WO2014140345A1

WO2014140345A1 - Methods for classification and treatment of hepatitis c

Info

Publication number: WO2014140345A1
Application number: PCT/EP2014/055208
Authority: WO
Inventors: Matthew Albert; Arnaud Fontanet; Stanislas Pol
Original assignee: Institut Pasteur; Institut National De La Sante Et De La Recherche Medicale (Inserm); Assistance Publique - Hopitaux De Paris; Universite Paris Descartes
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18

Abstract

Apolipoprotein H and a panel of biomarkers are disclosed as biomarkers relevant to Hepatitis C virus infections.

Description

METHODS FOR CLASSIFICATION AND TREATMENT OF HEPATITIS C FIELD OF THE INVENTION

The invention relates to a molecular classification of disease and particularly to molecular markers for Hepatitis C virus infection prognosis and methods of use thereof.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a major public health problem. Approximately 150 million people are infected worldwide. Most individuals infected with HCV progress to chronic HCV (cHCV) infection, which places these individuals at an increased risk for progressive liver fibrosis, cirrhosis and hepatocellular cancer.

Treatment for HCV infection carries serious side effects and therefore it is advantageous to identify patients whose HCV infection is likely to progress to chronic HCV and those who are likely to clear their HCV infection.

Genome wide association studies have identified an association between single nucleotide polymorphisms (SNPs) near the IL28B locus and spontaneous clearance of untreated HCV infection and clearance of HCV infection in peg-IFN/RBV treated patients. IL28B SNPs are considered to be the strongest predictor of viral clearance, and specifically the CC allele of the rs12979860 SNP confers a two to three fold higher rate of spontaneous viral clearance or sustained virological response after peg-IFN/RBV treatment as compared to either the CT or TT alleles.

While the IL28B locus is known to be involved in clearance of HCV infection, pQTL IL28B has been identified. A need to identify a pQTL IL28B exists. The lifecycle of HCV is dependent on host cell lipid metabolism, which plays a role in cell entry, viral RNA replication, and viral particle production and assembly. Circulating infectious virions associate with very low density lipoprotein-like particles, to form lipo-viral- particles (LVP). Host apolipoproteins are necessary for viral assembly and for the production of infectious virions. In addition, it has been proposed that the incorporation of host apolipoproteins into the LVP assists in viral entry.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is related to the discovery that levels of certain biomarkers, including apolipoprotein H (apoH), in the plasma of HCV patients is predictive of those patients that will clear their HCV infections. Accordingly, in one aspect of the invention, a method is provided for assessing the ability of a patient to clear HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from a patient infected with HCV to determine the level of apoH in the plasma sample; and (2) assessing the likelihood that the patient will clear their HCV infection based on the level of apoH.

In another aspect of the invention, a method is provided for predicting the progression of HCV infection in a patient suffering from HCV infection. Generally, the method includes at least the following steps: (1 ) determining the level of apoH in a biological sample taken from the patient; and (2) assessing the likelihood that the patient will develop chronic HCV infection based on the level of apoH.

In another aspect of the invention, a method is provided for treating HCV infection. Generally, the method includes at least the following steps: (1 ) obtaining a plasma sample from a patient identified as having chronic HCV infection; (2) determining the level of apoH in the plasma sample; and (3) determining the aggressiveness of the drug therapy prescribed based on the level of apoH in the sample.

The present disclosure is also related to the discovery that levels of a panel of biomarkers in the plasma of HCV patients are predictive of those patients that will clear their HCV infections. The panel of biomarkers is selected from a group consisting of two or more of the following: apoH, SHBG, IFGBP2, SAP, CathespinD, ErbB3, AFP, IGFBP1 , AXL, HGF receptor, Lpa, C3, IL6Rb, and Vitronectin.

Accordingly, in one aspect of the present application, a method is provided for assessing the ability of a patient to clear HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from a patient infected with HCV to determine the levels of the panel of biomarkers in the plasma sample; and (2) assessing the likelihood that the patient will clear their HCV infection based on the levels of the panel of biomarkers.

In another aspect of the present application, a method is provided for predicting the progression of HCV infection in a patient suffering from HCV infection. Generally, the method includes at least the following steps: (1 ) determining the levels of the panel of biomarkers in a biological sample taken from the patient; and (2) assessing the likelihood that the patient will develop chronic HCV infection based on the levels of the panel of biomarkers.

In another aspect of the present application, a method is provided for treating HCV infection. Generally, the method includes at least the following steps: (1 ) obtaining a plasma sample from a patient identified as having chronic HCV infection; (2) determining the levels of the panel of biomarkers in the plasma sample; and (3) determining the aggressiveness of the drug therapy prescribed based on the levels of a panel of biomarkers in the sample.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . Plasma apoH is associated with spontaneous viral clearance and is a pQTL of IL28B in acute HCV (aHCV) infection. ApoH was quantified from the plasma of acute HCV patients, and IL28B genotype at SNP position rs12979860 was determined as described in the methods. Comparisons were made using the apoH levels measured in the first sample collected post-intake for each acutely infected symptomatic patient, a time point when all patients were HCV RNA PCR positive. (A) Plasma apoH concentrations in patients that spontaneously cleared their virus (CL, n=15) and those that failed to clear their virus (NCL, n=10) are shown. Statistical analysis was performed using a Mann-Whitney (M-W) test (p-value = 0.017). (B) Plasma apoH levels were followed over time, again comparing CL (red circles) and NCL patients (black squares). A mixed model longitudinal regression analysis was performed (p-value < 0.0001 ). (C) The percent of patients who spontaneously cleared their virus was plotted according to quartile values of apoH plasma concentration (OJ = 41 .8-133 μg/mL, 0-25%; Q2 = 134-160 μξ/mL, 25-50%; Q3 = 161 -200 μg/mL, 50-75%; and Q4 = 201 -261 μ /mL, 75-100%). Chi-squared analysis was performed (p-value = 0.009). (D) The association between IL28B rs12979860 polymorphism and apoH levels was examined. Higher plasma apoH is observed in patients who carry the protective CC SNP genotype (n=16), as compared to those patients who had either the CT, or TT SNP variant (grouped together and depicted as "non-CC")(n=9). M-W analysis was performed (p = 0.014). Figure 2 shows virological response by gender for SVR (sustained virological response) and NR (non-responders) patients for the chronic double therapy (cHCV-dt) cohort. Figure 3 shows virological response by HCV genotype for SVR (sustained virological response) and NR (non-responders) patients for the chronic double therapy (cHCV-dt) cohort.

Figure 4 shows number of patients by fibrosis score for SVR (sustained virological response) and NR (non-responders) patients for the chronic double therapy (cHCV-dt) cohort.

Figure 5 shows number of patients by naive to treatment or previous treatment for SVR (sustained virological response) and NR (non-responders) patients for the chronic double therapy (cHCV-dt) cohort.

Figure 6 shows number of patients by IL28B polymorphism for SVR (sustained virological response) and NR (non-responders) patients for the chronic double therapy (cHCV-dt) cohort.

Figure 7. Plasma apoH is associated with response to treatment and is a pQJL of IL28B in chronic HCV double therapy (cHCV-dt) patients. Plasma concentration of apoH and IL28B SNP analyses were determined as described in the methods. (A) Baseline plasma concentration of apoH was evaluated as a determinant of sustained virologic response in patients that received peg-IFNa/RBV therapy (SVR, n=40; NR, n=101 ). Statistical analysis was performed using a M-W test (p-value = 0.002). (B) The percent of patients who achieved SVR was plotted according to quartile values of apoH plasma concentration (Q1 = 83-160 μ /mL; Q2 = 161 -194 μ /mL; Q3 = 195-242 μg/mL; and Q4 = 243-395 μg/mL). Chi-squared analysis was performed (p-value = 0.009). (C) Patients were segregated based on their being naive to prior treatment or having previously received a course of peg-IFNa/RBV therapy. ApoH levels were again evaluated in SVR (naive, n=1 3; previously treated, n=27), as compared to NR patients (naive, n=6; previously treated, n=94). Statistical analysis was performed using a M-W test (p-value = 0.03 and 0.02, respectively). (D and E) The association of IL28B variation and apoH levels was examined. Patients were stratified based on their encoding the protective CC allele (n=24), as compared to the "non-CC" allele (n=117). Statistical analysis was performed using a M-W test (p-value < 0.0001 ) (D). We additionally segregated patients to compare CC (n=24) vs. CT (n=77) vs. TT (n=40). Statistical analysis was performed using a Kruskal-Wallis (K-W) test (p-value < 0.0001 ) (E). Figure 8 shows apoH as a biomarker of response for apoH alone versus apoH plus usual clinical score (+/- IL28B) for response for the chronic double therapy (cHCV-dt) cohort.

Figure 9 shows odds ratio of response to treatment (adjusted on clinical factors +/- IL28B) with respect to apoH model for chronic double therapy (cHCV-dt) cohort.

Figure 10 shows apoH for prediction of response for the chronic double therapy (cHCV-dt) cohort.

Figure 11 . Plasma apoH is associated with early virologic response and is a pQTL of IL28B in HIV-HCV co-infected patients. Plasma concentration of apoH and IL28B SNP analyses were determined as described in the methods. (A) Plasma apoH levels were evaluated in HiV-HCV co-infected patients that achieved early virologic response to peg-IFNa/RBV therapy (EVR, n=26), as compared to those patients who did not exhibit a reduction in viral load after 12 weeks of therapy (NR, n=17). Statistical analysis was performed using a M-W test (p-value = 0.002) (B) The percent of patients who achieved EVR was plotted according to quartile values of apoH plasma concentration. Chi-squared analysis was performed (p-value = 0.002). (C). Patients were stratified based on their encoding the protective CC allele (n=6), as compared to the "non-CC" allele (n=33). Statistical analysis was performed using a M-W test (p- value = 0.05).

Figure 12. Pathways modeled and examined by uni- and multi-variate regression analysis. Schematic models by which IL28B and/or apoH may be impacting viral clearance are depicted. Model (A) depicts the univariate association between IL28B and viral clearance, independent of other factors; and model (B) depicts a similar, univariate association of apoH with viral clearance. Model (C) schematizes variations on an /L28B/apoH interdependent model as was assessed using multivariate logistic regression analyses. Odds ratios (OR) for a 50 pg/ml increase of plasma apoH concentration (in acute and chronic HCV) and ranking above or below the medial in HIV/HCV co-infection are given. The 95% confidence intervals are denoted in parentheses, and the p-value for the measured parameters (IL28B or apoH or both) are indicated.

Figure 13 shows virologic response at Week 12 for responders (R) and non-responders (NR) and gender distribution for each group in the chronic HCV infected, triple therapy (cHCV-tt) cohort. Response was determined by RNA < 1000 lU/ml. Figure 14 shows HCV genotype for responders (R) and non-responders (NR) in the cHCV-tt cohort. Patients were classified as positive for genotype 1 b (G1 b), genotype 1 a (G1 a), or not determined (ND).

Figure 15 shows Log 10 values of the p values of biomarkers and the median values for R and NR based on response at Week12 in the cHCV-tt cohort, where the cutoff for virologic response was RNA < 1000 lU/ml.

Figure 16 shows median level in responders (R), median level in non-repsonders (NR), and p value for apoH, SHBG, SAP, AFP; cathespin D, AXL, IL6Rb, C3, IFBP2, ErbB3, MCSF, Adiponectin, KLK7, TTR, Lpa, vWF, CD26activity, Haptoglobin, Vitronectin, HGFreceptor, CFHR1 , PSAf, TotallPI O, VKDPS, IGFBP1 , HCC4, HER2, CRP, TM, Testosterone^"!^", TIMP1 , and CD40 in the cHCV-tt cohort.

Figure 17 shows receiver operating characteristic (ROC) curve for ApoH, AFP, SHBG, SAP, AXL, and CathespinD in the cHCV-tt cohort. Figure 17 also shows area under ROC (AUROC), standard error, p value, and lower bounds and upper bounds of 95% confidence interval.

Figure 18 shows level of apoH compared with virologic response at W12 for R and NR in the cHCV-tt cohort. Figure 18 also shows level of apoH compared with IL28B for CC and non CC.

Figure 19 shows a radar plot of p-values for discriminating biomarkers EVR vs NR in the cHCV-tt cohort of Example 2, where virologic response is determined by a 2 log reduction in detectable viral RNA.

Figure 20 shows univariate analysis of the cHCV-tt patient population at 16W. The results from the test cohort (dotted black line, n=140) and the total cohort (solid black n=187) are represented. Additionally, we represent patient results stratified based on treatment (Teleprevir - solid grey line n=122 ; Boceprevir - dotted grey line n=65), indicating that in both groups ApoH is associated with response at W16. Data is represented as a radar plot with values indicating p values for biomarkers, liver function tests and clinical factors (Mann-Whitney test). FDR adjusted p-value <0.005 is considered significant. Biomarker results are sorted based on statistical significance measured within the total cohort, ordered in clockwise direction.

Figure 21 shows the association of apolipoprotein H with virologic response. (A) Results for univariate analysis of the 220 screened plasma proteins in the Test cohort are presented using a Dubai plot (Proteomic screens have become highly standardized, however analysis of the data has not been formalized in the same way as SNP-based genetic screens. Modeled after the "Manhattan plot" we introduce the "Dubai plot" as a means of visualizing high-content data as a function of statistical significance. ). Analytes were analyzed for their association with early virologic response (EVR16) and sustained virologic response (SVR) as compared to nonresponse (NR). Analytes were clustered according to their principal biological functions: cancer marker and other plasma proteins (violet), growth factors and tissue remodeling proteins (blue), apolipoproteins (pink), metabolic proteins and hormones (yellow) and cytokine/chemokines (brown), p-values were calculated using Mann- Whitney (MW) tests and adjusted to account for a False Discovery Rate (FDR). Data was represented as a function of the -log10 (p-value), with a p-value <0.0002 (i.e., q- value <0.05) being considered significant (dotted line). Results for EVR16 (circles) and SVR (squares) are shown for each analyte. Baseline concentrations of apoH are presented in Test and Replication cohorts for EVR16 and SVR (B) and for all patients stratified for treatment regimen (C): telaprevir vs. boceprevir. Significance was assessed using a Mann-Whitney test. (D) Median value of baseline apoH concentration for all patients (262.5 pg/ml) to evaluate the likehood of SVR. Results indicate that patients with apoH concentrations higher than 262.5 μg/ml have an odd ratio of 3.2 (1 .8- 5.9) to achieve SVR.

Figure 22 shows the association between apolipoprotein H and IL28B rs12979860 polymorphism. Baseline plasma apolipoprotein H (apoH) concentrations are presented based on IL28B rs12979860 polymorphism (CC or non-CC alleles) for all patients (A) and IL28B based stratification (B). ApoH (50 units increase indicating the smallest increased interval for interquartile change) and IL28B polymorphism were incorporated together in binary logistic regression for sustained virologic response. Odd ratio (OR), 95% confidence interval (CI) and p-values are shown (C).

Figure 23 shows that apolipoprotein H improves classification of treatment responders. A "clinical model" was established based on clinical data analysis and included prior response to PR, viral load and albumin for EVR16 and HCV sub- genotype, prior response to PR and albumin level for 5VR. We incorporated biomarkers concentrations as a continuous variable for the "biomarker-based model". Result of area under the receiver operator characteristic (AUROC) curve +/- 95% confidence interval is represented for EVR16 (A) and SVR (B). ROC curve of apoH- based model for SVR is presented. Se = sensitivity, Sp = specificity, PPV = positive predicted value and NPV = negative predictive value (C). Category-free net reclassification improvement (NRI>0) was used to assess accurate classification of patients based on the respective models for EVR16 (D) and SVR (E). AFP: alpha foeto protein; Apo CI: apolipopotein C-l, apoH: apolipoprotein H; IL6rb: soluble Interleukin 6 receptor beta; MSCF: Macrophage colony stimulating factor 1 ; TTR: transthyretin. Figure 24 shows the inhibition of viral replication by apolipoprotein H in HCVcc infected liver slices. (A) Freshly harvested liver tissue was cut into 350 μηι-thick slices (approximately 2.7x106 cells per slice) and inoculated with HCVcc Con1 supernatant (MOI=0.1 ). Cultures were treated with 150 pg/ml (open squares) or 300 Mg/ml (closed squares) of purified human apoH. Post-infection day 0, 1 , 5 or 10, liver slice cultures were lysed and intracellular HCV RNA was quantified by strand-specific RT-qPCR. The detection of negative strand HCV RNA confirmed active replication (data not shown). Positive strand RNA is presented as log10 [copies / pg RNAtotal / mg tissue], with values indicating the mean of triplicate experimental wells with error bars indicating standard error (SEM). Data was compared to untreated HCVcc infected liver slices (filled circles) using a paired student's t-test, ** indicates p< 0.001 . (B) Cell survival was evaluated as a function of lactate dehydrogenase (LDH) release. Results were normalized to the control HCVcc infection condition. Values indicate the mean of triplicate experimental wells with error bars indicating standard error (SEM). (C, D) Schematic representation of pre-incubation experiment. Liver slices or free virus were independently treated with 50 pg/ml of apoH. After indicated time intervals, the cells or virus were extensively washed and infection was initiated. As above, intracellular positive strand HCV RNA was quantified after 5 or 10 days post-infection. Values are expressed as mean of triplicate experimental wells +/- standard error (SEM). p-values were calculated using paired t-test, * indicates p < 0.05, ** indicates p< 0.001 .Results in Figure 24 are representative of two independent experiments with different liver donor samples.

DETAILED DESCRIPTION OF THE INVENTION

Levels of Apolipoprotein H circulating in the plasma of a patient infected with HCV are predictive of clearance of the HCV infection. Applications may include evaluation of patients with acute HCV (aHCV), chronic HCV (cHCV), and HIV/HCV co-infections, whether the patients are treated for HCV infection or are treatment naive. Further, the inventors have analyzed the relationship of apoH and the rs12979860 single nucleotide polymorphism (SNP) of the IL28B gene in patients with HCV infection. Specifically, the relationship between apoH and HCV has been analyzed and it has been discovered that apoH plasma level is a predictive marker for clearance of hepatitis C virus (HCV). This has been demonstrated in a cohort of acute HCV patients, a cohort of chronically infected HCV patients, a cohort of HIV/HCV infected patients, and a cohort of HCV infected patients with who carry the CC haplotype of the rs12979869 SNP of the IL28B gene. In all cases, the level of apoH correlates with early virologic response after initiation of HCV treatment and/or sustained virologic response.

The present application discloses apoH as a clinically important predictive marker for likelihood to clear HCV. This represents an important and medically useful discovery. This discovery enables the discrimination of patients, prior to treatment, into a group of patients that will likely clear their HCV infections regardless of treatment for HCV infection and a group of patients that will fail to spontaneously clear their HCV infections without therapeutic treatment. Determining that a patient is likely to clear their HCV infection may save them from expensive treatment with significant side effects. This diagnostic tool may also assist physicians in identifying patients who are unlikely to clear their HCV infections, in particular patients with cirrhosis, and thus may suggest those patients require earlier or more aggressive treatment.

One aspect of the invention is to assess the ability of an individual infected with HCV to clear HCV from the body based on the levels of apoH in the individual's body. In one embodiment, the individual is a patient seeking medical treatment. In one embodiment, the individual is a cirrhotic patient. In one embodiment HCV infection is diagnosed by a positive enzyme linked immune-assay (ELISA) test for presence of HCV antibodies. In one embodiment HCV infection is diagnosed by a positive Western Blot test for presence of HCV antibodies. In one embodiment HCV infection is diagnosed by a positive HCV recombinant immunoblot assay. In one embodiment HCV infection is indicated by a positive rapid antibody blood test for HCV. In one embodiment HCV infection is diagnosed by a positive PCR test for presence of HCV RNA. In one embodiment severity of HCV infection is measured by HCV viral load test using a quantitative PCR test. In one embodiment a cleared HCV infection is indicated by a positive HCV antibody test and a negative HCV PCR test. In one embodiment the level of apoH in the individual's is assessed by performing an antibody test on a biological sample from the patient. By "biological sample", it is herein referred to any sample that is taken from a subject, which includes but is not limited to, for example, blood, serum, plasma, sputum, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, tears, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal central nervous system tissue, buffy coat, saliva, or buccal swabs. Preferably, the biological sample is blood or is derived from blood, such as plasma or serum.. In yet another embodiment, apoH plasma levels may be tested by Western blot or ELISA.

One embodiment of the invention is to assess the ability of a patient with acute HCV to spontaneously clear HCV from circulation. Yet another embodiment of the invention is to assess the ability of a patient with chronic HCV to achieve sustained virologic response after initiation of HCV treatment. Yet another embodiment of the invention is to assess the ability of a treatment naive patient with chronic HCV to achieve sustained virologic response. As is well known to the person of skills in the art, the endpoint of HCV treatment is a sustained virologic response (SVR), which correlates strongly with a permanent clearance of the virus and effectively a cure. Sustained virologic response is usually defined as an undetectable HCV RNA level after several weeks or months of treatment discontinuation. Preferably, sustained virologic response is indicated by the absence of detectable RNA after 12 or 24 weeks of treatment discontinuation. There are, however, a number of intermediate viral endpoints— measurement of the HCV RNA level at specific time points during the course of HCV treatment— that inform the clinician about the patient's responsiveness to treatment and likelihood of SVR. One such intermediate viral endpoint which is particularly useful in the context of the invention is the Early Virologic Response (EVR). An early virologic response is defined in the art as an undetectable serum HCV RNA or a 2 logi₀ or greater drop in HCV RNA after several weeks or months of therapy. Preferably, the early virologic response is assessed after 1 to 4 months of therapy; more preferably after 12 weeks of therapy. Thus another embodiment of the invention is to assess the ability of a patient with HIV/HCV co-infections to achieve early virologic response after initiation of HCV treatment.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from the patient; and (2) assessing the likelihood that the patient will clear the HCV virus based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear an acute, symptomatic HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from a patient identified as having an acute, symptomatic HCV infection; and (2) assessing the likelihood that the patient will clear the HCV virus based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level is indicative of an increased likelihood for spontaneous clearance of HCV. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another aspect of the inventio n, a method is provided for assessing the ability of a patient to clear a chronic HCV. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from a patient identified as having chronic HCV infection; and (2) assessing the likelihood that the patient will achieve sustained virologic response to treatment for HCV infection based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In one embodiment a chronic HCV infection is indicated by a multiple positive HCV PCR tests taken over several weeks or months. In one embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 12 to 24 weeks after completion of therapy for HCV infection. In another embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 6 to 12 months after completion of therapy for HCV infection.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level is indicative of an increased likelihood for sustained virologic response to treatment of HCV. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV. In yet another embodiment, the patient is undergoing treatment with peg-IFN/RBV and a HCV non-structural protein 3 (NS3) inhibitor. Preferably, said inhibitor is telaprevir or boceprevir. In another aspect of the invention, a method is provided for assessing the efficacy of a treatment of HCV infection. Generally, the method includes at least the following steps: (1 ) determining the level of apoH in a biological sample from a patient identified as having chronic HCV infection; and (2) determining the likelihood that the patient will achieve early virologic response to their HCV infection based on the level of apoH in the sample. The method may comprise a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In one embodiment a chronic HCV infection is indicated by a multiple positive HCV PCR tests taken over several weeks or months. In one embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 12 to 24 weeks after completion of therapy for HCV infection. In another embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 6 to 12 months after completion of therapy for HCV infection.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV. In yet another embodiment, the patient is undergoing treatment with peg-IFN/RBV and a HCV non-structural protein 3 (NS3) inhibitor. Preferably, said inhibitor is telaprevir or boceprevir.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear HCV when the patient is co-infected with HIV and HCV. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from a patient identified as having HIV/HCV co-infection; and (2) assessing the likelihood that the patient will achieve early virologic response to HCV infection based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from a patient identified as having HCV infection.. In one embodiment HIV/HCV co-infection is indicated by a positive test for HIV in addition to a positive HCV antibody or PCR test. In one embodiment, early virologic response to treatment is indicated by a negative HCV PCR test or a 2- log decrease in HCV RNA as indicated by comparison to an earlier quantitative PCR test for HCV viral load, after 1 to 4 months of therapy for HCV infection, more preferably after 12 weeks of therapy.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level is indicative of an increased likelihood for early virologic response to treatment of HCV. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV.

In another aspect of the invention, a method is provided for predicting the progression of HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from a patient identified as having acute HCV infection; and (2) assessing the likelihood that the patient will develop chronic HCV infection based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level of apoH is indicative of a decreased likelihood for development of chronic HCV. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV.

In another embodiment, the patient has been genotyped at the rs12979860 IL28B locus.

In another aspect of the invention, a method is provided for treating HCV infection. Generally, the method includes at least the following steps: (1 ) assaying a biological sample from the patient to determine the level of apoH in the biological sample from a patient identified as having chronic HCV infection; and (2) determining the aggressiveness of the drug therapy prescribed based on the level of apoH in the sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level of apoH indicates a decrease in the aggressiveness of the therapy administered. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and an increased level of apoH indicates a delay in the administration of drug therapy. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and a basal or decreased level of apoH indicates an increase in the aggressiveness of the therapy administered.

In another embodiment, the level of apoH in the plasma sample is compared to a reference standard and a basal or decreased level of apoH indicates early administration of drug therapy. Preferably, said increase of apoH level is at least 25%, more preferably at least 50%.

Reference Standards

In many embodiments, the level of apoH in the plasma sample is compared to a reference standard. The reference standard used for any embodiment disclosed herein may comprise an average, mean, or median level of apoH in a control population. The control population may comprise healthy individuals, individuals infected with HCV, individuals who spontaneously cleared an HCV infection or individuals who did not spontaneously clear an HCV infection.

In some embodiments, the reference standard for apoH comprises the mean or median level of apoH in two or more individuals who spontaneously cleared an HCV infection. In related embodiments, individuals with a level of apoH greater than or equal to the reference level would be more likely to spontaneously clear an HCV infection. Therefore, an individual presenting with an HCV infection and an apoH level greater than or equal to the reference standard would be a candidate for less aggressive treatment, or delayed treatment or both. On the other hand, an individual presenting with an HCV infection and an apoH level less than the reference standard would be a candidate for more aggressive treatment, or earlier treatment or both. In another embodiment, the reference standard for apoH comprises the mean or median level of apoH in two or more individuals who did not spontaneously clear an HCV infection. In related embodiments, individuals with a level of apoH less than or equal to the reference level would be less likely to spontaneously clear an HCV infection. Therefore, an individual presenting with an HCV infection and an apoH level less than or equal to the reference standard would be a candidate for more aggressive treatment, or earlier treatment or both. On the other hand, an individual presenting with an HCV infection and an apoH level greater than the reference standard would be a candidate for less aggressive treatment, or delayed treatment or both.

Reference Therapy

In some embodiments, a patient is treated more or less aggressively than a reference therapy. A reference therapy is any therapy that is the standard of care for HCV infection treatment. The standard of care can vary temporally and geographically, and a skilled person can easily determine the appropriate standard of care by consulting the relevant medical literature.

In some embodiments, based on a determination that the level of apoH is a) greater than, b) less than, c) equal to, d) greater than or equal to, or e) less than or equal to a reference standard, treatment will be either 1 ) more aggressive, or 2) less aggressive than a standard therapy.

In some embodiments, a more aggressive therapy than the standard therapy comprises administering a drug earlier than in the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises administering more of a drug than in the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises administering a drug on an accelerated schedule compared to the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises administering additional drugs not called for in the standard therapy.

In some embodiments, a less aggressive therapy than the standard therapy comprises delaying administering a drug relative to the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering less of a drug than in the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering a drug on a decelerated schedule compared to the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering less drugs than called for in the standard therapy, for example, administering no drugs.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Treatment of Hepatitis C

A practitioner treats hepatitis C infection by taking actions to ameliorate the causes or symptoms of the infection in a patient. Treatment of HCV comprises administering therapy to a patient. Therapy may include: selecting and administering one or more anti-HCV drugs to the patient, adjusting the dosage of the anti-HCV drug, adjusting the dosing schedule of the drug, and adjusting the length of the therapy. Anti-HCV drugs are selected by practitioners based on the nature of the infection, the patient's response to the infection and the patient's response to the drug. The dosage of the anti-HCV drug can be adjusted as well by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, and the patient's response to the drug. The dosing schedule can also be adjusted by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, and the patient's response to the drug. Also, the length of the therapy can be adjusted by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, the patient's response to the drug. Also, the practitioner can select between a single drug therapy, a dual drug therapy, or a triple drug therapy. Also, the anti-HCV therapy can be adjusted by the practitioner based on whether the patient suffers from acute HCV infection, chronic HCV infection, or HIV/HCV co- infection

In one embodiment, the practitioner adjusts the therapy based on the patient's level of apoH compared to a reference level. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different combination of drugs. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting drug dosage. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy.

In one embodiment, therapy comprises the selection and administration of an anti- HCV drug to the patient by the practitioner. In one embodiment, the anti-HCV drug comprises antiviral IFN. In one embodiment, the anti-HCV drug comprises peglFNa. In one embodiment, the anti-HCV drug comprises peginterferon alfa-2a. In one embodiment, the anti-HCV drug comprises peginterferon alfa-2b. In one embodiment, the anti-HCV drug antiviral interferon comprises alfacon-1. In one embodiment, the drug comprises polymerase inhibitor. In one embodiment, the drug comprises protease inhibitor. In one embodiment, the anti-HCV drug comprises ribavirin (RBV). In one embodiment, the anti-HCV drug comprises telaprevir (TVR). In one embodiment, the anti-HCV drug comprises boceprevir (BOC). In one embodiment, the anti-HCV drug comprises sofosbuvir.

In another embodiment, therapy comprises the selection and administration of two anti-HCV drugs to the patient by the practitioner as part of dual therapy. In one embodiment the two dual therapy drugs are an interferon drug and ribavirin. In one embodiment the two dual therapy drugs are antiviral IFN and ribavirin. In one embodiment the two dual therapy drugs are peglFNa and ribavirin. In one embodiment the two dual therapy drugs are peginterferon alfa-2a and ribavirin. In one embodiment the two dual therapy drugs are peginterferon alfa-2b and ribavirin. In one embodiment the two dual therapy drugs are interferon alfacon-1 and ribavirin. In one embodiment the two dual therapy drugs are polymerase inhibitor and ribavirin. In one embodiment the two dual therapy drugs are sofosbuvir and ribavirin. In another embodiment, therapy comprises the selection and administration of three anti-HCV drugs to the patient by the practitioner as part of triple therapy. In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and a NS3 protease inhibitor. The NS3 serine proteinase or NS3 protease (NS3P) is a nonstructural hepatitis C protein responsible for proteolytic processing of other nonstructural viral proteins. Inhibitors of NS3 protease have been identified, see e.g. , Eley et al. (Clinical Pharm in Drug Dev, 2: 316-327, 2013), U.S. Patent Ser. No. 6,995, 174 or PCT application WO 2004/103996. In particular, telaprevir (TVR) and boceprevir (BOV), both NS3 protease inhibitors, have been approved by the FDA. Thus, in a preferred embodiment, the NS3 protease inhibitor of the invention is telaprevir (TVR) or boceprevir (BOV). In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peginterferon alfa-2b, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peginterferon alfa-2b, ribavirin, and boceprevir (BOC) . In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peginterferon alfa-2b, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and protease inhibitor.

In one embodiment where there is an increased level of apoH with respect to a reference level, treatment comprises a less aggressive therapy than a reference therapy. In one embodiment a less aggressive therapy comprises not administering drugs and taking a "watchful waiting" approach. In one embodiment a less aggressive therapy comprises delaying administration of anti-HCV drugs. In one embodiment a less aggressive therapy comprises selecting and administering less potent drugs. In one embodiment a less aggressive therapy comprises decreasing dosage of anti-HCV drugs. In one embodiment a less aggressive therapy comprises decreasing the frequency of the dose schedule. In one embodiment a less aggressive therapy comprises shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decreasing drug dosage. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage and decreasing dose schedule. In one embodiment, less aggressive therapy comprises decreasing drug dosage and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing dose schedule and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy.

In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy. In one embodiment a less aggressive therapy comprises delaying administration of anti-HCV drugs and selecting and administering a single therapy instead of a dual therapy. In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and selecting and administering less potent drugs. In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and decreasing dosage of anti-HCV drugs. In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and decreasing the frequency of the dose schedule. In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and selecting and administering less potent drugs and decreasing drug dosage. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy and selecting and administering less potent drugs and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, selecting and administering less potent drugs and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, decreasing drug dosage and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, decreasing drug dosage and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, decreasing dose schedule and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, selecting and administering less potent drugs, decreasing drug dosage, and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, selecting and administering less potent drugs, decreasing drug dosage, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, selecting and administering less potent drugs, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy, selecting and administering less potent drugs, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy. In one embodiment a less aggressive therapy comprises selecting and administering a single therapy instead of a dual therapy.

In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy. In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy and delaying administration of anti-HCV drugs. In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy and selecting and administering less potent drugs. In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy and decreasing dosage of anti-HCV drugs. In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy and decreasing the frequency of the dose schedule. In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs and decreasing drug dosage. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, decreasing drug dosage and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, decreasing drug dosage and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, decreasing dose schedule and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs, decreasing drug dosage, and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs, decreasing drug dosage, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering a dual therapy instead of a triple therapy, selecting and administering less potent drugs, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy.

In one aspect of the present application where there is a decreased level of apoH with respect to a reference level, treatment comprises a more aggressive therapy than a reference therapy. In one embodiment a more aggressive therapy comprises earlier administration of anti-HCV drugs. In one embodiment a more aggressive therapy comprises increased dosage of anti-HCV drugs. In one embodiment a more aggressive therapy comprises increased length of therapy. In one embodiment a more aggressive therapy comprises increased frequency of the dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing drug dosage. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage and increasing dose schedule. In one embodiment, more aggressive therapy comprises increasing drug dosage and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing dose schedule and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, increasing dose schedule, and increasing length of therapy.

In one embodiment a less aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy. In one embodiment a more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy and earlier administration of anti-HCV drugs. In one embodiment a more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy and increased dosage of anti-HCV drugs. In one embodiment a more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy and increased length of therapy. In one embodiment a more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy and increased frequency of the dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs and increasing drug dosage. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, increasing drug dosage and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, increasing drug dosage and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, increasing dose schedule and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs, increasing drug dosage, and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs, increasing drug dosage, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, increasing drug dosage, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a dual therapy instead of a single therapy, selecting and administering more potent drugs, increasing drug dosage, increasing dose schedule, and increasing length of therapy.

In one embodiment a more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy. In one embodiment a more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy and earlier administration of anti-HCV drugs. In one embodiment a more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy and increased dosage of anti-HCV drugs. In one embodiment a more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy and increased length of therapy. In one embodiment a more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy and increased frequency of the dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs and increasing drug dosage. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, increasing drug dosage and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, increasing drug dosage and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, increasing dose schedule and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs, increasing drug dosage, and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs, increasing drug dosage, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, increasing drug dosage, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering a triple therapy instead of a dual therapy, selecting and administering more potent drugs, increasing drug dosage, increasing dose schedule, and increasing length of therapy.

In another embodiment, the reference therapy comprises single therapy administered for about 24 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly. In another embodiment, the reference therapy comprises dual therapy administered for about 24 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses. In another embodiment, the reference therapy comprises triple therapy administered for about 36 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses and about 800 milligrams of boceprevir administered about three times a day about four weeks after the commencement of peglFN alfa 2a/RBV. In yet another embodiment, the reference therapy comprises triple therapy administered for about 36 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses and about 750 milligrams of telaprevir administered about three times a day about four weeks after the commencement of peglFN alfa 2a/RBV administration.

Panel of Biomarkers

Levels of a panel of biomarkers circulating in the plasma of a patient infected with HCV is predictive of clearance of the HCV infection. Applications may include evaluation of patients with acute HCV, chronic HCV, and HIV/HCV co-infections, whether the patients are treated for HCV infection or are treatment naive. Further, the inventors have analyzed the relationship of a panel of biomarkers and the rs12979860 single nucleotide polymorphism (SNP) of the IL28B gene in patients with HCV infection . Specifically, the relationship between a panel of biomarkers and HCV has been analyzed and it has been discovered that the plasma levels of a panel of biomarkers is a predictive marker for clearance of hepatitis C virus (HCV). This has been demonstrated in a cohort of acute HCV patients, a cohort of chronically infected HCV patients, a cohort of HIV/ HCV infected patients, and a cohort of HCV infected patients with who carry the CC haplotype of the rs12979869 SNP of the IL28B gene. The present application discloses a panel of biomarkers as a clinically important predictive marker for likelihood to clear HCV. This represents an important and medically useful discovery. This discovery enables the discrimination of patients, prior to treatment, into a group of patients that will likely clear their HCV infections regardless of treatment for HCV infection and a group of patients that will fail to spontaneously clear their HCV infections without therapeutic treatment. Determining that a patient is likely to clear their HCV infection may save them from expensive treatment with significant side effects. This diagnostic tool may also assist physicians in identifying patients who are unlikely to clear their HCV infections and thus may suggest those patients require earlier or more aggressive treatment.

One aspect of the present application is a panel of biomarkers that is predictive of clearance of HCV infection. In one embodiment, the panel of biomarkers, comprises any group of two or more biomarkers selected from the biomarkers, including but not limited to apoH, apoCI, SHBG, SAP, AFP, CathepsinD, AXL, IL6Rb, C3, IFGBP2, ErbB3, MCSF, Adiponectin, KLK7, TTR, Lp(a), vWF, CD26activity, Haptoglobin, Vitronectin, HGF receptor, CFHR1 , PSAf, TotallPI O, VKDPS, IGFBP1 , HCC4, HER2, CRP, TM, Testosterone T, TIMP1 , CD40, PAI1 , ApoB, Proinsulin 1 , Angiogenin, LAPTGFbl , MSLN, SDF1 , ASAT, Albumin, INR, Total Bilirubin, ALAT, viral genotype, and IL28B. In one embodiment, the panel of biomarkers may be any two of the biomarker analytes. In other embodiments, the panel of biomarkers may be any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, or all fifteen of the biomarkers.

In some embodiments, the panel of biomarkers comprises apoH and SHBG. In some embodiments, the panel of biomarkers comprises apoH and IGFBP2. In some embodiments, the panel of biomarkers comprises SHBG and IGFBP2. In some embodiments, the panel of biomarkers comprises SHBG, and SAP. In some embodiments, the panel of biomarkers comprises SHBG, and CathespinD. In some embodiments, the panel of biomarkers comprises SHBG, and ErbB3. In some embodiments, the panel of biomarkers comprises SHBG, and AFP. In some embodiments, the panel of biomarkers comprises IFGBP2 and SAP. In some embodiments, the panel of biomarkers comprises IFGBP2 and CathespinD. In some embodiments, the panel of biomarkers comprises IFGBP2 and ErbB3. In some embodiments, the panel of biomarkers comprises IFGBP2 and AFP.

In some embodiments, the panel of biomarkers comprises apoH, SHBG, and AFP. In some embodiments, the panel of biomarkers comprises apoH, SHBG, and SAP. In some embodiments, the panel of biomarkers comprises apoH, SHBG, and IGFBP2. In some embodiments, the panel of biomarkers comprises IFGBP2, SHBG, and SAP. In some embodiments, the panel of biomarkers comprises IFGBP2, SHBG, and CathespinD. In some embodiments, the panel of biomarkers comprises IFGBP2, SHBG, and ErbB3. In some embodiments, the panel of biomarkers comprises IFGBP2, SHBG, and AFP. In some embodiments, the panel of biomarkers comprises apoH, SHBG, and CathespinD. In some embodiments, the panel of biomarkers comprises apoH, SHBG, and ErbB3.

Preferably, the panel of biomarkers of the invention comprises the apoH biomarker. More preferably, the panel of biomarkers of the invention further comprises SHBG. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, and SAP. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, and CathespinD. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, and ErbB3. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, and AFP. In some embodiments, the panel of biomarkers comprises apoH, AFP, SHBG, SAP, AXL, and CathespinD. In some embodiments, the panel of biomarkers comprises apoH, AFP, SHBG, SAP, and HGFR. In some embodiments, the panel of biomarkers comprises apoH, AFP, ApoCI, IL6rb, MCSF, and TTR. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, SAP, CathespinD, ErbB3, and AFP. In some embodiments, the panel of biomarkers comprises apoH, SHBG, IFGBP2, SAP, CathespinD, ErbB3, AFP, IGFBP1 , AXL, HGF receptor, Lpa, C3, IL6Rb, and Vitronectin. In some embodiments, the panel of biomarkers comprises apoH, SHBG, ApoCI, IFGBP2, SAP, MCSF, TTR, CathespinD, ErbB3, AFP, IGFBP1 , AXL, HGF receptor, Lpa, C3, IL6Rb, and Vitronectin.

One aspect of the present application is to assess the ability of an individual infected with HCV to clear HCV from the body based on the levels of a panel of biomarkers in the individual's body. In one embodiment, the individual is a patient seeking medical treatment. In one embodiment HCV infection is diagnosed by a positive enzyme linked immune-assay (ELISA) test for presence of HCV antibodies. In one embodiment HCV infection is diagnosed by a positive Western Blot test for presence of HCV antibodies. In one embodiment HCV infection is diagnosed by a positive HCV recombinant immunoblot assay. In one embodiment HCV infection is indicated by a positive rapid antibody blood test for HCV. In one embodiment HCV infection is diagnosed by a positive PCR test for presence of HCV RNA. In one embodiment severity of HCV infection is measured by HCV viral load test using a quantitative PCR test. In one embodiment a cleared HCV infection is indicated by a positive HCV antibody test and a negative HCV PCR test. In one embodiment the levels of a panel of biomarkers in the individual is assessed by performing an antibody test on a biological sample taken from the patient. Convenient biological samples, as explained above, include but are not limited to, for example, blood, serum, plasma, sputum, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, tears, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal central nervous system tissue, buffy coat, saliva, or buccal swabs. Preferably, the biological sample is blood or is derived from blood, such as plasma or serum. In yet another embodiment, levels of a panel of biomarkers may be tested by Western blot or ELISA.

One embodiment of the invention is to assess the ability of a patient with acute HCV to spontaneously clear HCV from circulation. Yet another embodiment of the invention is to assess the ability of a patient with chronic HCV to achieve sustained virologic response after initiation of HCV treatment. Yet another embodiment of the invention is to assess the ability of a treatment naive patient with chronic HCV to achieve sustained virologic response. Yet another embodiment of the invention is to assess the ability of a patient with HIV/HCV co-infections to achieve early virologic response after initiation of HCV treatment.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear HCV infection. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having HCV infection; and (2) determining the likelihood that the patient will clear their HCV infection based on the levels of a panel of biomarkers in said sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In another aspect of the invention, a method is provided for assessing the ability of a patient to clear an acute, symptomatic HCV infection. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having acute HCV infection; and (2) determining the likelihood that the patient will clear their HCV infection based on the levels of a panel of biomarkers in said sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the levels of a panel of biomarkers in the biological sample are compared to a reference standard.

In another embodiment, the levels of a panel of biomarkers in the biological sample are compared to a reference standard and an increased level is indicative of an increased likelihood for spontaneous clearance of HCV.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear a chronic HCV. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having chronic HCV infection; and (2) determining the likelihood that the patient will achieve sustained virologic response to treatment for HCV infection based on the levels of a panel of biomarkers in said sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In one embodiment a chronic HCV infection is indicated by multiple positive HCV PCR tests taken over several weeks or months. In one embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for six to twelve months after completion of therapy for HCV infection.

In another embodiment, the levels of a panel of biomarkers in the biological sample are compared to a reference standard and an increased level is indicative of an increased likelihood for sustained virologic response to treatment of HCV.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV. In yet another embodiment, the patient is undergoing treatment with peg-IFN/RBV and a HCV non-structural protein 3 (NS3) inhibitor. Preferably, said inhibitor is telaprevir or boceprevir. In another aspect of the invention, a method is provided for assessing the efficacy of a treatment of HCV infection. Generally, the method includes at least the following steps: ; (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having chronic HCV infection; and (2) determining the likelihood that the patient will achieve early virologic response to their HCV infection based on the levels of a panel of biomarkers in the sample. The method may comprise a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In one embodiment a chronic HCV infection is indicated by a multiple positive HCV PCR tests taken over several weeks or months. In one embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 12 to 24 weeks after completion of therapy for HCV infection. In another embodiment, sustained virologic response to treatment is indicated by a negative HCV PCR test for 6 to 12 months after completion of therapy for HCV infection.

In another embodiment, the levels of a panel of biomarkers in the plasma sample are compared to a reference standard and an increased level is indicative of an increased likelihood for sustained virologic response to treatment of HCV.

In another aspect of the invention, a method is provided for assessing the ability of a patient to clear HCV when the patient is co-infected with HIV and HCV. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having HIV/HCV co-infection; and (2) determining the likelihood that the patient will achieve early virologic response to their HCV infection based on the levels of a panel of biomarkers in said sampte. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient. In one embodiment HIV/HCV co-infection is indicated by a positive test for HIV in addition to a positive HCV antibody or PCR test. In one embodiment, early response to treatment is indicated by a negative HCV PCR test or a 2-log decrease in HCV RNA as indicated by comparison to an earlier quantitative PCR test for HCV viral load, after one to four months of therapy for HCV infection.

In another embodiment, the levels of a panel of biomarkers in the biological sample are compared to a reference standard and an increased level is indicative of an increased likelihood for early virologic response to treatment of HCV.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV. In another aspect of the invention, a method is provided for predicting the progression of HCV infection. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having acute HCV infection; and (2) determining the likelihood that the patient will develop chronic HCV infection based on the levels of a panel of biomarkers in said sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the levels of a panel of biomarkers in the plasma sample is compared to a reference standard and increased levels of a panel of biomarkers is indicative of a decreased likelihood for development of chronic HCV.

In another embodiment, the patient is undergoing treatment with peg-IFN/RBV.

In another aspect of the invention, a method is provided for treating HCV infection. Generally, the method includes at least the following steps: (1 ) determining the levels of a panel of biomarkers in a biological sample from a patient identified as having chronic HCV infection; and (2) determining the aggressiveness of the drug therapy prescribed based on the levels of a panel of biomarkers in said sample. The method may also include a prior step of obtaining a biological sample, such as e.g. a plasma sample, from said patient.

In another embodiment, the levels of a panel of biomarkers in the plasma sample is compared to a reference standard and increased levels of a panel of biomarkers indicates a decrease in the aggressiveness of the therapy administered. In another embodiment, levels of a panel of biomarkers in the plasma sample is compared to a reference standard and increased levels of a panel of biomarkers indicates a delay in the administration of drug therapy.

In another embodiment, the levels of a panel of biomarkers in the plasma sample is compared to a reference standard and basal or decreased levels of a panel of biomarkers indicates an increase in the aggressiveness of the therapy administered. In another embodiment, the levels of a panel of biomarkers in the plasma sample is compared to a reference standard and basal or decreased levels of a panel of biomarkers indicates early administration of drug therapy.

Panel of Biomarkers Reference Standards

In many embodiments, the levels of a panel of biomarkers in the plasma sample are compared to a reference standard. The reference standard used for any embodiment disclosed herein may comprise average, mean, or median levels of a p in a control population. The control population may comprise healthy individuals, individuals infected with HCV, individuals who spontaneously cleared an HCV infection or individuals who did not spontaneously clear an HCV infection.

In some embodiments, the reference standard for a panel of biomarkers comprises mean or median levels of a panel of biomarkers in two or more individuals who spontaneously cleared an HCV infection. In related embodiments, individuals with levels of a panel of biomarkers greater than or equal to the reference level would be more likely to spontaneously clear an HCV infection. Therefore, an individual presenting with an HCV infection and levels of a panel of biomarkers greater than or equal to the reference standard would be a candidate for less aggressive treatment, or delayed treatment or both. On the other hand, an individual presenting with an HCV infection and levels of a panel of biomarkers less than the reference standard would be a candidate for more aggressive treatment, or earlier treatment or both. In another embodiment, the reference standard for a panel of biomarkers comprises mean or median levels of a panel of biomarkers in two or more individuals who did not spontaneously clear an HCV infection. In related embodiments, individuals with levels of a panel of biomarkers less than or equal to the reference level would be less likely to spontaneously clear an HCV infection. Therefore, an individual presenting with an HCV infection and levels of a panel of biomarkers less than or equal to the reference standard would be a candidate for more aggressive treatment, or earlier treatment or both. On the other hand, an individual presenting with an HCV infection and levels of a panel of biomarkers greater than the reference standard would be a candidate for less aggressive treatment, or delayed treatment or both.

Panel of Biomarkers Reference Therapy

In some embodiments, based on a determination that levels of a panel of biomarkers is a) greater than, b) less than, c) equal to, d) greater than or equal to, or e) less than or equal to a reference standard, treatment will be either 1 ) more aggressive, or 2) less aggressive than a standard therapy.

Panel of Biomarkers Treatment of Hepatitis C

A practitioner treats hepatitis C infection by taking actions to ameliorate the causes or symptoms of the infection in a patient. Treatment of HCV comprises administering therapy to a patient. Therapy may include: selecting and administering one or more anti-HCV drugs to the patient, adjusting the dosage of the anti-HCV drug, adjusting the dosing schedule of the drug, and adjusting the length of the therapy. Anti-HCV drugs are selected by practitioners based on the nature of the infection, the patient's response to the infection and the patient's response to the drug. The dosage of the anti-HCV drug can be adjusted as well by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, and the patient's response to the drug. The dosing schedule can also be adjusted by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, and the patient's response to the drug. Also, the length of the therapy can be adjusted by the practitioner based on the nature of the drug, the nature of the infection, the patient's response to the infection, the patient's response to the drug. Also, the practitioner can select between a single drug therapy, a dual drug therapy, or a triple drug therapy. Also, the anti-HCV therapy can be adjusted by the practitioner based on whether the patient suffers from acute HCV infection, chronic HCV infection, or HIV/HCV co- infection.

In one embodiment, the practitioner adjusts the therapy based on the patient's levels of a panel of biomarkers compared to a reference level. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different combination of drugs. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting drug dosage. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy.

In another embodiment, therapy comprises the selection and administration of two anti-HCV drugs to the patient by the practitioner as part of dual therapy. In one embodiment the two dual therapy drugs are an interferon drug and ribavirin. In one embodiment the two dual therapy drugs are antiviral IFN and ribavirin. In one embodiment the two dual therapy drugs are peglFNa and ribavirin. In one embodiment the two dual therapy drugs are peginterferon alfa-2a and ribavirin. In one embodiment the two dual therapy drugs are peginterferon alfa-2b and ribavirin. In one embodiment the two dual therapy drugs are interferon alfacon-1 and ribavirin. In one embodiment the two dual therapy drugs are polymerase inhibitor and ribavirin. In one embodiment the two dual therapy drugs are sofosbuvir and ribavirin. In another embodiment, therapy comprises the selection and administration of three anti-HCV drugs to the patient by the practitioner as part of triple therapy. In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are peginterferon alfa-2b, ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and telaprevir (TVR). In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are peginterferon alfa-2b, ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and boceprevir (BOC). In one embodiment the three triple therapy drugs are an interferon drug, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are antiviral IFN, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peglFNa, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peginterferon alfa-2a, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are peginterferon alfa- 2b, ribavirin, and protease inhibitor. In one embodiment the three triple therapy drugs are interferon alfacon-1 , ribavirin, and protease inhibitor.

In one embodiment where there is increased levels of a panel of biomarkers with respect to a reference level, treatment comprises a less aggressive therapy than a reference therapy. In one embodiment a less aggressive therapy comprises not administering drugs and taking a "watchful waiting" approach. In one embodiment a less aggressive therapy comprises delaying administration of anti-HCV drugs. In one embodiment a less aggressive therapy comprises selecting and administering less potent drugs. In one embodiment a less aggressive therapy comprises decreasing dosage of anti-HCV drugs. In one embodiment a less aggressive therapy comprises decreasing the frequency of the dose schedule. In one embodiment a less aggressive therapy comprises shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decreasing drug dosage. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage and decreasing dose schedule. In one embodiment, less aggressive therapy comprises decreasing drug dosage and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing dose schedule and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and decreasing dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage, decreasing dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, decreasing dose schedule, and shortening length of therapy.

In one aspect of the present application where there is decreased levels of a panel of biomarkers with respect to a reference level, treatment comprises a more aggressive therapy than a reference therapy. In one embodiment a more aggressive therapy comprises earlier administration of anti-HCV drugs. In one embodiment a more aggressive therapy comprises increased dosage of anti-HCV drugs. In one embodiment a more aggressive therapy comprises increased length of therapy. In one embodiment a more aggressive therapy comprises increased frequency of the dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing drug dosage. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage and increasing dose schedule. In one embodiment, more aggressive therapy comprises increasing drug dosage and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing dose schedule and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and increasing dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, increasing dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, increasing dose schedule, and increasing length of therapy.

In another embodiment, the reference therapy comprises single therapy administered for about 24 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly. In another embodiment, the reference therapy comprises dual therapy administered for about 24 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses. In another preferred embodiment, the reference therapy comprises triple therapy administered for about 36 weeks with about 180 micrograms of peglFN alfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses and about 800 milligrams of boceprevir administered about three times a day about four weeks after the commencement of peglFN alfa 2a/RBV. In yet another preferred embodiment, the reference therapy comprises triple therapy administered for about 36 weeks with about 180 micrograms of peglFN atfa-2a administered about once weekly and about 800 to about 1 ,400 milligrams of ribavirin administered daily in two divided doses and about 750 milligrams of telaprevir administered about three times a day about four weeks after the commencement of peglFN alfa 2a/RBV administration.

Examples

The present examples are derived from experiments involving four separate cohorts of HCV infected individuals. The first cohort (the "acute HCV" or "aHCV" cohort) consisted of 25 Egyptian patients with symptomatic acute HCV infection. The second cohort (the "chronic HCV double therapy" or "cHCV-dt" cohort) consisted of 42 previously banked samples from patients enrolled in an observational study and treated for their chronic HCV infection with peg-IFN-a2/ribavirin therapy, and 99 patients who were in the control arm of a separate trial, receiving placebo along with peg-IFN-a2/ribavirin. The third cohort (the HCV/HIV cohort) consisted of 43 HIV/HCV co-infected patients who participated in a trial for co-infected individuals who did not respond to a first round of PeglFN /RBV treatment. Finally, the fourth cohort (the "chronic HCV triple therapy" or "cHCV-tt" cohort) consisted of chronic HCV infected participants enrolled in a study measuring early virologic response after triple therapy with pegylated-interferon(peg-IFN)/ribavirin(RBV)/NS3 protease inhibitor (boceprevir or telaprevir). This chronic HCV triple therapy cohort included 651 patients recruited from 55 different centers.

Example 1

Example 1 details the recruitment, sample collection, monitoring, and analysis of three HCV cohorts: an acute HCV cohort (aHCV), a chronic HCV cohort receiving double therapy (cHCV-dt), and an HIV/HCV co-infected cohort. Acute HCV cohort

Patients with symptomatic acute hepatitis C infection were recruited from two "fever" hospitals, which specialize in identification and treatment of infectious disease in Cairo, Egypt. Inclusion criteria were symptomatic disease, indicated by fever or jaundice, duration of symptoms for < 21 days and elevated ALT > 3 times the upper limit of normal (ULN).

Acute HCV infection was defined using the detection of HCV RNA by reverse transcriptase polymerase chain and anti-HCV antibody (Ab) serology. Patients testing negative for anti-HCV Ab and positive for HCV RNA were defined as definitive acute hepatitis C. Patients with positive anti-HCV Ab serology and positive HCV RNA were considered probable acute hepatitis C if the ALT levels were > 10 times ULN and the patient reported a recent history of high-risk exposure to HCV. Patients concurrently infected with other hepatitis viruses (HAV, HBV and/or HEV) or serology indicating acute T. gondii, CMV and /or EBV infections, were excluded from the study. Patients enrolled in the study were monitored at The National Hepatology and Tropical Medicine Research Institute (NHTMRI) in Cairo, Egypt.

Spontaneous viral clearance was defined as the loss of serum HCV RNA in the absence of treatment during the first 6 months of infection and two consecutive negative viral RNA PCR tests. Antiviral treatment was offered to all aHCV patients who had not spontaneously cleared the virus within 6 months after the onset of symptoms. All protocols were reviewed and approved by the Institutional Review Board of the Ministry of Health and Population in Egypt and the NHTMRI ethics committee; all patients provided written informed consent. This study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.

Chronic HCV cohorts

Multi-analyte profiling and IL28B genotyping were performed using a combination of previously banked samples from patients enrolled in an observational study and treated for their chronic HCV infection with peg-IFN-a2/ribavirin therapy (n=42), and patients who were in the control arm of a separate trial, receiving placebo along with peg-IFN-a2/ribavirin (n=99). All patients were infected with HCV genotype 1 (HCVgl ) and were included irrespective of prior treatment history and/or response. Chronic HCV infection was identified by the presence of anti-HCV antibodies and detectable HCV RNA. An endpoint of sustained virologic response (SVR), defined as undetectable plasma HCV RNA for longer than 6 months after the termination of therapy, was used. Protocols for sample collection were approved by independent ethics committees at the participating study centers. The study conformed to the ethical guidelines of the Declaration of Helsinki, and all patients provided written informed consent.

HIV/HCV co-infected cohort

The 43 HIV/HCV co-infected patients included in this analysis all participated in a trial for co-infected individuals who did not respond to a first round of PeglFN/RBV treatment. In this cohort, two endpoints were evaluated: (1 ) early virological response (EVR), defined as undetectable or a minimum of 2-log decrease HCV RNA 12 weeks after the initiation of treatment; and (2) sustained virologic response (SVR), defined as above. Non-response was defined as a decrease in viral loads that did not reach a 2-log reduction during the first 12 weeks of therapy. All patients were infected with HCVgl (63%) or HCVg4 (37%), and the proportion of extensive fibrosis (>F3) was high (66%). This pilot study examined the impact of a double dose of Peg- IFN (360pg/w) + ribavirin (17mg/kg/d) dual therapy regimen, given in conjunction with hematological growth factors (EPO and G-CSF) and an adapted antiretroviral treatment course to promote SVR with a duration guided by the viral response.

Healthy donors

Healthy control plasma samples were collected from 63 anonymous blood bank donors, and confirmed negative for HCV, HBV and HIV infection.

Multi-analyte profiling

Plasma samples were clarified by high-speed centrifugation and analyzed using Luminex xMAP technology. Samples were measured by a diagnostic laboratory, for the measurement of 11 apolipoproteins, with all assays and lab procedures conforming to the guidelines set forth by the USA Clinical and Laboratory Standards Institute.

Genotyping

The IL28B genotype at SNP position rs12979860 was determined by real-time PCR using genomic DNA extracted from patient plasma samples

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 apolipoprotein longitudinal expression data according to spontaneous clearance status in the acute hepatitis C cohort. A random intercept was introduced for each patient to account for the correlations among observations at different time points. Logistic regression models were used to estimate the increase in the odds of clearance associated with IL28B variants (CC versus CT-TT combined) and apolipoprotein H plasma concentrations (introduced as a continuous variable in the model).

Models were built for each of the three cohorts: Patients with acute hepatitis C (n=25); mono-infected patients with chronic hepatitis C undergoing treatment (n=139); and patients co-infected with HIV and undergoing treatment (n=45). Variables (IL28 and apolipoprotein H) 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.

For the model with co-infected patients, odds-ratios for IL28B variants were calculated using median unbiased estimates with exact logistic regression models since all (9/9) IL28 "CC" variants cleared the virus under treatment (this would translate into infinite positive odds-ratio in classical logistic regression models). Since exact logistic regression cannot be performed with continuous variables, apolipoprotein H plasma concentrations were introduced in these models as a dichotomous variable (comparing patients above and under the median level).

Results

We genotyped and measured the levels of plasma apolipoproteins in three different HCV cohorts: (i) acute HCV patients enrolled in Cairo, Egypt (n=33) (aHCV); (ii) chronic HCV patients who received standard peglFN/RBV treatment, France (n=141 )(cHCV-dt); and (iii) HIV-HCV co-infected individuals treated with a double dose of peglFN and a standard dose of RBV (n=43).

RESULTS

Acute HCV cohort

The high incidence of HCV genotype 4 in Egypt (14.7% of the adult population is HCV seropositive) provides the unique opportunity to identify acute, symptomatic HCV patients, thus permitting the investigation of host factors that influence spontaneous viral clearance. Of the 25 patients examined, 15 spontaneously cleared their virus ("cleared", CL), and the remaining 10 individuals developed chronic HCV infection ("not cleared", NCL). Comparison of plasma apolipoprotein levels between the two patient groups revealed significantly elevated apolipoprotein H (apoH) in the CL individuals (p=0.017, Figure 1A), strongly associating this protein with viral clearance (Table 1 ). Acute HCV patients were followed for at least six months following the diagnosis of their infection. Longitudinal analysis demonstrated that apoH levels in the CL patients were consistently elevated over those observed in NCL individuals throughout the course of acute infection, regardless of viral clearance (p<0.0001 , Figure 1 B).

Furthermore, when patients were stratified according to quartile values of circulating plasma apoH, the percentage of patients undergoing spontaneous viral clearance increased as apoH levels increased (p=0.009, Figure 1 C). ApoH levels were not associated with either plasma viral load, or liver inflammation, as indicated by the plasma serum alanine transferase (ALT) concentration. We next examined the impact of IL28B polymorphism on apoH concentration. Interestingly, apoH levels were significantly elevated in those individuals encoding the protective CC IL28B haplotype (n=16), as compared to those expressing either the CT or TT variant (n=9, p=0.014, Figure 1 D). This strong association suggests that we have identified apoH as the first pQTL for the rs12979860 IL28B SNP associated with viral clearance. Results of the complete apolipoprotein profiling are shown in Table 1 .

Chronic HCV cohort receiving double therapy

We next examined a dataset from chronically infected HCV patients, pooling information from two chronic HCV cohorts who received standard peglFN/RBV treatment, a total of 141 individuals (cHCV-dt).

Of the 141 patients treated, 40 patients achieved sustained virologic response (SVR) and 101 were considered non-responders (NR) based on standard clinical criteria. The cHCV-dt samples were collected and analyzed from 141 patients with HCV genotype 1 and 4. The patients were both non-cirrhotic and cirrhotic and included treatment na^'ive and non responders to previous treatment. The gender of the patients was analyzed for this chronic cohort (Figure 2). Of the patients with sustained virologic response (SVR), 18 of the patients were female and 22 were male (Figure 2). Of the patients that were non-responders (NR), 30 were female and 71 were male (Figure 2). For the chronic cohort, the HCV genotype and virologic response of the patients was analyzed (Figure 3). For those patients with HCV genotype 1 a, 48 of the patients were NR and 19 of the patients were SVR (Figure 3). For those patients with HCV genotype 1 b, 49 were NR and 18 of the patients were SVR (Figure 3). For those patients with HCV genotype 4, 2 were NR and 2 were SVR (Figure 3). For the chronic cohort, the fibrosis score and virologic response were analyzed (Figure A). For patients with SVR, 30 were <F4 and 7 were F4 (Figure 4). For NR patients 72 were <F4 and 26 were F4 (Figure 4). For the chronic cohort, virologic response and treatment history were analyzed (Figure 5). For the SVR group, 13 patients were treatment naive and 26 had previous treatment history (Figure 5). For the NR group, 6 were treatment naive and 94 had previous treatment history (Figure 5). The type of IL28B polymorphism and virologic response were analyzed (Figure 6). For the SVR group, 20% had the IL28B TT polymorphism, 30% had the IL28B CC polymorphism, and 50% had the IL28B CT polymorphism (Figure 6). For the NR group, 32% had the IL28B TT polymorphism, 12% had the IL28B CC polymorphism, and 56% had the IL28B CT polymorphism (Figure 6).

Supporting our findings in aHCV patients, individuals who achieved viral clearance had higher baseline apoH plasma levels as compared to NR patients (p=0.002, Figure 7A). As our dataset was comprised of both treatment- naive and treatment- experienced patients, we were able to perform a stratified analysis by treatment history. Interestingly, the association of viral clearance and high plasma apoH concentration was maintained, regardless of past treatment (Figure 7B), therefore suggesting that apoH concentrations may predict the functional readiness of a patient to clear their virus. The apoH levels were not, however, associated with baseline viral load, infecting HCV genotype or the extent of fibrosis progression. Patients were segregated based on their being naive to prior treatment or having previously received a course of peg-IFNa/RBV therapy. (Figure 7C). P value was 0.03 for the naive group and 0.02 for the treated group. When patients were stratified based on their IL28B polymorphisms, we again observed that baseline apoH was higher in patients with the CC IL28B SNP (p<0.0001 , Figure 7D). It has been demonstrated that the protective effect of the C allele is recessive, only contributing to viral clearance when it is present as a homozygous pair. Investigating the impact of the different IL28B SNP variants on apoH concentration extended this observation to the relationship between IL28B and apoH, as there were no observed differences in the plasma apoH concentrations measured in patients encoding the CT versus TT SNP (Figure 7E). These data, in addition to those from aHCV infection, suggest that apoH is a protein Quantitative Trait Loci (pQTL) for the rs12979860 IL28B SNP. Furthermore, the association of apoH with viral clearance is a feature of both acute and chronic HCV infection.

To further characterize apoH as a biomarker of sustained virologic response in the cHCV-dt cohort, we calculated AUC values. The clinical score alone, apoH alone, and the clinical score + apoH were analyzed without the IL28B SNP and with the IL28B SNP (Figure 8). For patients without the IL28B SNP the AUC [95% CI] was 0.700 for clinical score alone, 0.668 for apoH alone, and 0.776 for clinical score + apoH (Figure 8). For patients with the IL28B SNP the AUC [95% CI] was 0.725 for clinical score alone, 0.668 for apoH alone, and 0.783 for clinical score + apoH (Figure 8). ApoH as a biomarker of response was analyzed for the association of apoH and response to treatment with the adjustment on clinical factors and +/- IL28B SNP (Figure 9). The response (model without IL28B) for OR[95% CI] was 1 .014 with a p value of 0.0005 (Figure 9). The response (model with IL28B) for OR[95% CI] was 1 .013 with a p value of 0.002 (Figure 9). ApoH for prediction of response was analyzed for reclassification of 9% of misclassified patients compared to typical clinical factors (Figure 10). For response (model without IL28B), in the NRI group, response equaled 0.3014; p=0.00658 (Figure 10). For response (model without IL28B), in the IDI group, response equaled 0.0942; p=0.00252 (Figure 10). For response (model with 1L28B), in the NRI group, response equaled 0.1602; p=0.07745 (Figure 10). For response (model with IL28B), in the IDI group, response equaled 0.0672; p=0.01471 (Figure 10).

HIV/HCV coinfected cohort

HCV infection and resulting liver disease is a considerable burden in the context of HIV/HCV co-infection. Co-infected patients show a reduced tolerance and response rate to peg-IFN/RBV therapy, and progression to fibrosis is more rapid than in mono- infected individuals. As prior studies have shown that the rs12979860 IL28B SNP correlates significantly with viral clearance in HIV/HCV-infected patients, we were interested to examine if our observed associations of apoH with viral clearance and IL28B SNPs would also remain consistent 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 toad at week 12 post-treatement initiation. Forty-three patients were identified for analysis; 26 achieved EVR, while 17 were classified as non-responders (NR).

As seen in both the aHCV and cHCV cohorts, plasma apoH levels were significantly elevated in HIV/HCV patients who achieved EVR (p=0.002, Figure 1 1 A). In concordance with mono-infected patients, the likelihood of achieving EVR was higher in patients with increased baseline plasma concentrations of apoH (p=0.002, Figure 1 1 B). Again, we found that apoH levels were not associated with HCV viral load, genotype or liver fibrosis. When stratified for IL28B, we established that in this third group of patients, we could find higher baseline levels of plasma apoH in IL28B CC patients, as compared to those with non-CC genotypes (p=0.05, Figure 1 1 C).

Comparative analysis of the three cohorts The results obtained in each of our patient groups were modeled in order to ascertain the relationship between IL28B polymorphisms, baseline apoH concentrations and the likelihood to achieve viral clearance (Figure 12). Three models were considered: (i) genetic variation in the IL28B locus leads to increased rates of viral clearance in acute HCV and chronic HCV with double therapy, as well as in HIV/HCV co-infection; (ii) an association between baseline levels of plasma apoH and viral clearance; and (iii) a model that accounts for both IL28B variation and apoH concentration as determinants of clearance. Based on the striking associations between baseline apoH and IL28B haplotype, it was important to consider whether they are independent predictors (model iii. ); or in the same causal pathway with IL28B being closer to the phenotype of viral clearance (model iii.) vs. apoH concentrations being closer to the phenotype of viral clearance (model iii.). In all three patient populations, the same result was obtained: uni-variate analysis showed statistical significance for IL28B (model i) or baseline apoH (model ii); and according to the multi-variate logistic regression IL28B and apoH were likely to be part of the same path with apoH being closer to the phenotype of viral clearance (model iii.). This conclusion is supported by the observation that the association between IL28B and viral clearance was of much lower magnitude, and no longer significant, after introducing apoH in the multivariate model, where as the association between apoH and viral clearance was much less affected by the introduction of IL28B in the model, and remained significant. Without being bound by theory, these data suggest that apoH is a stable predictor of viral clearance across three patient populations, and may be positioned in the pathway between the IL28B CC predictive allele and HCV viral clearance.

Example 2

Samples were obtained from chronic HCV infected participants enrolled in a study measuring early virologic response after triple therapy with pegylated- interferon(peg-IFN)/ribavirin(RBV)/NS3 protease inhibitor (boceprevir or telaprevir). This chronic HCV triple therapy (cHCV-tt) cohort included 651 patients recruited from 55 different centers. The patients were infected with HCV genotype 1 , were cirrhotic, and were non responders (NR) to previous treatment. 167 patients were analyzed for response at Week 12 (W12). The patient samples were analyzed for the levels of a panel of biomarkers comprising apoH, SHBG, SAP, AFP, Cathespin D, AXL, IL6Rb, C3, IFBP2, ErbB3, MCSF, Adiponectin, KLK7, TTR, Lpa, vWF, CD26activity, Haptoglobin, Vitronectin, HGFreceptor, CFHR1 , PSAf, TotallPI O, VKDPS, IGFBP1 , HCC4, HER2, CRP, TM, Testosterone!^", TIMP1 , and CD40. The levels of the biomarkers were analyzed at baseline (before treatment) with DiscoveryMAP technology (Myriad- RBM, Inc.). Sample preparation, multi-analyte profiling, and statistical analysis were performed in similar fashion as in Example 1.

The data for 167 patients was analyzed based on response at W12. For the 167 patients the virologic response at Week 12 was divided into response (R) and non responders (NR) (Figure 13). Virologic response was determined by RNA < 1000 lU/ml. Virologic response was further analyzed with respect to gender and of the R group, 31 % were female and 69% were male. For the NR group, 50% were female and 50% were male. The patients were also compared by HCV genotype (Figure 14). Of the R group, 57% of the patients were positive for the HCV 1 b genotype, 37% of the patients were positive for the HCV 1a genotype, 6% were not determined (Figure 14). For the NR group, 41 % of the patients were positive for the HCV 1 b genotype, 54% were positive for the HCV 1a genotype, and 5% were not determined (Figure 14). The Log 10 values of the p values of the biomarkers were calculated and compared and the median values for R and NR based on response at W12 were calculated and plotted (Figure 15). ApoH, SHBG, SAP, AFP, CathespinD, AXL, IL6Rb, C3, IGFBP2, ErbB3, MCSF, Adiponectin, KLK7, TTR, Lpa, vWF, CD26activity, Haptoglobin, Vitronectin, HGFreceptor, CFHR1 , PSAf, TotallPI O, VKDPS, IGFBP1 , HCC4, HER2, CRP, TM, TestosteroneT, TIMP1 , CD40, PAI1 , ApoB, ProinsulinT, and Angiogenin were analyzed.

The biomarker analytes were also analyzed with respect to discriminating between R and NR (Figure 16). The median R, median NR, and p value for each of the biomarkers was calculated. Four biomarkers resulted in p < 0.000118523 (apoH, SHBG, SAP, AFP; see Figure 16). Cathespin D, AXL, IL6Rb, C3, IFBP2, ErbB3, MCSF, Adiponectin, KLK7, TTR, Lpa, vWF, CD26activity, Haptoglobin, Vitronectin, HGFreceptor, CFHR1 , PSAf, TotallPIO, VKDPS, IGFBP1 , HCC4, HER2, CRP, TM, TestosteroneT, TIMP1 , and CD40.were also analyzed. (Figure 16).

The results for ApoH, AFP, SHBG, SAP, AXL, and CathespinD were analyzed by receiver operating characteristic (ROC) curve (Figure 17). ApoH demonstrated an area under ROC (AUROC) of 0.731 , a standard error of 0.044, a p value of 0.000, and a lower bound and upper bound of 95% confidence interval of 0.644 and 0.818, respectively. AFP demonstrated an AUROC of 0.354, a standard error of 0.051 , a p value of 0.005, and a lower bound and upper bound of 95% confidence interval of 0.254 and 0.455, respectively. SHBG demonstrated an AUROC of 0.341 , a standard error of 0.051 , a p value of 0.002, and a lower bound and upper bound of 95% confidence interval of 0.241 and 0.442, respectively. SAP demonstrated an AUROC of 0.686, a standard error of 0.045, a p value of 0.000, and a lower bound and upper bound of 95% confidence interval of 0.597 and 0.775, respectively. AXL demonstrated an AUROC of 0.342, a standard error of 0.049, a p value of 0.002, and a lower bound and upper bound of 95% confidence interval of 0.246 and 0.437, respectively. CathespinD demonstrated an AUROC of 0.333, a standard error of 0.049, a p value of 0.001 , and a lower bound and upper bound of 95% confidence interval of 0.237 and 0.429, respectively.

Finally, the level of apoH was measured and compared with virologic response at W12 for R and NR (Figure 18). The P value was calculated as p=0.0018. Additionally, the level of apoH was measured and compared with IL28B for CC and non CC (Figure 18). The P value was calculated as p=0.0299.

Example 3

Because triple therapy of chronic HCV infected individuals is a new therapy, there is uncertainty regarding the correct cutoff to be used to determine whether a patient has achieved virologic response. In Example 2, virologic response was determined by RNA < 1000 lU/ml, which was essentially below the limit of detection of the assay. In this example, we performed the same analysis as in Example 2, however using the current standard for early virologic response in HCV double therapy (2 log reduction in detectable RNA). The p-values for discrimination between early virologic response (EVR) and no response (NR) are shown in Figure 19. Table 2 tabulates the p-values for the biomarkers with the fourteen lowest p-values.

Example 4

187 of the patients from the cHCV-tt study in Example 2 were assessed for early virologic response at 16 weeks (16W) after commencement of therapy. 250 plasma proteins were screened at baseline, and correlated with early virologic response (EVR) at W16 (EVR determined by HCV RNA negative, i .e. below limit of detection). 75% of the patients were selected randomly to assess predictive biomarkers of EVR(i.e. ,test cohort), and then model accuracy was evaluated in the remaining 25% patients(i.e., validation cohort)

Univariate analyses of the "test" cohort demonstrated Sex Hormone Binding Glogulin(SHBG), Apolipoprotein H(ApoH), Alpha-FoetoProtein(AFP), Hepatocyte Growth Factor Receptor, Serum Amyloid P-component concentrations were associated with EVR (Figure 20). No clinical factor reached significance. In multivariate analysis, SHBG and ApoH remained significantly associated with EVR (p=0.003 and p=0.004, respectively). Additional correlation studies indicated that ApoH levels were independent of fibrosis score (i.e. , cirrhosis severity); whereas SHBG showed a strong association (p<0.001 ), as such we used ApoH in the building of a predictive model. The AUROC curve of the model was 0.73 (95%CI:0.61 -0.84) in the "test" cohort and 0.81 (95%CI:0.68-0.95) in the "validation cohort", in which the model based on ApoH had a good accuracy to predict EVR: sensitivity:94%, specificity:75%, positive predictive value:91 %, negative predictive value:82%.

Baseline concentration of ApoH is associated with EVR in difficult-to-treat HCV patients receiving triple therapy; and provides a strategy to improve the prediction of response.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Example 5

Patients with cirrhosis or other medical complications remain difficult to treat, as highlighted by response rates ranging from 42 - 52%^7"11. Pre-treatment predictors of SVR have not yet been identified for direct acting antiviral regimens^12,13.

In order to identify predictors of response to treatment, samples were obtained from patients enrolled in the French ANRS CUPIC cohort, which examined real life response rates and identified adverse effects of triple therapy in treatment- experienced cirrhotic patients. A screen of 220 plasma proteins, including all apolipoproteins, was conducted in order to identify correlates of response to combination therapy using pegylated interferon/ribavirin (PR) and HCV non-structural protein 3 (NS3) inhibitors (i.e. , telaprevir or boceprevir) in treatment-experienced cirrhotic patients from the ANRS CUPIC cohort. Apolipoprotein H (apoH) was identified to be the first protein biomarker to correlate with sustained virologic response (SVR) in this clinical setting. Notably, increased plasma concentration of apoH, used in combination with known clinical parameters, established a performant model with improved classification of patients as likely to achieve SVR (AUC=0.77, Se=66%, Sp=72%, NRI=39%). Moreover, mechanistic information indicating a previously unidentified role for apoH during viral entry was identified. A human liver slices HCV infection model was thus used to demonstrate that ApoH limits replication. These data support new strategies for the management of cirrhotic HCV patients and expand our understanding of how apolipoproteins intersect with HCV life cycle.

Patients and methods

Patients selection

Our national multicentre prospective cohort study was conducted in 56 French tertiary care centers, as an ancillary study of the French Early Access Program ANRS CO20-CUPIC (AO 2012-1, ClinicalTrials.gov number NCT01514890), from February 2011 to April 2012.

Patients were eligible for the study if they were older than 18 years and were chronically infected with HCV genotype 1 with compensated cirrhosis (Child-Pugh class A), and if they did not achieve a SVR to a prior standard bi-therapy combining peg-IFN/RBV (PR) with an indication to start an IFN-based triple therapy. The diagnosis of cirrhosis was made by liver biopsy or non invasive tests according to the French recommendations. Exclusion criteria were co-infection with HIV or HBV, renal insufficiency (defined by creatinin clearance <50 ml/mn) or organ graft. Written informed consent was obtained from each patient before enrolment. The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research and was approved by the "He de France IX" Ethics Committee (Creteil, France).

Study design

Patients were prospectively enrolled to receive either 12 weeks of telaprevir (750 mg every 8 hours) in combination with Peg-IFN-a2a and RBV then 36 weeks of Peg-IFN- a2a/RBV, or 4 weeks (lead-in phase) of Peg-IFN-a2b and RBV then 44 weeks of Peg- IFN/RBV and boceprevir (800 mg every 8 hours), according to the European label. Treatment was prescribed at the discretion of each investigator without randomization. The following clinical and biological data were assessed at baseline: age, gender, HCV subgenotype (1a or 1 b), previous nonresponse to PR (null response, partial response, relapse), liver function with AST, ALT, bilirubin, albumin, prothrombin time and platelets count. HCV viral load was measured at baseline, at week 16 of treatment and 24 weeks after the end of treatment.

Blood for each patient was collected at baseline, before initiation of IFNa based triple therapy, and plasma was harvested and frozen at -20 °C for further analysis. 220 plasma proteins concentration were screened using CLIA certified muti-analyte protein immune assay testing (Luminex technology), Discovery Map, RBM Inc. The plasma proteins concentrations were correlated to the virologic response at week 16 of treatment (early virologic response (EVR16)) and at weeks 24 after the end of treatment (sustained virologic response (SVR)).

Patients were tested for IL28B rs12979860 single nucleotide polymorphism. DNA samples were extracted from plasma and genotyped for the rs12979860 with TaqMan SNP genotyping assays (Applied Biosystems Inc. , Foster City, CA).

HCV-RNA levels were measured at baseline, after 16 weeks of therapy and after 24 weeks after the end of treatment with a real-time PCR-based assay, either COBAS AmpliPrep®/COBAS TaqMan® (Roche Molecular Systems, Pleasanton, California) with a lower limit of detection of 15 lU/mL, or m2000SP/m2000RT (Abbott Molecular, Des Moines, Illinois), with a lower limit of detection of 12 lU/mL Both assays have been validated for their accuracy in patients infected with HCV genotype 1 ³⁷'³⁸. Patients were considered as virologic responders if HCV RNA level was undetectable at each time point after initiation of treatment. From the 189 enrolled patients, 143 (76%) were early virologic responders at week 16 of treatment and 87 (46%) achieved sustained virologic response 24 weeks after end of treatment. All non early virologic responders at week 16 of treatment were non responders 24 weeks after end of treatment.

In vitro assays

Liver slices culture:

Liver slices were prepared from normal-appearing tissue within surgical liver resection specimens from two single donors. The patients used as liver donors had undergone partial hepatectomy for the therapy of metastatic colorectal cancer. They were seronegative for hepatitis B virus, and human immunodeficiency virus (Department of Digestive Surgery, Cochin Hospital, AP-HP, Paris, France). Experimental procedures were carried out in accordance with French laws and Regulations. Non-infected liver slices, obtained from human liver resection of the donors and cut in 350 μιη-thick slices (2.7x10⁶ cells per slice) were infected with HCVcc Con1 /C3 (genotype 1 b) supernatant [JFH1 -derived chimeric viruses whose structural proteins are encoded by the genotype 1 b-HCV sequence Con1] , [HCVcc: cell culture-grown hepatitis C virus], (MOI = 0.1 ) and cultivated for up to 10 days as previously described¹⁷.

Quantification of HCV RNA by real-time quantitative Reverse Transcription- Polymerase Chain Reaction (qRT-PCR):

The liver slices were washed three times in PBS at 4° C. RNA was extracted from three combined slices using Trizol reagent as described in protocol (Invitrogen, Cergy Pontoise, France). A strand-specific qRT-PCR technique to quantify the intracellular levels of positive and negative strand HCV RNA was carried out, over the time of the experiments with the quantification of 28SrRNA used as an internal standard to quantify HCV in total liver RNA, as previously described39.

Apolipoprotein H treatment:

The human liver slices were infected with HCVcc Con1 supernatant as previously described. Two different concentrations (150 μg/mL or 300 ug/mL) of human plasma purified apolipoprotein H (30-AB23, Fitzgerald, Acton, USA) were added either 24 hours before infection to culture medium, or in the same time of HCVcc infection or preincubated with the HCVcc supernatant for 2 hours before infection. The HCV RNA replication was measured at day 1 , day 5 and day 10 post-infection. All the experiments were performed in triplicates.

Cytotoxicity assays:

Using the cytoTox 96R Non-Radioactive Cytotoxicity Assay (Promega), we assessed the potential cytotoxicity of ApoH concentrations. The percentages of lactate deshydrogenase (LDH) leakage relative to carrier control were calculated as previously described¹⁷.

Statistical analysis

Clinical study:

Analyses were done in intention to treat. Comparisons between independent groups used the Mann-Whitney test for continuous variables or the Fisher's exact test for categorical variables and comparisons within-groups used the Wilcoxon signed-rank test. We randomly selected a Test (n=141 ) and a Replication (n=48) sample from the ANRS CUPIC cohort. All screened plasma protein concentrations were analyzed for early virologic response at week 16 of treatment (EVR16) and for sustained virologic response at week 24 (SVR) after the end of treatment in the Test sample. Biomarkers with p-value < 0.0002 in likelihood ratio testing in univariate analysis (FDR adjustment, i.e. q-value < 0.05) were considered significant and tested in the Replication sample for validation. Taking the entire data set, we next analyzed demographical data and following from our findings, we performed a binary logistic regression model to identify independent clinical predictors of EVR16 and SVR.

Selection of independent covariates was based on a backward elimination procedure, retaining covariates with p-value < 0.05. To test the accuracy of baseline plasma biomarker concentrations as predictors of virologic response, we next established a multivariate model ("clinical model") based on significant clinical covariables and evaluated the incorporation of each statistically significant biomarker ("protein biomarker-based model"), one by one, for prediction of EVR16 and SVR using Area Under Receiver Operating Characteristic (AUROC) curves and category-free net reclassification index (NRI>0) (evaluating the predictive power of the models based on sensitivity and specificity, and the improvement of patient classification (responder vs. non-responder) respectively)^15,16. In addition, we tested the independent association of the protein biomarkers with virologic response by incorporating statistically significant clinical data with each separate biomarker in a binary logistic regression model.

In vitro assay:

The human liver samples from two single donors were sliced and infected with HCVcc Con1 /C3. At different days of the kinetics, the results were obtained from the mean of the three slices culture (triplicates). Values are expressed as means +/- standard errors of the mean (SEM). The data were compared using the paired two-tailed Student's t-test or the Mann-Whitney test, p-value < 0.05 was considered significant. All statistical analyses were performed by using SPSS software, version 20 for Macintosh (SPSS, Chicago, Illinois).

Results

Baseline clinical parameters, prior response to PR therapy, IL28B single nucleotide polymorphism (rs12979860), and results of treatment with combination DAA regimens are reported in Table 3.

Plasma samples from our test cohort (n = 141 ) were screened using xMAP Luminex technology, which permitted quantification of 220 plasma proteins, including all known apolipoproteins. All assays were run under CLIA guidelines. Univariate analysis was performed, applying a false discovery rate (FDR) correction for multiple analytes testing.

Initial results were plotted on a Dubai plot, and indicated that six plasma proteins - alpha foeto-protein (AFP), apolipoprotein C-l (apoCI), apoH, soluble Interleukin 6 receptor beta (IL6rb), Macrophage Colony-Stimulating Factor 1 (MCSF) and transthyretin (TTR) were significantly associated with either early virologic response (EVR16), as defined by undetectable HCV RNA 16 weeks after initiation of treatment, or SVR12 (Figure 21 A and Table S1 ).We next screened a replication sampling of patients (n = 48) from the same cohort in order to validate these findings. Results indicated apoH to be the most stable predictor of EVR16 and SVR (Figure 21 B and Table S2). Taking the entire data set of 189 patients, we next stratified according to the selection of the NS3 PI utilized. Strikingly, higher apoH concentrations segregated treatment responsive patients irrespective of the treatment regimen. (Figure 21 C). Next, we evaluated the likelihood of SVR patients having higher levels of plasma apoH, utilizing the overall median apoH concentration of 262.5 μg ml as the cut- value. We calculated an odds ratio of 3.23 with a 95% confidence interval (CI) of 1 .8 - 5.9 (p = 1 .2 x 10^'4) for SVR patients to have baseline apoH concentration above the median value (Figure 21 D).

Next, available genetic and clinical data were analyzed in order to assess potential confounding factors. Following from prior observations that apoH plasma concentration associates with IL28B polymorphism, we examined the correlation in our patient population.

Despite our patients being prior non responders to standard PR, which effectively skewed the sample from a genetic perspective, we observed higher plasma apoH concentrations in patients with the IL28BCC alleles as compared to those being non- CC (p = 0.02, Figure 22A). While we acknowledge the new data indicating that the IFNA4-creating ss469415590[AG] allele is more closely associated with response to anti-viral therapy ¹⁴, 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 Western European descent (HapMap, r² = 0.92) ¹⁴, suggests that it would not impact the findings reported herein. What is clear from our data analysis is that when patients are first stratified based on being IL28B non-C/C, we still observe a significant correlation of higher apoH and better clinical outcome (Figure 22B) and when tested together in linear regression model, baseline apoH concentration and IL28B polymorphism correlated with SVR independently (Figure 22C).

We further identified prior response to PR, viral load, Aspartate aminotransferase (AST) level, platelets count, prothrombine time (PT) and albumin level as associated with EVR16 (p = 0.003, p = 0.04, p = 0.004, p = 0.03, p = 0.04 and p = 0.004, respectively) and HCV subgenotype, prior response to PR, IL28B polymorphism, AST level, platelets count, PT and albumin level as associated with SVR (p = 0.004, p< 0.00001 , p = 0.007, p = 0.005, p = 0.003, p = 0.001 and p = 0.002, respectively) (Table S3). These latter results are consistent with our patient sample being representative of the ANRS CUPIC cohort [CUPIC] . Following from these results, we performed multivariate analysis using a binary logistic regression model that incorporated statistically significant clinical data. Results for EVR16 indicated that prior response to PR (odd ratio (OR)_reiapse = 5.0, 95% confidence interval (CI) = 1.2 - 20.9, p = 0.02), viral load (OR = 0.44, 95% CI = 0.2 - 0.9, p = 0.02) and albumin level (OR = 1.14, 95% CI = 1.03 - 1.3, p = 0.009) were correlated with treatment outcome (Table S3). For SVR, we found that HCV sub-genotype 1 b (OR = 6.8, 95% CI = 2.4 - 19.5, p = 0.0003), prior response to PR (OR_{partial response} = 0.07, 95% CI = 0.01 - 0.9, p = 0.04) and albumin level (OR = 1 .21 , 95% CI = 1.06 - 1.4, p = 0.004) retained statistical significance (Table S3).

To examine the accuracy of baseline plasma concentrations of the six individual biomarkers as a predictor of early or sustained virologic response we established a multivariate "protein biomarker-based model." This was compared to a "clinical model" that was based on clinical data typically available to clinicians prior to initiating antiviral therapy: prior response to PR, viral load and albumin level for EVR16 and HCV sub-genotype, prior response to PR and albumin level for SVR. The two models were compared using area under receiver operating characteristic (AUROC) curves and category-free net reclassification improvement (NRI>0) for both EVR16 and SVR^15,16. These tests assess the predictive power of the models based on sensitivity and specificity, and the improvement of patient classification (responder vs. nonresponder) when a biomarker is incorporated into the model, respectively. From all tested biomarkers, results indicated that an apoH-based model had higher AUROC for prediction of EVR16: 0.82(95% CI = 0.75 - 0.89) and SVR: 0.77 (95% CI = 0.69 - 0.84) (Figure 23A-B), with the following parameters: sensitivity (Se) = 91%, specificity (Sp) = 26%, positive predictive value (PPV) = 80%, negative predictive value (NPV) = 48% for EVR16 and Se = 66%, Sp = 72%, PPV = 68%, NPV = 70% for SVR (Figure 23C). Importantly, the apoH-based model out-performed the "clinical model" with regard to accurate classification of patients with higher NRI>0 points for EVR16 (86%, 95% CI = 53 - 1 19%) and SVR (39%, 95% CI = 8 - 69%) (Figure 23D-E). In addition, baseline apoH concentration correlated independently with SVR when incorporated with clinical data in binary logistic regression model (Table S4). Our findings therefore indicate that baseline apoH plasma concentration offers improved accuracy for the prediction of EVR16 and SVR in cirrhotic patients receiving NS3 PI regimens.

To determine if our biomarker studies might be informative for HCV pathogenesis, we explored a possible mechanistic link between apoH and HCV replication using a recently described human liver slices infection model¹⁷. We exposed the liver slices to HCVcc Con1 (MOI 0.1 ), in the presence or absence of purified apoH. Liver slices were incubated and intracellular HCV RNA was assessed at interval timepoints - 1 , 5 or 10 days post-infection.

We confirmed efficient HCVcc replication by demonstrating a 6-log increase in negative-strand virus during the 10 day culture (data not shown). Strikingly, we observed a >3-log decrease in HCV positive strand RNA when the liver slices were infected in the presence of 150 - 300 g/ml apoH, doses that reflect the pathological concentrations in chronic HCV patients (Figure 24A). Notably, addition of apoH did not impact cell survival, as measured by lactate dehydrogenase (LDH) leakage into the culture supernatant (Figure 24B). Finally, we pre-incubated either the human liver slices or HCVcc with apoH, prior to initiating infection (Figure 24C). Results indicated a more significant decrease in viral replication when apoH was exposed to the infectious input HCVcc (Figure 24D). These data suggest that apoH, likely due to its ability to bind anionic phospholipids, may opsonize HCV and compromise entry. Moreover, during active replication, apoH may interfere with subsequent hepatocyte infection, thus limiting HCV spread.

These studies report the first predictive biomarker for virologic response in cirrhotic patients receiving NS3 protease inhibitor treatment regimens. While the treatment for HCV has improved, and is expected to further advance with newer DAAs, there remain challenges for the management of so called "difficult to treat" patients, who have only modest response to therapies and often suffer from more severe adverse effects [CUPIC]. Indeed, SVR results with the second wave HCV protease inhibitors (simeprevir, faldaprevir) is associated with similar results in difficult-to-treat patients (Quest-1 and Quest-2 or StatVerso studies, respectively). The identification and validation of apoH as a robust biomarker may support patient stratification and treatment optimization. Importantly, apoH has been previously reported to be associated with HCV clearance in three other clinical situations. Higher plasma apoH concentrations was found to associate with spontaneous clearance during the acute phase of infection, and with viral clearance in chronic HCV and co-infected HIV/HCV patients receiving standard PR [Laird et al, submitted]. Given its general association with viral clearance, we believe it to be more relevant than previously described biomarkers such as IL28B polymorphisms and plasma IP-10 concentrations, which seem to be predictive in settings of PR-only treatment regimens¹⁸. The general relevance of apoH concentrations is further reinforced by our demonstration that it serves to limit viral replication in a physiologically relevant liver slices model. While additional mechanistic studies will be required to integrate apoH into the infectious cycle, we suggest that viral opsonization by apoH may reduce the initial pool of infected hepatocytes during acute infection, and also limit viral spread in the setting of treatment-induced viral clearance.

ApoH (also known as B2-glycoprotein I) is synthetized by hepatocytes and belongs to the apolipoproteins family¹⁹. Despite a clear role for some apolipoproteins in HCV life cycle^4,20"26, apoH is the only one that has been consistently associated with HCV clearance under antiviral therapy^3,6,27 [Laird et al, submitted]. ApoH has been well characterized for its role in the pathogenesis of antiphospholipid syndrome, being a target antigen of anti- B2-glycoprotein I antibodies, and thought to regulate coagulation through its inhibition of prothrombinase activation^28,29. ApoH has also been reported to be involved in immune responsiveness. Notably, it has been demonstrated to limit bacterial sepsis secondary to lipopolysaccharide opsonization³⁰, and may also regulate antigen capture and phagocytosis of dying cells, again through its role as anopsonin^31"33. ApoH has been reported to bind virus, including rotavirus³⁴, human immunodeficiency virus³⁵ and hepatitis B surface antigen ³⁶, thus supporting its role as an inhibitor of HCV entry into hepatocytes.

In sum, we provide an exciting new player in the pathogenesis of HCV. ApoH is interesting both for its role in HCV spread, and also its potential general use as a predictive biomarker for response to antiviral therapy.

Table 1

ALT: alanine aminotransferase; AST: aspartate aminotransferase; EOT12(24): 12 (24) weeks after end of treatment; EVR: early viroiogic response; HCV: hepatitis C virus; IQR: interquartile range (1-3); INR: international normalized ratio for prothrombine time; NR: non response; NS3: non-structural protein 3; PR: standard PeglFN/ribavirin therapy; SNP: single nucleotide polymorphism; SVR12(24): sustained viroiogic response at 12 (24) weeks after end of treatment; W16: 16 weeks of treatment

* calculated using a Mann-Whitney test.

† calculated using Fisher exac test.

Supplemental Table 1

IQR: interquartile range (1-3); LDD: least detectable dose, p-values were calculated using a Mann-Whitney test, p-values < 0.0002 (False Discovery Rate adjustment, i.e. q-value < 0.05) were considered significant.

Supplemental Table 2

EVR,₆: early virologic response after 16 weeks of treatment; SVR: sustained virologic response at 24 weeks after end of treatment

p values were calculated using a Mann-Whitney test.

Supplemental Table 3

ALT: alanine aminotransferase; AST: aspartate aminotransferase; 95% CI : 95% confidence intervalle; EVR1 6: early virologic response after 16 weeks of treatment; HCV: hepatitis C virus; IQR: interquartile range (1 -3) ; N R: non response; OR: odd ratio; PR: standard PeglFN/ribavirin therapy; SNP: single nucleotide polymorphism; SVR: sustained virologic response at 24 weeks after end of treatment.

" - " : indicates variables used as reference for multivariate

analyses " NS " : non significant in multivariate analysis

Variables were first analysed in univariate analysis : *p-values were calculated using Mann-Whitney test and^†p-values were calculated using Fisher exact test. Statistically significant variables were then analysed in multivariate analysis using a binary logistic regression model to identify independent clinical predictors of EVR_i6 and SVR. Selection of independent covariates was based on a backward elimination procedure, retaining covariates with p-value < 0.05.

Supplemental Table 4

OR: odd ratio; 95% CI: 95% confidence interval; PR: standard PeglFN/ribavirin therapy; SVR: sustained virologic response at 24 weeks after end of treatment.

Each significant plasma protein biotnarker was incorporated (using the smallest increased interval of

values for interquartile change) with HCV sub-genotype, prior response to PR and Albumin in multivariate analysis using a binary logistic regression model based on a backward elimination procedure to evaluate their potential independent association with SVR. Increased Intervals of values were 3 units increase for alpha-foeto protein, 30 units increase for apolipoprotein C-I, 50 units increase for apolipoprotein H, 0.3 units increase for macrophage colony stimulating factor 1 and 50 units increase for soluble interleukin 6 receptor beta. Odd ratio, 95% CI and p value are shown.

Claims

1. A method of assessing the ability of a patient diagnosed with chronic HCV infection to clear hepatitis C virus comprising:

a) assaying a biological sample from the patient to determine the level of apoH in the biological sample from the patient; and

b) assessing the likelihood the patient will clear the hepatitis C virus based on the level of apoH.

2. The method of claim 1 , wherein the level of apoH is compared to a reference standard.

3. The method of any one of claims 1 or 2, wherein the patient has previously been treated or is currently undergoing treatment for HCV infection with triple therapy of peg-IFN/RBV and a HCV non-structural protein 3 (NS3) protease inhibitor.

4. The method of any one of claims 1 to 3, wherein said inhibitor is telaprevir or boceprevir.

5. The method of any one of claims 1 to 4, wherein the patient is identified as possessing the CC haplotype of the rs12979860 IL28B SNP.

6. The method any one of claims 1 to 5, wherein step a) further comprises determining the levels of apoCI, MCSF, TTR, AFP, and IL6R, and wherein the assessment of step b) is based on the levels of apoH, apoCI, MCSF, TTR, AFP, and IL6R.

7. The method of any one of claims 1 to 6, wherein the levels of apoH, apoCI,

MCSF, TTR, AFP, and IL6R are compared to a reference standard.

8. A method of treating hepatitis C infection comprising:

a) assaying a biological sample from a patient diagnosed with chronic HCV infection to determine the level of apoH in the biological sample; and b) administering to said patient a therapy based on the level of apoH.

9. A method of assessing the efficacy of a treatment of HCV infection comprising administering therapy, said method comprising:

a) assaying a biological sample from a patient diagnosed with chronic HCV infection to determine the level of apoH in said biological sample; and b) assessing the efficacy of said treatment based on the level of apoH.

10. The method of any one of claims 8 or 9, wherein the level of apoH is compared to a reference standard.

11 . The method of any one of claims 8 to 10, wherein the therapy is a triple therapy of peg-IFN/RBV and a HCV non-structural protein 3 (NS3) protease inhibitor.

12. The method of any one of claims 8 to 11 , wherein said inhibitor is telaprevir or boceprevir.

13. The method of any one of claims 8 to 12, wherein the patient is identified as possessing the CC haplotype of the rs12979860 IL28B SNP.

14. The method of any one of claims 8 to 13, wherein step a) further comprises determining the levels of apoCI, MCSF, TTR, AFP, and IL6R, and wherein the administration or the assessment of step b) is based on the levels of apoH, apoCI, MCSF, TTR, AFP, and IL6R.

15. The method of any one of claims 8 to 14, wherein the levels of apoH, apoCI, MCSF, TTR, AFP, and IL6R are compared to a reference standard.

16. The method of anyone of claims 1 to 15, wherein the patient is cirrhotic.