WO2017202855A1 - Méthode d'évaluation de l'effet anti-vhb - Google Patents

Méthode d'évaluation de l'effet anti-vhb Download PDF

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WO2017202855A1
WO2017202855A1 PCT/EP2017/062437 EP2017062437W WO2017202855A1 WO 2017202855 A1 WO2017202855 A1 WO 2017202855A1 EP 2017062437 W EP2017062437 W EP 2017062437W WO 2017202855 A1 WO2017202855 A1 WO 2017202855A1
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phh
culture medium
hbv
medium
infection
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PCT/EP2017/062437
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English (en)
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Lu Gao
Zhipeng YAN
Li Wang
Xiaoju NI
Qihui Zhu
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Publication of WO2017202855A1 publication Critical patent/WO2017202855A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10131—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the present invention relates to a method to screen an agent for treating or preventing HBV infection, an assay to evaluate the anti-HBV effect of an agent for treating or preventing HBV infection, a method to produce an infection system in vitro with robust HBV infection, and a medium used in the method.
  • Hepatitis B virus (HBV) infection is one of the most prevalent viral infections and is a leading cause of chronic hepatitis. It is estimated that worldwide, 2 billion people have evidence of past or present infection with HBV. Over 240 million individuals are currently chronically infected with HBV and are therefore at high risk to develop liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). There are data to indicate -800,000 deaths per year are directly linked to HBV infection ( Lozano, R. et al., Lancet (2012), 380 (9859), 2095-2128; Goldstein, S.T. et al., Int J Epidemiol (2005), 34 (6), 1329-1339).
  • FDA-approved treatments for chronic hepatitis B include two type 1 interferons (IFN) which are IFNalfa-2b and pegylated IFN alfa-2a and six nucleos(t)ide analogues (NAs) which are lamivudine (3TC), tenofovir disoproxil fumarate (TDF), adefovir (ADV), telbivudine (LdT), entecavir (ETV), and tenofovir alafenamide (TAF).
  • IFN interferons
  • TDF tenofovir disoproxil fumarate
  • ADV adefovir
  • LdT telbivudine
  • ETV entecavir
  • TAF tenofovir alafenamide
  • HBsAg hepatitis B surface antigen
  • IFN treatment is finite, but it is known to have severe side effects, and only a small percentage of patients showed a sustained loss of hepatitis B surface antigen (HBsAg), which is believed to be the ideal outcome of anti- HBV therapy.
  • NAs are inhibitors of the HBV reverse transcriptase, profoundly reduce the viral load in vast majority of treated patients, and lead to improvement of liver function and reduced incidence of liver failure and HCC.
  • the rate of sustained HBsAg loss is ⁇ 3 after 1 year of therapy and the HCC incidence in NA-treated patient is not eliminated (Ahmed, M. et al., Drug Discov Today (2015), 20 (5), 548-561; Zoulim, F. and Locarnini, S., Gastroenterology (2009), 137 (5), 1593-1608 el591-1592).
  • HBV chronic infection is caused by persistence of covalently closed circular (ccc)DNA, which exists as an episomal form in the nuclei of infected hepatocytes, because cccDNA is the transcriptional template for viral pregenomic RNA and mRNA. Only a few copies of cccDNA can establish or re-initiate viral replication. Therefore, a complete cure of chronic hepatitis B will require elimination of cccDNA or permanently silencing of cccDNA.
  • cccDNA is intrinsically very stable and currently available therapeutics could not eliminate cccDNA which is already present in the infected cells (Nassal, M., Gut (2015), 64 (12), 1972-1984; Gish, R.G.
  • HepaRG cells which were originally isolated from a female patient suffering from hepatocarcinoma and hepatitis C virus (HCV) infection, were shown to acquire an undifferentiated morphology after few passages. Differentiated HepaRG is permissive for HBV infection. However the infection is inefficient. Other disadvantages with the system include: culturing and differentiation steps prior to infection are time-consuming, and expression of a number of metabolism enzymes in differentiated HepaRG is different from primary human hepatocytes (Kanebratt K.P. and Andersson T. B., Drug Metab Dispos (2008), 36(7), 1444-1452).
  • Inducible pluripotent stem (iPS) cells were shown to be differentiated into hepatocyte-like cells when expressing a set of liver- specific transcription factors.
  • the hepatocyte-lrke cells could support HCV and HBV infection.
  • the phenotype of these cells is more similar to fetal hepatocytes but not adult human hepatocytes, and the infection is not robust (Shlomai A. et al., PNAS (2014), 111(33), 12193-12198).
  • the primary human hepatocyte In natural infection, the primary human hepatocyte (PHH) is the only cell type that is infected by HBV. Therefore an infection assay established in PHHs is redeemed as the most physiologically relevant in vitro model, and the gold standard system for anti-HBV drug evaluation.
  • primary fetal hepatocytes were shown to be susceptible for HBV infection up to 14 days, however fetal hepatocytes differ from adult hepatocytes in gene expression profiles.
  • Micropatterned coculture (MPCC) of PHH with stromal cells was shown to support HBV replication but infection was inefficient based on the levels of HBV DNA, antigens, and cccDNA.
  • mice such as urokinase-type plasminogen activator- severe combined immunodeficiency (uPA-SCID) mice (Ishida Y. et al., Am J Patho (2015), 185(5), 1275-85) or Fah-/- Rag2-/-I12rg-/- (FRG) triple gene knockout mice (Bissig K. D. et al., / Clin Invest (2010), 120(3), 924-30) could support robust HBV replication, but the in-life experiment is lengthy and liver perfusion procedure is complicated. A few laboratories have reported the establishment of HBV infection assay with cryopreserved PHH.
  • the invention includes the procedure to plate PHH, the procedure of establishing sandwich culture, and the PHH culture medium recipe.
  • the obtained viral DNA copy number in culture supernatant is 10 folds higher than the previously reported cryopreserved assay system(Zhou, M. et al., Journal of Virological Methods (2014), 207, 29-37; Yan, H. et al., eLife (2012), l:e00049; Shlomai, A. et al., PNAS (2014), 111(33), 12193-12198).
  • the assay handling is easier and with shorter turn-around than the other HBV infection systems.
  • the robust HBV infection assay enabled the establishment of a 96-well format assay with good signal to noise ratio and high reproducibility.
  • Object in the present invention is to provide a method to produce an in vitro infection system with robust HBV infection, a method to screen a candidate agent for the treatment or prophylaxis of HBV infection, an assay for evaluating a candidate agent for the treatment or prophylaxis of HBV infection.
  • the invention relates to a method to produce an in vitro infection system with robust HBV infection, comprising the steps of: a) obtaining primary human hepatocytes; b) culturing the primary human hepatocytes; c) infecting the primary human hepatocytes obtained in step b) with HBV in a PHH culture medium; and d) culturing the infected primary human hepatocytes obtained in step (c) with sandwich culture method in the PHH culture medium so as to provide the in vitro infection system with robust HBV infection; wherein the PHH culture medium comprises a mammal serum.
  • the present invention also relates to a method for screening a candidate agent for the treatment or prophylaxis of HBV infection, wherein the method comprises the steps of: i) obtaining an in vitro infection system with robust HBV infection produced according to the invention; and ii) adding the candidate agents into the in vitro infection system obtained in step i) and incubating in a PHH culture medium, wherein the PHH culture medium comprises a mammal serum, preferably comprises a fetal bovine serum, more preferably comprises a fetal bovine serum in an amount of 1% to 20% volume percent based on the volume of the PHH culture medium.
  • the PHH culture medium comprises a mammal serum, preferably comprises a fetal bovine serum, more preferably comprises a fetal bovine serum in an amount of 1% to 20% volume percent based on the volume of the PHH culture medium.
  • the present invention also relates to assay for evaluating a candidate agent for the treatment or prophylaxis of HBV infection, wherein the assay comprises: i) obtaining an in vitro infection system with robust HBV infection produced according to the invention; and ii) adding the candidate agents into the in vitro infection system obtained in step i) and
  • the PHH culture medium comprises a mammal serum, preferably comprises a fetal bovine serum, more preferably comprises a fetal bovine serum in an amount of 1% to 20% volume percent based on the volume of the PHH culture medium.
  • a or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound.
  • a compound refers to one or more compounds or at least one compound.
  • the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
  • variable can be equal to any integer value of the numerical range, including the end-points of the range.
  • variable can be equal to any real value of the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can be 0, 1 or 2 for variables which are inherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other real value for variables which are inherently continuous.
  • Phem an abbreviation for primary human hepatocyte, is a cell that can be infected by HBV in vivo, and commonly considered to be the ideal cellular model for HBV infection.
  • cryopreservation is a process where organelles, cells, tissues, extracellular matrix, organs or any other biological constructs are preserved by cooling to very low temperatures (Pegg, David E. (2007-01-01), Principles of cryopreservation, Methods in Molecular Biology (Clifton, N.J.) 368: 39-57.) (typically -80 °C using solid carbon dioxide or - 196 °C using liquid nitrogen).
  • cryopreserved PHH means the primary human hepatocytes that are preserved by cryopreservation.
  • Cryopreserved PHH is commercially available, e.g., from Life Technologies, Corning Gentest, IV AL (In Vitro ADMET Laboratories) or Bioreclamation IVT.
  • a “PHH culture medium” is a medium that is used for maintaining viability and biological activity of PHH.
  • the PHH culture medium usually comprises the essential medium, some essentiall factors, such as human epidermal growth factor, dexamethasone, human recombinant insulin that is needed for the growth of hepatocytes, and optionally some antibiotics such as penicillin and streptomycin.
  • the invention relates to method to produce an in vitro infection system with robust HBV infection, comprising the steps of: a) obtaining primary human hepatocytes; b) culturing the primary human hepatocytes; c) infecting the primary human hepatocytes obtained in step b) with HBV in a PHH culture medium; and d) culturing the infected primary human hepatocytes obtained in step (c) with sandwich culture method in said PHH culture medium so as to provide the in vitro infection system with robust
  • the PHH culture medium comprises a mammal serum.
  • the PHHs of the present invention obtained in step a) are fresh PHHs.
  • the PHHs obtained in step a) are cryopreserved PHHs that are thawed.
  • cryopreserved PHHs are thawed (preferably in pre-warmed thawing medium,e.g., VitroGRO HT Medium) for a time minutes, preferably ⁇ 4 minutes, more preferably ⁇ 3 minutes, still more preferably ⁇ 2 minutes, most preferably ⁇ 1 minutes.
  • pre-warmed thawing medium e.g., VitroGRO HT Medium
  • the PHHs in step a) are centrifuged at 60-100 rcf (relative centrifucal foce), preferably 70 rcf, for 3-5 mins, preferably 4 mins, at 18 °C-25°C, preferably 22°C.
  • 70 rcf for 4 minutes is optimal to spin down viable PHH. It is found in current invention that appropirate combination of spin speed and spin time is critical to minimize PHH loss and while keeping cell viability.
  • step a) after centrifugation, the PHHs are plated on a collagen coated plat in pre-warmed plating medium, e.g., InVitroGROTM CP Medium.
  • pre-warmed plating medium e.g., InVitroGROTM CP Medium.
  • the thawed cryopresered PHHs are obtained by the following steps:
  • the thawing medium or plating medium used in the present method includes any medium that is known to be used in thawing or plating hepatocytes cells.
  • the thawing medium or plating medium can be fomulated by the skilled person or commercially available.
  • the thawing medium or plating medium is In VitroGRO CP Medium.
  • the thawing medium and the plating medium are the same, e.g., thery are both InVitroGROTM CP Medium.
  • the thawing medium and the plating medium are
  • the thawing medium is pre-warmed to the temperature that is appropriate for the PHH growth, such as about 37 °C.
  • the plating medium is pre-warmed to the temperature that is appropriate for the PHH growth, such as about 37 °C.
  • the PHH culture medium used in the present invention comprises mammal serum, preferablly bovine serum, more preferablly fetal bovin serum.
  • the fetal bovine serum in the PHH culture medium is l%-20% volume percent based on the volume of the culture medium, preferably, 1%-10%, more preferably at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%, preferably, the fetal bovin serum in the PHH culture medium is about 5% to about 10%, more preferably, the fetal bovin serum in the PHH cultrue medium is about 10%.
  • the PHH culture medium used in the present invention comprises Dulbecco's Modified Eagle Medium /F12, a fetal bovine serum, human epidermal growth factor, dexamethasone, and human recombinant insulin, and optionally antibiotics.
  • the human epidermal growth factor in the PHH culture medium is in a concentration of 1 to 100 ng/ml, preferablyl0-50ng/ml, more preferably about 20ng/ml, based on the volume of the PHH culture medium.
  • the dexamethasone in the PHH culture medium is in a concentration of 4 to 400 ng/ml, preferably 100-400 ng/ml, more preferably, 150-300ng/ml, most preferably, 250ng/ml, based on the volume of the PHH culture medium.
  • the human recombinant insulin in the PHH culture medium is in a concentration of 50 to 500 ng/ml, preferably 100-400 ng/ml, more preferably 150-300 ng/ml, most preferably 250 ng/ml, based on the volume of the PHH culture medium.
  • the PHH culture medium used in the present invention comprises Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1 in volume ratio), at least 5% fetal bovine serum, 5 ng/ml human epidermal growth factor, 20 ng/ml dexamethasone, 250 ng/ml human recombinant insulin, and optionally 100 U/ml penicillin and 100 ⁇ g/ml
  • DMEM Dulbecco's Modified Eagle Medium
  • F12 1: 1 in volume ratio
  • fetal bovine serum 5 ng/ml human epidermal growth factor
  • 20 ng/ml dexamethasone 20 ng/ml dexamethasone
  • 250 ng/ml human recombinant insulin 250 ng/ml human recombinant insulin
  • the PHH culture medium used in the present invention comprises Dulbecco's Modified Eagle Medium [DMEM]/F12 (1: 1 in volume ratio), 10% fetal bovine serum, 5 ng/ml human epidermal growth factor, 20 ng/ml dexamethasone 250 ng/ml human recombinant insulin, and optionally 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, based on the volume of the PHH culture medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • F12 1: 1 in volume ratio
  • 10% fetal bovine serum 5 ng/ml human epidermal growth factor
  • 20 ng/ml dexamethasone 250 ng/ml human recombinant insulin 20 ng/ml dexamethasone 250 ng/ml human recombinant insulin
  • optionally 100 U/ml penicillin and 100 ⁇ g/ml streptomycin based on the volume of the PHH culture medium.
  • the PHH culture medium used in the present invention is supplemented with DMSO.
  • the DMSO suplemented in the PHH culture medium in the prsent invention is about 0.1% to 2%, preferably more than about 0.5 and less than about 1.5%, more preferably about 0.7%-1.2%, most preferably about 0.9% to 1.1%, based on the volume of the PHH culture medium.
  • DMSO free environmentm Compared to the medium with 2% DMSO (Zhou, M. et al., Journal of Viwlogical Methods (2014), 207, 29-37; Yan, H. et al., eLife (2012), l:e00049) or DMSO free effetm (Shlomai, A.
  • infecting PHH is performed at least about 6 hours after plating the PHH on a plate, preferably about 6-72 hours, more preferably about 10-24hours, most preferably about 24 hours.
  • removing the plating medium and replenishing the PHH with PHH culture medium is preformed about 2-6 hours before infecting PHH, preferably, about 4 hours.
  • sandwitch culture of the infected PHH is performed at least about 6 hours after infecting the PHH, preferably about 8-72 hours, more preferably about 10-24 hours, most preferably about 16 hours.
  • the cell seeding densitity obtained in step b) should be determined by the skilled person according to the status of the cell.
  • the cell seeding density should also be adjusted according to the plate to be seeded in.
  • the cell seeding density in the plate can be about 0.2-0.5 million cells (preferably about 0.25-0.45million cells, more preferably about 0.3-0.4 million cells, most preferably about 0.33-0.38 million cells or about 0.35 million cells) per well in a 24 well plate.
  • Optimal seeding density is critical for ensuring PHH attachment and achieve full confluence.
  • the cell seeding density in the plate can be about 2 x 10 4 to 8 x 10 4 cells (preferably about 3 x 10 4 to 7 x 10 4 cells, more preferably about 4 x 10 4 to 6 x 10 4 cells, most preferably about 5.5 x 10 4 to 6 x 10 4 or about 5.8 x 10 4 cells) per well in a 96- well plate.
  • the HBV used in the infection in step (c) is in the form of HBV patient serum or HBV virus.
  • HBV patient serum or HBV virus is in PHH culture medium (e.g., pre-warmed to the temperature appropriate to the PHH growth, e.g., 37°C).
  • the PHH culture medium is supplemented with DMSO.
  • the DMSO is suplemented in the PHH culture medium in the prsent invention in an amount of about 0.1% to 2%, preferably more than about 0.5 and less than about 1.5%, more preferably about 0.7% to 1.2%, most preferably about 0.9% to 1.1%, based on the volume of the culture medium.
  • the culture medium in the step d) is supplemented with DMSO, preferably the amount of the DMSO is 0.1% to 2% volume percent, more preferably 0.5% to 1.5% volume percent, preferably ⁇ 2% volume percent, based on the volume of the PHH culture medium.
  • collagenous matrix e.g., matrix gel
  • PHH culture medium is further supplemented with collagenous matrix (e.g., matrix gel), preferably, collagenous matrix (e.g., matrix gel) is about 0.1-0.4mg/ml, or about 0.1-0.3mg/ml, or about 0.2- 0.3mg/ml, or about 0.25 mg/ml, based on the volume of the culture medium.
  • the invention further relates to a medium used in the present invention to culture PHH, wherein the medium comprises a PHH culture medium compirsing mammal serum, preferably bovine serum, more preferably fetal bovine serum.
  • the fetal bovine serum in the PHH culture medium is l%-20% volume percent based on the volume of the culture medium, preferably at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%, preferablly, the fetal bovin serum in the PHH culture medium is about 5% to about 10%, more preferablly, the fetal bovin serum in the PHH cultrue medium is about 10%.
  • the present invention also relates to method for screening a candidate agent for the treatment or prophylaxis of HBV infection, wherein the method comprises the steps of:
  • the PHH culture medium comprises a mammal serum, preferably comprises a fetal bovine serum, more preferably comprises a fetal bovine serum in an amount of 1% to 20% volume percent based on the volume of the PHH culture medium, preferably, treating the cells obtained in step i) with serial dilutions of the agents diluted with the PHH culture medium; and optionally,
  • the present invention also relates to an assay for evaluating a candidate agent for the treatment or prophylaxis of HBV infection, wherein the assay comprises:
  • the PHH culture medium comprises a mammal serum, preferably comprises a fetal bovine serum, more preferably comprises a fetal bovine serum in an amount of 1% to 20% volume percent based on the volume of the PHH culture medium, preferably, treating the cells obtained in step i) with serial dilutions of the agents diluted with the PHH culture medium; and optionally
  • the time to treat the PHH with the agents should be adjusted according to the agent and can be determined by the skilled person.
  • the treatment lasts for at least 6 days, preferably 6-12 days, more preferably 9 days.
  • the medium comprising the candidate agents to be screened or evaluated should be refreshed, e.g., according to the agent and the cell status. In one embodiment, the medium comprising the agents to be evaluated is refreshed every 2-4 days, preferably every 3 days.
  • the HBsAg level is the extracellular HBsAg level, which is preferably measured with Chemiluminescence Immuno Assay. In one embodiment, the HBeAg level is the extracellular HBsAg level, which is preferably measured with Chemiluminescence Immuno Assay.
  • the HBV DNA is the extracted and determined with quantitative
  • FIG. 1 the viral DNA and antigen levels in HBV infected cryopreserved PHH growing in 24- well plate.
  • PHH lot AKB and PSM were infected with HBV purified from HepAD38 cell at 50 genomic equivalents (GE) per cell.
  • PreSl was added together with virus to block the entry of HBV and serve as the negative control.
  • Viral DNA was extracted from supernatant of cell culture, quantified using qPCR, and shown as copies per ⁇
  • FIG. 2 HBsAg and HBeAg levels in YJM cells infected by HBV for 27 days.
  • YJM cells were infected with HBV patient serum genotype C, GE number of 5 or 20.
  • PreSl was added together with virus to block the entry of HBV and serve as the negative control.
  • Extracellular HBsAg ( Figure 2A) and HBeAg ( Figure 2B) were measured.
  • Figure 3 the importance of specific ingredients in medium recipe and assay procedure to HBV infection in cryopreserved PHH.
  • the extracellular viral DNA and HBsAg were normalized to 1.
  • Figure 4 the signal to noise ratio, tool compound validation, and reproducibility of the 96-well plate format.
  • YJM cells were seeded at the density of 5.8xl0 4 cells per well in 96-well plate format, and infected with HBV at 100 GE per cell for 12 days.
  • Figure 4A Signal to noise ratio of HBsAg (left panel), HBeAg (middle panel) and viral DNA
  • IC 50 the concentration of a compound, which produces 50% of the inhibition effect for that target
  • DMEM/F12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
  • EDTA ethylenediaminetetraacetic acid
  • TAE tris-acetate electrophoresis
  • PHH primary human hepatocytes
  • CLIA Chemiluminescence Immuno Assay
  • PEG polyethylene glycol
  • Plateable Cryopreserved Female Human Hepatocyte or Plateable Cryopreserved Male Human Hepatocyte were purchased from Bioreclamation IVT, Cat# F00995-P and Cat# M00995-P (respectively).
  • Each lot of PHH such as lot AKB, PSM and YJM, is derived from an individual donor.
  • HBeAg or HBsAg were measured by using the HBeAg or HBsAg Chemiluminescence Immuno Assay (CLIA) Quantitative kit (Autobio) according to the manufacturer's direction.
  • CLIA Chemiluminescence Immuno Assay
  • HBV DNA from cell supernatants was extracted using the MagNA Pure 96 System
  • HBV-Forward 5'-AAGAAAAACCCCGCCTGTAA -3' (SEQ ID NO: l)
  • HBV-Reverse 5'-CCTGTTCTGACTACTGCCTCTCC -3' (SEQ ID NO:2)
  • HBV-Probe 5'-TARMA-CCTGATGTGATGTTCTCCATGTTCAGC-BHQ2-3' (SEQ ID NO:3)
  • Plasmid pBR322-HBV1.3 (SEQ ID NO: 4): 10-fold dilution from 2 x 10 6 copies / ⁇ to 2 copies / ⁇ ) was used as standard for qPCR. Quantification of extracted HBV DNA was performed using a LightCycler® 480 machine (Roche). 5 ⁇ of DNA sample was added to 10 ⁇ of PCR master mix (Roche, Cat. no. 04887301001), 0.5 ⁇ of 10 ⁇ forward primers, 0.5 ⁇ of 10 ⁇ reverse primer, 0.2 ⁇ .
  • PCR was performed using the following settings: pre-incubation (10 min, 95°C), and PCR (45 cycles with 10 sec at 95°C for denaturing, and 30 sec at 60°C for annealing and extension). DNA copy numbers were calculated from Cp values based on a HBV plasmid DNA standard curve.
  • Cell Counting Kit-8 (CCK-8, Dojindo) was performed to determine cell viability according to the manufacturer's instruction.
  • Cryopreserved PHH culture medium contains Dulbecco's Modified Eagle Medium (DMEM)/F12 (1: 1) (Gibco, Cat. 11320- 033),10% fetal bovine serum (Gibco Cat. 10099141), 100 U/ml penicillin, 100 ⁇ g/ml
  • DMEM Dulbecco's Modified Eagle Medium
  • F12 1: 1
  • fetal bovine serum Gibco Cat. 10099141
  • penicillin 100 ⁇ g/ml
  • HepAD38 is a HepG2 hepatoblastoma cell line that produce HBV virus under the regulation of tetracycline.
  • HepG2 2.2.15 cell line is also derived from HepG2 cell line that stably produce HBV particle. The viruses purified from HepAD38 or HepG2 2.2.15 cells were added to the cells at the desired inoculum together with 4% PEG
  • HBeAg was 383 NCU/ml and 176 NCU/ml in AKB and PSM cells respectively ( Figure 1C and IF).
  • PreSl 200nM PreSl was added together with the virus to block the viral entry and present throughout the experiment to block re-infection, DNA titer, HBsAg level, and HBeAg level dropped to detection limit level.
  • HBV DNA level in this study was more than 10-fold higher (Zhou, M. et al., Journal of Virological Methods (2014), 207, 29-37; Yan, H. et al., eLife (2012), l:e00049; Shlomai, A. et al., PNAS (2014), 111(33), 12193-12198).
  • the 96-well plate format was established and optimized based on 24-well plate format that is described above. Briefly, cryopreserved PHH (BioreclamationlVT, Lot YJM) was thawed at 37°C and gently transferred into pre-warmed InVitroGRO HT medium (BioreclamationlVT, Cat: PY-HMD-02). The mixture was centrifuged at 70 relative centrifugal force (RCF) for 4 minutes at RT, and the supernatant was discarded.
  • RCF relative centrifugal force
  • MW8000 (Sigma, Cat. P1458-50ML) and 1% DMSO (Sigma, Cat. D2650). 5.8 x 10 6 genomic equivalents of HBV were added into the medium. At 16 hours post-infection, the cells were gently washed with PBS and refreshed with PHH culture medium supplemented with 1% DMSO, and 0.25mg/ml matrix gel (Corning, Cat.

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Abstract

La présente invention concerne une méthode pour cribler un agent pour le traitement ou la prévention d'une infection par le virus de l'hépatite B (VHB), un dosage pour évaluer l'effet anti-VHB d'un agent pour le traitement ou la prévention d'une infection par le virus de l'hépatite B, une méthode pour produire un système d'infection in vitro avec une infection résistante au VHB.
PCT/EP2017/062437 2016-05-26 2017-05-23 Méthode d'évaluation de l'effet anti-vhb WO2017202855A1 (fr)

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WO2004011622A2 (fr) * 2002-07-30 2004-02-05 Massachusetts Institute Of Technology Procedes permettant d'injecter des hepatocytes primaires et de les mettre en culture sur boite de petrie et milieu correspondant
EP2067856A1 (fr) * 2006-09-26 2009-06-10 Toyo Boseki Kabushiki Kaisha Procede destine a permettre la proliferation du virus de l'hepatite, fibre creuse pour cultiver des cellules infectees par le virus de l'hepatite et leur utilisation
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