Sofosbuvir in the Treatment of Hepatitis E virus Infection: A Review of in vitro and in vivo Evidence

Abstract

Chronic hepatitis E virus (HEV) infection, which occurs almost exclusively in immunocompromised patients, if untreated may progress to cirrhosis and possibly hepatocellular carcinoma. The reduction of immunosuppression and/or administration of ribavirin is frequently curative but there remain many immunocompromised individuals whose HEV infection is refractory to these therapeutic strategies. Moreover, the haematological toxicity of ribavirin limits its use. Pegylated interferon has demonstrated success in a small number of patients with chronic HEV infection; however, the potentially increased risk of graft rejection associated with its use renders it unsuitable for many transplant recipients. Alternative therapeutic strategies are therefore required. This article reviews the in vitro and in vivo literature to date of the antiviral agent sofosbuvir (well established in the treatment of hepatitis C) in the treatment of HEV infection.

The hepatitis E virus (HEV) is a single-stranded ribonucleic acid (RNA) virus belonging to the Hepeviridae family.¹ Its transmission is primarily via the faecal-oral route and globally it is the commonest cause of acute viral hepatitis affecting an estimated 20 million individuals per year with over 3 million clinically apparent cases and 70,000 deaths.² It causes large scale epidemics in resource-limited settings in the regions of Latin America, Asia and Africa, typically caused by HEV genotypes 1 and 2.³ HEV genotypes 3 and 4 are considered zoonotic infections (with swine serving as the reservoir host); genotype 3 is responsible for locally acquired infections globally, whereas genotype 4 is predominantly identified in China and Japan.⁴ HEV genotypes 5 and 6 are largely confined to wild boar and genotypes 7 and 8 to camels, however, in an experimental infection model, HEV genotype 5 has demonstrated transmissibility to primates.^5,6

Acute HEV infection, which has a 2 to 10 week incubation period (5–6 weeks on average), is typically a self-limiting illness in the immunocompetent but rarely can cause a more severe illness including fulminant liver failure, particularly in individuals with pre-existing chronic liver disease and also in pregnant women; HEV genotype 1 has up to 25% mortality in pregnant women with the majority of deaths occurring in the 3rd trimester.^7,8

Chronic HEV infection (defined by many as HEV infection for at least 3 months although no universally accepted definition currently exists), which occurs almost exclusively in immunocompromised individuals,⁹ has been reported in patients with a range of underlying immunosuppressive conditions, including human immunodeficiency virus (HIV),¹⁰ solid organ transplantation,¹¹ haematopoietic stem cell transplantation¹² and in patients receiving immunosuppressive agents (including steroids and chemotherapy).¹³ The majority of chronic HEV infections are asymptomatic (with up to 32% reporting symptoms) with mild to moderate elevations in serum alanine aminotransferase.^3,9,14 Apart from a case report of a genotype 7 chronic HEV infection in a liver transplant recipient who regularly consumed camel products¹⁵ and a report of a genotype 1 chronic HEV infection in a child who had been successfully treated for acute lymphoblastic leukaemia,¹⁶ almost all reported cases of chronic HEV infection to date are genotype 3 and 4 (predominantly genotype 3 with no reported genotype 1 or 2 cases).^3,17 In a multi-centre (Europe and the USA) study by Kamar et al., 65.9% (56/85) of solid organ transplant recipients who acquired HEV developed a chronic infection (defined in their study as persistently elevated liver enzyme levels and PCR evidence of HEV in the serum and/or stool for at least 6 months) and progression to cirrhosis occurred in 14.3% of those with chronic HEV infection.¹⁴ This potential progression of liver disease (fibrosis/cirrhosis, which occurs to varying degrees) in untreated patients with chronic HEV causes significant morbidity and mortality thus warranting further pursuit of optimal treatment strategies.

Limitations of currently recommended therapy for chronic HEV infection

Where possible, the reduction in immunosuppression is the first strategy in order to try to achieve HEV clearance. In the study by Kamar et al. of chronic HEV in solid organ transplant recipients, 32% (18/56) of patients with chronic HEV achieved sustained viral clearance with reduction in immune suppression alone. This is an effective strategy in many patients but there still remains a significant number of patients in whom this strategy does not work and/or in whom significant reductions in immune suppression put the individual at a significantly higher risk of graft rejection. Ribavirin, the most extensively studied drug in chronic HEV infection, has been shown to achieve cure in approximately 65% of subjects treated for chronic HEV infection;¹⁸ however, anaemia is a frequent and significant side effect.¹⁹

Sofosbuvir

The nucleotide NS5B RNA-dependent RNA polymerase inhibitor oral prodrug sofosbuvir (formerly known as GS-7977/PSI-7977; Gilead Sciences, Foster City, CA, United States), which once metabolised to its active form is incorporated into hepatitis C virus (HCV) RNA by the NS5B polymerase acting as a chain terminator,²⁰ is well established as a highly effective agent and in the treatment of HCV infection and features in current HCV treatment international guidelines.^21,22 It has a well-established safety profile and high tolerability in patients treated for HCV (frequency of serious adverse events ranged from 1% to 8% and rates of treatment discontinuation due to adverse events range from 0% to 4.4% in phase III trials).²³ Recently, there has been an increasing interest in the potential use of this agent for chronic HEV infection, a condition for which current therapeutic options are extremely limited. It has been suggested that sofosbuvir likely acts on HEV replication by directly competing with nucleotides for incorporation by the HEV polymerase into the nascent RNA strand but further clarification is needed.²⁴ This article reviews the in vitro and in vivo literature on sofosbuvir against HEV and discusses the potential role for sofosbuvir as monotherapy and as combination therapy, primarily with ribavirin, in the treatment of chronic HEV infection.

Search strategy and selection criteria

An extensive literature search was performed using the MEDLINE/PubMed/PubMed Central, Embase and Google Scholar databases up until 01 March 2021. Combinations of the following keywords were used to identify relevant studies: ‘sofosbuvir’; ‘GS-7977’; ‘PSI-7977’; ‘NS5B inhibitor’; ‘DAA’; ‘hepatitis E’; ‘HEV’. References of identified studies were searched and the PubMed ‘similar articles’ tool was used to identify further potentially relevant studies. Additionally, clinicaltrials.gov and clinicaltrialsregister. eu were searched for unpublished studies.

All studies identified through this method were screened for relevance and were not restricted based on the date of publication, type of study (except for review articles), study setting, HEV genotype, type of drug/agent used in combination with sofosbuvir (where applicable) or duration of sofosbuvir and/or other agents co-administered. In vitro and clinical studies were included. Only publications in English were included for further evaluation. Figure 1 illustrates the schematic of publications identified, screened and included.

Figure 1.

Schematic of publications identified, screened and included.

in vitro studies of sofosbuvir in HEV infection

The early suggestions of a potential role for sofosbuvir in the treatment of HEV infection came from an in vitro study using a human hepatocellular carcinoma-derived cell line (S10-3) harbouring autonomously replicating genotype 3 HEV RNA (identified as HEV3 Rep/Neo).²⁵ A prior study had already demonstrated that HEV3 Rep/Neo cells treated with interferon-α (IFN-α) or ribavirin for 48 h resulted in a dose-dependent fall in HEV RNA levels relative to untreated cells (without compromising cell viability).²⁶ Dao Thi et al. subsequently demonstrated that HEV replication was efficiently inhibited when HEV3 Rep/Neo cells were treated with sofosbuvir but HEV RNA replication was not inhibited when HEV3 Rep/Neo cells where treated with the nucleoside reverse transcriptase inhibitor lamivudine or with the non-nucleoside HCV polymerase inhibitor nesbuvir.²⁵ This antiviral activity of sofosbuvir against HEV was also confirmed in a HEV replicon assay (RNA replication measured indirectly via the expression of a reporter gene in the viral genome) within the same study. Moreover, the investigators found an additive effect in suppressing HEV replication in the combined presence of RBV and sofosbuvir; the addition of ribavirin enabled maximal inhibition of HEV RNA replication at lower concentrations of sofosbuvir. In contrast to the findings from this study, another in vitro study by Wang et al. using human hepatoma (huh7) cells transfected with HEV genotype 1 and 3 RNA found that sofosbuvir (using concentrations ranging from 0.01 to 10 μmol/L; comparable to those used in the study by Dao Thi et al.) did not effectively inhibit HEV RNA replication after 48 h.²⁷ Even at the highest concentration of sofosbuvir (10 μmol/L), only a modest anti-HEV effect (a limited reduction in HEV RNA levels) was observed, whereas sofosbuvir was highly effective in inhibiting HCV RNA replication in huh7 cells in their study.

Wu et al. performed an in vitro study in which induced-pluripotent stem cell-derived hepatocyte-like cells, which have been previously demonstrated to be permissive to HEV,²⁸ were transfected with HEV (genotypes 1–4) and the inhibitory potency of ribavirin was examined alone and in combination with sofosbuvir.²⁴ Ribavirin inhibited the replication of all viruses (genotype 2 was the most sensitive [up to 75% inhibition] and genotype 1 the least sensitive [up to 45% inhibition]). The addition of sofosbuvir (1 μM) further inhibited the replication of all viruses with the exception of genotype 2, which may be related at least in part to the fact that this genotype was already significantly inhibited by ribavirin alone. Additionally, neither drug had any cytotoxic effects on the hepatocyte-like cells. These findings therefore suggest a potential synergistic effect of ribavirin with sofosbuvir against HEV replication. If proven to be the case this may be particularly beneficial in cases of haematological toxicity with ribavirin requiring dose reductions.

Netzler et al. assessed the in vitro efficacy of sofosbuvir (and 15 other compounds) at a fixed concentration of 10 μM in inhibiting the replication of a human HEV genotype 1 replicon (pSK-HEV-2-Luc).²⁹ Sofosbuvir (and 3 other antivirals tested; dasabuvir, NITD008 and GPC-N114) demonstrated >50% inhibition at the 10 μM dose. Sofosbuvir inhibited HEV genotype 1 replication in a dose-dependent manner with a half maximal effective concentration (EC₅₀) of 1.97 μM. This is in contrast to the findings by Dao Thi et al. and Wang et al. which reported EC₅₀ values of 1.20 μM against genotype 3 and > 10 μM against genotype 1 HEV replicons.^25,27,29 In addition, Netzler et al. assessed the effects of sofosbuvir on cell viability and found that no cytotoxic effects on Huh7 cells were observed (up to 100 μM).²⁹ Interestingly, in an in vitro experimental model of genotype 5 HEV infection (identified to date only in wild boar^30,31 with no reported human cases) by Li et al., the investigators not only demonstrated that it can be transmitted to primates but they also demonstrated that sofosbuvir moderately decreased the HEV genotype 5 copy number (i.e. only a partial effect) using a human hepatocarcinoma cell line (PLC/PRF/5) whereas ribavirin drastically reduced the HEV copy number.⁵

In vitro data for sofosbuvir against HEV therefore does suggest that sofosbuvir has at least some degree of antiviral activity against HEV, albeit relatively limited in comparison to its in vitro potency in inhibiting HCV replication. Synergistic effects against HEV replication have also been observed when given in addition to ribavirin. Additionally, where assessed, none of the in vitro studies reported cytotoxic effects associated with sofosbuvir.

Nishiyama et al. conducted an in vitro study in which they demonstrated that the combination of sofosbuvir with 4 different types of interferons had additive inhibitory effects on HEV growth in cultured cell lines and with no observed cytotoxic effects. Sofosbuvir in combination with interferon could be another further potential avenue for human studies in cases of refractory HEV infection in certain patient groups.

Sofosbuvir monotherapy

The human data for sofosbuvir as monotherapy in the treatment of HEV infection come from the recently published HepNet SofE pilot study.³² Nine immunocompromised patients (including patients who had undergone haematopoietic stem cell transplantation, solid organ transplantation and with common variable immune deficiency) with a genotype 3 chronic HEV infection received sofosbuvir monotherapy (400 mg/day) for 24 weeks in a prospective, open labelled, single-arm clinical trial. Seven patients had previously failed therapy with ribavirin and ribavirin was contraindicated in the other 2 patients. The investigators reported a median 1.1 log₁₀ IU/ml (580,000 IU/mL to 45,000 IU/mL) decline in HEV RNA levels 2 weeks into treatment with sofosbuvir. However, HEV RNA increased in most between 4 weeks and the end of treatment; at week 12, the median HEV RNA (at 100,000 IU/mL) was 0.76 log₁₀ IU/ml below baseline pretreatment levels and at week 24 (233,000 IU/mL) 0.40 log₁₀ IU/ml below the median pretreatment baseline HEV RNA level. At the end of follow up, 12 weeks after completing sofosbuvir, the median HEV RNA rose back to approximately baseline levels. The drug was generally tolerated although all patients reported at least 1 adverse event; the most frequently reported were infections (including gastroenteritis, sepsis and ocular toxoplasmosis) and these were deemed by the investigators more likely to be associated with immunosuppression than with sofosbuvir. None of the 9 patients achieved undetectable HEV RNA at the end of therapy and therefore although a small preliminary study, it is evident that sofosbuvir monotherapy at a dose of 400 mg/day, the dose licensed for its use in HCV infection, is ineffective in as a curative treatment option for chronic HEV infection. The fact that 5 of the 9 patients achieved at least a 1.0 log₁₀ IU/ml decline in HEV RNA on treatment suggests that sofosbuvir has moderate anti-HEV activity, which is line with the in vitro findings of sofosbuvir against HEV.^25,27 The presence or absence or resistance mutations was not reported in this study. Whether higher doses of sofosbuvir may lead to more potent and prolonged inhibition of HEV replication is unclear. For HCV infection, for which sofosbuvir is approved, the current guidelines recommend sofosbuvir in combination with agents of differing but complimentary antiviral activity rather than as monotherapy.^21,22 Sofosbuvir as monotherapy for chronic HCV infection achieved a sustained virological response at 12 and 24 weeks in 60% (6/10) of patients which although is significantly better than the findings of sofosbuvir against HEV in the HepNet SofE study is still highly suboptimal than the response rates seen (>95%) when sofosbuvir is used in combination with other antivirals for HCV.^21,33 Therefore, a combined antiviral approach is likely to be the most appropriate avenue for the future therapeutic anti-HEV strategies. Nevertheless the HepNet SofE study is important in providing in vivo human evidence for at least some degree of the activity of sofosbuvir against HEV and the duration of HEV infection in the patients included was sufficiently long (mean 29.1 months, range 3 to 89 months) that the observed declines in HEV could be somewhat attributed sofosbuvir rather than the natural declines that can occur in the acute phase of HEV infection.³² It is important to note that potential immune-mediated factors including changes to immunosuppressive therapy may have impacted upon responses in individual patients.

Sofosbuvir as combination therapy

There are no clinical trials of sofosbuvir as combination therapy in HEV infection. Human data come solely from one case series and several case reports (see Table 1). Van der Valk et al. reported a case of a 60-year-old patient with chronic lymphocytic leukaemia who was diagnosed with an acute HEV infection (genotype 3) treated with ribavirin (600 mg/day) but relapsed after stopping ribavirin on two occasions (HEV RNA became undetectable on ribavirin on both occasions).³⁴ He received a 3rd course of ribavirin (this time at a higher dose of 600 mg twice daily and adequate serum ribavirin trough levels were achieved) on which HEV RNA levels did not decline and the patient was found to be harbouring mutations (K1383N and D1384N) associated with ribavirin resistance (and also another ribavirin resistance-associated mutation G1634R was detected but only transiently).³⁵ Of note, a recent retrospective multi-centre European study of ribavirin for chronic HEV infection in solid organ transplant recipients found that pretreatment and de novo mutations (including patient with HEV harbouring K1383N, D1384N and G1634R mutations) did not have a negative impact on HEV clearance.¹¹ The patient reported by Van der Valk et al. experienced haematological toxicity with the higher ribavirin dose and this was therefore reduced to 400 mg twice daily.³⁴ As HEV RNA remained detectable (no signs of liver fibrosis on Fibroscan; Metavir F0–F1) sofobuvir 400 mg/day was commenced (in addition to ribavirin) and after 1 month HEV RNA levels declined from 1.27 × 10⁴ IU/mL to undetectable. A low-level viraemia, however, subsequently recurred and after cessation of ribavirin and sofosbuvir, which were given in combination for 8 weeks, HEV RNA returned to pretreatment levels. This case supports the in vitro data in suggesting that sofosbuvir has some degree of activity against HEV but did not have adequate potency to achieve the sustained viral suppression. This raises the question as to whether higher dosing of sofosbuvir, if demonstrated to be safe at higher dosages, may be able to achieve this.

Table 1.

Published Studies of Sofosbuvir in the Treatment of Hepatitis E virus Infection.

Study location	Year of treatment with sofosbuvir	Number treated	Age (s)	Gender	Medical history	HEV genotype	HEV treatment	Clinical outcome with sofosbuvir	Reported emergence of resistance (if any)	Adverse events
Sofosbuvir monotherapy
Germany32	2017–2019	9	Mean 44 (range 24–61) years	6 males and 3 females	Varied, including solid organ transplant, haematopoietic stem cell transplant and common variable immune deficiency Chronic HEV infection in all 9 patients	All genotype 3	Sofosbuvir for 24 weeks in all patients	Median 1.1 log10 IU/ml decline in HEV RNA levels 2 weeks after starting treatment 5/9 patients achieved a ≥1.0 log10 IU/ml fall in HEV RNA None of the patients achieved undetectable HEV RNA at completion of treatment	Presence/absence of resistance not reported	Generally tolerated but all patients reported ≥1 adverse effect; most common was infection (likely related to immune suppression One patient with common variable immune deficiency died from sepsis
Sofosbuvir as combination therapy
Netherlands34	2017	1	60	Male	Hematopoietic stem cell transplant for chronic lymphocytic leukaemia 2006, subsequently relapsed 2014. Retreated with chemotherapy and in remission in early 2015. HEV diagnosed January 2015 (no evidence of fibrosis on FibroScan) Chronic HEV infection	3	January 2015 – ribavirin 600mg/day given for 2 months May 2015 – ribavirin 600mg/day restarted due to HEV RNA relapse (HEV RNA became undetectable but then relapsed in September 2015 September 2015 – ribavirin dose increased to 600mg twice daily (adequate ribavirin trough levels were achieved) but no decline in HEV RNA. Ribavirin reduced to 400mg twice daily and sofosbuvir 400 mg/day added – both agents given for 8 weeks	One month after the addition of sofosbuvir HEV RNA became undetectable (1.27 × 104 IU/mL prior to sofosbuvir) but then recurrence of low level viraemia. After stopping ribavirin/sofosbuvir HEV RNA returned to pretreatment levels	G1634R mutation transiently detected on ribavirin monotherapy, K1383N and D1384N also detected (all associated with ribavirin resistance)	Haematological toxicity whilst on ribavirin 600 mg twice daily monotherapy requiring dose reduction No specific adverse events reported related to sofosbuvir
France35	2015	1	59	Male	HIV (virally suppressed but with CD4 count <100 cells/mm3), previous pneumocystis pneumonia, previous cerebral toxoplasmosis, hepatitis B virus coinfection, liver cirrhosis and diabetes mellitus Chronic HEV infection	3	Failed treatment with 50 months of ribavirin 1000 mg/day Failed treatment with intravenous immunoglobulins, pegylated interferon and ribavirin in 2015 Sofosbuvir 400 mg/day and ribavirin 1000 mg/day given for 12 weeks (starting in December 2015)	Initial >2 log10 copies/ml fall in HEV RNA on sofosbuvir/ribavirin but did not reach undetectable levels and rebounded to pretreatment levels after cessation of treatment	G1634R mutation detected	No specific adverse events were reported
France36	2016	1	49	Male	Cardiac transplant in 2011 for dilated cardiomyopathy Chronic HEV infection	3	July 2014 – started ribavirin 800 mg/day which was given for 3 months with HEV RNA undetectable at the end of treatment January 2015 – HEV RNA detected and ribavirin restarted at 1000 mg/day September 2015 – ribavirin increased to 1200 mg/day May 2016 – sofosbuvir 400 mg/day added to ribavirin for 6months On ribavirin monotherapy at the time of publication	Initial >2 log10 IU/ml fall in HEV RNA on sofosbuvir/ribavirin but did not reach undetectable levels and rebounded in second month. HEV RNA has remained detectable since	G1634R mutation detected	No specific adverse events were reported
Lebanon37	2017	1	57	Male	Kidney/pancreas transplant Chronic HEV infection	3	2013 – ribavirin 600 mg/day for 4 months 2015 - ribavirin 600 mg/day for 9 months 2017 – sofosbuvir 400 mg and ribavirin (initially 1200 mg for 4 weeks then reduced to 800 mg/day) in combination for 3 months then ribavirin continued for a further 1 month	HEV RNA undetectable at the end of completion of treatment and at 10 months post completion of treatment	Presence/absence of resistance not reported	Symptomatic anaemia with ribavirin requiring dose reduction from 1200 mg to 800 mg
Netherlands38	2017	3	25, 52 and 64	1 male and 2 females	All solid organ transplant recipients Chronic HEV infection in all 3 patients	All genotype 3	Sofosbuvir 400 mg in addition to ribavirin for 24, 12 and 8 weeks, respectively	Partial antiviral response in 2 patients following the addition of sofosbuvir No antiviral response in 1 patient after addition of sofosbuvir	G1634R in 1 patient with partial response K1383N and G1634R in 2nd patient with partial response Presence/absence of resistance not reported 3rd patient who had no response	Two patients had ribavirin dose reductions in view of anaemia
Germany39	2016	1	30	Male	Crohn's disease, short bowel syndrome, total parenteral nutrition-associated liver fibrosis, chronic renal insufficiency - underwent liver, pancreas, kidney, small bowel and right hemicolon transplantation in 2011 Chronic HEV infection	3	February 2015 – ribavirin monotherapy (600 mg–800 mg) Sofosbuvir 400 mg/day added to ribavirin after 21 months of continuous ribavirin monotherapy Sofosbuvir and ribavirin combination therapy continued for 6 months then sofosbuvir stopped and ribavirin monotherapy continued	Achieved fall in HEV RNA to below the lower limit of quantification following the addition of sofosbuvir but undetectable HEV RNA was not achieved Rebound in HEV RNA occurred after stopping sofosbuvir	4 ribavirin resistance mutations detected; K1383N, D1384N, V1479I, and G1634R	Required doses adjustments of ribavirin due to anaemia and chronic renal impairment
Switzerland40	2017	1	57	Male	Liver transplant in 1998 for alcoholic cirrhosis and hepatocellular carcinoma Previous diffuse large B cell lymphoma Chronic HEV infection	3	September 2016 – ribavirin started February 2017 to July 2017 – sofosbuvir 400mg/da given in combination with ribavirin July 2017 to February 2018 – ribavirin monotherapy August 2018 to time of publication in 2019 – ribavirin monotherapy	Achieved undetectable HEV RNA in plasma after addition of sofosbuvir (which was sustained whilst on sofosbuvir) but unable to maintain undetectable HEV RNA in stool Rebound in HEV RNA in plasma after stopping sofosbuvir	2 ribavirin resistance mutations detected; K1383N and G1634R	No specific adverse events were reported
Scotland41	2015	1	Not specified	Not specified	Liver transplant recipient Chronic HEV infection and chronic HCV infection	3	2015 – sofosbuvir 400 mg/day and daclatasvir 60 mg/day for 12 weeks	<2 log10 fall in serum HEV RNA on treatment HEV cure not achieved	Sequences of the HEV ORF-2 region pretreatment and 2 months into therapy demonstrated little sequence change variation	No specific adverse events were reported
Italy42	2016	1	57	Male	HCV (active) cirrhosis Acute HEV infection	3	March 2016 – started sofosbuvir 400 mg/day & ribavirin 800 mg/day	HEV RNA (& HCV RNA) undetectable by day 6 of treatment and still undetectable at day 20 Patient died due to septic shock	Presence/absence of resistance not reported	Death due to septic shock No specific adverse events reported prior to death
India43	2018	1	30	Male	Acute HEV infection with associated Guillain-Barre syndrome	1	2018 – sofosbuvir 400 mg/day & ribavirin 400 mg/day for 1 month	Achieved cure of HEV infection and complete neurological recovery	Presence/absence of resistance not reported	No specific adverse events were reported
Pakistan44	Not specified (published in 2019)	1	72	Male	Asthma Diabetes mellitus HEV, HCV and HBV coinfection of unknown duration – patient presented with jaundice and acute hepatitis	Not specified	12 weeks of combination antiviral therapy consisting of sofosbuvir 400 mg/day, ribavirin, tenofovir 300 mg/day and daclatasvir 600 mg/day	Achieved clearance of all 3 viruses in the blood which was sustained at 48 weeks post treatment completion	Presence/absence of resistance not reported	No specific adverse events were reported

Abbreviations: hepatitis E virus (HEV), hepatitis C virus (HCV), hepatitis B virus (HBV), Ribonucleic acid (RNA).

Ribavirin and sofosbuvir were also used for chronic HEV genotype 3 infection with neurological sequelae (and HEV RNA also detectable in cerebrospinal fluid at a level of 3.2 log₁₀ copies/ml) in a 59-year-old patient with HIV (virally suppressed but a persistently low CD4 count <100 cells/mm³), hepatitis B virus (HBV) coinfection,liver cirrhosis and diabetes mellitus.³⁶ He had failed treatment with 50 months of ribavirin (1000 mg/day). Intravenous immunoglobins and subsequent combination Peginterferon-alpha-2a/ribavirin therapy also failed to suppress HEV RNA. Interferon-alpha has been shown in vitro to have moderate but significant synergism with ribavirin against HEV.²⁶ The sequencing of his HEV revealed a G1634R substitution in the polymerase encoding gene (ORF-1). This mutation has been reported to improve the replicative capacity of HEV in human liver and decrease the efficacy of ribavirin.^35,37 He was subsequently treated with ribavirin (1000 mg/day) and sofosbuvir (400 mg/day) for 12 weeks. An initial >2 log₁₀ copies/ml fall in plasma HEV RNA was observed but this then plateaued and following cessation of treatment rebounded to pretreatment levels. Similar to the case by Van der Walk et al., the early decline in HEV RNA suggests at least an initial effect of the ribavirin/sofosbuvir combination but it is difficult to elucidate whether it was one/or both agents in combination with exerted this effect. This was complicated further by the presence of mutations associated with ribavirin resistance; it is unclear whether this combination regimen would have been more efficacious in the absence of this mutation. Another case by Todesco et al. reported similar findings.³⁸ A 49-year-old man who had previously undergone cardiac transplantation for dilated cardiomyopathy and who had a genotype 3 chronic HEV infection unsuccessfully treated with ribavirin (2 courses) and a reduction in immunosuppression, had sofosbuvir (400 mg/day) added to ribavirin (1200 mg/day). Despite a >2 log₁₀ IU/ml fall in HEV RNA in the first month, it increased again in the second month into treatment and remained elevated. At the time of publication, sofosbuvir had been stopped after 6 months and the patient remained on ribavirin monotherapy with persistently detectable HEV RNA. Similarly, in this patient, the G1634R mutation was identified. Again in this case, there was an early sharp drop in HEV RNA followed by a sudden rise which is concerning for the possible emergence of unidentified sofosbuvir resistant strains.

The successful use of sofosbuvir in combination with ribavirin was reported by Drinane et al. who reported of a case of a 57-year-old diagnosed with a genotype 3 infection (no signs of liver fibrosis on Fibroscan; Metavir F0–F1) 5 years following his kidney/pancreas transplant.³⁹ Reduction of immunosuppression, 4 months of ribavirin monotherapy (600 mg/day) and latter course of 9 months of ribavirin monotherapy had all failed to clear his HEV infection. He was finally treated with sofosbuvir (400 mg/day) and ribavirin (1200 mg/day for the first 4 weeks then reduced to 800 mg/day due to symptomatic anaemia) in combination. Baseline HEV RNA was 25,100 IU/mL. He received 3 months of sofosbuvir and 4 months ribavirin. HEV RNA was undetectable (<100 IU/mL) at the end of treatment and 10 months after completing treatment HEV RNA remained undetectable. This case therefore supports a role for sofosbuvir in combination with ribavirin although the lower doses of ribavirin used initially as monotherapy in this patient may have been contributory in his failure to achieve HEV RNA clearance with his prior treatment attempts. One must also note that treatment was stopped without confirming the absence of detectable HEV RNA in both blood and stool given the fact that persistent shedding of HEV RNA in stool is associated with higher relapse rates.^40,41

A case report by Schulz et al. described a 30-year-old male with Crohn's disease and short bowel syndrome, total parenteral nutrition-associated liver fibrosis and chronic renal insufficiency who had previously underwent a liver, pancreas, kidney, small bowel and right hemicolon transplant.⁴² He developed a chronic genotype 3 HEV infection which had failed to respond to 21 months of continuous ribavirin (600–800 mg/day) and therefore sofosbuvir 400 mg/day was added and after 4 weeks of sofosbuvir HEV RNA fell to below the lower limit of quantification but did remain detectable. His immunosuppression (tacrolimus) was also reduced. After 6 months of sofosbuvir and ribavirin, over which period HEV RNA remained detectable at very low levels, sofosbuvir was stopped and he continued on ribavirin monotherapy (600 mg) at which point he experienced a rebound in HEV RNA levels. The sequence analysis at 3 consecutive time points confirmed the presence of 4 mutations associated with ribavirin resistance (K1383N, D1384N, V1479I, and G1634R).^42,43 Therefore, again in this case, although the addition of sofosbuvir to ribavirin did demonstrate heightened inhibition of viral replication of this combination at the doses administered (a relatively low ribavirin dose due to anaemia and impaired renal function) failed to achieve viral eradication. Similar findings were observed in a case reported by Fraga et al. in which a 57 year old liver transplant recipient who was diagnosed with a genotype 3 chronic HEV infection and who failed to achieve undetectable HEV RNA levels in plasma despite 5 months of ribavirin monotherapy (doses not specified), had sofosbuvir (400 mg/day) added for a period of 24 weeks.⁴⁴ HEV RNA became undetectable in plasma shortly after the addition of sofosbuvir and remained undetectable over the 24 week period; however, HEV RNA was detectable in stool towards the end of the 24 week period (having become initially undetectable in stool after the addition of sofosbuvir). HEV RNA rebounded after stopping sofosbuvir at which time he was on ribavirin monotherapy and sequencing revealed the presence of the K1383N and G1634R mutations known to be associated with ribavirin failure.⁴³ Therefore, in both cases the addition of sofosbuvir exerted a significant antiviral effect but this was inadequate to achieve a sustained viral response. Ribavirin resistance mutations were detected in both cases.

A case series by van Wezel et al. in which three solid organ transplant recipients with genotype 3 chronic HEV infection and had failed on ribavirin therapy all received additional sofosbuvir (400 mg/day) but still failed to eradicate their HEV infection.⁴⁵ The first patient achieved undetectable plasma HEV RNA after the addition of sofosbuvir but HEV RNA remained detectable in stool, and after sofosbuvir and ribavirin were stopped at 24 weeks, HEV RNA became detectable again in plasma. The second patient had achieved undetectable HEV RNA in plasma on ribavirin monotherapy but persistently detectable HEV RNA in stool therefore sofosbuvir was added for 12 weeks but failed to clear HEV RNA from the stool. In the third patient, who had persistently detectable HEV RNA in plasma despite 7 months of ribavirin, 8 weeks of sofosbuvir with ribavirin was reported to have no additional antiviral effect. A partial response to treatment was seen in 2 of the 3 cases and in both cases mutations were detected (G6134R in the first patient and G6134R along with K1383N in the second patient). The presence or absence of mutations was not reported in the third patient in whom the addition of sofosbuvir had no additional antiviral response.

In view of previous intolerance to ribavirin, Donnelly et al. treated a liver transplant recipient with genotype 3 chronic HEV infection in addition to chronic HCV infection (genotype 3) with 12 weeks of sofosbuvir 400 mg/day in combination with daclatasvir (an NS5a inhibitor used in the treatment of HCV²¹) 60 mg/day.⁴⁶ This combination was ineffective in curing this patient's HEV infection (<2 log₁₀ fall in serum HEV RNA on treatment) but HCV RNA became undetectable 4 weeks into treatment. Of note, sequences of the ORF-2 region of HEV from pretreatment and at 2 months into antiviral treatment showed little sequence change variation. However, it is possible that there may have been other potentially significant sequence changes in other, unexamined regions of the genome. Unlike with sofosbuvir, there are currently no in vitro or in vivo data suggesting daclatasvir has any anti-HEV activity.

Wahid et al. reported a case of a 72-year-old man living in Pakistan and with a medical history of asthma and diabetes mellitus who presented with jaundice and who was diagnosed with an HEV, HCV and hepatitis B coinfection.⁴⁷ He received 12 weeks of combination antiviral therapy consisting of sofosbuvir (400 mg/day), ribavirin, tenofovir (300 mg/day) and daclatasvir (600 mg/day). He achieved clearance of all three viruses from the blood and this was sustained at 48 weeks post treatment completion. Choudhary et al. reported a case of acute HEV infection (genotype 1; HEV RNA 3.4 × 10³ IU/mL) with associated Guillain-Barre syndrome in a 30-year-old male (no underlying health conditions were reported) treated with 1 month of sofosbuvir (400 mg/day) and ribavirin (400 mg/day).⁴⁸ He achieved cure of his HEV infection as well as complete neurological recovery. Biliotti et al. reported a case of a 57-year man with HCV cirrhosis diagnosed with acute-on-chronic liver failure due to an acute HEV (genotype 3 infection).⁴⁹ He had an HCV RNA level of 803 IU/mL and HEV RNA level of 2285 copies/mL. He started treatment with sofosbuvir 400 mg/day and ribavirin 800 mg/day, and on day 6 of treatment, HCV RNA and HEV RNA were both undetectable. Both HCV RNA and HEV remained undetectable on day 20 of treatment; however, the patient died shortly after due to septic shock. On post-mortem examination, HEV RNA was undetectable in the liver, cardiac tissue and other muscle tissue. In all three of these cases, the patients (all immunocompetent) were deemed to have an acute HEV infection and therefore their recovery cannot be attributed with any degree of certainty to the antiviral therapy the received. Nevertheless, these cases do provide additional evidence that the drug combinations used (at the administered doses) can be well tolerated.

Discussion

In the studies and case reports/series described, the majority (76%, excluding one case where the gender was not reported) of patients who received sofosbuvir for their HEV infection were males which correlates with previously published data reporting that all four HEV genotypes (1–4) seen in humans are more likely to be clinically apparent in men.³ All of the patients who received sofosbuvir for chronic HEV had a genotype 3 infection. Interestingly, 3 patients were treated with antivirals for acute HEV infection, which is spontaneously cleared in most cases.⁵⁰ The efficacy of sofosbuvir or ribavirin can therefore not be ascertained from these cases.

Both the in vitro and in vivo data albeit limited, support the notion that sofosbuvir, at least to some degree, exerts an inhibitory effect on HEV replication and this is enhanced in the presence of ribavirin. However, sofosbuvir at a dose of 400 mg/day, apart from in one case report where it was used successfully in conjunction with ribavirin,³⁹ has not been successful in achieving a sustained virologic response as monotherapy or in combination with ribavirin. The fact that 5 of the 9 patients who received sofosbuvir 400 mg/day as monotherapy for chronic HEV infection in the HepNet sofE study experienced a ≥1.0 log₁₀ IU/ml fall in HEV RNA and 8 of the 9 patients who received sofosbuvir 400 mg/day as combination therapy with ribavirin for chronic HEV infection in the published case series and case reports had at least a partial antiviral response raises the question as to whether sofosbuvir at an increased dose may achieve improved response rates.

The highest dose of sofosbuvir that has been administered to human subjects was a single supratherapeutic dose of 1200 mg, 3x the normally administered dose, given to 59 healthy study participants.⁵¹ This was found to be safe; no significant differences in the frequency and severity of adverse events (including no clinically significant effect on the QT interval) were observed when compared to sofosbuvir 400 mg and to placebo. The effects of even higher doses of sofosbuvir or the dose of 1200 mg over a more prolonged period are unknown. Additionally, a particular challenge with treating chronic HEV infection is the fact that patients are universally immunocompromised and many additionally have impaired T-cell function. HEV-specific T-cell responses are known to be associated with control of HEV infection, however, HEV-specific T-cell responses are decreased in transplant patients for example.^52,53 Therefore, human immune-associated factors are likely to impact the response to antiviral treatment for chronic HEV infection to a greater degree than with chronic HCV infection.

Given the fact that ribavirin resistance-associated mutations (where assessed) were reported in 7 out of 7 cases in which sofosbuvir/ribavirin was given for chronic HEV infection raises an important question regarding whether better responses would be observed if sofosbuvir had been used from the outset prior to the emergence of ribavirin resistance (assuming ribavirin resistance was not transmitted). However, this then diminishes a potential role for sofosbuvir as salvage therapy in refractory cases. The early but only transient fall in HEV RNA in the presence of sofosbuvir observed in the majority of patients may have been due to treatment emergent variants conferring reduced susceptibility to sofosbuvir and this area requires further study.

The in vitro study by Nishiyama et al.⁵⁴ showed that the additive effect of interferons with sofosbuvir could be another strategy to be explored to add to the extremely limited anti-HEV armamentarium. However, given the earlier drive to move away from interferon-based therapy with HCV, in part due to its adverse effects including bone marrow toxicity and neuropsychiatric effects,⁵⁵ there may potentially be an unwillingness to investigate its clinical utility in HEV. If no other treatment options emerge or prove efficacious, then it may need to be a further explored avenue for refractory cases. Pegylated interferon has not been extensively studied or utilised in HEV infection in the same manner that it has been in HCV infection, and data regarding its use in HEV infections are extremely limited. Kamar et al reported cure in 2 of 3 liver transplant recipients with genotype 3 chronic HEV infection treated with 3 months of pegylated interferon-alpha-2a monotherapy.⁵⁶ Haagsma et al. reported successful use of pegylated interferon-alpha-2a monotherapy in 2 other liver transplant recipients with chronic HEV infections.⁵⁷ Therefore, in cases where ribavirin monotherapy has failed and/or mutations are detected associated with reduced efficacy of ribavirin, sofosbuvir in combination with pegylated interferon and possibly also in triple combination with ribavirin could be an option to be further explored albeit taking into consideration the potential adverse effects of these agents. A major consideration however is that pegylated interferon is generally contraindicated in a large proportion of solid organ transplant recipients (kidney, pancreas, heart and lung transplant recipients) due to a potentially increased the risk of transplant rejection.^3,11,50 A systematic review of antiviral therapy in chronic HEV found that pegylated interferon led to acute transplant rejection in 2/8 patients.¹⁸

Treatment of chronic HEV infection remains a challenge not only in terms of which agent(s) to use (where required) but also in terms of the timing of initiation of therapy. It is also complicated by the fact that many the vast majority of patients with chronic HEV infection have significant co-morbidities thereby limiting potential treatment options further. Based on the currently available data, there is no role for sofosbuvir either as monotherapy or as combination therapy in the treatment of chronic HEV infection. Its efficacy when used ab initio with ribavirin, interferon or both, or when used at higher doses for more prolonged periods, remains to be studied. At least until other potential therapeutic options for HEV infection emerge, further work on sofosbuvir as combination therapy is warranted.

Limitations

In vitro studies do provide important preliminary data to support a potential role for sofosbuvir in chronic HEV infection, however, the findings from the in vitro studies to date are not entirely consistent with each other and thus far do not translate into efficacy in clinical studies. Due to the highly different metabolism of sofosbuvir in rodents compared to humans (with only small quantities of the active form of sofosbuvir detected in liver of mice following sofosbuvir administration),²⁵ in vivo murine studies would not serve suitable avenue for further research in this area. Only one human prospective study of sofosbuvir in genotype 3 chronic HEV infection exists to date³² and this study only included 9 patients. There are no prospective clinical trials of sofosbuvir as combination therapy for chronic HEV infection, which is potentially the more promising strategy for its use. Additionally, there are no data for sofosbuvir in chronic HEV infection at doses other than the 400 mg/day dose recommended for HCV infection.

The majority of studies focused on genotype 3 HEV infections which account for the vast majority of chronic HEV infections. Although there are a small number of in vitro studies that have evaluated the antiviral activity of sofosbuvir against genotype 1 and 2 HEV strains, a potential role for sofosbuvir in genotype 1 or 2 severe/fulminant acute HEV infections in at risk groups such as pregnant patients or patients with underlying chronic liver disease has not been widely considered or investigated in clinical studies. Currently, there are no recruiting or active clinical trials for sofosbuvir or any other novel antivirals in the treatment of HEV.

Areas for further research

• •Studies into the safety of administering higher doses of sofosbuvir and further investigation into the role of sofosbuvir at higher doses as adjunctive therapy with ribavirin and possibly with pegylated interferon (taking into consideration the fact the pegylated interferon may be contraindicated in certain transplant recipients)
• •Studies to determine whether sofosbuvir use drives the selection of mutated HEV variants conferring reduced susceptibility to sofosbuvir and further characterisation of these variants (if identified)
• •Investigation of the use of sofosbuvir in combination with ribavirin in severe forms of genotype 1 HEV infections for example in pregnancy and in individuals with underlying chronic liver disease

Credit authorship contribution statement

Conceptualization, methodology, writing, review and editing by Temi Lampejo.

Conflicts of interest

The author has none to declare.

Funding

No specific funding was received for this study.