- Email: [email protected]
Apolipoprotein H expression is associated with IL28B genotype and viral clearance in hepatitis C virus infection
Accepted Manuscript Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection Melissa E. Laird, Amira Mohsen, Darragh Duffy, Rasha Mamdouh, Lenaig LeFouler, Armanda Casrouge, Mai El-Daly, Mona Rafik, Mohamed AbdelHamid, Alexandre Soulier, Jean-Michel Pawlotsky, Christophe Hézode, Isabelle Rosa, Philippe Renard, Mostafa K. Mohamed, Philippe Bonnard, Jacques Izopet, Vincent Mallet, Stanislas Pol, Matthew L. Albert, Arnaud Fontanet PII: DOI: Reference:
S0168-8278(14)00388-2 http://dx.doi.org/10.1016/j.jhep.2014.05.040 JHEPAT 5193
To appear in:
Journal of Hepatology
Received Date: Revised Date: Accepted Date:
18 November 2013 5 May 2014 25 May 2014
Please cite this article as: Laird, M.E., Mohsen, A., Duffy, D., Mamdouh, R., LeFouler, L., Casrouge, A., El-Daly, M., Rafik, M., Abdel-Hamid, M., Soulier, A., Pawlotsky, J-M., Hézode, C., Rosa, I., Renard, P., Mohamed, M.K., Bonnard, P., Izopet, J., Mallet, V., Pol, S., Albert, M.L., Fontanet, A., Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection, Journal of Hepatology (2014), doi: http:// dx.doi.org/10.1016/j.jhep.2014.05.040
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1 Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection. Laird, Melissa E.1,2, Mohsen, Amira3, Duffy, Darragh1,2, Mamdouh, Rasha4, LeFouler, Lenaig5, Casrouge, Armanda1,2, El-Daly, Mai6, Rafik, Mona 4, AbdelHamid, Mohamed6,7, Soulier, Alexandre8,9, Pawlotsky, Jean-Michel8,9, Hézode, Christophe9,10 , Rosa, Isabelle 9,10, Renard, Philippe 11, Mohamed, Mostafa K. 6, Bonnard, Philippe12,13 , Izopet, Jacques14,15, Mallet, Vincent16,17,18, Pol, Stanislas 16,17,18, Albert, Matthew L.1,2,18*, Fontanet, Arnaud5,19 *
1
Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
2
INSERM U818, Paris, France
3
Community Medicine Department, National Research Center, Cairo, Egypt
4
Faculty of Medicine, Ain Shams University, Cairo, Egypt
5
Emerging Disease Epidemiology Unit, Institut Pasteur, Paris, France
6
Liver Disease Research Unit, National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
7
Faculty of Medicine, Minia University, Egypt
8
National Reference Center for Viral Hepatitis B, C and D, Department of Virology,
Hôpital Henri Mondor, Université Paris-Est, Créteil, France 9
INSERM U955, Créteil, France
10
Department of Hepatology and Gastroenterology, Hôpital Henri Mondor,
Université Paris-Est, Créteil, France. 11
Department of Gastroenterology and Hepatology, Hôpital Victor Dupouy,
Argenteuil, France 12
Maladies Infectieuses et Tropicales, Hôpital Tenon (APHP), Paris, France
2 13
INSERM U-707, UPMC, Paris, France
14
Department of Virology, CHU Toulouse, Toulouse
15
INSERM U1043, IFR-BMT, Toulouse, France
16
Université Paris Descartes, Paris, France
17
Institut Cochin, INSERM (IMR-S1016), CNRS (UMR 8104), Paris France
18
Assistance Publique – Hôpitaux de Paris (APHP), Groupe Hospitalier Cochin Saint-Vincent de Paul, Unité d’Hepatologie, Paris, France.
19
Conservatoire National des Arts et Métiers, Paris, France
* indicates shared authorship
Co-Corresponding Authors: Pr. Arnaud Fontanet Unit of Emerging Disease Epidemiology Institut Pasteur, Paris, France Telephone : +33 1 40 61 37 66 Fax: +33 45 68 88 76 Email: [email protected]
Pr. Matthew L. Albert Unit of Dendritic Cell Immunobioogy Institut Pasteur & Inserm U818, Paris, France Telephone : +33 1 45 68 89 02 Fax: + 33 1 45 68 85 48 Email: [email protected]
3 Abstract Word Count : 239 Manuscript Word Count : 4694 Number of Figures : 4 Number of Tables : 1
List of Abbreviations: HCV: Hepatitis C virus VLDL: very low density lipoprotein Apo: apolipoprotein aHCV: acute Hepatitis C virus cHCV: chronic Hepatits C virus LVP: lipoviral particle ALT: serum alanine aminotransferase RNA: ribonucleic acid PCR: polymerase chain reaction MAP: multi-analyte profiling SNP: single nucleotide polymorphism IFNλ: Interferon lambda CL: cleared patient NCL: not cleared EVR: early virologic response SVR: sustained virologic response NR: non-responder HIV: Human Immunodeficiency virus
4 Conflict of Interest: To our knowledge, none of the authors listed maintain any financial interests that would serve as a conflict of interest for the publication of this manuscript.
Financial Support: This work was supported by ANRS grants 12210, 12199, 12135 (MLA, AF), the European Research Council Young Investigator Award (MLA), and the European FP7 project SPHINX (ID 261365). Chronic HCV patient samples were provided from an INSERM sponsored clinical trial and also from the control arm of the REALIZE trial as run by Tibotec (Trial N° VX-950-TiDP24-C216). HCV/HIV patient samples were provided as part of the ANRS HC 20 ETOC study (NCT00901524).
5
ABSTRACT
Background and Aims: HCV requires host lipid metabolism for replication, and apolipoproteins have been implicated in the response to treatment. Methods: We examined plasma apolipoprotein concentrations in three cohorts of patients: mono-infected patients with symptomatic acute hepatitis C (aHCV); those undergoing treatment for chronic hepatitis C (cHCV); and HIV/HCV co-infected patients being treated for their chronic hepatitis C. We also evaluated associations between apolipoproteins and IL28B polymorphisms, a defined genetic determinant of viral clearance. Results: Plasma apolipoprotein H (ApoH) levels were significantly higher in patients who achieved spontaneous clearance or responded to pegylated-interferon / ribavirin therapy. Strikingly, patients carrying the IL28B rs12979860 CC SNP correlated with the plasma concentration of ApoH in all three cohorts. Both ApoH and IL28B CC SNP were associated with HCV clearance in univariate analysis. Additional multivariate analysis revealed that the association between IL28B and HCV clearance was closely linked to that of Apo H and HCV clearance, suggesting that both belong to the same biological pathway to clearance. The association between IL28B CC SNP and ApoH was not observed in healthy individuals, suggesting that early postinfection events trigger differential ApoH expression in an IL28B allele dependent manner. Conclusions: This relationship identifies ApoH as the first induced protein quantitative trait associated with IL28B, and characterizes a novel host factor implicated in HCV clearance.
6 Keywords:
Hepatitis
C
virus,
Quantitative
trait
loci,
Lipid
metabolism,
Apolipoproteins, IL28B
INTRODUCTION Hepatitis C virus (HCV) represents a serious public health problem, infecting approximately 170 million people worldwide [1]. Around 70% of infected individuals will progress to chronic HCV infection, one third of which are at significantly increased risk of progressive liver fibrosis, cirrhosis and hepatocellular carcinoma. Recent advances in the deployment of first generation direct acting antivirals (DAAs), and the development of second generation DAAs, have been major steps forward in the eradication of HCV infection [2-5]. HCV is known to be critically dependent on host lipid metabolism. HCV proteins require intracellular association with lipid droplets for productive virion assembly [6], and circulating infectious virions associate with very low-density lipoprotein (VLDL)-like particles, referred to as lipoviral particles (LVP) [7]. Incorporation of host apolipoproteins (apos) into the LVP is postulated to assist in viral entry [7-9], and several apolipoproteins are necessary for viral assembly and the production of infectious virions [10-12]. Additionally, elevated lipid levels have been associated with the response to pegylated interferon α2 / ribavirin (PR) therapy during chronic HCV infection, suggesting a role for host proteins in viral clearance [13, 14]. Genome wide association studies (GWAS) have identified a striking association between single nucleotide polymorphisms (SNPs) near the IL28B locus and spontaneous clearance of untreated HCV infection, as well as response to PR therapy [15-18]. For the rs12979860 SNP within the IL28B locus, the “protective” CC genotype confers a 2- to 3-fold higher rate of spontaneous viral clearance or sustained
7 virologic response (SVR) following PR treatment, as compared to either the CT or TT haplotype [16-18]. Recently, a dinucleotide SNP in strong linkage disequilibrium with the rs12979860 SNP was shown to determine expression of a newly discovered gene product, IFN-λ4 [19, 20]. In addition higher plasma levels of ApoB have been reported to be associated with sustained virological response in patients carrying the rs8099917 responder genotype (located proximal to rs12979860) in IL28B [21]. In this study, we report ApoH as a correlate of both spontaneous clearance and response to therapy in HCV patients. Moreover, we observed that ApoH concentration was higher in subjects carrying the CC allele of the IL28B haplotype. This was confirmed across three independent patient cohorts infected with two different HCV genotypes (1 and 4). Based on multivariate analysis, we show that the association between IL28B and HCV clearance is closely linked to that of ApoH and HCV clearance, suggesting that both belong to the same biological pathway to clearance. These findings open new areas for HCV research and may prove critical to defining the mechanism behind the strong genetic association between IL28B and HCV clearance.
8
PATIENTS AND METHODS Patient, Cohort and Study Group Information Clinical study details, patient information, sample collection and monitoring of the three HCV cohorts are described in Table S1.
Multi-analyte profiling Plasma was collected from fasting patients in Vacutainer blood collection tubes containing Sodium Heparin (BD) and stored at -80°C. Plasma was clarified by highspeed centrifugation and analyzed using Luminex xMAP technology. The measurement of 11 apolipoproteins was performed by Myriad-Rules Based Medicine, (Austin, TX), according to guidelines set by the USA Clinical and Laboratory Standards Institute.
IL28B genotype determination IL-28B genotype at SNP position rs12979860 was determined by real-time PCR using genomic DNA extracted from frozen serum samples in conjunction with minor groove binder probes, [22] and as described in Supplemental Methods.
Statistical analyses Categorical and continuous data were compared across groups using Chi Square and Mann-Whitney tests, respectively and as indicated. Mixed linear regression models were used to analyze longitudinal apolipoprotein expression data according to spontaneous clearance status in the acute hepatitis C cohort.
9 Logistic regression models were used to estimate the increase in the odds of clearance associated with IL28B variants (CC versus CT-TT combined) and ApoH plasma concentrations (introduced as a categorical variable, in quartiles, in the model). Models were built for each of the three cohorts. Variables (IL28B SNP and ApoH) were tested in separate models first to estimate their individual effect on clearance, and then introduced simultaneously in the same model to estimate their independent effect on clearance. Since the HCV chronic cohort included both naïve and treated patients, odds-ratios were adjusted for history of treatment. For the model using HIV/HCV co-infected patients, odds-ratios for IL28B variants were calculated using median unbiased estimates with exact logistic regression models as all (9/9) IL28B CC variants cleared the virus under treatment (this would translate into an infinitely positive odds-ratio in classical logistic regression models).
10 RESULTS To address the potential impact of the rs12979860 IL28B SNP on host apolipoprotein levels and HCV clearance, the levels of plasma apolipoproteins and IL28B SNP genotypes were evaluated in three different HCV cohorts: (i) acute HCV (aHCV) patients in Cairo, Egypt (n=33), (ii) chronic HCV (cHCV) patients receiving standard pegIFN/RBV treatment (n=141), and (iii) HIV/HCV co-infected patients treated with a double dose of pegIFN and a standard dose of RBV (n=43). Summarized clinical characteristics of each cohort are provided in Table S1. The high incidence of HCV genotype 4 in Egypt (14.7% seropositivity) provides the unique opportunity to identify acute, symptomatic HCV patients, and potentially factors that influence spontaneous viral clearance [23-25]. Patients were recruited after presenting with symptoms to two fever hospitals in Cairo, as described in the Supplementary Information, and followed for at least six months after the onset of symptoms. Of the 33 aHCV patients examined, 19 spontaneously cleared their virus (“cleared”, CL), and the remaining 14 individuals developed chronic HCV infection (“not cleared”, NCL). We examined 11 apolipoproteins for differences between CL and NCL groups, and for associations with the IL28B SNP (Table S2). ApoCI was associated with the IL28B SNP, but did not correlate with viral clearance. Only ApoH demonstrated significant associations with both HCV clearance (p=0.005, Fig. 1A) and the CC IL28B allele. ApoH levels were significantly elevated in those individuals encoding the protective CC IL28B haplotype (n=19), as compared to those expressing either the CT or TT variant (n=14, p=0.03, Fig.
1C). Longitudinal
analysis demonstrated that ApoH levels in the CL patients remained consistently elevated compared to those observed in the NCL group in the six months following diagnosis (p<0.0001, Fig. 1B). ApoH levels were not associated with either plasma
11 viral load, or liver inflammation, as indicated by the plasma serum alanine transaminase (ALT) concentration (Fig. S1A, B). These results suggested a novel relationship between ApoH and the rs12979860 SNP, which we sought to confirm in distinct patient cohorts. We next examined chronically infected HCV patients, pooling information from our previously characterized cHCV cohort [26], and patients treated as part of the control arm of the REALIZE trial [27], a total of 141 individuals. Of the 141 patients treated, 40 achieved SVR and 101 were non-responders (NR), based on standard clinical criteria [28]. Supporting our findings in aHCV, individuals who achieved viral clearance had higher baseline ApoH plasma levels (samples were drawn prior to treatment), as compared to NR patients (p=0.002, Fig. 2A). Other apolipoproteins (Apo A1, B, C1, and D) showed differences between CL and NCL patients though only Apo B also associated with IL28B genotype (Table S3). As our dataset was comprised of treatment-naïve and treatment-experienced patients, we stratified patients by treatment history. The association of viral clearance and high plasma ApoH concentration was maintained, regardless of treatment history (Fig. 2B), thus suggesting that ApoH concentrations may predict the functional readiness of a patient to clear HCV. ApoH levels were not associated with baseline viral load, HCV genotype or the extent of fibrosis progression (Fig. S1C-E). However, baseline ApoH was higher in patients with the IL28B CC allele (p<0.0001, Fig. 2C,D). Finally, we extended our study to a third patient cohort. HIV/HCV co-infected patients show a reduced tolerance and response rate to PR therapy, and progression to fibrosis is more rapid than in HCV mono-infected individuals [29, 30]. As prior studies have shown that the rs12979860 IL28B SNP correlates significantly with viral clearance in HIV/HCV-infected patients [18, 31, 32], we examined if ApoH also
12 associated with viral clearance and the IL28B SNP in this disease setting. We analyzed data from an ongoing HIV/HCV co-infected cohort, basing our association analysis on the primary endpoint for viral clearance: an early virologic response (EVR) as defined by a greater than two log reduction of HCV RNA viral load at week 12 post-treatment initiation. Forty-three patients were identified for analysis: 26 achieved EVR, while 17 were classified as non-responders (NR). As previously seen in both the aHCV and cHCV cohorts previously, plasma ApoH levels were significantly elevated in HIV/HCV patients who achieved EVR (p=0.002, Fig. 3A), but were not associated with HCV viral load, genotype or liver fibrosis (Fig. S1F-H). No other apolipoproteins showed differences in this patient population (Table S4). Higher baseline levels of plasma ApoH were found in IL28B CC patients, compared to those with non-CC genotypes (p=0.05, Fig. 3B). Patients recently reached the SVR24 endpoint, and the association between plasma ApoH and SVR24 was found again; however due to low numbers of patients who achieved viral clearance (n = 8), the results did not achieve statistical significance between the two groups (SVR24 vs. NR, p = 0.08) (Fig. S2). The results obtained in each of our patient groups were modeled in order to ascertain the
relationship
between IL28B polymorphisms,
baseline ApoH
concentrations and viral clearance (Table 1). In all three patient populations, IL28B and baseline ApoH were significantly associated with HCV clearance in univariate analysis.
However, when introduced simultaneously into the same model, the
association between ApoH and viral clearance overrode that between IL28B and viral clearance. These data suggest that ApoH is a stable predictor of viral clearance across the three patient populations, and is situated within the pathway of the IL28B CC predictive allele and HCV viral clearance [33].
13 Two scenarios could explain the observed relationship between IL28B and apoH plasma concentrations. Either, circulating ApoH levels may be influenced by the genetic variation at the IL28B locus, serving as a predisposing predictor of enhanced viral clearance; or alternatively, in the context of HCV infection ApoH levels are differentially regulated with IL28B polymorphisms, In order to distinguish between these two possibilities, we assessed steady-state levels of plasma ApoH and IL28B SNP distribution in healthy donors (n=100), and observed no difference between haplotypes (Fig. 4A,B). These data suggest that differential ApoH expression is established early in the pathogenesis of HCV infection, with concentrations impacted by variation at the IL28B locus.
14
DISCUSSION As HCV is known to be critically dependent on host lipid metabolism for productive viral infection we examined the association of several apolipoproteins with viral clearance. We observed a significant, consistent correlation between elevated levels of plasma apoH concentrations and viral clearance in three different patient populations. Recent studies detailing the genetic variation within the IL28B locus prompted us to investigate possible associations between apolipoprotein expression and the well-characterized rs12979860 IL28B SNP. Interestingly, we demonstrated that increased levels of ApoH were associated with the protective CC IL28B SNP (rs 12979860). Strengthening the relevance of this finding was the confirmation of this association across two different genotypes (1 and 4), and three different disease states [34].
While
we
acknowledge
the
new
data
indicating
IFNλ4-creating
ss469415590[∆G] allele as being more closely associated with response to PR therapy [19, 20], we were unable to obtain these data for our patient cohorts. Nonetheless, the high level of linkage disequilibrium with rs12979860 IL28B SNP, especially in persons of European descent (HapMap, r2 = 0.92) [20], indicates that it would not impact the findings reported herein. Although several apolipoproteins have been identified to play critical roles in the HCV life cycle [7-12, 35], ApoH has not previously been linked to HCV replication or viral clearance. Briefly, ApoH (also known as β2-glycoprotein I or β2GPI) is synthesized by hepatocytes and is found on several classes of lipoprotein particles. ApoH has been principally studied in the context of coagulation, where it is thought to act as an inhibitor of the intrinsic coagulation cascade through its regulation of prothrombinase. Furthermore, the presence of anti-β2-GPI antibodies is
15 a criteria for antiphospholipid syndrome. Notably, HCV patients have been reported to harbor anti-β2-GPI antibodies [36] and it is well known that chronic HCV patients are at risk for coagulopathies [37]. ApoH can opsonize dying cells [38], and its ability to decorate lipoprotein particles and apoptotic cells is likely due to its avidity for phosphatidylserine or other anionic phospholipids [39]. ApoH has also been shown to bind the enveloped viruses [40, 41], and while data has not yet been reported, it can presumably bind to HCV lipoviral particles. Based on our data, we hypothesize that ApoH opsonization of HCV lipoviral particles may serve as an inhibitory mechanism, preventing the binding to entry receptors and accounting for enhanced viral clearance in patients with higher levels of ApoH. Alternatively, it is possible that ApoH influences HCV clearance secondary to its role as an activator of lipoprotein lipase (LPL) [42, 43], as prior data has shown that LPL inhibits HCV infection [44]. ApoH has also been reported as an acute phase reactant [45, 46]; however, the ApoH promoter does not contain interferon response elements, and there is no known association between IFN and ApoH induction. The development of novel HCV treatment regimens has progressed rapidly in recent years, from the standardization of triple therapy (IFN, ribavirin and direct-acting antiviral agents, DAA) ([47]), to the recent interferon-free regimens, which will transform the management of patients ([25]). Ongoing work in our laboratory is focused on investigating the impact of ApoH in the context of these new therapies, as well as the mechanism of how it is involved in viral clearance. Regarding the association between the IL28B CC variant (rs12979860 SNP) and apoH, it is interesting that no correlation was found in healthy donors. This suggests that ApoH concentration is an induced trait, with the trigger being HCV infection. Differences were observed in plasma ApoH levels depending on the
16 infection stage; with lower overall ApoH concentrations detected in acute infection, most notably among those moving towards chronic infection, and higher overall levels during chronic HCV. Our findings are also unique in that, for the extent of patient follow-up data examined, circulating ApoH levels remain elevated following HCV clearance in the acute HCV cohort (Fig. 1B). Future studies will be designed to characterize the kinetics of plasma ApoH expression following spontaneous viral clearance. While intriguing, comparisons of plasma ApoH levels between acute and chronic stages should be treated with caution as they are not based on longitudinal follow-up of the same individuals, and instead are from different study populations. Four recent studies have identified relationships between IL28B genotype and lipid metabolism [42-45]. In contrast to our findings, Sheridan and colleagues identified a significant association between ApoE levels, EVR and IL28B polymorphisms [48]. We believe that the work of Sheridan warrants additional investigation, particularly in light of the importance of ApoE for viral assembly [12], but we highlight several technical differences that could explain the conflict between our results. First, we assessed ApoE levels in patient plasma, whereas serum samples were used by Sheridan et al. Second, ApoE was measured using CLIA-certified sandwich immunoassays in our study, whereas Sheridan utilized automated nephelometric methods to quantify ApoE concentrations, the accuracy of which may be limited by the association of ApoE and HCV proteins in LVP. In the study of McCarthy et al., the authors demonstrated that subjects with the rs12979860 CC responder genotype had elevated low-density lipoprotein (LDL) cholesterol [49], however the association between LDL levels and viral clearance was substantially attenuated when IL28B polymorphisms were evaluated as possible confounding factors. A follow-up study indicated an interesting link between IL28B CC genotype
17 and a lower prevalence of steatosis [50]. In an independent study, it was shown that pre-treatment levels of LDL cholesterol were higher in patients with a favorable IL28B polymorphism, but again the association between LDL and HCV clearance was lost when regressing out the genetic differences between patient groups [51]. Thus, our data identify a host factor that is a likely part of the causal pathway to viral clearance as the IL28B polymorphism. With a growing body of evidence documenting that gene expression data may not directly correlate with protein abundance [52-55], the identification of proteinbased quantitative traits associated with genetic loci is gaining attention as a possible method to further our understanding of the association of genetic variability and disease outcome [56]. The identification of ApoH constitutes the first induced trait that associates with IL28B, and more interestingly with HCV clearance, in multivariate analyses across three unique HCV cohorts. These data support the necessity of further investigation into how IL28B is modulating expression of ApoH, and how ApoH may be influencing the viral life cycle and/or the antiviral immune response. Our study also further highlights the importance and value of clinical cohort studies for identifying potential mediators of disease clearance.
ACKNOWLEDGEMENTS We thank Stéphanie Thomas (INSERM) for project management support, Estelle Mottez of the Center for Human Immunology (CIH), Institut Pasteur for clinical coordination, and Sam Labrie (Myriad RBM) for Luminex analysis.
18
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20 genome-wide association study. Gastroenterology 2010;138:1338-1345, 1345 e1331-1337. [33] Greenland S, Robins JM. Identifiability, exchangeability and confounding revisited. Epidemiol Perspect Innov 2009;6:4. [34] Moonesinghe R, Khoury MJ, Janssens AC. Most published research findings are false-but a little replication goes a long way. PLoS Med 2007;4:e28. [35] Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007;5:453-463. [36] Matsuda J, Saitoh N, Gotoh M, Gohchi K, Tsukamoto M, Syoji S, et al. High prevalence of anti-phospholipid antibodies and anti-thyroglobulin antibody in patients with hepatitis C virus infection treated with interferon-alpha. Am J Gastroenterol 1995;90:1138-1141. [37] Sene D, Piette JC, Cacoub P. [Antiphospholipid antibodies, antiphospholipid syndrome and viral infections]. Rev Med Interne 2009;30:135141. [38] d'Angeac AD, Stefas I, Duperray C, Rucheton M, Graafland H, Montero JL, et al. Oxidation and biotinylation of beta 2 glycoprotein I glycan chains induce an increase in its affinity for anionic phospholipids similar to that obtained by the addition of anti-beta 2 glycoprotein I or anti-cardiolipin antibodies. J Immunol Methods 2005;300:160-178. [39] Balasubramanian K, Maiti SN, Schroit AJ. Recruitment of beta-2glycoprotein 1 to cell surfaces in extrinsic and intrinsic apoptosis. Apoptosis 2005;10:439-446. [40] Adlhoch C, Kaiser M, Hoehne M, Mas Marques A, Stefas I, Veas F, et al. Highly sensitive detection of the group A Rotavirus using Apolipoprotein Hcoated ELISA plates compared to quantitative real-time PCR. Virol J 2011;8:63. [41] Stefas I, Rucheton M, D'Angeac AD, Morel-Baccard C, Seigneurin JM, Zarski JP, et al. Hepatitis B virus Dane particles bind to human plasma apolipoprotein H. Hepatology 2001;33:207-217. [42] Nakaya Y, Schaefer EJ, Brewer HB, Jr. Activation of human post heparin lipoprotein lipase by apolipoprotein H (beta 2-glycoprotein I). Biochem Biophys Res Commun 1980;95:1168-1172. [43] Andreo U, Maillard P, Kalinina O, Walic M, Meurs E, Martinot M, et al. Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection. Cell Microbiol 2007;9:2445-2456. [44] Maillard P, Walic M, Meuleman P, Roohvand F, Huby T, Le Goff W, et al. Lipoprotein lipase inhibits hepatitis C virus (HCV) infection by blocking virus cell entry. PLoS One 2011;6:e26637. [45] Agar C, de Groot PG, Morgelin M, Monk SD, van Os G, Levels JH, et al. beta(2)-glycoprotein I: a novel component of innate immunity. Blood 2011;117:6939-6947. [46] Sellar GC, Keane J, Mehdi H, Peeples ME, Browne N, Whitehead AS. Characterization and acute phase modulation of canine apolipoprotein H (beta 2glycoprotein I). Biochem Biophys Res Commun 1993;191:1288-1293. [47] Zeuzem S, Andreone P, Pol S, Lawitz E, Diago M, Roberts S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med 2011;364:2417-2428. [48] Sheridan DA, Bridge SH, Felmlee DJ, Crossey MM, Thomas HC, TaylorRobinson SD, et al. Apolipoprotein-E and hepatitis C lipoviral particles in
21 genotype 1 infection: evidence for an association with interferon sensitivity. J Hepatol 2012;57:32-38. [49] Li JH, Lao XQ, Tillmann HL, Rowell J, Patel K, Thompson A, et al. Interferon-lambda genotype and low serum low-density lipoprotein cholesterol levels in patients with chronic hepatitis C infection. Hepatology 2010;51:19041911. [50] Tillmann HL, Patel K, Muir AJ, Guy CD, Li JH, Lao XQ, et al. Beneficial IL28B genotype associated with lower frequency of hepatic steatosis in patients with chronic hepatitis C. J Hepatol 2011;55:1195-1200. [51] Clark PJ, Thompson AJ, Zhu M, Vock DM, Zhu Q, Ge D, et al. Interleukin 28B polymorphisms are the only common genetic variants associated with lowdensity lipoprotein cholesterol (LDL-C) in genotype-1 chronic hepatitis C and determine the association between LDL-C and treatment response. Journal of Viral Hepatitis 2012;19:332-340. [52] Foss EJ, Radulovic D, Shaffer SA, Goodlett DR, Kruglyak L, Bedalov A. Genetic variation shapes protein networks mainly through non-transcriptional mechanisms. PLoS Biol 2011;9:e1001144. [53] Foss EJ, Radulovic D, Shaffer SA, Ruderfer DM, Bedalov A, Goodlett DR, et al. Genetic basis of proteome variation in yeast. Nat Genet 2007;39:1369-1375. [54] Futcher B, Latter GI, Monardo P, McLaughlin CS, Garrels JI. A sampling of the yeast proteome. Mol Cell Biol 1999;19:7357-7368. [55] Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999;19:1720-1730. [56] Melzer D, Perry JR, Hernandez D, Corsi AM, Stevens K, Rafferty I, et al. A genome-wide association study identifies protein quantitative trait loci (pQTLs). PLoS Genet 2008;4:e1000072.
22
FIGURE LEGENDS Fig. 1. Plasma ApoH is strongly associated with IL28B genotype and spontaneous viral clearance in acute HCV infection. (A) Plasma ApoH in patients that spontaneously cleared (CL, n=19) or failed to clear virus (NCL, n=14); MannWhitney p-value = 0.005. (B) Kinetics of plasma ApoH in CL () and NCL patients (), mean values and standard deviation are shown.
Mixed model longitudinal
regression analysis was performed (p < 0.0001). (C) Associations between IL28B rs12979860 polymorphisms and apoH were examined. Elevated plasma ApoH is observed in CC patients (n=19), compared to those with the CT or TT SNP (“nonCC”) (n=14); M-W p-value = 0.03.
Fig. 2. Plasma ApoH is strongly associated with IL28B genotype and response to IFN/Ribavirin treatment in chronic HCV. (A) Baseline plasma ApoH levels in patients that responded (SVR n=40) or failed to respond (NR n=101) to pegIFNα/RBV (M-W p-value = 0.002). (B) Patients were segregated by treatment history; SVR (naïve, n=13; treated, n=27), and NR (naïve, n=6; treated, n=94); (M-W p-value = 0.03 and 0.02, respectively). (C and D) Patients were stratified by IL28B genotype; CC (n=24) vs. “non-CC” (n=117) alleles (C), (M-W p-value < 0.0001). Further investigation compared CC (n=24) vs. CT (n=77) vs. TT (n=40) (D) (KruskalWallis p-value < 0.0001).
Fig. 3. Plasma ApoH is associated with IL28B genotype and early virologic response and in patients co-infected with HIV and HCV. (A) Plasma apoH was evaluated in HIV/HCV co-infected patients that achieved early virologic response to
23 peg-IFNα/RBV therapy (EVR, n=26), as compared to nonresponders (NR, n=17); a M-W test was performed (p-value = 0.002) (B). Patients were stratified based on expression of the protective CC allele (n=6), vs. the “non-CC” allele (n=33); analysis was performed using a M-W test (p-value = 0.05).
Fig. 4. Circulating levels of ApoH are independent of IL28B genotype in healthy donors. (A and B) Plasma ApoH levels were evaluated in 100 healthy donor plasma samples. ApoH levels remained equivalent regardless of the IL28B genotype of the healthy individual.
Table 1. Uni- and multivariate analysis of the association between IL28B, apo H, and viral clearance. Univariate OR (95% CI) Acute HCV cohort IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤135 136-166 167-197 ≥198 Chronic HCV cohort# IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤160 161-194 195-242 ≥243 HCV/HIV cohort *§ IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤136 136-213 214-283 ≥284
5.04 (1.13 – 22.5) 1 5.83 (0.70 - 48.9) 4.67 (0.53-40.9) 28.0 (2.07-379.2) 4.19 (1.38 – 12.7) 1 1.77 (0.52-6.01) 0.84 (0.20-3.56) 6.31 (1.87-21.3) 10.3 (1.46 - ∞) 1 1.00 (0.13-7.91) 7.07 (0.84-100.5) 13.5 (1.15-786.3)
P value 0.03
0.03
0.01
0.004
0.02
0.03
Adjusted-OR 2.80 (0.50 – 15.8) 1 3.62 (0.37-35.6) 3.47 (0.36-33.4) 17.9 (1.20-266.8) 2.77 (0.81 – 9.44) 1 1.64 (0.48-5.60) 0.69 (0.16-3.02) 4.64 (1.30-16.5) 1.69 (0.17 - ∞) 1 0.95 (0.10-9.67) 4.47 (0.41-76.0) 7.05 (0.45-471.1)
Adjusted-P value 0.24
0.22
0.23
0.02
0.54
0.13
* Outcome is spontaneous virological clearance in the acute HCV cohort, sustained virological response in the chronic HCV cohort, and early virological response in the HIV co-infected cohort. # One patient was taking Simvastatin but results are unchanged after excluding him from the analysis. § 6 (out of 43) patients were taking lipid lowering medication. No differences were observed (p=0.99) in ApoH levels between those patients under lipid treatment (mean 240ug/ml) compared to those without treatment (mean 239ug/ml). In addition no differences were observed when we removed these 6 patients from the global analysis.
S0168-8278(14)00388-2 http://dx.doi.org/10.1016/j.jhep.2014.05.040 JHEPAT 5193
To appear in:
Journal of Hepatology
Received Date: Revised Date: Accepted Date:
18 November 2013 5 May 2014 25 May 2014
Please cite this article as: Laird, M.E., Mohsen, A., Duffy, D., Mamdouh, R., LeFouler, L., Casrouge, A., El-Daly, M., Rafik, M., Abdel-Hamid, M., Soulier, A., Pawlotsky, J-M., Hézode, C., Rosa, I., Renard, P., Mohamed, M.K., Bonnard, P., Izopet, J., Mallet, V., Pol, S., Albert, M.L., Fontanet, A., Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection, Journal of Hepatology (2014), doi: http:// dx.doi.org/10.1016/j.jhep.2014.05.040
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1 Apolipoprotein H expression is associated with IL28B genotype and viral clearance in Hepatitis C virus infection. Laird, Melissa E.1,2, Mohsen, Amira3, Duffy, Darragh1,2, Mamdouh, Rasha4, LeFouler, Lenaig5, Casrouge, Armanda1,2, El-Daly, Mai6, Rafik, Mona 4, AbdelHamid, Mohamed6,7, Soulier, Alexandre8,9, Pawlotsky, Jean-Michel8,9, Hézode, Christophe9,10 , Rosa, Isabelle 9,10, Renard, Philippe 11, Mohamed, Mostafa K. 6, Bonnard, Philippe12,13 , Izopet, Jacques14,15, Mallet, Vincent16,17,18, Pol, Stanislas 16,17,18, Albert, Matthew L.1,2,18*, Fontanet, Arnaud5,19 *
1
Laboratory of Dendritic Cell Immunobiology, Institut Pasteur, Paris, France
2
INSERM U818, Paris, France
3
Community Medicine Department, National Research Center, Cairo, Egypt
4
Faculty of Medicine, Ain Shams University, Cairo, Egypt
5
Emerging Disease Epidemiology Unit, Institut Pasteur, Paris, France
6
Liver Disease Research Unit, National Hepatology & Tropical Medicine Research Institute, Cairo, Egypt
7
Faculty of Medicine, Minia University, Egypt
8
National Reference Center for Viral Hepatitis B, C and D, Department of Virology,
Hôpital Henri Mondor, Université Paris-Est, Créteil, France 9
INSERM U955, Créteil, France
10
Department of Hepatology and Gastroenterology, Hôpital Henri Mondor,
Université Paris-Est, Créteil, France. 11
Department of Gastroenterology and Hepatology, Hôpital Victor Dupouy,
Argenteuil, France 12
Maladies Infectieuses et Tropicales, Hôpital Tenon (APHP), Paris, France
2 13
INSERM U-707, UPMC, Paris, France
14
Department of Virology, CHU Toulouse, Toulouse
15
INSERM U1043, IFR-BMT, Toulouse, France
16
Université Paris Descartes, Paris, France
17
Institut Cochin, INSERM (IMR-S1016), CNRS (UMR 8104), Paris France
18
Assistance Publique – Hôpitaux de Paris (APHP), Groupe Hospitalier Cochin Saint-Vincent de Paul, Unité d’Hepatologie, Paris, France.
19
Conservatoire National des Arts et Métiers, Paris, France
* indicates shared authorship
Co-Corresponding Authors: Pr. Arnaud Fontanet Unit of Emerging Disease Epidemiology Institut Pasteur, Paris, France Telephone : +33 1 40 61 37 66 Fax: +33 45 68 88 76 Email: [email protected]
Pr. Matthew L. Albert Unit of Dendritic Cell Immunobioogy Institut Pasteur & Inserm U818, Paris, France Telephone : +33 1 45 68 89 02 Fax: + 33 1 45 68 85 48 Email: [email protected]
3 Abstract Word Count : 239 Manuscript Word Count : 4694 Number of Figures : 4 Number of Tables : 1
List of Abbreviations: HCV: Hepatitis C virus VLDL: very low density lipoprotein Apo: apolipoprotein aHCV: acute Hepatitis C virus cHCV: chronic Hepatits C virus LVP: lipoviral particle ALT: serum alanine aminotransferase RNA: ribonucleic acid PCR: polymerase chain reaction MAP: multi-analyte profiling SNP: single nucleotide polymorphism IFNλ: Interferon lambda CL: cleared patient NCL: not cleared EVR: early virologic response SVR: sustained virologic response NR: non-responder HIV: Human Immunodeficiency virus
4 Conflict of Interest: To our knowledge, none of the authors listed maintain any financial interests that would serve as a conflict of interest for the publication of this manuscript.
Financial Support: This work was supported by ANRS grants 12210, 12199, 12135 (MLA, AF), the European Research Council Young Investigator Award (MLA), and the European FP7 project SPHINX (ID 261365). Chronic HCV patient samples were provided from an INSERM sponsored clinical trial and also from the control arm of the REALIZE trial as run by Tibotec (Trial N° VX-950-TiDP24-C216). HCV/HIV patient samples were provided as part of the ANRS HC 20 ETOC study (NCT00901524).
5
ABSTRACT
Background and Aims: HCV requires host lipid metabolism for replication, and apolipoproteins have been implicated in the response to treatment. Methods: We examined plasma apolipoprotein concentrations in three cohorts of patients: mono-infected patients with symptomatic acute hepatitis C (aHCV); those undergoing treatment for chronic hepatitis C (cHCV); and HIV/HCV co-infected patients being treated for their chronic hepatitis C. We also evaluated associations between apolipoproteins and IL28B polymorphisms, a defined genetic determinant of viral clearance. Results: Plasma apolipoprotein H (ApoH) levels were significantly higher in patients who achieved spontaneous clearance or responded to pegylated-interferon / ribavirin therapy. Strikingly, patients carrying the IL28B rs12979860 CC SNP correlated with the plasma concentration of ApoH in all three cohorts. Both ApoH and IL28B CC SNP were associated with HCV clearance in univariate analysis. Additional multivariate analysis revealed that the association between IL28B and HCV clearance was closely linked to that of Apo H and HCV clearance, suggesting that both belong to the same biological pathway to clearance. The association between IL28B CC SNP and ApoH was not observed in healthy individuals, suggesting that early postinfection events trigger differential ApoH expression in an IL28B allele dependent manner. Conclusions: This relationship identifies ApoH as the first induced protein quantitative trait associated with IL28B, and characterizes a novel host factor implicated in HCV clearance.
6 Keywords:
Hepatitis
C
virus,
Quantitative
trait
loci,
Lipid
metabolism,
Apolipoproteins, IL28B
INTRODUCTION Hepatitis C virus (HCV) represents a serious public health problem, infecting approximately 170 million people worldwide [1]. Around 70% of infected individuals will progress to chronic HCV infection, one third of which are at significantly increased risk of progressive liver fibrosis, cirrhosis and hepatocellular carcinoma. Recent advances in the deployment of first generation direct acting antivirals (DAAs), and the development of second generation DAAs, have been major steps forward in the eradication of HCV infection [2-5]. HCV is known to be critically dependent on host lipid metabolism. HCV proteins require intracellular association with lipid droplets for productive virion assembly [6], and circulating infectious virions associate with very low-density lipoprotein (VLDL)-like particles, referred to as lipoviral particles (LVP) [7]. Incorporation of host apolipoproteins (apos) into the LVP is postulated to assist in viral entry [7-9], and several apolipoproteins are necessary for viral assembly and the production of infectious virions [10-12]. Additionally, elevated lipid levels have been associated with the response to pegylated interferon α2 / ribavirin (PR) therapy during chronic HCV infection, suggesting a role for host proteins in viral clearance [13, 14]. Genome wide association studies (GWAS) have identified a striking association between single nucleotide polymorphisms (SNPs) near the IL28B locus and spontaneous clearance of untreated HCV infection, as well as response to PR therapy [15-18]. For the rs12979860 SNP within the IL28B locus, the “protective” CC genotype confers a 2- to 3-fold higher rate of spontaneous viral clearance or sustained
7 virologic response (SVR) following PR treatment, as compared to either the CT or TT haplotype [16-18]. Recently, a dinucleotide SNP in strong linkage disequilibrium with the rs12979860 SNP was shown to determine expression of a newly discovered gene product, IFN-λ4 [19, 20]. In addition higher plasma levels of ApoB have been reported to be associated with sustained virological response in patients carrying the rs8099917 responder genotype (located proximal to rs12979860) in IL28B [21]. In this study, we report ApoH as a correlate of both spontaneous clearance and response to therapy in HCV patients. Moreover, we observed that ApoH concentration was higher in subjects carrying the CC allele of the IL28B haplotype. This was confirmed across three independent patient cohorts infected with two different HCV genotypes (1 and 4). Based on multivariate analysis, we show that the association between IL28B and HCV clearance is closely linked to that of ApoH and HCV clearance, suggesting that both belong to the same biological pathway to clearance. These findings open new areas for HCV research and may prove critical to defining the mechanism behind the strong genetic association between IL28B and HCV clearance.
8
PATIENTS AND METHODS Patient, Cohort and Study Group Information Clinical study details, patient information, sample collection and monitoring of the three HCV cohorts are described in Table S1.
Multi-analyte profiling Plasma was collected from fasting patients in Vacutainer blood collection tubes containing Sodium Heparin (BD) and stored at -80°C. Plasma was clarified by highspeed centrifugation and analyzed using Luminex xMAP technology. The measurement of 11 apolipoproteins was performed by Myriad-Rules Based Medicine, (Austin, TX), according to guidelines set by the USA Clinical and Laboratory Standards Institute.
IL28B genotype determination IL-28B genotype at SNP position rs12979860 was determined by real-time PCR using genomic DNA extracted from frozen serum samples in conjunction with minor groove binder probes, [22] and as described in Supplemental Methods.
Statistical analyses Categorical and continuous data were compared across groups using Chi Square and Mann-Whitney tests, respectively and as indicated. Mixed linear regression models were used to analyze longitudinal apolipoprotein expression data according to spontaneous clearance status in the acute hepatitis C cohort.
9 Logistic regression models were used to estimate the increase in the odds of clearance associated with IL28B variants (CC versus CT-TT combined) and ApoH plasma concentrations (introduced as a categorical variable, in quartiles, in the model). Models were built for each of the three cohorts. Variables (IL28B SNP and ApoH) were tested in separate models first to estimate their individual effect on clearance, and then introduced simultaneously in the same model to estimate their independent effect on clearance. Since the HCV chronic cohort included both naïve and treated patients, odds-ratios were adjusted for history of treatment. For the model using HIV/HCV co-infected patients, odds-ratios for IL28B variants were calculated using median unbiased estimates with exact logistic regression models as all (9/9) IL28B CC variants cleared the virus under treatment (this would translate into an infinitely positive odds-ratio in classical logistic regression models).
10 RESULTS To address the potential impact of the rs12979860 IL28B SNP on host apolipoprotein levels and HCV clearance, the levels of plasma apolipoproteins and IL28B SNP genotypes were evaluated in three different HCV cohorts: (i) acute HCV (aHCV) patients in Cairo, Egypt (n=33), (ii) chronic HCV (cHCV) patients receiving standard pegIFN/RBV treatment (n=141), and (iii) HIV/HCV co-infected patients treated with a double dose of pegIFN and a standard dose of RBV (n=43). Summarized clinical characteristics of each cohort are provided in Table S1. The high incidence of HCV genotype 4 in Egypt (14.7% seropositivity) provides the unique opportunity to identify acute, symptomatic HCV patients, and potentially factors that influence spontaneous viral clearance [23-25]. Patients were recruited after presenting with symptoms to two fever hospitals in Cairo, as described in the Supplementary Information, and followed for at least six months after the onset of symptoms. Of the 33 aHCV patients examined, 19 spontaneously cleared their virus (“cleared”, CL), and the remaining 14 individuals developed chronic HCV infection (“not cleared”, NCL). We examined 11 apolipoproteins for differences between CL and NCL groups, and for associations with the IL28B SNP (Table S2). ApoCI was associated with the IL28B SNP, but did not correlate with viral clearance. Only ApoH demonstrated significant associations with both HCV clearance (p=0.005, Fig. 1A) and the CC IL28B allele. ApoH levels were significantly elevated in those individuals encoding the protective CC IL28B haplotype (n=19), as compared to those expressing either the CT or TT variant (n=14, p=0.03, Fig.
1C). Longitudinal
analysis demonstrated that ApoH levels in the CL patients remained consistently elevated compared to those observed in the NCL group in the six months following diagnosis (p<0.0001, Fig. 1B). ApoH levels were not associated with either plasma
11 viral load, or liver inflammation, as indicated by the plasma serum alanine transaminase (ALT) concentration (Fig. S1A, B). These results suggested a novel relationship between ApoH and the rs12979860 SNP, which we sought to confirm in distinct patient cohorts. We next examined chronically infected HCV patients, pooling information from our previously characterized cHCV cohort [26], and patients treated as part of the control arm of the REALIZE trial [27], a total of 141 individuals. Of the 141 patients treated, 40 achieved SVR and 101 were non-responders (NR), based on standard clinical criteria [28]. Supporting our findings in aHCV, individuals who achieved viral clearance had higher baseline ApoH plasma levels (samples were drawn prior to treatment), as compared to NR patients (p=0.002, Fig. 2A). Other apolipoproteins (Apo A1, B, C1, and D) showed differences between CL and NCL patients though only Apo B also associated with IL28B genotype (Table S3). As our dataset was comprised of treatment-naïve and treatment-experienced patients, we stratified patients by treatment history. The association of viral clearance and high plasma ApoH concentration was maintained, regardless of treatment history (Fig. 2B), thus suggesting that ApoH concentrations may predict the functional readiness of a patient to clear HCV. ApoH levels were not associated with baseline viral load, HCV genotype or the extent of fibrosis progression (Fig. S1C-E). However, baseline ApoH was higher in patients with the IL28B CC allele (p<0.0001, Fig. 2C,D). Finally, we extended our study to a third patient cohort. HIV/HCV co-infected patients show a reduced tolerance and response rate to PR therapy, and progression to fibrosis is more rapid than in HCV mono-infected individuals [29, 30]. As prior studies have shown that the rs12979860 IL28B SNP correlates significantly with viral clearance in HIV/HCV-infected patients [18, 31, 32], we examined if ApoH also
12 associated with viral clearance and the IL28B SNP in this disease setting. We analyzed data from an ongoing HIV/HCV co-infected cohort, basing our association analysis on the primary endpoint for viral clearance: an early virologic response (EVR) as defined by a greater than two log reduction of HCV RNA viral load at week 12 post-treatment initiation. Forty-three patients were identified for analysis: 26 achieved EVR, while 17 were classified as non-responders (NR). As previously seen in both the aHCV and cHCV cohorts previously, plasma ApoH levels were significantly elevated in HIV/HCV patients who achieved EVR (p=0.002, Fig. 3A), but were not associated with HCV viral load, genotype or liver fibrosis (Fig. S1F-H). No other apolipoproteins showed differences in this patient population (Table S4). Higher baseline levels of plasma ApoH were found in IL28B CC patients, compared to those with non-CC genotypes (p=0.05, Fig. 3B). Patients recently reached the SVR24 endpoint, and the association between plasma ApoH and SVR24 was found again; however due to low numbers of patients who achieved viral clearance (n = 8), the results did not achieve statistical significance between the two groups (SVR24 vs. NR, p = 0.08) (Fig. S2). The results obtained in each of our patient groups were modeled in order to ascertain the
relationship
between IL28B polymorphisms,
baseline ApoH
concentrations and viral clearance (Table 1). In all three patient populations, IL28B and baseline ApoH were significantly associated with HCV clearance in univariate analysis.
However, when introduced simultaneously into the same model, the
association between ApoH and viral clearance overrode that between IL28B and viral clearance. These data suggest that ApoH is a stable predictor of viral clearance across the three patient populations, and is situated within the pathway of the IL28B CC predictive allele and HCV viral clearance [33].
13 Two scenarios could explain the observed relationship between IL28B and apoH plasma concentrations. Either, circulating ApoH levels may be influenced by the genetic variation at the IL28B locus, serving as a predisposing predictor of enhanced viral clearance; or alternatively, in the context of HCV infection ApoH levels are differentially regulated with IL28B polymorphisms, In order to distinguish between these two possibilities, we assessed steady-state levels of plasma ApoH and IL28B SNP distribution in healthy donors (n=100), and observed no difference between haplotypes (Fig. 4A,B). These data suggest that differential ApoH expression is established early in the pathogenesis of HCV infection, with concentrations impacted by variation at the IL28B locus.
14
DISCUSSION As HCV is known to be critically dependent on host lipid metabolism for productive viral infection we examined the association of several apolipoproteins with viral clearance. We observed a significant, consistent correlation between elevated levels of plasma apoH concentrations and viral clearance in three different patient populations. Recent studies detailing the genetic variation within the IL28B locus prompted us to investigate possible associations between apolipoprotein expression and the well-characterized rs12979860 IL28B SNP. Interestingly, we demonstrated that increased levels of ApoH were associated with the protective CC IL28B SNP (rs 12979860). Strengthening the relevance of this finding was the confirmation of this association across two different genotypes (1 and 4), and three different disease states [34].
While
we
acknowledge
the
new
data
indicating
IFNλ4-creating
ss469415590[∆G] allele as being more closely associated with response to PR therapy [19, 20], we were unable to obtain these data for our patient cohorts. Nonetheless, the high level of linkage disequilibrium with rs12979860 IL28B SNP, especially in persons of European descent (HapMap, r2 = 0.92) [20], indicates that it would not impact the findings reported herein. Although several apolipoproteins have been identified to play critical roles in the HCV life cycle [7-12, 35], ApoH has not previously been linked to HCV replication or viral clearance. Briefly, ApoH (also known as β2-glycoprotein I or β2GPI) is synthesized by hepatocytes and is found on several classes of lipoprotein particles. ApoH has been principally studied in the context of coagulation, where it is thought to act as an inhibitor of the intrinsic coagulation cascade through its regulation of prothrombinase. Furthermore, the presence of anti-β2-GPI antibodies is
15 a criteria for antiphospholipid syndrome. Notably, HCV patients have been reported to harbor anti-β2-GPI antibodies [36] and it is well known that chronic HCV patients are at risk for coagulopathies [37]. ApoH can opsonize dying cells [38], and its ability to decorate lipoprotein particles and apoptotic cells is likely due to its avidity for phosphatidylserine or other anionic phospholipids [39]. ApoH has also been shown to bind the enveloped viruses [40, 41], and while data has not yet been reported, it can presumably bind to HCV lipoviral particles. Based on our data, we hypothesize that ApoH opsonization of HCV lipoviral particles may serve as an inhibitory mechanism, preventing the binding to entry receptors and accounting for enhanced viral clearance in patients with higher levels of ApoH. Alternatively, it is possible that ApoH influences HCV clearance secondary to its role as an activator of lipoprotein lipase (LPL) [42, 43], as prior data has shown that LPL inhibits HCV infection [44]. ApoH has also been reported as an acute phase reactant [45, 46]; however, the ApoH promoter does not contain interferon response elements, and there is no known association between IFN and ApoH induction. The development of novel HCV treatment regimens has progressed rapidly in recent years, from the standardization of triple therapy (IFN, ribavirin and direct-acting antiviral agents, DAA) ([47]), to the recent interferon-free regimens, which will transform the management of patients ([25]). Ongoing work in our laboratory is focused on investigating the impact of ApoH in the context of these new therapies, as well as the mechanism of how it is involved in viral clearance. Regarding the association between the IL28B CC variant (rs12979860 SNP) and apoH, it is interesting that no correlation was found in healthy donors. This suggests that ApoH concentration is an induced trait, with the trigger being HCV infection. Differences were observed in plasma ApoH levels depending on the
16 infection stage; with lower overall ApoH concentrations detected in acute infection, most notably among those moving towards chronic infection, and higher overall levels during chronic HCV. Our findings are also unique in that, for the extent of patient follow-up data examined, circulating ApoH levels remain elevated following HCV clearance in the acute HCV cohort (Fig. 1B). Future studies will be designed to characterize the kinetics of plasma ApoH expression following spontaneous viral clearance. While intriguing, comparisons of plasma ApoH levels between acute and chronic stages should be treated with caution as they are not based on longitudinal follow-up of the same individuals, and instead are from different study populations. Four recent studies have identified relationships between IL28B genotype and lipid metabolism [42-45]. In contrast to our findings, Sheridan and colleagues identified a significant association between ApoE levels, EVR and IL28B polymorphisms [48]. We believe that the work of Sheridan warrants additional investigation, particularly in light of the importance of ApoE for viral assembly [12], but we highlight several technical differences that could explain the conflict between our results. First, we assessed ApoE levels in patient plasma, whereas serum samples were used by Sheridan et al. Second, ApoE was measured using CLIA-certified sandwich immunoassays in our study, whereas Sheridan utilized automated nephelometric methods to quantify ApoE concentrations, the accuracy of which may be limited by the association of ApoE and HCV proteins in LVP. In the study of McCarthy et al., the authors demonstrated that subjects with the rs12979860 CC responder genotype had elevated low-density lipoprotein (LDL) cholesterol [49], however the association between LDL levels and viral clearance was substantially attenuated when IL28B polymorphisms were evaluated as possible confounding factors. A follow-up study indicated an interesting link between IL28B CC genotype
17 and a lower prevalence of steatosis [50]. In an independent study, it was shown that pre-treatment levels of LDL cholesterol were higher in patients with a favorable IL28B polymorphism, but again the association between LDL and HCV clearance was lost when regressing out the genetic differences between patient groups [51]. Thus, our data identify a host factor that is a likely part of the causal pathway to viral clearance as the IL28B polymorphism. With a growing body of evidence documenting that gene expression data may not directly correlate with protein abundance [52-55], the identification of proteinbased quantitative traits associated with genetic loci is gaining attention as a possible method to further our understanding of the association of genetic variability and disease outcome [56]. The identification of ApoH constitutes the first induced trait that associates with IL28B, and more interestingly with HCV clearance, in multivariate analyses across three unique HCV cohorts. These data support the necessity of further investigation into how IL28B is modulating expression of ApoH, and how ApoH may be influencing the viral life cycle and/or the antiviral immune response. Our study also further highlights the importance and value of clinical cohort studies for identifying potential mediators of disease clearance.
ACKNOWLEDGEMENTS We thank Stéphanie Thomas (INSERM) for project management support, Estelle Mottez of the Center for Human Immunology (CIH), Institut Pasteur for clinical coordination, and Sam Labrie (Myriad RBM) for Luminex analysis.
18
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22
FIGURE LEGENDS Fig. 1. Plasma ApoH is strongly associated with IL28B genotype and spontaneous viral clearance in acute HCV infection. (A) Plasma ApoH in patients that spontaneously cleared (CL, n=19) or failed to clear virus (NCL, n=14); MannWhitney p-value = 0.005. (B) Kinetics of plasma ApoH in CL () and NCL patients (), mean values and standard deviation are shown.
Mixed model longitudinal
regression analysis was performed (p < 0.0001). (C) Associations between IL28B rs12979860 polymorphisms and apoH were examined. Elevated plasma ApoH is observed in CC patients (n=19), compared to those with the CT or TT SNP (“nonCC”) (n=14); M-W p-value = 0.03.
Fig. 2. Plasma ApoH is strongly associated with IL28B genotype and response to IFN/Ribavirin treatment in chronic HCV. (A) Baseline plasma ApoH levels in patients that responded (SVR n=40) or failed to respond (NR n=101) to pegIFNα/RBV (M-W p-value = 0.002). (B) Patients were segregated by treatment history; SVR (naïve, n=13; treated, n=27), and NR (naïve, n=6; treated, n=94); (M-W p-value = 0.03 and 0.02, respectively). (C and D) Patients were stratified by IL28B genotype; CC (n=24) vs. “non-CC” (n=117) alleles (C), (M-W p-value < 0.0001). Further investigation compared CC (n=24) vs. CT (n=77) vs. TT (n=40) (D) (KruskalWallis p-value < 0.0001).
Fig. 3. Plasma ApoH is associated with IL28B genotype and early virologic response and in patients co-infected with HIV and HCV. (A) Plasma apoH was evaluated in HIV/HCV co-infected patients that achieved early virologic response to
23 peg-IFNα/RBV therapy (EVR, n=26), as compared to nonresponders (NR, n=17); a M-W test was performed (p-value = 0.002) (B). Patients were stratified based on expression of the protective CC allele (n=6), vs. the “non-CC” allele (n=33); analysis was performed using a M-W test (p-value = 0.05).
Fig. 4. Circulating levels of ApoH are independent of IL28B genotype in healthy donors. (A and B) Plasma ApoH levels were evaluated in 100 healthy donor plasma samples. ApoH levels remained equivalent regardless of the IL28B genotype of the healthy individual.
Table 1. Uni- and multivariate analysis of the association between IL28B, apo H, and viral clearance. Univariate OR (95% CI) Acute HCV cohort IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤135 136-166 167-197 ≥198 Chronic HCV cohort# IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤160 161-194 195-242 ≥243 HCV/HIV cohort *§ IL28B (CC vs non-CC) Baseline apo H (μg/ml) ≤136 136-213 214-283 ≥284
5.04 (1.13 – 22.5) 1 5.83 (0.70 - 48.9) 4.67 (0.53-40.9) 28.0 (2.07-379.2) 4.19 (1.38 – 12.7) 1 1.77 (0.52-6.01) 0.84 (0.20-3.56) 6.31 (1.87-21.3) 10.3 (1.46 - ∞) 1 1.00 (0.13-7.91) 7.07 (0.84-100.5) 13.5 (1.15-786.3)
P value 0.03
0.03
0.01
0.004
0.02
0.03
Adjusted-OR 2.80 (0.50 – 15.8) 1 3.62 (0.37-35.6) 3.47 (0.36-33.4) 17.9 (1.20-266.8) 2.77 (0.81 – 9.44) 1 1.64 (0.48-5.60) 0.69 (0.16-3.02) 4.64 (1.30-16.5) 1.69 (0.17 - ∞) 1 0.95 (0.10-9.67) 4.47 (0.41-76.0) 7.05 (0.45-471.1)
Adjusted-P value 0.24
0.22
0.23
0.02
0.54
0.13
* Outcome is spontaneous virological clearance in the acute HCV cohort, sustained virological response in the chronic HCV cohort, and early virological response in the HIV co-infected cohort. # One patient was taking Simvastatin but results are unchanged after excluding him from the analysis. § 6 (out of 43) patients were taking lipid lowering medication. No differences were observed (p=0.99) in ApoH levels between those patients under lipid treatment (mean 240ug/ml) compared to those without treatment (mean 239ug/ml). In addition no differences were observed when we removed these 6 patients from the global analysis.