HEPATOLOGY ELSEWHERE EDITORSHartmut Jaeschke, Tucson, AZKevin Mullen, Cleveland, OHDarius Moradpour, Lausanne, Switzerland

“Strong Reasons Make Strong Actions” —The Antiviral Efficacy of NS3/4A ProteaseInhibitors

Hinrichsen H, Benhamou Y, Wedemeyer H, Reiser M,Sentjens RE, Calleja JL, et al. Short-term antiviral efficacy ofBILN 2061, a hepatitis C virus serine protease inhibitor, inhepatitis C genotype 1 patients. Gastroenterology 2004;127:1347-1355. (Reprinted with permission of American Gas-troenterological Association.)

AbstractBackground: Novel, potent and well-tolerated hepatitis C (HCV)drugs are still needed. BILN 2061 is a potent and specific inhibitor ofHCV serine protease in vitro. Pre-clinical toxicology data and studiesin healthy volunteers supported the administration of BILN 2061 topatients with HCV infection. Methods: The antiviral efficacy, phar-macokinetics and tolerability of 25, 200 and 500 mg bid BILN 2061given as monotherapy for two days in 31 patients infected withchronic genotype 1 HCV infection and with minimal liver fibrosis(Ishak score 0-2) were assessed in a placebo controlled double blindpilot study. In two, subsequent, placebo controlled studies of similardesign, 200 mg bid BILN 2061 was administered for two days to 10patients with advanced liver fibrosis (Ishak score 3-4) and to 10 pa-tients with compensated cirrhosis (Ishak 5-6). Results: Viral RNAreductions of 2-3 log10 copies/mL were achieved in most of the pa-tients. There was a trend towards a higher number of patients receiv-ing 500 mg BILN 2061 achieving a viral RNA reduction >3 log10copies/mL as compared to patients receiving 25 mg BILN 2061. Ad-vanced fibrosis or compensated cirrhosis did not affect the antiviralefficacy of BILN 2061. BILN 2061 was well tolerated in all studies.Conclusions: BILN 2061 is a well tolerated and very active compound,which reduced serum viral RNA concentrations after two days oftreatment in patients infected with genotype 1 HCV independentfrom the degree of fibrosis. Nevertheless further clinical trials are onhold pending resolution of animal toxicity issues.

CommentsMuch of the progress that has been made in the control of

hepatitis C since the discovery of the virus in the late 1980scan be related to the availability of virus-specific tests forhepatitis C virus (HCV) infection. However, the develop-ment of specific antiviral therapies will be an equally signifi-cant benefit resulting from the discovery of HCV. Anexample of this is the NS3/4A protease inhibitor, BILN2061, which was evaluated clinically in studies reported re-cently by Hinrichsen and colleagues.1 BILN 2061 is the firstof what is likely to be a number of specific small moleculetherapeutics to be shown to possess antiviral activity in clin-

ical trials.2 Almost certain to follow are novel compoundstargeting the viral RNA-dependent RNA polymerase,NS5B, as well as additional compounds that inhibit theNS3/4A serine protease.3 These compounds will come fromdrug discovery programs that have benefited from a wealthof information on the structure and function of the criticalenzymatic activities of the nonstructural proteins of HCV,4-6

as well as the development of cell-based systems supportingthe replication of HCV RNA replicons.7

The NS3/4A protease has an essential role in the life cycleof the virus. It folds into two distinct and largely independentdomains, with the N-terminal domain containing the serineprotease activity, and the larger C-terminal domain contain-ing RNA helicase and nucleotide triphosphatase (NTPase)activities.5 The protease domain is stabilized by a tight, non-covalent interaction with a peptide strand from the NS4Aprotein, which interacts with residues within the N-terminalsegment of the protease to form a distorted 8-stranded �-bar-rel.4 The mature NS3/4A serine protease is one of two virallyencoded protease activities that are required for processing ofthe viral polyprotein (Fig. 1). It cleaves in cis at the NS3/4Ajunction, and then subsequently in trans at the NS5A/5B,NS4A/4B and NS4B/5A junctions,8,9 thereby generatingthe nonstructural proteins essential for HCV RNA replica-tion.7 Significant differences exist in the rate of proteolysis ateach site of trans cleavage, and the proteolytic processing ofthe polyprotein appears to occur through a series of carefullyregulated cleavage reactions that are essential for viral repli-cation. The NS3 protein is also an integral part of the viralRNA replicase complex,10 and thus is likely to carry outadditional functions required for viral RNA replication. Aprecise role has not been established for the helicase activityof NS3/4A, but this activity is essential for replication of thevirus.11 Thus, it is not surprising that pharmacological inhi-bition of the protease activity should stop replication of thevirus. Furthermore, since the RNA genome of HCV canonly be maintained within cells through a process of contin-ued replication, it is not likely to persist for long once repli-cation is suppressed. This is an important distinction fromthe human immunodeficiency virus (HIV), which integratesits genome into the host cell DNA, assuring its survival de-spite shutdown of replication.

When the crystallographic structure of the complete bi-functional NS3/4A molecule was solved,5 the C-terminal 6residues of NS3 were found to be closely associated through

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extensive hydrogen bonding with the P-side protease activesite in what appeared to be a product complex arising fromcis-cleavage at the NS3/4A junction. (Conventional nomen-clature assigns the designations NH2-P4-P3-P2-P1-P�1-P�2-P�3-P�4-COOH to the amino acid residues flanking theprotease cleavage site, where scission occurs between the P1and P�1 residues.) This was consistent with prior evidencethat short peptides representing the P-side products of thepolyprotein trans-cleavage reactions have significant (low�mol/L) activity in inhibiting the NS3/4A protease.12,13 Al-though the relatively open, solvent-exposed nature of thesubstrate-binding groove of the NS3/4A protease has madedevelopment of specific inhibitors very challenging, theseshort oligopeptides were the early lead compounds fromwhich peptidomimetic inhibitors of the protease haveevolved. BILN 2061 emerged from an intensive develop-ment program involving iterative structure-activity determi-nations of compounds containing chemical modifications ofthe side-chains of an oligopeptide inhibitor. BILN 2061 ischaracterized as a macrocyclic peptidomimetic because of anintramolecular link created between the P1 and P3 side-chains that significantly enhances its drug-like properties.2

BILN 2061 is extraordinarily active in inhibiting the repli-cation of genotype 1a/b HCV RNA replicons in culturedcells, possessing a half-maximal effective inhibitory concen-tration (IC50) of 3 to 4 nmol/L. In contrast, the concentra-tion required to cause nonspecific cytotoxicity is about10,000-fold higher.2

BILN 2061 was the first specific inhibitor of NS3/4A tobe evaluated clinically. Stunning antiviral efficacy was dem-onstrated in a phase 1 trial involving the administration of 4doses of 200 mg each over a period of two days to relativelyhealthy persons with genotype 1 infection.2 A dramatic de-

crease of more than 2 to 3 log10 in the quantity of viral RNAin the blood was noted within 48 hours of the first dose ofBILN 2061 in each of 10 patients receiving the drug in thisproof-of-concept study. This significantly exceeds the anti-viral response expected even with daily dosing of interferonand ribavirin,14 vividly demonstrating the therapeutic poten-tial of specific NS3/4A protease inhibitors. The additionalclinical studies reported by Hinrichsen et al.1 suggest thatBILN 2061 possesses equivalent antiviral efficacy in patientswith advanced fibrosis, and even compensated cirrhosis.This is an important observation, because extensive fibrosiscould limit the efficacy of a drug. In addition, patients withadvanced fibrosis and cirrhosis may have difficulty toleratingconventional therapy with interferon and ribavirin, althoughthese are precisely the patients most in need of antiviral ther-apy. Also of note, more than half of the patients studied byHinrichsen et al.1 had previously received interferon treat-ment for hepatitis C but for one reason or another had failedto achieve a sustained antiviral response. The antiviral re-sponses to BILN 2061 in this group were similar to those ininterferon-naive patients, confirming that the response tosmall molecule inhibitors of the protease will not be influ-enced by prior unsuccessful interferon-based treatment.

What accounts for the remarkable antiviral efficacy ofBILN 2061? Is it due entirely to the ability of the compoundto inhibit the processing of the viral polyprotein? Or could itreflect in part a reversal of a blockade imposed by the viralprotease on signaling pathways that lead to the induction ofendogenous interferon responses? Recent studies indicatethat the NS3/4A protease effectively blocks the phosphory-lation of interferon regulatory factor 3 (IRF-3) that normallyoccurs in response to virus infection.15 IRF-3 is a latent tran-scription factor that is constitutively expressed in the cyto-plasm. Virus infections induce its specific C-terminalphosphorylation, causing it to dimerize and translocate tothe nucleus, where it plays a central role in inducing theexpression of type 1 interferons (�/�) (Fig 2).16

Virus infections appear to activate IRF-3 via two distinctsignaling pathways: one is triggered by an interaction of dou-ble-stranded RNA (dsRNA) with Toll-like receptor 3(TLR3),17 and another is triggered by the binding of a cellu-lar DEAD-box helicase, retinoic acid-inducible gene I(RIG-I) to structured viral RNA.18 Both pathways also leadto activation of the transcription factor nuclear factor �B(NF-�B), which is similarly blocked by NS3/4A.19,20 Al-though events appear to vary in different cell types, theNS3/4A protease is capable of disrupting both the TLR3 andRIG-I pathways.19,20 In both cases the inhibition is reversedin vitro when cells are treated with a peptidomimetic inhib-itor of the NS3/4A protease.15,20 This suggests that the inhi-bition of signaling is due to NS3/4A-mediated proteolysis ofone or more cellular signaling molecules. The cellular mole-

Fig. 1. Schematic showing proteolytic processing of the polyproteinexpressed by HCV. Two cellular proteases, signal peptidase and signalpeptide peptidase, contribute to the processing of the N-terminal seg-ment of the polyprotein that contains the core, E1, E2, and p7 proteins.Processing of the downstream nonstructural proteins is carried out in cisand in trans by the NS2/3 and NS3/4A proteases, as indicated.

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cule cleaved by the protease within the TLR3 pathway ap-pears to be TRIF (Toll-IL receptor containing inducer ofinterferon, also known as TICAM-1), an adaptor proteinthat links TLR3 to the downstream TBK1/IKK� kinases.20

In contrast, the putative cellular substrate within the RIG-Ipathway has yet to be identified.

Although many questions remain to be answered, thedisruption of these signaling pathways is likely to contributeto the ability of HCV to establish persistent infections. Res-toration of the patency of these signaling pathways by BILN2061 or similar protease inhibitors would restore the abilityof infected cells to generate endogenous interferon responsesthat may play critical roles in limiting virus replication. Suchan effect could be additive or synergistic with the direct an-tiviral effects of the compound, providing what has beendescribed as a “double-whammy” for hepatitis C.21 The ex-tent to which this effect may contribute to the striking anti-viral efficacy of BILN 2061 is difficult to estimate. However,recent nonhuman primate studies with a nonpeptidic inhib-itor of the NS3/4A protease, GW00014X, demonstrated anantiviral effect against GB virus B (GBV-B) that was surpris-ingly robust given the levels of drug achieved in blood rela-tive to the IC50 of the compound.22 The GBV-B protease isclosely related to that of HCV23 and is also able to disruptIRF-3 signaling (K. Li, R. Rijnbrand, and S.M. Lemon, un-published observations, 2004). As Shakespeare put it,“strong reasons make strong actions,” and this indirect effectof protease inhibitors may be important to their antiviralefficacy in vivo. However, the actual clinical benefit derived

from reversing this major immune evasion mechanism ofHCV may only become apparent when comparative clinicalstudies are done with NS3/4A and NS5B inhibitors.

The results reported by Hinrichsen et al.1 and those re-ported earlier by Lamarre et al.2 provide solid proof of theantiviral efficacy in humans of NS3/4A inhibitors such asBILN 2061. These early clinical results hold great promisefor the future, but there are significant hurdles that must beovercome before such drugs enter clinical practice. First, theformulation used in the studies done by Hinrichsen et al.1

would be difficult to adapt for general use, as BILN 2061 wasadministered in a 3-mL solution of polyethylene glycol 400and ethanol due to solubility concerns. Second, the very spe-cific nature of this small molecule therapeutic renders it lessactive against genotype 2 and 3 viruses. In contrast to thestriking antiviral results described above in genotype 1 infec-tions, only 4 of 8 patients with genotype 2 or 3 infection whoreceived four 500-mg doses of BILN 2061 over 48 hoursresponded with a decrease in viral RNA greater than 1log10.24 A significant proportion of patients with chronichepatitis C in the United States and Europe are infected withthese viruses, and it remains to be shown whether otherNS3/4A protease inhibitors will have broader spectrum ac-tivity.

An additional problem is certain to be the emergence ofresistance. This happens rapidly when cells containing repli-con RNAs are exposed to both the antiviral compound andcontinued G418 selection.25,26 Resistance can be broad, as itis with substitutions at Ala156 in the NS3 protein, for exam-ple. A Thr156 substitution confers high-level resistance notonly to the macrocyclic compound BILN 2061, but also to asecond inhibitor from a different chemical series and a non-peptidic, pyrollidine-5,5-trans-lactam inhibitor (M.K. Yiand S.M. Lemon, unpublished data, 2004). A Ser156 sub-stitution has also been shown to cause resistance to yet afourth, chemically unrelated, compound, VX-950.26 TheThr156 substitution significantly impairs both the cis- andtrans-cleavage activities of NS3/4A and results in a corre-sponding reduction in the replication capacity of HCV rep-licons. However, these effects can be reversed by second-sitemutations within the protease that maintain the drug resis-tance phenotype (M.K. Yi and S.M. Lemon, unpublisheddata, 2004). Thus, it seems clear that resistance will be amajor concern clinically, as it has been with inhibitors of theHIV protease.27 The emergence of resistance could be re-duced by concomitant use of interferon, or perhaps a secondantiviral targeting a different HCV protein such as the NS5Bpolymerase. Thus, when protease inhibitors enter clinicalpractice, it is likely that they will be used as one componentof a multiple drug regimen. Significantly, the combinationof an NS3/4A inhibitor and interferon has also been shownrecently to be synergistic in a replicon-based assay.28

Fig. 2. The NS3/4A protease disrupts signaling pathways that nor-mally lead to activation of IRF-3 and NF-�B, and then to induction ofinterferon-� (IFN-�) synthesis, in response to virus infections. IFN-� inturn induces the expression of IRF-7, which activates IFN-� synthesis andsubsequently downstream interferon-stimulated gene (ISG) expression byautocrine and paracrine mechanisms utilizing the Jak-STAT pathway. Seetext for details.

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The major immediate obstacle to further clinical devel-opment of BILN 2061, however, appears to be its poten-tial cardiac toxicity.1 Although the drug proved safe in theshort-term clinical studies reported by Hinrichsen et al.,1

longer term animal toxicology tests employing signifi-cantly higher doses of the compound revealed cardiac ab-normalities that have apparently precluded furtherclinical trials. This is the point at which the public’s accessto information concerning BILN 2061 becomes limited,because details of the abnormalities encountered, the pos-sible underlying mechanisms, and potential approaches toovercoming such toxicity have not been disclosed byBILN 2061 developer Boehringer-Ingelheim (Canada).Until such time as they are revealed, it will remain uncer-tain whether the excitement generated by these first clin-ical trials of BILN 2061 will be justified by its ultimatelicensing (or the licensing of a BILN 2061 derivative). Ifnot, the field will need to wait for a chemically unrelatedinhibitor of the NS3/4A protease to make it all the waythrough the drug development pipeline.

STANLEY M. LEMON, M.D.MINKYUNG YI, PH.D.KUI LI, M.D., PH.D.Center for Hepatitis ResearchInstitute for Human Infections & ImmunityUniversity of Texas Medical BranchGalveston, TX

References1. Hinrichsen H, Benhamou Y, Wedemeyer H, Reiser M, Sentjens RE,

Calleja JL, et al. Short-term antiviral efficacy of BILN 2061, a hepatitis Cvirus serine protease inhibitor, in hepatitis C genotype 1 patients. Gastro-enterology 2004;127:1347-1355.

2. Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger G, Bonneau P, etal. An NS3 protease inhibitor with antiviral effects in humans infected withhepatitis C virus. Nature 2003;426:186-189.

3. Ni ZJ, Wagman AS. Progress and development of small molecule HCVantivirals. Curr Opin Drug Discov Devel 2004;7:446-459.

4. Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer MD, Landro JA, et al.Crystal structure of the hepatitis C virus NS3 protease domain complexedwith a synthetic NS4A cofactor peptide. Cell 1996;87:343-355.

5. Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. Molecular viewsof viral polyprotein processing revealed by the crystal structure of thehepatitis C virus bifunctional protease-helicase. Structure Fold Des 1999;7:1353-1363.

6. Bressanelli S, Tomei L, Roussel A, Incitti I, Vitale RL, Mathieu M, et al.Crystal structure of the RNA-dependent RNA polymerase of hepatitis Cvirus. Proc Natl Acad Sci U S A 1999;96:13034-13039.

7. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science 1999;285:110-113.

8. Hijikata M, Kato N, Ootsuyama Y, Nakagawa M, Shimotohno K. Genemapping of the putative structural region of the hepatitis C virus genome by invitro processing analysis. Proc Natl Acad Sci U S A 1991;88:5547-5551.

9. Tomei L, Failla C, Santolini E, De Francesco R, La Monica N. NS3 is aserine protease required for processing of hepatitis C virus polyprotein.J Virol 1993;67:4017-4026.

10. Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, etal. Identification of the hepatitis C virus RNA replication complex inHuh-7 cells harboring subgenomic replicons. J Virol 2003;77:5487-5492.

11. Kolykhalov AA, Mihalik K, Feinstone SM, Rice CM. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3� non-translated region are essential for virus replication in vivo. J Virol 2000;74:2046-2051.

12. Ingallinella P, Altamura S, Bianchi E, Taliani M, Ingenito R, Cortese R, etal. Potent peptide inhibitors of human hepatitis C virus NS3 protease areobtained by optimizing the cleavage products. Biochemistry 1998;37:8906-8914.

13. Llinas-Brunet M, Bailey M, Fazal G, Goulet S, Halmos T, LaPlante S, et al.Peptide-based inhibitors of the hepatitis C virus serine protease. BioorgMed Chem Lett 1998;8:1713-1718.

14. Bekkering FC, Stalgis C, McHutchison JG, Brouwer JT, Perelson AS.Estimation of early hepatitis C viral clearance in patients receiving dailyinterferon and ribavirin therapy using a mathematical model. HEPATOL-OGY 2001;33:419-423.

15. Foy E, Li K, Wang C, Sumter R, Ikeda M, Lemon SM, et al. Regulation ofinterferon regulatory factor-3 by the hepatitis C virus serine protease. Sci-ence 2003;300:1145-1148.

16. Hiscott J, Grandvaux N, Sharma S, Tenoever BR, Servant MJ, Lin R.Convergence of the NF-kappaB and interferon signaling pathways in theregulation of antiviral defense and apoptosis. Ann N Y Acad Sci 2003;1010:237-248.

17. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of dou-ble-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature 2001;413:732-738.

18. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miy-agishi M, et al. The RNA helicase RIG-I has an essential function indouble-stranded RNA-induced innate antiviral responses. Nat Immunol2004;5:730-737.

19. Foy E, Li K, Sumpter R, Jr., Loo MY, Johnson C, Wang C, et al. Controlof antiviral defenses through hepatitis C virus disruption of RIG-I signal-ing. Proc Natl Acad Sci U S A 2005, in press.

20. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon ACM, Ikeda M, et al.Immune evasion by hepatitis C virus N3/4A protease-mediated cleavage ofthe TLR3 adaptor protein TRIF. Proc Natl Acad Sci U S A 2005, in press.

21. Farley S. A double whammy for hep C. Nat Rev Drug Discov 2003;2:419.22. Bright H, Carroll AR, Watts PA, Fenton RJ. Development of a GB virus B

marmoset model and its validation with a novel series of hepatitis C virusNS3 protease inhibitors. J Virol 2004;78:2062-2071.

23. Scarselli E, Urbani A, Sbardellati A, Tomei L, De Francesco R, Traboni C.GB virus B and hepatitis C virus NS3 serine proteases share substratespecificity. J Virol 1997;71:4985-4989.

24. Reiser M, Hinrichsen H, Benhamou Y, Sentjens R, Wedemeyer H, CallejaL, et al. Antiviral effect of BILN 2061, a novel HCV serine proteaseinhibitor, after oral treatment over 2 days in patients with chronic hepatitisC, non-genotype 1. HEPATOLOGY 2003;38(Suppl. 1):221A. Abstract 136.

25. Lu L, Pilot-Matias TJ, Stewart KD, Randolph JT, Pithawalla R, He W, et al.Mutations conferring resistance to a potent hepatitis C virus serine proteaseinhibitor in vitro. Antimicrob Agents Chemother 2004;48:2260-2266.

26. Lin C, Lin K, Luong YP, Rao BG, Wei YY, Brennan DL, et al. In vitroresistance studies of hepatitis C virus serine protease inhibitors, VX-950and BILN 2061: structural analysis indicates different resistance mecha-nisms. J Biol Chem 2004;279:17508-17514.

27. Kuritzkes DR. Preventing and managing resistance in the clinical setting. JAcquir Immune Defic Syndr 2003;34(Suppl 2):S103-S110.

28. Lin K, Kwong AD, Lin C. Combination of a hepatitis C virus NS3-NS4Aprotease inhibitor and alpha interferon synergistically inhibits viral RNAreplication and facilitates viral RNA clearance in replicon cells. AntimicrobAgents Chemother 2004;48:4784-4792.

Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20610Conflict of interest: Nothing to report.

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Hepatitis C Virus and the Threshold ofNatural Killer Cell Inhibition

Reprinted abstracted with permission from Khakoo SI,Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, etal. HLA and NK Cell Inhibitory Receptor Genes in Re-solving Hepatitis C Virus Infection. Science 2004;305:872-874. Copyright 2004 AAAS. http://www.sciencemag.org

AbstractNatural killer (NK) cells provide a central defense against viral

infection by using inhibitory and activation receptors for major his-tocompatibility complex class I molecules as a means of controllingtheir activity. We show that genes encoding the inhibitory NK cellreceptor KIR2DL3 and its human leukocyte antigen C group 1(HLA-C1) ligand directly influence resolution of hepatitis C virus(HCV) infection. This effect was observed in Caucasians and AfricanAmericans with expected low infectious doses of HCV but not inthose with high-dose exposure, in whom the innate immune responseis likely overwhelmed. The data strongly suggest that inhibitory NKcell interactions are important in determining antiviral immunity andthat diminished inhibitory responses confer protection against HCV.

CommentsThe innate immune response is the first line of defense

against infecting pathogens. Natural killer (NK) cells playan important part in this response as they recognize andlyse infected cells and produce inflammatory cytokinescapable of inhibiting replication of specific viral patho-gens and activating the adaptive immune response.

NK cells integrate signals from both activating andinhibitory receptors. Because inhibition dominates overactivation, the threshold for NK cell activation is lowest inthe absence of a ligand for an inhibitory receptor.1,2 Twodifferent kinds of inhibitory receptors are known, theCD94:NKG2A receptor, a C-type lectin, and a group ofthe killer cell immunoglobulin-like receptors (KIRs).Whereas CD94:NKG2A and its ligand HLA-E are highlyconserved, KIRs and their ligands HLA-A, HLA-B, andHLA-C are highly polymorphic. In fact, the KIR system iseven more diverse than the HLA system due to its allelicpolymorphism3 and due to different KIR expression onindividual NK cell clones.4 Thus, almost all persons differin their KIR genotypes, and within each person KIRs aredifferentially expressed on individual NK cell clones.

Although the KIR system was initially thought to consist ofonly inhibitory NK cell receptors, further research revealed anumber of activating NK cell receptors encoded by the samegene locus on chromosome 19q13.4. Information on the acti-vating or inhibitory nature of each receptor is included in itsname. Each KIR consists of either two or three extracellularimmunoglobulin domains (2D or 3D) and either short (S) or

long (L) cytoplasmatic tails.5 As illustrated in Fig. 1, KIRs withshort cytoplasmatic tailsmediate activationviaDAP12(DNAXactivation protein of 12 kd) and immunoreceptor tyrosine-based activation motifs (ITAM), whereas KIRs with long tailstransmit inhibitory signals via immunoreceptor tyrosine-basedinhibition motifs (ITIM). Because inhibitory signals dominateover activating ones,6 the general rule that NK cell activation isinduced by the absence of the ligand for an inhibitory receptorstill holds true.

Because of the highly polymorphic nature of KIR andHLA systems it has long been speculated that specific geno-types might influence the response to pathogens and contrib-ute to the outcome of infections. The recent study byKhakoo et al. provides evidence for an epidemiological asso-ciation between specific KIR/HLA compound genotypesand the outcome of hepatitis C virus (HCV) infection.7 Inthis study, the investigators analyzed 352 subjects who hadspontaneously cleared HCV and 685 subjects who had de-veloped persistent HCV infection. Subjects who were ho-mozygous for KIR2DL3 and group 1 HLA-C alleles weremore likely to recover from HCV infection than patientswith any other KIR/HLA compound genotype. Interest-ingly, the association between KIR2DL3:HLA-C1 andHCV clearance was statistically significant only for thosesubjects who had been infected via injection drug use (n �524) or via accidental needlestick (n � 19) and not for thosewho had been transfused with blood products, i.e., erythro-cytes (n � 41) or clotting factors (n � 453). Therefore, theauthors hypothesize that genetically influenced NK cell re-sponsiveness has the strongest impact on infection with sup-posedly low amounts of HCV, i.e., in the nontransfusedgroup, and speculate that NK cell responses may have beenoverwhelmed by the large amount of transmitted HCV inthe transfused group.

Now, what is so special about HLA-C1:KIR2DL3 ho-mozygosity? Group 1 HLA C allotypes such as Cw*1,Cw*3, Cw*7, Cw*8 are characterized by a serine at posi-tion 77 and asparagine at position 80. In contrast, group2 HLA C allotypes such as Cw*2, Cw*4, Cw*5, Cw*6,Cw*15 are characterized by an asparagine at position 77and lysine at position 80. Group 1 HLA C-allotypes areligands for KIR2DL2 and KIR2DL3 and group 2 HLA Callotypes bind to KIR2DL1. Khakoo et al. postulate thatHLA-C1:KIR2DL3 homozygosity is the least inhibitorycombination. This is because homozygosity for HLA-C1requires the absence of HLA-C2, the ligand forKIR2DL1. Furthermore, homozygosity for KIR2DL3 re-quires the absence of KIR2DL2, because KIR2DL3 andKIR2DL2 are located on the same locus. If HLA-C1:KIR2DL3 is the least inhibitory compound genotype, theNK cell activation threshold should be lower in thesepatients and facilitate eradication of HCV infection. This

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interpretation is in line with a prior report by Fanning etal., who describe an association between the group 2HLA-C allele Cw*04 and a chronic course of infection ina single source outbreak of HCV.8 It would be interestingto determine the KIR haplotypes in this single sourceoutbreak to see whether the protective nature of the HLA-C1:KIR2DL3 compound genotype can be confirmed.

Along the same line, it would also be interesting toanalyze the large cohort by Khakoo et al. for a role of theadaptive immune response in the outcome of hepatitis C.All patients studied by Khakoo et al. displayed HCV an-tibodies indicating activation of the adaptive immune sys-tem. Moreover, a separate analysis of the U.S. subgroup ofthis cohort9 as well as other studies support a role forMHC class II alleles in the outcome of hepatitis C (re-cently reviewed by Yee10). All previous studies includefewer patients with spontaneous viral clearance than thestudy by Khakoo et al., and it would therefore be inter-esting to determine in the large cohort of the present study

whether HLA class II alleles present an independent prog-nostic factor for the outcome of infection.

As this epidemiological report shows a potential rolefor NK cell responses in the outcome of HCV infection,the study by Khakoo et al. opens several interesting ave-nues for further investigation. An immediate question re-lates to the functional basis for the observedepidemiological association. The authors hypothesizethat the interaction between KIR2DL3 and group 1 HLAC allotypes results in the weakest inhibition of NK cells.Although it has been reported that group 1 HLA C allo-types display weaker binding affinity to KIR2DL3 than toKIR2DL2 and KIR2DL1, these studies have used KIR-Igfusion proteins and did not study intact NK cells.11 Withrespect to functional assays of NK cell inhibition, the dataare even less well defined as KIR proteins were overex-pressed in KIR-negative NK cells using the vaccinia virussystem and KIR2DL2 exerted only mildly stronger inhi-bition in cytotoxicity assays than KIR2DL3.11 Finally, itis also unknown whether the degree of NK cell inhibitionis influenced by the peptides that are displayed on theHLA-C molecules. Although some studies succeeded inidentifying individual peptides that modulated NK cellfunction in the context of HLA-B*270512,13 andCw*0304,14 Cw*415 and Cw*7,16 it still appears unlikelythat all HLA-C allotypes of a given group present thesame peptides because of differences in individual peptidebinding motifs.17 Functional analyses of NK cell inhibi-tion and activation in KIR and HLA-typed subjects aretherefore necessary to unravel the specific immunologicalmechanisms that may explain these epidemiological ob-servations.

Several other observations by Khakoo et al. also war-rant further investigation. HLA-C1:KIR2DL3 was onlyassociated with HCV clearance if exposure occurred viainjection drug use or accidental needlestick and not if itoccurred via transfusion. The authors explain this findingwith the innate immune response being overwhelmed bythe large amount of virus transmitted via transfusion. Assmall animal models of HCV infection do not exist, thishypothesis is difficult to prove experimentally. Experi-mental infection of chimpanzees typically results in rapidincrease of HCV serum titers within the first week afterinfection and delayed induction of cellular immune re-sponses.18 Thus, viral replication appears to outpace theimmune response after high-dose HCV infection.Whereas a systematic comparison of high and low-doseHCV infections has not been performed in chimpanzees,recent human studies demonstrated that exposure toHCV via needlestick transiently activates the adaptive cel-lular immune response even in the absence of clinicallyevident infection.19 It is therefore conceivable that low-

Fig. 1. Structure of KIR2DL1, 2DL2, 2DL3 and 3DS1. The KIRs arenamed according to their number of extracellular domains (D) and thelength of the cytoplasmatic tail (long � L, short � S). Long tails containimmunoreceptor tyrosine-based inhibition motifs (ITIMs) and transmitinhibitory signals. Short cytoplasmic tails are associated with the DNAXactivation protein of 12 kd (DAP12) that includes immunoreceptortyrosine-based activation motifs (ITAMs) and transmits activating signals.Note that the interaction between 3DS1 and Bw4 is hypothetical and hasnot yet been formally demonstrated.

676 HEPATOLOGY ELSEWHERE HEPATOLOGY, March 2005

dose exposure may also activate NK cells as part of theinnate immune response. In addition, other factors suchas HCV quasispecies diversity and the number of repeatedexposures may play a role. Regarding quasispecies diver-sity an HCV-contaminated transfusion product likelycontains a higher number of HCV quasispecies than arelatively small amount of blood from a contaminatedneedle, and this difference might be further amplified bythe “bottleneck phenomenon,” i.e., by preferential repli-cation of a small subpopulation of quasispecies bestadapted to the new host. Regarding the number of expo-sures hemophiliacs and injection drug users are likely tohave multiple exposures, whereas blood transfusions andaccidental needlestick injuries are more likely a one-timeexposure. Thus, there might be additional factors thatinfluence the immune response and contribute to the out-come of HCV infection.

It is also of interest that a statistically significant associa-tion between HLA-C1:KIR2DL3 homozygosity and HCVclearance was found in Caucasians, and that the same trend,albeit not statistically significant, was found in African Amer-icans.7 Although the lack of statistical significance in AfricanAmericans is possibly due to the smaller number of AfricanAmericans (n � 106) in the study, it is also known thatAfrican Americans clear HCV infection less frequently thanCaucasians and that the distribution of HLA class I and classII haplotypes differs among ethnic groups.20,21 Indeed, Thioet al. described HCV clearance to be associated with differentHLA class II alleles in African Americans and Caucasiansnoticing for example a stronger association of HLA-DQB1*0301 with viral clearance in African Americans thanin Caucasians.9 Thus, future studies should address the dif-ferential impact of genetic markers of NK cell responses inindividual ethnic groups.

In summary, the study by Khakoo et al. provides epide-miological evidence that the outcome of HCV infection maybe influenced by various degrees of genetically predeter-mined NK cell inhibition and activation. Their data shouldstimulate functional studies to identify the underlying mech-anisms and further epidemiological studies on specific ethnicsubgroups and routes of HCV transmission.

GOLO AHLENSTIEL

BARBARA REHERMANN

Liver Diseases Section, NIDDK,National Institutes of Health, DHHS,Bethesda, MD

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Copyright © 2005 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.20622Conflict of interest: Nothing to report.

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