassen wir den aktuellen Stand der Therapieentwicklungen für die chronische Virushepatitis mit dem Fernziel der Heilung zusammen.

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Affiliations 1 Department für Infektiologie, Molekulare Virologie, Universitätsklinikum Heidelberg, Heidelberg 2 Deutsches Zentrum für Infektionsforschung (DZIF), Standort Heidelberg 3 Abteilung Virus-Assoziierte Karzinogenese, Deutsches Krebsforschungszentrum Heidelberg 4 Institut für Virologie, Technische Universität München 5 Helmholtz Zentrum München 6 Deutsches Zentrum für Infektionsforschung (DZIF), Standort München

ABSTR AC T Bibliography DOI https://doi.org/10.1055/a-0824-1576 Z Gastroenterol 2019; 57: 61–73 © Georg Thieme Verlag KG, Stuttgart · New York ISSN 0044-2771 Correspondence Prof. Dr. Ralf Bartenschlager Department für Infektiologie, Molekulare Virologie, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 344, 69120 Heidelberg, Germany Tel.: ++ 49/62 21/56 42 25 Fax: ++ 49/62 21/56 45 70 [email protected] Z US A M M E N FA SS U N G Persistente Infektionen mit Hepatitis-Viren sind ein wichtiges medizinisches Problem, das in der Öffentlichkeit häufig wenig Beachtung findet. Daraus resultiert eine massive Unterdiagnose dieser Infektionen. Nach jüngsten Schätzungen der Weltgesundheitsorganisation ist die Zahl der Todesfälle aufgrund einer Virushepatitis mittlerweile höher als die durch Malaria und HIV-Infektionen und vergleichbar mit der durch

*

Persistent infection with hepatitis viruses is a major medical burden that unfortunately is often ignored in the general public. This results in a massive under-diagnosis of these infections. According to the world health organization, the number of people dying every year from the consequence of hepatitis virus infection has surpassed the death rate caused by malaria and HIV and is equivalent to the one of tuberculosis. These numbers call for more intense efforts to devise more effective, ideally curative therapies that must become as widely accessible as they are e. g. for HIV infected individuals. A major step forward has been the development of curative antiviral therapy for chronic hepatitis C. In the case of hepatitis D virus, new drug candidates have been developed that should receive conditional approval very soon. For chronic hepatitis B, development is lagging behind but thanks to a re-emerging interest of pharmaceutical industry that was triggered by the success of hepatitis C therapy and the availability of novel infection systems, new attempts towards curative approaches for this highly prevalent infection are undertaken. Here we summarize the current status of therapy development for chronic viral hepatitis that ultimately aims at cure.

These authors contributed equally.

Bartenschlager R et al. Towards curative therapy… Z Gastroenterol 2019; 57: 61–73

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Übersicht

▶ Table 1 Overview about the properties of hepatitis viruses and the diseases they can cause. HAV

HBV

HCV

HDV

HEV

family

Picornaviridae

Hepadnaviridae

Flaviviridae

Deltavirus

Hepeviridae

replication strategy

RdRp; cytoplasmic

RT; nuclear cccDNA

RdRp; cytoplasmic

cellular RNA pol II; HBV dependent

RdRp; cytoplasmic

course of infection

acute self-limiting; no chronicity

chronicity 95 – 10 %; age dependent

55 – 85 %; age independent

> 90 % persistence in super-infection

acute self-limiting; rarely chronic (pre-disposition)

selective therapy

none

virosuppressive

curative

none

none

vaccine

yes

yes

no

yes (HBV vaccine)

only in China

Introduction Hepatitis viruses are a heterogeneous group of human pathogens that share a pronounced hepatotropism, but differ remarkably in their biological properties (▶ Table 1) such as virion morphology and the replication strategy. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are mainly transmitted by the fecal-oral route and cause acute self-limiting infections and only rarely, they cause prolonged or even chronic infections (e. g. during immunosuppression after transplantation) [1, 2]. In contrast, infections with the hepatitis B virus (HBV), the hepatitis C virus (HCV) and the hepatitis D virus (HDV) are transmitted parenterally by bloodblood contact. HBV and probably also HDV are additionally transmitted by the sexual route [3]. A hallmark of these hepatitis viruses is their propensity to become chronic. While HCV persists in up to 75 % of cases, HBV persistence very much depends on the age of an individual at transmission. Perinatal HBV infections almost uniformly become chronic whereas adults clear the infection in 90 – 95 % of cases. Although HDV prevalence is highly variable between different countries, overall up to 10 % of chronic hepatitis B patients are co-infected with HDV [4]. The clinical consequences of HBV-HDV coinfection can be dramatic because it causes the most severe form of viral hepatitis. HBV and HCV infections show a broad spectrum of clinical manifestations reaching from asymptomatic infection to inflammatory liver disease, i. e. viral hepatitis that can be accompanied by liver failure or result in liver cirrhosis and the development of hepatocellular carcinoma (HCC) [5]. Liver disease observed in HBV infection is much exacerbated by co- or super-infection with HDV; accordingly, HBV-/ HDV co-infected individuals have a very high risk to develop serious liver damage within a short period of time. Interestingly, HDV is a satellite virus relying on HBV that serves as helper by providing the surface glycoproteins to form infectious virions. Although in the absence of the HBV encoded envelope proteins, hepatocytes can be infected with HDV, and replicate the viral RNA, infectious virus particles cannot be released. Under those conditions HDV remains strictly intracellular and may only spread by division of the HDV infected cell [6]. Infections with hepatitis viruses are amongst the most prevalent ones worldwide [7]. Overall, the number of deaths caused by viral hepatitis (> 1.2 mio per year) has surpassed that caused by all

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other virus infections such as HIV and has reached the level of deaths caused by tuberculosis [7]. Importantly, in contrast to all the other infectious diseases where the number of deaths is steadily declining, the number of deaths caused by viral hepatitis is continuously increasing. For HBV it is estimated that around two fifth of the world population has had an infection with this virus. Of these, 257 million were unable to clear the infection and are persistent virus carriers; around 880 000 individuals die every year from the consequence of HBV infection to a large part because the disease is widely under-diagnosed [8]. It is estimated that 15 – 25 million HBV carriers are co-infected with HDV [4]. In the case of HCV around 71 million people are chronically infected, but most people are unaware of their infection that is predominantly asymptomatic. Infection rates of HAV very much depend on sanitary conditions and in areas with low hygiene standards around 90 % of children have had HAV infection before the age of 10 [9]. In the case of HEV around 20 million individuals are infected annually, half of them showing symptoms [7]. HEV infections are zoonotic and transmission often occurs via consumption of pork meat or meat products, especially for HEV genotype 3 that is most prevalent in the Western world. Long term sequelae of HBV, HCV and HDV infection are liver cirrhosis and hepatocellular carcinoma (HCC). Importantly, around 50 % of all liver cancer cases worldwide are associated with HBV (including coinfection with HDV) and an additional 25 % with HCV infection [10]. Although viral hepatitis is a major risk factor for the development of liver cirrhosis and liver cancer, the likelihood for these disease manifestations very much depends on cofactors, the most important ones being obesity and excessive alcohol consumption. While in the case of HCV liver cancer (notably HCC) follows prior liver cirrhosis, in chronic HBV infection HCC can develop also in non-cirrhotic patients, arguing for a more direct oncogenic effect of this virus [5]. Hereby, the 5-year cumulative HCC risk ranges from 0.1 % – 0.3 % in inactive carriers, 0.6 % – 2.4 % in chronic hepatitis B patients and 9.7 % – 15.5 % in cirrhotics, with the rates being higher in patients from East Asia as compared to Europe [11]. Despite important advances in clinical care and prevention of viral hepatitis, the high disease burden requires further public health, scientific and clinical efforts. While for HCV curative thera-



RdRp, RNA-dependent RNA polymerase; RT, reverse transcriptase; cccDNA, covalently closed circular DNA; RNA pol II, DNA-dependent RNA polymerase II.

Towards curative therapy for chronic hepatitis B Therapies controlling HBV HBV is a DNA virus replicating in a rather complicated fashion by reverse transcription of a pregenomic RNA intermediate (▶ Fig. 1). The virus expresses only one protein with enzymatic activities, the Pol protein that acts as primer for reverse transcription and has in addition reverse transcriptase as well as ribonuclease H (RNase H) activities. These are required to synthesize new HBV DNA genomes from the RNA pregenome that is degraded during second strand DNA synthesis. Thus, HBV polymerase has been a primary target for drug development [12]. Consequently, six nucleos(t)ide analogues (NA) have been FDA-approved that all target the reverse transcriptase activity of HBV Pol (▶ Table 2). Three of those, entecavir, tenofovir disoproxil and its improved version, tenofovir alafenamide, are in the mainstay of current therapies and widely used due to their high barrier to resistance [13]. By using next-generation prodrugs of such NAs (e. g. Tenofovir exalidex, CMX157, AGX-1009, Besifovir, and Lagociclovir valactate), current attempts aim at improving antiviral efficacy, oral bioavailability, and long-term tolerability. In general, NAs are very well tolerated and efficiently control virus replication to levels undetectable in peripheral blood by DNA-based detection methods. Long term NA treatment is associated with a partial restoration in HBV-specific T-cell functions, regression of inflammation and a slow-down of disease progression. However, NA therapies rarely lead to cure and life-long treatments is mostly required [14]. Less than 5 % of chronic hepatitis B patients worldwide have access to antiviral drugs and, even in Europe and in the US, treatment indication is limited to those who have developed inflammatory liver disease. Less than 1 % of HBeAg-negative and no more than 10 % of HBeAg-positive patients achieve anti-HBs (hepatitis B surface antigen) seroconversion after 3 – 5 years of continuous NA therapy [13], a rate that does not significantly exceed that of spontaneous control of HBV infection. Thus patients still face the stigma of chronic infection. In addition, despite long-term NA therapy there is a 5 – 7-fold increased risk to develop HCC, especially in patients with liver cirrhosis [15, 16], but also a 5-year cumulative HCC incidence


rate of 5 – 8 % in Caucasian patients without cirrhosis [17]. Caucasian patients receiving long-term entecavir or tenofovir therapy and having compensated liver disease have an overall eight-year survival similar to that of the general population, but only if they do not develop HCC [17]. Because of these limitations there is an urgent demand for alternative and ideally curative treatments. Alternative strategies to develop directly acting antivirals against HBV target the assembly of the viral nucleocapsid, the viral RNAse H or RNA translation, the latter by using antisense oligonucleotides or small inferring RNAs (siRNAs) (▶ Table 2). Other steps in the viral life cycle that are targeted are entry into hepatocytes e. g. by the receptor-targeting peptide Myrcludex B or neutralizing antibodies, or limiting envelope protein release from infected cells e. g. by nucleic acid polymers (summarized in [18, 19]). A number of capsid assembly modifiers or inhibitors [20] are under development and two of them, developed by Novira Therapeutics Inc and by Arbutus Biopharma, have already entered phase Ib/IIa studies. It will be exciting to see whether these drugs that prevent the formation of mature capsids and virions will be superior to NAs. It is important to note, however, that NAs and capsid assembly modifiers as well as RNA translation inhibitors act at late steps in the viral life cycle and do not target the covalently closed circular (ccc) HBV DNA genome that the virus deposits in the nucleus of infected cells to establish and sustain persistence [21, 22].

Is HBV cure possible at all? Sterilizing HBV “cure” would require eliminating all HBV infected hepatocytes – even those carrying integrated HBV-DNA – or at least purge all HBV DNA from infected cells. This, however, seems not realistic since even after spontaneous resolution of HBV infection low level persistence is observed for decades [23]. This indicates on the one hand a functional rather than a complete cure, and on the other hand that the immune system is able to keep HBV in check. Taking this into account, an expert panel from Europe and the USA discussing options to measure HBV cure proposed to define “functional cure” of HBV as clearance of circulating HBsAg, ideally followed by a seroconversion to anti-HBs, which is a status when antibodies against HBsAg become detectable in the blood [24]. Achieving functional cure would parallel spontaneous HBV control and seems a much more realistic goal for a new therapy. The goal of the world health organization (WHO) is to eliminate HBV as a major public health threat until 2030. A recent study predicts that only a combination of a better vaccination coverage, innovations in prevention of mother-to-child transmission, and ambitious population-wide testing and treatment will allow reaching this goal [25]. Barriers to implementing these measures and associated costs are high, but could be largely reduced if a curative treatment would exist [25]. Thus, there is a high medical need for the development of new antiviral strategies to achieve at least a functional cure of HBV and reduce HBV-related disease burden. This will most likely require the combination of antiviral therapies that includes an immune stimulatory approach.

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pies have been developed there is no vaccine available (▶ Table 1). In the case of HBV a prophylactic vaccine is available for decades that also protects against HDV infection, but its world-wide coverage is insufficient. For the huge number of patients with chronic HBV infection and at high risk to develop liver cirrhosis or HCC, a finite, curative treatment is lacking. Last but not least, for HDV infection no specific therapy has been approved yet. This calls for more intense efforts to develop novel preventive and therapeutic concepts for viral hepatitis. In this review we will briefly summarize the recent progress that has been made toward curative antiviral drugs for those viruses that cause chronic hepatitis with the aim to address an important unmet medical need.

▶ Fig. 1 Schematic of the replication cycles of HCV, HBV and HDV. Upon entry of HCV (top sector), viral RNA is translated at the ER and a membranous replication factory is formed (membranous web). There, viral RNA is amplified and packaged into nucleocapsids that bud into the ER. Enveloped virions are secreted out of the cell. In the case of HBV (lower right sector), upon entry in an NTCP dependent manner, the partially double stranded viral DNA genome is delivered into the nucleus where it is converted into the cccDNA form persisting as nuclear episome. Viral (v)RNAs are transcribed and used for protein synthesis. Within the nucleocapsids the viral RNA pre-genome is reverse transcribed (the red dot indicates the co-packaged P protein) and virions are formed by budding into the ER lumen. Virions are secreted from the cell, along with subviral particles (SVPs) that lack a nucleocapsid and are therefore non-infectious. SVPs are composed of the same envelope proteins as infectious HBV particles. In the case of HDV (lower left sector), the virus enters hepatocytes probably in the same way as HBV does. The viral RNA genome is delivered into the nucleus and used for the production of mRNAs that are translated in the cytoplasm. From there the two HDV proteins are imported into the nucleus to form RNPs. These are exported into the cytoplasm where they acquire their envelope via budding using HBV envelope glycoproteins. HDV particles are released by the secretory pathway, along with HBV virions and SVPs in case of those cells that are co-infected with both viruses.

Enhancing antiviral immunity to cure HBV infection Different attempts are undertaken to break HBV persistence by stimulating innate immunity against the virus or inducing a vigorous adaptive anti-viral immune response. Interferon-α2 (IFN-α2) is the only immune modulator licensed for the therapy of chronic hepatitis B. It can achieve HBV cure in a minority of HBV infected patients but has severe side effects and thus is not widely used any-

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more [13]. Cytokines released after stimulation of innate immunity can control HBV infection [26] and even purge HBV cccDNA from infected cells [27, 28]. Therefore, toll-like receptor 7 agonists were developed to stimulate innate immunity in chronic HBV infection. Although preclinical data looked very promising, first clinical applications were rather disappointing since no antiviral effect was observed [29]. Currently, a second generation of innate immune



Übersicht

virus

target

mode-of-action

drug

status of development

HBV

viral polymerase

inhibition of reverse transcriptase

Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir disoproxil, Tenofovir alafenamid

approved

Tenofovir exalidex

phase III trial

inhibition of RNAseH

only experimental compounds

preclinical

inhibition of capsid formation

diverse classes

phase I/II trials

capsid

HDV

HCV

HEV

viral mRNA

RNA translation inhibitors

siRNAs, antisense oligonucleotides

phase I/II trials

cccDNA

transcriptional silencing e. g. by HBx inhibitors

only experimental compounds

preclinical

HBsAg? Unknown cellular factor(s)?

reduction of HBsAg level by unknown mechanism

nucleic acid polymers (e. g. REP2139)

phase II trials

NTCP

entry inhibition

Myrcludex B

phase II clinical trials

interferon receptors

stimulation of antiviral defense; immune activation?

IFN-α; peg-IFN-α; IFN-λ

approval of IFN-α and peg-IFN-α for hepatitis B

immune checkpoints

limiting T cell exhaustion/ depletion

anti-PD1, anti-PDL1, anti-CTLA4

approved for cancer therapy

pattern recognition receptors

innate immune activation

agonists of TLR-7, TLR-8, STING; Rig-I activation

phase II trials (TLR-7), remaining drugs preclinical or phase I

adaptive immunity

activation of B and T cell responses

diverse therapeutic vaccines

phase I – III clinical trials

NTCP

entry inhibition

Myrcludex B

phase II clinical trials (combination with Tenofovir); phase II clinical trials (combination with IFN-α)

delta antigen

prenylation inhibition

Lonafarnib

phase II clinical trial (combination with ritonavir)

HBsAg? Unknown cellular factor(s)?

reduction of HBsAg level by unknown mechanism

REP2139

phase II (combination with pegIFN-α2a)

immune activation

stimulation of antiviral defense

pegIFN-α; IFN-λ

approval of pegIFN-α

NS3

inhibition of serine-type protease

Telaprevir, Boceprevir, Simeprevir, Asunaprevir, Glecaprevir, Voxilaprevir, Grazoprevir, Paritaprevir

approved combination therapies: glecaprevir/ pibrentasvir; voxilaprevir/velpatasvir/sofosbuvir; elbasvir/ grazoprevir; paritaprevir/ombitasvir ± dasabuvir, combined with ritonavir boost no longer used in Germany/EU. Telaprevir, Boceprevir, Simeprevir taken from the market; Asunaprevir approved only in Asia.

NS5A

inhibition of viral replication and assembly of infectious virus particles

Daclatasvir, Ledipasvir, Pibrentasvir, Elbasvir, Velpatasvir, Ombitasvir

approved combination therapies see above; in addition velpatasvir/sofosbuvir; ledipasvir/sofosbuvir

NS5B

inhibition of the RNA-dependent RNA polymerase (non-nuc) or chain termination (nucs)

Beclabuvir, Dasabuvir (non-nuc), Sofosbuvir (nuc)

combination therapies: see above

viral polymerase

chain termination hypermutation and perhaps additional mechanism(s)

Sofosbuvir Ribavirin

phase II clinical trial off-label

Non-nuc, non-nucleosidic inhibitor; nuc, nucleos(t)ide.

stimulants is being developed targeting other pattern recognition receptors and we will see clinical results in the coming years. The aim is to trigger innate immunity affecting HBV replication and


activating also adaptive immunity. However, broad stimulation of the innate immune system may not be specific enough to treat hepatitis B and bears the risk of side effects.

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▶ Table 2 Overview about available therapies and therapies under development for chronic hepatitis virus infection.

In contrast, stimulating the adaptive immune system against HBV antigens is specific and could mimic what happens during spontaneous resolution of HBV infection: the emergence of neutralizing antibodies as well as CD4 and CD8 T cell responses [30]. One peculiarity of HBV is the production of high amounts of subviral particles which are released from infected cells and detected in the blood as HBsAg (▶ Fig. 1). Antibodies against HBsAg are induced by vaccination and protective, but patients with chronic hepatitis B do not produce them in detectable amounts. In addition, T cells against HBV are scarce and at least partially dysfunctional [31]. We assume that high level HBV antigen production tolerizes adaptive immunity against the virus (reviewed in [32]), but in the absence of robust CD8 + T cell responses the infection will not be controlled. Unfortunately, currently available therapy with NAs neither affects the production of HBV proteins nor HBsAg titers. In contrast, translation inhibitors like siRNAs or antisense oligonucleotides, but also nucleic acid polymers that affect HBsAg release might support the induction of adaptive immunity by reducing HBsAg production [33]. Along these lines, three promising immune therapy strategies stimulating or supporting adaptive immunity are currently under development: the redirection of T cells against HBV-infected cells, checkpoint inhibitors and therapeutic hepatitis B vaccines. These approaches will be discussed briefly. Because HBV-specific T cells are so scarce in chronic hepatitis B, attempts are undertaken to redirect T cells that are not HBV-specific towards infected cells and activate them to become anti-HBV effector cells. This approach referred to as T cell therapy is very successfully used for the treatment of hemato-oncologic malignancies. The most natural way to redirect T cells is T cell receptors cloned from donors that have cleared the infection [34, 35]. Such T-cell receptor grafted T cells have been used in patients and found to be safe [36], but the approach has to be individualized to a patient’s HLA haplotype. Alternative approaches that are HLA-independent and thus more broadly applicable are the use of T-cell engager antibodies [37] or chimeric antigen receptors, referred to as “CAR”s, that have been shown to remove HBV from infected cell cultures and to be safe in vivo in transgenic mice [38, 39]. These approaches are currently about to enter clinical trials for HBV-associated HCC. Although T-cell redirection is an interesting concept, a major down-side is the complex patient management and the high cost, limiting applicability of this approach for a large number of chronically infected individuals. More broadly applicable alternatives are checkpoint inhibitors and therapeutic vaccination. Checkpoint inhibitors such as antiPD1 or anti-PD ligand 1 antibodies are licensed drugs that have been developed to overcome immune tolerance in cancer patients. They are currently discussed as anti-HBV therapy but since HBV-specific T cells are scarce, but only partially dysfunctional, checkpoint inhibitors bear the risk to induce undesired immune responses. Therefore, only clinical trials using low doses of checkpoint inhibitors have been initiated so far [30]. Therapeutic vaccination has been shown to efficiently reduce HBV replication and even cure HBV in animal models, but failed in several clinical trials (summarized in [30, 40]). Perhaps the most promising results thus far have been obtained with a therapeutic HBV vaccine candidate comprising recombinant yeast-

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derived HBsAg complexed with hepatitis B immunoglobulin (HBIG) [41]. This regimen induced HBsAg seroconversion in patients receiving the highest vaccine dose, but statistical significance could not be reached in any of the study arms. In addition, DNA-based genetic vaccines are under development, some of them showing promising results in first clinical studies with chronic hepatitis B patients [42, 43]. However, a better understanding of the antiviral immune response, rapidly growing clinical experience with immunotherapies in cancer treatment, and a more rational vaccine design raise new hope that this approach can be improved. Alternative developments include multi-epitope vaccines, heterologous prime-boost vaccination or a combination of therapeutic vaccination with check point inhibition (▶ Table 2). For instance, a combination of the NA entecavir with a DNA-based prime-boost therapeutic vaccine and antibodies against PD ligand-1 was able to cure chronic HBV infection established after neonatal infection of woodchucks with the woodchuck hepatitis B virus [44]. Heterologous prime-boost vaccination using either two different components inducing T cell immunity (i. e. DNA or viral vectors) or combining protein-based and vector-based vaccine components are currently prepared for clinical evaluation. Although these regimens admittedly are complicated to develop, preclinical results are more promising than those achieved with homologous vaccines [40]. One example is the TherVac B strategy consisting of a protein-prime with particulate HBsAg and core antigen, and a boost using a modified vaccinia virus Ankara (MVA) vector. TherVac B was shown to overcome the strong immune tolerance in HBV transgenic mice expressing low to medium, however not high, viral antigen load [45]. Several clinical trials with HBV-specific therapeutic vaccines have been initiated and we can expect important information to devise the best strategies to achieve at least a functional HBV cure.

Towards curative therapy for chronic hepatitis C HCV replication cycle and reasons for efficient elimination HCV is a positive strand RNA virus encoding a single polyprotein that is cleaved by cellular and viral proteases into 10 different products. In contrast to HBV, HCV has no nuclear phase and replicates exclusively in the cytoplasm where it induces a membranous replication organelle (designated membranous web) that serves to coordinate the different steps of the viral life cycle, i. e. RNA translation, RNA replication, virion assembly and virus particle release (▶ Fig. 1). Since HCV has no persistence reservoir, i. e. a ‘place to hide’, continuous virus replication is required to sustain chronicity. In addition, replication dynamics are very high (around 10e12 virus particles are produced and cleared per day in a patient; [46, 47]) and therefore, an efficient block of viral replication translates into a rapid decline of viremia. This results in a rapid shrinking of the virus population in a patient and thus, the number of virus variants, which reduces the likelihood for the selection of replication competent drug resistant variants. For all



Übersicht

Development of curative therapy for chronic hepatitis C Initial treatment of chronic hepatitis C relied on the use of IFN-α, later on the combination of pegylated IFN-α (pegIFN-α) and ribavirin [48, 49]. However, this therapy had numerous side-effects and many patients were not eligible. Moreover, therapy success very much depended on the genotype of the infecting virus and, for reasons that are still not known, a distinct polymorphism in the IFN-λ gene locus (reviewed in [50]). Given the high medical need intensive efforts to develop IFN-free antiviral therapy were undertaken and a high number of new drugs have been developed. These are targeting three viral factors: (1) the serine-type protease residing in nonstructural protein 3 (NS3); (2) the NS5A replicase and assembly factor; (3) the NS5B RNA-dependent RNA polymerase (▶ Table 2) [51]. The first direct acting antiviral drugs (DAAs) approved for chronic hepatitis C were targeting the NS3 protease [52, 53]. However, owing to a low genetic barrier drug resistant HCV variants rapidly developed and therefore, these first generation NS3 protease inhibitors had to be given in combination with pegIFN-α and ribavirin. Although the sustained viral response (SVR), i. e. absence of HCV RNA 24 weeks after cessation of therapy, could be increased compared to pegIFN-α/ribavirin combination therapy, the number and severity of side effects was higher and many patients had to terminate treatment prematurely. This limitation could be overcome by second and third generation protease inhibitors having fewer side effects, a much higher resistance barrier and covering most, or even all, HCV genotypes [54], thus allowing IFN-α/ribavirin free treatment schedules (▶ Table 2). The second DAA class targets NS5A, a multifunctional protein involved in HCV RNA replication and assembly of infectious virus particles. Initially, this target was not under consideration by drug developers because it lacks enzymatic activity and appeared non-druggable. However, by using high-throughput screens and a cell-based replication assay (the HCV replicon system [55]) a novel class of DAAs was developed that selected for resistance in NS5A and therefore was designated NS5A inhibitor [56]. Most remarkable was the unprecedented antiviral potency with EC50 concentrations of 1 – 2 pM as determined with a genotype 1b replicon (EC50 is the drug concentration required to reduce viral replication by 50 %). Clinical trials confirmed this high antiviral potency and mechanistic studies demonstrated that NS5A-targeting DAAs inhibit both the formation of the HCV replication factory in the cytoplasm of infected cells and the assembly of infectious virions [57, 58]. This dual mode-of-action might be the reason for the high antiviral potency. The first-in-class NS5A inhibitor, Daclatasvir, rapidly selected for drug resistant HCV variants and had a strong bias towards HCV genotype 1 [59]. However, second and third generation NS5A inhibitors very much overcome these weaknesses [60]. The third drug class targets the NS5B RNA-dependent RNA polymerase of HCV, which is the catalytic engine of the viral replicase. Similar to the treatment of HIV, two subclasses of NS5B inhibitors have been developed [51]: on the one hand, non-nucleosi-


dic inhibitors, binding to the enzyme and blocking its catalytic activity, with Dasabuvir being the only drug of this kind that has been approved [61]; on the other hand, the nucleotide Sofosbuvir was developed that does not inhibit the NS5B polymerase but instead is accepted by the enzyme as substrate and incorporated into the newly synthesized HCV RNA [62]. Owing to its chemical properties, Sofosbuvir causes a chain termination, i. e. subsequent nucleotides can no longer be added once Sofosbuvir has been incorporated into the growing RNA chain [51]. The main advantage of Sofosbuvir is its extremely high resistance barrier and thus far, only very rarely a resistance-conferring mutation has been identified in treated HCV-infected patients but owing to high fitness cost (i. e. reduced replication capacity), this mutant rapidly reverts to wildtype (e. g. [63]). For that reason Sofosbuvir is the only HCV-specific DAA that has been given without combination with another DAA. However, the decline of viremia is relatively slow under these conditions and therefore, Sofosbuvir is given in combination with an NS5A inhibitor or with an NS5A and an NS3 protease inhibitor as triple combination (▶ Table 2). In retrospect, NS5A inhibitors are the most remarkable drug class as they have emerged out of the blue by using a cell-based screening assay that was unbiased and covered all intracellular aspects of the HCV replication cycle [64]. Initial hits had rather moderate potency and it required tenacity and courage to follow up this drug class targeting a protein lacking enzymatic activity and having unknown functions. Pioneering work conducted by Min Gao and colleagues led to the first-in-class NS5A inhibitor and demonstrated the high clinical value of this drug class and the complexity of the drug target [64, 65]. These results spurred major interest in other pharmaceutical companies and laid the ground for the development of further NS5A inhibitors that became a cornerstone to treat chronic hepatitis C (▶ Table 2).

A need for a vaccine for global control of HCV infection? IFN-free therapy has been implemented in 2014 and has rapidly become the standard of care. This treatment is safe and highly efficacious reaching virus elimination rates of more than 95 %. For those reasons, it can be given to patients with advanced liver disease and even to patients after liver transplantation to eliminate re-infection of the liver graft, which was inevitable in the absence of this treatment [66]. Moreover, an increasing number of patients that are listed for liver transplantation because of chronic hepatitis C can be taken off the list because of liver function improvement as a result of virus elimination and thus, inflammation cessation [67, 68]. These unprecedented achievements raise enormous expectations to eradicate HCV on a global scale. However, to reach this ambitious goal major hurdles have to be overcome calling for intense efforts in the public health sector, but also to continue basic research with the ultimate goal to develop a vaccine that can prevent at least chronicity of HCV infection [69]. The fact that reinfection with HCV is common (because of the lack of a protective immunity even after acute self-limiting infection) and the strong rise of new infections (according to WHO, 1.75 million in 2015) argue that global elimination of HCV is unlikely to happen without a vaccine. Moreover, access to antiviral treatment, limited treatment capacity in most

67


these reasons, an efficient block of HCV replication has a high chance to eliminate the virus from an infected individual.

Übersicht

Towards curative therapy for chronic hepatitis D HDV is a defective virus that relies on the helper function of HBV to propagate its genome in its host population (so far only humans [70, 71]) (▶ Fig. 1). Resembling, and possibly derived from, a plant viroid (i. e. a small circular RNA genome) HDV can spread within an infected liver through cell division [6], thereby circumventing the extracellular enveloped form of a virion. HDV infection is necessarily associated with an HBV infection; however, the interaction of both viruses within their host is complex and not yet fully understood. Clinically noticeable is the suppression of HBV replication by HDV in vitro [72] and in patients [73]. HBV fulfills its helper function by provision of its self-assembly competent envelope proteins, which allows HDV to package its ribonucleoprotein complex (RNP) for extracellular spread (▶ Fig. 1). Accordingly, HDV (after having acquired an HBV envelope from an HBsAg expressing hepatocyte) can enter naïve or possibly already infected hepatocytes by a similar pathway than HBV (using heparan-sulfate proteoglycans and sodium taurocholate co-transporting polypeptide (NTCP) as receptors), initiate RNA replication, genome editing, production of large and small HDAg and RNP packaging, but cannot exit the cell in the absence of HBV envelope proteins (for detailed reviews on HDV replication see [70, 71, 74]). Supplementation of HBV envelope proteins and formation of virions occurs when HBV RNA is expressed within the same cell thereby providing all three envelope proteins (L-, M-, and S-protein). These can be produced from the episomal cccDNA or replication-deficient HBV integrates which, although sometimes mutated, have recently been shown to constitute the major source of serum HBsAg in chronically infected HBeAg-negative patients [75]. The implication is that HDV can uncouple virion production in chronically HBV infected patients from cccDNAmediated transcription of HBV RNAs. Moreover, clonal expansion of such HBsAg-expressing hepatocytes during liver regeneration could provide an increasingly growing replication space for HDV following hepatocyte mitosis. The therapeutic implications of this situation are discussed below but would entail that elimination of cccDNA in chronically HBV infected patients may not necessarily result in a clearance of HDV RNA from the liver. Another peculiarity of HDV replication is its dependence on essential cellular host factors (e. g. RNA polymerase II, host ligases or the farnesyl transferase) [76] restricting the development of specifically acting antiviral drugs (▶ Fig. 1, ▶ Table 2). The only viral factor that could be therapeutically addressed is the HDAg or the two ribozymes, which are essential for HDV RNA replication [77]. Unfortunately, no such antiviral drug has been developed so far.

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Therapeutic implications for treatment of HDV infection Since HDV virion release relies on the synthesis of the HBV surface proteins the reduction/suppression of HBsAg (e. g. by shRNAmediated suppression of HBV RNAs or HBsAg release inhibitors) would presumably also affect HDV replication. This possibility has not been investigated in clinical trials so far; however, it should be considered in those trials with HBV-/HDV co-infected patients aiming at HBsAg suppression. Treatment of patients with NAs like Tenofovir or Entecavir barely affects HBsAg levels in patients, because these drugs neither deplete cccDNA substantially, nor do they influence HBsAg synthesis from integrated HBV DNA. Accordingly, NAs show no significant effects on HDV release in vitro (F. Lempp and S. U., unpublished) and on HDV serum levels in clinical trials [78]. However, NAs can at least control the possible contribution of HBV-induced liver disease in HBV-/HDV co-infected individuals [79]. IFN-α treatment results in HBsAg loss in a small percentage (< 10 %) of HBV infected patients, which also holds true for patients that are co-infected with HDV [80]. In these patients, which predominatly express low levels (< 1000 IU/ml) of HBsAg, IFN-α – by a poorly understood mechanism – induces a reactivation of the immune response, leading to viral clearance, HBsAg negativation and anti-HBs seroconversion. However, IFN-α also shows direct antiviral effects on HDV replication in patients [81]. The mechanism of action of IFNs is currently under investigation [81]. IFN-α had been used in HDV infected individuals already in the 1980 s [82]. In a large systematic study (the HIDIT-I study), one year of IFN-α therapy in combination with the nucleoside analogue Adefovir, suppressed HDV serum RNA levels in about 30 % of patients to non-detectable levels [78]; however, late relapses were observed in > 50 % of patients, showing that SVR could not be achieved [78, 83]. Treatment prolongation with IFN-α to 96 weeks (the HIDIT-II study) had no significant benefit for the patients regarding HDV relapse after treatment cessation, indicating that the antiviral effect of IFN-α alone is insufficient to eliminate HDV RNA in the liver. Nevertheless, IFN-α is presently used “off label” in eligible patients [79]. There is consensus that sustained loss of HBsAg, associated with anti-HBs seroconversion is the most desirable therapeutic outcome for both HBV mono-infected and HBV/HDV co-infected patients. However, at present this goal is far from being achievable since it requires continuous suppression of HBsAg expression from cccDNA, or loss of cccDNA, and the elimination of HBsAg expressing cells containing HBsAg-encoding integrates [24]. Novel experimental HBV targeting drugs primarily aiming at silencing of cccDNA or its elimination will probably have limited effect on HDV replication and HDV-related pathogenesis when only partial reduction of HBsAg level is achieved. Thus, HBV independent, targeted approaches to control HDV are desirable to address the medical need of chronic hepatitis D patients. Luckily, accelerated approval procedures for novel drug candidates (e. g. Orphan drug assignment and Prime Eligibility Status by FDA and EMA or Breakthrough Therapy designation by the FDA) allow faster accessibility of novel HDV specific drugs for HDV infected patients.



HCV endemic countries, high treatment costs, and the large number of undiagnosed infections (estimated to be around 80 %) clearly illustrate that we are far away from “game over”. Thus, the jury is still out whether global eradication of HCV can be reached by pure antiviral therapy and calls on activities to develop a prophylactic HCV vaccine.

Based on detailed insights into the replication cycle of HDV two drugs with well-defined modes of actions are now entering phase III/registration trials (▶ Table 2). The first one is the entry inhibitor Myrcludex B, which specifically blocks HDV and HBV entry into hepatocytes via the commonly used NTCP-receptor pathway [84] (▶ Fig. 1). Myrcludex B is a peptide derived from the NTCP-binding domain of the HBV L-surface protein. It is presently administered subcutaneously [84]; however oral availability of the peptide is achievable [85]. A recently finished phase II clinical trial (Myr-202) including 120 patients, receiving three different doses (2 mg, 5 mg or 10 mg) of Myrcludex B for 24 weeks in combination with Tenofovir [86], demonstrated profound reductions of HDV serum RNA levels in all three Myrcludex B arms, which was not the case with patients receiving Tenofovir alone [86, 87]. Moreover, ALT normalization was observed in all Myrcludex B treated patients consistent with a reduction in liver stiffness and reduced inflammatory activity. Importantly, and consistent with its mode of action as an entry inhibitor, a strong reduction of HDV infected hepatocytes was found, accompanied by a correlating intrahepatic decline of HDV RNA [88]. The HDV serum RNA decline followed zero-order elimination-kinetics indicating a continuous loss and possible elimination of infected hepatocytes upon long-term treatment even when ALT levels were already normalized. Cessation of Myrcludex B treatment after 24 weeks in the course of this clinical trial led to a rebound of HDV serum RNA to almost base line levels. Nevertheless, mathematical modelling revealed that the majority of HDV-infected patients should be able to eliminate the virus after treatment for > 2 years. Given these encouraging results and the results of a pilot study using Myrcludex B in combination with IFN-α, demonstrating strong synergistic effects on HDV serum RNA levels and remarkable effects on HBsAg [89], a follow up trial (Myr-203) including 60 patients has been started. The interim results showed that administration of Myrcludex B for 48 weeks alone and in combination with peg-IFNα was safe. Furthermore, the strong synergy of IFN-α and Myrcludex B on HDV RNA decline was confirmed. Remarkably, profound HBsAg declines in a substantial number of patients were observed providing first evidence that entry inhibition by Myrcludex B, in combination with peg-IFNα, bears curative potential for chronic HDV and HBV infection [90]. The second drug that has successfully passed several phase II dose finding studies is Lonafarnib, which efficiently suppresses the release of HDV RNPs by inhibiting the cellular farnesyl transferase required for prenylation and subsequent envelopment of the viral RNP-complex [91]. Lonafarnib has been initially developed as an oral drug to treat different forms of cancer [91, 92]. Since it inhibits the essential cellular enzyme farnesyltransferase, which is required for prenylation of e.g c-Ras, particular attention has been addressed to permit long-term tolerability in HDV infected patients. In an initial phase IIa clinical trial with dosing of up to 300 mg Lonafarnib daily, significant reductions of serum HDV RNA could be achieved after 4 weeks of treatment [93]. In three follow up studies (LOWR-HDV-2, LOWR-HDV-3 and LOWR-HDV-4), combination of Lonafarnib with Ritonavir (inhibiting P450-mediated degradation of the drug) allowed tolerable dose reductions and


prolongation of treatment duration for up to 6 months [94 – 96]. These studies confirmed the antiviral activity of the drug in a higher number of patients with still substantial reductions of HDV serum RNA levels and normalization of ALT levels. According to its mode of action Lonafarnib does not block intracellular HDV RNA replication, but results in an accumulation of HDAg and HDV replicative intermediates in vitro [97] (Lempp et al. unpublished). The clinical consequences of this effect are presently unclear, but it might enhance the turnover rate of infected hepatocytes. Although the pathological consequences cannot be foreseen, induced hepatocyte turnover might be an important aspect for possible combination therapies with IFN-α. Cessation of treatment with the drug is associated with a fast rebound of serum HDV RNA leading in some cases to immunological flares with post-treatment viral clearance [98]. Beside these two most advanced drug candidates, two other molecules with more complex modes of action are under clinical investigation. The first one is pegIFN-λ, initially developed to treat chronic hepatitis B patients [99, 100]. Interims results at week 24 of a phase IIa multicenter clinical trial involving 33 HDV/HBV co-infected patients treated with two doses of pegIFN-λ (LIMT-1HDV) indicate antiviral activity of this cytokine. Responses with > 2 log 10 reduction of HDV serum RNA levels were observed in 50 % of patients. This result supports the concept that exogenously applied IFN-λ, although induced by HDV replication itself [81], can still repress HDV RNA replication. The anti-HDV activity of pegIFN-λ was comparable to pegIFN-α [101], but it remains to be determined whether long-term treatment with pegIFN-λ is an alternative to IFN-α. The final reports are expected in 2019. The second drug with a yet poorly defined mode-of-action is the nucleic acid polymer (NAP) REP2139 [102]. It is one representative of a diverse group of NAPs, which display a broad spectrum of antiviral activities against HIV, HSV or LCMV [103]. NAPs are active against duck hepatitis B virus infection in vivo [104] and have subsequently been tested in chronically HBV infected patients (for a summary of conference presentations see http:// replicor.com/science/conference-presentations/). The results of these trials demonstrate that NAPs induce pronounced reductions of serum HBsAg levels in patients. The detailed mechanism of action underlying this HBsAg reduction is not known since in vitro studies do not confirm the observation in patients [105]. In the Rep-301 study patients received 500 mg REP 2139 intravenously once per week for 15 weeks, followed by a combination of 250 mg intravenous REP 2139 and 180 μg s. c. pegIFN-α2a for 15 weeks, and a subsequent monotherapy with 180 μg pegIFNα2a once per week for 33 weeks. A strong reduction of serum HDV RNA levels was observed, accompanied by a strong HBsAg antigen decline in a subset of patients [106]. Long-term follow up studies revealed that “functional remission of HBV/HDV infection” can be achieved [106]. Interestingly responders to the drug combination showed strong ALT-flares indicating activation of the adaptive antiviral immune response [107 – 109]. In summary, four drug candidates show promising antiviral effects with Myrcludex B and Lonafarnib being the most advanced ones for the treatment of chronic HDV infection. Given that these two molecules received Orphan drug designation by FDA and EMA, Lonafarnib the Fast Track Status by FDA, and Myrcludex B

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Novel treatment options for HDV infected patients

Übersicht

Towards curative therapy for chronic hepatitis E Infections with HEV were originally thought to occur only in certain developing countries, but this perception has changed profoundly because it has become clear that HEV is endemic in most high-income countries where it is transmitted primarily as zoonotic infection. HEV is an RNA virus that has been molecularly cloned for the first time in 1990 [110]. The HEV genome possesses 3 open reading frames, one of them encoding for an RNA-dependent RNA polymerase. We now know 8 different genotypes that are prevalent in different geographical regions such as Asia (genotype 1, 4), Africa and Mexico (genotype 2) and Europe (genotype 3). HEV genotypes 3 and 4 are widely distributed in different animal species such as pigs, boar or deer and may also be transmitted as a zoonosis via contaminated meat not thoroughly cooked. Most HEV infections are acute self-limiting and infected individuals mount an immune response that is however, not sterilizing. In addition, rapid progression to liver disease can occur in a fraction of patients [111, 112]. More importantly, HEV infection can also persist, especially in patients that are immunocompromised as a consequence of HIV infection or after organ transplantation. Even in those patients the majority of infections is asymptomatic (except for fatigue) or associated with rather moderate, yet persistent, abnormalities of liver function tests. Apart from the liver, HEV infection appears to affect other organs. Guillain-Barré syndrome, neuralgic amyotrophy, glomerulonephritis, cryoglobulinemia, pancreatitis, lymphoma, thrombopenia, meningitis, thyroiditis and myocarditis have been observed in the context of hepatitis E. Given these clinical symptoms, treatment of chronic HEV infection is required (▶ Table 2). In solid organ transplant recipients a decrease of immunosuppression should be considered, which leads to virus clearance in around one-third of these patients [113, 114]. Alternatively, pegIFN-α has been used with success to treat a small number of liver transplant recipients (e. g. [115]), but in many patients it is contraindicated. An alternative is ribavirin monotherapy that has been used in solid-organ transplant recipients with a reported SVR of 78 % [116]. Therefore, EASL recommends a 12-week ribavirin treatment of patients with an HEV replication that persists for more than 3 months. An extension of this treatment for an additional 3 months might be considered for those patients that still contain HEV RNA after the end of therapy [117]. However, there are also contraindications for ribavirin, which calls for alternative drugs. One of them might be sofosbuvir that has shown some effect in chronic hepatitis E [118]. Currently, sofosbuvir is studied in a phase II clinical trial [119] and first results are expected for March 2019.

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Summary During the last decade, antiviral therapy of viral hepatitis has undergone a major change. We have seen a revolution in the treatment of chronic hepatitis C that can easily be controlled by all oral IFN-free antiviral treatment. For HDV infection, new drugs with high potency and the potential for a cure will be approved soon. As collateral reaction to the success of hepatitis C treatment, the interest in curative therapy for chronic hepatitis B has been awakened in academia and industry. With increasing knowledge about the viral replication cycle, made possible more recently by the discovery of the entry receptor [120] and the availability of infection-based cell culture systems as well as insights into the specifics of the immune defect caused by HBV, promising new treatment modalities have been developed that are in clinical development. We can have justified hope that they will significantly increase SVR rates also for chronic hepatitis B. Given the steady increase of deaths caused by viral hepatitis, such novel approaches are urgently needed.

Conflict of interest R.B. is inventor of HCV replicons and holds rights in HCV replicon technology. S.U. is co-inventor and patent holder of MyrcludexB. U.P. is inventor and hold rights of a heterologous prime-boost therapeutic hepatitis B Vaccine using MVA vectors and bi- and trispecific antibodies against HBV.

Acknowledgment We are most grateful to Diane Schad and Stefan H. E. Kaufmann at the Max Planck Institute for Infection Biology in Berlin for provision of ▶ Fig. 1, Ilka Rebhan for help in finalizing ▶ Fig. 1 and Catherine Moreau for excellent editorial assistance. Work in the authors’ laboratories was supported by the Deutsche Forschungsgemeinschaft (TRR179, TP9, TP14, TP 15 and TP18). U.P. was additionally supported by the European Union Horizon 2020, Hepcar Consortium, and by the Helmholtz Association via the ‘proof-of-concept’ platform.

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