WO2018229733A1 - Immuno-biomarqueurs distinguant la réactivité de la non-réactivité pendant des traitements immunothérapeutiques - Google Patents

Immuno-biomarqueurs distinguant la réactivité de la non-réactivité pendant des traitements immunothérapeutiques Download PDF

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WO2018229733A1
WO2018229733A1 PCT/IB2018/054463 IB2018054463W WO2018229733A1 WO 2018229733 A1 WO2018229733 A1 WO 2018229733A1 IB 2018054463 W IB2018054463 W IB 2018054463W WO 2018229733 A1 WO2018229733 A1 WO 2018229733A1
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vaccine
biomarkers
immuno
combination
patients
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Bin Wang
Gan Zhao
Xuan Zhou
Weidong Zhao
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BEIJING ADVACCINE BIOTECHNOLOGY Co Ltd
Fudan University
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BEIJING ADVACCINE BIOTECHNOLOGY Co Ltd
Fudan University
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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
    • G01N33/5761—Hepatitis B
    • 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
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • 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
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]

Definitions

  • the present invention relates to the field of biomedicine, in particular to Immuno-biomarkers, their uses and methods for distinguishing a patient who is responsive to a vaccine from a non-responsive one during immunotherapeutic treatments of chronic hepatitis B patients.
  • HBV hepatitis B virus
  • HBV specific T cells are deleted, dysfunctional or become exhausted in chronic hepatitis B (CHB) patients.
  • CHB chronic hepatitis B
  • Several immunotherapeutic approaches have been developed to restore the T cell functions [4].
  • Wen et al. have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex with alum adjuvant as a therapeutic vaccine (YIC), aiming to enhance the uptake of HBsAg-Ab complex by antigen presenting cells (APC) through their Fc receptors [5, 6].
  • APC antigen presenting cells
  • efficacy has been demonstrated among CHB patients [7, 8].
  • the YIC has completed a phase Ilia clinical trial (trial number: 2002L0038), and a phase 1Mb trial is underway [9].
  • cytokines and chemokines were examined the expression of cytokines and chemokines in the plasma.
  • Applicants used a Boolean logic method to analyze the detection of multiple cytokines/chemokines, such as a combination of IL-10, IL-33 and MIP-1a, a combination of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 and a combination of IL- 13, IFN-a, and IL-18.
  • the method differentiated seroconverted from non-converted YlC-immunized subjects.
  • the invention provides immuno-biomarkers and their uses, discloses a method for distinguishing HBeAg Seroconverted from non-converted Individuals in chronic hepatitis B patients, especially for those treated with a vaccine such as an antigen-antibody complex therapeutic vaccine.
  • a vaccine such as an antigen-antibody complex therapeutic vaccine.
  • the invention provides a combination of biomarkers comprising a plurality of immuno-biomarkers associated with immune function that differentiates a responding patient from a non-responding one against a vaccine.
  • the immuno-biomarkers associated with immune function are three or more biomarkers selected from the group consisting of MIP-1 a, SDF-1 a, IL-27, IL- ⁇ , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, IL-11 , Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL-35, IL-1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG, Gran
  • the vaccine is a hepatitis B vaccine.
  • the vaccine is a chronic hepatitis B vaccine.
  • the vaccine is an HBsAg-HBIG therapeutic vaccine (YIC).
  • the vaccine is a hepatitis B vaccine in combination of GM-CSF.
  • the immuno-biomarkers when the vaccine is an HBsAg-HBIG therapeutic vaccine (YIC), the immuno-biomarkers comprise IL-10, IL-33 and MIP-1 a. More preferably, the immuno-biomarkers consist of IL-10, IL-33 and MIP-1a.
  • the immuno-biomarkers when the vaccine is a hepatitis B vaccine in combination of GM-CSF, the immuno-biomarkers either comprise IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 or comprise IL-13, IFN-a, and IL-18. More preferably, the immuno-biomarkers either consist of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 or consist of IL-13, IFN-a, and IL-18.
  • the combination of biomarkers can differentiate a responding patient from a non-responding one against the vaccine.
  • the combination of biomarkers can differentiate a responding patient from a non-responding one against the vaccine before the vaccination.
  • the combination of biomarkers can differentiate a responding patient from a non-responding one against the vaccine during the vaccination.
  • the present invention reveals a method for evaluating efficacy of a vaccine on a patient.
  • the method comprises the steps of a) obtaining a biological sample of each of the patients including the patient from a patient pool including both seroconverted patients and non-seroconverted ones; b) quantifying concentrations of a combination of immuno-biomarkers of claim 1 in the biological sample for each of the patients; c) generating a profile of the immuno- biomarkers from the concentrations of the immuno-biomarkers of each of the patients by using a model; d) determining in the profile whether each of the immuno- biomarkers of each of the patients is expressed or not expressed, wherein one immuno-biomarker of each of the patients is expressed if its concentration is above a cut-off concentration and otherwise it is unexpressed; e) setting every possible combination of Boolean states as a criterion to distinguish converted patients from non-converted; and f) classifying the patient as a converted one (or a responding one
  • the vaccine is a hepatitis B vaccine.
  • the vaccine is a chronic hepatitis B vaccine.
  • the vaccine is an HBsAg-HBIG therapeutic vaccine (YIC).
  • the vaccine is a hepatitis B vaccine in combination of GM-CSF.
  • the model is a Boolean modeling.
  • the cut-off concentration is 0.1 pg/ml.
  • the immuno-biomarkers when the vaccine is an HBsAg- HBIG therapeutic vaccine (YIC), the immuno-biomarkers comprise IL-10, IL-33 and MIP-1 a. More preferably, the immuno-biomarkers consist of IL-10, IL-33 and M IP- la.
  • the immuno-biomarkers when the vaccine is a hepatitis B vaccine in combination of GM-CSF, the immuno-biomarkers either comprise IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 or comprise IL-13, IFN-a, and IL-18. More preferably, the immuno-biomarkers either consist of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 or consist of IL-13, IFN-a, and IL-18.
  • the biological sample is obtained from the patient before vaccination by the vaccine.
  • the biological sample is obtained from the patient during vaccination by the vaccine.
  • the biological sample comprises cerebrospinal fluid, whole blood, blood serum, plasma, urine, saliva, other bodily fluid, breath, condensed breath, or an extract, purification, or dilution of any of these.
  • the quantifying step may use a technique comprising llluminex, flow cytometry, Chemiluminescence, Electroluminescence, Photoluminescence, Bioluminescence, ELISA, CyTOF, or combinations thereof.
  • the quantifying step uses a technique comprising SELDI (-TOF) spectrometry, MALDI (-TOF) spectrometry, a 1-D gel-based analysis, a 2-D gel-based analysis, mass spectrometry (MS), liquid chromatography (LC), reverse phase liquid chromatography (RP-LC), size permeation chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography, FPLC, HPLC, UPLC, other LC-based techniques, other LC-MS-based technique, or combinations thereof.
  • SELDI SELDI
  • MALDI MALDI
  • the quantifying step uses a method comprising an immunological method, a biosensor method, a microanalytical method, a microengineered method, a microseparation method, an immunochromatography method, or combinations thereof.
  • the present invention reveals a kit for differentiating a responding patient from a non-responding one against a vaccine.
  • the kit comprises a biosensor capable of detecting and/or quantifying the combination of biomarkers as defined in this application.
  • FIG. 1 (a) and FIG. 1 (b) are a set of graphs showing a baseline expression of 14 cytokines/chemokines in the HBeAg seroconverted (FIG. 1 (a)) and non-seroconverted (FIG. 1 (b)) patients.
  • Heat map images are shown for I L- ⁇ , IFN- a, TNF-a, IFN- ⁇ , IL-2, IL-7, IL-15, IL-21 , IL-33, IL-10, MIG, IP-10, MIP-1a, ⁇ -1 ⁇ in pretreatment plasmas from 44 HBeAg seroconverted patients and 49 HBeAg non- seroconverted.
  • FIG. 2(a), FIG. 2(b), FIG. 2(c), FIG. 2(d), FIG. 2(e), FIG. 2(f) and FIG. 2(g) are a systematic diagram showing a scheme of Boolean modeling to distinguish HBeAg seroconverted patients from the non-converted ones based on the baseline immune status.
  • FIG. 2(a) Each patient has a cytokine/chemokine expressing profile measured by the Luminex system.
  • FIG. 2(c) The immune status depicted by means of a series of 0/1 s are assembled for all 93 CHB patients and a Boolean state pool is generated.
  • FIG. 2(d) Patients who had HBeAg-seroconverted after the 76-week YIC treatments are represented with "+", and non-seroconverted ones are represented with A Boolean state is considered as a unique immune characteristic state.
  • FIG. 1 The immune status depicted by means of a series of 0/1 s are assembled for all 93 CHB patients and a Boolean state pool is generated.
  • FIG. 2(d) Patients who had HBeAg-seroconverted after the 76-week YIC treatments are represented with "+", and
  • FIG. 2(f) The numbers of HBeAg seroconverted and non-converted patients predicted by these combinations are counted.
  • FIG. 2(g) The optimal combinational pattern used to differentiate converted patients from non-converted ones is based on the sensitivity and specificity according to the formulas described.
  • FIG. 3(a), FIG. 3(b), FIG. 3(c) and FIG. 3(d) are a set of graphs showing virological changes in the actual and predicted HBeAg converted and in the actual and predicted non-converted groups during the 76-week YIC phase III clinical trial.
  • the predicted groups were distinguished by the cytokine combination analysis.
  • the changes in plasma levels of HBeAg (FIG. 3(a)), HBV DNA (FIG. 3(b)), HBsAg (FIG. 3(c)) and HBe Ab (FIG. 3(d)) are shown.
  • FIG. 4(a), FIG. 4(b), FIG. 4(c) and FIG. 4(d) are a set of graphs showing that Boolean Model gives the smallest 2-factor ROC for the predicted endpoint.
  • FIG. 4(a) Boolean flow diagram, IL-10 and MIG at week 0 predicted sAg seroconversion
  • FIG. 4(b) IL-13 and GRO-a at week 0 predicted sAg decreasing
  • FIG. 4(c) IL-23 and MIP-1 b at weeks 12 predicted sAg seroconversion
  • FIG. 4(d) IL-18 and IFN-a at weeks 12 predicted sAg decreasing.
  • FIG. 5(a) and FIG. 5(b) are a set of graphs showing dynamic variation of Jmax for multiple factorial model.
  • FIG. 5(a) Jmax of 4-8 factors random combinations.
  • FIG. 5(b) Jmax of 3 factor random combination.
  • FIG. 6(a), FIG. 6(b), FIG. 6(c) and FIG. 6(d) are a set of graphs showing the comparison result of virological data of converted patients with the multiple-factor predicted data by the combination of IL-10, IL-13, IL-23, IFN-a, IL-18, and MIG.
  • Actual sAg concentration dynamic changes of converted patients compared with predicted data (FIG. 6(a)) with week 0 as starting point and sAg seroconversion as end point;
  • FIG. 6(b) with week 0 as starting point and sAg decreasing as end point;
  • FIG. 6(c) with week 12 as starting point and sAg seroconversion as end point;
  • FIG. 6(d) with week 12 as starting point and sAg decreasing as end point.
  • FIG. 7(a), FIG. 7(b), FIG. 7(c) and FIG. 7(d) are a set of graphs showing the comparison result of virological data of non-converted patients with IL- 13, IFN-a, and IL-18 multiple-factor predicted data.
  • Actual sAg concentration dynamic changes of non-converted patients compared with predicted data (FIG. 7(a)) with week 0 as starting point and sAg seroconversion as end point;
  • FIG. 7(b) with week 0 as starting point and sAg decreasing as end point;
  • FIG. 7(c) with week 12 as starting point and sAg seroconversion as end point;
  • FIG. 8 is a set of graphs showing the receiver operating characteristic curves of TNF-a, MIP-1a, ⁇ -1 ⁇ , MIG, and IP-10.
  • Receiver operating characteristic (ROC) curves were performed for TNF-a (AUC, 0.604), ⁇ -1 ⁇ (AUC, 0.559), IP-10 (AUC, 0.478), MIG (AUC, 0.454), and MIP-1a (AUC, 0.565).
  • AUC areas under the curves.
  • FIG. 9(a), FIG. 9(b) and FIG. 9(c) are a set of diagrams showing the selection of final prediction biomarkers from 14 cytokines/chemokines.
  • FIG. 9(a) Among 14 cytokines/chemokines, TNF-a, ⁇ -1 ⁇ , MIG and IP-10 were highly expressed in almost all CHB patients, did not contribute to discrimination between converted and non-converted patients, and were excluded from subsequent analysis.
  • FIG. 9(b) The remaining 10 factors were set as Boolean nodes, representing the immune status before YIC treatments. However, seven of these factors made only minor contribution towards discrimination in the combined analysis.
  • FIG. 9(c) In contrast, IL-10, IL-33 and MIP-1a, singly and in combination, were strongly associated with HBeAg seroconversion after YIC treatments.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
  • biomarker refers to a specific substance or biochemical in the body that has a particular molecular feature to make it useful for diagnosing and measuring the progress of disease or the effects of treatment.
  • immuno-biomarker refers to a specific substance or biochemical in the body that has a particular molecular feature to make it useful for diagnosing and measuring the progress of immuno-response of a patient with a disease or during a treatment.
  • sample refers to any animal tissue or fluid containing, e.g., polynucleotides, polypeptides, antibodies, metabolites, and the like, including cells and other tissue containing DNA and RNA. Examples include adipose, blood, cartilage, connective, epithelial, lymphoid, muscle, nervous, sputum, and the like.
  • a sample may be solid or liquid and may be DNA, RNA, cDNA, bodily fluids such as blood or urine, cells, cell preparations or soluble fractions or media aliquots thereof, chromosomes, organelles, and the like.
  • biological sample refers to any sample from a patient for diagnosis and analysis, and the non-limiting examples comprise cerebrospinal fluid, whole blood, blood serum, plasma, urine, saliva, other bodily fluid, breath, condensed breath, or an extract, purification, or dilution of any of these.
  • animal refers to a human or other animal, including avian, bovine, canine, equine, feline, hircine, murine, ovine, and porcine animals.
  • the animals that are compared are animals of the same species and possibly of the same race or breed.
  • a "companion animal” is any domesticated animal, and includes, without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like.
  • the animal is a human or a companion animal such as a canine or feline.
  • 3x refers to a frequency of administration as three times.
  • 3x GM-CSF means GM-CSF is administrated three times.
  • composition refers to any chemical or biological compound or substance, or a mixture or combination of two or more such compounds or substances, intended for use in the medical diagnosis, cure, treatment, or prevention of disease or pathology.
  • hepatitis B virus or "HBV,” as used herein, is used to describe the virus (serum hepatitis virus) which produces viral hepatitis type B in humans. This is a viral disease with a long incubation period (about 50 to 160 days) in contrast to Hepatitis A virus (infectious hepatitis virus) which has a short incubation period.
  • the virus is usually transmitted by injection of infected blood or blood derivatives or merely by use of contaminated needles, lancets or other instruments. Clinically and pathologically, the disease is similar to viral hepatitis type A; however, there is no cross-protective immunity.
  • Viral antigen (HBAg) is found in the serum after infection.
  • the term "seroconversion,” as used herein, refers to a time period during which a specific antibody develops and becomes detectable in the blood. After seroconversion has occurred, the disease can be detected in blood tests for the antibody. During an infection or immunization, antigens enter the blood, and the immune system begins to produce antibodies in response. Before seroconversion, the antigen itself may or may not be detectable, but the antibody is, by definition, absent. During seroconversion, the antibody is present but not yet detectable. Any time after seroconversion, the antibodies can be detected in the blood, indicating a prior or current infection.
  • HBeAg seroconversion is often used as an important milestone in the natural history of chronic hepatitis B virus, usually accompanied by clinical hepatitis disease remission and a good prognosis. Thus, the clinical doctors and researchers often turn to "whether patients HBeAg seroconversion after treatment" to determine whether treatment is effective as of the main indicators.
  • the inventive combination of immuno-biomarkers or the related methods can be used to predict efficacy of a vaccine such as a HBV vaccine by distinguishing a seroconverted patient from a non-converted one during a therapeutic vaccine treatment.
  • a vaccine such as a HBV vaccine
  • the combination of immuno-biomarkers or the related methods can be used to predict efficacy of a vaccine such as a HBV vaccine by distinguishing a seroconverted patient from a non-converted one during a therapeutic vaccine treatment.
  • a vaccine such as a HBV vaccine
  • the treatment or prevention followed by the inventive combination of immuno-biomarkers or related methods can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the cancer can be any cancer, including any of the cancers associated with any of the tumor antigens described herein.
  • mammal refers to a warm-blooded vertebrate animal such as a human, dog or cat or the like.
  • mammal includes rodents such as rodent, rabbit, feline, canine, swine, and cattle.
  • the term "vaccine,” as used herein, refers to a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in a production of antibodies or simply in the activation of certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes.
  • the vaccine composition can be a composition for prophylactic purposes or for therapeutic purposes, or both.
  • the vaccine is a hepatitis B vaccine. In one preferred embodiment, the vaccine is a chronic hepatitis B vaccine.
  • the present invention reveals a combination of immuno-biomarkers that can be used to differentiate a responding patient against a hepatitis B vaccine or a chronic hepatitis B vaccine from a non-responding patient against the same vaccine.
  • the combination of immuno- biomarkers of the present invention can be used wither before or during vaccination by the hepatitis B vaccine or chronic hepatitis B vaccine.
  • the terms "administer” or “administration,” as used herein, refers to their usual and ordinary meaning in the art of treating a patient with a substance such as a vaccine or a composition.
  • co-administration and “concomitant administration” as used herein are synonymous and refer to administering two substances or two compositions to a patient in such a manner and with such timing that both substances, or both compositions, reside in the patient's body at the same time.
  • the co-administration may be simultaneous or sequential in time, and the coadministered substances or compositions may be administered to a patient at the same time, or separately but near in time, or on the same day, or otherwise in a way that results in substantial overlap of the residence periods for the respective substances or compositions in the body.
  • the administration e.g., parenteral administration, may include subcutaneous administration, intramuscular administration, transcutaneous administration, intradermal administration, intraperitoneal administration, intraocular administration, intranasal administration and intravenous administration.
  • the vaccine or the composition according to the invention may be administered to an individual according to methods known in the art. Such methods comprise application e.g. parenterally, such as through all routes of injection into or through the skin: e.g., intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal, or subcutaneous. Also, the vaccine may be applied by topical application as a drop, spray, gel or ointment to the mucosal epithelium of the eye, nose, mouth, anus, or vagina, or onto the epidermis of the outer skin at any part of the body. Other possible routes of application are by spray, aerosol, or powder application through inhalation via the respiratory tract.
  • parenterally such as through all routes of injection into or through the skin: e.g., intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal, or subcutaneous.
  • the vaccine may be applied by topical application as a drop, spray
  • the particle size that is used will determine how deep the particles will penetrate into the respiratory tract.
  • application may be via the alimentary route, by combining with the food, feed or drinking water e.g. as a powder, a liquid, or tablet, or by administration directly into the mouth as a: liquid, a gel, a tablet, or a capsule, or to the anus as a suppository.
  • a combination of biomarkers comprising a plurality of immuno- biomarkers associated with immune function that differentiates a responding patient from a non-responding one against a vaccine.
  • the combination of biomarkers comprising a plurality of immuno-biomarkers associated with immune function works best against a hepatitis B vaccine, more preferably against a chronic hepatitis B vaccine.
  • the combination of biomarkers may include a plurality of different immuno-biomarkers.
  • the combination of biomarkers may include a plurality of different immuno-biomarkers selected from the group consisting of M IP- la, SDF-1a, IL-27, IL- ⁇ , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, IL-11 , Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL-35, IL-1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1 a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG, Granulocyte-colony stimulating factor (G-CSF or GCSF), C-X-C motif chemokine receptor 1 (CXCR1), CXCR2, CXCR3, C
  • the combination of biomarkers may include three or more different immuno-biomarkers selected from the group consisting of MIP-1 a, SDF-1 a, IL-27, I L- 1 , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, IL-1 1 , Eotaxin, IL- 12p70, IL-13, IL-17A, IL-31 , IL-35, IL-1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1 a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG, Granulocyte-colony stimulating factor (G-CSF or GCSF), C-X-C motif chemokine receptor 1 (CXCR1), CXCR2, CXCR
  • the combination of biomarkers may include IL-33, IL-10, and MIP-1 a.
  • the combination of biomarkers consists to IL-33, IL-10, and MIP-1 a.
  • the combination of biomarkers may include IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 for predicting sAg seroconversion and sAg decreasing.
  • the combination of biomarkers consists of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 for predicting sAg seroconversion and sAg decreasing.
  • the combination of biomarkers may include IL-13, IFN-a, and IL-18 to predict non sAg seroconversion and non-sAg decreasing.
  • the combination of biomarkers consists of IL-13, IFN-a, and IL-18 to predict non sAg seroconversion and non-sAg decreasing.
  • the present invention is applicable to any hepatitis B vaccine or any combination of vaccine with additional components (e.g., immune enhancers).
  • the combination of biomarkers may include a plurality of different immuno-biomarkers when a different hepatitis B vaccine or a different chronic hepatitis B vaccine is used.
  • the combination of biomarkers may include a plurality of different immuno-biomarkers selected from the group consisting of MIP-1a, SDF-1a, IL-27, I L- ⁇ , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, IL-11 , Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL-35, IL- 1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1 a, IL-23, IL-15, IL-18, IL-21 , IL
  • the objective of the invention is to predict efficacy of a vaccine (e.g., any known HBV vaccine) against chronic hepatitis B and to distinguish a seroconverted patient from a non-converted one before or during a therapeutic vaccine treatment.
  • a vaccine e.g., any known HBV vaccine
  • a patient is determined to be non- responding to a specific chronic hepatitis B vaccine, one could further provide the patient a different chronic hepatitis B vaccine or related immunotherapeutic pharmaceutical compositions to treat chronic hepatitis B or prevent the patient from chronic hepatitis B.
  • hepatitis B vaccine or chronic hepatitis B vaccine may be used to treat chronic hepatitis B or prevent the patient from chronic hepatitis B.
  • Applicants use an HBsAg-HBIG therapeutic vaccine (YIC) as an exemplary hepatitis B vaccine (See Example 1).
  • YIC HBsAg-HBIG therapeutic vaccine
  • Applicants use GM-CSF or a hepatitis B vaccine along with GM-CSF as exemplary hepatitis B vaccines (See Example 2).
  • Applicants use chronic hepatitis B virus as an example. Applicants envision that the methods of the present invention may be used to predict efficacy of other vaccine for the treatment of many diseases or viruses such as herpes virus, HPV, HIV, Merkel cell virus, influenza virus and RSV.
  • diseases or viruses such as herpes virus, HPV, HIV, Merkel cell virus, influenza virus and RSV.
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function comprising three or more biomarkers selected from the group consisting of MIP-1a, SDF-1a, IL-27, I L- ⁇ , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, IL-11 , Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL-35, IL- 1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1 a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG, Granulocyte-colony stimulating factor (G-CSF or GCSF), C-X-C motif chemokine receptor 1 (CXCR1), C
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function comprising three or more biomarkers selected from the group consisting of IL- ⁇ , IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , IL-2, IL-7, IL-15, IL- 21 , IL-33, IL-10, MIG, IP-10, MIP-1a and ⁇ -1 ⁇ .
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function comprise IL-33, IL-10, and MIP-1 a.
  • the immuno- biomarkers of the combination of the biomarkers associated with immune function consists of IL-33, IL-10, and MIP-1a.
  • Example 1 shows the methods of identifying the immuno-biomarkers of IL-33, IL-10, and MIP-1a as the decisive factors associated with responsiveness among CHB patients before and during YIC treatment.
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function comprises IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6.
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function consists of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6.
  • the combination of the biomarkers of IL- 10, IL-13, IL-23, IFN-a, IL-18 and MIG6 may be used to predict HBsAg seroconversion and decreasing.
  • Example 2 shows the methods of identifying the immuno-biomarkers of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 as the decisive factors associated with responsiveness among CHB patients before and during the treatment of a hepatitis B vaccine and GM-CSF.
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function comprises IL-13, IFN-a, and IL-18 to predict non-HBsAg seroconversion and non-decreasing.
  • the immuno-biomarkers of the combination of the biomarkers associated with immune function consists of IL-13, IFN-a, and IL-18.
  • the combination of the biomarkers of IL- 13, IFN-a, and IL-18 may be used to predict non-HBsAg seroconversion and non- HBsAg decreasing.
  • Example 2 shows the methods of identifying the immuno- biomarkers of IL-13, IFN-a, and IL-18 as the decisive factors associated with responsiveness among CHB patients before and during the treatment of a hepatitis B vaccine and GM-CSF.
  • the combination of biomarkers comprising a plurality of immuno-biomarkers associated with immune function can differentiate a responding patient from a non-responding one against a hepatitis B vaccine.
  • the combination of biomarkers comprising a plurality of immuno- biomarkers associated with immune function can distinguish a seroconverted patient from a non-converted one before or during a therapeutic vaccine treatment.
  • the combination of biomarkers comprising a plurality of immuno-biomarkers associated with immune function can distinguish a responding patient from a non-responding one against a chronic hepatitis B vaccine.
  • the combination of biomarkers comprising a plurality of immuno- biomarkers associated with immune function can distinguish a seroconverted patient from a non-converted one before or during a therapeutic vaccine treatment by using a chronic hepatitis B vaccine.
  • the vaccine is an HBsAg-HBIG therapeutic vaccine (YIC).
  • YIC HBsAg-HBIG therapeutic vaccine
  • Example 1 shows the prediction and differentiation of a responding patient from a non-responding one by using the combination of biomarkers before or during vaccination of an HBsAg-HBIG therapeutic vaccine (YIC).
  • the combination of biomarkers can differentiate a responding patient from a non-responding one against the vaccine before the vaccination.
  • the combination of biomarkers can predict efficacy of a vaccine before the vaccine is administered to a patient.
  • the combination of biomarkers can differentiate a responding patient from a non-responding one against a vaccine during the vaccination.
  • the combination of biomarkers can provide critical information of efficacy of different vaccines on a patient so that one could better design a scheme for treating the patient.
  • the present invention reveals a method for evaluating efficacy of a vaccine on a patient. Specifically, the present invention reveals a method for evaluating efficacy of a chronic hepatitis B vaccine on a patient.
  • the method comprises the steps of a) obtaining a biological sample of each of the patients including the patient from a patient pool including both seroconverted patients and non-seroconverted ones; b) quantifying concentrations of a combination of immuno-biomarkers of claim 1 in the biological sample for each of the patients; c) generating a profile of the immuno-biomarkers from the concentrations of the immuno-biomarkers of each of the patients by using a model; d) determining in the profile whether each of the immuno-biomarkers of each of the patients is expressed or not expressed, wherein one immuno-biomarker of each of the patients is expressed if its concentration is above a cut-off concentration and otherwise it is unexpressed; e) setting every possible combination of Boolean states as a criterion to distinguish converted patients from non-converted; and f) classifying the patient as a converted one (or a responding one) to the vaccine if the patient's Boolean state consists with the criterion
  • a biological sample from each of the patients including the patient from a patient pool including both seroconverted patients and non-seroconverted ones is obtained.
  • the non-limiting examples may include cerebrospinal fluid, whole blood, blood serum, plasma, urine, saliva, other bodily fluid, breath, condensed breath, or an extract, purification, or dilution of any of these.
  • concentrations of a combination of immuno-biomarkers as discussed above in the biological sample are quantified for each of the patients.
  • the step of quantification may use any known technique such as llluminex, flow cytometry, Chemiluminescence, Electroluminescence, Photoluminescence, Bioluminescence, ELISA, CyTOF, or combinations thereof.
  • the step of quantification may use any known technique and non-limiting examples of the techniques may comprise SELDI (-TOF) spectrometry, MALDI (-TOF) spectrometry, a 1-D gel-based analysis, a 2-D gel- based analysis, mass spectrometry (MS), liquid chromatography (LC), reverse phase liquid chromatography (RP-LC), size permeation chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography, FPLC, HPLC, UPLC, other LC-based techniques, other LC-MS-based technique, or combinations thereof.
  • SELDI SELDI
  • MALDI MALDI
  • MS mass spectrometry
  • LC liquid chromatography
  • RP-LC reverse phase liquid chromatography
  • size permeation chromatography gel filtration chromatography
  • ion exchange chromatography affinity chromatography
  • FPLC FPLC
  • HPLC HPLC
  • UPLC other LC-based techniques
  • the quantifying step may use any known method of diagnosis and/or analysis.
  • the non-limiting examples may comprise an immunological method, a biosensor method, a microanalytical method, a microengineered method, a microseparation method, an immunochromatography method, or combinations thereof.
  • the concentration of each of the immuno-biomarkers is quantified for each of the patients.
  • the concentrations of the following immuno-biomarkers may be quantified for each of the patients: MIP-1a, SDF-1a, IL-27, ⁇ _-1 ⁇ , IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL- 10, IL-1 1 , Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL-35, IL-1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL-9, TNF- ⁇ , GRO-alpha, IL-1a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG, Granulocyte-colony stimulating factor (G-CSF or GCSF), C-X
  • a profile of the immuno-biomarkers is generated from the concentrations of the immuno-biomarkers of all the patients by using a model.
  • the model is Boolean modeling. It is determined in the profile whether each of the immuno-biomarkers of each of the patients is expressed or not expressed, wherein one immuno- biomarker of each of the patients is expressed if its concentration is above a cut-off concentration and otherwise it is unexpressed.
  • Example 1 provides detail information of Boolean modeling.
  • a Boolean model may be adapted to construct the correlation between immune biomarkers and final seroconverstion.
  • expression data of immune biomarkers may be first transformed into 1 (Expressed) or 0 (Not expressed) by comparing their concentrations with a cut-off concentration.
  • the cut-off concentration is the detection limit of the system used. In one specification, the cut-off concentration is 0.1 pg/ml, which is the detection limit of the Luminex system.
  • Boolean state of the biomarkers was generated from each patient as a serial of 1/0s to form a Boolean state pool. Even further, a Boolean states pool was generated for all the patients including the HBeAg-seroconverted and non-converted ones.
  • each patient's position on the Boolean state pool is rearranged to achieve better separation between the seroconverted ones and non-converted ones. Every possible combination of Boolean states is set as a criterion to distinguish converted patients from non- converted.
  • ROC receiver operating characteristic curves
  • ROC performance may be analyzed with a commercial software such as Origin 8.0 graphing software.
  • the patient may be classifies as a converted one (or a responding one) to the vaccine if the patient's Boolean state consists with the criterion state and as a non-converted one (or a non-responding one) otherwise.
  • the patients with Boolean states that were not consistent with the criterion states may be classified as non-converted patients.
  • the present method also includes the step of grouping the patients according to multiple expression statuses of immuno-biomarkers, wherein the expression statuses include expression of each of the immuno-biomarkers, expression of any two of the immuno-biomarkers, expression of any three of the immuno-biomarkers, . . . and expression of all the immuno-biomarkers; and the step of correlating different groups of expression status with whether the corresponding patients are converted or non-converted to determine sensitivity and specificity of the predication.
  • Example 1 provides detailed information of the steps of grouping and correlating.
  • a specific combination of immuno-biomarkers may be chosen for each of the vaccine.
  • Example 1 the specific combination of immuno-biomarkers of IL-33, IL-10, and MIP-1a is chosen when YIC is used as a vaccine. Further, as shown in Example 2, the specific combination of immuno- biomarkers of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 may be used to predict HBsAg seroconversion and decreasing before and during the treatment of a hepatitis B vaccine and 3x GM-CSF.
  • Example 2 the specific combination of immuno-biomarkers of IL-13, IFN-a, and IL-18 to predict non- sAg seroconversion and non-sAg decreasing before and during the treatment of a hepatitis B vaccine and 3x GM-CSF.
  • the present invention provides methods of identifying a specific combination of immuno-biomarkers against a vaccine such as a chronic hepatitis B vaccine to evaluate efficacy of the vaccine on a patient. Further, the present invention provides a specific combination of immuno-biomarkers against a vaccine such as a chronic hepatitis B vaccine and related methods to distinguish a patient's responsiveness versus non-responsiveness during immunotherapeutic treatments of chronic hepatitis B. Specifically, the specific combination of immuno-biomarkers identified in the present invention allows one to distinguish a seroconverted patient from a non-converted one before or during a therapeutic vaccine treatment.
  • the cut-off concentration is the detection limit of the system used. In one specification, the cut-off concentration is 0.1 pg/ml, which is the detection limit of the Luminex system.
  • a patient is classified as a responding one to the related hepatitis B vaccine provided that the patient's Boolean state consists with any of the criterion states and as a non-converted one (or a non-responding one) otherwise.
  • the patient's Boolean state consists with any of the criterion states and as a non-converted one (or a non-responding one) otherwise.
  • the patient's Boolean state consists with any of the criterion states and as a non-converted one (or a non-responding one) otherwise.
  • the patient's Boolean state does not consists with any of the criterion states and one single biomarkers or some of the specific immuno-biomarkers of the patient in the profile may be expressed.
  • a patient who is classified as a responding one to the related hepatitis B vaccine may have certain combinations of the specific immuno-biomarkers of the patient in the profile expressed.
  • a patient who is classified as a non-responding one to the related hepatitis B vaccine may have certain combinations of the specific immuno-biomarkers of the patient in the profile expressed.
  • the present invention is applicable when the vaccine is a hepatitis B vaccine. In one preferred embodiment, the present invention is applicable when the vaccine is a chronic hepatitis B vaccine.
  • Example 1 shows one exemplary case when the vaccine is an HBsAg-
  • HBIG therapeutic vaccine (YIC).
  • Example 2 shows another exemplary case when the vaccine is a hepatitis B vaccine with 3x GM-CSF.
  • the specific combination of immuno-biomarkers comprises IL-33, IL-10, and MIP-1 a.
  • the specific combination of immuno-biomarkers consists of IL-33, IL-10, and MIP-1a.
  • the specific combination of immuno- biomarkers of IL-10, IL-13, IL-23, IFN-a, IL-18 and MIG6 may be used to predict HBsAg seroconversion and decreasing before and during the treatment of a hepatitis B vaccine and GM-CSF.
  • a specific combination of immuno-biomarkers of IL-13, IFN-a, and IL-18 may be used to predict non sAg seroconversion and non-sAg decreasing before and during the treatment of a hepatitis B vaccine and 3x GM-CSF.
  • the present invention provides a method of treating or preventing hepatitis B, specifically chronic hepatitis B by combining the specific combination of immuno-biomarkers with a known vaccine or an existing vaccine treatment.
  • the present invention provides a method of identifying a specific combination of immuno-biomarkers to distinguish a patient who responds to a vaccine from a non-responsive one during immunotherapeutic treatments of hepatitis B, preferably during immunotherapeutic treatments of chronic hepatitis B.
  • the present invention provides a method of treating or preventing hepatitis B, specifically chronic hepatitis B by combining the specific combination of immuno-biomarkers as discussed herein with a known hepatitis B vaccine, specifically a chronic hepatitis B vaccine or treatment.
  • a known hepatitis B vaccine specifically a chronic hepatitis B vaccine or treatment.
  • Any known or existing vaccine composition against hepatitis B, specifically against chronic hepatitis B would be applicable to the present invention.
  • Non-limiting examples of vaccine compositions are discussed as follow: [00130] Vaccine Composition
  • the vaccine compositions in the present invention may be prophylactic (prevent infection) and may also be therapeutic.
  • the vaccine composition comprises immunogenic antigen(s) (such as protein antigen) and is combined with "pharmaceutically acceptable carrier(s)" which does not induce carrier-specific antibodies.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as aluminum hydroxide, dextrose, mannitol, trehalose, cyclodextrin, carrier proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates such as oil droplets or liposomes and/or any combinations of them. These vectors are well known to the general technical persons in this field.
  • Antigens can also be conjugated to bacterial toxins (toxoids such as diphtheria, tetanus, cholera, diphtheria CRM 197) or carrier proteins (L1 from HPV16 or HPV18, VP1 from Merkel's cell virus, Ovalbumin, keyhole limpet hemocyanin).
  • bacterial toxins toxoids such as diphtheria, tetanus, cholera, diphtheria CRM 197
  • carrier proteins L1 from HPV16 or HPV18, VP1 from Merkel's cell virus, Ovalbumin, keyhole limpet hemocyanin.
  • adjuvants that enhance the effect of the immunogenic composition may include, but are not limited to: (1) alum, such as aluminum hydroxide, aluminum phosphate, Aluminum sulfate and the like; (2) oil-in-water emulsion formulations such as (a) MF59 (see WO90 / 14837), (b) SAF, and (c) RibiTM Adjuvant System (RAS); (3) Freund complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (4) TLR agonists, eg.
  • alum such as aluminum hydroxide, aluminum phosphate, Aluminum sulfate and the like
  • oil-in-water emulsion formulations such as (a) MF59 (see WO90 / 14837), (b) SAF, and (c) RibiTM Adjuvant System (RAS)
  • RibiTM Adjuvant System RibiTM Adjuvant System
  • CFA Freund complete adjuvant
  • IFA Freund's incomplete adjuvant
  • cytokines such as interleukins (such as IL-1 , IL- 2 and IL- 4, IL-5, IL-6, IL-7, IL-12 and the like), tumor necrosis factor (TNF) and the like
  • TNF tumor necrosis factor
  • Vaccine compositions include immunogenic compositions such as antigens, pharmaceutically acceptable carriers, and adjuvants, typically contain diluents such as water, saline, glycerol, buffer, and the like.
  • diluents such as water, saline, glycerol, buffer, and the like.
  • auxiliary substances such as wetting, emulsifying agents, pH buffering substances and the like may also be included.
  • vaccines including immunogenic compositions, contain an immunologically effective amount of an immunogenic polypeptide, as well as the other desired components described above.
  • An "immunologically effective amount” means that the amount administered to a subject as a single dose or as part of a continuous dosages is effective for treatment or prophylaxis. The amount can vary depending on the health and physiology of the individual, the type of individual (e.g., human), the ability of the individual's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, the medical physician's assessment of medical condition, and other related factors. It is expected that this amount will be within a relatively wide range and can be determined by routine experimentation.
  • the vaccine composition or the immunogenic composition can be formulated as an injection, for example as a liquid solution or suspension; it can also be produced as a solid form suitable for preparation as a solution or suspension before injection.
  • the formulation can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
  • the vaccine composition of the present invention may be monovalent or multivalent.
  • the composition can be administered directly to a subject.
  • the subject may be a human or non-human mammal, preferably a human.
  • the composition of the invention can be administered directly to the individual using any known methods. These vaccines are usually administered using the same routes as used by conventional vaccines and/or mimicking the pathogen's infection.
  • Routes of administration of the pharmaceutical composition or vaccine composition of the invention may include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, intranasal, vaginal, oral or other parenteral routes of administration. If desired, the route of administration may be combined or adjusted according to the condition of the disease.
  • Vaccine compositions can be administered in a single dose or in multiple doses and can include the administration of booster doses to elicit and/or maintain immunity.
  • the vaccine should be given in a "therapeutically effective amount,” that is, the amount of vaccine is sufficient to elicit an immune response in the chosen route of administration and can effectively promote the protection of the host against viral infections.
  • each dose contains the protein antigen from about O. ⁇ g to about 1000C ⁇ g, preferably about ⁇ g to about 10C ⁇ g, and more preferably about 1 C ⁇ g to about 5C ⁇ g.
  • Antibody titers and other reactions in the subject can be used as standard methods to determine the optimal dosage for a particular vaccine.
  • the level of immunity can be used to determine if a booster dose is needed. After assessing antibody titers in serum or T cell functions of PBMC, booster doses may be required.
  • Administration of an adjuvant and/or an immunostimulant may increase the immune responses to antigens of the invention.
  • the preferred method is to administer the immunogenic composition by injection from the subcutaneous or intramuscular route.
  • the present invention reveal a kit for differentiating a responding patient from a non-responding one against a vaccine.
  • the kit can differentiate a responding patient from a non-responding one against a hepatitis B vaccine, preferably against a chronic hepatitis B vaccine.
  • the kit comprises a biosensor capable of detecting and/or quantifying the combination of biomarkers as defined herein.
  • Biomarkers distinguish HBeAg seroconverted from non-converted individuals in chronic hepatitis B patients treated with a therapeutic vaccine
  • ABSTRACT
  • Cytokine assays of host immune responses to vaccination can indicate vaccine efficacy.
  • assays of the cytokine status of infected individuals prior to therapeutic vaccination might provide a guide to vaccine therapeutic efficacy. If so, cytokine analysis might be used to select appropriate patients for therapeutic vaccination.
  • Data were obtained from a panel of 14 cytokine/chemokine assays that were done during a phase III clinical trial of HBsAg- HBIG therapeutic vaccine (YIC) treatment of chronic hepatitis B (CHB) patients. Summarized assay results were compared between patients who responded by HBeAg seroconversion and non-responders.
  • Plasma samples had been collected at baseline and weeks 12, 24, 40, 52, and 76 after initial vaccine injection, tested for HBeAg, anti-HBe, HBV DNA, and HBsAg and stored at -80 °C.
  • Patient's consent for retrospective serum analysis had been obtained in the phase Ilia clinical tria.
  • Plasma samples from a total of 44 patients who had HBeAg-seroconverted in the trial and from 49 patients randomly picked from those who had not seroconverted were thawed, mixed by vortexing, and centrifuged to remove particulates prior to the cytokine/chemokine assays.
  • Frozen plasma samples used for the main analysis had been taken on the day before treatment (baseline).
  • cytokines/chemokines were measured using a human MILLIPLEX MAP kit (Millipore) that assays ⁇ _-1 ⁇ , IFN- ⁇ , TNF- ⁇ , IFN- ⁇ , IL-2, IL-7, IL- 15, IL-21 , IL-33, IL-10, MIG, IP-10, ⁇ -1 ⁇ , ⁇ -1 ⁇ . Plates were read with a Luminex instrument according the manufacturer's instruction (Luminex 200, Austin Luminex, USA). Concentrations of each target were analyzed using MILLIPLEX Analyst 5.1 software (Merck Millipore Darmstadt, Germany) according to the manufacturer's standard curve. The concentrations of various cytokines/chemokines were displayed as heat maps by MutiExperiment Viewer 4 software (TM4 Group; Dana Farber Cancer Institute, Boston, MA).
  • a Boolean model was adapted [15].
  • expression data of immune biomarkers were first transformed into 1 (Expressed) or 0 (Not expressed) by cut-off 0.1 pg/ml, which is the detection limit of the Luminex system.
  • the boolean state of the biomarkers was generated from each patient as a serial of 1/0s.
  • ALT alanine aminotransferase
  • Host factors particularly anti-viral and associated immune responses, are the keys to be considered.
  • the combined effects of pro-inflammatory, inflammatory, anti-inflammatory and chemo-attracting factors determine the outcome and efficacy of the host immune responses.
  • TNF-a, MIP-1 a, ⁇ -1 ⁇ , MIG and IP-10 appeared as possible markers to distinguish between the groups, and among these five, the IP-10 and MIG were the most abundant factors according to the assay.
  • the other 9 factors were below detectable limits in both groups except for a few patients.
  • the Boolean model uses logical values ('on' (1) or Off (0)) to determine if the factor is active/present or not.
  • the on/off events can be linked to multiple factors by a biological association or functional role fitted into a regulatory network, which can consequently be encoded by Boolean operations on the network nodes.
  • the scheme of Boolean modeling is detailed in FIG. 2(a)-FIG. 2(g).
  • TNF-a, ⁇ -1 ⁇ , MIG and IP-10 were highly expressed by most of the patients, made no contribution to discriminate between converted and non-converted patients and were therefore excluded (See FIG. 9).
  • the baseline status could distinguish the group of patients who subsequently converted after the YIC treatments from those who did not.
  • Six comparisons contributed to distinguishing the converted from the non-converted patients. These were: (1-3) single expression of either IL-10 alone, IL-33 alone, or MIP-1a (0.1-20 pg/ml) alone, (4) dual expressions of IL-10 and MIP1 a, (5) triple expressions of IL- 10, IL-33 and MIP1a, and (6) all 10 cytokines expressed (FIG. 9 and Table 3).
  • IL-33 For instance, four patients had only IL-33 detectable prior to the YIC treatments and all four had HBeAg seroconverted at the endpoint; none of the patients in the HBeAg non-seroconverted group had expressed IL-33. This result suggested that the IL-33 expression may reflect a patient more likely becoming an HBeAg seroconverted individual if he finished the YIC treatments. However, the number of patients who expressed IL-33 alone was limited and more study would be needed to confirm this point.
  • cytokine/chemokines at baseline level used to discriminate the HBeAg seroconverted patients from the non-converted ones are listed and the numbers of patients within these combinations are shown.
  • MIP-1 a ( ⁇ 20 pg/mL) 0.70 0.32
  • IL-33 0.14 0.80 sensitivity and specificity of IL-10, IL-33, MIP-1a 0.59 0.80 single biomarker ( ⁇ 20 pg/mL) including MIP-1 a ( ⁇ 20 pg/mL), IL-10 10 cytokines all or IL-33 and the optimal expressed combinations were calculated according to the sensitivity and specificity calculation formulas described.
  • Host factors particularly anti-viral and associated immune responses, are key factors to be considered as determinants of immunotherapeutic efficacy. Analysis of the combined contribution of pro-inflammatory, inflammatory, antiinflammatory and chemo-attracting factors is required.
  • IL-33 a member of the IL-1 cytokine family, binds to the ST2 receptor and regulates the NF- kB signaling pathway.
  • IL-33 can enhance both innate and adaptive immune responses. Endogenous IL-33 has been found to act as an alarmin and be necessary for induction of potent CD8 + T cell (CTL) responses to virus infections in mice [17].
  • CTL CD8 + T cell
  • IL-10 is generally considered a central immunoregulatory cytokine, especially in maintaining immune tolerance in chronic virus infections [18, 19], a beneficial role of IL-10 expression in the earlier HBeAg seroconversion is also observed, suggesting that the IL-10 may be bifunctional during the course of HBV infection [20].
  • Chemokine MIP-1 a is involved in the recruitment and activation of NK cells, T cells and macrophages through binding to its receptors CCR5, CCR3 and CCR1 [21].
  • CCR5 is a key regulator of immune responses to HBV infection, hepatic fibrosis and HCC progression [22-24], and has been observed to be decreased on the peripheral blood cytotoxic T lymphocytes of CHB patients [25].
  • CCR5 is a key regulator of immune responses to HBV infection, hepatic fibrosis and HCC progression [22-24]
  • Boolean modeling has previously been successfully applied in analysis of biological complex networks at the level of genes, proteins, cells and populations [26-28]. Boolean analysis can be applied to simplify the multi-factor profiles and display them in a variety of ways [29, 30]. In this study, the same principle was used after simplifying the state of these cytokine/chemokine nodes as 1 (ON) if it was detectable and as 0 (OFF) if undetectable based on the limitations of the test kit employed. Any node that signaled in this network and seemed disconnected could indicate either a positive or a negative mediator since the mechanisms in such networks are still vague.
  • this Boolean model method and results offered limited insights into the mechanism of HBeAg seroconversion by YIC treatment in CHB patients. More data, to be collected in future clinical trials, may verify its use for selecting the most appropriate CHB patients for YIC treatment.
  • All patients were randomly divided into four groups: the first group of 23 people continued oral adefovir dipivoxil (ADV) once a day; the second group of 17 people was injected IFN-a intramuscularly every two days on the basis of the first group; the third group of 17 people was injected GM-CSF intramuscularly once a day for 3 days on the basis of the second group.
  • the fourth group of 19 people received hepatitis B vaccine after 3 needles GM-CSF. All patients stopped treatments after 48 weeks except follow-up oral ADV to 72 weeks.
  • Luminex 200 including MIP-1 alpha, SDF-1 alpha, IL-27, IL-1 beta, IL-2, IL-4, IL-5, IP-10, IL-6, IL-7, IL-8, IL-10, Eotaxin, IL-12p70, IL-13, IL-17A, IL-31 , IL- 1 RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1 beta, IFN-a, MCP-1 , IL -9, TNF-beta, GRO-alpha, IL-1a, IL-23, IL-15, IL-18, IL-21 , IL-22, MIG.
  • the blank hole density was set up as the cutoff value, the detected samples were higher than the cutoff value was defined as positive result (1), and lower than or equal to the cutoff value was defined as negative result (0).
  • the 34 factor (REF: EPX340-12167-901) and MIG (REF: EPX01 A- 10285-901) kits were purchased from Invitrogen.
  • the predictive model effect was validated by comparing the actual end- point virological data with the predicted data.
  • the combination of low IL-10 and high MIG represent 50% of sAg seroconversion patients (FIG. 4(a)), as well as low IL-13 and high GRO-alpha combination can distinguish patients with a decrease in 54.5% sAg (FIG. 4(b)), with the patient's baseline 35 factors expression level as starting points and sAg seroconversion or sAg decreased as endpoints.
  • low IL-13 and high MIP- 1 b can distinguish 70% of sAg seroconversion patients (FIG. 4(c)), and high IL-18 and high IFN-a can distinguish 86.4% sAg decreased patients (FIG. 4(d)) through 35 factors at weeks 12 expression level as starting points and sAg seroconversion or sAg decreased as endpoints.
  • HIV nonprogressors preferentially maintain highly functional HlV-specific CD8+ T cells, Blood 107 (12) (2006) 4781 ⁇ 1789.

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Abstract

L'invention concerne une combinaison de biomarqueurs comprenant une pluralité d'immuno-biomarqueurs associés à une fonction immunitaire, qui différencie un patient répondant d'un patient non-répondant vis-à-vis d'un vaccin. La combinaison de biomarqueurs comprenant une pluralité d'immuno-biomarqueurs associés à la fonction immunitaire sont au moins trois biomarqueurs choisis dans le groupe constitué par MIP-1α, SDF-1α, IL -27, IL-1β, IL-2, IL-4, IL-5, IP-10, IL-6, IL -7, IL-8, IL-10, IL-11, éotaxine, IL-12 p70, IL-13, IL-17A, IL -31, IL-35, IL-1RA, RANTES, IFN-g, GM-CSF, TNF-a, MIP-1β, IFN-a, MCP-1, IL-9, TNF-β, GRO-α, IL-1α, IL-23, IL-15, IL-18, IL-21, IL-22, MIG, le facteur de stimulation des colonies de granulocytes (g-CSF ou GCSF), le récepteur 1 de chimiokine à motif C-X-C (CXCR1), CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CXCR8, CXCR9, CXCR10, CXCR11, CXCR12, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCL9, CCR10, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CXCL1, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13 et VEGF.
PCT/IB2018/054463 2017-06-16 2018-06-18 Immuno-biomarqueurs distinguant la réactivité de la non-réactivité pendant des traitements immunothérapeutiques Ceased WO2018229733A1 (fr)

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