WO2013174988A1 - Methods for predicting and monitoring treatment response in hcv- and hcv/hiv-infected subjects - Google Patents

Description

METHODS FOR PREDICTING AND MONITORING TREATMENT RESPONSE IN HCV- AND HCV/HIV-INFECTED SUBJECTS

FIELD OF THE INVENTION:

The present invention relates to methods and compositions for predicting and monitoring treatment response in HCV- and HCV/HIV-infected subjects.

BACKGROUND OF THE INVENTION:

Infection by hepatitis C virus (HCV) and HIV shared common routes of transmission and thus co-infection with the two viruses is frequent [1]. HCV infection is seen in 15-30% of HIV-infected patients in Western Europe, with around 25 000 HIV-infected individuals also chronically infected by HCV in France [1, 2]. Since the introduction of highly active antiretroviral therapy (HAART), HCV-related liver disease has become a leading cause of morbidity and mortality in HIV-infected individuals [3-5].

Faster progression to cirrhosis was observed in patients co-infected with HCV and

HIV by comparison with individuals infected with HCV alone [6]. HIV/HCV co-infection is also associated with higher HCV viral levels in serum [7]. Finally, HAART -related adverse events are more frequent in HCV/HIV co-infected individuals, which increase difficulty to optimally treat HIV infection in those patients [8-11].

HCV eradication is therefore a suitable objective in HIV/HCV co-infected individuals.

Current standard therapy for HCV/HIV co-infected patients is PeglNFa in combination with ribavirin (RBV). Combination of RBV with PeglNFa has resulted in an approximately 50% rate of recovery in patients infected with HCV genotype 1 [12, 13]. HCV/HIV co-infected individuals have a lower response to therapy [14-17]. Interferons directly activate immune cells and have multiple functions limiting virus replication.

The effect of PeglNFa on cytokine production and cell activation is probably highly complex throughout multiple regulatory networks and with large inter-individual differences. Therapy efficacy against HCV seems to be dependent on the patient's immunological status at baseline.

By contrast, data on the impact of PeglNFa-RBV treatment, which is an active immunotherapy, on T cell activation and cytokine network remains poorly characterized. Likewise immunological changes associated to sustained virological response needs to be explored. Thus, the association between multiple cytokines, T cells activation and treatment outcomes are largely unknown. Characterizing and monitoring the immunological determinants of sustained virological response among HIV/HCV co-infected patients, particularly after PeglNFa-RBV treatment initiation may be helpful in optimizing therapeutic outcome.

SUMMARY OF THE INVENTION:

The present invention relates to methods and compositions for predicting and monitoring treatment response in HCV- and HCV/HIV-infected subjects. DETAILED DESCRIPTION OF THE INVENTION:

The HCV treatment responsiveness was investigated by inventors in HCV- and HCV/HIV-infected subjects using microbeads-based multiplex immunoassay to simultaneously measure secretion level of 25 cytokines alongside with the level of CD8⁺ T cell activation. Immunological response to HCV treatment was evaluated at baseline and after initiation of PeglNFa-RBV therapy. The inventors found that IL-8, IP- 10 and MCP-1 pretreatment levels were higher in the non-responder subjects. The inventors also demonstrated that cytokine levels change in serum samples obtained before and 4 weeks after initiation of treatment. The inventors demonstrated that MCP-1, IL-4, IL-6, IL-7 and IP- 10 levels statistically increase in responder patients after four weeks of therapy whereas their values decreased or remained stable in the non-responder patients. The inventors also found that the IL-8 and MIP-Ιβ concentration levels remain stable or increase in responder subjects whereas their values tend to decrease in non-responder subjects four weeks after initiation of treatment by comparison with pretreatment levels. Therefore, measuring cytokines levels before and 4 weeks after initiation of HCV treatment constitutes new approaches for monitoring HCV or HCV/HIV treatment efficacy and for maintaining or reorienting such a treatment.

Definitions The term "subject" denotes a mammal. In a preferred embodiment of the invention, a subject refers to any subject (preferably human) afflicted with HCV infection. In a particular embodiment, the subject may also be infected by HIV. The term "HCV infection" has its general meaning in the art and refers to a subject infected by hepatitis C virus.

The term "HCV/HIV co-infection" has its general meaning in the art and refers to a subject co-infected by hepatitis C virus and human immunodeficiency virus.

The term "interferon-alpha" or "IFN-alpha" refers to a family of related polypeptides that inhibit viral replication and cellular proliferation and modulate immune response. The term "IFN-alpha" includes naturally occurring IFN-alpha; synthetic IFN-alpha; derivatized IFN-alpha (e. g., PEGylated IFN-alpha, glycosylated IFN-alpha, and the like); and analogs of naturally occurring or synthetic IFN-alpha; essentially any IFN-alpha that has antiviral properties, as described for naturally occurring IFN-alpha. Suitable interferons alpha include, but are not limited to, naturally-occurring IFN-alpha (including, but not limited to, naturally occurring IFN-alpha2a, IFN-alpha2b) or recombinant interferon alpha. The term "IFN-alpha" also encompasses derivatives of IFN-alpha that are derivatized (e. g., are chemically modified) to alter certain properties such as serum half-life. As such, the term "IFN-alpha" includes glycosylated IFN-alpha; IFN-alpha derivatized with polyethylene glycol ("PEGylated IFN-alpha" or "PEG-IFN"); and the like. PEGylated IFN-alpha, and methods for making same, is discussed in, e. g., U. S. Patent Nos. 5,382, 657; 5,981, 709; and 5,951, 974. PEGylated IFN-alpha encompasses conjugates of PEG and any of the above-described IFN- alpha molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon,Hoffman La-Roche, Nutley, N. J.), interferon alpha 2b (Intron, Schering-Plough, Madison, N. J. ), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferons alpha (Infergen, InterMune, Inc. , Brisbane,Calif).

The term "ribavirin" denotes the l-B-D-ribofuranosyl-l,2,4-triazole-3-carboxamide compound. Ribavirin is a nucleoside analog available from ICN Pharmaceuticals, Inc., Costa Mesa,Calif., and is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U. S. Pat. No. 4,211,771.

The term "HCV treatment" relates to any type of HCV therapy undergone by the HCV-infected subjects. Typically, said treatment may be a PeglNFa and ribavirine treatment, a protease inhibitor treatment, a replicase inhibitor treatment, a polymerase inhibitor treatment, a helicase inhibitor treatment, a therapeutic vaccine-based treatment, a RNA interference-based treatment, an entry inhibitor treatment, an immunotherapy treatment, an immunomodulatory treatment or a combination of any of these treatments. The term "HCV/HIV treatment" relate to any type of HCV/HIV therapy undergone by the HCV/HIV co-infected subjects, including highly active antiretroviral therapy (HAART), interferon, peginterferon-a (PeglNFa), ribavirin. Typically, said treatment may be a PeglNFa and ribavirine treatment.

The term "responder" refers to a subject with a sustained virological response (SVR) when serum HCV RNA was undetectable 24 weeks after completing treatment. The term "responder" refers to HCV infected or HCV/HIV subject that will respond to HCV or HCV/HIV treatment. The term "non-responder" refers to a subject without a sustained virological response (Non-SVR) when HCV RNA decrease was < 2 log copies/mL at week 12 or when HCV RNA was detectable at the end of treatment. The term "non-responder" refers to HCV infected or HCV/HIV subject that will not respond to HCV or HCV/HIV treatment.

The term "IL-8" has its general meaning in the art and refers to interleukin-8 (IL-8). The term "MCP-1" has its general meaning in the art and refers to monocyte chemoattractant protein-1 (MCP-1).

The term "MIP-Ιβ" has its general meaning in the art and refers to macrophage inflammatory protein (ΜΙΡ-1β).

The term "IL-4" has its general meaning in the art and refers to interleukin-4 (IL-4).

The term "IL-6" has its general meaning in the art and refers to interleukin-6 (IL-6). The term "IL-7" has its general meaning in the art and refers to interleukin-7 (IL-7).

The term "IP- 10" has its general meaning in the art and refers to interferon gamma- inducible protein 10 (IP- 10). Predicting methods during the course of the treatment

In one embodiment, the present invention relates to a method for determining whether a HCV-infected subject is a responder or is a non-responder to a HCV treatment which comprises the steps of:

i) measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ι β, MCP-1, IL-4, IL-6, and IP-10 in a blood sample obtained from said subject before treatment,

ii) measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1, IL-4, IL-6, and IP-10 in a blood sample obtained from said subject after 1, 2, 3 or 4 weeks of treatment,

iii) and comparing the expression levels measured at step ii) with the expression levels measured at step i) wherein a difference between said expression levels is indicative that said subject is a responder or a non-responder.

In a particular embodiment, the cytokine expression level of step ii) is measured after 4 weeks of treatment.

Typically, when the expression level of IL-7 determined at step ii) is higher than the expression level of IL-7 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of IL-7 determined at step ii) is equal or lower than the expression level of IL-7 determined at step i), then it is concluding that the subject is a non-responder to treatment. Typically, when the expression level of IL-8 determined at step ii) is higher than the expression level of IL-8 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of IL-8 determined at step ii) is equal or lower than the expression level of IL-8 determined at step i), then it is concluding that the subject is a non-responder to treatment.

Typically, when the expression level of MIP-Ι β determined at step ii) is higher than the expression level of MIP-Ιβ determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of MIP-Ιβ determined at step ii) is equal or lower than the expression level of MIP-Ιβ determined at step i), then it is concluding that the subject is a non-responder to treatment.

Typically, when the expression level of MCP-1 determined at step ii) is higher than the expression level of MCP-1 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of MCP-1 determined at step ii) is equal or lower than the expression level of MCP-1 determined at step i), then it is concluding that the subject is a non-responder to treatment.

Typically, when the expression level of IL-4 determined at step ii) is higher than the expression level of IL-4 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of IL-4 determined at step ii) is equal or lower than the expression level of IL-4 determined at step i), then it is concluding that the subject is a non-responder to treatment.

Typically, when the expression level of IL-6 determined at step ii) is higher than the expression level of IL-6 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of IL-6 determined at step ii) is equal or lower than the expression level of IL-6 determined at step i), then it is concluding that the subject is a non-responder to treatment.

Typically, when the expression level of IP- 10 determined at step ii) is higher than the expression level of IP- 10 determined at step i), then it is concluding that the subject is a responder to treatment, and accordingly, when the expression level of IP- 10 determined at step ii) is equal or lower than the expression level of IP- 10 determined at step i), then it is concluding that the subject is a non-responder to treatment.

In some embodiments, the expression levels of 2, 3, 4, 5, 6, or 7 cytokines are measured.

In a particular embodiment, the expression level of IL-8 and ΜΙΡ-Ιβ, IL-8 and MCP-1 or MIP-Ιβ and MCP-1 are measured. In a particular embodiment, the expression level of IL-8, MIP-Ι β and MCP-1 are measured.

In a particular embodiment, the expression levels of IL-8, ΜΙΡ-Ιβ, MCP-1, IL-4, IL-6, IL-7 and IP- 10 are measured.

Predicting methods before the initiation of the treatment

In a further embodiment, the present invention relates to a method for determining whether a HCV-infected subject will be a responder or a non-responder to a HCV treatment which comprises the steps of:

(i) measuring the expression level of at least one cytokine selected from the group consisting of IL-8, ΜΙΡ-Ιβ, MCP-1, IL-4, IL-6, IL-7 and IP-10 in a blood sample obtained from said subject before the treatment,

(ii) comparing the expression level measured at step i) with a reference value,

(iii) detecting differential in the cytokine expression level between the blood sample and the reference value is indicative that said subject will be a responder or a non-responder.

In a particular embodiment, the present invention comprises measurement of IL-8 expression level.

A "reference value" can be a "threshold value" or a "cut-off value". Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the cytokine expression levels obtained according to the method of the invention with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the cytokine expression level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders to HCV or HCV/HIV treatment. In one embodiment of the present invention, the threshold value may also be derived from cytokine expression level (or ratio, or score) determined in a blood sample derived from one or more subjects who are non-responders to HCV or HCV/HIV treatment. Furthermore, retrospective measurement of the cytokine expression levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

In one embodiment of the invention, the reference value may consist in expression level measured in a blood sample associated with a responder subject or in a blood sample associated with a non-responder subject.

Typically, when the expression level determined for IL-8, MCP-1 or IP- 10 is lower than the corresponding reference value it is concluded that the HCV-infected subject will be a responder to HCV treatment, and accordingly, when the expression level determined for IL-8, MCP-1 or IP- 10 is higher than the corresponding reference value it is concluded that the HCV-infected subject will be a non responder to HCV treatment.

In some embodiments, the expression levels of 2, 3, 4, 5, 6, or 7 cytokines are measured.

In a particular embodiment, the expression levels of IL-8 and IP- 10, IL-8 and MCP-1 or IP- 10 and MCP-1 are measured.

In a particular embodiment, the expression levels of IL-8, IP- 10 and MCP-1 are measured.

In a particular embodiment, the expression levels of IL-8, ΜΙΡ-Ιβ, MCP-1, IL-4, IL-6, IL-7 and IP- 10 are measured. In another particular embodiment, a score which is a composite of the expression levels of the different cytokines may be also determined and compared to a reference value wherein a difference between said score and said reference value is indicative whether said subject is a responder or a non-responder to HCV or HCV/HIV treatment. In a particular embodiment, the score may be generated by a computer program.

Methods for determining the expression level of a cytokine Methods for measuring the expression level of a cytokine in a blood sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a protein including, but not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, ELISA, Luminex, ELISPOT and enzyme linked immunoabsorbant assay.

Said analysis can be assessed by contacting the blood sample with a binding partner capable of selectively interacting with the cytokine present in the blood sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1 , IL-4, IL-6, and IP- 10. In another embodiment, the binding partner may be an aptamer.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Ko filer and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-cytokine, single chain antibodies. Antibodies useful in practicing the present invention also include anti-cytokine fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to cytokine. For example, phage display of antibodies may be used. In such a method, single- chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. 1997. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Re 186, Rel88.

The afore mentioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads.

In a particular embodiment, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said cytokine(s). A blood sample containing or suspected of containing said cytokine(s) is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

In one embodiment, an Enzyme-linked immunospot (ELISpot) method may be used. Typically, the blood sample is transferred to a plate which has been coated with the desired anti-cytokine capture antibodies. Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted.

In one embodiment, when multi- cytokine expression measurement is required, use of beads bearing binding partners of interest may be preferred. In a particular embodiment, the bead may be a cytometric bead for use in flow cytometry. Such beads may for example correspond to BD™ Cytometric Beads commercialized by BD Biosciences (San Jose, California). Typically cytometric beads may be suitable for preparing a multiplexed bead assay. A multiplexed bead assay, such as, for example, the BD^(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens. Typically, beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. A number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target-specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No. 0 126,450), and beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655). Both one-dimensional and two- dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD^<-™-⁾ Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex^(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex¹'™^ microspheres (Bangs Laboratories, Fisher, Ind.). An example of a two-dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749-1756). The beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fiuorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fiuorophores for use in the red, violet or UV laser (e.g. Pacific blue, pacific orange). In another particular embodiment, bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized.

Kits of the invention The invention also relates to a kit for performing the methods as above described, wherein said kit comprises means for measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1 , IL-4, IL-6, and IP- 10 that are indicative of subject responder to HCV or HCV/HIV treatment. Typically the kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.

Methods of treatment

The method of the invention allows to define a subgroup of subjects who are responder or non responder to the HCV treatment.

A further object of the invention relates to a method for the treatment of HCV- infection in a subject in need thereof comprising the steps of:

a) determining whether a HCV-infected subject is a responder or a non-responder to HCV treatment by performing the method according to the invention,

b) administering the HCV treatment, if said subject has been considered as a responder.

A further object of the invention relates to a PeglNFa and ribavirine for use in the treatment of HCV infection in a subject in need thereof, wherein the subject was being classified as responder by the method as above described.

The invention will be further illustrated by the following examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention. EXAMPLE:

Multicytokine response to Peg-Interferon-a-based therapy strongly influences HCV responsiveness in HIV co-infected patients.

Material & Methods Patient samples

Thirty patients co-infected by HCV and HIV and followed at Montpellier University Hospital were included in this study after providing written informed consent. Ethics approval was obtained from our Institutional Review Board. Chronic hepatitis was proved by presence of serum HCV antibodies and detectable viral RNA. HCV genotype, HCV and HIV-1 viral loads, CD4+ T cell count, and liver enzyme levels were all determined using standard procedures.

Based on the treatment effect, HCV/HIV co-infected patients were ranged into two distinct groups: i) those with a sustained viro logical response (SVR) when serum HCV RNA was undetectable 24 weeks after completing therapy, and ii) those without a sustained viro logical response (Non-SVR) when HCV RNA decrease was < 2 log copies/mL at week 12 or when HCV RNA was detectable at the end of treatment (Table 1). Patient relapsing in 24 weeks period after therapeutic cessation were exclude from the study.

All (n=30) R (n=19) NR (n=ll)

Age 45 44 47

CD4 T (cells/μΐ) 350 (50-1076) 378(128-1076) 328 (50-715)

Nadir CD4 T

(cells/μΐ) 122.5 (32-598) 124 (33-598) 119.5 (32-324)

ALAT (UI/1) 48.5 (13-172) 40.5 (13-172) 56.5 (24-133)

ASAT (UI/1) 40 (20-224) 37 (20-224) 54 (31-106)

CV VHC SO

(Ul/ml) 915000 311000 4830000

CV VHC S4 (UI/1) 27000 270 1215500

CV VIH SO (UI/1) 0 0 0

Gen VHC 1/2/3/4 19/1/6/1 9/1/6/0 10/0/0/1

Ratio CD8CD38

S4/S0 1.34 (0.15-4.5) 1.27 (0.15-4.5) 1.46 (0.8-4.3)

Table 1: Patient characteristics

Routine laboratory testing

The inventors detected antibodies to HCV in serum samples via third generation enzyme-linked immunoassays (Cobas; Roche Laboratories). Serum levels of HCV RNA were detected using the COBAS AMPLICOR assays (Roche Diagnostic Systems), which amplify HCV RNA by reverse transcriptase-polymerase chain reaction. The lower limit of the assay was 50 IU/mL. HCV genotype was determined using INNO-LiPA HCV II test (Innogenetics). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were quantified using standard methods.

Quantitation of serum cytokine concentrations

Cytokines and chemokines were quantified in serum samples obtained before initiation of treatment, and 4 weeks after the start of PeglNFa-based therapy. A multiplexed microbead assay was used in this purposed according to manufacturer's instructions (cytokine twenty- five-plex kit, Life Technologies Ltd, Paisley, UK) and a FIDIS™ apparatus (BMD, Marne La Vallee, France). Twenty-five cytokines were quantified: IL-Ι β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40/70, IL-13, IL-15, IL-17, EOTAXIN, GM-CSF, IFN-a, IFN-γ, IP-10, MCP-1 , MIG, MIP-la, ΜΙΡ-Ι β, RANTES, and TNF-a. Data were analyzed using the MLX-Booster program (BMD) and standard curves. Mean concentrations (pg/ml) of cytokines and chemokines were all superior to the detection limits, defined as the mean background value plus 2 SD.

Analysis of CD8 /CD38^{bri ht} T cells

Activation of CD8⁺ T cells was assessed by flow cytometry analysis on EDTA-treated fresh whole blood using FC 500 apparatus (Beckman Coulter, Miami, Florida). The expression of CD38^bnght on CD8⁺ T cells was explored by using two-color staining with anti- CD8 and anti-CD38 conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively (Beckman Coulter) as previously described [18]. The positive threshold for CD38^bright analysis was established using the CellQuant CD38/CD8 kit for quantitation of CD38 cell surface expression (BioCytex, Marseille, France) and was defined as 8,500 CD38 binding sites/cells. The CD8⁺/CD38^bnght values were expressed as the percentage of CD38^bnght cells from the CD8⁺ T cell populations. Statistical Analysis

The Mann- Whitney U test and Kruskal-Wallis test were used to analyze continuous variables where appropriate. The Friedman test was used to evaluate changes in serum cytokine levels over time . A P value < .05 was considered statistically significant. Multivariate analyses were performed using a stepwise logistic regression model. Statistical analyses were done using SPSS software version 18.0J.

Results

Clinical characteristics of patients at baseline

Of the 30 HCV/HIV co-infected patients receiving peglFNa and RBV therapy, 19 (63%) have SVR with prolonged suppression of HCV plasma RNA after therapeutic cessation and 11 (37%) have non-SVR. Before treatment initiation, the median AST and ALT levels were significantly lower in the SVR group than those in the non-SVR group (37 vs 54 IU/L). Likewise the inventors observed a lower HCV RNA viral load in the SVR group compared to the non-SVR group (311000 vs 4830000 IU/L).

Pretreatment levels of soluble and cell inflammatory markers

Twenty five cytokines secretion levels were quantitated in serum samples prior to anti- HCV therapy by multiplex assays (Table 2). Two of soluble markers were statistically associated with therapeutic response at baseline. IP- 10 levels were higher in the non-SVR group than in the SVR group: 64.3 (33.0) pg/ml vs 132.0 (58.9) pg/ml, respectively, P = 0.021 . Individuals from the non-SVR group had also higher MCP-1 pretreatment concentrations: 347.9.3 pg/ml (91.2) vs 442.6 (145.7) pg/ml, respectively, P = 0.0009. Serum concentrations of IL-8 observed in patients of the non-SVR group also tend to be higher than those observed in the SVR group: 22.5 (73.8) pg/ml vs 128.1 (707.8) pg/ml, respectively P = 0.106.

SVR pg/ml NSVR pg/ml p-value

Cytokines mean [CV] mean [CV] (Wilcoxon)

ILlbeta 12.9 [34.24] 10.84 [4.904] 0.093

IL1RA 1556 [198] 1506 [123] 0.779

IL2 6.296 [2.427] 5.866 [2.628] 0.425

IL2R 837 [812.27] 884.1 [155.6] 0.101

IL4 8 [177.95] 8 [173.5] 0.338

IL5 2.352 [0.589] 2.352 [0] 0.616 IL6 4.666 [2.25] 5 [23.017] 0.353

IL7 18.91 [2.38] 20.88 [4.32] 0.142

IL8 22.51 [73.85] 128.1 [707.79] 0.106

IL10 3.608 [1.18] 3.092 [2.061] 0.777

IL12p40.70 321.5 [55.8] 318.3 [56.5] 0.948

IL13 11 [4.125] 11 [2.755] 0.702

IL15 33.21 [9.48] 33.21 [3.16] 0.863

IL17 24.89 [9.34] 24 [8] 0.73

TNFa 8.866 [2.606] 8.866 [1.167] 0.762

IFNg 5.268 [1.371] 6.146 [0.878] 0.621

GM.CSF 18.46 [9.23] 15.38 [13.84] 0.482

MlPla 119.4 [1.8] 120.6 [6.8] 0.262

MlPlb 150.4 [24.3] 150.4 [23.6] 0.88

IP10 64.27 [32.98] 132 [58.89] 0.021

MIG 458.6 [5.9] 458.6 [4] 0.497

EOTAXIN 75.54 [30.71] 72.96 [29.4] 0.813

RANTES 10540 [6413] 9350 [10922] 0.683

MCP1 347.9 [91.2] 442.6 [145.7] 0.009

IFNa 143 [21.3] 143 [1.4] 0.477

Table 2: Pretreatment Cytokine concentration in serum from SVR and NSVR patients

Cytokine changes after therapeutic initiation of HC V treatment

As a consequence of PeglNFa administration, IFN-a rose in the two groups. This enhancement of INF-a in serum was not different in SVR vs. non-SVR patients. PeglNFa therapy induced also a significant increased of the levels of 7 immune soluble markers in serum of SVR and non-SVR patients after four weeks of treatment: IL-Ιβ, IL-1RA, IL-2, IL- 5, IL-12, IL-13, and Eotaxin. By contrast, five cytokines and chemokines levels rose only in the SVR group: IL-4, IL-6, IL-7, IP-10, and MCP-1. Finally, when results were analyzed without consideration of the therapeutic HCV response, the inventors also observed an increase in IL-10 and RA TES concentration (Table 3).

Comparison of the cytokine level changes induced by PeglNFa therapy in the SVR and the non-SVR group.

The ratios of cytokines concentrations at four weeks and at baseline were compared in SVR and non-SVR patients (Table 3). A robust increased of MCP-1 and ΜΙΡ-1β concentration was induced by the PeglNFa-based therapy in the SVR group whereas the value decreased or remained stable in the non-SVR group. Likewise in most of patients responding to anti-HCV treatment, the IL-8 concentration remains stable after one month period of PeglNFa-based therapy, whereas it is declined in non-SVR patients.

All patients SVR NSVR

p- p-

Cytokine Median [95%IC] value Median [95%IC] value Median [95%IC] p-value

ILlbeta 2.05 [1.21 -4.86] 2e-04 4.42 [1.15-6.17] 0.0053 1.32 [1.11 -4.09] 0.0068

IL1RA 1.38 [1.3 - 1.54] le-04 1.43 [1.32 - 1.63] 3e-04 1.35 [0.99 - 1.57] 0.0537

IL2 1.3 [1.07 - 1.54] 0.0066 1.3 [1 - 1.65] 0.0546 1.29 [1 -6.1] 0.042

IL2R 1.26 [0.79 - 6.03] 0.2621 1.38 [0.83 - 8.44] 0.0955 0.93 [0.42 - 7.78] 0.8311

IL4 16.73 [1.19 - 19.42] 0.0104 16.81 [1.2- 19.6] 0.0058 1.21 [0.49-21.1] 0.7596

IL5 1.34 [1.23 - 1.47] <0.001 1.31 [1.16- 1.52] 0.001 1.37 [1.18- 1.77] 0.0067

IL6 1.65 [1.23 - 7.08] 0.0013 2.11 [1.33 - 10.58] 0.001 1.23 [0.69 - 6.36] 0.5195

IL7 1.12 [1.02- 1.5] 0.0237 1.14 [1.03 - 1.63] 0.0124 1.03 [0.89-4.1] 0.6247

IL8 0.95 [0.56-2.39] 0.9032 1.26 [0.68-3.13] 0.49 0.58 [0.09-2.9] 0.1748

IL10 1.43 [1.02 -2.35] 0.0417 1.67 [0.92-3.15] 0.0948 1.21 [0.89 - 3.45] 0.2061

IL12p40.70 1.44 [1.31 - 1.61] <0.001 1.48 [1.29- 1.8] <0.001 1.42 [1.22- 1.66] 0.001

IL13 1.99 [1.61 - 8.89] <0.001 2.24 [1.63-9.6] 8e-04 1.66 [1.38 - 12.04] 0.0038

IL15 1.13 [0.95 - 1.34] 0.1812 1.19 [0.97- 1.49] 0.08 0.97 [0.81 -4.72] 0.8984

IL17 1.22 [0.99-4.76] 0.0799 1.9 [0.99-5.18] 0.0663 1.04 [0.76-4.74] 0.8385

TNFa 0.86 [0.76- 1.12] 0.2286 0.94 [0.78- 1.21] 0.8596 0.77 [0.68 - 14.39] 0.0674

IFNg 1.04 [0.78- 1.21] 0.5784 1.03 [0.78 - 1.22] 0.732 1.06 [0.73 - 1.41] 0.6889

GM.CSF 1.24 [0.96- 1.57] 0.0712 1.34 [0.89-2] 0.1025 1.16 [0.8- 1.44] 0.624

MlPla 1.06 [0.74- 1.16] 0.7096 1.08 [0.73 - 1.24] 0.6026 0.99 [0.64- 1.16] 0.9188

MlPlb 1.08 [0.71 -2.54] 0.7922 1.37 [0.84- 5.52] 0.1232 0.56 [0.1 - 1.09] 0.083

IP10 1.46 [1.18- 1.94] 6e-04 1.6 [1.19-2.56] 0.0033 1.24 [0.91 - 1.96] 0.1016

MIG 1.07 [0.77 - 1.09] 0.3762 1.04 [0.76- 1.1] 0.5062 1.08 [0.73 - 1.24] 0.6247

EOTAXIN 1.95 [1.53 -2.45] <0.001 2.24 [1.57 -2.98] <0.001 1.55 [1.16-2.23] 0.0068 RANTES 1.33 [1.01 - 1.89] 0.0427 1.31 [0.97 - 3.19] 0.0874 1.4 [0.73 - 2.04] 0.2783

MCP1 1.23 [1.04 - 1.47] 0.0145 1.42 [1.2 - 1.97] le-04 0.87 [0.69 - 1.24] 0.5771

IFNa 4.89 [3.85 - 6.12] <0.001 5.06 [3.83 - 6.71] <0.001 4.6 [2.95 - 8.51] 0.001

Table 3: Cytokines fold changes after therapeutic initiation of HCV treatment. Ratios of S4/S0 concentrations are indicated overall and according to the HCV response (SVR and NSVR) for each cytokines

Changes in T cell activation over the 3 months period of therapeutic initiation.

Pretreatment CD8⁺ T cell activation was more elevated in non-SVR patients compared to patients belonging to the SVR group. Under PeglNFa-based therapy, an increased of CD38^bnght expression was observed at the surface of CD8 T⁺ cells in the two groups of patients. This is the direct effect of the immunologically active therapy on T cell activation. However, no significant differences in the increase of CD8⁺ T cell activation were observed between the two groups of patients suggesting that this marker was not associated with a better PeglNFa immune response. REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for determining whether a HCV-infected subject is a responder or is a non- responder to a HCV treatment which comprises the steps of:

i) measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1 , IL-4, IL-6, and IP-10 in a blood sample obtained from said subject before treatment,

ii) measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1 , IL-4, IL-6, and IP-10 in a blood sample obtained from said subject after 1, 2, 3 or 4 weeks of treatment,

iii) and comparing the expression levels measured at step ii) with the expression levels measured at step i) wherein a difference between said expression levels is indicative that said subject is a responder or a non-responder.

2. The method according to claim 1 wherein the expression level of at least one cytokine of step ii) is measured after 4 weeks of treatment.

3. The method according to any one of claims 1 or 2 wherein said method comprises measuring the expression level of IL-8, MIP-Ιβ and MCP-1.

4. A method for determining whether a HCV-infected subject will be a responder or a non-responder to a HCV treatment which comprises the steps of:

(i) measuring the expression level of at least one cytokine selected from the group consisting of IL-8, ΜΙΡ-Ιβ, MCP-1 , IL-4, IL-6, IL-7 and IP-10 in a blood sample obtained from said subject before the treatment,

(ii) comparing the expression level measured at step i) with a reference value,

(iii) detecting differential in the cytokine expression level between the blood sample and the reference value is indicative that said subject will be a responder or a non-responder.

5. The method according to claim 4 wherein said method comprises measuring the expression level of IL-8, IP-10 and MCP-1.

6. The method according to any one of claims 1, 2 or 4 wherein said method comprises measuring the expression level of IL-8.

7. The method according to any one of claims 1 to 6 wherein said subject is also infected by HIV.

8. A kit for performing the methods according to any one of claims 1 to 7 wherein said kit comprises means for measuring the expression level of at least one cytokine selected from the group consisting of IL-7, IL-8, ΜΙΡ-Ιβ, MCP-1, IL-4, IL-6, and IP-10.

9. A method for treating HCV-infection in a subject in need thereof comprising the steps of:

a) determining whether a HCV-infected subject is a responder or a non-responder to HCV treatment by performing the method according to any one of claims 1 to 7,

b) administering the HCV treatment, if said subject has been considered as a responder.