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linics and Research in Hepatology and Gastroenterology (2013) 37, 10—16

Available online at

www.sciencedirect.com

INI REVIEW

epatitis C virus and lipids in the era of directcting antivirals (DAAs)avid A. Sheridana,∗, R. Dermot G. Neelyb, Margaret F. Bassendinea

Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United KingdomDepartment of Clinical Biochemistry, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom

vailable online 5 September 2012

ummary The six different HCV-genotypes have marked differences in response to therapyith pegylated interferon-� and ribavirin. The introduction of the direct acting antiviral (DAA)rotease inhibitors, telaprevir and boceprevir in combination with pegylated interferon-� andibavirin has become the new standard of care for genotype 1 infection. Several host factorsssociated with response to pegylated interferon-� and ribavirin are not as important in pre-icting response to triple therapy, and yet low-density lipoprotein cholesterol (LDLC) and statinse remain important associations of outcome with DAAs. This review focuses on the clinicalssociations between lipids and treatment response to interferon based antiviral treatments.

e consider how understanding the interactions of HCV and host lipid metabolism remains rel-vant in the era of DAAs for genotype 1 infection and for treatment of non-genotype 1 chronicepatitis C.

2012 Elsevier Masson SAS. All rights reserved.

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epatitis C virus (HCV) chronically infects around 170 mil-ion people worldwide. The treatment for chronic hepatitis

virus (CHC) has been combination pegylated interferon- and ribavirin (PR). There are six major HCV-genotypeshich have markedly different response rates to interferon

IFN) based therapies. There is currently no explanationor the HCV-genotype specific differences in treatment out-

ome. There is some evidence to suggest that interferonensitivity correlates with the relative evolutionary agesf the HCV-genotypes [1] (Fig. 1), although this remains

∗ Corresponding author. Institute of Cellular Medicine, Leechuilding, Newcastle University, Newcastle upon Tyne, NE2 4HH,nited Kingdom. Tel.: +44 1912 228 782; fax: +44 1912 220 723.

E-mail address: david.sheridan@ncl.ac.uk (D.A. Sheridan).

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210-7401/$ – see front matter © 2012 Elsevier Masson SAS. All rights rettp://dx.doi.org/10.1016/j.clinre.2012.07.002

ontroversial [2]. Pang et al. hypothesised that immuneelection was a significant driving force in the divergencef the HCV-genotypes [1], but other host factors includinghe interactions with lipids may have exerted a selectiveressure on the evolution of HCV. HCV is known to interactith host lipid metabolism in a genotype specific manner [3].hus patients infected by HCV-genotype 3 have striking dif-erences in lipid metabolism compared to HCV-genotype 1.CV-genotype 3 is associated with viral steatosis and low LDLholesterol (LDLC) concentration, whereas HCV-genotype 1s associated with insulin resistance and higher LDLC con-entrations, and yet HCV-genotype 3 is more sensitive to

FN based therapy.

Considerable advances have been made in defining theactors that predict poor response to PR in HCV-genotype

infection (Table 1). These include IL28B genotype [4—6],

served.

HCV and lipids in the era of DAAs 11

Figure 1 A schematic representation of the relative evolutionary ages of the major hepatitis C virus (HCV) genotypes with caninehepacivirus (CHV) as a common ancestor [2]. The relative evolutionary age of HCV correlates with sustained virological response(SVR) to previous interferon-based standard of care (pegylated interferon-� and ribavirin).

Adapted from Pang et al., 2009 [1].

Table 1 Reported host and viral factors associated with outcome to Pegylated interferon-� and ribavirin alone and in combi-nation with direct acting antivirals, telaprevir and boceprevir, in treatment of hepatitis C virus genotype 1 infection.

Dual therapyPeginterferon-� + Ribavirin

Triple TherapyProtease Inhibitor + Peginterferon-� + Ribavirin

Factor Effect on SVR Telaprevir Trials Boceprevir Trials

Naive Experienced Naive Experienced

Age Increasing age reduced SVR[15]

No [67] Not reported No [9] Not reported

Male Male had lower SVR [68] No [67] No [69]

IL28Brs12979860rs8099917

CC Predicts SVR [4—6,70] Not reported No [71] Yes Yes [9]

ISGs/IP10 High ISGs reduced SVR [72] Not reported Not reported Not reported Not reported

Insulin resis-tance/HOMAIR

IR Reduced SVR [59—61] No effect [67] Not reported Not reported Not reported

LDL cholesterol Low LDL reduces SVR[14—16,73]

Yes [67] Yes [17] Not reported Not reported

Statin use Associated with higher SVR[16]

Not reported Not reported Yes [9] Not reported

Fibrosis stage Cirrhosis reduced SVR [68] Yes [67] Yes if prior nullor partial EVR;No if priorrelapse [13]

Yes [9] Yes [12]

LVP High LVP ratio reduced EVR[8,45]

Unknown Unknown Unknown Unknown

Viral load VL < 400,000 IU/mLassociated with SVR [74]

No [67] Not reported Yes but lessimportant [69]

Not reported

BMI High BMI reduced SVR [75] No [67] Not reported No [69] Not reported

ApoE High apoE reduced EVR [8] Unknown Unknown Unknown Unknown

Abbreviations: ISG: interferon stimulated genes; IP10: interferon gamma inducible protein 10 kDa; HOMA IR: homeostasis model assess-ment of insulin resistance; LDL: low density lipoprotein; LVP: lipoviral particles; BMI: body mass index; SVR: sustained virological response;EVR: early virological response.

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igh interferon stimulated genes (surrogate marker IP10),nd metabolic factors including LDLC and insulin resistance.he current licensed direct acting antivirals (DAAs) (thentiproteases boceprevir and telaprevir) are administereds triple therapy in combination with PR in patients withCV-genotype 1 with substantial increases in sustained viro-

ogical response (SVR) (reviewed in [7]), comparable tohose of HCV-genotype 3 treated with PR alone. Severalactors identified as important predictors of response toR remain important in these triple therapy regimens. Weave recently reported an association of lipids with sensi-ivity to IFN in HCV-genotype 1 [8]. In this mini-review weriefly address the role of lipids in the HCV lifecycle and innterferon sensitivity.

actors associated with response toeginterferon-� + ribavirin and triple therapy

ost and viral factors known to be associated with responseo PR alone and combination triple therapy with boceprevirnd telaprevir in treatment naïve and experienced patientsith HCV-genotype 1 infection are summarised in the table.ata on factors associated with SVR in response to bocepre-ir and telaprevir has been reported from post hoc analysesf the landmark phase 3 trials in treatment naïve (Sprint-

[9], Advance [10], Illuminate [11]) and PR treatmentxperienced (Respond-2 [12], Realize [13]) patients withCV-genotype 1. ‘Real world’ data from use of these agents

n routine clinical practice is not yet available. Many fac-ors associated with response to dual therapy (PR) do notppear to be significant predictors of response to triple ther-py including insulin resistance, BMI, male sex, age and viraload. Initial data also suggest that IL28B genotype may beess important in telaprevir-based regimens than in bocepre-ir or PR alone.

LDLC concentration is an independent predictor of SVRo dual PR therapy in HCV-genotype 1 [14—16]. Interest-ngly, LDLC remained a significant independent predictor ofVR in post hoc analysis of the telaprevir-based REALIZErial [17]. Statin use in HCV-genotype 1 has been associ-ted with increased SVR in dual PR therapy [16]. Givenhat other host factors appear less important in predict-ng response to triple therapy, it is striking that LDLCemains significantly associated with response to telapre-ir triple therapy in both naïve and experienced patients.lthough similar data on LDLC and boceprevir have noteen reported, statin use was associated with increasedesponse to boceprevir triple therapy in naïve patients9].

hy is low-density lipoprotein cholesterolavourable to treatment response?

he continued association of LDLC and treatment outcomeo triple therapy (PR + DAAs) implies that factors involved inegulation of plasma LDLC concentrations affect the inter-

ction of HCV and the host. These interactions may occurt the stages of entry, replication, assembly or remod-lling in the vascular compartment. The major steps ofhe HCV lifecycle have been unravelled by experiments

ntsD

D.A. Sheridan et al.

tilising Japanese Fulminant Hepatitis virus 1 (JFH1), aenotype 2a strain of HCV that is able to replicate in theepatoma (Huh7) cell line (HCVcc) [18]. This model sys-em has allowed the steps of viral entry and replication toe studied in vitro, but has inherent limitations to study-ng HCV-lipid interactions in vivo, because the Huh7 celline does not process very low density lipoproteins (VLDL)ormally [19]. HCV entry in the liver is thought to be a mul-istage process with initial capture of viral particles fromhe blood by sinusoidal endothelial cells and concentra-ion at the hepatocyte basolateral surface. Internalisationf HCVcc involves interaction of the HCV envelope glyco-roteins E1/E2 with a number of cell surface receptorsncluding SRB1, tetraspanin CD81, claudin 1 and occludinreviewed in [20]). The role of the low-density lipoproteineceptor (LDLR) remains controversial. LDLR is required as

source of cholesterol, essential for viral replication inuh7 cells but is not required for entry of HCVcc [21]. How-ver, in vivo evidence suggests that lipoprotein receptorsre more likely to be important. LDLR is required for per-issive uptake of HCV by primary hepatocytes but not foruh7 cells [22]. It is possible that chronic HCV infectionlters regulation of LDLR in vivo. Indeed we have foundhat plasma levels of proprotein convertase subtilisin/kexinype 9 (PCSK9), a proprotein convertase that regulates LDLRecycling and has been shown to inhibit HCV entry in vitro23], are lower in patients with HCV-genotype 3 infection24]. Recently, the Niemann-Pick C1 like 1 (NPC1L1) recep-or has been identified as another HCV entry co-factor [25].CP1L1 can be inhibited by ezetimibe, therefore ezetemibe

s an attractive agent for further trials as an HCV entrynhibitor [26]. Targeting lipoprotein receptors involved inhe multistep process of HCV entry remains an importanttrategy to prevent recurrence following liver transplanta-ion and may be also important in vaccine design (reviewedn [27]).

Replication and assembly of infectious HCV is intimatelynterwoven with intracellular lipid droplets and produc-ion of VLDL. HCV core associates with the surface ofntracellular lipid droplets [28—30] and recruits viral non-tructural proteins including NS5A and NS5B to distributeround core coated lipid droplets [31] as the replica-ion complex. New viral particles are co-assembled withLDL [32,33]. The main structural component of VLDL ispolipoprotein B (apoB). Suppression of apoB by smallnterfering RNA (siRNA) inhibits both VLDL and HCVcc pro-uction [34] but does not inhibit replication [35]. NS5Also recruits intracellular apolipoprotein-E (apoE) whichs required for the production of infectious virus [36].MG CoA reductase inhibitors (statins) have been shown

n vitro, to have some antiviral efficacy on HCV repli-ation that is synergistic with IFN [37—39]. It should beoted however that concentrations of fluvastatin requiredo inhibit HCV replication in vitro were ten times higherhan plasma concentrations achieved by dosing fluvastatin0 mg daily for 4 weeks [37], therefore making sufficientosing to achieve antiviral efficacy problematic in vivo. Ithould also be noted that telaprevir alters the pharmacoki-

etics of atorvastatin [40]. Therefore, it is unlikely thatargeting interactions of HCV and apoB/apoE will impactignificantly on HCV replication with comparable efficacy toAAs.

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HCV and lipids in the era of DAAs

Hepatitis C virus association with lipoproteinsin the vascular compartment

HCV particles in the circulation are associated with lipopro-teins. Association with lipoproteins of varying triglycerideand cholesterol composition determines the density dis-tribution of HCV RNA in plasma [41]. HCVcc particles oflow-density have higher specific infectivity in vitro [31,42].Low-density HCV particles associated with apoB containinglipoproteins (VLDL, IDL, LDL) have been termed ‘lipoviralparticles’ (LVP) [43,44]. We have shown that levels of LVP(HCV RNA at density < 1.07 g/L) in the fasting state in HCV-genotype 1 infection were correlated with insulin resistance[45] and plasma apolipoprotein-E concentration [8]. In vitroHCV particles have been shown to be enriched in apoEand possess a unique lipid composition [46]. This is sup-ported by a recent study showing that LVP have elevatedand steady levels of apoCIII/IV which reduces lipoproteinlipase mediated inhibition of HCV infection [47]. Moreover,the association of HCV with lipoproteins in the vascularcompartment appears to be highly dynamic. Very low-density HCV particles (d < 1.025 g/L) increase on average26 fold after a high fat meal [48]. These particles arethen rapidly cleared, possibly by the VLDL remnant path-way. Postprandial HCV particles can be associated with VLDLand chylomicrons [48,49]. The rapid postprandial increaseof HCV co-associated with triglyceride rich lipoproteins islargely contributed by transfer within the circulation [48].Furthermore, it has been reported that there is an excessof nucleocapsid free LVP in vivo [50], which are HCV RNA‘empty’ sub-viral apoB/HCV envelope composites. There-fore, HCV induced alterations in VLDL composition and thedynamic nature of HCV particles in the circulation may bea crucial factor in permitting infectivity and facilitatingescape from antibody mediated clearance [51]. These fac-tors may also contribute to the lack of efficacy of antibodymediated vaccines to HCV [52].

Hepatitis C virus-genotype specific differencesin lipid metabolism

It is apparent that there are HCV-genotype specific dif-ferences in lipid and metabolic phenotypes betweenHCV-genotypes 1 and 3. HCV-genotype 3 in particular is asso-ciated with low LDLC, apoB and hepatic steatosis, thought tobe due to viral inhibition of microsomal triglyceride transferprotein (MTP) [53]. Our data supports reduced endogenouscholesterol synthesis in both HCV-genotype 1 and genotype3. However, in HCV-genotype 3 there is evidence of selectivepreservation of desmosterol, and of increased LDLR activity,suggesting that HCV-genotype 3 is having similar effects onLDLC as statin therapy [24]. Functionally this supports theconcept that HCV-genotypes 1 and 3 utilise different lipidpathways as the preferred portals of entry into the liver.

ApoE is a likely ligand for highly infectious LVP to bind toLDLR because apoE are enriched on low-density HCV par-

ticles [44,46,54]. ApoE is distributed in plasma betweentriglyceride rich lipoproteins (TRLs, i.e. chylomicrons andVLDL remnants) and HDL. The distribution of apoE is iso-form dependent. ApoE4 is distributed more in TRLs, whereas

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poE3 and E2 preferentially associate with HDL. The infec-ivity of HCV in vitro is influenced by apoE isoforms, withhe E2 isoform that has low affinity for binding to the LDLRaving reduced infectivity [55]. This is supported by an ear-ier report of an association of the apoE2 allele with reducedisk of chronic HCV infection [56].

We have found that genotype 1 LVP positively correlateith plasma apoE concentration [8] and insulin resistance

45], whereas genotype 3 LVP inversely correlate with apoEnd HDL/apoA1 [57]. Individual host metabolic factors andiral genotype specific variations in the association of HCVith lipoproteins (e.g. apoE and apoC’s), whether at theoint of assembly and secretion, or following intravascularransfer, is therefore likely to influence the predominanteceptor (SRB1, CD81, NPC1L1 or LDLR) that mediatesptake and infectivity in vivo. Additional complexity inivo is introduced by competition between LVP and nativeipoproteins for these receptors as portals of entry. Usef lipid modulating agents as adjuncts to antiviral therapyherefore needs to be evaluated in HCV-genotype specifictudies.

linical studies of lipid modulation in hepatitis virus

number of retrospective and prospective studies havehown an association of increased LDLC and favourableesponse to PR therapy [8,58]. In HCV-genotype 1 infection,tatins have shown limited antiviral efficacy when used asonotherapy [59—63], although in small numbers appearell tolerated [64]. Statin use in combination with PR may

mprove outcome. In a retrospective study of US veterans65], statin use in diabetics and non diabetics was associatedith increased SVR, and statin use was also associated with

ncreased SVR in a sub-group in the Ideal study [16]. How-ver, these associations need to be interpreted with caution,ecause the favourable IL28B CC genotype is also associatedith increased LDLC [8,58,66]. The association of IL28B andDLC is dependent on HCV infection and is not associatedfter viral clearance [58]. It is possible that reported statinse may be a reflection of high LDLC, and LDLC is a surrogatendicator of IL28B genotype and interferon stimulated genexpression rather than statins enhancing interferon sensitiv-ty per se. The possible mechanisms of the co-association ofDLC, IL28B and interferon sensitivity have not been eluci-ated. We have identified an association of IL28B and apoE,ndirectly mediating levels of infectious lipoviral particlesn genotype 1 infection [8]. We have found an associationf IL28B CC with lower serum apoE levels, and higher ‘non-VP’, in addition to increased LDLC and apoB [8]. Targetingipids therefore remains an attractive possibility as a noveldjunctive therapy to enhance or restore interferon sensitiv-ty in vivo [8]. It could be that lipid modulating therapy thatims to raise LDLC and/or lower apoE may increase inter-eron sensitivity, by reducing levels of infectious lipoviralarticles, and reducing LDLR as a portal of viral entry.

onclusions

he intimate interaction of HCV and lipid pathways con-inues to provide possible therapeutic avenues in the post

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AA era, because of the association of lipids with interferonensitivity. Remarkably LDLC and statin use are associatedith favourable outcomes in the phase 3 DAA trials. Fur-

her data of factors associated with non-response to DAAsn ‘real world’ use needs to be reported. A mass of initro data emphasises the importance of lipid metabolismo the viral lifecycle, and there are striking differences inipid metabolism between the HCV-genotypes. Appropriatelyargeted lipid modulating drugs therefore remain attrac-ive candidates as adjunctive agents in IFN null responderso potentially restore IFN sensitivity, block entry to pre-ent HCV recurrence post liver transplantation and targetntravascular remodelling which may be relevant for vaccinefficacy. Understanding of HCV-genotype specific perturba-ions in lipid pathways is therefore vital to devise and tailoripid modulating agents in appropriate clinical trials.

isclosure of interest

he authors declare that they have no conflicts of interestoncerning this article.

Funding: Funding supported by the Medical Researchouncil (G0502028) and the Newcastle upon Tyne Healthcareharity.

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