WO2014046045A1 - Méthode de détection du vhc dans le sang, et procédé permettant de déterminer l'effet d'un traitement anti-vhc - Google Patents

Méthode de détection du vhc dans le sang, et procédé permettant de déterminer l'effet d'un traitement anti-vhc Download PDF

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WO2014046045A1
WO2014046045A1 PCT/JP2013/074859 JP2013074859W WO2014046045A1 WO 2014046045 A1 WO2014046045 A1 WO 2014046045A1 JP 2013074859 W JP2013074859 W JP 2013074859W WO 2014046045 A1 WO2014046045 A1 WO 2014046045A1
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hcv
hepatitis
virus
protease
blood
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PCT/JP2013/074859
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Japanese (ja)
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浩文 宇都
博仁 坪内
誠一 馬渡
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国立大学法人鹿児島大学
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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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a

Definitions

  • the present invention relates to a blood HCV detection method and an anti-HCV treatment effect determination method.
  • Hepatitis C virus is a positive-strand single-stranded RNA virus belonging to the Flaviviridae family, and is known to cause human hepatitis by using humans as a unique host.
  • HCV genotypes are classified into genotypes 1 to 6, and they are further classified into a plurality of subtypes (Non-patent Document 1).
  • the RNA genome of HCV is a precursor protein (polyprotein) consisting of four structural protein regions (Core, E1, E2, p7) and six nonstructural protein regions (NS2, NS3, NS4A, NS4B, NS5A, NS5B). ) Is encoded (Non-Patent Document 2). After translation, the precursor protein is hydrolyzed by protease and viral protease in the host cell to become 10 kinds of mature proteins.
  • ⁇ Current hepatitis C treatments mainly include antiviral treatment by drug administration for 24 to 72 weeks to eliminate HCV.
  • serum HCV RNA is negative after the administration period is completed and serum HCV RNA is negative, and the serum HCV RNA is negative until week 24, the therapeutic effect is sustained virologically effective ( sustained virological response (SVR).
  • SVR sustained virological response
  • Current treatment for hepatitis C is ultimately aimed at this SVR.
  • serum HCV RNA was negative at the end of drug administration, but if the serum HCV RNA subsequently became positive, the therapeutic effect is judged to be relapse.
  • serum HCV RNA does not become negative, it is determined that the treatment is invalid.
  • HCV RNA is quantitatively detected and used as an index of the amount of HCV in order to determine the effect of antiviral therapy.
  • Amplicor method and TaqMan method are used for detection of HCV RNA, but more sensitive HCV detection method is required in order to confirm virus negation more strictly and early.
  • NS3 / 4A an enzyme composed of NS3 and NS4A of HCV, has serine protease activity, plays an important role in HCV replication, and is one of the targets of direct-acting antiviral agents (DAA) (Non-patent Documents 3 and 4, Patent Document 1, etc.).
  • DAA direct-acting antiviral agents
  • NS3 / 4A protease inhibitor one of DAA, has dramatically improved the therapeutic effect on HCV in recent years, and peginterferon + ribavirin + NS3 / 4A protease inhibitor triple therapy was said to be incurable In patients with chronic hepatitis C who have HCV genotype 1 and high viral load, it has a high virus elimination effect.
  • the mechanism of antiviral action of NS3 / 4A protease inhibitors has not been fully elucidated.
  • Non-Patent Document 5 HCV acts to suppress host immune responses in persistent infection with HCV
  • Non-Patent Document 6 HCV carrier with persistent normal ALT
  • Non-Patent Documents 7 to 10 The present inventors have attempted to identify a protein that can be mainly a marker for liver cancer from the serum of patients with liver diseases by a technique using proteomics (Non-Patent Documents 7 to 10). However, the relationship between these proteins in vivo and the pathology of patients with liver disease has not been fully elucidated.
  • An object of the present invention is to provide a method for detecting hepatitis C virus in the blood of a human subject with higher sensitivity.
  • the present inventors have found that NS3 / 4A protease of HCV cleaves complement C4 within the ⁇ chain, and the amount of C4 ⁇ cleaved fragment generated thereby is HCV infected.
  • the present inventors have found that the presence of HCV in the blood and the change in the amount of HCV after anti-HCV treatment (effect of antiviral treatment) are well reflected, thereby completing the present invention.
  • the present invention includes the following.
  • a method for detecting hepatitis C virus (HCV) in blood of a subject comprising detecting a C4 ⁇ -cleavage fragment in a blood sample produced by cleavage of HCV NS3 / 4A protease in complement C4.
  • the C4 ⁇ -cleavage fragment is obtained by cleaving NS3 / 4A protease between cysteine at position 1583 and serine at position 1584, or between cysteine at position 1590 and alanine at position 1591 in the amino acid sequence shown in SEQ ID NO: 1.
  • [5] A method for monitoring the amount of hepatitis C virus in a subject, comprising measuring hepatitis C virus over time using the method according to [3] or [4] above.
  • a method for determining an antiviral therapeutic effect comprising measuring hepatitis C virus using the method described in [4] or [5] above, and using the measurement result as an index of the antiviral therapeutic effect .
  • a hepatitis C virus detection reagent comprising an antibody that binds to a C4 ⁇ cleaved fragment produced by cleavage of HCV NS3 / 4A protease of complement C4.
  • the C4 ⁇ -cleavage fragment is obtained by cleaving NS3 / 4A protease between cysteine at position 1583 and serine at position 1584 or between cysteine at position 1590 and alanine at position 1591 in the amino acid sequence shown in SEQ ID NO: 1.
  • a kit for detecting hepatitis C virus comprising the reagent for detecting hepatitis C virus according to [7] or [8] above.
  • HCV in the blood of an HCV-infected person can be detected with higher sensitivity, and can be advantageously used for determining the effect of antiviral treatment.
  • FIG. 1 shows the analysis result of C4 cleavage by HCV NS3 / 4A protease.
  • FIG. 1A shows the results of SDS-PAGE
  • FIG. 1B shows the position of the N-terminus of the C4 ⁇ -cleavage fragment on the amino acid sequence of the precursor protein of complement C4. The sequence shown in FIG. 1B is shown in SEQ ID NO: 3.
  • FIG. 2 shows the cleavage pattern of complement C4 by HCV NS3 / 4A protease.
  • FIG. 3 shows an SDS-PAGE image showing C4 ⁇ -cleaved fragments Frag F1, F2, and F3 generated by C4 cleavage with HCV NS3 / 4A protease.
  • FIG. 1 shows the analysis result of C4 cleavage by HCV NS3 / 4A protease.
  • FIG. 1A shows the results of SDS-PAGE
  • FIG. 1B shows the position of the N-terminus of the C4 ⁇ -cle
  • FIG. 4 shows the protein concentration of each band shown in FIG. 3 for samples (1) to (6) in optical density.
  • FIG. 5 shows the effect of HCV NS3 / 4A protease on complement function (hemolysis). Data were measured in triplicate and expressed as mean ⁇ standard deviation.
  • the test groups with C4 addition amounts of 12.5 ⁇ g / ml, 6.3 ⁇ g / ml, and 3.1 ⁇ g / ml are shown from the left. “No C4” indicates a control without addition of C4.
  • FIG. 6 shows the effect of an HCV NS3 / 4A protease inhibitor on the complement function-reducing action of HCV NS3 / 4A protease. C4 was added at 6.3 ⁇ g / ml.
  • FIG. 7 is a photograph showing detection results of C4 ⁇ -cleaved fragments Frag F1, F2 in the blood of HCV-infected and non-HCV-infected people.
  • FIG. 8 is a diagram showing the results of measuring the C4 ⁇ cleaved fragments in the serum of HCV-infected persons undergoing antiviral treatment over time.
  • a type 1 SVR case SVR case of a genotype 1 HCV infected person
  • a type 2 SVR case a genotype 2 HCV infected person SVR case
  • Western blot results and HCV-RNA measurements A, C, E
  • B, D, F concentration ratio
  • the complement system plays a central role in innate immunity and helps eliminate pathogens.
  • the complement system is composed of many small proteins in the blood and is activated by three biochemical processes: the classical pathway, the mannose-binding lectin pathway, and the alternative pathway.
  • Complement C4 one of the components that make up the complement system, is activated in the classical and mannose-binding lectin pathways.
  • C4 is a glycoprotein with a molecular weight of 198,000 and has a unit structure in which three polypeptide chains ( ⁇ chain, ⁇ chain, ⁇ chain) are cross-linked by disulfide bonds (FIG. 2).
  • C4 precursor polypeptide One precursor polypeptide encoded by the human C4 gene (C4 precursor polypeptide) is cleaved post-translationally to complement C4 ⁇ chain, complement C4-A ⁇ chain (complement C4 ⁇ chain), and complement C4 ⁇ chain Produces.
  • the amino acid sequence of the precursor polypeptide encoded by the C4 gene has been determined.
  • SEQ ID NO: 1 For example, in the typical amino acid sequence (SEQ ID NO: 1) of the human C4 precursor polypeptide disclosed under the accession number P0C0L4 (version: P0C0L4.1) in the NCBI Protein Database (UniProtKB / Swiss-Prot), 1 to Signal peptide at position 19, Complement C4 ⁇ chain at positions 20-675, Propeptide at positions 676-679, Complement C4 ⁇ chain at positions 680-1446, Propeptide at positions 1447-1453, and Complements at positions 1454-1744 Body C4 ⁇ chain.
  • the amino acid sequence from positions 1454 to 1744 of SEQ ID NO: 1 corresponding to the complement C4 ⁇ chain is shown in SEQ ID NO: 2.
  • a cleavage fragment generated by cleavage of HCV-derived NS3 / 4A protease from this complement C4 ⁇ chain in the human body well reflects the abundance of HCV in the blood. This is based on the surprising findings found by the present inventors.
  • the present invention relates to a method for detecting a C4 ⁇ cleaved fragment in a blood sample derived from a subject and detecting hepatitis C virus (HCV) in the blood of the subject based on the detection result.
  • HCV hepatitis C virus
  • blood sample refers to blood (whole blood) collected from a subject whose blood HCV should be detected, or serum or plasma separated therefrom by a conventional method, and serum is more preferable.
  • the blood sample is preferably stored at a refrigeration temperature (2 to 8 ° C.) or lower, and when HCV detection is performed several days after blood collection, it may be stored frozen (eg, ⁇ 20 ° C. or ⁇ 80 ° C.). Further preferred.
  • the subject in the present invention is preferably a human subject who needs to examine the presence of HCV in the blood or the amount of HCV.
  • the subject may be a person suspected of having HCV infection.
  • the subject may also be a person who has been infected with HCV (HCV-infected person) or a person who has been infected with HCV (a person who has been infected with HCV and healed).
  • HCV carriers who have normal ALT levels (approximately 30-40 IU / L or less, usually 30 IU / L or less) that indicate liver function status (HCV carriers with persistent normal ALT) PNALT)) or an HCV-infected patient who has an abnormally high serum ALT level and develops acute hepatitis C or chronic hepatitis C, cirrhosis or liver cancer.
  • the subject may be infected with any HCV of genotype 1, 2, 3, 4, 5 or 6.
  • the subjects also have high viral load cases (more than 5.0 log IU / ml in real-time PCR method such as TaqMan method), but low viral load cases (less than 5.0 log IU / ml in real-time PCR method such as TaqMan method). There may be.
  • antiviral treatment refers to treatment performed for the purpose of eliminating hepatitis C virus in a patient.
  • Antiviral treatment is usually a process of administering a drug over a predetermined drug administration period of 24 to 72 weeks to reduce HCV RNA to below the detection limit. However, it has an unacceptable side effect and low response to treatment. Therefore, drug administration may be interrupted or discontinued before HCV RNA falls below the detection limit.
  • “patient on antiviral treatment” means a patient who is in the period of administration of a drug for antiviral treatment (normally, but not limited to 24 to 72 weeks).
  • the “patient after completion of antiviral treatment” specifically means a patient who has received antiviral treatment but has not received antiviral treatment at the time of collection of a blood sample.
  • Patient after completion of antiviral treatment '' is a patient who is in the follow-up period after completing the drug administration period for antiviral treatment and negative HCV RNA, SVR was achieved at the end of the follow-up period This includes patients, patients who relapsed at the end of the follow-up period (against HCV once became positive again), and patients who discontinued drug administration for antiviral treatment due to side effects or poor therapeutic response.
  • end of antiviral treatment means that drug administration for antiviral treatment has been completed or stopped for a predetermined period, and does not necessarily mean that HCV in the blood of the patient has disappeared. .
  • Antiviral treatments include, but are not limited to, interferon therapy (interferon- ⁇ or interferon- ⁇ monotherapy, or peginterferon monotherapy), pegylated interferon (eg, PEGylated IFN ⁇ -2a or PEGylated IFN ⁇ -2b). ) + Ribavirin combination therapy, peginterferon + ribavirin + NS3 / 4A protease inhibitor (eg telaprevir, boceprevir, TMC435, MK7009, and BI201335), and NS5A inhibitor or NS5B inhibitor + protease inhibitor (NS3 Inhibitors, etc.) combination therapy and the like.
  • interferon therapy interferon- ⁇ or interferon- ⁇ monotherapy, or peginterferon monotherapy
  • pegylated interferon eg, PEGylated IFN ⁇ -2a or PEGylated IFN ⁇ -2b.
  • Ribavirin combination therapy eg, peginterferon
  • C4 ⁇ cleaved fragments in the blood sample of the subject are detected, preferably quantitatively detected (measured).
  • the C4 ⁇ cleavage fragment is generated by cleaving the ⁇ chain of host complement C4 by HCV-derived NS3 / 4A protease in vivo. More specifically, the C4 ⁇ cleaved fragment is obtained by converting the C4 ⁇ chain located at positions 1454 to 1744 in the C4 precursor polypeptide consisting of the base sequence represented by SEQ ID NO: 1 by HCV NS3 / 4A protease to SEQ ID NO: 1.
  • the ⁇ chain of complement C4 is a 32 kDa protein, which becomes a 17 kDa protein and a 15 kDa protein when cleaved by HCV NS3 / 4A protease, and these are “C4 ⁇ cleaved fragments”.
  • proteins having molecular weights of 32 kDa, 17 kDa, and 15 kDa mean proteins that are separated as having molecular weights of about 32 kDa, about 17 kDa, and about 15 kDa as analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • a 17 kDa protein also referred to herein as a 17 kDa protein, which is a C4 ⁇ cleavage fragment, is typically cleaved between the cysteine at position 1583 and the serine at position 1584 as the N-terminal amino acid sequence.
  • the 17 kDa protein that is a C4 ⁇ cleaved fragment may consist of the sequence from positions 1584 to 1744 or the sequence from positions 1591 to 1744 of the amino acid sequence shown in SEQ ID NO: 1, or one or several numbers within the sequence. It may be an active C4 ⁇ chain fragment consisting of an amino acid sequence in which one (for example, 2 to 10) amino acids have been deleted, substituted or added.
  • the 17 kDa protein which is a C4 ⁇ cleaved fragment, is an amino acid that exhibits 90% or more, preferably 95% or more sequence identity with the sequence of 1584 to 1744 or the sequence of 1591 to 1744 of the amino acid sequence shown in SEQ ID NO: 1. It may be an active C4 ⁇ chain fragment consisting of a sequence.
  • a 15 kDa protein also referred to herein as a 15 kDa protein
  • that is a C4 ⁇ cleaved fragment may typically have “EAPKVVEE” (SEQ ID NO: 9) as the N-terminal amino acid sequence.
  • the 15 kDa protein that is a C4 ⁇ cleaved fragment may consist of the sequence of positions 1454 to 1583 or the sequence of positions 1454 to 1590 of the amino acid sequence shown in SEQ ID NO: 1, or one or several numbers within the sequence. It may be an active C4 ⁇ chain fragment consisting of an amino acid sequence in which one (for example, 2 to 10) amino acids have been deleted, substituted or added.
  • the 15 kDa protein that is a C4 ⁇ cleaved fragment is an amino acid sequence that shows 90% or more, preferably 95% or more sequence identity with the sequence of positions 1454 to 1583 or the sequence of positions 1454 to 1590 of the amino acid sequence shown in SEQ ID NO: 1. It may be an active C4 ⁇ chain fragment.
  • Detection and measurement of C4 ⁇ cleaved fragments in a blood sample of a subject can be performed using protein quantification methods well known to those skilled in the art.
  • protein quantification methods include, but are not limited to, Western blotting using SDS-polyacrylamide gel electrophoresis, ELISA, affinity binding electrophoresis, and the like.
  • Western blotting a protein sample derived from a blood sample is first separated by gel electrophoresis according to the molecular weight of the protein. For this gel electrophoresis, sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (SDS-PAGE) is generally used.
  • SDS sodium dodecyl sulfate
  • SDS-PAGE sodium dodecyl sulfate
  • proteins are treated with an anionic surfactant, SDS, and a reducing agent, mercaptoethanol, to break down the higher-order structure of the protein, so that all proteins have a negative charge. Separation is performed based on the difference in the molecular weight of the protein due to the sieving effect. Remove the protein-separated gel, transfer the separated protein to a membrane such as PVDF, remove the surfactant and reducing agent, perform blocking treatment, and then introduce the labeled antibody that binds to the C4 ⁇ cleaved fragment Can cause.
  • an enzyme such as horseradish peroxidase
  • a fluorescent dye such as a fluorescent dye
  • chemical chromophore or the like can be used.
  • an anti-C4 polyclonal antibody or an anti-C4 ⁇ cleaved fragment monoclonal antibody can be used as an antibody that binds to the C4 ⁇ cleaved fragment.
  • the anti-C4 polyclonal antibody can be prepared by a conventional method using human complement C4 as an antigen, but a commercially available product can also be used.
  • the anti-C4 ⁇ -cleaved fragment monoclonal antibody can be prepared by a conventional method using a C4 ⁇ chain or a 17-kDa protein or a 15-kDa protein that is a C4 ⁇ -cleaved fragment as an antigen.
  • the protein can be quantified by image analysis of the signal of the obtained protein band.
  • the concentration of the 17 kDa protein may be calculated as the sum of the measured values of the two 17 kDa proteins that are the above-mentioned C4 ⁇ cleaved fragments.
  • the concentration of the 15 kDa protein may be calculated as the sum of the measured values of the 15 kDa protein, which are the above two C4 ⁇ cleaved fragments. Only one of the 17 kDa protein concentration and the 15 kDa protein concentration may be calculated, or both the 17 kDa protein and the 15 kDa protein concentration may be calculated.
  • the C4 ⁇ cleaved fragment (17kDa protein or 15kDa protein) in the blood sample of the subject is statistically significant compared to the blood sample of a healthy person (one who is negative for HCV RNA and does not have liver damage). Detected at a high concentration indicates the presence of HCV in the blood of the subject, ie, infection with HCV. C4 ⁇ cleaved fragments are hardly detected in blood samples from healthy individuals. The concentration of C4 ⁇ cleaved fragments in the blood sample correlates with the HCV concentration in the subject's blood. Therefore, by measuring the concentration of the C4 ⁇ cleaved fragment in the blood sample, the relative level of the amount of HCV in the blood in the subject's living body can be examined.
  • “measurement of the amount of hepatitis C virus (HCV)” includes not only calculating the absolute amount of HCV in a sample but also calculating the relative level of HCV amount (concentration) in vivo. .
  • the C4 ⁇ cleaved fragment is used as a direct detection target. Can be detected.
  • Such a correlation between the level of C4 ⁇ cleaved fragments in blood and the level of HCV means that HCV has produced an amount of C4 ⁇ cleaved fragments according to the amount of persistent infection. This is because HCV cleaves complement C4 within the ⁇ chain by the action of its NS3 / 4A protease, thereby inhibiting complement activation, reducing complement function and attenuating the immune response of the human host. This is thought to be due to maintaining persistent infection.
  • the method of the present invention can be used for examining the presence or absence of HCV infection in a subject when the subject is suspected of having HCV infection.
  • the subject is an HCV-infected person or a person who has a history of HCV infection, to examine the amount of HCV in the subject's blood, or whether HCV infection in the subject's blood has been eliminated and the HCV infection has been cured Can be used to investigate.
  • HCV detection by conventional HCV RNA measurement together with HCV detection of the present invention based on detection of C4 ⁇ cleaved fragments.
  • HCV RNA measurement can be performed by real-time PCR methods such as amplicore HCV measurement method and TaqMan HCV measurement method.
  • the amount of HCV in the subject can be monitored by measuring the amount of HCV in the subject over time using the above-described HCV detection method.
  • the subject is preferably an HCV infected person or a person with a history of infection.
  • the subject is also preferably a patient during or after completion of antiviral treatment for HCV.
  • Many HCV-infected persons become chronic and develop chronic hepatitis C, but 20-30% of HCV chronically infected persons are known to have normal ALT levels and no obvious liver damage despite persistent infection. ing.
  • ALT normal persistent HCV-infected persons often show mild or mild chronic hepatitis histologically, and fibrosis may progress.
  • HCV ALT normal persistent HCV infected subjects
  • the change in HCV is monitored by measuring the above-mentioned C4 ⁇ cleaved fragments in blood samples collected regularly or irregularly Often, this is useful for objectively examining the deterioration or improvement of the liver disorder.
  • the HCV in the patient's blood is highly sensitive by measuring the C4 ⁇ cleaved fragment in the blood sample regularly or irregularly. May be monitored. The monitoring makes it possible to detect early HCV re-growth (relapse) that has fallen below the detection limit, and to immediately resume antiviral treatment.
  • the change in the amount of HCV in the blood of the patient may be monitored with high sensitivity.
  • the monitoring makes it possible to examine the implementation of another antiviral treatment while confirming fluctuations in the amount of HCV, and to implement it if necessary.
  • “measurement with time” means that measurement is performed continuously or irregularly.
  • HCV in the blood of the subject is detected and measured by detecting and measuring a C4 ⁇ -cleavage fragment in the blood sample of the subject who is a patient during or after the antiviral treatment. And measuring the result of the measurement as an index to determine the effect of antiviral treatment in the patient. If the amount of C4 ⁇ cleaved fragments in the blood sample of a patient during or after antiviral treatment falls below the detection limit during the drug administration period and remains below the detection limit over the follow-up period It can be determined that the antiviral treatment was effective and that the patient achieved SVR.
  • the follow-up period is generally 24 weeks from the expiration of the drug administration period.
  • the follow-up period can be shorter than 24 weeks.
  • the antiviral treatment effect is determined to be “relapse”. The It is known that the incidence of subsequent liver cancer is significantly reduced even in cases of relapse, so even if it is determined to be relapse, the progression of liver damage is suppressed for the antiviral treatment administered The effect can be expected to some extent.
  • the amount of C4 ⁇ cleaved fragments in a patient's blood sample is clearly reduced during antiviral treatment but does not fall below the detection limit at the completion of the prescribed drug administration period, antiviral treatment is not possible during that drug administration period. It can be determined that the effect was not sufficient (“insufficient effect”). In this case, the drug administration period may be extended and antiviral treatment may be continued further. If the amount of C4 ⁇ cleaved fragments in the patient's blood sample does not show a clear decrease during antiviral treatment and does not fall below the detection limit when the drug administration period is completed, the effect of the antiviral treatment is determined to be “invalid” Is done.
  • the treatment may be determined to be invalid and antiviral treatment may be discontinued during the drug administration period.
  • Antiviral treatment often has side effects, so it would be advantageous if an invalid treatment could be determined early.
  • Changes in the amount of C4 ⁇ cleaved fragments in a blood sample of a patient during or after completion of antiviral treatment correlate well with the effect of such antiviral treatment (change in viral load).
  • the C4 ⁇ cleaved fragment in the blood sample is significantly reduced, it is suggested that the amount of HCV in the blood of the subject is also significantly reduced. If no significant decrease in C4 ⁇ cleaved fragments is observed in the blood sample, it is suggested that the amount of HCV in the subject's blood is not sufficiently reduced.
  • SVR may occur in patients who have achieved an “early virologic response (EVR)” in which the viral load decreases or disappears by 2 log 10 or more by 12 weeks after the start of antiviral treatment
  • EMR early virologic response
  • Higher long-term antiviral treatment may be effective in achieving SVR in cases where HCV is significantly reduced at 12 weeks but remains and then disappears by, for example, 24 weeks (late responder) Has been reported.
  • the effect of the antiviral treatment was delayed for the subject. It can be determined that the drug is sex, and a longer drug administration (for example, 72 weeks in total) can be performed by adding a drug administration period.
  • the present invention also detects a C4 ⁇ -cleaved fragment in a blood sample derived from a subject, and detects hepatitis C virus (HCV) in the blood of the subject based on the detection result, thereby detecting HCV infection in the subject. Also provided are methods of diagnosing the presence or absence or HCV retention level of a subject. Furthermore, the present invention uses the above-described HCV detection method to measure the amount of HCV in a subject who is infected with HCV over time, and by monitoring the amount of HCV in the subject, the treatment policy for hepatitis C in the subject is determined. And a method for treating hepatitis C using the method.
  • HCV hepatitis C virus
  • the present invention also provides an antibody that binds to the C4 ⁇ -cleaved fragment produced by HCV NS3 / 4A protease cleavage (preferably an antibody that specifically binds to the C4 ⁇ -cleaved fragment), which can be suitably used in the HCV detection method and the like.
  • a reagent for detecting hepatitis C virus is also provided.
  • An antibody that binds to a C4 ⁇ -cleaved fragment for example, an antibody that specifically binds to a C4 ⁇ -cleaved fragment, may be prepared according to a conventional method for producing polyclonal or monoclonal antibodies.
  • the C4 ⁇ cleaved fragment or C4 ⁇ chain that can be used as an antigen during antibody production is as described above.
  • Anti-C4 polyclonal antibodies can also be used as antibodies that bind to C4 ⁇ cleaved fragments.
  • a commercially available anti-C4 polyclonal antibody can also be used.
  • hepatitis C virus detection reagents are inert additives such as carriers, solvents (water, buffers, etc.), excipients, pH adjusters, isotonic agents, preservatives, etc. May be included.
  • the present invention also provides a hepatitis C virus detection kit comprising such a hepatitis C virus detection reagent.
  • the hepatitis C virus detection kit may further contain an ELISA or Western blot analysis reagent, an SDS-PAGE reagent, and the like.
  • the HCV NS3 / 4A protease used in the following examples was purchased from AnaSpec (Fremont, CA, USA), and HCV Core and HCV NS5 from ProSpec (Israel). This HCV NS3 / 4A protease is derived from the HC-J4 strain of genotype 1b. Core and NS5 are derived from genotype 1 HCV strains. Isolated human complement (C1, C2) from Hycult Biotech (Netherlands), human complement C4 and C4 deficient guinea pig serum (C4 deficient guinea pig serum; C4dGPS) from Sigma Aldrich (St. Louis) Purchased.
  • C1, C2 Human complement C4 and C4 deficient guinea pig serum
  • C4dGPS Sigma Aldrich
  • HCV NS3 / 4A protease inhibitor VX-950 (Telaprevir) was purchased from Selleck Chemicals (Houston). Veronal buffer was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), sheep erythrocytes were purchased from Japan Biotest Laboratories (Tokyo, Japan), and hemolysin was purchased from Denka Seken (Tokyo, Japan).
  • SPSS software SPSS Inc., Chicago, IL, USA was used for statistical analysis. The test was conducted using Mann-Witit-U's test or Tukey 's ⁇ ⁇ test, and p ⁇ 0.05 was considered statistically significant.
  • Example 1 Analysis of the action of NS3 / 4A protease on complement C4 How the NS3 / 4A protease, an HCV nonstructural protein, affects complement C4, which is part of the host immune response To investigate, complement C4 was mixed with HCV NS3 / 4A protease, HCV Core or HCV NS5, and tested to see if C4 was cleaved.
  • complement C4 (4 ⁇ l), either HCV NS3 / 4A protease, HCV Core or HCV NS5 (4 ⁇ l) and 30 mM m dithiothreitol (DTT) -containing assay buffer (SensoLyte (registered trademark) 490 HCV protease assay kit, AnaSpec) 12 ⁇ l was mixed and incubated at 30 ° C. for 30 minutes.
  • the above assay buffer (16 ⁇ l) was mixed with C4, HCV NS3 / 4A protease, Core or NS5 (4 ⁇ l) alone and incubated in the same manner.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the protein detected by CBB staining was subjected to N-terminal peptide sequencing by Nippi Co., Ltd. (Tokyo, Japan), and the amino acid sequence of the N-terminal region was analyzed.
  • the results are shown in FIG.
  • the solution in which C4 is incubated with NS3 / 4A protease contains about 100 kDa protein and about 75 kDa protein, as well as about 17 kDa protein (Frag F1 and Frag F2 in Fig. 1) and about 15 kDa protein (Frag F3). ) was detected (FIG. 1A).
  • a solution in which C4 was incubated with HCV Core or NS5 a protein of about 100 kDa and a protein of about 75 kDa were detected, but a protein of about 17 kDa and about 15 kDa were not detected (FIG. 1A).
  • An approximately 32 kDa protein detected in a solution of C4 incubated with HCV Core or NS5 and a solution of C4 alone was not detected in a solution of C4 incubated with NS3 / 4A protease.
  • the 100 kDa protein is C4 ⁇ chain (N-terminal amino acid sequence is NVNFQKAI; SEQ ID NO: 4), the 75 kDa protein is C4 ⁇ chain (N-terminal amino acid sequence is KPRLLLFS; SEQ ID NO: 5), and the 32 kDa protein is C4 ⁇ chain ( The N-terminal amino acid sequence was shown to be EAPKVVEE; SEQ ID NO: 6).
  • the N-terminal amino acid sequence of the 17 kDa protein is the amino acid sequence SAEVCQCA (1584-1591; SEQ ID NO: 7) for Frag F1, and the amino acid sequence AEGKCPRQ (1591-1598 for Crag4 for Frag F2; sequence Consistent with number 8), both were shown to be within the C4 ⁇ chain (FIG. 1B).
  • the N-terminal amino acid sequence of the 15 kDa protein (Frag F3) matched the amino acid sequence EAPKVVEE (positions 1454-1461; SEQ ID NO: 9) in C4 (FIG. 1B).
  • HCV NS3 / 4A protease is between cysteine (C) at position 1583 and serine (S) at position 1584 in the amino acid sequence of the C4 precursor protein (SEQ ID NO: 1), or cysteine at position 1590 (C ) And alanine (A) at position 1591, it was shown to cleave the C4 ⁇ chain to produce a protein fragment of about 17 kDa and a protein fragment of about 15 kDa (FIG. 2).
  • Example 2 Analysis of C4 cleavage inhibitory effect of NS3 / 4A protease inhibitor It was examined whether NS3 / 4A protease inhibitor inhibits C4 cleavage by NS3 / 4A protease.
  • HCV NS3 / 4A protease inhibitor (VX-950) is dissolved in dimethyl sulfoxide (DMSO), and then with the above-mentioned 30 mM DTT-containing assay buffer so as to have various concentrations (0, 1, 10, 100, and 500 ⁇ M). Diluted to 12 ⁇ l.
  • NS3 / 4A protease (4 ⁇ l) was added to this HCV NS3 / 4A protease inhibitor and preincubated for 30 minutes at 30 ° C., then C4 (4 ⁇ l) was added and incubated at 30 ° C. for 30 minutes.
  • the stained images of the separated C4 ⁇ chain (32 kDa), 17 kDa and 15 kDa protein bands were quantified using image processing software image J (NIH) (optical density analysis).
  • FIG. 3 electrospray image
  • FIG. 4 relative protein concentration
  • NS3 / 4A protease inhibitor VX-950 inhibits C4 cleavage by NS3 / 4A protease.
  • This result also confirmed that the 17 kDa protein and the 15 kDa protein are fragments of the C4 ⁇ chain.
  • Example 3 Analysis of the effect of HCV NS3 / 4A protease on complement function
  • sheep erythrocytes Ab-sensitized sheep sensitized to hemolysin erythrocyte (EA) was sequentially mixed with complement to reproduce complement activation by the classical pathway, and the effect of HCV NS3 / 4A protease on complement function was tested by the erythrocyte hemolysis test as follows. The erythrocyte hemolysis test based on the binding of sensitized sheep erythrocytes and complement was followed (Krych-Goldberg M., et al., J. Biol.
  • Veronal buffer (GVB) containing 2% gelatin was used for dilution of erythrocytes and complement. All operations were performed on ice.
  • EAC1 sensitized sheep erythrocytes
  • NS3 / 4A protease is a solution containing 20 mM Tris-HCl (pH 8.0), 20% glycerol, 100 mM KCl, 1 mM DTT and 0.2 mM EDTA (pH 7.5) at various concentrations (0, 6.3, 12.5, and 25 ⁇ g / ml).
  • Complement C4 was prepared with GVB at various concentrations (0, 3.1, 6.3, and 12.5 ⁇ g / ml).
  • Complement C4 (5 ⁇ l) at each concentration and NS3 / 4A protease (10 ⁇ l) at each concentration were mixed with 6 mM DTT-containing GVB (5 ⁇ l) (2 mM DTT in the mixture), incubated at 30 ° C.
  • EAC1 Complement intermediate EAC1-C4 was prepared by mixing with 100 ⁇ l and incubating at 30 ° C. for 15 minutes. After washing twice with GVB, 1 ⁇ l of C2 (0.1 mg / ml) was added and incubated at room temperature for 4 minutes to prepare complement intermediate EAC1-C4-C2. Further, after washing twice with GVB, 150 ⁇ l of C4-deficient guinea pig serum (C4dGPS) diluted 80-fold to 30 ⁇ l of EAC1-C4-C2 was added and incubated at 37 ° C. for 30 minutes.
  • C4dGPS is used as a source of C3 and terminal complement components C5-C9 because it contains complement components other than C4.
  • a sample to which 150 ⁇ l of distilled water was added instead of C4dGPS was also prepared.
  • the obtained reaction solution was centrifuged, the absorbance of the supernatant was measured at 415 nm, and the degree of hemolysis was calculated by the following formula.
  • Hemolysis (test sample OD415-GVB OD415) / (distilled water added sample OD415-GVB OD415) The results are shown in FIG. In the presence of NS3 / 4A protease, the degree of hemolysis was significantly reduced in a NS3 / 4A protease concentration-dependent manner compared to the absence of NS3 / 4A protease (control). Similar results were obtained at any C4 concentration.
  • NS3 / 4A protease inhibited erythrocyte lysis (hemolysis) caused by complement activation via the classical pathway through interaction with C4. This indicates that HCV NS3 / 4A protease decreases host complement function in a concentration-dependent manner.
  • Example 4 Inhibition of decrease in complement function by NS3 / 4A protease inhibitor NS3 / 4A protease (10 ⁇ l) diluted to 12.5 ⁇ g / ml was diluted with 100 ⁇ M NS3 / 4A protease inhibitor VX950 (diluted with 6 mM DTT-containing GVB) After mixing with 5 ⁇ l and incubating at 30 ° C. for 30 minutes, it was mixed with C4 (5 ⁇ l) diluted to 6.3 ⁇ g / ml and incubated at 30 ° C. for 30 minutes. Subsequently, 100 ⁇ l of EAC1 prepared in the same manner as in Example 3 was mixed and incubated at 30 ° C.
  • Example 5 Detection of C4 ⁇ -cleaved fragments in the blood of HCV-infected persons
  • the C4 ⁇ -cleavage fragments generated in the cleavage of complement C4 by NS3 / 4A protease found in Example 1 are contained in the serum of HCV-infected persons. I examined whether or not.
  • HCV antibody positive and HCV-RNA positive liver cancer patient
  • HCV antibody positive and HCV RNA positive HCV carrier
  • chronic hepatitis C patient HCV antibody positive and HCV RNA positive (HCV carrier) chronic hepatitis C patient
  • hepatitis B virus (HBV) surface antigen (HBsAg) positive Blood was collected from chronic hepatitis B patients (HBV carriers) and healthy individuals. Written informed consent was given at the time of blood collection. Serum separated from blood was stored at -80 ° C.
  • the separated protein was transferred to a PVDF membrane and reacted with a 1000-fold diluted goat anti-human C4 polyclonal antibody (MP Biomedicals, LLC-Cappel Products, Solon, OH, USA) for 1 hour at room temperature. Furthermore, after reacting with 1000-fold diluted donkey anti-goat IgG-horseradish peroxidase (HRP) (Santacruz, CA, USA) for 1 hour at room temperature, add ECL Prime Western blot detection reagent (GE Healthcare Life Sciences, Sweden) Then, image analysis was performed using ChemiDoc XRS (Bio-Rad, CA). The resulting protein band concentration was quantified with Image J.
  • HRP donkey anti-goat IgG-horseradish peroxidase
  • Figure 7 shows an example of the SDS-PAGE analysis results obtained.
  • the 17 kDa protein was hardly observed in the serum of chronic hepatitis B patients (CBH) and healthy subjects (Normal), but hepatoma patients infected with HCV (HCC ( C)) and serum of patients with chronic hepatitis C (CHC) were detected at high concentrations.
  • HCC HCC
  • CHC chronic hepatitis C
  • Example 6 Judgment of antiviral therapeutic effect using C4 ⁇ cleaved fragment amount as an index Collecting blood during treatment while peginterferon + ribavirin combination therapy as antiviral treatment for HCV positive patients (HCV infected patients) Then, the 17 kDa protein (C4 ⁇ cleaved fragment) in the serum was detected in the same manner as in Example 5. Serum HCV-RNA was quantified by real-time PCR.
  • Figures 8A and B show SVR cases of genotype 1 HCV-infected persons (administered for 72 weeks with combination therapy) before starting antiviral treatment (before IFN), 1 week after administration (1w), and 6 weeks after administration After (6w), 8 weeks after starting administration (8w), 18 weeks after starting administration (18w), 28 weeks after starting administration (28w), 17kDa protein (Frag F1, F2) and HCV in the serum after administration -A detection image of RNA (A) and a 17 kDa protein concentration (B) are shown.
  • the HCV-RNA level was below the detection threshold ( ⁇ ) after 8 weeks, but the 17 kDa protein level continued to decrease after 8 weeks and was almost undetectable after the end of treatment. became.
  • 8C and D show SVR cases (24 weeks in combination therapy) of genotype 2 HCV infected patients before the start of antiviral treatment (before IFN), 1 day after administration (1d), 2 weeks after administration (2w), 6 weeks after start of administration (6w), 13 weeks after start of administration (13w), detection results of 17 kDa protein (Frag F1, F2) and HCV-RNA in the serum after administration (C), and A 17 kDa protein concentration (D) was indicated.
  • the HCV-RNA level was below the detection threshold (-) after 13 weeks, but the 17 kDa protein level continued to decrease after 13 weeks and was almost undetectable after the end of treatment. became.
  • the amount of the 17 kDa protein (C4 ⁇ cleaved fragment) reflects the amount of HCV in the blood of the subject and is an indicator of the antiviral effect (HCV reduction effect) in HCV-infected patients who have received antiviral treatment. It was shown that. HCV NS3 / 4A protease is thought to maintain persistent infection by cleaving C4, thereby inhibiting complement activation and attenuating host immune responses.
  • the present invention is useful for detecting hepatitis C virus (HCV) in a blood sample of a subject with high sensitivity and determining the effect of antiviral treatment for an HCV-infected patient using the measurement result as an index.
  • C4 ⁇ cleaved fragments may be useful as new serum markers that can determine the effects of antiviral treatment early.

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Abstract

Cette invention concerne une méthode de détection du virus de l'hépatite C (VHC) dans le sang d'un sujet d'essai, la méthode comprenant la détection d'un fragment C4γ dans un échantillon de sang, généré par clivage par la protéase NS3/4A du VHC du complément C4.
PCT/JP2013/074859 2012-09-18 2013-09-13 Méthode de détection du vhc dans le sang, et procédé permettant de déterminer l'effet d'un traitement anti-vhc WO2014046045A1 (fr)

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EP3545308A4 (fr) * 2016-11-25 2020-06-24 The Government of the United States of America, as represented by the Secretary of the Navy Système d'édition de protéome et biomarqueur d'infection par veev

Citations (4)

* Cited by examiner, † Cited by third party
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JP2006308533A (ja) * 2005-05-02 2006-11-09 Mcbi:Kk 新規肝がんバイオマーカーおよび該バイオマーカーを用いた肝がんの検出方法
JP2008514723A (ja) * 2004-10-01 2008-05-08 バーテックス ファーマシューティカルズ インコーポレイテッド Ｈｃｖｎｓ３−ｎｓ４ａプロテアーゼの阻害
WO2009051259A1 (fr) * 2007-10-18 2009-04-23 Miyazaki Prefectural Industrial Support Foundation Biomarqueur pour le diagnostic d'une maladie hépatique
JP2010507093A (ja) * 2006-10-16 2010-03-04 ザ ユニバーシティ オブ ノッティンガム バイオマーカー

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008514723A (ja) * 2004-10-01 2008-05-08 バーテックス ファーマシューティカルズ インコーポレイテッド Ｈｃｖｎｓ３−ｎｓ４ａプロテアーゼの阻害
JP2006308533A (ja) * 2005-05-02 2006-11-09 Mcbi:Kk 新規肝がんバイオマーカーおよび該バイオマーカーを用いた肝がんの検出方法
JP2010507093A (ja) * 2006-10-16 2010-03-04 ザ ユニバーシティ オブ ノッティンガム バイオマーカー
WO2009051259A1 (fr) * 2007-10-18 2009-04-23 Miyazaki Prefectural Industrial Support Foundation Biomarqueur pour le diagnostic d'une maladie hépatique

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3545308A4 (fr) * 2016-11-25 2020-06-24 The Government of the United States of America, as represented by the Secretary of the Navy Système d'édition de protéome et biomarqueur d'infection par veev

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