WO2012107584A1 - Procédés de prédiction des résultats d'une infection par le virus de l'hépatite - Google Patents

Procédés de prédiction des résultats d'une infection par le virus de l'hépatite Download PDF

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WO2012107584A1
WO2012107584A1 PCT/EP2012/052359 EP2012052359W WO2012107584A1 WO 2012107584 A1 WO2012107584 A1 WO 2012107584A1 EP 2012052359 W EP2012052359 W EP 2012052359W WO 2012107584 A1 WO2012107584 A1 WO 2012107584A1
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tgf
cells
patient
hepatitis
level
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Brigitte Autran
Sawsan HARFOUCH
Marc-Antoine VALANTIN
Assia Samri
Marguerite GUIGUET
Ioannis THEODOROU
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Universite Pierre et Marie Curie
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to predictive methods to evaluate the outcome of an infection by a hepatitis virus.
  • hepatitis viruses are believed to exist, and include the A, B, C, D, E, F, G and cryptogenic hepatitis viruses.
  • the hepatitis viruses are spread through a number of different virus families. Of these viruses, hepatitis C virus (HCV, a flavivirus) and hepatitis B virus (H BV, a hepadnavirus) pose the greatest public health problem in industrialized countries.
  • HCV hepatitis C virus
  • H BV hepatitis B virus
  • Both hepatitis C and B are bloodborne diseases, although both viruses may also be transmitted perinatally and via sexual contact. Hepatitis C and B can each give rise to acute and chronic infections. A relatively low level of mortality is due to acute C and B hepatitis (primarily due to fulminant hepatitis).
  • HCV liver transplants in the United States.
  • HCV infections are symptomatic in the acute phase, which typically presents with symptoms such as jaundice, fatigue, abdominal pain and/or loss of appetite.
  • Subclinical infections can be detected using diagnostic testing for viral antigens and/or DNA.
  • acute HCV infections are substantially less common than acute HBV infections, by a factor of approximately ten.
  • the prevalence of chronic HCV infection is two to three times greater than that for chronic HBV infection.
  • HCV infection carries a much greater risk of the development of chronic liver disease and liver failure.
  • Interferon a2a, interferon a2b and interferon a con-1 are currently used for the treatment of chronic hepatitis virus infection, optionally in combination with ribavirin (an antiviral drug).
  • the invention provides in vitro methods for predicting hepatitis outcome in patients infected with a hepatitis virus, more particularly for determining a susceptibility to non-spontaneous viral clearance or to a non-response to a treatment.
  • a subject of the invention is an in vitro method for predicting whether a patient affected with a viral hepatitis will cure spontaneously, which method comprises determining the level of TGF- ⁇ produced by T cell that are specific for the hepatitis virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ , in a biological sample from the patient, wherein the patient is predicted to cure spontaneously when said level is not significantly higher, compared to a control group.
  • Another subject of the invention is an in vitro method for predicting whether a patient affected with a viral hepatitis will be responsive to an antiviral hepatitis treatment, such as a treatment with an interferon and/or an anti-viral nucleoside, e.g. ribavirin, or direct anti-viral agents (DAAs), which method comprises determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ , in a biological sample from the patient, wherein the patient is predicted to be responsive to said treatment when said level is not significantly higher compared to a control group.
  • an antiviral hepatitis treatment such as a treatment with an interferon and/or an anti-viral nucleoside, e.g. ribavirin, or direct anti-viral agents (DAAs)
  • DAAs direct anti-viral agents
  • the method comprises determining the level of TGF- ⁇ , produced by T cells, upon stimulation with a hepatitis virus antigen.
  • the method may comprise stimulating Peripheral Blood Mononuclear Cells (PBMC) obtained from the biological sample of the patient with a hepatitis virus antigen, detecting TGF- ⁇ Din the culture supernatant, and comparing with the level of TGF- ⁇ ⁇ produced in the absence of hepatitis virus antigen.
  • PBMC Peripheral Blood Mononuclear Cells
  • the method comprises determining the level of T cells that produce TGF- ⁇ , upon stimulation with a hepatitis virus antigen.
  • the method comprises stimulating Peripheral Blood Mononuclear Cells (PBMC) obtained from the biological sample of the patient with a hepatitis virus antigen, and quantifying the subset of cells that produce TGF- ⁇ , e.g. by flow cytometry.
  • PBMC Peripheral Blood Mononuclear Cells
  • kits for evaluating the outcome of an infection by a hepatitis virus comprising (i) means for determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis C virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ , in a biological sample from the patient, and optionally (ii) instructions for use.
  • the method of the invention makes it possible to classify patients who are likely to cure, either spontaneously or after standard treatment versus patients who are likely to progress to liver fibrosis.
  • New candidate drugs, or liver transplant, may be proposed to the latter.
  • Figures 1A and 1 B are graphs that show comparison of HCV-specific CD4+ ( Figure 1A) and CD8+ ( Figure 1 B) TGF- ⁇ - T cell Percentages (presented after subtraction of background), between HD (healthy donors), RE (patients with recovery) and VI patients (patients who remained viremic).
  • Figure 2 is a graph that shows the association trend between anti-HCV TGF- ⁇ producing T cells and progress of liver disease (12 patients).
  • the inventors have shown that patients infected with HCV who have progressed toward chronic hepatitis have a dramatically high level of CD4+ or CD8+ T cells that produce TGF- ⁇ .
  • they provide an in vitro method for predicting whether a patient affected with a viral hepatitis will cure, or is likely to cure, spontaneously, or whether a patient affected with a viral hepatitis will be, or is likely to be, responsive to an antiviral treatment, which method comprises determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ , in a biological sample from the patient. Definitions
  • the term "patient” or “subject” refers to any individual capable of being tested.
  • it is a human being, but the term includes any other mammal, such as dogs, cats, rodents, cattle, horses, monkeys, etc.
  • the patient can be tested irrespective of the sex or age thereof.
  • it is a human being, including males or females. It may be a child, an adolescent, or an adult of any age.
  • the patient may be may be asymptomatic, or may show early or advanced signs of hepatitis.
  • the patient may be an individual predisposed to cure spontaneously from hepatis infection, in particular an individual carrying one or more mutations in the gene encoding IL-28B.
  • TGF- ⁇ refers to transforming growth factor beta that is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. Many cells synthesize ce peptide and almost all of them have specific receptors for this peptide. Any of TGF- ⁇ , TGF-p2, and TGF-p3 is encompassed, since they all function through the same receptor signaling systems.
  • the present invention preferably assays TGF- ⁇ or T cells that are specific for a hepatitis virus and that produce TGF- ⁇ TGF- ⁇ is produced as a large protein precursor (containing 390 amino acids) that is proteolytically processed to produce a mature peptide of 112 amino acids. Mature TGF- ⁇ , more particularly mature TGF- ⁇ , is preferably assessed.
  • predicting refers to the determination of a likelyhood for a patient infected with a hepatitis virus to cure spontaneaously or after treatment.
  • the method is performed before initiation of therapy>.
  • to cure preferably means that viral clearance is observed. Viral clearance and alleviation of the symptoms may be observed within 1 week to 12 months, generally within 3 to 6 months after infection.
  • responsive means that the method of the invention assesses whether the patient is likely to be respond to an antiviral treatment, such as a treatment including or consisting of, an interferon and/or an anti-viral nucleoside, such as ribavirin.
  • an antiviral treatment such as a treatment including or consisting of, an interferon and/or an anti-viral nucleoside, such as ribavirin.
  • Interferon a2a and even preferably pegylated Interferon a2a, combined with ribavirin, is currently the standard treatment.
  • the antiviral treatment may comprise any of the below mentioned drugs.
  • Treatments of hepatitis virus include: (1) Interferon and ribavirin; (2) Substrate-based NS3 protease inhibitors (WO 98/22496); (3) Non-substrate-based inhibitors such as 2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al., Biochemical and Biophysical Research Communications, 238:643-647 (1997); Sudo K., et al.
  • S-ODN Antisense phosphorothioate oligodeoxynucleotides complementary to sequence stretches in the 5' non-coding region (NCR) of the virus, or nucleotides 326-348 comprising the 3' end of the NCR and nucleotides 371-388 located in the core coding region of the HCV RNA;
  • Inhibitors of IRES-dependent translation (12) Nuclease-resistant ribozymes; and (13) Miscellaneous compounds including 1-amino-alkyloyclohexanes (U.S. Pat. No.
  • telaprevir VX-950
  • SBSS03034 Boceprevir
  • 2- Polymerase inhibitors of 2' and 3' substituted ribonucleoside analogues such as Valopicitabine, a prodrug of the nucleoside analogue 2-Cmethylcytidine (NM283) (Pierra C J med chem.
  • RNA-dependent RNA polymerase inhibitors such as benzimidazole derivatives JTK-109 and JTK-003 (Tomei L. J Virology 2004, 78(2): 938-946).
  • 3- Immune modulators including the Toll-like receptor agonists such as isatoribine (TLR7) (Horsmans Y, Hepatology 2005, 42:724-731), resiquimod (TLR7 and 8) (Pockros PJ, Hepatology 2007, 47: 174-182), and CPG10101 (TLR9) (McHutchison JG, Hepatology 2007, 46:1341-1349).
  • spontaneous means that the patient is not treated with any antiviral drug against the hepatitis virus, such as those mentioned above.
  • the patient is not treated with an interferon and/or an anti-viral nucleoside, such as ribavirin.
  • biological sample refers to any biological sample originating from a patient.
  • samples include biological fluids and tissue biopsies.
  • the sample may be blood, serum, saliva, urine or sperm. More preferably, the biological sample is a blood sample.
  • control refers to a basal value corresponding to the average of the values obtained with the biological sample from healthy individuals not infected with a hepatitis virus. It may be a statistical reference value.
  • a level "not significantly higher, compared to a control value” generally means that the value is in the limits of normal values or that no statistically significant increase, for example the increase, if any, is less than two standard deviations above the mean of the concentration of TGF- ⁇ in the healthy individuals.
  • a level " significantly higher, compared to a control value” generally means that the value shows a statistically significant increase, for example the increase is at least than two standard deviations above the mean of the concentration of TGF- ⁇ in the healthy individuals.
  • level » means concentration of TGF- ⁇ produced by T cells that are specific of a hepatitis virus and that produce TGF- ⁇ , or concentration or quantity of T cells that are specific of a hepatitis virus and that produce TGF- ⁇ .
  • T cells that are specific of a hepatitis virus and that produce TGF- ⁇ includes CD4+ or CD8+ T cells.
  • the T cells may present other markers such as CD3, CD2.
  • the patient to test The patient to test:
  • the patient is infected with any hepatitis virus, preferably hepatitis C virus (HCV), or hepatitis B virus (HBV), of any genotype or strain.
  • hepatitis virus preferably hepatitis C virus (HCV), or hepatitis B virus (HBV), of any genotype or strain.
  • the patient is infected with hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • the patient is co-infected with a HIV virus.
  • the patient is co-infected with both HIV and HCV.
  • the patient is affected with an acute viral hepatitis.
  • the level of TGF- ⁇ and/or of T cells that are specific for the hepatitis virus and produce TGF- ⁇ is determined in a biological sample taken from the patient within three months, preferably within two months, after an acute viral hepatitis is diagnosed.
  • the method may also be useful when the patient already suffers from chronic hepatitis.
  • the patient is predicted to cure spontaneously or to be responsive to a treatment with an interferon and/or an anti-viral nucleoside, e.g. ribavirin, or direct anti-viral agents (DAAs).
  • an interferon and/or an anti-viral nucleoside e.g. ribavirin, or direct anti-viral agents (DAAs).
  • DAAs direct anti-viral agents
  • an application might be that if the patient has suffered from an acute hepatitis, the physician may then propose to wait for a few months (typically 3 months), to let the possibility for the patient to recover spontaneously, without any treatment. If the patient does not show any sign of recovery after said period without treatment, then a treatment with an interferon and/or an anti-viral nucleoside, e.g. ribavirin, or direct anti-viral agents (DAAs) may be proposed, since the patient is likely to respond to said treatment.
  • an interferon and/or an anti-viral nucleoside e.g. ribavirin
  • DAAs direct anti-viral agents
  • TGF- ⁇ produced by T cell that are specific for the hepatitis virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ in a biological sample from the patient is undetectable or not significantly higher than the normal values from a control group that may be a healthy uninfected group, then the patient is predicted neither to cure spontaneaously nor with a treatment with an interferon and/or an anti-viral nucleoside, e.g. ribavirin.
  • alternative therapeutic options are to be contemplated, e.g. direct anti-viral agents (DAAs) or any innovative treatment.
  • DAAs direct anti-viral agents
  • the method of the invention comprises determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis virus, or the level of T cells that produce TGF- ⁇ upon stimulation with a hepatits virus antigen. It generally includes a step of comparing the level of T cells that produce TGF- ⁇ , or of TGF- ⁇ produced by T cells, either in absence or in presence of a hepatitis virus antigen, such as a virus protein, peptide, or a virus particle or vector. In a particular embodiment, the comparison may be performed with a hepatitis virus antigen, vs a non-viral or a non-hepatitis virus antigen. It should be underlined that determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis virus does not mean determining the plasmatic level of non specific TGF- ⁇ .
  • Determining the level of TGF- ⁇ produced by T cells that are specific of a hepatitis virus may be performed by various methods.
  • the method may involve stimulating Peripheral Blood Mononuclear Cells (PBMC), or purified CD4+ or CD8+ T cells, obtained from the biological sample of the patient, with a hepatitis virus antigen such as a virus protein, peptide, or a virus particle or vector.
  • PBMC Peripheral Blood Mononuclear Cells
  • CD4+ or CD8+ T cells obtained from the biological sample of the patient
  • a hepatitis virus antigen such as a virus protein, peptide, or a virus particle or vector.
  • it may involve isolating and optionally culturing the CD4+ or CD8+ T cells either before or after stimulation by a HCV antigen.
  • assays of the invention thus utilize antibodies directed to TGF- ⁇ .
  • antibodies are well known in the art.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • the biological sample can be optionally treated in a prior step, or brought directly into contact with at least one capture antibody.
  • the method can be carried out according to various formats well known to those skilled in the art: in solid phase or in homogenous phase; in one step or in two steps; in a competition method, by way of non-limiting examples.
  • the capture antibody is immobilized on a solid phase.
  • a solid phase use may be made of microplates, in particular polystyrene microplates, such as those sold by the company Nunc, Denmark. Use may also be made of solid particles or beads, paramagnetic beads, such as those provided by Dynal or Merck-Eurolab (France) (under the trademark EstaporTM), or else polystyrene or polypropylene test tubes, etc.
  • An immunoassay format for detecting TGF- ⁇ by competition is also possible.
  • Other immunoassay modes can also be envisioned and are well known to those skilled in the art.
  • Anti-TGF- ⁇ antibodies of use with the present invention may be purchased from commercial sources or may be generated using art-recognized methods. It will be appreciated that any antibody directed to TGF- ⁇ will be useful in assays of the invention.
  • antibodies used with assays of the invention recognize epitopes of the extracellular portion of membrane-bound TGF- ⁇ .
  • the anti- TGF- ⁇ antibodies are monoclonal antibodies.
  • the method of the invention involves quantifying TGF ⁇ that is produced or released by the hepatitis virus- specific T cells, preferably within a time window of between 4 to 36h after stimulation with a hepatitis virus antigen.
  • assays of the invention detect both membrane-bound and soluble TGF- ⁇ .
  • TGF- ⁇ is a known assay for the quantification of active TGF ⁇ and utilises a TGF- ⁇ responsive cell containing an expression vector having a TGF- ⁇ response element (the PAI-1 promoter) and a structural region encoding an indicator molecule, for example luciferase.
  • TGF- ⁇ present in a test sample induces activation of the PAI-1 promoter. This results in transcription and the expression of the indicator molecule.
  • Measurement of the amount of indicator molecule determines the amount of TGF- ⁇ responsible for the induced activation.
  • the method of the invention may comprise determining the level of other markers of treatment outcome, but preferably not IP-10.
  • assaying the level of TGF- ⁇ produced by T cells upon stimulation by a hepatitis virus antigen only is sufficient, and is alone useful to evaluate the outcome of the infection in the patient.
  • the method further comprises detecting polymorphisms, especially single-nucleotide polymorphisms (SNPs) in IL-28b gene (see Thomson et al, Gastroenterology. 2010, 139(1): 120-9, and international patent application WO2011/013019).
  • SNPs single-nucleotide polymorphisms
  • the method of the invention comprises determining the level of T cells that are specific for the hepatitis virus and that produce TGF- ⁇ .
  • the first steps may be identical as those described above, by stimulating Peripheral Blood Mononuclear Cells (PBMC) obtained from the biological sample of the patient, with a hepatitis virus antigen, e.g. a virus protein, peptide, or a virus particle or vector.
  • PBMC Peripheral Blood Mononuclear Cells
  • the subset of cells that produce TGF- ⁇ is then quantified, e.g. by flow cytometry, e.g. by following a standard protocol of intracytoplasmic cytokine staining (see e.g. Prussin and Metcalfe, Journal of Immunological Methods, 1995, 188: 117-128).
  • assays of the invention analyze antibody staining of cells using flow cytometry. Such flow cytometry methods are well known and well- characterized in the art.
  • assays of the invention include the steps of applying an anti- TGF- ⁇ antibody to a population of cells and detecting whether the antibody is able to stain the cells.
  • Cells stained by the antibody express TGF- ⁇ on their surface. Detection of such stained cells can be accomplished using methods known in the art.
  • cells stained for surface TGF- ⁇ are detected using flow cytometry. It will be appreciated that stains for both membrane-bound and intracellular TGF- ⁇ can be utilized in accordance with the present invention.
  • Kits may be useful to perform such methods.
  • a kit for evaluating the outcome of an infection by a hepatitis virus i.e. for predicting whether a patient affected with a viral hepatitis will cure spontaneously, or whether a patient affected with a viral hepatitis will be responsive to an antiviral hepatitis treatment, comprising (i) means for determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis C virus and/or the level of T cells that are specific for the hepatitis virus and produce TGF- ⁇ , in a biological sample from the patient, and optionally (ii) instructions for use.
  • the means for determining the level of TGF- ⁇ produced by T cells that are specific for the hepatitis C virus and/or the level of T cells may include reagents, such as anti-TGF- ⁇ antibodies, or staining agents, optionally in combination with a hepatitis virus antigen, such as a hepatitis virus antigenic peptide.
  • Example 1 Lack of TGF- ⁇ production by Hepatitis C virus-specific T cells during HCV acute phase is associated with HCV clearance.
  • Acute HCV infection was prospectively diagnosed on liver enzymes increase during routine monitoring of HIV infection and confirmed by two consecutive positive HCV-RNA detections in patients previously HCVRNA or HCV-Ab-negative within the past 8 months. Patients were prospectively studied for 15 months from diagnosis. Patients who did not spontaneously clear HCV 3 months after diagnosis were proposed a treatment with pegylated-IFN-a-2a and ribavirin at standard dosages for 6 months. Rapid virologic response was defined when HCV RNA became undetectable after 4 weeks of treatment (RVR). Sustained virologic response (SVR) defined by a negative PCR 6 months (M) after treatment discontinuation.
  • PBMCs Peripheral blood mononuclear cells
  • Enzyme-linked immunospot (ELISPOT) assays were performed as described in Schnuriger, et al, 2009, AIDS, 23:2079-2089. Cryopreserved PBMCs (2*105 per well) were incubated in duplicates in ELISpot plates coated with anti-IFN-g antibody, with 10 ⁇ g/ml of HCV peptide pool (Core, Env1 , NS2, and NS4) in medium with 10% fetal calf serum for 20 h, in the presence or absence of blocking monoclonal antibodies (MAbs) or their isotype controls, anti-IL-10 and anti-TGF- ⁇ , -2, and -3 (clone DM) or immunoglobulin G1 (lgG1) and lgG2b isotype controls (R&D Systems, Minneapolis, MN) simultaneously added to the wells at the optimized concentration (10 ⁇ g/ml). Phytohemagglutinin (Sigma-Aldrich, St. Louis, MO;
  • SFC spot-forming cell
  • PBMCs Cryopreserved PBMCs were incubated overnight with 5 ⁇ g/ml of HCV peptide pool (Core, Env1 , NS2, and NS4), with brefeldin-A added 1 h later. Positive and negative controls were LPS, SEB, or PMA/lonomycine and medium alone.
  • Cells stimulated with core and NS2 and those stimulated with Env1 and NS4 were pooled together just before staining with anti- CD3/PB, anti-CD8/APC-CY7, anti-CD161/PE-CY5, anti-CD27/FITC (Becton-Dickinson/BD), anti-CD4/PE-CY7, and anti-CD45RA/ECD (Beckman Coulter/BC), anti-CCR7/APC or Alexa647 (R&D or BD), then fixed, permeabilized and stained with anti-IFN-g/ Alexa 700 (BD) and anti-TGF- ⁇ /PE (IQ Products).
  • TGF- ⁇ secreting HCV-specific T-cells are induced during acute HCV-HIV coinfection:
  • T cells producing one of those cytokines IL-10 or TGF- ⁇
  • IL-10 production was not detectable even after the control positive stimulation, we concentrated our effort on TGF- ⁇ production.
  • TGF- ⁇ production in T cells after stimulation with a pool of HCV core, NS2, NS4, and Env1 peptides together, but not against HIV P24 used as control.
  • TGF- ⁇ anti-HCV TGF- ⁇ producing T-cells in 9 out of the 23 tested patients (39 %).
  • the source of TGF- ⁇ production was both CD4+ and CD8+ T-cells, with median values 0.58 %
  • HCV-specific TGF- ⁇ - cells were detectable in 4 of the 4 patient's samples in whom anti-TGF- ⁇ + anti-IL-10 mAbs could restore IFN- ⁇ responses, and in 3 of the 6 patient's samples in whom no restoration was observed.
  • TGF- ⁇ - cells In order to determine the phenotype of the TGF- ⁇ - cells, we analysed the expression of T cell differentiation markers, and of CD161. We found that CD4+TGF ⁇ + cells displayed, half a central memory CM (CD45RA-CCR7+) 40% (IQR: 12-66) and half an effector memory EM (CD45RA-CCR7-) 46% (IQR: 26-82) phenotype. In contrast, CD8+TGF ⁇ + cells were mainly EM with median expression 67% (IQR: 63-72). CD4+ and CD8+TGF ⁇ + cells were both CD161-, with median proportion of 76% (IQR: 67-87), and 76% (IQR: 72-84) in CD4+ and CD8+TGF-3+ cells, respectively.
  • TGF- ⁇ production in 22 patients at advanced time points of follow up treated patients were examined post treatment at M12 and M15 (except for two patients at M1 and M5 of treatment), untreated patients were re-examined between M9 and M12 of follow-up.
  • TGF- ⁇ producing T cells remained undetectable in the 13 RE patients, except in patient 6006 with TGF- ⁇ producing cells maintained even after eradicating the virus. With other view, TGF- ⁇ producing T cells were undetectable in 14 patients at baseline and remained so within follow-up, except in two VI patients in whom these cells became detectable at M 12 for 6204 and M1 of treatment for 6206.
  • TGF- ⁇ - cells phenotype the profile did not change a lot.
  • the median expression was 39% (IQR: 24-50) for CM, and 39% (IQR: 29-62) for EM.
  • the median expression of EM cells was 58% (IQR: 34-66), with an augmentation of effector phenotype Eff (CD45RA+CCR7-), 24% (IQR: 16-31).
  • TGF- ⁇ + cells remained predominantly of CD161- phenotype.
  • Table-1 Patients' characteristics:
  • HCV-specific TGF-B+ cells HCV-specific TGF-B+ cells
  • Groupe 1 ID Treatment Outcome Time point %CD4+TGF-B+ %CD8+TGF-B+ point %CD4+TGF-B+ %CD8+TGF-B+
  • N non responder to treatment.
  • Example 2 Lack of influence of the IL-28b gene polymorphism on the predictive value of the HCV-specific TGF- ⁇ production by T cells in acute hepatitis
  • TGF- ⁇ assay alone appears as the most potent predictor of recovery, whether it is spontaneous or treatment induced.
  • Example 3 TGFp producing anti-HCV responses in Chronic Hepatitis C infected and HIV coinfected patients
  • the inventors have then analysed the frequency of TGF- ⁇ producing T cells in response to core, NS2 or NS4 during chronic phase of HCV infection in patients coinfected with HIV. The inventors have further determined the impact of those cells on the progress of the infection, HCV viral load and liver disease stage. The same analysis was performed in monoinfected patients in HCV chronic phase to assess the impact of HIV on the frequency of TGF- ⁇ producing T cells.
  • Median CD4 T cell rate was 448/mm3 (IQR: 373-710). All these patients were treated for HIV infection.
  • Median HIV viral load was 20 copies/mL (IQR: 20-20).
  • Median ALAT rate was 51 Ul/L (IQR: 36-109).
  • HCV genotype was mainly genotype 1 (10 patients) and genotype 4 (10 patients). Genotype 3 was observed in 2 patients, genotype 2 in 1 patient and genotype 6 in a last patient.
  • the stage of the liver disease after a median infection period of 17 was mostly moderate fibrosis (1 1/24 patients) and cirrhosis (9/24 patients), 1 patient only had a minimal fibrosis and 3 patients had a severe fibrosis.
  • Table 3 patients' characteristics patients HCV/HIV
  • Period of infection (years) median 17 (IQR: 6-24)
  • CD4/mm3 median 448 (IQR: 373-710) VL HIV copies/mL median 20 (IQR: 20-20)
  • VL HCV lU/mL log median 5,9 (IQR: 5,9-6,61)
  • the inventors have analysed the frequency of TGF- ⁇ producing CD4 and CD8 T cells in response to a stimulation with 3 HCV peptide pools (core, NS2, and NS4).
  • Anti HCV T cells were detected at rate in the group of patients (frequency was considered positive when superior to 0.1 for CD4+TGF ⁇ +, and superior to 0.12 for CD8+TGF ⁇ +).
  • Anti-HCV T cells with production of TGF- ⁇ were detected against at least 1 peptide pool peptidique in 7/24 patients coinfected (29%) (Table 4).
  • TGF- ⁇ producing T cells were CD4+ and/or CD8+.
  • Medians of percentages in anti-NS4 T cells are : 0.2 (Range: 0.04-0.27) for CD4+TGF ⁇ + T cells, and 0.1 (Range: 0-0.22) for T CDS+TGF- ⁇ + ⁇ cells . All medians are shown in Table 4.
  • Frequency of anti-HCV TGF- ⁇ producing T cells was independent of CD4 T cellcounts , HIV viral load or HCV viral load, and HCV genotype.
  • the inventors have observed that the patients with anti-HCV TGF- ⁇ producing T cells tend to be at a more advanced stage of the liver disease since Cirrhosis and severe fibrosis were observed in 6/7 (86%) patients with TGF- ⁇ - cells, versus 6/17 (35%) in patients with no such cells (Figure 2).
  • Table 4 anti-HCV TGF- ⁇ producing T cells in patients coinfected with HIV and HCV, (after substracting background noise).
  • NT non tested. Percentages in bold are considered positive.

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Abstract

L'invention concerne un procédé in vitro permettant de prévoir si un patient atteint d'une hépatite virale guérira spontanément, ou s'il réagira à un traitement antiviral contre l'hépatite ; ledit procédé consistant à déterminer le niveau de TGF-⊐β produit par des cellules T qui sont spécifiques au virus de l'hépatite et/ou le niveau des cellules T qui sont spécifiques au virus de l'hépatite et qui produisent TGF-⊐β, dans un échantillon prélevé sur le patient.
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